critical choices in hplc

Welcome

Selecting Column Stationary Phases and Dimensions

Gradient HPLC Factors to Consider

The Fundamentals of HPLC Detectors

Video IntroductionLaura Bush

Column SelectionTony Edge and Dawn Watson

Gradient MethodsDwight R Stoll and

Scott Fletcher

Detectors Scott Fletcher

Table of contentsTOC

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in HPLC

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INTR

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To take full advantage of the interactive featuers of this PDF be sure to view it in Adobe Acrobat Reader

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Choosing a high performance liquid chromatography (HPLC) stationary phase requires an understanding of the chemistry of both the stationary phase and the molecules that are being separated This article highlights some of the key criteria to be aware of when selecting a column including specifications about the columnrsquos physical parameters such as length and diameter and also an understanding of the chemistry highlighting primary and secondary interactions with the stationary phase and support substrate material Additionally this article discusses the dependence of retention factor on the mobile-phase pH and how acids and bases are affected Ultimately consideration of a columnrsquos physical characteristics combined with a thorough understanding of the stationary-phase chemistry is essential for achieving the best separation

When considering the mode of chromatography that should be employed for a given separation it is necessary to understand some basic chemistry In general the stationary phase is designed to retain the analyte with the mobile phase providing additional retention by having limited solubility of the analyte In reversed-phase chromatography the stationary phase is less polar than the mobile phase therefore less-polar molecules will be attracted to the stationary phase and the polar mobile phase will have limited solubility resulting in a greater retention of hydrophobic analytes on the stationary phase The difference in the retention of different analytes based on this chemistry between the analyte stationary phase and mobile phase will determine the quality of the separation

One physiochemical parameter that is very useful when considering the retention of an analyte on a reversed-phase HPLC column is the solubility or the log partition coefficient (log P) of the analyte

4

Comparison of Reversed-Phase Selectivity of Solid-Core HPLC Columns

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Optimizing Chromatographic Results with Mobile-Phase Preheating

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log Poctwat = log [solute]octanol

[solute] un-ionized water( ) [1]

SELECTING COLUMN STATIONARY PHASES AND DIMENSIONSTony Edge and Dawn Watson

5

The log P value determines how soluble the compound is larger positive numbers indicate that the compound is more hydrophobic and less water soluble and negative numbers indicate that the compound is quite polar In the case of ionizable analytes the distribution coefficient (log D) provides a better estimate of the analyte solubility as it takes into account all forms of the analyte molecule (ie ionized and unionized Equation 2) Log D is pH-dependent hence when it is measured the pH at which the measurement was carried out must be specified

In general the more carbon atoms present in a molecule the greater the value of log P and in turn the greater the retention under reversed-phase separation conditions The shape of the molecule can also affect analyte solubility with straight-chain molecules in general having larger log P values hence greater retention is seen for branched chain molecules Furthermore the greater the saturation of the carbon-carbon bonds the greater the log P value and hence a greater retention will be observed In general aliphatic compounds exhibit greater retention than compounds with induced dipoles which have greater retention than compounds containing permanent dipoles which have greater retention than weak bases weak acids and strong acids It should be noted at this point that most molecules have many different functionalities which can make the exact interpretation quite tricky

For a separation to occur the high performance liquid chromatography (HPLC) column must be able to differentiate between similar molecules As has already been stated this can be difficult to judge because there may only be small differences between molecules mdash perhaps a difference of one carbon unit or perhaps two or three differences that could cancel each other out in terms of the overall retention It is necessary therefore to consider the analytes that will be analyzed and how to maximize the differences in interactions between the analytes and the stationary phase The most predominant modes of interactions when using a reversed-phased column are hydrophobic dipolendashdipole and πndashπ interactions

There are other parameters to consider other than the chemistry between the stationary phase and the analyte For a separation to occur effectively the column has to have sufficient available surface area to load the sample In addition the pH temperature and pressure can and do have an effect on the selectivity of the separation mechanism and also on the robustness of the assay

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[2]log Doctwat = log ( )[solute]octanol

[solute] ionized water [solute] neutral

water+

SELECTING COLUMN STATIONARY PHASES AND DIMENSIONS

6

Column SpecificationsColumn manufacturers will generally provide information regarding the following aspects of an HPLC column

The nature of the solid support This is the material to which the bonded phase is attached most commonly silica Silica particles can be fully porous superficially porous or nonporous The silica particle type will have an effect on the chromatography and can affect the efficiency of the separation mechanism

Bonded phase This is the chemistry of the moiety that is bonded to the silica surface Bonded phases are typically based on an alkyl or phenyl group and it is the interaction between the bonded phase and the analytes that primarily drives the separation mechanism

Particle size Particle size is measured as the average diameter of the column packing particles Manufacturers will also report the distribution of the size of the particles used to pack the column In general smaller particles and tighter particle-size distributions will give sharper and hence more efficient chromatography

Particle shape (irregular and spherical) Irregularly shaped particles can be less expensive but they provide separations with poor efficiency because of the way they pack into a column It is much easier to pack a column with regularly shaped particles than it is with irregularly shaped particles Irregularly shaped particles are also prone to shearing which creates fines that can block columns causing both chromatographic and instrument-based problems such as poor peak shapes and increased back pressure

Pore size The majority of the stationary phase exists within the silica pore structure therefore the analytes have to access the pores to interact with the bulk of the bonded stationary phase This means that the pore size needs to be appropriate because a big molecule will not fit into small pore For small molecules the pore size should be about 150 Aring or less Larger molecules (gt2000 Da) need bigger pores of 300 Aring The larger the pores the smaller the surface area which means that the analytes will have less bonded phase with which to interact

Surface area Columns with high surface area may exhibit greater retention loading capacity and resolution However low-surface-area columns have their advantages They equilibrate between runs more easily which can be particularly useful in gradient HPLC Also the reduced porosity results in better kinetics meaning that there is less dispersion in the column

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7

Temperature limit Manufacturers will often quote an upper temperature limit which is the highest temperature at which the column can be operated without damaging the stationary phase

pH range This is the working pH range of the column Pure silica has a working pH range of 25ndash75 outside of this range the silica will be hydrolyzed At low pH hydrolysis of the silyl ether linkage between the bonded phase and silica surface can also occur resulting in a loss of both retention and loading capacity The bonded phase can act as a protective covering for the silica but in general at high pH the silica surface will eventually hydrolyze These problems can both be exacerbated when operating at higher temperatures especially as the temperature limit of the column is reached

Endcapping The endcapping process covers surface silanol species which would otherwise cause unwanted secondary interactions and poor peak shape particularly when analyzing polar or ionizable species To endcap a column the surface silanols are reacted with a small silylating reagent such as trimethylchlorosilane which produces an endcapped trimethylsilyl (TMS) species as shown in Figure 1

Carbon load Carbon load () describes the amount of ligand bonded to the surface It also describes the background carbon load that is present if using unmodified silica In general the higher the carbon load the lower the number of surface silanols It should be noted that that not all C18 columns will have the same percent carbon and columns with different endcapping groups cannot be compared because endcap groups contain different numbers of carbon atoms

Surface coverage Surface coverage is a better measure of retention or the hydrophobicity of a column It is defined as the mass of stationary phase per unit area which is bonded to the support and is expressed in units of micromolm2 As can be seen in Figure 2 with high surface coverage there are fewer free surface silanols with which analytes can interact to cause unwanted secondary interactions If there is lower surface coverage there will be more surface silanol groups available to the analyte which will ultimately result in different interactions between the analyte and stationary phase However in some cases such interactions could be advantageous if a change in selectivity is desired for a separation

Secondary InteractionsSilica is often referred to as type A or type B silica or type 1 and 2 silica The difference between the two types relates to the manufacturing process and the resulting purity of the silica produced Type 1 silica is manufactured by

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Figure 1 Diagram showing various bonded phase groups including the tri-methylsilyl (TMS) group resulting from endcapping with trimethylchlorosilane

Si mdash O mdash Si

Si mdash O mdash Si

Si mdash O mdash Si

Si mdash O mdash Si

Si mdash O mdash H

Si mdash O mdash H

Si mdash O mdash H

O

O

O

O

O

O

HO

Lone acidic silanol

C8 bonded phase

C8 bonded phase

TMS group

8

polymerizing a metal silicate molecule which results in high metal content in the final silica that is produced The metal atoms will tend to migrate to the surface where they are energetically favored At the surface they affect the acidity and hence the reactivity of the silica increasing the strength of the secondary interactions which is very noticeable with basic compounds Type 2 silica is produced using an organosilicate monomer and therefore has less metal content this type of silica is less acidic and less reactive toward basic compounds It is not possible to say that one of these types of silica is better than another unless the analytes are also discussed in the same context

As well as type 1 and type 2 silicas there are also different forms of silanol groups that exist at the surface Different types of silanol species on the surface can interact to different degrees For example acidic lone silanols will cause the most peak tailing with basic analytes A hydrated silanol will not induce much interaction because it is lower in energy Some examples of the different forms of surface silica are shown in Figure 3

Types of Solid SupportAdvancements in solid support are helping ensure faster and more efficient HPLC They include the following supports

Corendashshell Corendashshell particles have a solid silica core and a porous outer layer In comparison to traditional fully porous silica supports they produce faster and more efficient chromatography They also have a narrow size distribution which can contribute to increased chromatographic efficiency

Monolithic silica rods Monolithic silica rods allow for high-speed separation with good resolution and shorter analysis time These supports contain macropores that are greater than 50 nm in diameter and mesopores that are 2ndash50 nm in diameter This structure allows separations to be performed at very low back pressures and at high mobile-phase linear velocities or with samples that are viscous Monolithic silica rods are also good for direct injection of dirty samples of plasma or food extracts Because of the increased flow rate analysis time is also reduced

Fully porous silica (traditional silica) Fully porous silica has a high surface area and excellent mechanical strength It can be used as a support material for normal-phase chromatography and with surface modification it can be used for reversed-phase chromatography As previously stated one of the major drawbacks of silica is its susceptibility to hydrolysis at pH extremes One way manufacturers have overcome this problem is to use organosilica hybrids An organo group grafted into the silica layers makes them more resistant to

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Figure 2 Diagrams showing high surface coverage with high ligand density (upper diagram) and low surface coverage with low ligand density (lower diagram)

Si

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

O

O O

H3C ndash Si ndash CH3

CH3


CH3

H3C ndash Si ndash CH3 H3C ndash Si ndash CH3 H3C ndash Si ndash CH3 H3C ndash Si ndash CH3


Si

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

O

O O

CH3


CH3CH3

High surface coverage ndash High ligand density

Low surface coverage ndash Low ligand density

Figure 3 Silica surface silanol groups

Vicinal hydrated

Bridged (vicinal)

Surface metal ion

Lone acidic

Geminal

Metal activated

9

dissolution at high pH and this characteristic will extend the column life and applicability in applications that require the use of high pH

Porous graphitic carbon This is a unique chemistry phase Porous graphitic carbon is composed of flat sheets of hexagonally arranged carbon atoms consequently it has no surface silanols and therefore unwanted interactions will not occur Porous graphitic carbon phases have total pH stability meaning that they can be used over the full pH range This wide applicability of pH makes them ideal for the analysis of compounds where extreme pH levels are required to drive the separation This capability is very good for the separation of strong acids and bases where the neutral form of the molecule may be required to increase retention which requires extremes of pH This phase is very versatile and can be used in reversed-phase LC normal-phase LC and hydrophilic interaction chromatography (HILIC) and for LCndashmass spectrometry (MS) applications

Dependence of Retention Factor on pHThe pH of the mobile phase is an important parameter for the retention of acidic and basic compounds As one changes the pH (Figure 4) it is possible to change the ionization state of acidic and basic molecules this renders them more or less polar which in turn affects their retention time For basic compounds at a low pH the base can accept a proton to become positively charged As the pH increases the protons in the surrounding environment are removed until eventually all the basic protons within the analyte are abstracted leaving a neutral species When the molecule is charged there is little retention but as pH increases the neutral form of the molecule becomes apparent and retention is increased

The opposite situation occurs for acids which are proton donors At low pH the neutral form of the molecule exists and hence the molecule will exhibit greater retention As the pH is increased above the analyte pKa any acidic protons will be removed from the analyte to produce a negatively charged species that exhibits less retention in comparison to its neutral counterpart

A good rule of thumb for determining the extent of analyte ionization is the 2 pH rule For acids at 2 pH units above the analyte pKa the analyte will exist in the ionized (negative) form Conversely for basic moieties adjusting the pH 2 pH units below the pKa will produce the ionized (positive) species Therefore for ionizable molecules retention can be altered and controlled by changing the pH of the mobile phase

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Figure 4 Plot showing the dependence of retention factor for various pharma-ceutical compounds on pH Mobile phase 35 acetonitrile 65 20 mM buffer

pH

log

kprime

10

100

1

012 4 6 8 10 12 140

AcetaminophenDoxepin

IbuprofenImipramine

Nortriptyline Lidocainep-Toluamide

Figure 5 Structures of various stationary phases and the associated analyte interactions

Si

O

Si

O

Si Si

OH

N

C

O O O O

AlkylDispersive

Phenylπ-π interactions

CyanoElectrostatic

dipole

SilicaH-bonding

Nonpolar Polar

10

Initial Column Selection and Analyte Functional GroupsWe sometimes make the assumption that there is only one mode of interaction in chromatography when actually there are multiple modes of interactions that can occur simultaneously within a column It is important to understand where those different modes of interactions come from and that on some occasions a separation scientist may want a particular interaction to drive a separation and on other occasions that interaction may be undesirable Thus it is not possible to say that a particular column is good or bad without describing the type of compounds that are being separated

So how do we go about selecting our column given that there are no really bad columns To answer this we need to be able to fingerprint the retention mechanisms of a column and better understand how they interact with the molecules that we are trying to separate

AnalytendashStationary Phase InteractionsA variety of modes of interaction potentially can exist between analytes and the stationary phase

Dispersive forces These forces exist in all molecules and are the major retention mechanism for alkyl phases Retention is proportional to the hydrophobicity of the molecule This means that the more hydrophobic the molecule the longer the retention time

Charge-transfer (π-π) interactions Charge-transfer interactions are prevalent in both unsaturated and aromatic compounds and greater retention is possible for these compounds when a phase is used that exhibits these types of interactions

Hydrogen bonding and dipolendashdipole interactions As the polarity of the analyte molecule is increased different retention mechanisms need to be investigated such as hydrogen bonding and dipolendashdipole interactions A polar analyte interacts with the stationary phase through hydrogen bonding or a dipolendashdipole interaction Figure 5 illustrates the interactions based on phases and modes

Column Selection and CharacterizationA change in selectivity can help change the retention mechanism and the elution order of analytes Figure 6 shows separations obtained using three phases cyano phenyl and C8 Differences can be seen in retention order particularly for

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Figure 6 Chromatograms showing the shift in selectivity obtained using cyano phenyl and C8 stationary phases

CN phase

C8 phase

Time (min)0

1

1

1

2

2

2

3

3

3

4

4

45

5

5

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6

6

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7

7

5 15 20

Phenyl phase

Figure 7 Column characterization plot H = hydrophobicity S = steric or shape effect A = hydrogen bond acidity B = hydrogen bond basicity C(28) = silanol ionization at pH 28 C(70) = silanol ionization at pH 70

C(70)10

C(28)

H10

B

S

A

11

the compounds that are eluted first Some compounds are not eluted at the same retention time from the various stationary phases and a degree of orthogonality appears among these different phases

We have talked about different modes of interactions but how can we start to quantify those modes The Physical Quantitation Research Institute (PQRI) has been trying to gain a better understanding of the different interactions that molecules can have with the stationary phase The radar plot shown in Figure 7 was generated for a Hypersil Beta Basic C18 column This is the fingerprint or characterization of this particular column To get this information it is necessary to test individual columns under the same conditions using identifiable test probes throughout the testing regime

Column ComparisonUsing the PQRI method of fingerprinting columns it is possible to compare and contrast different column chemistries to assess which retention mechanisms dominate and can be exploited to differentiate between differences in analyte molecules Figure 8 illustrates the difference between type A and type B silica (both from the same manufacturer) The type A silica is made with sodium silicate monomer which has a high metal content this metal content increases the acidity of the surface silanols and thus may promote secondary interactions with basic analytes

In comparison the type B silica is manufactured from an organosilicate which has a very low metal concentration As a consequence the surface silanol activity at pH 28 is markedly different With the more acidic silanols greater interaction of positively charged analytes can occur whereas with the high-purity silica these types of interaction will be reduced

Common Stationary-Phase TypesSome common stationary phases used in chromatography include the following

C18 or octadecylsilane (ODS) This stationary phase is potentially the most retentive alkyl phase and is used for 70ndash80 of all applications

Silica Silica is used for normal-phase chromatography or HILIC This stationary phase is ideal for polar molecules

Cyano Cyano phases can be run in both normal-phase and reversed-phase modes but care must be taken when switching between these two modes to ensure that both the column and HPLC system are suitably equilibrated with the new mobile-phase composition

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Figure 8 Column characterization plots for type A and type B columns (left) and two type B columns See Figure 7 for symbol identification

C(70)10

C(28)

H10

B

S

A

C(70)10

C(28)

H10

B

S

A

Type A Type B Both Type B

12

Amino Amino columns offer a variety of modes of interaction In HILIC mode amino or amide phases are very good for sugar analysis but they can also be run in reversed-phase and normal-phase modes

Phenyl and pentafluorophenyl (PFP) These stationary phases are predominantly used for analyzing polar and moderately polar compounds

Diol Diol phases are commonly used in reversed-phase and normal-phase separations but are being used more frequently as HILIC phases

Anion exchange These stationary phases are good when trying to retain organic acids

Porous graphitic carbon Porous graphitic carbon can be used for normal-phase and reversed-phase separations as well as in HILIC applications These phases are very good for separating extremely polar compounds

Physical Properties of ColumnsThe physical properties of a column need to be considered when selecting a column for a particular application Some of these properties are

Particle size A smaller particle size equates to better resolution however there is a compromise the smaller the particle size the higher the back pressure in a column Efficiency is inversely proportional to particle size therefore if particle size is decreased efficiency will increase

Length Increasing the length of the column increases resolution however by doubling the column length (which will double analysis time and increase the cost of the column) a gain in resolution of only 14 times is achieved It also should be noted that increasing column length can alter analyte selectivity under gradient elution conditions

Internal diameter Reducing the internal diameter of the column reduces the flow rate that is required to reach the optimum linear velocity If the absolute flow rate is maintained the back pressure will increase as column diameter decreases

Maximize sensitivity The sensitivity of an analytical separation can be improved by adjusting various column and method parameters including reducing the column length and internal diameter using smaller particle sizes (to increase the efficiency of the separation) minimizing extracolumn volumes and increasing the flow rate Sensitivity can also be increased by decreasing the background noise from other matrix components by using appropriate sample preparation techniques

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Mass loading considerations The amount of sample that can be loaded onto a column is dependent on the column dimensions and stationary phase type Loading an excess of sample onto a column will result in poor peak shapes (broad peaks change in apex retention time and fronting or tailing peaks) and will ultimately decrease resolution

Peak capacity This parameter is important in modern HPLC and describes the number of components that can be successfully separated with a given column under gradient conditions Peak capacity (P) is calculated using equation 3 The peak capacity can be optimized by changing the gradient time as a function of flow rate

where tg is the gradient time and w is average peak width

SummaryIt has been shown that numerous parameters pertaining to the stationary phase and dimensions of an HPLC column should be considered to select the correct column for a particular application

This article is based on the LCGCndashCHROMacademy web seminar ldquoCritical Choices in HPLC mdash Selecting Column Stationary Phase and Dimensionsrdquo presented on March 20 2014 by Tony Edge and Dawn Watson

Tony Edge PhD is a Scientific Advisor for Chromatography Consumables at Thermo Fisher Scientific in Stockport UK

Dawn Watson PhD is a CHROMacademy Technical Expert with Crawford Scientific in Strathaven Lanarkshire UK

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P asymp 1 + [3]tgw


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High-performance liquid chromatography (HPLC) separations using gradient elution generally are more powerful than those performed using isocratic elution Gradient elution is more complex however This article provides the essential information for understanding gradient elution and how to use it including how to account for dwell volume determine the washout volume calculate peak elution and column reequilibration times minimize drifting baselines and how to implement an isocratic hold It also explains the various type of pumps used in gradient separations and how to test the performance of your formed gradient It also explains the benefits of running a scouting gradient which is the most important step in developing any method to account for the wide polarity of analytes Lastly tips are provided for effective method transfer of gradient methods

Isocratic Versus Gradient ElutionFirst we are going to compare isocratic and gradient elution from the perspective of characteristics of these separations Figure 1 shows an example of an isocratic separation of a relatively simple mixture using a mobile phase composed of 30 acetonitrile the strong solvent Some of the hallmarks of an isocratic separation are that the early-eluted peaks are not resolved nearly as well as the peaks eluted midway through the analysis We see increasing peak widths with increasing retention time one of the phenomena that accompanies increasing width is decreasing peak height which leads to poorer detection limits and resolution for later-eluted analytes We also have a relatively long analysis time because of the late elution of the highly retained compounds and especially with complex samples we have the potential for contamination of the column itself by the strong retention of highly retained components in the sample

If we then look at a typical gradient elution chromatogram shown in Figure 2 the key difference compared to the isocratic elution is that the solvent composition is changed during the run In this case we are starting

GRADIENT HPLC

Factors to ConsiderBy Dwight R Stoll and Scott Fletcher

Tune Your Mixing Volume for Gradient Generation

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Eliminating Delays Caused by Column Wash and Recondi-tioning in Gradient Methods

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initially at 20 acetonitrile in the mobile phase and then moving to 60 in a linear gradient over 30 min One of the key differences that results is that we have improved resolution for both the early- and late-eluted compounds Also when we have analytes with very diverse chemistries we have increased or improved detection capabilities because now the later-eluted compounds have much narrower peak widths and therefore much higher peak heights

We also have an increased ability to separate complex samples mainly because we can spread the peaks out better and because on average they have narrower widths This approach can translate to a shorter analysis time Because the mobile phase has the ability to elute strongly retained compounds at the end of the run column deterioration from the retention of those compounds is avoided

One of the potential downsides of gradient elution is that the instrumentation required tends to be more expensive There is also a potential for precipitation of buffer salts at the interface where the two solvents are mixed to produce the gradient and for a change in mobile-phase composition over time Reequilibration of the column following the gradient separation inevitably increases analysis time and differences between the pumping systems used in different instruments can cause difficulty when transferring methods

Gradient Elution ApplicationsSome of the common applications of gradient separations include rapid ldquoscouting runsrdquo during method development to get a sense for how the compounds in the sample are behaving Gradient elution is also very effective for removal of strongly retained compounds and interfering compounds in the sample This is the major reason why many chromatographers use gradient elution mdash it is just too risky to perform isocratic work on a sample that you donrsquot know very well because some of the analytes may remain in the column

We also use gradient elution with low-concentration analytes particularly when those compounds are dissolved in a weak solvent such as in the case of using reversed-phase LC with a weak solvent like water For example it is possible to inject extremely large volumes of sample into a reversed-phase column and essentially preconcentrate or focus the analyte at the inlet of the column which can significantly improve detection limits

It is also true that for large molecules such as polymers of various kinds including peptides and small proteins retention has a very strong dependence on the composition of the mobile phase In these cases gradient elution is required otherwise it is very difficult to elute these compounds from the column which can lead to irreversible retention of those compounds This relationship is exemplified

Figure 1 An example of an isocratic separation of a relatively simple mixture of herbicides using a mobile phase composed of 30 acetonitrile in water where the solvent composi-tion stays the same over the entire run Peaks 1 = tebuthiuron 2 = prometon 3 = prometryne 4 = atra-zine 5 = bentazon 6 = propazine 7 = propanil 8 = metolachlor

0

3

12 4

5

6

78

Time (min)

25 50 75

Figure 2 Example of a gradient elution chromatogram of the same sample mixture analyzed in Figure 1 where a 20ndash60 acetonitrile gradient is used during the run

Time (min)

0 5 10

1

2

3

45

6

7

8

15 20 25 30

GRADIENT HPLC

Factors to Consider

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in Figure 3 which is a plot of log of retention factor k versus the composition of the mobile phase expressed as a ratio Φ

As can be seen in the figure for a rather small simple molecule like benzene the retention of that molecule is reduced as we increase the amount of organic solvent in the mobile phase but that change is rather slow compared to a peptide like enkephalin which has a much steeper slope For a small protein like lysozyme this dependence becomes very strong and with a small change in the concentration of organic solvent in the mobile phase the compound is either very highly retained or not retained at all So this dependence of the retention of these molecules on the mobile-phase composition is very important

Benefits of Gradient ElutionAs mentioned one of the major benefits of gradient elution is the fact that narrow peaks are obtained where the peak width is nominally independent of the retention time So letrsquos investigate this advantage in greater detail A significant factor is the focusing of the analyte band at the inlet of the column Figure 4 includes plots of two analytes and shows how they are affected during a gradient separation below the column diagram The top one shows the distance that the analytes travel in the column as a function of time and the bottom plot shows the retention as a function of time

These two plots provide different perspectives on how the analytes are behaving inside the column But the conclusion is that when the elution strength of the mobile phase is low the analytes come into the column and basically stick at the column inlet mdash they have very high retention and very low velocity As the elution strength of the mobile phase increases the retention of those compounds goes down as shown in the lower graph in Figure 4 and at the same time their velocity increases

A secondary effect that contributes to the narrow peak width is that the mobile-phase composition in the column close to the analyte band is weaker than the solvent composition thatrsquos coming behind the band Thus the mobile phase that follows the analyte through the column tends to have a slightly higher elution strength which tends to give the analyte molecules in the tail of the peak a higher velocity whereas the solutes on the leading edge of the peak have slightly higher retention and lower velocity These factors again compress the band somewhat and also lead to narrow peak widths

Figure 3 A plot of retention factor versus the composition of the mobile phase showing that larger molecules are more sensitive than small molecules to changes in the percentage of the organic components

Leucine enkephalins = 11

Benzenes = 27

Lysozymes = 40

014

1

10

100

018 022 026 030 034 038 042

k

ϕ

GRADIENT HPLC

Factors to Consider

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Gradient Delivery Pumps High-Pressure Binary PumpsBoth high- and low-pressure pumping systems are used for gradient separations The first type a high-pressure binary pumping system is shown in Figure 5 In the lower left and right parts of this figure are two independent pump heads One of them is pulling in solvent such as water from a bottle going through a degasser and the other one is pulling in a second solvent such as acetonitrile or methanol The solvent or mobile phase is then pumped out of these two pump heads and mixed in a low-volume mixing chamber where it goes through a secondary mixture chamber and a pulse-dampening device to minimize pressure fluctuations during the flow through the column

Itrsquos important to emphasize that the solvents are mixed under high-pressure conditions This pump design is typically characterized by a low internal mixing volume which is a very important factor with respect to gradient dwell volume which is the volume in the system from the point where the gradient is formed to the top of the column But on the other hand they tend to be more complicated designs and typically are more expensive to purchase

Low-Pressure Quaternary and Ternary PumpsIn contrast the second approach is to use a low-pressure gradient pumping system Figure 6 shows schematic diagrams of low-pressure quaternary and ternary systems Functionally there is no difference between them the choice just depends on how many solvent options you need for producing the gradients A ternary system can mix up to three solvents to produce the mobile phase and a quaternary system can mix up to four solvents to produce the mobile phase In this case the mixing of the fluids happens before the point where the pressure of the fluid is elevated to actually push it through the column

The proportioning valve is frequently a bank of solenoid valves that open and close at specified intervals to allow packets of solvent to enter the mixing point Figure 6 shows that these packets of solvent enter a single piece of tubing going from the mixing point to the pump head itself as these packets of solvent travel through the pumping system they are gradually mixed up to the point where they enter the analytical column Similar to the high-pressure system there is also a pulse dampening unit and a secondary mixing chamber but the important point here is that the solvent mixing happens at low pressure before it reaches the pump head itself However because there is a greater volume of solvent between the mixing point and the analytical column there is a larger gradient dwell volume

Figure 4 The focusing effect of an analyte as it moves through a column The upper plot shows the distance that the analyte travels through the column as a function of time and the lower plot shows the retention as a function of time

Time (min)

End

End

Start

14 min

20

10

00 10 20

22 min

Start

0 10 20 30 40 50 60 70 80 90Organic

modifier ()

Dis

tan

ce (

cm)

k

100

GRADIENT HPLC

Factors to Consider

GR

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19

Low-pressure systems tend to cost less than high-pressure mixing systems Mixing at low pressure can lead to complications however such as extremes in flow rate or gradient composition and can cause other potential problems related to outgassing of the solvents

Testing the Gradient Performance When operating a pumping system designed for gradient elution itrsquos important to be aware of what tests can be used to characterize the performance of the system and troubleshoot problems These gradient performance tests can be used to troubleshoot or evaluate the performance of specific components of the pumping system and also to compare different pumping systems in terms of the accuracy and precision of the gradient profile that is produced

There are many different ways to test a systemrsquos gradient performance Most pumping systems have a built-in test that can be run using the instrument software One of the most common tests is shown in Figure 7 in which a step gradient begins and ends at 0 of the B solvent With a solvent mixture composed of solvents A and B a gradient is run from 0 to 100 B in steps of 10 B passing it through a system where the analytical column has been replaced with a restriction capillary such as a long length of narrow tubing

This test can be done in different ways with various solvents used as solvents A and B One common way to conduct this test is to use pure water for A and then for B to use water spiked with some compound that absorbs UV light such as acetone or benzyl alcohol

One good approach is to use a 5050 mixture of methanol and water for these tests If you use pure water or a pure organic solvent sometimes the test

Figure 5 Schematic of a high-pressure binary pump

Low-volumemixing chamber

To autosampler

Pulse damperSecond mixing chamber

GRADIENT HPLC

Factors to Consider

GR

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T M

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20

molecule will adsorb onto various instrument components There are other considerations too In the case of biological applications for example you should use mobile phases that are similar to the mobile phases that actually are going to be used in your application And when your mobile phases consist of highly aqueous solutions benzyl alcohol may not be soluble enough in such cases acetone uracil or thiourea would all be good alternatives

Figure 7 is also an indicator of how the mobile-phase mixture is detected at the detector The signal actually changes as a function of flow rate and given that we know that we are programming it in 10 steps we can get some sense for how the solvent mixing system is performing This can then be used as a way of troubleshooting or characterizing the performance of this system

Calculating Dwell VolumeAnother important factor for characterizing the pumping system is calculating the gradient dwell volume because differences in the dwell volume can cause significant changes in selectivity and resolution when transferring a method from one instrument to another The dwell volume is measured in a similar way to gradient performance mdash using an A and a B solvent where the B solvent is spiked with some compound that absorbs UV light Then a gradient is run from 0 to 100 B in a linear fashion (not using steps as in the determination of gradient performance) The goal is to determine the length of the delay between telling the instrument to start making the gradient and when the gradient or the change in solvent composition arrives at the detector This delay time is called the dwell time The delay volume which is the volume of solvent that has to go through the system before the solvent change actually reaches the detector is equal to the delay time multiplied by the flow rate

Accounting for Dwell VolumeFigure 8 shows that differences in gradient dwell volume between instruments can have an impact on resolution particularly for closely eluted pairs of compounds as shown by the improvement in resolution of 16 to 12 between systems A and B One way to account for two systems that have very different gradient delay or dwell volumes is to make the system with the lower dwell

GRADIENT HPLC

Factors to Consider

Figure 6 Examples of low-pressure pumping systems schematics of a ternary pump (left) and a quaternary pump (right)

Ternary pumps

To autosampler

Quaternary pumps

Proportioning valvePulse damper

Outlet valve

Inlet valve

Figure 7 Plots of absorbance and B versus time for a two-solvent step-gradient test of pump performance (1)

Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21

volume act like the system with the higher dwell volume by deliberately programming into the pumping system control an isocratic hold at the beginning of the run to effectively mimic the high gradient delay volume

Washout VolumeSo far we have discussed the characteristics of the gradient profile that we can test by carrying out the composition steps and looking at what happens at the detector We also talked about the dwell volume which is the delay of the gradient actually arriving at the column Letrsquos now turn our attention to what happens at the end of the gradient

Typically a scouting type of gradient proceeds from 10 to 90 B during the run At the end of the gradient we make a step change from 90 B back down to 10 B to equilibrate the system and column for the next injection of sample and the next gradient elution Chromatographers should be aware that there is also a delay in that process caused by the washout volume in the system Although a step change is made from 90 down to 10 it doesnrsquot happen immediately

This is exemplified in Figure 9 which shows the delay when using two solvents A and B where B is spiked in this case water spiked with acetone If a step change from 100 B to 0 B is made at time 0 we see that there is a slight delay and then an exponential flush of the B solvent out of the system

This delay is measured using an approach similar to that used to measure the dwell volume and for the purpose of discussion we characterize this washout volume by looking at the time it takes for the B solvent to be 97 flushed out of the system This washout volume becomes important in determining or estimating how much time we should allow for reequilibration of the analytical column because we want to make sure that the analytical column is prepared for the next run by flushing the final mobile phase composition out and refilling it with whatever solvent composition we are using at the start of the gradient elution run

System A Dwell volume = 05 mL Gradient = 1 Bmin

System B Dwell volume = 50 mL Gradient = 1 Bmin

0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163

Figure 8 Differences in gradient dwell volume between instruments can have an impact on analysis time

GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20

Flow rate 1mLminA WaterB 01 acetone in waterDetection 254 nm

Figure 9 Graphical display of washout time which is the delay in time from when the pumping system is programmed to change the solvent composition relative to when the composition actually changes Adapted with permission from reference (2)

GR

AD

IEN

T M

ETH

OD

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22

We can devise a way to systematically determine times that we should use for these various factors when transferring a method from one system to another With respect to washout volume we can look at the ratio of the washout volumes on the two systems (see Figure 10) Equation 1 can be used to readjust our expectations for how much time we need to allow for the last segment in the gradient on the new system

New segment time = original segment time X (original system washout volumenew system washout volume) [1]

Essential Gradient ParametersLetrsquos now turn our attention to optimizing essential gradient parameters and in particular the benefits of running a scouting gradient A scouting gradient is probably the most important step in developing any method and makes it possible to account for the wide polarity of analytes

When we donrsquot know how many compounds or the types of compounds we are looking for we need to understand the range of analyte polarities during the method development process (the essential gradient parameters are shown in Figure 11) so that we can encompass and retain as many of those analytes as possible And to give ourselves the best chance of capturing these analytes we use a scouting gradient for the most nonpolar analytes that starts at 5 B and goes up to 100 B (that is100 organic mobile phase) this gradient elutes the most highly retained nonpolar (hydrophobic) analytes and also provides the best chance of retaining the more polar hydrophilic analytes The information that we gather from this initial scouting gradient is helpful in determining whether a gradient is needed or whether the method should be run isocratically

Isocratic runs will provide the best resolving power for analytes of similar polarties and the best indication of whether the analytes are interacting with the stationary phase as much as possible So a scouting gradient run may indicate that an isocratic run is recommended or it might suggest the use of a gradient run because of the differing polarity of analytes However it will be extremely difficult to pick an isocratic mobile-phase composition that will retain the highly polar analytes and not retard the more hydrophobic analytes so much that the peaks broaden or remain bound onto the stationary phase If the scouting run is advising the use of an isocratic mobile phase it can also tell us what mobile-phase composition to use and if a gradient approach is suggested it will indicate whether we can actually increase our initial and final organic compositions or perhaps decrease them to save time

GRADIENT HPLC

Factors to Consider

Figure 10 Plots showing how the washout volume can impact the transfer of a method from one system to another

Gradient dwell New system

Wash out time New system Wash out time

Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20

Gradient profileOriginal system

Gradient slopeNew system

Gradient slopeOriginal system

Gradient profileNew system

Programmedgradient

Figure 11 Essential gradient parameters to be considered in optimizing a method

Reequilibration

Time

Conditioning

Initial isocratichold

PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23

Calculating Peak ElutionThe initial approach to use when calculating peak elution is to determine the percentage difference between the first and the last peak retention times using the following equation

Peak elution range = ([tf ndash ti]tG) X 100 [2]

where tf and ti are the final and initial retention times respectively and tG is the total time during which the eluent composition is changing If that difference is 25 or greater then we typically recommend using a gradient whereas if it is less than 25 an isocratic run is usually optimal If the analytes are eluted significantly below the 25 threshold of the gradient we want to know what isocratic portion to run To identify that portion there are a couple of further calculations that can be used to better understand the average retention time mdash that is the retention time in the middle of the peak elution window We also need to calculate the rate of change of the organic component of the mobile phase (the speed at which the mobile-phase composition is changing every minute) For example in the method described previously if we change from 95 aqueous down to 0 over 20 min the rate is about 475min This rate can be calculated by dividing the difference between the initial and final B by the time of the gradient We can then use these two values to carry out further optimization studies of the gradient parameters For the sake of clarity these equations will not be described but instead we will provide a general overview of the optimization procedure

Initially we need to know the percentage of organic solvent in the isocratic mobile phase It can be determined by adding the initial B to the amount that the organic composition has increased by the time a peak is eluted or by the time the middle of that peak is eluted if itrsquos an isocratic elution If we then multiply the average retention time by the rates of change of B the summation of that plus the initial concentration tells us what mobile-phase composition the pumps are pumping which is a very useful parameter to know

However that composition is not what is passing through the column We therefore need to account for the delay or dwell volume The way we do that is to convert the dwell volume back to a time by dividing dwell volume by the flow rate and then multiplying that value by the rate of change in units of B per minute Then by subtracting the B value obtained from the previous calculation from what the pumps are pumping we can determine what mobile-phase composition is passing through the column at the time the analytes are detected Because the analytes have passed through the column and have been detected we subtract 10 Essentially we are calculating what mobile-phase composition is passing through the column when the middle of that peak grouping is eluted and then we take away 10

GRADIENT HPLC

Factors to Consider

Figure 12 Optimization based on changing the eluent composition of the first peak in a chromatogram

0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50

Initial B ndash 5Final B ndash 100Bmin ndash 19Gradient time ndash 50 min

Initial B ndash Eluent compostion of first peak ndash 10B



GR

AD

IEN

T M

ETH

OD

S

24

If we are optimizing the parameters for a gradient analysis we repeat the same calculation twice but rather than using the average peak retention time we use the retention time of the first peak to be eluted and then we calculate when the last peak is eluted When we use the initial peak retention time we obtain the initial B and when we use the final retention time we obtain the final B

An example of this appears in Figure 12 which shows a series of chromatograms with values for the initial B ranging from 5 to 40 These chromatograms are showing just the first portion of that gradient As the initial B is increased the selectivity remains fairly constant but the resolution is degrading and the peaks are getting broader If the gradient is overly compressed the analytes donrsquot have sufficient time to interact with the stationary phase

Figure 13 shows the same chromatograms but in this case the final B has been optimized As the final B is reduced from 100 through 60 down to 40 B the gradient time decreases from 60 min to 35 min to 20 min respectively The peaks and peak spacing remain in proportion and constant primarily because we are keeping the rates of change the same Thus as we reduce the final B we reduce the gradient time accordingly

To scale a gradient the average retention factor k must be calculated We typically canrsquot have a retention factor for a gradient because we are always changing the mobile-phase composition so we use an average retention factor

k = tG FS∆ΦVm [3]

where F is the flow rate S is the slope of a plot of log k vs Φ ∆Φ is the fractional change in the organic composition during the gradient and Vm is the column volume

We typically use the same range as with an isocratic separation looking for a retention factor somewhere between 2 and 10 with conventional HPLC systems However for modern ultrahigh-pressure liquid chromatography (UHPLC) columns values of 05ndash5 are fairly typical

To estimate S we use the following equation

S = 025MW05 [4]

So we take the square root of the molecular weight of the analyte which really drives its S value and then we multiply it by 025 As a rule of thumb if you work on anything less than a 1000 Da in size an S value of 5 is a very good starting point

GRADIENT HPLC

Factors to Consider

Figure 13 Optimization based on changing the eluent composition of last peak in a chromatogram (Note that only the first 14 min of each separation is shown)

0 5 10

0 5 10

0 5 10

Initial B ndash 10Final B ndash 100 Bmin ndash 15Gradient time ndash 60 min



Figure 14 Chromatograms showing the effect of gradient slope on resolution and selectivity

100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25

Equation 3 can be rearranged to account for tG which can be very useful if you are actually trying to calculate what a gradient time should be With a known flow rate an S value of 5 a ∆Φ of 095 and a column volume that has been calculated using the standard column volume calculation we can then use a k value of 5 because we know what we are looking for And for a standard 150 mm x 46 mm id column with a flow rate of 2 mLmin we obtain a k value of 5 which will result in a tG of about 20 min

Figure 14 emphasizes what can happen when the rate of change is too fast or the slope of the line is too steep If the gradient time is too short there is too much compression of the analyte elution window Alternatively if we make the slope too shallow we are wasting time as can be seen with the tG = 40 chromatogram where there is a significant dead time in the separation

When analyzing a multiple-component sample you will find that analytes can be affected to a different degree by changes in the gradient time Itrsquos not always the case that reducing the gradient time will improve resolution or increasing the gradient time will improve resolution mdash depending on the composition of a sample the optimal gradient time can be found somewhere in the middle which is contrary to the results obtained with isocratic separations In gradient separations changing the gradient time can also change the selectivity which in turn changes the resolution Arbitrarily changing the gradient time can affect the separation of your samples both positively and negatively

Column Reequilibration TimesHistorically column reequilibration has been discussed in terms of column volumes and multiple column volumes A general rule of thumb for column reequilibration is expressed as equation 5

Required reequilibration time = 2(Vd + Vm)F [5]

Where Vd is the dwell volume of the system This rule of thumb is an incredibly useful guide for estimating the reequilibration time that is required post-gradient An important parameter to remember is that a run time is not purely the gradient time it is a summation of the gradient time plus reequilibration time It should always be determined empirically Although equation 5 provides a good estimate for the required reequilibration time you should always ensure that your analytes are not affected by insufficient equilibration Irreproducible retention times can be caused by giving the column insufficient reequilibration time before the next injection

GRADIENT HPLC

Factors to Consider

Figure 15 Chromatograms showing the effect of changing flow rate and gradient time on selectivity and sensitivity

0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30

Initial B ndash 10Final B ndash 90Bmin ndash 1333Gradient time ndash 60 minFlow rate ndash 05 mLminColumn length ndash 150 mmColumn id ndash 46 mm



Rs = 216

Rs = 199

Rs = 166

Figure 16 Plots showing differences in baseline absorbance when using methanol and acetonitrile as the organic solvent in a gradient run

GR

AD

IEN

T M

ETH

OD

S

26

Method TransferNow we are going to discuss method transfer and translation in terms of flow rate length and column internal diameter Previously we talked about gradient time and column flow rates Changes in the flow rate can affect resolution and selectivity If you want to maintain selectivity k should remain the same for the analytes and therefore resolution is maintained as much as possible If the flow rate is doubled for example the same k value (sometimes referred to as B value) can be maintained by halving the gradient time If you want to maintain selectivity the equation must be balanced by making a proportional change to the gradient time as we did for the flow rate and vice versa

Figure 15 shows that as we go from a 60-min gradient in the top run to 15 min in the middle run and down to 6 min with the bottom run the resolution will be affected This order of magnitude reduction in run time can be accounted for and selectivity can be maintained by ramping up the flow rate by an order of magnitude Yes the efficiency has been lost but selectivity is good and actually the resolution will be quite adequate in most cases

Changes in Column LengthColumn length doesnrsquot play as important a part in gradient analysis as it does in isocratic analysis because by the time the analytes reach the end of a 10ndash15 cm column they are actually residing purely in the mobile phase As the mobile-phase strength increases during a run the analyte interactions with the stationary phase will decrease and as result they are traveling through the column at the same velocity as the mobile phase So the column length isnrsquot as important as it is in isocratic separations where the analytes are continually partitioning in and out of the stationary phase as they move though the column For that reason separation or selectivity in gradient separations is driven by an analytersquos affinity for the mobile phase as the mobile-phase composition changes

How to Minimize Drifting BaselinesWhen there is an increase in absorbance or a change in the refractive index of the more strongly absorbing solvents the baseline will rise or drop during a gradient run This change in baseline absorbance will have an impact on the ability to integrate precisely for quantification purposes and it is one of the reasons acetonitrile is often a preferred solvent The plot of absorbance against time in a gradient run shown in Figure 16 demonstrates that methanol is fairly strongly absorbing whereas the absorbance is fairly stable with acetonitrile over the same time period

GRADIENT HPLC

Factors to Consider

Figure 17 Plot of peak capacity against the ratio of gradient time (tG) and the unretained peak time (t0) showing the optimal range Adapted with permission from reference (3)

g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27

Peak Capacity Peak capacity is a term that has gained favor in recent years predominantly because of the power of modern UHPLC systems which can resolve a greater number of peaks in a gradient separation Peak capacity is defined as the ratio of the gradient time and the average peak width of the first and last eluted peak added to 1 which gives us the theoretical number of peaks that can be resolved It is our experience that the practical empirical number of peaks that can be resolved is an order of magnitude lower than the theoretical number However it is a good way of understanding the efficiency of a separation

The gradient length for optimum peak capacity should be neither too short nor too long Figure 17 is a plot of peak capacity against the ratio of gradient time (tG) and the unretained peak time (t0) often known as the holdup time The optimal range is the highlighted blue zone where the peak capacity is highest Very long gradients provide little increase in peak capacity

The Impact of Gradient ProfilesThere is no question that the gradient profile can affect certain peaks as exemplified by the two critical peak pairs shown in Figure 18 There is almost baseline resolution between the peak pairing 1 and only very poor resolution of peak pair 2 The segmented gradient used for this separation allows control over early and later portions of the gradient but there are no really hard and fast rules for when to implement the segment change

So what happens when we slow the gradient down Figure 19 shows the initial gradient at the top and the gradient slowed down on the bottom In this example the critical peak pair 2 is resolved by the slower gradient but peak pair 1 is still fairly problematic A much better approach is to incorporate an isocratic hold and isocratic segments within the gradient

GRADIENT HPLC

Factors to Consider

Figure 19 Adjusting the gradient shown in Figure 18 to optimize separation of critical peak pair 1

0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1

Figure 18 Chromatogram obtained using a 5ndash95 B gradient The critical peak pairs 1 and 2 are unresolved

0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28

By using the method described earlier we can calculate the mobile-phase composition where those peaks are being eluted Letrsquos take a look at the critical peak pair 1 in Figure 20 By subtracting approximately 10 and incorporating an isocratic hold and turning off the separation for peak pair 2 we can improve the separation We calculated that the peak pair 1 could be best resolved at 52 B and in this case if we subtract 12 those peaks are pulled apart very nicely We typically use an isocratic hold of two to three column volumes as an initial approximation

A good place to start is 10 less than where each critical peak pair is eluted and hold for two to three column volumes If that hold time is not long enough hold for slightly longer If the mobile phase is too strong try using a lower B This approach is a little more complex than using a traditional linear gradient from 5 to 95 or 100 B but it is not that complex using the calculation described earlier it is very easy and straightforward to implement

Summary of Gradient Elution Method DevelopmentThe method development optimization process for a gradient separation can be summarized in the following stepsbull Run a blank gradient to ensure there are no problems with baseline driftbull Run a scouting gradient (5ndash100 B) and estimate initial and final B or begin

with a 20-min gradient with k = 5 when F = 2 mLmin for a typical 46 x 150 mm column

bull Optimize gradient steepness for the conditions found from the scouting gradient

bull Perform the separation and repeat to ensure correct column reequilibrationbull Vary the gradient time to assess the effect on the analysis (vary by twofold or

more) and note any changes in the resolution of critical pairsbull Initial and final B may need to be adjustedbull If further optimization is required vary the solvent type and then the column

chemistrybull Gradient steepness should be reoptimized following any changes in solvent

or columnbull For ionizable analytes variation in pH or temperature should be investigated

before changing column chemistrybull Complex gradients can be used if required to reduce analysis time or to

affect retention and selectivitybull After conditions have been optimized using the steps above the analysis

time can be reduced by varying the flow rate column length or particle size Keep k constant when changing the column flow rate or length to maintain selectivity

Figure 20 Chromatograms showing the benefits of incorporating an isocratic hold within the gradient elution of the sample from Figure 18

0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

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ETH

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29

GRADIENT HPLC

Factors to Consider

bull Final adjustment of the reequilibration time can be made to optimize overall analysis time optimize the separation empirically noting any changes in retention behavior

bull Ensure that dwell and washout volumes have been taken into consideration

References(1) S Marten A Knoumlfel and P Foumlldi LCGC Europe 21(7) 371ndash379 (2008)(2) A Schellinger D Stoll P Carr J Chromatogr A 1064 (2005) 143ndash156(3) M Gilar AE Daly M Kele UD Neue and JC Gebler J Chromatogr A 1061 183ndash192 (2004)

This article is based on the LCGCndashCHROMacademy web seminar ldquoGradient HPLC mdash 10 Things You Absolutely Need to Knowrdquo presented on June 19 2014 by Dwight R Stoll and Scott Fletcher

Dwight R Stoll PhD is an Assistant Professor in the Department of Chemistry at Gustavus Adolphus College in St Peter Minnesota

Scott Fletcher is a technical business development manager at Crawford Scientific in Strathaven Lanarkshire UK and a senior tutor for LCGCrsquos CHROMacademy

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ECTO

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31

A variety of detectors may be used with high performance liquid chromatography (HPLC) This article explains the operating principles and the strengths and weaknesses of various types of detectors including UVndashvisible diode array refractive index and fluorescence detectors as well as novel detection approaches such as evaporative light scattering detection charged aerosol detection and electrochemical detection

The Ideal DetectorLetrsquos start by considering the properties of the ldquoidealrdquo detector for high performance liquid chromatography (HPLC) Ideally we would like to detect the presence of everything in a sample independent of anything else thatrsquos going on in the background of either the mobile or stationary phase For example we might have a situation where we would like to detect as many of the analytes in our separation as we possibly can Alternatively in a slightly different scenario we might need more-selective detection when we want to measure only the solutes of interest and ldquomake invisiblerdquo the presence of matrix components that we are not interested in measuring

Obviously we would like the detector to be stable and for its performance not to vary with changes in temperature or mobile phase In a perfect world we would also like to be able to detect very low concentrations of analytes We also want our detector to have certain physical properties that will not negatively affect the separation procedure For example we donrsquot want the detector cell to increase the volume because this will cause dispersion of our chromatographic peaks and thus will not only make it more difficult to maintain the quality of the separation but also to ensure sensitivity and detection capability

On the other hand we also would like to be able to detect the narrow peaks that are associated with increasingly high performance forms of chromatography such as ultrahigh-pressure LC (UHPLC) where the

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THE FUNDAMENTALS OF

HPLC DetectorsBy Scott Fletcher

DET

ECTO

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32

peak volumes may be extremely small If the detector response time is too slow it may miss very sharp peaks that arise between the detector observation periods And finally we would like the detector to be robust and easy to optimize

Detector Figures of MeritItrsquos important to understand the terminology and the figures of merit used in detector technology One important concept is selectivity If we use a nonselective detector such as a refractive-index (RI) detector the property of the analytes we wish to monitor must be as universal as possible so that we can detect the presence of whatever is eluted from the column irrespective of its structure or physical properties Nonselective detectors are not very common however because itrsquos very difficult to monitor one property covering all analyte molecules one may encounter

Selective detectors on the other hand respond to a specific property of the analyte Letrsquos take a UV detector as an example A UV detector requires interaction between the UV radiation and the molecules of interest If there is no UV activity and the UV light just passes straight through the sample then as far as the detector is concerned nothing is present

The sensitivity of a detector defines how easily it can detect very small signals above the background noise At low analyte levels the signal will be very erratic and unstable and will be difficult to measure with a high degree of precision or accuracy This is important because when you first optimize a detector you typically set it up so the noise level is minimal In addition sensitivity affects the detection of your analytes Itrsquos universally recognized that you cannot confidently assign a signal unless itrsquos at least three times the average noise value In fact to be rigorous with analytical quantitation itrsquos also generally accepted that the limit of quantitation should be an order of magnitude greater than the noise

Letrsquos now focus on the linear range of the detector In a perfect world we would like our detector to be linear forever in all directions In other words it would have the capability of detecting one molecule of our substance above the noise and then continue to be able to detect increasing quantities of that molecule and never run out of linearity even if we have an infinite number of molecules reaching the detector This scenario is not very realistic and in the real world the detector gets to a point where it canrsquot respond proportionately to any more analyte signal We need to know when that occurs otherwise the detector wonrsquot be counting the molecules correctly This can potentially be very problematic not just in measuring the concentration of a molecule but also in assigning the size of a contaminant peak such as an impurity because we are making an assumption that the contribution of the analyte is proportional to the area of the peak

THE FUNDAMENTAL S OF

HPLC Detectors

DET

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33

Additionally if we try to detect above the linear range of the detector then we overestimate the quantity of any impurities because we are not counting the area of the main peak proportionately compared to the increase in the height of the impurities

When detector signal is plotted against concentration the slope is typically used to determine the sensitivity of the method and the intercept indicates the degree of error within the method which is a direct result of the background response However this is an area of much debate when we start talking about what constitutes the limit of detection and the minimal detectible amount against the signal-to-noise ratio

Table I shows the typical selectivity and sensitivity of seven commonly employed detectors As can be seen the most selective detection methods typically are the most sensitive When we require that a detector be more selective we are effectively demanding an increase in the specificity of detection parameters and itrsquos very unlikely that all of these criteria would be met by anything in the general background noise In fluorescence for example you just donrsquot set the wavelength at which your compound absorbs you also effectively couple that with the emission wavelength And the chances are extremely unlikely that any given interfering molecule will have the same set of coupled conditions as the analyte Similarly with electrochemical detectors you can set the parameters of the detector to observe only the electrochemical effect of the molecule of interest which will often be in a range that other background contaminants are not responsive to

But for a nonspecific nonselective detector such as an RI detector noise temperature and environmental changes may affect its performance so it is quite difficult to measure very small changes in concentration Additionally with some detectors particularly with low-selectivity detectors such as RI itrsquos very difficult to eliminate all the background effects that affect detection capability

UVndashvis Detection Letrsquos now turn our attention to UVndashvisible or UVndashvis detection by first explaining what happens in the flow cell Figure 1 is a diagram of a generic UVndashvis flow cell showing the liquid flow from the chromatograph arriving at the cell and passing through the collimated light of the UVndashvisible source which is in line with the detector We can use this principle to measure the difference between what is going into the cell at the front end and what is passing through the cell and being detected at the back end This difference in the transmission of light can be converted into an absorbance signal which is shown here as the chromatogram This peak will be proportional to the concentration so the more analyte


HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary

Mobile-phase flow to waste second detector

or fraction collector

Mobile-phase flow from

column

Collimated light from UVndashvis source

Chromatogram

Figure 1 Schematic of a typical UVndashvis flow cell

Table I Selectivity and sensitivity for various HPLC detection methods

Detection method Selectivity Sensitivity

Refractive index Low 1ndash5 microg

Conductivity Low 10ndash50 ng

UVndashvis Medium 05ndash10 ng

Electrochemical High 50ndash500 pg

Fluorescence High 10ndash100 pg

Evaporative light scattering Low 01ndash10 ng

Charged aerosol Low 01ndash10 ng

DET

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34

molecules of a substance that pass through the cell the more light is absorbed and therefore the less that comes out at the back end which results in a larger peak appearing in the chromatogram

UVndashvis Detectors QuantitationTo apply UV-vis detection to quantitative analysis we first think about the fact that absorbance is proportional to the analyte concentration The Beer-Lambert law basically tells us that absorbance is proportional to the concentration of the sample and the pathlength of the sample cell However the pathlength is typically fixed in the detector Thus we are effectively suggesting the Beer-Lambert law in fact says that absorbance is proportional to the concentration of the sample passing through the cell

However if there are any interferences present in the sample or if the concentration becomes sufficiently high some of the light will be scattered rather than being absorbed and as a result the law starts to break down With UV detectors installed on modern HPLC and UHPLC systems the peak absorbance should be in the order of 15 absorbance units (AU) or lower Once the absorbance exceeds that range the Beer-Lambert law may not apply and you may start to see nonlinear effects So a general rule of thumb is to keep the absorbance below 15 AU by either reducing the concentration or the amount of injected sample

The molar absorption coefficient is a measurement of how strongly a molecular species absorbs light at a given wavelength This is a very useful property because it allows us to translate this light absorption back to the concentration of a sample once we have calibrated the measurement using a reference material If we donrsquot know the concentration we can calculate it using a standard and then compare it with an unknown concentration based on its being the same molecule under the same conditions However in the real world we often donrsquot know the value of the molar absorption coefficient and we have to make the assumption that there will be an equal response from each component of a sample based on the likelihood that for similar structural features molar absorption coefficients are also similar

We tend to use peak area for quantitation as opposed to peak height because in the real world peaks donrsquot always behave perfectly and peak area is a much more robust measurement than peak height For that reason peak area is a much better measurement to use because it is more tolerant of changes in the actual chromatographic separation

ChromophoresUV chromophores give the molecule its UV activity This activity is typically electronic in nature so the more mobile the electrons in the conjugated


HPLC Detectors

DET

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RS

35

system are the easier it is to see good UV activity Additionally more highly conjugated molecules will tend to absorb higher wavelengths which translate to lower energies of UV radiation A general rule of thumb is that some solvents particularly acetonitrile are transparent to UV light at 190 nm With methanol and some other common solvents it is difficult to detect them below 220 nm So broadly speaking to avoid seeing any significant effect from the background we should work above the 210ndash220 nm range particularly when running gradients where a changing composition in the background of the solvent could lead to a sizeable baseline drift

Variable-Wavelength UVndashvis DetectorsIn variable-wavelength UVndashvis detectors the wavelength of interest is selected by moving a monochromator We start with a polychromatic light source which is a mixture of all wavelengths and effectively filter out the wavelength that we are interested in using a diffraction grating The grating allows only the wavelength of interest to pass through the flow cell which will give us information based specifically on the absorption of that particular wavelength of light This capability is very useful when analyzing a suite of samples that donrsquot have the same molecular template and that would otherwise not be detected if other sample components were present

UVndashvis Detection Advantages and DisadvantagesLetrsquos sum up the advantages and disadvantages of UVndashvis detectors They are very sensitive and can be used for quantitation of unknown molecules In addition they are ideally suited for gradient elution and respond to many analytes providing they absorb at that wavelength Their disadvantages are that no structural information is generated absorption is dependent on solution conditions and response factors have to be calculated particularly when it comes to impurity quantification However UVndashvis detectors are suitable for small organic molecules such as aromatic hydrocarbons and for analyte molecules with double bonds because in such cases you are likely to see plenty of UV activity

Diode-Array DetectionLetrsquos now take a look at diode-array detection (DAD) With these detectors you are looking at all wavelengths that are passing through the flow cell instead of just one wavelength as occurs with a UVndashvis detector There is no wavelength separation before the detection process The detector determines which wavelengths are missing from the original input light source (in other words which wavelengths were absorbed by the sample) after absorption has taken place So with diode-array detectors you donrsquot just get an absorption signal from your solute at a specific wavelength you actually get real-time spectra from the molecule These principles are presented schematically in Figure 2 which shows


HPLC Detectors

Figure 2 Schematic of a diode-array detector and chromatograms showing how it can be used for detection at single or multiple wavelengths

Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

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36

that DAD can be used for detection at single or multiple wavelengths where spectra can be dynamically obtained and stored for peak purity analysis library searching or extraction of signals

This advantage of looking at multiple wavelengths is probably the biggest reason why there has been such an increase in the use of diode arrays particularly if your analyte molecule has a unique spectrum because it becomes a way of identifying individual molecules Of course if your molecules have very similar spectra the benefits are not so obvious However even if this is the case there is no downside to using a diode-array detector because it can just be used as a variable-wavelength detector albeit with some sensitivity compromises Additionally the cost of diode-array detectors has gone down and they have become much more affordable

Itrsquos worth spending some time to understand how the response rate is optimized for a diode-array detector Basically the faster you make the response time the faster the ability to respond to whatever species is coming through the cell and the more likelihood of increasing peak sensitivity However as the response time goes down the noise also goes up so the overall sensitivity that results from using a higher response factor may not be any better than using a lower response factor and may even be worse in some cases Thus to get the best signal-to-noise ratio these parameters have to be optimized based on the chromatographic separation conditions and the detection capability required Generally speaking on modern UPHLC instruments where you are using very efficient chromatography and getting peaks that are 2ndash3 s in width you rarely get any better response frequency than 40 measurements per second which means you donrsquot have to use anything faster than a response coefficient of 40 Hz Modern detectors go up to 240 Hz but as soon you go higher than 40 Hz you can start to run into problems with noise

Another important capability of diode-array detectors is that we can use a reference wavelength to get a better understanding of what is going on in the cell without the sample being present For example if you want to compensate for background shifts caused by the mobile phase or other sample components another wavelength or range of wavelengths can be selected to investigate those effects in the reference cell enabling you to compensate for changes in the sample Generally speaking a reference wavelength or wavelength range is chosen that does not interfere with the absorbance of the analyte molecule as shown in Figure 3

The biggest advantage with diode-array detectors is that simultaneous multiwavelength detection can be carried out very quickly By careful setup of a DAD system you can detect and display all wavelengths at once even if


HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm

Bandwidth at 50 peak height

Analytical wavelength

Anisic acidOptimum Slit 8 mm (16) Signal 25530 Ref 340100

Reference bandwidth100 nm

Reference wavelength(290 nm + 50 nm)

340 nm

Figure 3 Spectrum of an analyte molecule (anisic acid) showing how a diode-ar-ray detector can be used monitor both the analytical wavelength and a reference wavelength at the same time

Figure 4 Schematic of a typical fluorescence detector

Emission monochromator

Excitation monochromator

Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

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37

you donrsquot want to look at all spectral information For this purpose the most important settings on a DAD are the detection wavelength and the bandwidth For example you can choose a detection wavelength such as 250 nm and set the bandwidth to 70ndash80 nm In this way you will actually be detecting everything that absorbs light at wavelengths ranging from 210 to 290 nm This can be problematic with quantitation in a mixture but it gives you the best chance of detecting any unknown components in the sample

However caution should be exercised when using diode-array detectors for the estimation of peak purity Itrsquos true that diode-array detectors can detect the presence of one component that is coeluted with another one However that detection relies on there being a significant difference in the spectra If the coeluted peaks have structural features that are very similar to those of the main molecule or to another solute in your mixture itrsquos highly likely that you wonrsquot see a significant difference in the spectra and therefore the peak will look pure when actually there is an impurity present But you can search the spectra against library reference spectra and in this way DAD can be used as a semiqualitative tool to confirm the identity of some components that have very characteristic UV spectra Additional limitations of diode-array detectors are that sensitivity is usually lower than that of a single-wavelength detector and these detectors are also susceptible to lamp fluctuations

Fluorescence DetectionA schematic of a fluorescence detector is shown in Figure 4 The radiation source is typically a xenon arc flash lamp which flashes every 3 micros producing a continuous spectrum of light from 200 nm to 900 nm Radiation from the lamp is focused by the first lens then reflected by the mirror onto the excitation monochromator grating which disperses and reflects the emitted radiation The light is then split in the flow cell to allow light to reach both the reference diode and photomultiplier tube Before the light reaches the emission monochromator a cutoff filter removes light below a certain wavelength to reduce noise from first-order scatter and second-order stray light The emission monochromator determines the wavelength range of light reaching the photomultiplier tube where the incident photons hit the photocathode and generate electrons thus multiplying the signal

The most important parameters to optimize in a fluorescence detector are the excitation and emission wavelengths The excitation wavelength can be taken from the excitation spectrum obtained on a spectrofluorimeter The optimum emission wavelength is dependent on the particular instrument and compound

Fluorescence detectors can be extremely sensitive but they detect only


HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum

Figure 5 Example excitation and emission spectra (left) and a diagram of electronic transitions (right) for an analyte

Figure 6 Schematic of a typical refractive-index detector

Purge valve 2 Purge valve 1

Waste

DET

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38

molecules that fluoresce Unfortunately not many molecules fluoresce so these detectors have limited applicability The types of molecules that fluoresce can be broken down into organic and inorganic molecules and some that intrinsically fluoresce such as the fluorophores The most common one is fluorescein which is typically used as a fluoro tag Because of its sensitivity as a fluorescence tag it is fairly common to actually bind it to analytes to detect and measure compounds that donrsquot naturally fluoresce In addition to fluorescein other common fluoro tags include fluorescent dyes such as acridine and also fluorescent proteins There are also inorganic fluorophores such as lanthanide-based probes and also CdSe-based quantum dots

As mentioned above the sensitivity of any detector is not only related to the intensity of the peak height but also the intensity of the signal noise Very often the noise drives down sensitivity and ultimately impacts the detection limit Figure 5 exemplifies this for a fluorescence detector Here is a great example using a second-order filter We have a specific excitation wavelength It can be seen from the electronic transitions that photons travel from the ground state to the excited state and then relax back down to the ground state This occurs at approximately 450 nm where we actually measure the signal So it is actually the emission spectrum and not the excitation response that gives us the second-order separation of the peak from the interference and the background signal In this example it can be seen that the excitation wavelength is within the UV range while the emission spectrum is much broader less defined and usually far more practical to measure

The main advantage of fluorescence detectors is that not only do you achieve good selectivity (because only a small handful of molecules fluoresce) but you also get high sensitivity which means that only small sample volumes are required But of course the selectivity of these detectors can actually be a disadvantage because of the fact that not many compounds naturally fluoresce In addition this type of detector can be affected by temperature because of the energy required and the additional collisions that take place and because wersquore looking at excitation and relaxation And both the excitation and emission wavelengths have to be optimized you cannot just label the excitation and emission wavelengths to be used as is typically done with a UV detector Also these settings tend be very detector-specific with fluorescence detection both the excitation and emission wavelengths have to be set on every different instrument

Refractive-Index DetectionFigure 6 shows a schematic that explains how an RI detector works We see that there are two cells On the right hand side we can see the light path passing


HPLC Detectors

Figure 7 Diagrams showing the basis of refractive-index detection

PhotomultiplierEluent only

Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier

Figure 8 Schematic of an evaporative light scattering detector

Column effluent

Single output

Analyte

AmplifierLight source

Light-scattering cell

Nebulizer gas (air or nitrogen)

Drift tube(heated-zoneevaporation stage)

Photomultiplier tube or photodiode

Nebulizer

Nebulizerchamber

DET

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through two cells We have a reference and a sample cell Before the analysis both cells are flushed with the mobile phase When the injection is made the valve is rotated and column effluent then passes through the sample cell with the reference cell being filled with just the mobile phase This technique relies on comparing the degree of bending or refracting the light between the mobile phase and the mobile phase containing the sample So when only pure mobile phase is coming from the column that light is perfectly balanced and there is no signal As soon as anything different is eluted from the column and into the flow cell the degree by which the light is bent changes the change in refractive index can be caused by a sample compound or just by a change in the mobile phase This process is shown in Figure 7

The main advantage of a refractive-index detector is that it detects everything so it is considered a universal detector Therefore it is particularly good for the detection of nonionic compounds analytes that do not have a UVndashvisible chromophore and molecules that do not fluoresce However it is the least sensitive of all detectors Another major drawback is that RI detection cannot be used for gradient LC separations because the changes in the mobile-phase composition make it impossible for the detector to compare the column effluent to a reference Another limitation of RI detectors is that they take a long time to equilibrate So if you are analyzing a polar compound by hydrophilic interaction liquid chromatography (HILIC) mode using an RI detector it has to be allowed to equilibrate for the better part of a week between runs Even then it might only work in the evenings and on weekends because these detectors are so temperature sensitive that with people coming in and out of the laboratory and air conditioning going on and off the detector signal is very unstable Thermocouples are used to compensate for these temperature changes but they are only partially effective

Evaporative Light Scattering Detection Evaporative light scattering detection (ELSD) and charged aerosol detection (CAD) are very similar in nature With these approaches the column effluent travels out of the column and then is nebulized using an inert gas to produce an aerosol similar to the initial process of electrospray ionization (ESI) mass spectrometry The mobile phase is evaporated into droplets to produce nonvolatile particles of the analytes As the light hits these particles the light is scattered to various degrees the amount of scattering is determined by the particle size so the larger the particle size the greater the scattering of light This principle is depicted in Figure 8

ESLD is an excellent approach for analyzing many nonvolatile species so it is fairly universal in its applicability It has very broad applicability almost as broad as that


HPLC Detectors

Figure 9 Diagram of a charged aerosol detection system

HPLCcolumneluent

Nebulizer and impactor

Gasinlet

Dryingtube

ElectrometerCharge is drawn o and measured by a sensitive electrometer

Signal outSignal is directlyproportional to quantity of analyte in sample

CollectorAnalyte particlestransfer their charge

Secondary gas stream positively chargedby a high-voltage platinum corona wire Positive charged

transferred to analyte particlesby charged opposing secondary gas steam

Ion trapNegatively chargedion trap removes high-mobility particles

Large droplets to waste

DET

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40

of the refractive-index detector In addition it can be used for analytes that donrsquot have any chromophoric properties and unlike an RI detector it can be used for gradient separations Its biggest drawback however is the fact that you canrsquot use it for volatile samples because they will be lost via evaporation in amongst the mobile phase

Additionally the mobile phase must be volatile for this technique to work although this is not a huge drawback Another challenge with these detectors is that the signal does not respond linearly to the concentration

Charged Aerosol DetectionA similar type of detection to ESLD is charged aerosol detection (CAD) which uses a nebulized inert gas to produce an aerosol to evaporate off the mobile phase An impactor is used to remove large particles but rather than looking at light scattering as occurs in ESLD we are looking at charge transfer processes A stream of charged gas (N2) is used to collide with the analytes and the charge is transferred to the analytes The particles pick up charge according to their surface area and as they enter the collector and electrometer the signal is measured This process is shown schematically in Figure 9 The benefits of this approach are that it covers a broad range of analytes and compounds with good selectivity and it provides reasonably high sensitivity with good dynamic range meaning that it can quantitatively respond to small components in the presence of much larger ones in the same run In addition like ELSD itrsquos also compatible with gradient elution However it has similar limitations with volatile analytes

Electrochemical DetectionThe last type of detection method we are going to look at is electrochemical detection (ECD) which is shown in Figure 10 There are many variations of this detection approach However they all have one thing in common They measure the property of an electrical current using three electrodes a working electrode a counter electrode and a reference electrode

There are a number of different electrochemical detectors available on the market The most common and the one that has the widest range in terms of applicability is the conductivity detector which measures the magnitude of the current within an applied electric field It can be used with any organic or inorganic compounds that are ionic in nature including cations anions zwitterions strong acids and strong bases

Another type of ECD is the DC amperometric detection which looks at an oxidation or reduction reaction taking place on the surface of an electrode


HPLC Detectors

Figure 10 Schematic of an electrochemical detector

Workingelectrode

Reference electrode

Counterelectrode

DET

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Typical samples that are applicable to this type of detection include phenol hydroxybenzene catechol dihydroxybenzene and similar types of aromatic functional groups Other sample matrices that lend themselves to amperometric detection are catecholamine dopamine and epinephrine

A variation on the DC amperometric detection approach is integrated and pulsed amperometric detection However it works slightly differently with regard to the electronics It also detects the current but measures the current by integration during a repeated potential versus time waveform It is applied via a standard or background current in a square-post wave so itrsquos the frequency of the pulsing that is typically measured This approach is well suited to the analysis of carbohydrates and related molecules where good sensitivity and linearity can be achieved Figure 11 gives examples of the types of molecules and functional groups that are well-suited to electrochemical detection

Summing up the relative pros and cons of ECD it is highly selective with good sensitivity and a linear range of approximately five orders of magnitude with a very fast response time However the analytes have to be electrochemically active Electrode fouling is also fairly common so some sample types are not really suited for ECD because of this limitation But applications like catecholamine natural products and neurotransmitters lend themselves nicely to electrochemical detection

This article is based on the LCGCndashCHROMacademy web seminar ldquoHPLC Detectors mdash What Where When and Howrdquo presented on January 23 2014



HPLC Detectors

Figure 11 Structures of molecules and functional groups well-suited for electrochemical detection

Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase

2014 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific and its subsidiaries Specifications terms and pricing are subject to change Not all products are available in all countries Please consult your local sales representative for details

The Only Universal LC Detector Your Lab Will Ever NeedSee What Other Detectors Are MissingCharged aerosol detection is a revolutionary technology that will change the way you view

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Improve Inter-Analyte ResponseAn analytersquos response to charged aerosol detection does not depend on optical properties

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chemical derivatization are not essential for detection Charged aerosol detection is a

mass-sensitive technique that measures any non-volatile and many semi-volatile analytes

Variance in inter-analyte relative response is minimal whether analyzing small molecules

or proteins And this technique is gradient compatible

0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen

Diclofenac Progesterone

Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone

Six pharmaceutical agents with an excipient (citric acid) were fully resolved using gradient reversed-phase HPLC and their responses measured first by UV detection and then by charged aerosol detection As can be seen UV detection significantly underestimates the levels of most analytes

Unbiased Universal Detection Charged aerosol detection has the flexibility to be used for a broad range of analytes in

many different matrices opening new opportunities for broad discovery and enhanced

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GRADIENT HPLC

Factors to Consider

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Benzenes = 27

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Factors to Consider

GR

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20

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00 10 20

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GRADIENT HPLC

Factors to Consider

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GRADIENT HPLC

Factors to Consider

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GRADIENT HPLC

Factors to Consider


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Quaternary pumps


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0

50

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25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

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(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






CO

LUM

N S

ELEC

TIO

N




4


SPONSORED

Click to view PDF


SPONSORED

Click to view PDF




5





CO

LUM

N S

ELEC

TIO

N



water+


6








CO

LUM

N S

ELEC

TIO

N


7







CO

LUM

N S

ELEC

TIO

N



Si mdash O mdash Si

Si mdash O mdash Si

Si mdash O mdash Si

Si mdash O mdash Si

Si mdash O mdash H

Si mdash O mdash H

Si mdash O mdash H

O

O

O

O

O

O

HO

Lone acidic silanol

C8 bonded phase

C8 bonded phase

TMS group

8







CO

LUM

N S

ELEC

TIO

N



Si

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

O

O O


CH3


CH3



Si

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

O

O O

CH3


CH3CH3




Vicinal hydrated

Bridged (vicinal)

Surface metal ion

Lone acidic

Geminal

Metal activated

9






CO

LUM

N S

ELEC

TIO

N



pH

log

kprime

10

100

1

012 4 6 8 10 12 140


IbuprofenImipramine



Si

O

Si

O

Si Si

OH

N

C

O O O O

AlkylDispersive


CyanoElectrostatic

dipole

SilicaH-bonding

Nonpolar Polar

10








CO

LUM

N S

ELEC

TIO

N



CN phase

C8 phase

Time (min)0

1

1

1

2

2

2

3

3

3

4

4

45

5

5

6

6

6

7

7

7

5 15 20

Phenyl phase


C(70)10

C(28)

H10

B

S

A

11









CO

LUM

N S

ELEC

TIO

N



C(70)10

C(28)

H10

B

S

A

C(70)10

C(28)

H10

B

S

A


12











CO

LUM

N S

ELEC

TIO

N


13








CO

LUM

N S

ELEC

TIO

N

P asymp 1 + [3]tgw
















copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All r

ight

s re

serv

ed A

ll tra

dem

arks

are

th

e pr

oper

ty o

f The

rmo

Fish

er S

cien

tific

and

its s

ubsid

iarie

s

GR

AD

IEN

T M

ETH

OD

S

15




GRADIENT HPLC



SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

GR

AD

IEN

T M

ETH

OD

S

16








0

3

12 4

5

6

78

Time (min)

25 50 75


Time (min)

0 5 10

1

2

3

45

6

7

8

15 20 25 30

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

17








Benzenes = 27

Lysozymes = 40

014

1

10

100

018 022 026 030 034 038 042

k

ϕ

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

18






Time (min)

End

End

Start

14 min

20

10

00 10 20

22 min

Start

0 10 20 30 40 50 60 70 80 90Organic

modifier ()

Dis

tan

ce (

cm)

k

100

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

19








To autosampler


GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






5





CO

LUM

N S

ELEC

TIO

N



water+


6








CO

LUM

N S

ELEC

TIO

N


7







CO

LUM

N S

ELEC

TIO

N



Si mdash O mdash Si

Si mdash O mdash Si

Si mdash O mdash Si

Si mdash O mdash Si

Si mdash O mdash H

Si mdash O mdash H

Si mdash O mdash H

O

O

O

O

O

O

HO

Lone acidic silanol

C8 bonded phase

C8 bonded phase

TMS group

8







CO

LUM

N S

ELEC

TIO

N



Si

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

O

O O


CH3


CH3



Si

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

O

O O

CH3


CH3CH3




Vicinal hydrated

Bridged (vicinal)

Surface metal ion

Lone acidic

Geminal

Metal activated

9






CO

LUM

N S

ELEC

TIO

N



pH

log

kprime

10

100

1

012 4 6 8 10 12 140


IbuprofenImipramine



Si

O

Si

O

Si Si

OH

N

C

O O O O

AlkylDispersive


CyanoElectrostatic

dipole

SilicaH-bonding

Nonpolar Polar

10








CO

LUM

N S

ELEC

TIO

N



CN phase

C8 phase

Time (min)0

1

1

1

2

2

2

3

3

3

4

4

45

5

5

6

6

6

7

7

7

5 15 20

Phenyl phase


C(70)10

C(28)

H10

B

S

A

11









CO

LUM

N S

ELEC

TIO

N



C(70)10

C(28)

H10

B

S

A

C(70)10

C(28)

H10

B

S

A


12











CO

LUM

N S

ELEC

TIO

N


13








CO

LUM

N S

ELEC

TIO

N

P asymp 1 + [3]tgw
















copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All r

ight

s re

serv

ed A

ll tra

dem

arks

are

th

e pr

oper

ty o

f The

rmo

Fish

er S

cien

tific

and

its s

ubsid

iarie

s

GR

AD

IEN

T M

ETH

OD

S

15




GRADIENT HPLC



SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

GR

AD

IEN

T M

ETH

OD

S

16








0

3

12 4

5

6

78

Time (min)

25 50 75


Time (min)

0 5 10

1

2

3

45

6

7

8

15 20 25 30

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

17








Benzenes = 27

Lysozymes = 40

014

1

10

100

018 022 026 030 034 038 042

k

ϕ

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

18






Time (min)

End

End

Start

14 min

20

10

00 10 20

22 min

Start

0 10 20 30 40 50 60 70 80 90Organic

modifier ()

Dis

tan

ce (

cm)

k

100

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

19








To autosampler


GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






6








CO

LUM

N S

ELEC

TIO

N


7







CO

LUM

N S

ELEC

TIO

N



Si mdash O mdash Si

Si mdash O mdash Si

Si mdash O mdash Si

Si mdash O mdash Si

Si mdash O mdash H

Si mdash O mdash H

Si mdash O mdash H

O

O

O

O

O

O

HO

Lone acidic silanol

C8 bonded phase

C8 bonded phase

TMS group

8







CO

LUM

N S

ELEC

TIO

N



Si

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

O

O O


CH3


CH3



Si

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

O

O O

CH3


CH3CH3




Vicinal hydrated

Bridged (vicinal)

Surface metal ion

Lone acidic

Geminal

Metal activated

9






CO

LUM

N S

ELEC

TIO

N



pH

log

kprime

10

100

1

012 4 6 8 10 12 140


IbuprofenImipramine



Si

O

Si

O

Si Si

OH

N

C

O O O O

AlkylDispersive


CyanoElectrostatic

dipole

SilicaH-bonding

Nonpolar Polar

10








CO

LUM

N S

ELEC

TIO

N



CN phase

C8 phase

Time (min)0

1

1

1

2

2

2

3

3

3

4

4

45

5

5

6

6

6

7

7

7

5 15 20

Phenyl phase


C(70)10

C(28)

H10

B

S

A

11









CO

LUM

N S

ELEC

TIO

N



C(70)10

C(28)

H10

B

S

A

C(70)10

C(28)

H10

B

S

A


12











CO

LUM

N S

ELEC

TIO

N


13








CO

LUM

N S

ELEC

TIO

N

P asymp 1 + [3]tgw
















copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All r

ight

s re

serv

ed A

ll tra

dem

arks

are

th

e pr

oper

ty o

f The

rmo

Fish

er S

cien

tific

and

its s

ubsid

iarie

s

GR

AD

IEN

T M

ETH

OD

S

15




GRADIENT HPLC



SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

GR

AD

IEN

T M

ETH

OD

S

16








0

3

12 4

5

6

78

Time (min)

25 50 75


Time (min)

0 5 10

1

2

3

45

6

7

8

15 20 25 30

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

17








Benzenes = 27

Lysozymes = 40

014

1

10

100

018 022 026 030 034 038 042

k

ϕ

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

18






Time (min)

End

End

Start

14 min

20

10

00 10 20

22 min

Start

0 10 20 30 40 50 60 70 80 90Organic

modifier ()

Dis

tan

ce (

cm)

k

100

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

19








To autosampler


GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






7







CO

LUM

N S

ELEC

TIO

N



Si mdash O mdash Si

Si mdash O mdash Si

Si mdash O mdash Si

Si mdash O mdash Si

Si mdash O mdash H

Si mdash O mdash H

Si mdash O mdash H

O

O

O

O

O

O

HO

Lone acidic silanol

C8 bonded phase

C8 bonded phase

TMS group

8







CO

LUM

N S

ELEC

TIO

N



Si

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

O

O O


CH3


CH3



Si

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

O

O O

CH3


CH3CH3




Vicinal hydrated

Bridged (vicinal)

Surface metal ion

Lone acidic

Geminal

Metal activated

9






CO

LUM

N S

ELEC

TIO

N



pH

log

kprime

10

100

1

012 4 6 8 10 12 140


IbuprofenImipramine



Si

O

Si

O

Si Si

OH

N

C

O O O O

AlkylDispersive


CyanoElectrostatic

dipole

SilicaH-bonding

Nonpolar Polar

10








CO

LUM

N S

ELEC

TIO

N



CN phase

C8 phase

Time (min)0

1

1

1

2

2

2

3

3

3

4

4

45

5

5

6

6

6

7

7

7

5 15 20

Phenyl phase


C(70)10

C(28)

H10

B

S

A

11









CO

LUM

N S

ELEC

TIO

N



C(70)10

C(28)

H10

B

S

A

C(70)10

C(28)

H10

B

S

A


12











CO

LUM

N S

ELEC

TIO

N


13








CO

LUM

N S

ELEC

TIO

N

P asymp 1 + [3]tgw
















copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All r

ight

s re

serv

ed A

ll tra

dem

arks

are

th

e pr

oper

ty o

f The

rmo

Fish

er S

cien

tific

and

its s

ubsid

iarie

s

GR

AD

IEN

T M

ETH

OD

S

15




GRADIENT HPLC



SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

GR

AD

IEN

T M

ETH

OD

S

16








0

3

12 4

5

6

78

Time (min)

25 50 75


Time (min)

0 5 10

1

2

3

45

6

7

8

15 20 25 30

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

17








Benzenes = 27

Lysozymes = 40

014

1

10

100

018 022 026 030 034 038 042

k

ϕ

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

18






Time (min)

End

End

Start

14 min

20

10

00 10 20

22 min

Start

0 10 20 30 40 50 60 70 80 90Organic

modifier ()

Dis

tan

ce (

cm)

k

100

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

19








To autosampler


GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






8







CO

LUM

N S

ELEC

TIO

N



Si

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

O

O O


CH3


CH3



Si

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

OH

OSi

O

OSi

OH

OSi

OH

OSi

O

O O

CH3


CH3CH3




Vicinal hydrated

Bridged (vicinal)

Surface metal ion

Lone acidic

Geminal

Metal activated

9






CO

LUM

N S

ELEC

TIO

N



pH

log

kprime

10

100

1

012 4 6 8 10 12 140


IbuprofenImipramine



Si

O

Si

O

Si Si

OH

N

C

O O O O

AlkylDispersive


CyanoElectrostatic

dipole

SilicaH-bonding

Nonpolar Polar

10








CO

LUM

N S

ELEC

TIO

N



CN phase

C8 phase

Time (min)0

1

1

1

2

2

2

3

3

3

4

4

45

5

5

6

6

6

7

7

7

5 15 20

Phenyl phase


C(70)10

C(28)

H10

B

S

A

11









CO

LUM

N S

ELEC

TIO

N



C(70)10

C(28)

H10

B

S

A

C(70)10

C(28)

H10

B

S

A


12











CO

LUM

N S

ELEC

TIO

N


13








CO

LUM

N S

ELEC

TIO

N

P asymp 1 + [3]tgw
















copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All r

ight

s re

serv

ed A

ll tra

dem

arks

are

th

e pr

oper

ty o

f The

rmo

Fish

er S

cien

tific

and

its s

ubsid

iarie

s

GR

AD

IEN

T M

ETH

OD

S

15




GRADIENT HPLC



SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

GR

AD

IEN

T M

ETH

OD

S

16








0

3

12 4

5

6

78

Time (min)

25 50 75


Time (min)

0 5 10

1

2

3

45

6

7

8

15 20 25 30

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

17








Benzenes = 27

Lysozymes = 40

014

1

10

100

018 022 026 030 034 038 042

k

ϕ

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

18






Time (min)

End

End

Start

14 min

20

10

00 10 20

22 min

Start

0 10 20 30 40 50 60 70 80 90Organic

modifier ()

Dis

tan

ce (

cm)

k

100

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

19








To autosampler


GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






9






CO

LUM

N S

ELEC

TIO

N



pH

log

kprime

10

100

1

012 4 6 8 10 12 140


IbuprofenImipramine



Si

O

Si

O

Si Si

OH

N

C

O O O O

AlkylDispersive


CyanoElectrostatic

dipole

SilicaH-bonding

Nonpolar Polar

10








CO

LUM

N S

ELEC

TIO

N



CN phase

C8 phase

Time (min)0

1

1

1

2

2

2

3

3

3

4

4

45

5

5

6

6

6

7

7

7

5 15 20

Phenyl phase


C(70)10

C(28)

H10

B

S

A

11









CO

LUM

N S

ELEC

TIO

N



C(70)10

C(28)

H10

B

S

A

C(70)10

C(28)

H10

B

S

A


12











CO

LUM

N S

ELEC

TIO

N


13








CO

LUM

N S

ELEC

TIO

N

P asymp 1 + [3]tgw
















copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All r

ight

s re

serv

ed A

ll tra

dem

arks

are

th

e pr

oper

ty o

f The

rmo

Fish

er S

cien

tific

and

its s

ubsid

iarie

s

GR

AD

IEN

T M

ETH

OD

S

15




GRADIENT HPLC



SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

GR

AD

IEN

T M

ETH

OD

S

16








0

3

12 4

5

6

78

Time (min)

25 50 75


Time (min)

0 5 10

1

2

3

45

6

7

8

15 20 25 30

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

17








Benzenes = 27

Lysozymes = 40

014

1

10

100

018 022 026 030 034 038 042

k

ϕ

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

18






Time (min)

End

End

Start

14 min

20

10

00 10 20

22 min

Start

0 10 20 30 40 50 60 70 80 90Organic

modifier ()

Dis

tan

ce (

cm)

k

100

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

19








To autosampler


GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






10








CO

LUM

N S

ELEC

TIO

N



CN phase

C8 phase

Time (min)0

1

1

1

2

2

2

3

3

3

4

4

45

5

5

6

6

6

7

7

7

5 15 20

Phenyl phase


C(70)10

C(28)

H10

B

S

A

11









CO

LUM

N S

ELEC

TIO

N



C(70)10

C(28)

H10

B

S

A

C(70)10

C(28)

H10

B

S

A


12











CO

LUM

N S

ELEC

TIO

N


13








CO

LUM

N S

ELEC

TIO

N

P asymp 1 + [3]tgw
















copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All r

ight

s re

serv

ed A

ll tra

dem

arks

are

th

e pr

oper

ty o

f The

rmo

Fish

er S

cien

tific

and

its s

ubsid

iarie

s

GR

AD

IEN

T M

ETH

OD

S

15




GRADIENT HPLC



SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

GR

AD

IEN

T M

ETH

OD

S

16








0

3

12 4

5

6

78

Time (min)

25 50 75


Time (min)

0 5 10

1

2

3

45

6

7

8

15 20 25 30

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

17








Benzenes = 27

Lysozymes = 40

014

1

10

100

018 022 026 030 034 038 042

k

ϕ

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

18






Time (min)

End

End

Start

14 min

20

10

00 10 20

22 min

Start

0 10 20 30 40 50 60 70 80 90Organic

modifier ()

Dis

tan

ce (

cm)

k

100

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

19








To autosampler


GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






11









CO

LUM

N S

ELEC

TIO

N



C(70)10

C(28)

H10

B

S

A

C(70)10

C(28)

H10

B

S

A


12











CO

LUM

N S

ELEC

TIO

N


13








CO

LUM

N S

ELEC

TIO

N

P asymp 1 + [3]tgw
















copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All r

ight

s re

serv

ed A

ll tra

dem

arks

are

th

e pr

oper

ty o

f The

rmo

Fish

er S

cien

tific

and

its s

ubsid

iarie

s

GR

AD

IEN

T M

ETH

OD

S

15




GRADIENT HPLC



SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

GR

AD

IEN

T M

ETH

OD

S

16








0

3

12 4

5

6

78

Time (min)

25 50 75


Time (min)

0 5 10

1

2

3

45

6

7

8

15 20 25 30

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

17








Benzenes = 27

Lysozymes = 40

014

1

10

100

018 022 026 030 034 038 042

k

ϕ

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

18






Time (min)

End

End

Start

14 min

20

10

00 10 20

22 min

Start

0 10 20 30 40 50 60 70 80 90Organic

modifier ()

Dis

tan

ce (

cm)

k

100

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

19








To autosampler


GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






12











CO

LUM

N S

ELEC

TIO

N


13








CO

LUM

N S

ELEC

TIO

N

P asymp 1 + [3]tgw
















copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All r

ight

s re

serv

ed A

ll tra

dem

arks

are

th

e pr

oper

ty o

f The

rmo

Fish

er S

cien

tific

and

its s

ubsid

iarie

s

GR

AD

IEN

T M

ETH

OD

S

15




GRADIENT HPLC



SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

GR

AD

IEN

T M

ETH

OD

S

16








0

3

12 4

5

6

78

Time (min)

25 50 75


Time (min)

0 5 10

1

2

3

45

6

7

8

15 20 25 30

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

17








Benzenes = 27

Lysozymes = 40

014

1

10

100

018 022 026 030 034 038 042

k

ϕ

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

18






Time (min)

End

End

Start

14 min

20

10

00 10 20

22 min

Start

0 10 20 30 40 50 60 70 80 90Organic

modifier ()

Dis

tan

ce (

cm)

k

100

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

19








To autosampler


GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






13








CO

LUM

N S

ELEC

TIO

N

P asymp 1 + [3]tgw
















copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All r

ight

s re

serv

ed A

ll tra

dem

arks

are

th

e pr

oper

ty o

f The

rmo

Fish

er S

cien

tific

and

its s

ubsid

iarie

s

GR

AD

IEN

T M

ETH

OD

S

15




GRADIENT HPLC



SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

GR

AD

IEN

T M

ETH

OD

S

16








0

3

12 4

5

6

78

Time (min)

25 50 75


Time (min)

0 5 10

1

2

3

45

6

7

8

15 20 25 30

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

17








Benzenes = 27

Lysozymes = 40

014

1

10

100

018 022 026 030 034 038 042

k

ϕ

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

18






Time (min)

End

End

Start

14 min

20

10

00 10 20

22 min

Start

0 10 20 30 40 50 60 70 80 90Organic

modifier ()

Dis

tan

ce (

cm)

k

100

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

19








To autosampler


GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory




















copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All r

ight

s re

serv

ed A

ll tra

dem

arks

are

th

e pr

oper

ty o

f The

rmo

Fish

er S

cien

tific

and

its s

ubsid

iarie

s

GR

AD

IEN

T M

ETH

OD

S

15




GRADIENT HPLC



SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

GR

AD

IEN

T M

ETH

OD

S

16








0

3

12 4

5

6

78

Time (min)

25 50 75


Time (min)

0 5 10

1

2

3

45

6

7

8

15 20 25 30

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

17








Benzenes = 27

Lysozymes = 40

014

1

10

100

018 022 026 030 034 038 042

k

ϕ

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

18






Time (min)

End

End

Start

14 min

20

10

00 10 20

22 min

Start

0 10 20 30 40 50 60 70 80 90Organic

modifier ()

Dis

tan

ce (

cm)

k

100

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

19








To autosampler


GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






GR

AD

IEN

T M

ETH

OD

S

15




GRADIENT HPLC



SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

GR

AD

IEN

T M

ETH

OD

S

16








0

3

12 4

5

6

78

Time (min)

25 50 75


Time (min)

0 5 10

1

2

3

45

6

7

8

15 20 25 30

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

17








Benzenes = 27

Lysozymes = 40

014

1

10

100

018 022 026 030 034 038 042

k

ϕ

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

18






Time (min)

End

End

Start

14 min

20

10

00 10 20

22 min

Start

0 10 20 30 40 50 60 70 80 90Organic

modifier ()

Dis

tan

ce (

cm)

k

100

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

19








To autosampler


GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






GR

AD

IEN

T M

ETH

OD

S

16








0

3

12 4

5

6

78

Time (min)

25 50 75


Time (min)

0 5 10

1

2

3

45

6

7

8

15 20 25 30

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

17








Benzenes = 27

Lysozymes = 40

014

1

10

100

018 022 026 030 034 038 042

k

ϕ

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

18






Time (min)

End

End

Start

14 min

20

10

00 10 20

22 min

Start

0 10 20 30 40 50 60 70 80 90Organic

modifier ()

Dis

tan

ce (

cm)

k

100

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

19








To autosampler


GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






GR

AD

IEN

T M

ETH

OD

S

17








Benzenes = 27

Lysozymes = 40

014

1

10

100

018 022 026 030 034 038 042

k

ϕ

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

18






Time (min)

End

End

Start

14 min

20

10

00 10 20

22 min

Start

0 10 20 30 40 50 60 70 80 90Organic

modifier ()

Dis

tan

ce (

cm)

k

100

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

19








To autosampler


GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






GR

AD

IEN

T M

ETH

OD

S

18






Time (min)

End

End

Start

14 min

20

10

00 10 20

22 min

Start

0 10 20 30 40 50 60 70 80 90Organic

modifier ()

Dis

tan

ce (

cm)

k

100

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

19








To autosampler


GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






GR

AD

IEN

T M

ETH

OD

S

19








To autosampler


GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






GR

AD

IEN

T M

ETH

OD

S

20





GRADIENT HPLC

Factors to Consider


Ternary pumps

To autosampler

Quaternary pumps


Outlet valve

Inlet valve


Time (min)0

0

50

0

25

50

100

75

100

150

500

20 40 60 80 100

Ab

sorb

ance

(m

AU

)

B

Flow rate (mLmin)

0125

02500550

GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






GR

AD

IEN

T M

ETH

OD

S

21








0

0

5

5 10 15 20

10 15 20

RS = 697

RS = 591RS = 119

RS = 163


GRADIENT HPLC

Factors to Consider

-16000 02 04 06

Time (min)

Ab

sorb

ance

(m

AU

)

09 10

-140

-120

-100

-80

-60

-40

-20

-0

20



GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






GR

AD

IEN

T M

ETH

OD

S

22






GRADIENT HPLC

Factors to Consider




Original system

Time (min)

Co

mp

osi

tio

n o

r re

spo

nse

80

20





Programmedgradient


Reequilibration

Time

Conditioning


PurgingFinal B

Initial B

tg

B

GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






GR

AD

IEN

T M

ETH

OD

S

23






GRADIENT HPLC

Factors to Consider


0 5 10 15

10 20 30 40 50 60 70 80

10 20 30 40 50





GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






GR

AD

IEN

T M

ETH

OD

S

24









S = 025MW05 [4]


GRADIENT HPLC

Factors to Consider


0 5 10

0 5 10

0 5 10





100 B

100 B

100 B

tg = 5 tg = 20

tg = 40tg = 10

0 B

0 B0 B

00 10 20 30 40

10

ShallowSteep

100 B

GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






GR

AD

IEN

T M

ETH

OD

S

25







GRADIENT HPLC

Factors to Consider


0 5 10 15 20

10 20 30 40 50 60 70 80 90

10 20 30




Rs = 216

Rs = 199

Rs = 166


GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






GR

AD

IEN

T M

ETH

OD

S

26





GRADIENT HPLC

Factors to Consider


g

190

170

150

130

110

90

70

5020 40 60 80 100 120 140 1600

Optimal range

tgt0

P

GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






GR

AD

IEN

T M

ETH

OD

S

27





GRADIENT HPLC

Factors to Consider


0 5 10 15

(33)

(51)

(88)

0 5 10 15 20 25

(5)

(95)

1

2

2

1


0 5 10 15 20 25

(5)

(95)

21

GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






GR

AD

IEN

T M

ETH

OD

S

28














0 10 20 30

(5)

(95)

(52)

(5)

(40) (40)

(95)

1

1

2

2

GRADIENT HPLC

Factors to Consider

GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






GR

AD

IEN

T M

ETH

OD

S

29

GRADIENT HPLC

Factors to Consider














Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory













Giving you more

copy 2

014

Ther

mo

Fish

er S

cien

tific

Inc

All

right

s re

serv

ed

All

trad

emar

ks a

re th

e pr

oper

ty o

f The

rmo

Fish

er

Sci

entifi

c In

c a

nd it

s su

bsid

iarie

s





DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






DET

ECTO

RS

31






SPONSORED

Click to view PDF


SPONSORED

Click to view PDF

THE FUNDAMENTALS OF


DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






DET

ECTO

RS

32







HPLC Detectors

DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






DET

ECTO

RS

33







HPLC Detectors

Inlet capillary

Flow cell window

Detector diode

Outlet capillary




column


Chromatogram











DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






DET

ECTO

RS

34








HPLC Detectors

DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






DET

ECTO

RS

35






HPLC Detectors


Achromatic lens

Holmiumfilter

Detectorflow cell

Opticalslit

Diodearray

Vis lamp

Grating

254 nm

240 240320 nm240 nm

320 nm

320 nm

254 +380 nm

240 +320 nm

UV lamp

Eλ1 Eλ2

DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






DET

ECTO

RS

36







HPLC Detectors

50

40

30

20

10

220 240 260 280 300 320 340 360 380 4000

Ab

sorb

ance

(m

AU

)

Wavelength (nm)

30 nm






340 nm





Mirror

Photomultiplier

Lens

Lens

Flow cellPhotodiode

Xenon flash lamp

DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






DET

ECTO

RS

37







HPLC Detectors

40353025

201510

50

250 300 350 400 450 500 550 600

200

nm

Internal conversion

250

nm

Exci

tati

on

Flu

ore

scen

ce

Ground state So

S2

S1

No

rm

Wavelength (nm)

Excitationspectrum

Emissionspectrum




Waste

DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






DET

ECTO

RS

38






HPLC Detectors



Eluent only

Eluent + sample

Lamp

Lamp

Photomultiplier


Column effluent

Single output

Analyte






Nebulizer

Nebulizerchamber

DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






DET

ECTO

RS

39






HPLC Detectors


HPLCcolumneluent


Gasinlet

Dryingtube








DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






DET

ECTO

RS

40








HPLC Detectors


Workingelectrode

Reference electrode

Counterelectrode

DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






DET

ECTO

RS

41







HPLC Detectors


Phenol

Catechol

Quinol

Quinone

Thiol

Carbohydrate

Glycoside

Nucleoside

Hydroxy

MethoxyAmine

Nucleobase





levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory










levels







0

0

2

600

4 6 8 10 12 14 16 18-50

pA

mAU

Minutes

Charged aerosol

UV

-2

25

Citric acid

Phenylalanine

Theophylline

Propranolol

Naproxen


Citric acid

Phenylalanine

Propranolol

Naproxen

Diclofenac

Progesterone




routine analysis


bull Biomolecules








for any laboratory






critical choices in hplc

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