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Chromatography Method Theory, Paper chromatography and TLC

CBB4032 Chemical Analysis Asna M.Z.
Content: A. Lecture: Chromatography theory Paper chromatography Thin Film chromatography B. Video session: 1. GC sample preparation – two organic extraction 2. Introduction to chromatography C. Quiz 1

crystallization or fractionated extraction .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.

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 .

Smobile  Sstationary for solutes S. or distribution coefficient (Nerst partition coef. for a dynamic equilibrium.  The partition coefficient. can be deduced G = .RT ln K . Kd for solutes between stationary and mobile phase.Chromatographic separation theory All chromatographic separation is governed by partition coefficient. variation of standard free energy. for Cm Cs transformation. K)  Kd = [S]stat/[S]mob Knowing the T of experiment.

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. . A quantity of 0.Example  The Kd of an organic salt between hexane and water is 90.

L is column length  Ave linear rate of solute migration for a chromatography. 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 .Chromatography theory If the solute. v’ = L/tR  And v’ = u x fraction time spend in mobile phase   .

VR volume of analyte present in mobile phase.Retention parameters • Retention times tR is time taken for a solute to elute from a column • Retention volume. t’R is the difference between tR and tm . 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.

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  = K’1/K’s ( will be greater than unity) where K’1 = larger Kd value & K’s = smaller kd value .

R used to quantify separation between two compounds .Resolution factor • Resolution factor.

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. mobile phase and Temp  Linear dispersion 1 measured by the variance 12 increase with distance of migration. (total column length) then L2 = H x L. so N = L2/ L2 = t2R/ 2  . stat phase . C are constant from column. B.Chromatography theory Column efficiency – described by the Van Deemter equation in term of flow rate . if the distance is L.

55 (tR/w ½ )2 or N = 16(tR/wb)2 since N = tR2/ 2 W½ =2. w½ Ideal chromatogram peak has a Gaussian shape if the peak height is h. showing retention time. tR and width at half height. w½ is 2.35 measured at ½h and 50 % of peak area.  represent half width of the peak at 60.35 and Wb = 4 Eluent C o l u m n L h Detector .Gaussion peak      Schematic gas chromatogram. then the width at half height.6 % and the base peak w=4 4 represent volume of peak (contain 95 % of injected compound) N = 5.

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 .Selection guide for all different chromatographic techniques for liquid mobile phase as a function of molar mass.

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. For each compound it can be worked out using the formula:   . 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.

plastic or aluminum plate Inert material added to enhance cohesion of particles of stationary phase.Thin-layer chromatography         Thin layer chromatography TLC is a planar chromatography similar principle as paper chromatography Thin layer stationary phase 100-200m Normally based on silica or alumina deposited on rectangular glass. 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 .

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 W XYZ Solvent .TLC Measurement of ratio to front value.

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