Chap 5 Chromatography

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Rezaul KarimEnvironmental Science and TechnologyJessore University of Science and Technology

Instrumental Technique forEnvironmental AnalysisChapter Chromatography


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Chapter content

Historical Background; 1,2 Chromatography: A Separation Technique;

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Classification of Chromatography 3

Basic Chromatography Concepts: 2Chromatogram; Distribution Coefficient;Retention Volume, Capacity Factor,Theoretical Plates, Selectivity

Physical Forces and Interactions;2

Modes of Separation; 2

Stationary Phases Versus Mobile Phases; 2

Detectors

Applications of Chromatography 32


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Reference

1. Francis Rouessac and Annick Rouessac,2007, Chemical Analysis-ModernInstrumentation Methods and Techniques,2ndEdition, John Wiley & Sons Ltd, The

Atrium, Southern Gate, England2. J.R.J. Pare and J.M.R. Belanger 1997

instrumental methods in food analysis,techniques and instrumentation in analyticalchemistry- volume 18, Elsevier science .Amsterdam, the Netherlands

3. Skoog, Holler & Crouch 2007, InstrumentalAnalysis, Brooks Cole Cengage Learning,USA.

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Introduction

Chromatography is a powerful separationmethod that finds applications in allbranches of science,.

Chromatography was invented, theRussian botanist Mikhail Tswett.

Various plant pigments such aschlorophylls and xanthophylls were

separated by passing solutionsof thesecompounds through a glass columnpacked with finely divided calciumcarbonate.

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The separated species appeared ascolored bands on the column.

the name for the method (Greek chromameaning "color and graphein meaning"writing").

The tremendous impact in 1952 - Nobel

Prize in Chemistryawarded to A. J P,Martin and R. LM, Synge for theirdiscoveries in the field,.

Many of the Nobel Prizes awarded sincethat time have been based on work inwhich chromatography played a vitalrole.


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Chromatography as

extraction Chromatography operates on the

same principle as extraction, but

one phase is held in place while the

other moves past it. In the Figure, a solution containing

solutes A and B placed on top of a

column are packed with solidparticles and filled with solvent.

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The idea behind chromatography: solute A, with a greater affinity than

solute B for the stationary phase, remains on the column longer.


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Fluid entering the column is called eluent.

Fluid emerging from the end of the column

is called eluate The mobile phase

the solvent moving through the column

is either a liquid or a gas.

The stationary phase the one that stays in place inside the

column

is most commonly a viscous liquidchemically bonded to the inside of acapillary tube or onto the surface of solidparticles packed in the column.


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When the outlet is opened, solutes A andB flow down into the column.

Fresh solvent is then applied to the top ofthe column and the mixture is washeddown the column by continuous solventflow.

If solute A is more strongly adsorbed

than solute B on the solid particles, thensolute A spends a smaller fraction of thetime free in solution.

Solute A moves down the column more

slowly than solute B and emerges at thebottom after solute B. We have just separated a mixture into its

components by chromatography.


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Theory

Chromatography is the process by whichthe components of a mixture can beseparated.

Chromatography is a physico-chemical

method of separation of componentswithin mixtures, liquid or gaseous, in thesame vein as distillation, crystallization, orthe fractionated extraction.

It has become one of the primary analyticalmethods for the identification andquantification of compounds in the gaseousor liquid state.

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The basic principle is based on theconcentration equilibrium of thecomponents of interest, between twoimmiscible phases. One is called the stationary phase, because it

is immobilized within a column or fixed upona support.

The second, called the mobile phase, isforced through the first.

The phases are chosen such that

components of the sample have differingsolubilities in each phase.

The differential migrations ofcompounds lead to their separation.


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Basic chromatographic

process

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A basic experiment in chromatography.

(a) The necessary ingredients (C, column; SP, stationary phase;

MP, mobile phase; and S, sample);

(b) introduction of the sample; (c) start of elution;

(d) recovery of the products following separation


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A vertical hollow glass tube (thecolumn) is filled with a suitable finely

powdered solid, the stationary phase.At the top of this column is placed a small

volume of the sample mixture to beseparated into individual components.

The sample is then taken up bycontinuous addition of the mobi lephase.

The mobile phase goes through thecolumn by gravity, carrying the variousconstituents of the mixture along with it.

This process is called elut ion.


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Elution chromatography

If the components migrate at differentvelocities, they will become separated fromeach other and can be recovered, mixed withthe mobile phase

Each component of a mixture will beassociated with a specific retention time.This type of chromatography is termed elutionchromatography.

Columns are either packed or open tubular.

A packed column is filled with particles ofstationary phase.

An open tubular column is a narrow, hollowcapillary with stationary phase coated on theinside walls.

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Classification of

Chromatographic Methods The first classification is based on the

physical means by which the stationary

and mobile phases are brought into contact.

In co lumn chromatography, the stationaryphase is held in a narrow tube through which

the mobile phase is forced under pressure.

In planar chrom atography, the stationary

phase is suppo r ted on a flat plate or in theinterstices of paper; here, the mobile phase

moves through the stationary phase by capillary

action or under the influence of gravity.

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one based on the typesof mobile and

stationary phases and the kinds ofequilibriainvolved in the transfer of

solutes between phases.

Three general categories: gas chromatography (GC),

liquid chromatography (LC), and

Supercritical fluid chromatography (SFC).


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The sample components

and the phases In adsorption chromatography, the

sample is attracted to the surface of thephase, usually to the surface of a solidstationary phase.

In absorption chromatography/partition chromatography, the samplediffuses into the interior of the

stationary phase. Most chromatographic separations are a

combination of both adsorption andabsorption phenomena.

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the difference between "-

gram & "-graph"??

gram = a record

-graph = an instrument for recording

Examples:

i. A telegraphis the piece of equipment used tosend the message, sometimes known as atelegram.

ii. An electrocardiographPRODUCES an

electrocardioogram. The e'graph recordselectrical voltage of the heart.

Analogy:

chromatograph ,,,,,,,,,,,,,,,,,,chromatogram

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The chromatogram The chromatogram is the

representationof the variation, with

time (rarely volume), of the amount of

the analyte in the mobile phase exitingthe chromatographic column.

It is a curve that has a baseline which

corresponds to the trace obtained in theabsence of a compound being eluted.

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etention time Retent ion t im e tR

It represents the time elapsed from the sampleintroduction to the detection of the peak maximumon the chromatogram.

A constituent is characterizedby its tR In an ideal case, tR is independent of the quantity

injected.

Hold-up t ime or dead t im e tM, formerly designated t0

It is the time required for the mobile phase topassthrough the column.

The differencebetween the retention time andthe hold-up time is designated by the adjustedretent ion t ime of the compound, tR.

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Distribution CoefficientAlso known Nernst partition coefficient It corresponds to the distribution of the

analyte (X)between the stationary (s)and mobile (m) phases, as it elutesthrough the column.

Kx = [X]s/[X]m= CS/CM

= Molar conc. of the solute in thestationary phase / Molar concentration ofthe solute in the mobile phase

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Retention volume The total volume of the mobile phase (Vr)

required to elute an analyte is the sum of thevolume of the mobile phase (Vm)and thevolume of the mobile phase which flows while

the analyte is held immobile (Vs*Kx). Vr= Vm+ Vs*Kx

the value of Vr = F*tr , where

F is the flow rate of the mobile phase (volume/time)

and the retention time (tr) is the time required for an

analyte to elute completely through the column(from injector to detector).

The dead volume, Vm=F*to25


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Capacity factor Capacity factor or Retention factor, kx It is defined bythe total number of moles of

x in the stationary phase overthe totalnumber of moles of x in the mobile phase.

kx = mS/ mM =Vs*[X]s / Vm*[X]m = Kx*Vs / Vm = CS/CM

Vs is the volume of the stationary phase withinthe column and

Vm is the volume of the mobile phase.

k= t'R / tM= (tRtM)tM

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Gaussian profileAll molecules of a given component do not

move at an identical velocity.

The centre of the elution band represents

the average retention time of the analyte. Some molecules travel slower, some

faster.

This variation in velocity is a result of thenumber of sorption and desorption of the

analytes and is what gives the detected

signal its Gaussian profile.

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Theoretical plates The number of theoretical plates (N) is a

quantitative measure of the efficiency of acolumn and can be obtained directly from thechromatogram: N = 16(tr/tw)

The theoretical plate model assumes thecolumn to be made of a series of plates.

The distributionof the analyte between themobile and stationary phase occurs at each

plate. Therefore, the higher the number of plates, the

better the separation since more sorption-desorption cycles occur.

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Column efficiency

Column efficiency can be expressedas Height Equivalent to aTheoretical Plate value (HETP or H-value):

H = L/N

This equation has the units of lengthsince L corresponds to the column

length. Thus, a column with a low H value is

preferred to a high value.

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Separation / selectivity

factor The selectivity or separation factor (S) refer to

a system's ability to distinguish betweentwo components

It is governed by the distribution of the

analyte between the mobile and stationaryphases.

A and B correspond to two different analytes.

S = (trB-t0)/(trA-t0) = kB / kA

As S decreasestowards a value of 1, theseparation becomes more difficult.

The only way to modify this parameter is tochange one or both phases.

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Resolution The resolution (R) is the degree of

separation between two components. This kind of separation is essential in

preparative chromatography where pure

compounds are required. R = 2 (trB- trA) / (tWA+ tWB)

R = 1/4 N (S- 1/S) (k'/1 + k') This expression shows that resolution is a

function of three factors which can beadjusted independently: the columnefficiency (N), the selectivity (S) and thecapacity (k').

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Names Symbol of

experimental

quantity

Determined from

Migration time, unretained

species

tM Chromatogram

Retention time, species A and B (tR)A, (tR)B Chromatogram

Adjusted retention time for A (t'R)A (t'R)A = (tR)A - tM

Peak widths for A and B tWA, tWB. Chromatogram

Length of column packing L Direct measurement

Volumetric flow rate F Direct measurement

Linear flow velocity u F and column dimensions

Stationary-phase volume Vs Packing preparation data

Concentration of analyte in

mobile ans stationary phase

CM, CS Analysis and preparation data


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Necessary Formula

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Math Problem

Substances A and B have retention times of 16.40and 17.63 min. respectively, on a 30.0-cm column.An unretained species passes through the columnin 1.30 min. The peak widths (at base) for A andBare 1.11 and 1.21 min. respectively.

Calculatea. the column resolution.

b. the average number of plates in the column,

c. theplate height,

d. the length of column required to achieve a resolution

of 1.5.e. the time required to elute substance B on the column

that gives an R, value of 1.5, And

f. the plate height required for a resolution of 1.5 on theoriginal 30-cm column and in the original time.

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A Column resolution:

R = 2 (trB- trA) / (tWA+ tWB)

Here, trB = 17.63 , trA = 16.40, and

tWA = 1.11, tWB = 1.21

then, R = 2 ( 17.63-16.40) (1.11+1.21) = 1.06

B the average number of plates in

the column : N = 16(tr/tw)2

NA = 16 (16.40/1.11)2 = 3493

NB = 16 (17.63/1.21)2 = 3397,

Naver = (NA+NB)/2 = 3445

C the plate height : H = L/N Here L = 30.0, then

H = 30.0 / 3445 = 8.7 x 10-3

D the length of column required to

achieve a resolution of 1.5.

k and S do not change greatly

with increasing N and L.

Then, R1/R2= N1/N2

or1.06 /1.50 = 3445 / N2

or N2= 6.9 x 103

and L = N2.H

= 6.9 x 103 x 8.7 x 10-3 = 60

E the time required to elute

substance B on the column that

gives an R, value of 1.5

tr1/tr2= (R1/R2)2then

17.63/tr2= (1.06/1.50)2

Or tr2 = 36 min.

f the plate height required for a

resolution of 1.5

Tr2/tr2= (R1/R2)2 H1/H2

then H2= (R1/R2)2 H1

=8.7 x 10-3 * (1.06/1.50)2

= 4.3 x 10-3


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Physical Forces andInteractions

Ionic Interactions Ions of the same charge are repulsed whereas ions

of opposite charge are attracted.

These forces are long-range and relatively strong.

Ionic interactions can also take place between ions.

Hydrogen Bonding bond can be formed between molecules containing a

hydrogen atom bonded to an electronegative atomlike oxygen or nitrogen.

Some can receive and donate a hydrogen atom. e,g.

alcohols, amines and water. Hydrogen bonds are relatively strong forces.

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Van der Waals Forces: three types Dipolar forces can be compared to magnetic

forces.

The dipole-induced dipole phenomenon canbe compared to a magnet attractingnonmagnetic iron is dependant on the polarisability of a molecule e.g.

larger molecules generally have greater polarisability.

induced dipole-induced dipole, consist ofweak forces that exist even in mono-atomicgases that are symmetrical and non-polar.

Charge Transfer

two molecules or ions combine bytransferring an electronfrom one to the other.

This process occurs mostly in gaschromatography


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Modes of Separation

Adsorption chromatography. A solid stationary phase and a liquid or gaseous mobile phase

are used.

Solute is adsorbed on the surface of the solid particles.

The more strongly a solute is adsorbed, the slower it travels throughthe column.

Partition chromatography. A liquid stationary phase is bonded to a solid surface , silica

(SiO2) in gas chromatography.

Solute equilibrates between the stationary liquid and the mobilephase.

Ion-exchange chromatography

Anions such as SO3or cations such as N(CH3)3arecovalently attached to the stationary solid phase, usually a resin. Solute ions o f the opposite charge are attracted to the stationary

phase. The mobile phase is a liquid.

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Size exc lus ion chromatography. this technique separates molecules by size, with the

larger solutes passing through most quickly.

the liquid or gaseous mobile phase passes through aporous gel.

Large molecules stream past without entering thepores.

Small molecules take longer to pass through the columnbecause they enter the gel.

Affinity chromatography. It employs specific interactions between one kind of

solute molecule and a second molecule that iscovalently attached to the stationary phase.

The immobilized molecule might be an antibodyto a

particular protein. When a mixture containing a thousand proteins is

passed through the column, only the one protein thatreacts with the antibody binds to the column.


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Stationary Phases versusMobile Phases The mobile phase can be

a liquid (as in LC) or

a gas(as in GC).

the stationary phase can be a liquid or asolid. In effect, there are three stationary phase

configurations: a solid packed into a column (as in SEC),

a solid coating the surface of a flat, plane material(as in thin layer chromatography - TLC) and

a liquid coated on the inside wall of an open tube(LLC).

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Application : chromatography

It can be employed for qualitativeidentification and quantitative

determination of separated species.

Qualitative Analysis Quantitative Analysis

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Qualitative Analysis

A chromatogram provides only a singlepiece of qualitative information about

each species.

Chromatograms can be used to deriveadditional data.

Spectral wavelength can not be

revealed by chromatography, unlikeby asingle IR, NM resonance, or mass

spectrum.

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Quantitative Analysis

a comparison of either the height or thearea of the analyte peak with that of one

or more standards.

Analyses Based on Peak Heigh t Analyses Based on Peak Areas

Cal ibration and Standards

The Internal-Standard Method The Area-Norm alizat ion Method

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Chap 5 Chromatography

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