CHROMATOGRAPHY

M.PRASAD NAIDUMsc Medical Biochemistry,

Ph.D Research scholar.

CHROMATOGRAPHYHistorical details

•1903: Michael Tswett (or Tsvet) a Russian botanist separated the pigments in green plant leaves using a glass column

packed with 2 μm inulin and ligroin as mobile phase

•Coined the term chromatography - color writing - to describe the separation of the pigments into colored bands along the length of the column

•Later used CaCO3 or sucrose as the column packing material

•Not much work done until the late 1930’s and 1940’s

•1941: Archer John Porter Martin & Richard Laurence Millington Synge published a paper on liquid partition chromatography that set the stage for gas liquid chromatography

•Studied the amino acid composition of wool

•Invented a process where a liquid is firmly bound to a finely granulated solid phase packed in a glass column

•H2O firmly bound to SiO2 - silica gel

•A second liquid immiscible with the bound H2O is percolated through the column - CHCL3

•Solute analyte molecules are partitioned between the stationary phase and the mobile phase

Historical details

•Partition chromatography

•Problem: the packing of silica gel columns is not very reproducible in its properties

•Martin and Synge invented a new method in which H2O is firmly bonded to filter paper

•Solute analyte molecules are partitioned between a moving liquid phase and the H2O bound to the cellulose surface

•Very reproducible elution times are obtained using paper chromatography

•1952: Martin and Synge were awarded the Nobel prize in chemistry for the invention of column partition chromatography, and particularly paper chromatography

•1938: Izmailov & Schreiber worked out a procedure where the solid stationary chromatographic phase is distributed as a

thin film on a glass plate

•Thin Layer Chromatography (TLC) was not much used until 1956 when it began to be used

ChromatographyHistorical details

•Gas chromatography was invented in Austria but was not much used until 1956

•1956: Martin & James applied gas chromatography to the separation of acids and amines

•Small particles are coated with a non-volatile liquid and packed into a tube which can be heated

•Analytical mixtures are injected into a heated inlet tube and volatilized

•The mixture is driven through the packed column by a compressed gas

•Interactions between the components of the mixture and the liquid stationary phase causes separation into zones as

the analytes are passed through the column

•Major recent advances involve the elimination of the packing

•Coat the inside of a fused silica capillary - 250 μm ID - tube with a liquid film - 0.10 - 0.50 μm thickness

•Most recently, 530 μm ID tubes - megabore capillaries - are being used

ChromatographyDefinition of chromatography: The differential migration of sample components dissolved in a mobile phase produced by selective retention by a stationary phaseClassification of chromatographic methods

Chromatography

Mobile Phase Gas Liquid

Stationary phase Liquid Solid Bonded Liq. Liq Solid Bonded Liq. Ion Ex Polymer Solid

Process (part’n) (adsorp’n) (adsorp’n- (part’n) (adsorp’n) (adsorp’n- (ion (part’n part’n) HPLC part’n) exc’ng) sieving) paper HPLC exclusion chromat

Conditions associated with each kind of chromatography•Gas-liquid partition chromatography involves establishing an equilibrium between components in the gaseous mobile phase and the liquid stationary phase

ChromatographyConditions associated with each kind of chromatography

•Gas-solid adsorption chromatography involves surface adsorption of analytes dissolved in the gas mobile phase using solids such as alumina, molecular sieves or porous silica•Gas bonded-liquid chromatography makes use of molecules that are chemically bonded to a solid support, either silica beads or the wall of a fused silica capillary tube

•The interaction between components dissolved in the stationary phase and the stationary phase involves both partitioning and adsorption

•Liquid-liquid partition chromatography involves a partitioning of components dissolved in the mobile phase between the mobile phase and the stationary liquid phase

•This is the process often associated with High Performance Liquid Chromatography (HPLC), paper chromatography and TLC

if silica gel is used•Liquid-solid adsorption chromatography involves an adsorption of analytes dissolved in the liquid mobile phase on the surface of the stationary phase; the stationary phase can be any adsorbent material such as alumina or silica

ChromatographyConditions associated with each kind of chromatography

•Liquid bonded-liquid chromatography involves silica beads (3-5 μm diameter) to which molecules are chemically bonded

•The interaction between analyte molecules in the liquid mobile phase and the stationary phase involves both partitioning and adsorption•This is now the usual method for carrying out HPLC

•Normal phase HPLC uses a polar stationary phase and a non-polar liquid mobile phase•Reversed phase HPLC uses a non-polar stationary phase and a polar liquid mobile phase

•Ion exchange chromatography makes use of natural or synthetic zeolites or synthetic or organic or inorganic polymer resins as the stationary phase

•The interaction between ions dissolved in the liquid mobile phase and the stationary phase involves an exchange of ions associated with the stationary phase

ChromatographyConditions associated with each kind of chromatography

•Ion exchange chromatography•Organic cation exchange resins involve crosslinked polystyrene

containing either SO3- or COO- functional groups with an

associated cation

•Organic anion exchange resin involve

•crosslinked polystyrene containing NH3+

•functional groups with an associated anion

C

H

C

H

H

C

H

C

H

H

Na+SO3

- SO3-Na

+

C

H

C

H

H

C

H

C

H

H

Cl-NH3 NH3

+Cl

-

The affinity of dissolved ions for the resin varies with the ion and the composition of the

solution

ChromatographyConditions associated with each kind of chromatography

•Exclusion chromatography makes use of a uniform, highly porous,

non-ionic gel•Small molecules are retained in the pores of the gel, large

molecules are not retained - they are excluded

•Separation involves sorting molecules by size

•Gel permeation chromatography involves use of polymers that swell in

organic liquids - polyacrylamide gels

•Gel filtration chromatography involves the use of polymers that swell

in water such as Sephadex, a polysucrose polymer

•For liquid chromatography, differences in the affinity of solute

analytes for the stationary phase can be controlled by

controlling the physical and chemical properties of not only the

stationary phase but also the mobile phase•Gradient elution liquid chromatography involves changing the composition of the mobile phase during the elution process

•Such liquid properties as the polarity, ionic strength and pH can be varied

•Isocratic liquid chromatography involves constant mobile phase

composition

ChromatographyChromatographic column theory of packed columns

•The differential migration of analytes in the mobile phase separates components into zones that move at different rates along the column length

t0

t1

t3

t4

t5

2 components

A&B mobile phase front

detector

Observations

•Bands move down the column with different velocities

relative to the mobile phase front

•Zones get wider the longer they remain in

the column

•There is a normal distribution of molecule concentration along the length of the column

ChromatographyChromatographic column theory of packed columns

•Resolution measures how well zones are separated compared to their overlap

•Determined by the standard deviation (σ) in column length over which the bands are distributed•Determined by the retention time: how long it takes a zone center to elute

0 time

tr1

tr2

w1 w2

tr

wt

R

www peaks, close for

wwt2

2wwtt

R

R

21

21

R

21

2R1R

W is determined by column efficiency

•Function of flow rate, packing particle size, column diameter

ΔtR determined by column selectivity

•Function of the nature of the mobile and stationary phases

•Function of relative amounts of mobile and stationary phases

•Function of how efficiently the stationary phase is used as

measured by the capacity factor - k’

ChromatographyChromatographic column theory of packed columns

•The effect of column efficiency and column selectivity on resolution

Poor resolution because of poor column efficiency

Good resolution because of good column efficiency,

although column selectivity is not great

Good resolution because of good column selectivity,

although column efficiency is poor

Poor resolution because of poor column selectivity,

although column efficiency is good

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