Chromatography Method Theory, Paper chromatography and TLC

CBB4032 Chemical Analysis Asna M.Z.

Principle of Chromatography
A process to separate a mixture in liquid or gaseous state For qualification and quantification of compound Principle based on concentration equilibrium of component of interest between two immiscible phases fixed to the column and mobile phase The phases must have different solubilities toward the component of interest A physico-chemical separation similar as distillation, crystallization or fractionated extraction

Consist of a column, stationary phase, mobile phase and sample Sample is eluted by a continuous addition of mobile phase for separation Sample is separated together with mobile phase and components in sample migrate at different velocities is recovered.

Basic of chromatography process

Chromatography analysis

Chromatographic separation theory

Kd = [S]stat/[S]mob Knowing the T of experiment, for Cm Cs transformation, variation of standard free energy, can be deduced G = - RT ln K

Example

The Kd of an organic salt between hexane and water is 90. A quantity of 0.1 mol of salt is dissolved in 100 cm3 of water. Predict how many moles of the salt will remain within aqueous phase following extraction by using 100 cm3 of aliquots of hexane to extract the salt from the aqueous phase.

After extraction,

Chromatography theory
If the solute, S spend some time in mobile phase and part of the time in mobile phase its rate of progress is governed by Kd. The ave linear rate of movement of mobile phase expressed as u = L/ tmob , L is column length Ave linear rate of solute migration for a chromatography, v = L/tR And v = u x fraction time spend in mobile phase

Retention parameters
Retention times tR is time taken for a solute to elute from a column Retention volume, VR volume of analyte present in mobile phase, VR = tR x F ; with constant F, flowrate Hold-up time, tm or dead time is time for mobile phase to pass through the column Adjusted retention time, tR is the difference between tR and tm

Retention factor of two compounds

Chromatography theory

Capacity factor parameter used to compare the relative rate of solute migration along column
K = (tR tmob) /tmob

Selectivity factor (separation factor) for two solutes

Resolution factor
Resolution factor, R used to quantify separation between two compounds

Chromatography theory
Column efficiency described by the Van Deemter equation in term of flow rate ,u H = L/N = A + B/u + Cu H is height equivalent of a theoretical plates N is number of theoretical plates L is the length of column A, B, C are constant from column, stat phase , mobile phase and Temp Linear dispersion 1 measured by the variance 12 increase with distance of migration, if the distance is L, (total column length) then L2 = H x L, so N = L2/ L2 = t2R/ 2

Gaussion peak

Schematic gas chromatogram, showing retention time, tR and width at half height, w Ideal chromatogram peak has a Gaussian shape if the peak height is h, then the width at half height, w is 2.35 measured at h and 50 % of peak area, represent half width of the peak at 60.6 % and the base peak w=4 4 represent volume of peak (contain 95 % of injected compound) N = 5.55 (tR/w )2 or N = 16(tR/wb)2 since N = tR2/ 2 W =2.35 and Wb = 4

Eluent C o l u m n

Detector

Selection guide for all different chromatographic techniques for liquid mobile phase as a function of molar mass, solubility and polarity of a compounds to be separated
IC Ionic HPLC ion pair Water soluble HPLC Normal phase Nonionic HPLC Reverse phase HPLC normal phase Polar HPLC reverse phase Organosoluble HPLC reverse phase Sample Nonpolar HPLC normal phase SEC gel filtration HPLC reverse phase

Molar mass <2000

Water soluble

Molar mass > 2000

IC

Organosoluble

SEC gel permeation

Chromatographic classification

Liquid phase
Liquid/solid chromatography Ion chromatography Size exclusion chromatography Liquid/liquid chromatography

Gas phase
Gas/liquid chromatography Gas/solid chromatography

Supercritical fluid chromatography

Paper chromatography
The paper a fixed phase and solvent as mobile phase. Ink mixture used is separated into separated component on white paper at difference distance from original point The distance travelled relative to the solvent is called the Rf value. For each compound it can be worked out using the formula:

Thin-layer chromatography

Thin layer chromatography TLC is a planar chromatography similar principle as paper chromatography Thin layer stationary phase 100-200m Normally based on silica or alumina deposited on rectangular glass, plastic or aluminum plate Inert material added to enhance cohesion of particles of stationary phase. Mobile phase are water, or mixture of aqueous alcohol/water/ethanoic acid Used for qualitative analysis of non-volatile mixture compound such as dyes and pharmacheuthicals Chemist used TLC to find impurities of synthetic samples Sample used can be visualize by iodine staining

TLC
Measurement of ratio to front value, Rf Substance A:

A B C Solvent front A B C

= X/W

Substance B:
= Y/W

Substance C:
= Z/W

Stationary Phase plate Initial pencil

XYZ

Solvent

References
Daniel C Harris, Exploring Chemical Analysis, W.H. Freeman And Company, 2ed 2000, QD75.2.H368 Francis Rouessac and Annick Roussac, Chemical Analysis -modern instrumentation methods and techniques, Wiley, 2007 QD 79.I5.R681 Seamus P.J. Higson, Analytical Chemistry, Oxford 2004