Chromatography Method

8/2/2019 Chromatography Method

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Chromatography Method

Theory, Paper chromatographyand TLC

CBB4032 Chemical AnalysisAsna M.Z.

Content:A. Lecture:Chromatography theory

Paper chromatographyThin Film chromatographyB. Video session:

1. GC sample preparation two organic extraction2. Introduction to chromatography

C. Quiz 1


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Principle of Chromatography

A process to separate a mixture in liquid orgaseous state

For qualification and quantification of compound

Principle based on concentration equilibrium ofcomponent of interest between two immisciblephases fixed to the column and mobile phase

The phases must have different solubilities towardthe component of interest

A physico-chemical separation similar asdistillation, crystallization or fractionatedextraction


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Basic of chromatographyprocess

Consist of a column, stationary phase, mobile phase andsample

Sample is eluted by a continuous addition of mobile phasefor separation

Sample is separated together with mobile phase andcomponents in sample migrate at different velocities isrecovered.


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Chromatography analysis


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Chromatographic separationtheory

All chromatographic separation is governed bypartition coefficient, Kd for solutes betweenstationary and mobile phase, for a dynamicequilibrium, Smobile Sstationary for solutes S.

The partition coefficient, or distribution coefficient(Nerst partition coef, K)

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


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Example

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

After extraction,


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Chromatography theory

If the solute, S spend some time in mobile phaseand part of the time in mobile phase its rate ofprogress is governed by Kd.

The ave linear rate of movement of mobile phaseexpressed 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


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Retention parameters

Retention times tR is time taken for a soluteto 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 differencebetween tR and tm


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Retention factor of twocompounds


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Chromatography theory

Capacity factor parameter used tocompare the relative rate of solutemigration along column

K = (tRt

mob) /t

mob

Selectivity factor (separation factor) fortwo solutes

= K1/Ks ( will be greater than unity)

where K1 = larger Kd value & Ks = smaller kd value


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Resolution factor

Resolution factor, R used to quantifyseparation between two compounds


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Chromatography theory Column efficiency described by the Van

Deemter equation in term of flow rate ,uH = 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 variance12 increase with distance of migration, if the

distance is L, (total column length) then

L2 = H x L,

so N = L2

/L2

= t2R/

2


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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 2.35measured 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 W

b= 4

h

Co

lumn

L

Eluent

Detector


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Selection guide for all different chromatographic techniques for liquid mobilephase as a function of molar mass, solubility and polarity of a compounds to beseparated

Sample

Molar mass2000

Water soluble

SEC gelfiltration

HPLC reversephase

IC

OrganosolubleSEC gel

permeation


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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


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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 fromoriginal point

The distance travelled relative to the solvent is calledthe Rf value. For each compound it can be worked outusing the formula:


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Thin-layer chromatography

Thin layer chromatography TLC is a planarchromatography similar principle as paperchromatography

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 aqueousalcohol/water/ethanoic acid

Used for qualitative analysis of non-volatile mixturecompound such as dyes and pharmacheuthicals

Chemist used TLC to find impurities of syntheticsamples

Sample used can be visualize by iodine staining


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TLC

Measurement ofratio to frontvalue, Rf

Substance A:

= X/W Substance B:

= Y/W

Substance C:= Z/W

A B C

Solvent

A B C

StationaryPhaseplate

Initial pencil

Solvent front

X Y Z

W


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References

Daniel C Harris, Exploring Chemical

Analysis, W.H. Freeman And Company,2ed 2000, QD75.2.H368

Francis Rouessac and AnnickRoussac, Chemical Analysis -modern

instrumentation methods andtechniques, Wiley, 2007 QD 79.I5.R681

Seamus P.J. Higson, AnalyticalChemistry, Oxford 2004

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Chromatography Method