HPLCPresented by:

AMOL PATILM.S. (Pharm.) IInd sem.Department of Pharm. AnalysisNIPER, MOHALI

DEFINITION CHROMATOGRAPHY

• Separation of a mixture into individual components.

• The separation uses a Column (stationary phase) and Solvent (mobile phase).

• The components are separated from each other based on differences in affinity for the mobile or stationary phase.

• The goal of the separation is to have the best RESOLUTION possible between components.

3

Invention of Chromatography by M. Tswett

Ether

CaCO3

Chlorophyll

ChromatoChromatography

ColorsColors

Milestones in Chromatography

Chromatography (from Greek :chromatos -- color , "graphein" -- to write)

• 1903: Russian botanist Mikhail Tswett separates plant pigments

• 1938: Russian scientists Izmailov and Shraiber use “drop chromatography”, later perfected as Thin Layer Chromatography (TLC) by Kirchner in the U.S.

• 1952: Martin and Synge receive Nobel Prize for “invention of partition chromatography” or plate theory to describe column efficiency

• 1966: HPLC was first named by Horvath at Yale University but HPLC didn’t “catch on” until the 1970s

• 1978: W.C. Stills introduced “flash chromatography”, where solvent is forced through a packed column with positive pressure

Late 1970s/early 1980s• Instrumentation developed for high pressure solvent

delivery: pumps, autosamplers, diode array detectors• More uniform packing material produced for columns

20 years later• Nothing really “new”, but by returning to the basic theory of

chromatography, even better columns are on the market: smaller particle sizes which yield faster separations, but require hardware to withstand higher pressures.

UPLC 2004

Types of Liquid Chromatography

Gravity Chrom.Tsvett, 1903

Flash Chrom.1978

HPLC 1952 UPLC 2004(TLC) PaperChrom.

Chromatography - Classification• Basis of shape

• Column Chromatography – Open column, flash, vacuum• Planar Chromatography – TLC, HPTLC, OPLC, Centrifugal TLC

• Mode of Separation• Adsorption (NPC, LSC)– separates molecules based on polarity, least polar

eluting first• Partition - (RPC, LLC) – Separates molecules based on combination of

solubility parameters, partition coefficients, and polarity, most polar eluting first

• Ion exchange – Separates molecules on basis of molecular charge• Size exclusion (GPC, GFC) – separation based on molecular size, largest

eluting first • Affinity – based on affinity with ligand

• Basis of Mobile Phase• Liquid Chromatography – LLC, LSC• Gas chromatography – GLC, GSC

Various forms of Chromatography• Column Chromatography

• Prep Column Chromatography• Flash Chromatography (FC)• Vacuum liquid Chromatography (VLC)

• Ion Exchange Chromatography• Gel Chromatography

• Gel Filtration (GFC)• Gel Permeation (GPC)

• Pressure Liquid Chromatography• Low-Pressure LC • Medium Pressure LC (MPLC)• High Pressure LC (HPLC)

• Normal Phase and Reversed Phase Chromatography

• Liquid-liquid Chromatography• Countercurrent Chromatography (CCC)• Droplet Countercurrent Chromatography (DCCC)

• Planar Chromatography• Preparative Thin Layer Chromatography (PTLC)• Centrifugal TLC• Overpressure layer Chromatography (OPLC)

• Gas Chromatography• Gas Liquid Chromatography• Gas Solid Chromatography

Liquid Chromatography • Liquid Chromatography (LC) is a chromatographic

technique in which the mobile phase is a liquid.• LC is a much older technique than GC, but was

overshadowed by the rapid development of GC in the 1950’s and 1960’s.

• LC currently the dominate type of chromatography and is even replacing GC in its more traditional applications.

• Advantages of LC compared to GC:• 1.) LC can be applied to the separation of any compound that is soluble in a liquid phase.• ‚ LC more useful in the separation of biological compounds, synthetic or natural • polymers, and inorganic compounds

• 2.) Liquid mobile phase allows LC to be used at lower temperatures than required by GC• ‚ LC better suited than GC for separating compounds that may be thermally

labile

• 3.) Retention of solutes in LC depend on their interaction with both the mobile phase and • stationary phase.• ‚ GC retention based on volatility and interaction with stationary phase• ‚ LC is more flexible in optimizing separations change either stationary or

mobile phase

• 4.) Most LC detectors are non-destructive• ‚ most GC detectors are destructive• ‚ LC is better suited for preparative or process-scale separations

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MOBILE PHASE LIQUID

Liquid-LiquidChromatography (Partition)

Liquid-SolidChromatography (Adsorption)

Liquid Solid

Normal Phase Reverse Phase Normal Phase Reverse Phase

Mobile Phase - NonpolarStationary phase - Polar

Mobile Phase - PolarStationary phase - Nonpolar

FORMAT

STATIONARYPHASE

•HPLC stands for “High-performance liquid chromatography” (sometimes referred to as High-pressure liquid chromatography).

•High performance liquid chromatography is a powerful tool in analysis, it yields high performance and high speed compared to traditional columns chromatography because of the forcibly pumped mobile phase.

HPLC is a type of liquid chromatography where the sample is forced through a column that is packed with a stationary phase composed of irregularly or spherically shaped particles, a porous monolithic layer , or a porous membrane by a liquid (mobile phase) at high pressure.

INTRODUCTION

Parameters used in HPLC

Dead volume , Dead Time

Dead volume is the total volume of the liquid phase in the chromatographic column.

The dead time of a column it is the elution time of an unretained component. It is also equal to the time required to flush one column volume of eluant through the column:

t0 = V0/F

Vm/F = tm

where V0 (Vm) is the volume of liquid contained in the column, and F is the flow rate.

 

 

COLUMN DIMENSIONS (I.D. x

Length (mm))  

VOID VOLUME

(ml)                         2.1   x   100       0.24          2.1   x   150       0.37          2.1  x    250       0.61          2.1   x   300       0.73                           4.6   x   100       1.16          4.6   x   150       1.75          4.6   x   250       2.90          4.6   x   300       3.49                         10.0   x   100       5.50        10.0   x   150       8.25        10.0   x   250       13.75        10.0   x   300       16.49                     

Retention Time (tR)

• Retention time is a measure of the amount of time a solute spends in a

column. It is the sum of the time spent in the stationary phase and the mobile phase.

• The time elapsed between the injection of the sample components into the column and their detection is known as the Retention Time (tR).  

• The retention time is longer when the solute has higher affinity to the stationary phase due to its chemical nature.  For example, in reverse phase chromatography, the more lypophilic compounds are retained longer.  

• Therefore, the retention time is a property of the analyte that can be used for its identification.

• retention time (adjusted) (t'R) - the retention value obtained by subtracting the dead time from the total retention time.

• What is Difference Between Retention Time and Relative Retention Time?

• The RT for a compound is not fixed as many factors can influence it even if the same GC and column are used.

These include:

• The gas flow rate• Temperature differences in the oven and column• Column degradation• Column length

• These factors can make it difficult to compare retention times. Even if you use the same GC just a few days apart, there can be small differences in the retention time of a compound

•Relative Retention Time - method 1

•The use of the relative retention time (RRT) reduces the effects of some of the variables that can affect the retention time. RRT is an expression of a sample’s retention time relative to the standard’s retention time.

RRT = Standard R• current method used by the United States Pharmacopeia and the European Pharmacopeia for calculation of RRT

RRT = (t2 - t0) / (t1 - t0)

•t0 = void time

•t1 = retention time of a standard

•t2 = retention time of an unknown

Relative Retention Time Calculation - method 2•This is an older version used by European Pharmacopeia up to 2002. It can occasionally be found in current literatures. In this method, the void time is not taken into account for the RRT calculation. You should be aware of this method, but it should not be used.

RRT = t2 / t1

•Regardless of which method was used, if the RRT is greater than one, the unknown elutes later than the standard. If the RRT is smaller than one, the unknown elutes before the standard. If the RRT is one, the unknown and the standard elute at the same time.

Retention Factor (k), Capacity Factor (k’)

•  Retention factor (k) is defined as the ratio of time an analyte is retained in the stationary phase to the time it is retained in the mobile phase.

• The k’value for a given analytes will be determined by its relative affinity

for the stationary phase and mobile phase.• It is calculated from RetentionRetention Time divided by the Time divided by the

time for an unretained peak{t0}time for an unretained peak{t0}

• As k’ grows larger, its effect reaches a limit at a value of about 10.

• Since k’ depends on retention time, longer columns eventually have a diminished effect on resolution.

Retention factor is much less than

unity, elution occur so rapidly that

the accurate determination of

retention factor is difficult.

Retention factor is larger

somewhere 20-30,the elution times

are unusually long.

Ideally separation of solute carried

out for which the retention factor are

between 2 and 10.

Influencing the Capacity Factor k’

• Mobile Phase Strength -• altering the mobile phase strength also alters the

retention of the analytes.

• Bonded Phase Functionality (Reverse Phase) -• As the bonded phase hydrophobicity increases

(increasing alkyl chain length, etc.) so will the retention of the analytes.

• Temperature -• As temperature increases, the retention time

decreases. This does not necessarily result in poorer separation because of the other factors in the resolution equation.

Retentivity (k’) decreases 2 - 3 fold for each Retentivity (k’) decreases 2 - 3 fold for each 10% increase in mobile phase strength.10% increase in mobile phase strength.

Mobile Phase Strength

Mobile Phase Strength vs. k

Temperature Effect on k

Temperature Effect on Capacity Factor

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

20ºC 25ºC 30ºC 35ºC 40ºC 45ºC

Column Temperature

k'

MP

EP

PP

BP

Besides shorter run times – usually a good thing – an increase in temperature can have other beneficial effects. As the temperature increases, the viscosity of the mobile phase will decrease, resulting in lower pressures. Lower mobile-phase viscosity also improves diffusion in the chromatographic system, giving narrower peaks. Simultaneously, shorter retention times will generate narrower peaks. many cases, an increase in temperature can have multiple benefits – if adverse selectivity changes are not encountered. Additionally, decreased solvent viscosity at elevated temperature (1-2% per °C) leads to lower back pressure

Summary of k’ Effects

• A larger value of k’ means better resolution...to a certain extent (k’ = 10 maximum)

• Increasing the mobile phase strength decreases k ’

• Increasing the temperature decreases k’, but may not result in a “bad” separation based on the other factors affecting resolution

Selectivity Factor, α

• The selectivity or separation factor represents the ratio of any two adjacent k’ values, thereby describing the relative separation of adjacent peaks. This relationship is expressed as:

If α = 1, two components are perfectly overlapping

Effect of α on Overall Resolution

• Remember the resolution equation?

• Let’s only look at the part involving α

• And see how much resolution will improve with small changes in α

)]'1/('[]/)1[(4/1 kkNR s

]/)1[(4/1 sR

Effect of α on Overall Resolution

• For an α value of 1.1, the contribution of the selectivity term is• (1.1 – 1) / 1.1 = 0.09

• For an α value of 1.4, the contribution of the selectivity term is• (1.4 – 1) / 1.4 = 0.29

• So...a very small change in α leads to a more than THREE-FOLD increase in the contribution to resolution.

]/)1[(4/1 sR

Effect of α on Overall Resolution

• As α grows larger, its effect reaches a limit at a value of about 5.

• Since α depends on components’ retention factor k’, longer columns eventually have a diminished effect on resolution.

Influencing the Selectivity Factor α

• Mobile Phase Type -• The importance of the type of interactions between the

mobile phase and analytes is critical to the optimization of the selectivity of a system.

• Column Type -

• The bonded phase functionality can be selected by its chemical nature to provide better selectivity in an analytical method.

• Temperature -• Selective interactions between analyte molecules and the

stationary phase may not become evident until a critical temperature is attained.

• At elevated temperature however, the solute transfer from the mobile phase to the stationary phase is more efficient. This results in high efficiency, even at elevated flow rates.

• The low back pressure allows the use of smaller particle sizes and/or longer columns to increase efficiency and resolution.

The Theory of Chromatography The Theory of Chromatography

The Theory of Chromatography

There are two theories to explain chromatography

Plate theory - older; developed by Martin & Synge in 1941

Rate theory - currently in use• Proposed by van Deemter in 1956• Accounts for the dynamics of the separation

Column Efficiency, N• Column efficiency is generally expressed in terms of

theoretical plate number

• A theoretical plate in many separation processes is a hypothetical zone or stage in which two phases, such as the stationary and mobile phases of a substance, establish an equilibrium with each other.

Plate Theory - Martin & Synge 1954 Nobel Laureates

• View column as divided into a number (N) of adjacent imaginary segments called theoretical plates

• Within each theoretical plate analyte(s) completely equilibrate between stationary phase and mobile phase

ColumnTheoretical plate

Greater separation occurs with: –greater number of theoretical plates (N) –as plate height (H or HETP) becomes smaller

H = L / N where L is length of column, N is number of plates, and H is height of plates or height equivalent

to theoretical plate (HETP)

N can be Estimated Experimentally from a Chromatogram

•N = 5.55 tR2/ w1/2

2 = 16 tR2/ w2

where:

tR is retention timew1/2 is width at h0.5 w is width measured at baseline

N is a ratio of tR and σ of Wb which is 4σ

Choice of Column Dimensions: maximum efficiency

• Nmax = 0.4 * L/dp

where:Nmax - maximum column efficiencyL - column lengthdp - particle size

• So, the smaller the particle size the higher the

efficiency!

Calculation of Theoretical Plates

N = A(tN = A(trr /W) /W)22

WW AA MethodMethod Width measured atWidth measured atWWii 4 4 InflectionInflection Inflection point (60.7% of Inflection point (60.7% of

peak height)peak height)

WWhh 5.54 ½ Height 5.54 ½ Height 50% of peak height 50% of peak height

WW3s3s 9 9 3s 3s 32.4% of peak height 32.4% of peak height

WW4s4s 16 16 4s4s 13.4% of peak height 13.4% of peak height

WW5s 5s 2525 5s 5s 4.4% of peak height 4.4% of peak height

WWbb 16 16 Tangent Tangent Baseline, following tangent Baseline, following tangent

drawingdrawing

Constants A are different at each peak width, assuming a perfect Gaussian shape.

Real-world peaks often have tailing, so widths measured at the lower part of the peak more accurately reflect the tailing when calculating N.

Calculation of Efficiency, N

• Width measured at the baseline after tangent lines are drawn on the peak. Used when tailing is minimal.

Width measured at 4.4% Width measured at 4.4% of peak height, no of peak height, no tangents drawn. Used tangents drawn. Used when tailing is significantwhen tailing is significant..

Effect of N on Overall Resolution

• Since the contribution of N to resolution is a square root, doubling N from 5000 to 10,000 only increases the contribution to resolution by 41%.

NR s 4/1

Plates N Contribution 5,000 70.7 - - - - 10,000 100 41% 20,000 141.4 100%

Effect of N on Overall Resolution

• Note that there is no flattening of the curve like with k’ and α.

• Resolution will continue to increase as theoretical plates increase.

Influencing the Efficiency, N

• Particle Size -• The smaller the particle size and the narrower the range of the

particle size distribution, the more efficient the column.

• Packing Type -• Totally porous particles will also have greater efficiency than

solid or pellicular-shaped packings, due to the additional surface area attributable to the pores.

• Mobile Phase Viscosity -• As mobile phase viscosity increases, molecular movement

through the mobile phase is inhibited.

• Temperature -• For reverse phase chromatography, an increase in efficiency, N,

may be realized as column temperature is increased.

Flow rates and Longer column

Effect of Particle Size on N

Column Diameter (mm)

Column Length (cm)

Particle Size (µm)

4 Peak Width (µL)

Theoretical Plates per centimeter

10 25 10 1118 333

4.6 25 10 237 333

4.6 25 5 167 667

4.6 10 5 106 667

4.6 10 3 82 1111

4.6 3 3 45 1111

3 10 5 45 667

2 25 10 45 333

2 25 5 32 667

2 10 5 20 667

2 10 3 15 1111

1 25 10 11 333

1 25 5 8 667

1 25 3 6 1111

1 10 5 5 667

1 10 3 4 1111

Smaller particle sizes result in higher numbers of theoretical plates

Definition of Resolution

Resolution is a measure of how well two peaks are separated from each other.It is calculated as the difference in retention time of two eluting peaks divided by the average width of the two peaks at the baseline.

63

tR1 tR2

W1 W2

W1/2h,1 W1/2h,2 h1/2

• (1) Retention time for the twopeaks will be a function ofretention factor (k’).

•(2) The selectivity ( αα ) will alsoaffect the retention timevalues for the two peaks.

•(3) Peak width will be a functionof column efficiency (N) andasymmetry (Asym).

• Resolution: The Relative Effectiveness of k’, α, and N

The Impact of Efficiency on Resolution

PARAMETER INFLUENCED BY TARGET VALUE Efficiency, N Column, System

Flowpath Configuration

Minimum of 400 Theoretical Plates per

centimeter Capacity Factor, k’

Mobile Phase Strength

1.0 - 10

Selectivity, Mobile Phase Type,

Stationary Phase Type

1.1 - 2

Resolution, Rs All of the Above 1.3 - 1.5 or Greater

Review of Factors

Relative Influence of All Factors on Resolution

Parameter Parameter ChangeChange

NN k’k’ αα RRss

StandardStandard 10,00010,000 22 1.11.1 1.521.52

+10% N+10% N 11,00011,000 22 1.11.1 1.591.59

-25% N-25% N 7,5007,500 22 1.11.1 1.311.31

-50% N-50% N 5,0005,000 22 1.11.1 1.071.07

-60% N-60% N 4,0004,000 22 1.11.1 0.960.96

-75% N-75% N 2,5002,500 22 1.11.1 0.760.76

+10% k’+10% k’ 10,00010,000 2.22.2 1.11.1 1.561.56

+10% +10% αα 10,00010,000 22 1.21.2 2.782.78

Note that changing α a very small amount has the biggest effectα a very small amount has the biggest effect

• In the rate theory, a number of different peak dispersion processes were proposed and expressions were developed that described

• the contribution of each of the processes to the total variance of the eluted peak

• the final equation that gave an expression for the variance per unit length of the column

Takes account of the time taken for the solute to equilibrate between the stationary and mobile phase (unlike the plate model, which assumes that equilibration is infinitely fast)

The resulting band shape or a chromatographic peak is therefore affected by the rate of elution

The Rate Theory of ChromatographyThe Rate Theory of Chromatography

• The first comprehensive approach to Peak dispersion in a Chromatographic Column was taken by Van Deemter .

• Van Deemter et al. assumed that there were four band spreading processes responsible for peak dispersion, namely,

• multi-path dispersion• longitudinal diffusion • resistance to mass transfer in the mobile phase• resistance to mass transfer in the stationary phase

• Van Deemter derived an expression for the variancethe overall variance per unit length of the column. Furthermore, as the individual dispersion processes can be assumed to be random and noninteracting, the total variance per unit length of the column can beobtained from a sum of the individual variance contributions.

• Longitudinal Diffusion• Solute continuously diffuses away from the concentrated center of its zone• The greater the flow rate, the less time is spent in the column and the less longitudinal

diffusion occurs• The longer the solute band remains in the column, the greater will be the extent of

diffusion. The time the solute remains in the column is inversely proportional to the mobile phase velocity, so, the dispersion will also be inversely proportional to the mobile phase velocity.

• Van Deemter et al. derived the following expression for the variance contribution by longitudinal diffusion, (sL2 ), to the overall variance/unit length of the column.

• Because longitudinal diffusion in a gas is much faster than in a liquid, the optimum flow rate in gas chromatography is higher than in liquid chromatography

Term B

H = A + B/u + u [CM +CS]

Van Deemter model

Longitudinal diffusion

B = 2γ DM

DM: molecular diffusion coefficientB term dominates at low u, and is more important in GC than LC since DM(gas) > 104 DM(liquid)

One of the main causes of band spreading is DIFFUSIONThe diffusion coefficient measures the ratio at which a substance moves randomly from a region of higher concentration to a region of lower concentration

where (Dm) is the diffusivity of the solute in the mobile phase,(u) is the linear velocity of the mobile phase,and gamma is a constant that depended on the quality of the packing.

Nonequilibrium (Resistance to MassTransfer Term)• This term comes from the finite time required for

the solute to equilibrate between the mobile andstationary phases

• Some solute is stuck in the stationary phase, butthe remainder in the mobile phase moves forwardresulting in spreading of the zone

• The slower the flow rate, the more completeequilibration is and the less band broadeningoccurs

H = A + B/u + u [CM +CS]

Van Deemter model

Term C (Resistance to mass transfer) BandwidthStationary

phase

Mobilephase

Elution

Broadened bandwidth

Slow equilibration

The analyte takes a certain amount of time to equilibrate between the stationary and mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase, the worse the broadening becomes.

Multiple Flow Paths(Eddy Diffusion)

Term A- molecules may travel unequal distances- independent of u- depends on size of stationary particles or coating (TLC)

H = A + B/u + u [CM +CS]

time

Eddy diffusionMP moves through the column which is packed with stationary phase. Solute molecules will take different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths.

• It follows, that those molecules taking shorterpaths will move ahead of the mean and those that take the longer paths will lag behind the mean.

• This will result in a differential distance traveled(dl) this differential flow of the solute moleculesresults in band dispersion.

λ– dimensionless constant characteristic ofpackingd p– the particle diameter

The A term can be minimized by:• Reducing the particle size of the packing• Packing the column more uniformly(spherical particles of similar size

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre If peak is not symmetrical- shows Fronting or Tailing

The asymmetry value (Asym) for a peak is a measure of the divergence of the peak from a perfect Gaussian peak shape. Peaks with asymmetry values > 1.0 are said to be “tailing”, those with asymmetry values < 1.0 are said to be “fronting”.

FRONTING

Due to saturation of S.P & can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites & can be eliminated by support pretreatment.

• Peak with poor symmetry can result in,

• The Tailing Factor is defined by the USP as the distance from the front edge of the peak to the back edge, divided by the distance from the front edge to the centerline, with all distances measured at 5% of the maximum peak height.

• The Asymmetry Factor is defined by ASTM as the distance from the centerline to the back edge divided by the distance from the centerline to the front edge, with all distances measured at 10% of the maximum peak height.

Peak Tailing

• The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention. In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase.

• However, polar interactions with any ionised residual silanol groups residing on the silica support surface are also common. Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups, producing tailing peaks. This is illustrated in the figure shown below at a mobile phase pH >3.0.

How to Avoid Peak Tailing• Operate at a lower pH. As Silanol groups are acidic, secondary

interactions can be minimised by performing the chromatographic separation at a lower pH, thereby ensuring the full protonation of such ionisable residual silanol groups;

• this may lead to a decrease in retention time is ionisable basic analytes are present in the sample - this may need to be mitigated by a reduction in the organic modifier content of the mobile phase. Standard silica should not be used under pH 3 as this may induce silica dissolution. Agilent ZORBAX Stable Bond (SB) columns are designed to operate at low pH (<3).

• The five component mix of basic drug compounds in the chromatograms below consist of Phenylpropanolamine, Ephedrine, Amphetamine, Methamphetamine and Phenteramine (Peaks 1-5) respectively.

• The first chromatographam was obtained at a mobile phase pH of 7.0, and under these separation conditions Peak 4, Methamphetamine, shows an Asymmetry of 2.35. Lowering the mobile phase pH reduces basic analyte interaction with residual silanol groups, thereby eliminating excessive peak tailing. The second chromatographic trace was acquired with a mobile phase pH of 3.0, and the Asymmetry of Methamphetamine under these separation conditions was reduced to 1.33.

Use a highly deactivated column or End capping

Chemical reagents typically used in the end-capping process include;•Trimethylchlorosilane (TMCS)•Hexamethyldisilazane (HMDS)

Work at High pH when analyzing basic compound

• From a practical point of view it is generally usually only possible to use pH to suppress acid ionisation, as base ionisation requires pH values typically greater than 8, at which pH silica dissolution can occur.

• However, Agilent ZORBAX Extend HPLC Columns are able to operate extended pH, the silica surface being protected from dissolution by the use of bi- and tridentate ligands. These ligands are bidentate and are bridged using proprietary chemistry, prior to their application to the stationary phase. Their bridged structure affords steric protection against hydrolysis of the silica surface.

b a T N

0.5 0.5 1.00 1850

0.6 0.4 1.50 1520

0.7 0.3 2.33 1160

0.8 0.2 4.00 790

0.9 0.1 9.00 410

Asymmetric peaks have fewer theoretical plates, and the more asymmetric the peak the smaller the number of theoretical plates. For example, the following table gives values forNfor a solute eluting with a retention time of 10.0 min and a peak width of 1.00 min.

InstrumentationPrinciple Pattern An Example

Detector

Thermostatted Column Compartment

Autosampler

Binary Pump

Vacuum DegasserSolvent Cabinet

Solvent Reservoirs

Controller

HPLC systemHPLC system

INSTRUMENTATION OF HPLC

Solvent storage bottleGradient controller and mixing unitDe-gassing of solventsPumpPressure gaugePre-columnSample introduction systemColumnDetector Recorder

Solvent delivery systems

• Continuously provide eluent (solvent).

• Provide accurate mobile phase compositions.• Includes solvent reservoirssolvent reservoirs, inlet filterinlet filter, and degassing facilitiesdegassing facilities which works

in conjugation

Inlet Filters

• Stainless Steel or glass with 10 micron porosity.

• Removes particulates from

solvent.

107

Solvent Filters

Solvent Inlet Filer Stainless Steel or

glass with 10 micron porosity.

Removes particulatesfrom solvent.

Precolumn Filter Used between the injector and

guard column. 2 to 0.5 micron Removes particulates from

sampleand autosampler wear debris.

Must be well designed to preventdispersion.

Guard column

Injector

Analytical Column

Precolumn Filter

Solvent Inlet Filter

Elution Modes in HPLC

• Isocratic elution– performed with a single solven or constant solvent mixture.

• Gradient elution–

continuous change of solvent composition to increase eluent strength.

• In isocratic elution, peak width increases with retention time linearly with the number of theoretical plates. This leads to the disadvantage that late-eluting peaks get very flat and broad.

• Best for simple separations

• Gradient elution decreases the retention of the later-eluting components so that they elute faster, giving narrower peaks . This also improves the peak shape and the peak height

• Best for the analysis of complex samples

111

High- / Low-Pressure Gradient System

High-pressure gradient

Mixer

Low-pressure gradient unit

Low-pressure gradient

Mixer

HPLC System Types

• High Pressure Gradient• Multiple pumps are used with a mixer after the

pumps

• Low Pressure Gradient• Solvents are mixed before the pump

113

Advantages and Disadvantages of High- / Low-Pressure Gradient Systems

• High-pressure gradient system• High gradient accuracy• Complex system configuration (multiple pumps

required)

• Low-pressure gradient system• Simple system configuration• Degasser required

Isocratic elution

Gradient elution

DEGASSING OF SOLVENTS:

• Several gases are soluble in organic solvents, when high

pressure is pumped, the formation of gas bubbles increases

which interferes with the separation process, steady

baseline & shape of the peak.

• Hence de-gassing is very important and it can be done by

various ways.

123

(i) Vacuum filtration:

De-gassing is accomplanished by applying a

partial vacuum to the solvent container.

But it is not always reliable & complete.

(ii) Helium Purging:

Done by passing Helium through the solvent.

This is very effective but Helium is expensive.

(iii) Ultrasonication:

Done by using ultrasonicator which converts ultra

high frequency to mechanical vibrations.

PUMP:

• The solvents or mobile phase must be passed through a

column at high pressures at up to 6000 psi(lb/in²) or 414 bar.

• As the particle size of stationary phase is smaller (5 to 10µ) the

resistance to the flow of solvent will be high.

• That is, smaller the particle size of the stationary phase the

greater is the resistance to the flow of solvents.

• Hence high pressure is recommended.

• Today the majority of modern chromatographs are fitted with

reciprocating pumps fitted with either pistons or diaphragms

126

The term "reciprocating" describes any continuously repeated backwards and forwards motion.

Widely used (~90% in HPLC system) type of pump.

Pulse damper Pulse damper is used to make the flow pulse free.

Solvent is sucked during back stroke and gets deliver to the column in forward stroke.

Flow rates of eluent can be set by adjusting piston displacement in each stroke.

128

Plunger Reciprocating Pump

motor and cam

plunger

plunger seal

check valvepump head

5 - 50µL

out

in

Mobile phase

check valve

• Solvent is pumped back and

front by a motor driven piston

• Two ball check valves which

open & close which controls the

flow

• The piston is in direct contact

with the solvent

• Small internal volume 35-400μL

• High output pressure up to

10,000 psi

• Ready adaptability to gradient

elution and constant flow rate

Advantages:Advantages:Small internal volume (35-400 µL)High output pressures up to 10,000 psi.Smart adaptability to gradient elution.Constant flow rates independent of column back column back pressure, solvent viscosity and temperaturepressure, solvent viscosity and temperature. .

Twin Piston Pump

The twin piston pumps with short stroke are among the most commonly used pumps for HPLC. Both pump heads are switched in series, whereby the piston in the first pump head delivers a specific volume per stroke. One pump is being filled while the other is delivering the solvent.

The twin piston pumps with short stroke are among the most commonly used pumps for HPLC. Both pump heads are switched in series, whereby the piston in the first pump head delivers a specific volume per stroke. One pump is being filled while the other is delivering the solvent.

The Pneumatic Pump

Air enters port (1) and applies a pressure to the upper piston that is directly transmitted to the lower piston. If connected to a liquid chromatograph, or column packing manifold, liquid will flow out of the left hand side non-return valve as shown in the diagram which continues until the maximum movement of the piston is reached. At the extreme of movement a micro switch is activated and the air pressure transferred from port (1) to port (2).

The piston now moves in the opposite direction and draws mobile phase through the right hand non-return valve refilling the cylinder with solvent. Again on reaching the limit of the piston movement a second micro switches the air supply from port (2) back to port (1) and the process repeated. The refilling process is extremely fast and, if an appropriate pulse dampener is used, the outlet pressure remains reasonably constant during the refill cycle.

Advantages:Advantages:Not very costly.Provide pulse free flow.

Disadvantages:Disadvantages:

Not suitable for gradient elution.Flow rate depends upon column back pressure, and viscosity.Small capacity for filing of solvent.

04/15/23 134

DISPLACEMENT PUMP / SYRINGE PUMP

WORKING

Consist of screw or plunger which revolves continuously driven by motor.

Rotatory motion provides continuous movement of the mobile phase which is propelled by the revolving screw at greater speed and pushes solvent through small needle like outlet.

Consist of large syringe like chamber of capacity 250 – 500 ml.

Double syringe pumps have also been developed in which one piston is delivering the solvent to the column while other one is refilled from the reservoir.

SAMPLE INJECTOR SYSTEM:

• Several injector devices are available either for manual or auto injection of the sample.

(a)Manual injection(Rheodyne/Valco injectors)

(b)Automatic injection

(i) Septum Injector

(ii)Stop Flow Injector

(iii)Rheodyne InjectorRheodyne Manual injector

(i)Septum Injector:

These are used for injecting the sample through a rubber septum.

This kind of injectors cannot be commonly used , since the septum has to withstand high pressures.

(ii)Stop Flow(On Line):

In this type the flow of mobile phase is stopped for a while & the sample is injected through a valve.

Rheodyne Injector

Automatic Injector System

• Also know as Autosampler.Autosampler.• Programmed based sample delivery system.• User loads vials filled with sample solution into the

autosampler tray (100 samples).• Autosampler automatically

1. Measures the appropriate sample volume,

2. Injects the sample,

3. Flushes the injector to be ready for

the next sample, etc., until all sample

vials are processed.• Also controls the sequence of samples for

injection from vials.140

HPLC Column [Column: is a heart of chromatography system]

C18 4.6 x 250 mm 5m 300oA

Stationary Phase

Dimension

Particle Size

Pore Size

                                                                       

                    

Water/Methanol

Column dimension (size), particle size and pore size, stationary phase

GUARD COLUMN:

Guard columns are placed anterior to the separating

column.

This protects and prolongs the life & usefulness of the

separating column.

They are dependable columns designed to filter or

remove:-

particles that clog the separating column,

compounds and ions that could ultimately cause

‘baseline drift’, decreased resolution, decreased

sensitivity and create false peaks.142

Compounds that may cause precipitation upon

contact with the stationary or mobile phase.

Compounds that may co-elute and cause

extraneous peaks & interfere with the detection

and quantification.

These columns must be changed on a regular

basis in order to optimize their protectiveness.

143

144

Normal Phase / Reversed Phase

Stationary phase

Mobile phase

Normal phase

High polarity(hydrophilic)

Low polarity(hydrophobic)

Reversed phase

Low polarity(hydrophobic)

High polarity(hydrophilic)

Separation Column for Reversed Phase Chromatography

• C18 (ODS) type

• C8 (octyl) type

• C4 (butyl) type

• Phenyl type • Cyano type

Si -O-Si

C18 (ODS)

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH3

Reversed Phase Chromatography

• Stationary phase: Low polarity• Octadecyl group-bonded silical gel (ODS)

• Mobile phase: High polarity• Water, methanol, acetonitrile• Salt is sometimes added.

Effect of Chain Length of Stationary Phase

C18 (ODS)

Strong

C8

C4

Medium

Weak

148

Separation Design

149

Separation Design

149

HPLC Column Dimensions

Most columns are constructed of stainless steel for highest pressure

resistance. PEEK™ [an engineered plastic] and glass, while less pressure tolerant,

may be used when inert surfaces are required for special chemical or biological applications.

Column Examples

A glass column wall offers a visual advantage. The flow has been stopped while the sample bands are still in the column. You can see that the three dyes in the injected sample mixture have already separated in the bed; the yellow analyte, traveling fastest, is just about to exit the column.

HPLC Columns:

1.Column Configuration2.Packing Material3.Bonded Phases

HPLC Column Configuration

HPLC Support Particles

Most often silica or silica gel (hydrated silicon-oxygen)

HPLC Support Particles

Micropellicular Particle:

Solid core

Thin outer skin of stationary phase

1.5 – 2.5 μm sizes

Very low surface area

Fast separation, low capacity

HPLC Support Particles

Porous Micro-sphere:

Most common

Large surface area

Variety of pore sizes

High capacity

HPLC Support Particles

Perfusion Particle:

Least common

Very large pores (0.5 μm)

High flow rate (fast)

Macromolecules (proteins)

HPLC Support Particles

Bonded Stationary Phases: R groups

Bonded Stationary Phases: R groups

Normal Phase separation of sugars using an amino bonded stationary phase and 75:25 acetonitrile:water

1. fructose2. glucose3. sucrose4. maltose

Bonded Stationary Phases: R groups

Bonded Stationary Phases

Size Exclusion

Stationary phase consists of porous particles with controlled pore diameter

Separation based on size (not MW)

No chemical interactions involved

Bonded Stationary Phases

Each Size Exclusion column has a defined range of separation

HPLC DETECTORSHPLC DETECTORS

The Detector is the“ “ Eye” ” of the HPLC System

Detector Analyte /attributes sensitivity Nature

UV-VisWorks with all molecules containing

chromophores absorbing UV-Visng

Specific

PDA Works for all wavelengths in UV-vis ng

FluorescenceCompound with native fluorescence

or fluorescence tagfg-pg

Conductivity Anions, cations, organic acids,

surfactantsng

Radio activity Radioactive labeled compounds ng-pg

HPLC Detectors

Detector Analyte /attributes sensitivity Nature

Refractive IndexTemperature sensitive; polymers, sugars, triglycerides, incompatible

with gradients0.1-10 µg

Universal

Evaporative light Scattering(ELSD)

Uniform response; nonvolatile to semi volatile compounds; compatible to

gradientsng

Electrochemical Redox reactions pg

Corona CADCan detect non UV absorbing

chromophoresng

Mass Spectrometer Definite analyte identification pg

Both universal and specific IR Works with all molecules Mg

Detection Condition Requirements

• Sensitivity• The detector must have the appropriate level of

sensitivity.

• Selectivity• The detector must be able to detect the target

substance without, if possible, detecting other substances.

• Adaptability to separation conditions• Operability, etc.

Types of detectors

• Bulk property detectors

1- Principle: Sense the difference in refractive index between column eluent and reference beam of pure mobile phase

2- Universal, not sensitive (limit of detection 1 µg)

3-Difficult to do gradient elution work

4- Must have good control of temperature of the instrument and composition of mobile phase

• Solute property detectors

1-UV/Vis spectrophotometer• Most popular• Only detects solute that absorb UV/Vis radiation• Mobile phase should not absorb radiation• Absorption of radiation by solute is a function of

concentration according to Beer Lambert’s law• Limit of detection is sub ng

Uv- visible detector

SenstiveWide linear range Principle :

• Lambert-Beer’s law:

A= ε b c

• At constant cell thickness and constant wavelength a linear connection between the Absorbance A and the Concentration C is achieved.

Types of uv-visible detectors

A. Fixed wavelength detectors

B. Variable wavelength detectors

C.Photodiode array detectors

Fixed wavelength detectors

Operates only at 1 wavelength (254nm) Best overall precision is noted for peak area

measurements as compared to variable wavelength detectors.

Uses a discrete source: • Low pressure mercury lamp (253.6 nm)

176

Optical System of UV-VIS Absorbance Detector

Sample cell

Reference cell

Photodiode

Photodiode

Ein

EinEin

Grating

D2 / W lamp

Eoutl

Optical System of Photodiode Array Detector

Sample cell

Photodiode arrayPhotodiode array

Grating

D2 / W lamp

A single photodiodemeasures the absorbance forthe corresponding wavelengthat a resolution of approx. 1 nm.

Photodiode array detectors

Earlier, they suffered from sensitivity problems, which has been solved by advanced flow cell design using fiber optics technology to extend path length without increasing noise or chromatographic band dispersion

Performs a simultaneous measurement of absorption as a function of analysis time and over a chosen wavelength range, so we get UV spectrum for each eluted peak

Used for method development where λmax of impurities in drug are unknown

Every compound can be quantitated at its λmax , so useful for trace analysis

Fluorescence detectorsDuring fluorescence detection, the sample is

irradiated with UV light of suitable wavelength and excited for emission of long

wavelength of visible region.The Xenon lamp continuously sends light of

325 - 410 nm.Provides LOD values 100 times lower than

absorption detectorsHighly selective

A UV/UV-VIS detector monitors the absorption of light with a specified wavelength. However, some substances absorb light at one wavelength, and then emit light called fluorescence at another wavelength.

This is a phenomenon in which a substance absorbs light to reach a high-energy level and then emits light to return to its original level. Such a substance has specific wavelengths of light that it absorbs (excitation wavelengths) and emits (emission wavelengths).

Figure 1 shows a FL detector optical system. While a UV/UV-VIS detector detects light that has passed through the flow cell, an FL detector detects fluorescence emitted in the direction to the exciting light.

• Refractive Index• For samples with little or no UV Absorption

• Alcohols, sugars, saccharides, fatty acids, polymers• Best results when RI of samples is very different

from RI of mobile phase• Flow cell is temperature controlled with a double

insulated heating block.

• REQUIRES isocratic separations• REQUIRES low pulsation pumps

A differential refractometer (DRI), or refractive index detector (RI or RID) is a detector that measures the refractive index of an analyte relative to the solvent. They are considered to be universal detectors because they can detect anything with a refractive index different from the solvent, but they have low sensitivity.

Principles of operation:When light leaves one material and enters another it bends, or refracts. The refractive index of a material is a measure of how much light bends when it enters. Differential refractometers contain a flow cell with two parts: one for the sample and one for the reference solvent. The detector measures the refractive index of both components. When only solvent is passing through the sample component the measured refractive index of both components is the same, but when an analyte passes through the flow cell the two measured refractive index are different. The difference appears as a peak in the chromatogram.

RI detector measures change in reflex index. A glass cell is divided into two chambers (cells). The effluent from LC column flow through the “sample cell”, while other cell called “reference cell” is filled with only mobile phase. When the effluent going through the sample cell does not contain any analyte, the solvent inside both cells are the same (Figure 1A). When a beam is irradiate on the cells, the observed beam will be straight in this case. However, in a case the effluent contains any components other than mobile phase; bending of the incident beam occurs due to the reflex index difference between the two solvents (Figure 1B). By measuring this change, the presence of components can be observed.

Differential Refractive Index Detector (Deflection-Type)

Light

Sample cell

Reference cell

Light-receiving unit

Conductivity detectors

Electric conductivity measurement of a solution is a method of detecting ions in the solution.

After the targeted ions are eluted, the change in electric current is detected, with a constant voltage imposed between the electrodes. A conductivity detector is employed as a detector in an ion chromatograph, which is a system dedicated to measuring ions. This detector is used mainly to measure inorganic ions and small organic substances, including organic acids and amines.

The electrical conductivity of the mobile phase is used as characteristic for detection.

Universal technique for ionic solutes and are used mainly for the separation of ions in water or polar eluents

Since the conductivity can change approx. 2 % per °C, conductivity detector are often equipped with automatic temperature compensation

An ECD is used to measure components displaying oxidation-reduction reactions, and detects electric currents generated by these reactions. The ECD has high selectivity, because the necessary voltage to cause an oxidation-reduction reaction depends on the component. The ECD has also high sensitivity, and is often used for measurement of biogenic substances such as catecholamine.

Electrochemical detectors

• Electrochemical detection can performed either in oxidative mode or in reductive mode depending in part on the analyte,

• Oxidative mode preferred coz-1) Operate and run routine samples2) Maintain the working electrode surface activity3) Avoid some preparative step need for detection • Reductive step also give poor signal to noise

ratio due reduction of dissolved oxygen in the solution

• Choice of oxidative vs reductive mode depend on the large part on the type of analyte as given in next slide

`

192

ELSD can outperform traditional detectors when analysing non-

chromtophoric samples by HPLC

Traditional HPLC detectors such as UV and RI have limited

capabilities

UV and RI are not compatible with a wide range of solvents

RI detection is not gradient compatible

Different analytes produce different UV responses depending

on their extinction co-efficient

ELSDs can detect anything that is less volatile than the mobile

phase

ELSD is universal and compatible with a wide range of solvents

193

Evaporative Light Scattering Detector

Unique Method of Detection

Three steps:

Nebulization

Mobile Phase Evaporation

Detection

195

Schematic representation of ELS

Step 1: Nebulization

Column effluent passes through nebulizer needle

Mixes with nitrogen gas

Forms dispersion of droplet

Step 2: Mobile Phase Evaporation

Droplets pass through a heated zone (Drift tube) Mobile phase evaporates from the sample particle Dried sample particles remain

Step 3: Detection

Sample particles pass through an optical cell Sample particles interrupt laser beam and

scatter light Photodiode detects the scattered light

ELSD – A Powerful Detector for HPLC

Universal Sensitive Gradient Compatible

AN ELSD IS AN EFFECTIVE REPLACEMENT OR A PERFECT COMPLEMENT TO EXISTING LC DETECTORS

RI UV Fluorescence

Four Reasons to Replace an RI with an ELSD:

1. Better sensitivity

2. Gradient compatible

3. Stable baselines

4. No solvent front peaks

201

DETECTOR LIMITATIONS:

Currently CAD appears to be most powerful and versatile detector based on its applicability.

A prerequisite for the use of CAD is, similar to MS detection, the use of volatile mobile phases.Hence its ease of use, make the CAD a powerful detector for research purpose, method development and routine analysis.

Dixon and Peterson - Corona – Charged Aerosol Detector

Impurity control AMINOGLYCOSIDES:

• Identification and control of impurities by UV - Spectrophotometry, mass spectrometry and pulsed amperometric detection (PAD).

METHOD DISADVANTAGE

• UV – Spectrophotometry

• Mass spectrometry

• Pulsed amperometric detection (PAD).

• Lack of a suitable chromophor, its requires high

Analyte concentrations and a low detection

wavelength (about 200 nm).

• Requires suitable specific reference materials for the

quantification of the impurities.

• Technically very difficult and Non - oxidizable

compounds are not detected.

• Methods developed for separation of impurities could be coupled to MS detection.

• Mobile phase compatible with HPLC – CAD methods must be volatile.

• By using different types of columns and mobile phases to analyze amino glycosides in pharmaceutical preparations.

• LOQs – 4ng on column for streptomycin,

- 100ng on column for netilmicin sulphate.

MAILLARD REACTION:

• Memantine (primary amine) - Alzheimer’s disease.• Primary amine reacts with reducing sugars in complex pathway –

“Maillard reaction”.• Degradation products from MR, reported in pharmaceutical

products.• Determine MR impurities using HPLC – CAD, by Hydro – RP

column and mobile phase was capable of separating 4 MR impurities.

• Detection by CAD allowed to quantification of impurities without a chromophor at levels 0.02 – 0.03%.