I NTRODUCTION TO C HROMATOGRAPHY PHR 310: P HARMACEUTICAL A NALYSIS - II

W HAT IS C HROMATOGRAPHY ? Greek word : Chroma ( colour) Graphein (to write). Chromatography provides a way to identify unknown compounds and separate mixtures

H ISTORY The Russian botanist Mikhail Tsvet coined the term chromatography in 1906 to describe his experiments in separating different colored constituents of leaves by passing an extract of the leaves through a column

Chromatography is a physical method of separation in which components to be separated are distributed between two phases Stationary phase (S. P.): The phase which does not move Mobile phase: The phase that moves through S.P in a definite direction. D EFINITION

C HROMATOGRAPHY Separates components in mixture: Based on - Polarity - Ionic strength - Size

C HROMATOGRAPHY Mobile phase : The liquid or gas that flows through a chromatography system, moving the components to be separated at different rates over the stationary phase. Stationary phase : Phase in which mobile phase is forced through it. It may be Solid, liquid coated on solid, etc. The stationary phase is the substance which is fixed in a column or on a flat plate or surface. The mobile phase is passed through the stationary phase where the sample interacts and is separated. Examples are silica gel, aluminum oxide or cellulose. Mobile and stationary phases are chosen in a manner so that analyte will distribute itself between the two phases

P RINCIPLE OF C HROMATOGRAPHY : 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. Affinity, in turn, is dictated by two properties of the molecule: ‘Adsorption’ and ‘Solubility’. The various components of the mixture travel at different speeds through the stationary phase causing them to separate. So that the separation is actually based on differential partitioning between the mobile and stationary phases.

The speed of a migrating sample component depends on whether the component has an affinity for the stationary or mobile phase. This affinity can be of different types such as: 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 later from the column.

T ERMINOLOGY The analyte is the substance to be separated during chromatography. The sample is the analyte analyzed in chromatography. It may consist of a single component or it may be a mixture of components. Elution: The process of washing out a compound through a column using a suitable solvent. The eluent is the fluid that entering the column and carring the analyte. The eluate is the mobile phase leaving the column.

T ERMINOLOGY The retention time is the characteristic time takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. A chromatograph is equipment that enables a sophisticated separation, e.g. gas chromatographic or liquid chromatographic separation. Chromatogram: It is the visual output of the chromatograph. In the case of an optimal separation, different peaks on the chromatogram correspond to different components of the separated mixture.

T ERMINOLOGY The detector refers to the instrument used for qualitative and quantitative detection of analytes after separation.

C HROMATOGRAM Detector signal (conc. of Sample) vs. retention time or volume of mobile phase time or volume Detector Signal 12

Chromatograms If a detector that responds solute concentration is placed at the end of the column and its signal is plotted as function of time (or of volume of the added mobile phase), a series of peaks is obtained. Such a plot is called a chromatogram, useful for both qualitative and quantitative analysis. - positions of peaks on the time axis may serve to identify the components of the sample - areas under the peaks provide a quantitative measure of the amount of each component. Concentration of sample Vs Time (Volume of mobile phase)

Based on the physical means of bringing the stationary and mobile phases into contact chromatographic methods can be classified into 2 broad categories. 1. Planar Chromatography: Here the stationary phase is supported on a flat plate or in the fibres of a paper. Here the mobile phase moves through the stationary phase by capillary action or by gravity. E.g.: Paper chromatography, Thin layer chromatography (TLC).

2. Column Chromatography: Here the stationary phase is packed in a test tube / boiling tube / glass column of any convenient size. The substances to be separated are introduced onto the top of a column and pass through the column at different rates that depend on the affinity of each substance for the adsorbent and for the solvent or solvent mixture.

T YPES OF C HROMATOGRAPHY According to purpose 1. Analytical chromatography: It is used to determine the existence and possibly also the concentration of analyte(s) in a sample. It is done in smaller amount of material. 2. Preparative chromatography: It is used to purify sufficient quantities of a substance for further use, rather than analysis. It is done in larger amounts of material.

T YPES OF C HROMATOGRAPHY On the basis of physical state of stationary and mobile phase. - liquid-solid chromatography - liquid-liquid chromatography - gas-solid chromatography - gas-liquid chromatography

T YPES OF C HROMATOGRAPHY On the basis of method of separation liquid chromatography can be classified into: 1. Adsorption chromatography 2. Partition chromatography 3. Ion Exchange chromatography 4. Molecular/size exclusion chromatography 5. Affinity chromatography

T YPES OF C HROMATOGRAPHY … Paper HPLCGas Thin layer Column

A DSORPTION C HROMATOGRAPHY The stationary phase is solid on which the sample components are adsorbed. The mobile phase may be a - liquid (liquid-solid chromatography) or, - gas (gas-solid chromatography) The components distribute between the two phases through the combination of - adsorption & - desorption e.g. Column chromatography, TLC

Adsorption chromatography is based on the adsorption of solute molecules onto the surface of a solid (stationary phase). This attachment or interaction depends on the polarity of solutes. E.g.: Column chromatography (CC), Thin Layer chromatography (TLC), High Performance Liquid chromatography (HPLC), etc.

P ARTITION C HROMATOGRAPHY The stationary phase is a liquid supported on an inert solid The mobile phase is a - liquid (liquid-liquid partition chromatography) or, - gas ( gas-liquid partition chromatography)

Here a thin film is formed on the surface of a solid support by a liquid stationary phase. Solute equilibriates between the mobile phase and the stationary liquid. i. Normal Phase– analyte is nonpolar organic; stationary phase is MORE polar than the mobile phase. ii. Reverse Phase– analyte is polar organic; stationary phase is LESS polar than the mobile phase. E.g.: TLC, Paper Chromatography

Paper chromatography is a type of partition chromatography in which the stationary phase is a layer of solvent adsorbed on a sheet of paper.

I ON E XCHANGE C HROMATOGRAPHY

Ionic mixtures can be separated by this technique. Here the stationary phase is composed of ion-exchange resins and 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.  Anionic stationary phases: used for cation separation  Cationic stationary phases: for anion separation

Solute ions of the opposite charge in the mobile phase attach to the resin by electrostatic forces. Ion exchange columns are of 2 types. 1. Cation exchange columns, contains (–)ve ly charged groups. 2. Anion exchange columns, contains (+)ve ly charged groups. E.g.: CC, HPLC.

M OLECULAR /S IZE E XCLUSION C HROMATOGRAPHY Also known as Gel permeation or Gel filtration. Separation based on size. Here no interactions occur between the stationary phase and the solute molecules. Stationary phase is polymeric substance containing numerous pores of molecular dimension. Larger molecules that will not fit into the pores remain in the mobile phase and are not retained. Small molecules get trapped in pores & take longer to get out

A FFINITY C HROMATOGRAPHY : This technique also requires the interaction between the solute molecules and active sites on the stationary phase. According to the theory of this technique, those compounds whose shape matches perfectly to the shape of the stationary molecules, bind well to the stationary phase. This interaction is of covalent type and thus taking longer period to elute where as other compounds elute earlier.

A FFINITY C HROMATOGRAPHY A method of separating biochemical mixtures Based on a highly specific biological interaction such as that between - antigen and antibody, - enzyme and substrate, or - receptor and ligand. Stationary phase is typically a gel matrix, often of agarose.

This technique is mainly used in antibody testing assays. Test sample contains a mixture of proteins and immobilized ( stationary) molecule is an antibody to some specific protein.

A PPLICATION OF C HROMATOGRAPHY It is a technique for  separating mixtures of compounds  identifying unknown compounds  establishing the purity or concentration of compounds  monitoring product formation in the pharmaceutical and biotechnology industries. It is widely used by forensic teams to analyse blood and urine samples for drugs.

S TATIONARY PHASES Alumina: Aluminum oxide - Surface is highly polar - Capable of adsorbing practically all polar molecules. - Judicious selection of the eluent allows to separate any pairs of solute on alumina column. - Adsorbent power of alumina is markedly affected by it’s water content, with freshly dried alumina having the highest adsorbent activity. - Adsorbent power of alumina is specified by its ability to retard the migration of certain dyestuffs through a column prepared with the alumina. Grades I-V

S TATIONARY PHASES Silicic acid or silica gel, SiO 2 - can be activated by heating or prewashing the bed with an anhydrous solvent to remove adosorbed water. Some others stationary phase: 1. Fuller’s earth (hydrous aluminium silicate) 5. Potassium carbonate 2. Activated charcoal6. Talc 3. Magnesium oxide7. Starch 4. Calcium carbonate