CHROMATOGRAPHYCHROMATOGRAPHY

HANIM AWABHANIM AWAB(C18(C18--105)105)

Department of ChemistryDepartment of Chemistry

1

Department of ChemistryDepartment of ChemistryFaculty of ScienceFaculty of Science

UTMUTM

FromFrom Greek words: Greek words: CHROMA CHROMA -- color color GRAPHYGRAPHY -- writewrite

Describes a variety of separation techniques Describes a variety of separation techniques based on thebased on the distributiondistribution (partitioning)(partitioning) of of

CHROMATOGRAPHYCHROMATOGRAPHYPart 1 (Introduction)Part 1 (Introduction)

based on thebased on the distributiondistribution (partitioning)(partitioning) of of analyteanalyte (solute)(solute) between a mobile phasebetween a mobile phase(solvent)(solvent) and aand a stationary phasestationary phase

Founded in 1906 by Micheal Tswett Founded in 1906 by Micheal Tswett (A (A Russian botanist who separated plant extracts on Russian botanist who separated plant extracts on a column packed with fine calcium carbonate)a column packed with fine calcium carbonate)

To separate two or more compounds in a To separate two or more compounds in a mixture based on the differences in the mixture based on the differences in the property of each individual substance property of each individual substance

The properties include polarity, solubility, The properties include polarity, solubility, ionic strength, and sizeionic strength, and size

Most useful analytical method for separation, Most useful analytical method for separation, identification, and quantitation of chemical identification, and quantitation of chemical

PURPOSE OF CHROMATOGRAPHYPURPOSE OF CHROMATOGRAPHY

identification, and quantitation of chemical identification, and quantitation of chemical compounds compounds (today)(today)

The sample molecules (analytes/solutes) The sample molecules (analytes/solutes) are are physically separatedphysically separated by by distributingdistributingthemselves between two phases: themselves between two phases: (a) stationary phase(a) stationary phase(b) mobile phase(b) mobile phase

Stationary phase:Stationary phase:-- A solid packed into a columnA solid packed into a column

BASIC PRINCIPLES BASIC PRINCIPLES

-- A solid packed into a columnA solid packed into a column-- A solid coating the surface of aA solid coating the surface of a

flat, plane material flat, plane material -- A liquid supported on a solid A liquid supported on a solid -- A liquid supported on theA liquid supported on the

inside wall of an open tubeinside wall of an open tube

Mobile phase:Mobile phase:A gas, a liquid or a supercritical fluidA gas, a liquid or a supercritical fluid

1.1. The The solutesolute (analyte, sample)(analyte, sample) is is introduced onto the stationary introduced onto the stationary phase, which interacts and phase, which interacts and retards (holds back) the retards (holds back) the analytes differentlyanalytes differently

2.2. ElutionElution is process of washing is process of washing components through the column components through the column by adding fresh mobile phase by adding fresh mobile phase (eluent, solvent)(eluent, solvent): :

-- The The eluenteluent carries with it the carries with it the -- The The eluenteluent carries with it the carries with it the components for which it has components for which it has affinity affinity

-- Changing the eluent changes Changing the eluent changes the analytes affinity for the the analytes affinity for the stationary phase, resulting in stationary phase, resulting in separation of components separation of components

-- The mobile phase that exit the The mobile phase that exit the column is called the column is called the eluateeluate

3.3. The The chromatogramchromatogram is the recordedis the recorded tracingtracing of the of the resulting resulting output signaloutput signal vsvs time (or eluent volume)time (or eluent volume)

Chromatographic processes are classified Chromatographic processes are classified according to the according to the typetype of interactions (or of interactions (or equilibration process involved) and equilibration process involved) and governed by the type of stationary phase governed by the type of stationary phase

AdsorptionAdsorption

Classification (Types) of Classification (Types) of Chromatographic Separation (Techniques)Chromatographic Separation (Techniques)

AdsorptionAdsorption

PartitionPartition

IonIon--exchangeexchange

AffinityAffinity

Gel Permeation or SizeGel Permeation or Size--exclusionexclusion

Adsorption ChromatographyAdsorption Chromatography

The solute and solvent The solute and solvent compete for active sites on compete for active sites on the adsorbentthe adsorbent

The active sites ( ) of the The active sites ( ) of the stationary phase stationary phase (eg silanols (eg silanols of silica) of silica) interact with the interact with the functional groups of the functional groups of the compounds to be separated compounds to be separated

Eluent flowEluent flow

compounds to be separated compounds to be separated ( ) by nonpolar interaction, ( ) by nonpolar interaction, van der Waals forces or van der Waals forces or hydrophobic interactionshydrophobic interactions

As relative adsorption of the As relative adsorption of the mobile phase is increased mobile phase is increased (by manipulating the eluent),(by manipulating the eluent),adsorption of the solute is adsorption of the solute is decreased, thus eluteddecreased, thus eluted

Partition ChromatographyPartition Chromatography

Solutes are partitioned between Solutes are partitioned between two immiscible liquids two immiscible liquids

One of the liquids is held by a One of the liquids is held by a solid inert support and acts as solid inert support and acts as the stationary phasethe stationary phase

The system is manipulated by The system is manipulated by changing the nature of the two changing the nature of the two liquidsliquids

NormalNormal phase chrom: the phase chrom: the more more polarpolar liquid is used as the liquid is used as the stationary phasestationary phase, the less polar , the less polar liquid is used as eluent liquid is used as eluent

ReversedReversed phase chrom: the phase chrom: the less less polarpolar liquid is made the liquid is made the stationary phasestationary phase and the more and the more polar liquid is used to elute polar liquid is used to elute componentscomponents

Ion Exchange ChromatographyIon Exchange Chromatography

Separation is based on the Separation is based on the difference in the affinity of difference in the affinity of charged stationary phase for charged stationary phase for charged speciescharged species

The stationary phase (ion The stationary phase (ion exchanger) contains fixed exchanger) contains fixed charged functional groups and charged functional groups and exchangeable counterions exchangeable counterions

Eluent Eluent flowflow

exchangeable counterions exchangeable counterions

Ions (or charged sites on large Ions (or charged sites on large molecules) exchange with molecules) exchange with counterions (same charge with counterions (same charge with ion) of the stationary phase ion) of the stationary phase

Components attached to the ion Components attached to the ion exchanger are eluted by exchanger are eluted by changing mobile phase propertychanging mobile phase property

Size Exclusion Chromatography (SEC)Size Exclusion Chromatography (SEC)

SEC separate molecules based on size differencesSEC separate molecules based on size differences

Stationary phase contains porous material that Stationary phase contains porous material that has pores comparable to the size of components has pores comparable to the size of components

Smaller sized molecules are retained longer in the Smaller sized molecules are retained longer in the pores, thus eluted later (Large sized molecules pores, thus eluted later (Large sized molecules are unretarded by the pores, thus eluted earlier)are unretarded by the pores, thus eluted earlier)

SEC requires calibration using a series of known SEC requires calibration using a series of known (standard) solutes (standard) solutes

Affinity ChromatographyAffinity Chromatography

Separation is based on Separation is based on specific and reversible specific and reversible interaction between a interaction between a solute molecule and a solute molecule and a ligand immobilized on the ligand immobilized on the stationary phasestationary phase

A ligand that specifically A ligand that specifically interacts with the molecule interacts with the molecule to be isolated is to be isolated is to be isolated is to be isolated is immobilized on the support immobilized on the support materialmaterial

Molecules that are Molecules that are complementary to the complementary to the ligand are adsorbed, other ligand are adsorbed, other components are elutedcomponents are eluted

Adsorbed molecules are Adsorbed molecules are eluted via exchange in the eluted via exchange in the mobilemobile--phase compositionphase composition

THEORY OF CHROMATOGRAPHIC SEPARATIONTHEORY OF CHROMATOGRAPHIC SEPARATION

Shape Shape of a chromatographic peak is based on a of a chromatographic peak is based on a Gaussian curveGaussian curve (( is standard deviation, is standard deviation, wwii is width is width at the inflection point, at the inflection point, wwhh is the width at half height, is the width at half height,

and and wwbb is the width at the baseline intercept)is the width at the baseline intercept)

Characteristics of the ChromatogramCharacteristics of the ChromatogramPlot of detector response vs retention time of peakPlot of detector response vs retention time of peak

10% ketinggian

to

t

t R2

R1

w1/2

10% Peak

tt0 0 or tor tm m the void volume (or dead volume) is the the void volume (or dead volume) is the retention timeretention time of unretained compound of unretained compound

ttR1R1 and and ttR2R2 retention times for separated retention times for separated components 1 and 2components 1 and 2

ww11 and and ww22 width at base peak of componentswidth at base peak of components

w w

a b10% ketinggian

puncakSuntikan

1 2

Injection 10% Peak Height

Retention time (tRetention time (tRR))Time elapsed between injection of sample Time elapsed between injection of sample and emergence of peak maximumand emergence of peak maximum

Mobile phase holdup time (tMobile phase holdup time (t00 or tor tMM) ) Time required for an injected solvent or any Time required for an injected solvent or any other unretained compound to traverse the other unretained compound to traverse the

Chromatographic Definitions & CalculationsChromatographic Definitions & Calculations

other unretained compound to traverse the other unretained compound to traverse the column column (aka column dead volume or void volume)(aka column dead volume or void volume)

Adjusted retention time (tAdjusted retention time (tRR) ) ttRR = t= tRR -- ttoo

Difference between analyte retention time Difference between analyte retention time and column void volumeand column void volume

Retention factor (aka Capacity factor), kRetention factor (aka Capacity factor), kA measure of how long a sample is retained A measure of how long a sample is retained with respect to the dead volumewith respect to the dead volume

k = (tk = (tRR-- ttoo)/t)/too

Resolution, Rs Resolution, Rs Degree of separation of two components Degree of separation of two components leaving the columnleaving the column

21

R2R1s

ww

)t2(tR+

=

Sample ProblemSample Problem

Given the following data and figure, calculate RsGiven the following data and figure, calculate Rs

dData:Data:d = 1.8 cmd = 1.8 cmww11 = 0.8 cm= 0.8 cmww = 0.9 cm= 0.9 cm

w1 w2

ww22 = 0.9 cm= 0.9 cm

RRss = 2 = 2 ------------------------------ = = -------------------------- = 2.1= 2.1d 3.6 cmd 3.6 cm

(w(w11 + w+ w22 ) 1.7 cm) 1.7 cm

21

R2R1s

ww

)t2(tR+

=

RRss < 1 (Unresolved, bad separation)< 1 (Unresolved, bad separation)RRss = 1 (98% separation)= 1 (98% separation)RRss = 1.50 (99.4% separation)= 1.50 (99.4% separation)RRss >1 sufficient for accurate quantification>1 sufficient for accurate quantification

Separation factor (Selectivity factor) Separation factor (Selectivity factor)

For two species A and B, the selectivity factor:For two species A and B, the selectivity factor:

= (t= (tR(B)R(B) -- tt00)/ (t)/ (tR(A)R(A) -- tt00))= (t= (tR(B)R(B) -- ttR(A)R(A))) = k= kBB -- kkAA

ttRR (B) 3.8 (B) 3.8 -- 1.0 min 1.0 min = = ________________ = = ______________________________ = 1.4= 1.4

ttRR (A)(A) 3.0 3.0 -- 1.0 min1.0 min

Column efficiency or Number of plates (NColumn efficiency or Number of plates (N))A measure of the broadening of the sample A measure of the broadening of the sample peak as it passes through the columnpeak as it passes through the column

N = 16 (tN = 16 (tRR/w/wbb))2 2 or N = 5.54or N = 5.54 (t(tRR /w/whh))

2 2

where, wwhere, wb b is bandwidth of peak (aka base peakwidth) is bandwidth of peak (aka base peakwidth) wwhh is the width of peak at half heightis the width of peak at half heighthh

Height Equivalent to a Theoretical Plate or Height Equivalent to a Theoretical Plate or Plate Height (HETP or H)Plate Height (HETP or H)Compares column efficiency of different Compares column efficiency of different lengths lengths

H = L /NH = L /N

A solute with a retention time of 407 s has a width A solute with a retention time of 407 s has a width at the base of 13 s on a column 12.2 m long. Find at the base of 13 s on a column 12.2 m long. Find the plate height and number of platesthe plate height and number of plates

SAMPLE PROBLEM SAMPLE PROBLEM

N

LH

w

16tN

2

2

r== ,

0.78mm101.57

12.2m

N

LH

101.5713

40716

w

16tN

4

4

2

2

2

2r

=

==

=

==

General Elution ProblemsGeneral Elution Problems

Problem of elution is the compromise Problem of elution is the compromise between elution time and resolutionbetween elution time and resolution

Arises whenever samples contain species with Arises whenever samples contain species with widely different partition ratioswidely different partition ratios

In case of good separations of the more In case of good separations of the more strongly held species, strongly held species, lack of resolutionlack of resolutionamong weakly retained species is observed among weakly retained species is observed among weakly retained species is observed among weakly retained species is observed

In case of satisfactory separations of the In case of satisfactory separations of the weakly retained compounds, severe weakly retained compounds, severe band band broadeningbroadening and long retention times are and long retention times are encountered for the strongly bound speciesencountered for the strongly bound species

Often solved in Often solved in LCLC by gradient elution and GC by gradient elution and GC by temperature programming by temperature programming

Separation of mixtures depend on:Separation of mixtures depend on:

width of solute peaks (we want narrow width of solute peaks (we want narrow peaks) for peaks) for efficiencyefficiency

spacing between peaks (we want large spacing between peaks (we want large spacing) for selectivityspacing) for selectivity

Parameters that effect resolution:Parameters that effect resolution:Parameters that effect resolution:Parameters that effect resolution:

Column efficiency Column efficiency -- more N gives narrower more N gives narrower peaks and better Rspeaks and better Rs

Capacity factor, Capacity factor, -- if low k analytes will if low k analytes will not be retained enough for good separationnot be retained enough for good separation

Selectivity Selectivity -- if analytes are very similar if analytes are very similar (k1 (k1 k2), then difficult to separatek2), then difficult to separate

(a)

(b)

N = 250, Rs = 0.8 N = 250, Rs = 0.8

N = 250, Rs = 1.8N = 250, Rs = 1.8

Poor resolutionPoor resolution

Illustration of the effects of N and Illustration of the effects of N and on resolution on resolution

Same efficiencySame efficiencyIncreased RsIncreased Rs

(c) N = 1100, Rs = 1.8N = 1100, Rs = 1.8

Increased efficiencyIncreased efficiencySame RsSame Rs

Same efficiencySame efficiencyIncreased RsIncreased Rs