ChromatographyChromatographyGeneralGeneral

Chromatographic ProcessChromatographic Process

Chromatographic SystemsChromatographic Systems

Chromatographic TechniquesChromatographic Techniques

TLC/PC

HPLC

GC/SFC

PC-Paper Chrom

Chromatography – Separation MechanismChromatography – Separation Mechanism

AdsorptionAdsorption PartitionPartition Ion - Exchange & Ion - InteractionIon - Exchange & Ion - Interaction Size ExclusionSize Exclusion Affinity (antibody-antigen interactions; Affinity (antibody-antigen interactions;

chemical interaction; attraction)chemical interaction; attraction) Complexation - ChelationComplexation - Chelation Ion – exclusion (Separation of weak acids) Ion – exclusion (Separation of weak acids)

Sorption problems

ABsorption ADsorption

Different sorptions explained

Chromatograhy – Mechanism of SeparationChromatograhy – Mechanism of Separation

Adsorption Partition Ion exchange

Chromatography – Mechanism of SeparationChromatography – Mechanism of Separation

Affinity Size Exclusion

Chromatogram – Basic ParameterChromatogram – Basic Parameter

tR = retention timetm = dead time

H

1/2HW1/2

unretained

Chromatographic TheoriesChromatographic Theories

Adjusted retention time: Adjusted retention time: ttR’R’ = t = tRR – t – tMM

Plate theoryPlate theory – distillation – plate – distillation – plate numbernumber

N = 5.54[(tN = 5.54[(tRR – t – tMM)/w)/w1/21/2]]22

Plate height Plate height H = L/NH = L/N

This theory did not include interaction of analytes This theory did not include interaction of analytes with stationery phasewith stationery phase

Chromatography – Peak Broadening

Chromatographic TheoriesChromatographic Theories

Rate Theory Rate Theory – kinetic factors – van – kinetic factors – van DeemterDeemter

H = B/u + Cu (+ A)H = B/u + Cu (+ A)

Where:Where: u – velocity of mobile phaseu – velocity of mobile phaseB – effect of molecular diffusion B – effect of molecular diffusion C – Resistance to mass transferC – Resistance to mass transferA – Spreading related to different A – Spreading related to different

distance traveled by distance traveled by molecules molecules in packed columnsin packed columns

Chromatography – Packing Effect on BroadeningChromatography – Packing Effect on Broadening

Chromatography - EquilibriumChromatography - Equilibrium

AAmobilemobile A Astationarystationary

Van Deemter factors:Van Deemter factors:

Molecular diffusion (B)Molecular diffusion (B) – in mobile – in mobile phasephase

proportional to time analyte spends in proportional to time analyte spends in a columna column

affected by diffusion coefficient of affected by diffusion coefficient of analyte in mobile phaseanalyte in mobile phase

affected by temperature and pressureaffected by temperature and pressure not important in LC – low diffusion not important in LC – low diffusion

coefficientcoefficient inversely affected by mobile phase inversely affected by mobile phase

velocityvelocity

Van Deemter factors:Van Deemter factors:

Resistance to mass transfer (C):Resistance to mass transfer (C): Mass transfer in mobile and stationary Mass transfer in mobile and stationary

phasephase Lack of equilibrium – moving phaseLack of equilibrium – moving phase Affected by thickness of liquid phaseAffected by thickness of liquid phase Affected inversely by the diameter of Affected inversely by the diameter of

particles or inner diameter of capillary particles or inner diameter of capillary columncolumn

Lower at higher temperatures (viscosity)Lower at higher temperatures (viscosity)

Van Deemter factors:Van Deemter factors:

Conclusions:Conclusions: Minimum value for H is achieved Minimum value for H is achieved

when:when: stationery phase thickness is minimalstationery phase thickness is minimal column packed with the smallest particlescolumn packed with the smallest particles capillary columns have the smallest capillary columns have the smallest

internal diameterinternal diameter mobile and stationary phases have low mobile and stationary phases have low

viscosity and high diffusion coefficientviscosity and high diffusion coefficient

Chromatography – van Deemter PlotChromatography – van Deemter Plot

Mobile phase velocity

Multipath effect

Mass transfer

Diffusion (Longitudinal)

Pla

te h

eigh

t (c

m)

H

Cu

A

B/u

Chromatography - ResolutionChromatography - Resolution

Response

tR1 tR2tR

Wb1

R = 2(tR1 – tR2)/Wb1 – Wb2

Wb2

Baseline resolution for Gaussianshape peaks = 1.5

100%

Chromatography - ResolutionChromatography - Resolution

Resolution equation where separation Resolution equation where separation parameters are included:parameters are included:

RRss = ½ = ½ xx ( (-1/-1/+1) +1) xx k’ k’22/1+k’/1+k’22xx (L/h) (L/h)1/21/2

Where:Where: – selectivity factor (separation) – selectivity factor (separation) = t= tR1R1/t/tR1R1

k’ – migration term, capacity factor; k’ – migration term, capacity factor;

k’ = mk’ = mss/m/mmm

L – column lengthL – column lengthh – plate height h – plate height

Chromatography - ResolutionChromatography - Resolution

ChromatographyChromatography

Qualitative AnalysisQualitative Analysis Retention data – RT; RRetention data – RT; Rff; RRT; Kovacs ; RRT; Kovacs

IndexIndex

Quantitative AnalysisQuantitative Analysis Peak area and height usually proportional Peak area and height usually proportional

to the amount of componentto the amount of component CalibrationCalibration Internal Standard method Internal Standard method External Standard methodExternal Standard method Area Normalization method Area Normalization method

Chromatogram – Basic ParameterChromatogram – Basic Parameter

tR = retention timetm = dead time

H

1/2HW1/2

unretained

IS α

- C

hol

esta

ne

Ch

oles

tero

l

1

2

3

4

5

RRT1 = RT1/RTIS

RRT2 = RT2/RTIS

RRT3 = RT3/RTIS

Accurate to e few digits (2) at fourthDecimal Point

ENDEND

Chromatography - MethodologyChromatography - Methodology

Peaks Broadening