KENYATTA UNIVERSITY

SBC SBC 370: Advanced Biochemistry

Tech. Department of Biochemistry & Biotechnology

ojolapatrick@gmail.com Dr. P. Ojola

Semester-II 2018/2018

Introduction to Chromatography Definition Chromatography is a separation technique based on the different interactions of compounds with

two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium.

Components: mobile phase: a solvent that flows through the supporting medium stationary phase: a layer or coating on the supporting medium that interacts with the

analytes supporting medium: a solid surface on which the stationary phase is bound or coated

The analytes interacting most strongly with the stationary phase will take longer to pass through the system than those with weaker interactions. These interactions are usually chemical in nature, but in some cases physical interactions can also be used.

Types of Chromatography

1.) The primary division of chromatographic techniques is based on the type of mobile phase

used in the system:

Type of Chromatography Type of Mobile Phase

Gas chromatography (GC) gas

Liquid chromatograph (LC) liquid

2.) Further divisions can be made based on the type of stationary phase used in the system:

Gas Chromatography

Name of GC Method Type of Stationary Phase

Gas-solid chromatography solid, underivatized support

Gas-liquid chromatography liquid-coated support

Bonded-phase gas chromatography chemically-derivatized support

Types of Chromatography

Liquid Chromatography

Name of LC Method Type of Stationary Phase

Adsorption chromatography solid, underivatized support

Partition chromatography liquid-coated or derivatized support

Ion-exchange chromatography support containing fixed charges

Size exclusion chromatography porous support

Affinity chromatography support with immobilized ligand

3.) Chromatographic techniques may also be classified based on the type of support material used in the system:

Packed bed (column) chromatography

Open tubular (capillary) chromatography Open bed (planar) chromatography

Theory of Chromatography 1.) Typical response obtained by chromatography (i.e., a chromatogram): chromatogram - concentration versus elution time

Wh

Wb

Where: tR = retention time tM = void time Wb = baseline width of the peak in time units Wh = half-height width of the peak in time units

Inject

Note: The separation of solutes in chromatography depends on two factors: (a) a difference in the retention of solutes (i.e., a difference in their time or volume of elution (b) a sufficiently narrow width of the solute peaks (i.e, good efficiency for the separation system) A similar plot can be made in terms of elution volume instead of elution time. If volumes are

used, the volume of the mobile phase that it takes to elute a peak off of the column is referred to as the retention volume (VR) and the amount of mobile phase that it takes to elute a non-retained component is referred to as the void volume (VM).

Peak width & peak position determine separation of peaks

2.) Solute Retention: A solute’s retention time or retention volume in chromatography is directly related to the strength

of the solute’s interaction with the mobile and stationary phases. Retention on a given column pertain to the particulars of that system: - size of the column - flow rate of the mobile phase Capacity factor (k’): more universal measure of retention, determined from tR or VR.

k’ = (tR –tM)/tM or k’ = (VR –VM)/VM capacity factor is useful for comparing results obtained on different systems since it is

independent on column length and flow-rate.

The value of the capacity factor is useful in understanding the retention mechanisms for a solute, since the fundamental definition of k’ is:

k’ is directly related to the strength of the interaction between a solute with the stationary and

mobile phases. Moles Astationary phase and moles Amobile phase represents the amount of solute present in each phase

at equilibrium. Equilibrium is achieved or approached at the center of a chromatographic peak.

k’ = moles Astationary phase

moles Amobile phase

When k' is # 1.0, separation is poor When k' is > 30, separation is slow When k' is = 2-10, separation is optimum

A simple example relating k’ to the interactions of a solute in a column is illustrated for partition chromatography:

A (mobile phase) A (stationary phase)

KD

where: KD = equilibrium constant for the distribution of A between the mobile phase and stationary phase

Assuming local equilibrium at the center of the chromatographic peak:

k’ = [A]stationary phase Volumestationary phase

[A]mobile phase Volumemobile phase

k’ = KD

Volumestationary phase

Volumemobile phase

As KD increases, interaction of the solute with the stationary phase becomes more favorable and the solute’s retention (k’) increases

k’ = KD

Volumestationary phase

Volumemobile phase

Separation between two solutes requires different KD’s for their interactions with the mobile and stationary phases since peak separation also represents different changes in free energy

DG = -RT ln KD

3.) Efficiency: Efficiency is related experimentally to a solute’s peak width. - an efficient system will produce narrow peaks - narrow peaks smaller difference in interactions in order to separate two solutes Efficiency is related theoretically to the various kinetic processes that are involved in solute

retention and transport in the column - determine the width or standard deviation (s) of peaks

Wh

Dependent on the amount of time that a solute spends in the column (k’ or tR)

Chromatographic Techniques General principles; -Basis of all chromatography- is partition or distribution coefficient (Kd)

-It describes how cpd a distributes btw 2 immiscible phases eg A & B phase

-Coefficient value is a constant at particular temp.

Kd= Conc in phase A

conc in Phase B

-effective Kd; is the total amount as distinct from conc, of a substance present in one

phase divided by total amount present in other phase

-ie. The Kd multiplied by ration of volumes of the two phases present;

-eg. If Kd of cpd btw A & B is 1, and this cpd is distributed 10cm3 of A and 1cm3 of B,

conc, in the phases is same, but total qnt of cpd in A is 10x more as in B

-All chromat systems have stationary phase- (solid, gel, liquid/gas), immobilized & the

mobile phase (liquid/gas) that flows thro, stationary

-choice of the phases is made so that cpds to be separated have different Kd

-This can be achieved by setting up;

a. Adsorption equilibrium btw stationary solid & immobile liquid phase

adsorption and hydrophobic chromatography

b. Partition equilibrium btw stationary liquid and mobile liquid/gas phase

partition, reverse-phase, ion-pair, chiral, gas-liquid, countercurrent

chromatographies

c. Ion-exchange equilibrium btw stationary ion-exchanger and mobile electrolyte phase

ion-exchange & chromatofocussing

d. Equilibrium btw liquid trapped in pores of stationary porous structure & mobile

liquid phase

exclusion or gel chromatography

e. Equilibrium btw stationary immobilised ligand & mobile liquid phase

affinity,immunoaffinity,lectin affinity,metalchelate, dye ligand, covalent

chromatography

Modes of Chromatography

-Column chromat;

stationary phase attached to suitable matrix (inert, insoluble support), packed into

glass/metal column mobile phase passed thro column by gravity or use of pump or

applied gas pressure

Commonly used method

-Thin –layer chromat;

.stationary phase attached to suitable matrix coated thinly on glass, plastic or metal

foil plate

.mobile liquid passes across thin-layer plate, held either horizontal/vertically by

capillary

.The method has advantage over column ie. Large qnt samples can be sample

simultaneously

-Paper chromat;

.stationary liquid phase supported by cellulose fibre of paper sheet

. Like in thin-layer, with similarities, mobile phase passes along paper sheet by

gravity feed or capillary action

. One of oldest method

column chromatography, thin-layer and paper chromatography

Column Chromatography

-Principle depicted by

considering column packed

with solid granular stationary

phase of height 5 cm

sorrounded by mobile liquid

phase

-typical column chromat,

system using liquid mobile

phase has column, mobile

phase reservior and delivery

system , detector- identifies

separated cpds (analyte) as

effuents

-Liquid column can be sub-divided according to pressure generated within column during

separation

Low pressure liquid chromatography pressures of < 5 bar (LPLC)

Medium pressure liquid chromatography pressure btw 6-50 bar (MPLC)

High pressure liquid chromatography pressures >50 bar (HPLC)

-MPLC & HPLC have similarities in equipment, procedures & resolution hence the term High

performance liquid chromatography for both of them

-Time taken by analyte to emerge from column is called retention time

-Vol, of mobile phase required for elute analyte is called elution/retention volume

-These are related to flow rate

-Success of particular chromatography system is judged by ability for good resolution and

determined by;

1.selectivity; measure of inherent ability of system to discriminate btw structurally related

cpds

-This influenced by chemical nature of mobile & stationary phases

2. efficiency; measure of diffusion effects that occur in column to cause peak broadening & overslap -influenced by physical parameters of mobile & stationary phases and by qlt of packing of column

3. Capacity; measure of qnt of material that can be resolved without causing peaks to overlap, irrespective of action of gradient elution

Sample Preparation

-Preliminary clean up of sample needs to be done;

-extraction & purification of components from cell homogenate oftenly a cplx multistage process

Solvent extraction

-simplest and most commonly used clean up method

-based on fact that organic cpds are extractable from aqueous mixture by extraction with low

boiling water-immiscible solvent eg. Diethylether or dichlomethane

-this tech. is replica of application of partition coefficient

-weak electrolytes cpds like acids & bases exist in ionised/unionised states

-And addition of anhydrous salts eg. Na2SO4& Mg2SO24 are added before extraction to remove

water extracted by use of diethylether/dichloromethane

Solid phase extraction -Alternative solvent extraction

-advantage over simple solvent method; exhibit greater selectivity

-test soln passed thro small (few mm in length) disposable column packed with relatively large

particles of bonded silica

-they adsorb analyte and allow interfering cpds pass thro

-Preliminary attention must be given to particular bonded silica selected & test sample which is

treated with agents like trichloroacetic acid, perchloric acid, or organic solvent

eg. Aceonitrite to deproteinise it to limit proteins binding to the analyte

-analyte pH adjusted to maximise retention time

- -once test solution is passed thro, column by either gravity the column is washed with water and

the adsorbent analyte recovered via elution using methanol/acetonitrile

Column switching

-more sophisticated procedure of sample preps

-suits the analysis of analytes in very low conc, in cplx mixture by HPLC

-Test solution applied to preliminary short column similar to one in solid phase extraction

-Once test analyte is adsorbed & impurities washed thro, column, analyte eluted with suitable

organic solvent, column effluent transferred to analytical HPLC column

-problem with the technique, unless all interfering cpds are eluted, from preliminary column

before adsorption analyte taken to analytical column, they will accumulate in analytical column

-this reduce its resolving power

Supercritical fluid extraction (SFE)

-it exploits fact that’s gases eg. CO2 exist as liquid at certain critical conditions.

-eg for CO2 conditions are 31.1ºC and 7.38 Mpa

-this can be used as extraction solvent, behaves as low polarity solvent comparable to hexane

-CO2 can be turned to gas thus simplifying recovery of extracted analyte

Matrix materials

-they support stationary phase & their selection is critical to success of chromatography

-must have mechanical stability for good flow rates & minimise pressure drops along column

-good chemical stability; fxnal grps to facilitate attachment of stationary phase

-high capacity ie density of fxnal grps to minimise bed vol.

-needs to be available in range of particle size

-others require a matrix with porous structure with correct size and shape

-surface of matrix must be inert to minimise non-selective adsorption of analytes

Six commonly used matrices

-Agarose; polysacch, made of D-galactose & 3,6-anhydro-1-galactose

-have good flow properties & high hydrophobicity but never to be allowed to try due

their irreversible chemical change

-commercial example, sepharose & bio-gelA

-Cellulose; polysacch, made of β-1-4 linked glucose units

-in matrix, its cross-linked with epichlorohydrin, extent of this linkage determines

pore size

-available in bead, microgranular & fibrous forms

- has good pH stability & flow properties, -highly hydrophilic

-Dextran; polysacch, of an α-1-6 linked glucose units

-in matrix, tis cross-linked with epichlorohydrin but less stable to acid hydrolysis than

cellulose

-stable upto pH 12 & is hydrophilic, -commercial eg. Sephadex

-Polyacrylamide; polymer of acrylamide crosslinked with N,N’-methylene-bisacrylamide

-stable in pH range 2-11

-commercials, Bio-gel P

-Polystyrene; polymer of styrene cross-linked with divinylbenzene

-the matrix have good stability over all pH ranges & are mostly used for exclusion

& ion-exchange

- have low hydrophilicities

-Silica; polymeric of orthosilic acid & many silanol (Si-OH) grps make it hydrophilic

- but excess silanol can be removed treatment with trychloromethylsilane

-silica stability is confined to pH 3-8

High performance Liquid Chromatography

principle -resoving power of chromatographic column increases with column length & number of

theoritical plates/unit

-The smaller the size of particle of stationary phase, the better the resolution

-But the smaller the size particle, the greater is the resistance to flow of mobile phase

-creating back pressure on column sufficient to damage matrix of stationary phase, reducing

eluent flow &impairing resolution

Sept. 16, 2009 GMS BI 555/755 Lecture 3. 26

High performance liquid chromatography

• Chromatographic resolution increases as the size of the chromatographic beads decreases

• The pressure required to push solvent (mobile phase) through the packed beads increases as their size decreases

• High performance liquid chromatography (HPLC): a pumping system that pushes solvent through a column packed with small beads (app. 5 microns).

• All tubing, columns, made of stainless steel to withstand pressures up to 2500 psi or so.

• Higher performance and cost than conventional low pressure liquid chromatography.

• Typically one begins with low pressure chromatography to concentrate the protein and then switches to HPLC for later steps

•Available with any chromatography mode •Many models and manufacturers

-but new smaller particle size, stationary phase been developed & can withstand pressure

-hence better resolution in chromatography

-HPLC column is steel made is ~ 15-20 cm length with 1-4mm diameter generally

Matricess & stationary phases

- 3 forms of column packing materials available based on a rigid structure

a. Microporous support- micropores ramify thro, the particle which are ~ 5-10μm in diameter

b. Pellicular (superficially porous), porous particles coated onto inert solid core like glass

bead ~ 40 μm in diameter

c. Bonded phases, stationary phase is chemically bonded on inert support like sillica

-in adsorption, adsorbents like silica & alumina are available as microporous or pellicular

-pellicular has high efficiency but low sample capacity

-in partition chromat, stationary phase may be coated on inert microporous or pellicular support

-one disad, of suppors coated with liquid phases;developing mobile phase may graduallywash off

liquid phase

-many types of ion-exchanger for HPLC are available; the cross-linked microporous polystyrene

resins are used or pellicular resinsin

-stationary phase of exclusion chromat,are porous silica, glass, polystyrene or polyvinylacetate

beads

-these are used when eluting solvent is in organic system

-semi-rigid gels eg.sephadex, Bio-gel-P, and non rigid gelseg, sepharose & bio-Gel-A are limited

in use for HPLC since can withstand only low pressures

--supportr for affinity chromat. Are similar to those of exclusion

-spacer arm and ligand attached to the supports by chemical means as in Low pressure affinity

chromat.

Applications of HPLC

-separation of proteins, oligopeptides

-

Adsorption

-Principle; certain solid materials (adsorbents) can hold molecules at their surface

-The adsorption involves weak, no-ionic attractive van der waaals’ forces & H-bonding at

specific sites

-The sites discriminate btw molecules of eluent/analyte in the mixture depending on strength of

attachment

-As eluent continue passing thro, column differences in binding strength make the difference

-Strenght of binding of particular analyte depends on its functional grps

-OH-grp & aromatic grps increase interaction with adsorption surface

-Aliphatic grps of different size differ only slightly in their interaction

-Adsorption chromat, is influenced more by presence of specific grps than simple molecular size

since only a specific grp not whole molecule

-typical adsorbent eg silica with silanol (Si-OH), which are slightly acidic can interact with polar

fxnal grps of eluent. Good for basic material

-Other commonly used adsorbents; alumina (basic good for acidic particles)& carbon.

-selection of correct eluent (mobile phase) is essential for good resolution it affects capacity

factor of analytes

-alcohols is good for analytes with OH- grps, acetone/esters good for analytes with carboxyl

grps

-hydrocarbons (hexane, heptane & toluene) for those with predominantly non-polar

Partition chromatography -based on differences in capacity ratio (K’) & distribution coefficients (Kd) of analyte using liquid

stationary & mobile phase

-can be subdivided into Liquid-Liquid chromat-(liquid stationary phase attached to matrix

physically) eg. Chromat, where water stationary phase supported by cellulose, starch or silica

-and bonded-phase liquid chromat-(stationary phase is covalently attached to matrix)

-Surplus Si-OH removed by capping with chlorotrimethylsilane to improve qlt of chromat

Ion-exchange chromatography

-relies on attraction btw oppositely charged particle

-Proteins & amino acids have ionisable grps and they may have –ve /+ve charge can be utilised in

separating mixtures of the cpds

-Net charge of the cpds is dependent on their pKa & pH of solution

-ion-exchange are carried in columns packed with ion-exchanger (two types- cation and anion)

-cation; posses –vely charged grps that attract +vely charged cation (are called acidic exchangers

because their –ve charges results from ionisation of acidic grps)

-anion exchangers have +vely charged grps & attract anions (basic exchangers)

-Ion-Exchanger mechanism thought to be composed of 5 steps

(i) Diffusion of ion to exchanger surface. Occurs very quickly in homogeneous solution.

(ii) Diffusion of ion thro, matrix structure of exchanger to exchanger site this dependent on

degree of cross-linkage of exchanger & conc, of solution

-this process thought to be feature controlling rate of whole ion-exchange process

Sept. 16, 2009 GMS BI 555/755 Lecture 3. 32

Charge-based separations: ion exchange chromatography

• Ion exchange entails loading a protein mixture at a given pH in a low salt concentration buffer and washing the unbound material through the column

• For cation exchange, positively charged proteins bind to the column, negatively charged proteins pass through

• For anion exchange, negatively charged proteins bind the column, positively charged ones pass through • Bound proteins are eluted by a gradient of increasing salt concentration • Weakly bound proteins elute at a relatively low salt concentration, tightly bound ones at higher salt

concentration • Proteins are focused by binding to the column. Thus, large volumes of solution may be applied to the

column.

(iii) Exchange of ions at exchange site, thought to occur instantaneously & is the equilibrium of

the process

(iv) Diffusion of exchanged ion thro, the exchanger to surface

(v) Selective desorption by eluent & diffusion of molecule into the external eluent

-selective desorption of bound ion is achieved by changes in pH and /or ionic conc, or affinity

elution(where ion that has greater affinity for exchanger than has the bound ion is introduced into

the system)

Exclusion (permeation ) chromatography

-separation done on basis of molecular size & shape

-uses molecular sieve of porous material eg. grp of polymeric organic cpds with 3-D network of

pores confering gel properties

-gel filtration describes separation of molecules of varying molecular size utilizing these gels

-porous glass granules also have been used (controlled –pore glass chromat.)

-exclusion/permeation, describes all molecular separation using molecular sieves

-the principle of this technique; A column of gel particles or porous glass granules is in

equilibrium with suitable mobile phase for molecules to be separated

-large particles are excluded from the pores but thro, interstitial spaces and appear in effluent first

-Smaller particles will distribute btw mobile phase inside & outside molecular sieve, & pass thro,

column at slower rate & appear last in effluent

Sept. 16, 2009 GMS BI 555/755 Lecture 3. 36

Size based separations: Gel-filtration chromatography

• AKA size exclusion chromatography • Smaller proteins experience a higher mobile

phase volume because they are able to enter pores of the stationary phase beads.

• The elution volume is inversely related to the molecular weight

• Chromatographic resolution depends on a number of factor including bead diameter, pore size, salt concentration, column volume, flow rate

• Other factors constant, resolution increases with column volume

• Protein must be freely soluble • Detergents necessary for membrane proteins • Typically 0.15 M salt necessary to prevent

non-specific interactions between proteins and beads.

• Volume of protein injected onto column must be <2% of column volume.

• Eluant is diluted

Sept. 16, 2009 GMS BI 555/755 Lecture 3. 38

Gel filtration/Size exclusion

Gel filtration: principles and methods, GE Healthcare

applications 1.Purification; of biological macromolecules by facilitating separation from larger & smaller.

Viruses, proteins, enzymes, hormones, Ab, nucleic acid & polysaccharides separation use this

2. Relative molecular mass Determination; elution Vol, of globular proteins are determined

largely by their RMM

elusion Vol, rate is an approximate linear fxn of log, of RMM, hence construction of

calibration curve, with proteins with similar shape & known RMM enable RMM value of other

particles even in crude form be estimated

3. Solution conc,; soln of high RMM substances can be concentrated by addition of dry sephadex

G-25 (coarse)

-water & low RMM cpds are absorbed by swelling gel, but those of high

RMM remain in soln

-after 10 min, gel is removed by centrifugation, leaving high RMM cpds in soln

whose conc, has increased but pH & ionic strenght not altered

4. Desalting; use of sephadex G-25, solns of high Rmm cpds may be desalted., they move with

void Vol, but low Rmm cpds are distributed btw mobile & stationary phases hence move slowly

-this tech. is faster & efficient than dialysis. Used in removal of phenols from N. acids preps., NH2SO4 from protein preps, salt from eluted cpds from ion-exchange column

5. protein-binding studies; exclusion is among the methods used to study reversible binding of

ligand to macromolecule eg. Protein, including receptor proteins

- sample of protein/ligand mixture is applied to column of suitable gel with previuos

equilibration with a soln of the ligand of same conc., as that in the mixture

-Sample eluted with buffer in std way & conc, of ligand & protein in effluent determined

--early fraction will contain unbound ligand, but subsequent appearance of the protein result in

increased in total qnt of ligand (bound & unbound)

Affinity Chromatography

-unlike other forms of chromat. & electrophoresis, centrifugation, it doesn’t rely on differences in

physical properties

-it exploits unique property of extreme specificity of biological interaction for separation to

occur

-hence capable of giving absolute purification from cplx cpds in single process

-was originally developed for enzyme but extended to nucleotides, N. acid, immunoglobulins,

memb, receptore, whole cell & fragment

-cpd to be isolated should be reversible bind to specific ligand attached to insoluble matrix

Sept. 16, 2009 GMS BI 555/755 Lecture 3. 42

Affinity Chromatography • A protein may be purified based on

its binding to a chemical group or another protein

• Immobilize the ligand on a chromatographic bead (many standard chemistries available)

• Pass the protein mixture through the column

• Wash non-bound material through the column

• Elute the bound proteins either using a competing ligand, an increasing salt concentration, or other denaturatn

• Examples • Avidin-biotin • Poly-His tags binding to

immobilized metal columns • Protein A/G binding to

antibodies • Growth factor binding to

heparin columns • Transcription factor binding to

immobilized DNA seq • Purification of recombinant

fusion proteins (GST, GFP, FLAG)

-mixture of to be isolated cpds is added to immobilsed ligand and only the cpd bind to ligand

-detail preliminary knowledge of structure & biological specificity is required of to be isolated

cpd

-eg enzyme, ligand may be substrate, a reversible inhibitor or allosteric activator

-Conditions chosen must be optimum to those of enzyme-ligand binding

-after binding dynamic situation is created where conc, of cplx & binding strenght increase

-eluetion is done by pH /ionic change and the cpd is removed from the ligand and collected

matrix

-must contain suitable & efficient chemical grps which ligand can be covalently coupled & be

stable under attachment conditions

-stable during binding of macromolecule & its subsequent elution

-interact only weakly with other macromolecule to minimise non-specific adsorption

-should exhibit good flow properties

-number of different spacer arms are used

-eg. 1,6 diaminohexane, 6-aminohexanoic acid and 1-4, bis-(2,3-epoxypropoxy) butane

-they must process a second fxnal grp to which ligand may be attached by conventional

organosynthetic procedure

-this involve use of succinic anhydride & water-soluble carbodiimide fi

Application -enzymes & proteins , immunoglobulins been purified by the method

-technique only limited by availability of immobilised ligand

-nucleic acid also; eg mRNA is isolated by selective hybridization on poly(U)-sepharose 4B by

exploiting its polyA tail

-Immobilised ssDNA can be used to isolate cRNA & DNA

Metal chelate (immobilised metal affinity) chromatography

-special form of affinity chromat.where immobilised metal ion eg Zn2+, Hg2+ ,Cd2+, Cu2+ or

transition metal like Ni2+, Mn2+ Co2+

-these used to bind proteins selectively by rxn with imidazole grp of His, thiols grp of Cyst &

indole grps of Tryp residues

-immobilisation of protein involves formation of coordinate bond that must be sufficiently stable

-this allows protein attachment and during elution of non-binding contaminating cpds

-release of protein involves low pH use destabilizing protein –metal cplx or use of EDTA

Covalent chromatography

-developed to separate thiol (-SH) containing proteins

-exploits their interactionwith immobilised ligand containing disulphide grp

-commonly use ligand is disulphide 2’-pyridyl grp attached to agarose matrix

Gas-Liquid chromatography -based on partitioning cpds btw liquid & gas phase

-widely used for qnltive & qntive analysis of large number of cpds due to its sensitivity,

reproducibility & speed of resolution

-most valuable for relatively low polarity cpds

-stationary phase of high b.p liquid material eg. Silicone grease supported on inert granular solid

-the material packed into narrow coiled coil glass/steel column 1-3 m lond and 2-4mm internal

diameter

-where inert carrier gas (mobile phase) eg. N2, He, or argon pass

-column put in oven for high temp, for to be separated cpds tkept in vapour state

-basis of separation is the difference in partition coefficients of volatilised cpds btw liquid & gas

phases as cpds carried thro, column by carrier gas

-as cpds leave column , pass thro, detector linked via amplify to chart recorder

application

-confined to volatile, non-polar cpds that need not derivatisation

-analyte xterized by their retention time relative to a std refernce cpd.

Thin layer chromatography (TLC)

-Thin layer of stationary phase formed on suitable flat surface eg. Glass, foil or plastic

-since layer is thin, mvt of mobile phase across the layer is by simple capillary action

-as mobile phase moves it transfers any analyte placed on the layer at rate determined by

distribution coefficient, Kd

-btw stationary & mobile phases

-analyte mvt stop either when mobile phase(solvent) reach end of layer & capillary action flow

stops or when plate is removed from mobile phase reservior

-Mvt of analyte is expressed by its retadation factor , Rf

Rf = distance moved by analyte from origin

distance moved by solvent from origin

-efficeincy of thin-layer plate is expressed by its number of theoritical plates, N & plate height H

N = 16 (dA)2

(w)2

dA- is distance moved by analyte from origina and w is

width of sop

H = dA

N

- Capacity factor, K’ for analyte is

K’ =dm = 1-RF dm is distance moved by solvent from origin

dA RF

Paper chromatography -principle is similar to thin layer

-cellulose fibre of chromatography paper act as support matrix for stationary phase

-stationary phase may be water, non-polar material eg. Liquid parafin/impregnated particles of

solid adsorbent

-papers are of different running xteristics eg slow, medium, fast.

Selection of a chromatographic system