2

Chromato-graphy / -Chromato-graphy / -graph / -gram / -graphergraph / -gram / -grapher

• Chromatography:Chromatography: Separation Separation techniquetechnique

• Chromatograph:Chromatograph: InstrumentInstrument• Chromatogram:Chromatogram: Obtained Obtained

“Picture/Spectra”“Picture/Spectra”

• Chromatographer:Chromatographer: PersonPerson

Invention of Invention of Chromatography by M. Chromatography by M.

TswettTswettEther

CaCO3

Chlorophyll

ChromatoChromatography

ColorsColors

Comparing Comparing Chromatography to the Chromatography to the

Flow of a River... Flow of a River...

Base

Water flow

Light leaf

Heavy stone

5

Mobile Phase / Mobile Phase / Stationary PhaseStationary Phase

• A site in which a moving A site in which a moving phase (phase (mobile phasemobile phase) and ) and a non-moving phase a non-moving phase ((stationary phasestationary phase) make ) make contact via an interface contact via an interface that is set up.that is set up.

• The affinity with the The affinity with the mobile phase and mobile phase and stationary phase varies stationary phase varies with the solute. with the solute. SeparationSeparation occurs due to occurs due to differences in the speed differences in the speed of motion.of motion.

Strong

Weak

Mobile Mobile phasephase

StationarStationary phasey phase

7

Separation Process and Separation Process and

ChromatogramChromatogram for Column for Column ChromatographyChromatography

Ou

tpu

t co

nce

ntr

ati

on

Time

ChromatogramChromatogram

8

Pump

Sample injection unit(injector)

Column

Column oven(thermostatic

column chamber)

Detector

Eluent (mobile phase)

Drain

Data processorDegasser

Flow Channel Diagram for High Flow Channel Diagram for High Performance Liquid Performance Liquid

ChromatographChromatograph

11

ChromatogramChromatogram

tR

t0

Inte

nsi

ty o

f d

ete

ctor

sig

nal

Time

Peak tR : Retention time

hA

t0 : Non-retention time

A : Peak areah : Peak height

Retention Factor, Retention Factor, kk

tR

t0

Str

en

gth

of

dete

ctor

sig

nal

Time

tR: Retention timet0: Non-retention time

0

0R

t

ttk

13

Theoretical Plate Theoretical Plate Number, Number, NN

W

W1/2

H1/2

H

2

.21

R

R

/

R

W

t

W

2

2

2

545

16

Area

Ht

tN

14

Resolution, Resolution, RRSS

2,2/11,2/1

RR

21

RRS

12

12

18.1

)(21

hh WW

tt

WW

ttR

tR1 tR2

W1 W2

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

16

Objectives of Objectives of PretreatmentPretreatment

• To improve sensitivity and selectivityTo improve sensitivity and selectivity• To improve the accuracy of To improve the accuracy of

quantitative valuesquantitative values• To protect and prevent the To protect and prevent the

deterioration of columns and analytical deterioration of columns and analytical instrumentsinstruments

• To simplify measurement operations To simplify measurement operations and proceduresand procedures

• To stabilize target substancesTo stabilize target substances

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Filtration and Centrifugal Filtration and Centrifugal SeparationSeparation

• In general, filter every In general, filter every sample before injection!sample before injection!

• It is convenient to use a It is convenient to use a disposable filter with a disposable filter with a pore diameter of pore diameter of approx. 0.45 approx. 0.45 µµm.m.

• Centrifugal separation Centrifugal separation is applicable for is applicable for samples that are samples that are difficult to filter.difficult to filter.

Filter Syringe

18

DeproteinizationDeproteinization

• PrecipitationPrecipitation– Addition of organic solvent (e.g., Addition of organic solvent (e.g.,

Acetonitrile)Acetonitrile)– Addition of acid (e.g., Trichloroacetic Addition of acid (e.g., Trichloroacetic

acid, Perchloric acid)acid, Perchloric acid)– Addition of heavy metal or neutral Addition of heavy metal or neutral

salt(Ammonium Sulfate)salt(Ammonium Sulfate)

• UltrafiltrationUltrafiltration

19

Solid Phase ExtractionSolid Phase Extraction

(1)Conditioning

(2)Sample addition

(3)Rinsing

(4)Elution

Solvent with low elution strength

Solvent with high elution strength

Target componentUnwanted components

20

Pre-Column Pre-Column DerivatizationDerivatization

• OPA Reagent (Reacts with Primary OPA Reagent (Reacts with Primary Amines)Amines)

o-phthalaldhyde(OPA)

+ R-NH2 N-R

S-R’

R’-SH

NO2

2,4-dinitrophenylhydrazine(2,4-DNPH)

+

CHO

CHO

2,4-DNPH (Reacts with Aldehydes and Ketones)

O2N

NHNH2

C=OR

R’NO2

O2N

NHN=C

H+

R

R’

HPLC ApplicationsHPLC Applications

Chemical

Environmental

Pharmaceuticals

Consumer Products

Clinical

PolystyrenesDyesPhthalates

TetracyclinesCorticosteroidsAntidepressantsBarbiturates

Amino Acidsvitaminshomocysteine

BioscienceProteinsPeptidesNucleotides

LipidsAntioxidantsSugars

Polyaromatic HydrocarbonsInorganic Ionsherbicides

23

Qualitative AnalysisQualitative AnalysisIdentification based on retention timeIdentification based on retention time

Identification based on retention factor and response

Identification based on sample spiking

Spectral Peak Identification by PDA The use of selective detectors and spectrometers can greatly increase the confidence in the peak assignment. Detector systems such as Diode Array UV Spectrometers are able to record unique spectra for each peak within the sample chromatogram. The spectra may be recorded in ‘real time’ as the eluent can be directly introduced into the detector system.

Peak Purity

Peak purity can be established by taking the ratio of two signals (wavelengths) across the peak(s) of interest. If the peak is pure, then the ratio of the two signals should be constant across the peak. If the peak is impure, then the ratio between the two signals (wavelengths) will change across the peak as the spectral differences caused by the interfering peak change the signal ratio.

Spectral Characterization Mass Spectrometric detectors can be configured to produce fragmentation patterns that can be assigned to analyte moieties, so building up a ‘picture’ of the analyte molecule. The spectral peaks and patterns combined with the molecular (pseudomolecular) weight acquired can be used to characterise the analyte molecule.

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Quantitative AnalysisQuantitative Analysis

• Quantitation performed with peak Quantitation performed with peak area or height.area or height.

• Calibration curve created Calibration curve created beforehand using a standard.beforehand using a standard.

• - External standard method- External standard method– Absolute calibration curve methodAbsolute calibration curve method– Internal standard methodInternal standard method– Standard addition methodStandard addition method

Peak Height or Peak Area

Peak area is mostly used. In HPLC peaks may be tailed. In this case, peak heights may vary (although area will remain constant), area vales are more repeatable. A disadvantage of peak-area methods is that they are more affected by neighboring peaks. For trace analysis, when the peak of interest is very small, use peak height for calculations, this reduces the error sustained in small changes in peak start and end time variation.

Area %/ Height % (Normalization)

External Standard Quantitation The ESTD procedure uses absolute response factors. The response

factor is normally calculated as amount / area of the analyte in the calibration sample. Response factors are normally viewed as a single point calibration curve, using the origin to determine the second point for the regression line – this approach is often called ‘Single Point Calibration’.

37

Calibration Curve for Calibration Curve for Absolute Calibration Absolute Calibration

Curve MethodCurve Method

C1

C4

C3

C2

ConcentrationArea

A1

A2

A3

A4C1 C2 C3 C4

A1

A2

A3

A4

Concentration

Peak a

rea

Calibration curveCalibration curve

A line of best fit (regression line) is used to join the points of the curve obtained. The line of best fit is usually given a ‘Correlation Coefficient’ which is the square root of the regression coefficient and gives a measure of how well the data points fit a straight line. The intercept of the regression equation indicates systematic error – a large positive or negative value may indicate an inherent error within the sample preparation, concentration or analysis .The slope of the line indicates the analytical ‘sensitivity’.

The Matrix EffectThe matrix effect problem occurs when the unknown sample contains many impurities.If impurities present in the unknown interact with the analyte to change the instrumental response or themselves produce an instrumental response, then a calibration curve based on pure analyte samples will give an incorrect determinationSuggested method:1. Internal Standard Method2. Standard Addition Methods

Internal Standard Method

Internal standards are used to adjust for variations in analytical response due to instrumental and/or matrix effects and variations in the amount of sample due to variable injection volumes.•It must have similar chemical properties to the target substance.•Its peak must appear relatively near that of the target substance.•It must not already be contained in the actual samples.•Its peak must be completely separated from those of other sample components. •It must be added at a concentration that will produce a peak-area or peak height ratio of about unity with compound(s) of interest• It must be stable; unreactive with sample components, column packing, or mobile phase• It is desirable for it to be commercially available in high purity•Are added in the concentration range 0.3 – 0.5 portion of the expected MAXIMUM analyte concentration

Internal standards are used to adjust for variations in analytical response due to instrumental and/or matrix effects and variations in the amount of sample due to variable injection volumes.•It must have similar chemical properties to the target substance.•Its peak must appear relatively near that of the target substance.•It must not already be contained in the actual samples.•Its peak must be completely separated from those of other sample components. •It must be added at a concentration that will produce a peak-area or peak height ratio of about unity with compound(s) of interest• It must be stable; unreactive with sample components, column packing, or mobile phase• It is desirable for it to be commercially available in high purity•Are added in the concentration range 0.3 – 0.5 portion of the expected MAXIMUM analyte concentration

Drug Substance Internal StandardCholesterol compounds 19-hydroxycholesterolPhenolic acid 2,3,4 trihydroxybenzoic acidThuringiensin Adenosine MonophosphateOxolinic Acid Nalidixic AcidChlormadinone Acetate Medroxyprogesterone-17-AcetateAcetylsalicylic Acid Salicylic AcidSorbitol Methyl NonadecanateSimvastatin LovastatinDiazepam And Oxazepam Prazepam

43

Calibration Curve for Calibration Curve for Internal Standard Internal Standard

MethodMethod

C1

C4

C3

C2

Concentration Area

A1

A2

A3

A4C1/CIS C2 /CIS C3 /CIS C4 /CIS

A1/AIS

A2 /AIS

A3 /AIS

A4 /AIS

Concentration of target substance / Concentration of

internal standard

Are

a f

or

targ

et

sub

stan

ce /

Are

a f

or

inte

rnal st

an

dard

Calibration curveCalibration curveTarget

substanceInternal standard

CIS

CIS

CIS

CIS

AIS

AIS

AIS

AIS

44

Advantages of Advantages of Internal Standard Internal Standard

Method (1)Method (1)• Not affected by inconsistencies in injection Not affected by inconsistencies in injection

volume.volume.

10 µL injected

9 µL injected

CX / CIS

AX / AIS

XIS

XIS

Same Same area ratioarea ratio

45

Advantages of Advantages of Internal Standard Internal Standard

Method (2)Method (2)• Not affected by the pretreatment Not affected by the pretreatment

recovery rate.recovery rate.

100% recovery

rate

90% recovery rate

CX / CIS

AX /

AIS

XIS

XIS

Same Same area ratioarea ratio

Standard Addition Standard Addition MethodsMethods

Better method to use when matrix effects can be Better method to use when matrix effects can be substantialsubstantial

Standards are added directly to aliquots of the Standards are added directly to aliquots of the sample, therefore matrix components are the same.sample, therefore matrix components are the same.

Procedure:Procedure:

•Obtain several aliquots of sample (all with the Obtain several aliquots of sample (all with the same volume).same volume).

• known and unknown are the same analyteknown and unknown are the same analyte

•Spike the sample aliquots ==> add different Spike the sample aliquots ==> add different volume of standards with the same concentration volume of standards with the same concentration to the aliquots of sampleto the aliquots of sample

•Dilute each solution (sample + standard) to a fixed Dilute each solution (sample + standard) to a fixed volumevolume

•Measure the analyte concentrationMeasure the analyte concentration

48

Standard addition usually involves the addition of an

increasing increments of a standard solution to sample

aliquots of the same size (spiking).

49

Glucose in blood serum is Glucose in blood serum is determined spectrophotometrically by determined spectrophotometrically by the formation of a the formation of a colored complex colored complex with with o-toludineo-toludine. . Six identical Six identical 50.0 50.0 L samples L samples of of blood serum were treated with blood serum were treated with increasing amount of known increasing amount of known standardsstandards of glucose. of glucose. The following results were obtained:The following results were obtained:What is the glucose concentration in What is the glucose concentration in the blood serum samples.the blood serum samples.

Example of Standard Addition Data

The magnitude of the x-intercept is the concentration of the glucose in the original solution. To understand why this is so, consider the absorbance at the following two values:•at x = 0, the value of y is the absorbance of the solution with no added standard (i.e., it corresponds to the concentration of silver that we ultimately want).•at the x-intercept, there is no absorbance.Thus, the magnitude of the difference between x=0 and the x-intercept is the concentration of silver that is needed to produce the signal for the original solution of interest! Our job now is to determine the x-intercept.

The magnitude of the x-intercept is the concentration of the glucose in the original solution. To understand why this is so, consider the absorbance at the following two values:•at x = 0, the value of y is the absorbance of the solution with no added standard (i.e., it corresponds to the concentration of silver that we ultimately want).•at the x-intercept, there is no absorbance.Thus, the magnitude of the difference between x=0 and the x-intercept is the concentration of silver that is needed to produce the signal for the original solution of interest! Our job now is to determine the x-intercept.

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The value of glucose in the original sample is found by The value of glucose in the original sample is found by determiningdetermining the x-intercept by extrapolation, i.e., the x-intercept by extrapolation, i.e., finding the value of x finding the value of x when y = 0. when y = 0.

Or by using the values shown by the Linear Fit and the form of Or by using the values shown by the Linear Fit and the form of the the linear equationlinear equation

y = mx + by = mx + b0 = 0.006580 = 0.00658xx + 0.229 + 0.229

Solving for x, Solving for x, xx = -0.229/0.00658 = = -0.229/0.00658 = - 34.8- 34.8 g.g.

The amount of glucose in unknown glucose sample, that had The amount of glucose in unknown glucose sample, that had no standard glucose was added, is no standard glucose was added, is 34.80 34.80 g.g.

Since the volume was 50.0 Since the volume was 50.0 L, the [Glucose] = 34.8 L, the [Glucose] = 34.8 g / 50 g / 50

L orL or

1 1 g/g/L. This could also be expressed as [Glucose] = 0.696 L. This could also be expressed as [Glucose] = 0.696

mg/mLmg/mL

Microporous Silica Particles

Bonded Stationary Phase Bonded Stationary Phase

New generation of organo-silane material incorporates ethylene bridges into porous silica. Tetraethoxysilane + Bis(tetraethoxysilyl)ethane = Polyethoxysilane. • Provide pH stability from 1-12 • Five times more durability than earlier hybrids. •The homogenous surface offers some steric selectivity.

Ethylene Bridged Hybrid [BEH] HPLC Column.

Ethylene Bridged Hybrid [BEH] HPLC Column.

BEH C18 BEH ShieldRP18 BEH C8 BEH Phenyl BEH HILIC

BEH Amide

Ligand Type

Trifunctonal C18

Monofunctional Embedded Polar

Trifunctional C8

Trifunctional Phenyl-Hexyl

Unbonded BEH Particle

Trifunctional Carbamoyl

Ligand Density

3.1μmol/m2 3.3μmol/m2 3.2μmol/m2 3.0μmol/m2 n/a 7.5 μmol/m2

Carbon Load

18% 17% 13% 15% Unbonded 12%

Endcap Style

Proprietary TMS Proprietary Proprietary N/A None

pH Range 1-12 2-11 1-12 1-12 1-9 1-11

Temp.Limit 60 oC 45oC 60 oC 60 oC 45 oC 90 oCPore Diameter

130AO 130AO 130AO 130AO 130AO 130AO

Surface Area

185m2/g 185m2/g 185m2/g 185m2/g 185m2/g 185m2/g

Particle Sizes

1.7,2.5,3.5,5μm

1.7,2.5,3.5,5μm 1.7,2.5,3.5,5μm

1.7,2.5,3.5,5μm

1.7,2.5,3.5,5μm

1.7,2.5,3.5,5 μm

Charged Surface Hybrid columnWhen the silica surface, including both inner-pore area and outer-pore surface, is covalently modified by silyl ligands bearing charges, an opposite charges—can be permanently attached to the outer-surface by electrostatic attraction while being excluded from entering the pores. As a result, a material with a charged inner-pore surface and an oppositely-charged exterior surface can be created. CSH 130 C18= BEH 130 Base particle + low level of basic moieties + tri functional C18 / End cap. •Positive surface charge and Acidic pH. •Used for impurity profiling in stability indicating methods and/or forced degradation studies .The advantages of CSH Technology include:· Superior peak shape for basic compounds· Increased loading capacity· Rapid column equilibration after changing mobile phase pH· Improved batch to batch reproducibility· Exceptional stability at low and high pH.

Charged Surface Hybrid columnWhen the silica surface, including both inner-pore area and outer-pore surface, is covalently modified by silyl ligands bearing charges, an opposite charges—can be permanently attached to the outer-surface by electrostatic attraction while being excluded from entering the pores. As a result, a material with a charged inner-pore surface and an oppositely-charged exterior surface can be created. CSH 130 C18= BEH 130 Base particle + low level of basic moieties + tri functional C18 / End cap. •Positive surface charge and Acidic pH. •Used for impurity profiling in stability indicating methods and/or forced degradation studies .The advantages of CSH Technology include:· Superior peak shape for basic compounds· Increased loading capacity· Rapid column equilibration after changing mobile phase pH· Improved batch to batch reproducibility· Exceptional stability at low and high pH.

High strength silica columnWhen compared to ethylene-bridge-containing BEH and CSH particles, the higher silanophilicity of the 100% silica HSS particle offers significant advantages including increase polar compound retention and significantly different selectivity. Additionally, the HSS particle possesses the mechanical strength necessary to tolerate UPLC pressures up to1034 bar.It is designed to provide different selectivity for basic compounds when compared to traditional high coverage, fully endcapped C18 chemistries. This is due to the increased silanol activity on the silica particle surface when bonded at intermediate ligand densities with no endcapping.

High strength silica columnWhen compared to ethylene-bridge-containing BEH and CSH particles, the higher silanophilicity of the 100% silica HSS particle offers significant advantages including increase polar compound retention and significantly different selectivity. Additionally, the HSS particle possesses the mechanical strength necessary to tolerate UPLC pressures up to1034 bar.It is designed to provide different selectivity for basic compounds when compared to traditional high coverage, fully endcapped C18 chemistries. This is due to the increased silanol activity on the silica particle surface when bonded at intermediate ligand densities with no endcapping.

-synonyms: solid core particles, Fused-core particles-Most recent available particle size is 1.7 m (core 1.25 m, layer 0.23 m)

Solid Core HPLC Column

Solid CorePorous silica

Core shell columns and Van Deemter

Van Deemter equation: H = A +B/u + CuA-value (Eddy dispersion) - narrow particle size distribution in addition to an enhanced roughness of their surface compared to porous particles, leading to a smaller A-coefficient by about 40%.B-value (Longitudinal diffusion) - Compared to totally porous particles, the Fused-Core particles have a much shorter diffusion path because of the solid core.; 20% decrease in comparison with porous particles.C-value (Mass transfer resistances) – solid core, impenetrable by analytes cause shorter diffusion path – C-value is reduced.

Vendors and Available phasesVendor Column/product name

Average particle diameter (μm)

Shell thickness (μm)

Stationary phase chemistry

Advanced Material Technology

Halo 2.7 0.50C18, C8, HILIC, RP-amide, phenylhexyl, pentafluorophenyl

Advanced Material Technology

Halo Peptide-ES 160 Å

2.7 0.50 C18

Agilent Poroshell 300 5 0.25 C18, C8, C3Agilent Poroshell 120 2.7 0.50 EC-C18, SB-C18

Sigma–Aldrich Ascentis Express 2.7 0.50C18, C8, HILIC, RP-amide, phenylhexyl, pentafluorophenyl

Sigma–AldrichAscentis Express Peptide-ES 160 Å

2.7 0.50 C18

Phenomenex Kinetex2.61.7

0.350.23

C18, XB-C18, C8, HILIC, pentafluorophenyl

Macherey-Nagel Nucleoshell 2.7 0.5 RP-18, HILIC

Thermo Scientific Accucore 2.6 0.50C18, aQ, RP-MS, HILIC, phenylhexyl, pentafluorophenyl

Sunniest SunShell 2.6 0.5 C18

Commercially not available

Eiroshell1.71.71.7

0.350.250.15

C18

2D HPLCChromatographic technique in which the injected sample is separated by passing through two different separation stages. This is done by injecting the eluent from the first column onto a second column. Typically the second column has a different separation mechanism, so that bands that are poorly resolved from the first column may be completely separated in the second column. (For instance, a C18 column may be followed by a phenyl column.) Alternately, the two columns might run at different temperatures. The second stage of the separation must be run much faster than the first, since there is still only a single detector. Can also adopt in GC, CE, SEC,SFC. Multidimensional Protein Identification Technology (MudPIT) is a separation technique which utilized two chromatography techniques back to back and can be coupled to silica capillary. Typically the column consist of strong cation exchanger (SCX) followed by reversed phase (RP) material.

2D HPLCChromatographic technique in which the injected sample is separated by passing through two different separation stages. This is done by injecting the eluent from the first column onto a second column. Typically the second column has a different separation mechanism, so that bands that are poorly resolved from the first column may be completely separated in the second column. (For instance, a C18 column may be followed by a phenyl column.) Alternately, the two columns might run at different temperatures. The second stage of the separation must be run much faster than the first, since there is still only a single detector. Can also adopt in GC, CE, SEC,SFC. Multidimensional Protein Identification Technology (MudPIT) is a separation technique which utilized two chromatography techniques back to back and can be coupled to silica capillary. Typically the column consist of strong cation exchanger (SCX) followed by reversed phase (RP) material.