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WHAT IS CHROMATOGRAPHY? A TECHNIQUE FOR SEPARATING MIXTURES INTO THEIR COMPONENTS IN ORDER TO ANALYZE, IDENTIFY, AND PURIFY THE MIXTURE OR COMPONENTS.

Chromatography (from Greek chroma "color and  graphein "to write") is the collective term for a set of laboratory techniques for the separation of mixtures. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases.

DEFINITION

Analyze : To examine the mixture or structure or something especially by separating it into its parts.

Purify : To make something pure by removing substances that are not wanted out of another substances that contains it.

Identify : To recognize something and to determine the identity of mixture.

PRINCIPLE OF CHROMATOGRAPHY

The speed of a migrating sample component depends on whether the component has an affinity for the stationary or mobile phase. This affinity appears via various actions: Adsorption, Partition, Ion exchange, etc . Components that have a higher affinity for the Mobile phase compared with the stationary phase migrate more rapidly, while components that have a higher affinity for the Stationary phase are eluted from the column later.

CLASSIFICATION

On the basis of Stationary phase & Mobile phase

ON THE BASIS OF MECHANISM

CHROMATOGRAPHY

ADSORPTION

ION EXCHANGE

PARTITION

AFFINITY

CHROMATOGRAPHY MECHANISM

01. Surface Adsorption The stationary phase of this particular technique

is a solid material on which the sample compounds are adsorbed. Mobile phase is either a liquid (solid-liquid chromatography) or a gas (gas-solid chromatography).. Adsorption chromatography is based on the interaction between the solute molecules and active sites on the stationary phase. This attachment or interaction depends on the polarity of solutes. 

02. ION EXCHANGE

This is to separate ionic mixtures. The stationary

phase of this technique is an ion-exchange resins

where as the mobile phase is a buffered aqueous

solution. Anions or cations can covalently attach on

to the resin. The resin can be either cationic or

anionic. Solute ions of the opposite charge in the

mobile phase attach to the resin by electrostatic

forces.  Ion exchange columns are of two types.

Those are cation exchange columns (contains

negatively charged groups) and Anion exchange

columns (contain positively charged groups).

03. SIZE EXCLUSION

This separation technique is based on the molecular size. The stationary phase is a chemically inert material such as gels, porous inorganic solids, porous glass etc. Mobile phase is a water or aqueous solution which will serves only as a carrier for the analyte. The stationary phase of this chromatography is a porous material. This technique usually applies to larger molecules or macromolecules like proteins and industrial polymers. Also the stationary phase should not interact with the molecules. 

04. AFFINITY BASED

 According to the theory of this technique, the shape of the compounds in blue color matches perfectly to the shape of the arrow head. Thus they are bound well to the stationary phase taking longer period to elute where as other compounds (yellow and dark blue) elute first. Thus the interaction is between one type of solute molecule in the test sample and the substance in the stationary phase (blue arrow).This reaction is a covalent interaction. This is mainly used in antibody testing assays. Test sample contains a mixture of proteins and immobilized molecule is an antibody to some specific protein. 

Components that have a higher affinity for the Mobile phase compared with the stationary phase migrate more rapidly, while components that have a higher affinity for the Stationary phase are eluted from the column later.

VISUALIZATION Chromatography is a technique used to separate

and quantify the components in a complex chemical mixture. In this technique, the mixture is introduced into a column filled with small particles. The different compounds in the mixture have different affinities for the particles (i.e., they participate in different types and strengths of intermolecular interactions). As solvent flows through the column, compounds with weaker affinities for the particles move down the column faster than compounds with stronger affinities. Thus, the various compounds in the mixture are separated based on their relative affinities for the solid particles.

01. U V VISUALIZATION This is one of the most appropriate method

of visualization in chromatography. TLC plates normally contain a flouroscent indicator which makes the tlc plate glow green under UV light of wavelength 254 nm. By apply the UV light on the plate dark purple colour or bluish spots appear .

02. IODINE CHAMBER

A second method of visualization is accomplished by placing the plate into iodine vapors for a few minutes. Most organic compounds will form a dark-colored complex with iodine. It is good practice to use at least two visualization techniques in case a compound does not show up with one particular method. The iodine method must be done after the UV method because the compound may react with the iodine, possibly changing the results. Put a TLC plate inside, keep the plate for 2 sec to 10 min inside and then take the plate out. Watch the color change. Pick the best moment to circle spots - they might soon disappear.

03. DETECTING AGENT

This is a brief selection of the many available TLC visualization techniques. Each title is the type of compounds or structure which can be detected with the specific reageBefore spraying, plates should be well dried in the hood of residual solvents and components. Amines and organic acids used in the mobile phases may adversely affect the visualization reaction being attempted. If heating to remove these components is done, consideration should be given so that loss of components or their decomposition is avoidednt.

EXAMPLE

Ninhydrin For detection of amino acids, amines, amino

sugars. Spray with a solution of 0.2g ninhydrin in

100ml sulphuric acid.Nitric acid / ethanol For detection of amines and alkaloids Spray with a solution of 50 drops 65% nitric

acid in 100ml ethanol and heat to 110 C until spots appear.

DIFFERENCE BETWEEN UV & IODINE METHOD

UV Detection Iodine Detection

Need of UV chamber

Spot appear only in presence of Ultra violet radiation.

High molecular wt. sample can detect.

Need of Iodine pellets

Spot appear in saturated iodine chamber but disappear after a time period.Only low molecular wt. sample can be detect.

LIMIT OF DETECTION & QUANTITATION

Sensitivity, Analytical Sensitivity, Functional Sensitivity, Lower Limit of Detection, , LoD, and LoQ are terms used to describe the smallest concentration of a measure and that can be reliably measured by an analytical procedure. To provide a standard method for determining LoB, LoD and LoQ, Clinical and Laboratory Standards Institute (CLSI) has published the guideline EP17, Protocols for Determination of Limits of Detection and Limits of Quantitation.

•LoD is the lowest analyte concentration likely to be reliably distinguished from the LoB and at which detection is feasible.

•LoD is determined by utilising both the measured LoD and test replicates of a sample known to contain a low concentration of analyte. LoD = LoB + 1.645(SD low concentration sample)

•LoQ is the lowest concentration at which the analyte can not only be reliably detected but at which some predefined goals for bias and imprecision are met. The LoQ may be equivalent to the LoD or it could be at a much higher concentration.