ChemLec- Chromatography Techniques

Chromatography

Colour Writing.

-It is new physical technique of separation, identification, and purification of components of a mixture.

International Union for Pure and Applied Chemistry

 defined Chromatography as - a physical method of separation in which the components to be separated are, distributed between two phases, one of which is stationary phase while the other is mobile phase, moves in define direction

chromatography

is a separation technique that uses the size, shape, chemical properties or charge of molecules in a sample to separate the sample into its constituent components

-It is often used to detect one, or a number of, components in a complex mixture.

Mikhail Tsvet

The  first true chromatography is usually attributed to the Russian-Italian botanist ________

fractionation, petroleum

• Tsvet applied his observations with filter paper extraction    to    the    new    methods    of column__________that had been developed in  the  1890s  for  separating  the  components of __________

Mobile Phase

Solvent moving through the column

Adsorbent

Substance that stays fixed inside the column

Eluent

Fluid entering the column

eluate

Fluid exiting the column (that is collected in flasks)

Elution

The process of washing out a compound through a column using a suitable solvent

Analyte

Mixture whose individual components have to be separated and analyzed

analytical chromatography

can  be  used  to  purify  compounds ranging from milligram to gram scale.

differential affinities

(strength of adhesion) of the various components  of  the  analyte  towards  the  stationary  and mobile phase results in the differential separation of the components.

adsorption

the property of how well a component of the mixture sticks to the stationary phase.

solubility

• the property of how well a component of the mixture dissolves in the mobile phase

slower

Higher the adsorption to the stationary phase, the _______ the molecule will move through the column.

faster

Higher the solubility in the mobile phase, the ______ the molecule will move through the column.

N.A Izmailov, M.S Scheiber

Use of thin layer chromatography was first reported by two Russian scientists, _______ and ______ Later this technique was developed further by other scientist.

mobile phase

which  consists  of  a  solvent  that  moves through the stationary phase, is used to move the components of the sample up through the stationary phase

TLC Plates

 preferably ready made with a stationary phase: These are stable and chemically inert plates, where a thin layer of stationary phase is applied on its whole surface layer. The stationary phase on the plates is of uniform thickness and is in a fine particle size.

TLC Chamber

This is used for the development of TLC plate. The chamber maintains a uniform environment inside for proper development of spots. It also prevents the evaporation of solvents, and keeps the process dust free.

Mobile Phase

This comprises of a solvent or solvent mixture The mobile phase used should be particulate-free and of the highest purity for proper development of TLC spots. The solvents recommended are chemically inert with the sample, a stationary phase.

Filter paper

this is moistened in the mobile phase, to be placed inside the chamber. This helps develop a uniform rise in a mobile phase over the length of the stationary phase.

Archer J.P Martin, Richard Synge

paper chromatography were invernte by

Sir Thompson, JT Way

ion exchanged chromatography was originally introduced by

ion- exchange chromatography

Method was used to treat clays with the salts, resulting in the extraction of ammonia in addition to release of calcium

zeolites

Compounds  knows  as  ________  were  introduced  to separate   individual   ions   or   electrically   charged particles.

ion exchange

is the most widely used chromatographic method for the separation and purification of charged  biomolecules    such    as    polypeptides,    proteins,  polynucleotides, and nucleic acids.

separation and purification of blood

components such as albumin, recombinant growth factors and enzymes.

biotechnology

Analytical applications such as quality control and process monitoring

Food and clinical research

to study wheat varieties and the  correlation  of  proteinuria  with  different  renal diseases.

Fermentation

Cation  exchange  resins  are  used  to monitor the fermentation process during ß-galactosidase production.

gel permeation chromatography

is an established cleanup technique that separates (size exclusion) high-molecular-weight compounds such as proteins, fats, and sugars from relatively low molecular weight compounds such as pesticides in animal and plant matrices

affinity chromatography

can be defined as a liquid chromatographic method in which a biological agent or biomimetic ligand is used for the selective retention of complementary compounds.

gas chromatography

s a separation technique capable of separating highly complex mixtures based primarily upon differences of boiling point/ vapor pressure and of polarity.

superficial fluid chromatography

•  is a variant of high-performance liquid chromatography (HPLC) in which a supercritical fluid is used to replace the liquid mobile phase.

capillary electrophoresis

• is the principal method utilized by forensic biologists to detect genetic variation. 

• Separation and detection of nucleic acids can be achieved rapidly and reliably, providing sufficient resolution for genotyping the large range of markers currently utilized for forensic casework and research

High performance liquied chromatography

a chromatographic technique similar to GC that involves the migration of a lipid mixture through a column containing a stationary phase. However, in HPLC the mobile phase is a liquid instead of a gas. HPLC

TLC Plate

a thin piece of aluminum coated on one side with the stationary phase (this phase does not move)

affinity chromatography

this form of _______ was originally used by Starkenstein in 1910 for the purification of amylase through the use of starch as a solid support.