CHROMATOGRAPHIC TECHNIQUES

PRESENTED BY BHAVANA SHIVANKAR

M.Sc.- II SEMDEPARTMENT OF CRIMINOLOGY AND FORENSIC SCIENCE

CHROMATOGRAPHY IUPAC Defined chromatography as, It is a physical method of separation in which the

components to be separated are distributed between two phases, one is stationary while the other moves.

Chromatography was invented by Mikhail Tswett, a botanist in 1906. He had successfully separated chlorophyll, xanthophylls and other colored substances by percolating vegetable extract through a column of calcium carbonate.

Paper chromatography was first introduced by German scientist Christian Freidrich Schonbein.

A.J.P. Martin, was awarded the Nobel prize for his work in developing liquid-liquid and paper chromatography in 1941 and recognized for the development of gas chromatography.

HISTORICAL BACKGROUND

Conti…

In 1952, A.P.J.Martin and Richard Laurence Millington Synge was awarded Nobel Prize for invention of partition chromatography.

PRINCIPLE

Chromatography is based on the principle of selective distribution of the components of a mixture between stationary (stagnant) and mobile phase(moving). Stationary phase can a solid or a liquid while the mobile phase can be a liquid or gas.

CLASSIFICATION OF CHROMATOGRAPHIC METHODS

Based upon the physical means by which stationary and mobile phases are brought into contact;

1. Planar chromatography2. Column chromatography

CLASSIFICATION OF CHROMATOGRAPHIC METHODS

Based upon mobile and stationary phase and kind of equilibrium established between two phases.

1. Gas chromatography2. Liquid chromatography Based upon the mechanism of separation1. Adsorption2. Partition

PLANAR CHROMATOGRAPHY

In planar chromatography, the stationary phase is supported on a flat plate or in the interstices of paper; here the mobile phase moves through the stationary phase by capillary action or under the influence of gravity.

In this the position of migrated spots on chromatograms are indicated by retention or retardation factor (Rf).

Rf= distance travelled by solute from baseline distance travelled by solvent from baseline

PLANAR CHROMATOGRAPHY

Paper chromatography

Thin layer chromatography

HPTLC(HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY)

The advanced form of TLC is HPTLC. It is a validated method for qualitative and quantitative analysis. Instrument works on sophisticated software which ensure the highest degree of usefulness, reliability and reproducibility.

HPTLC

INSTRUMENTATION

(A)

(C)

(B)

Fig. (A) application of sample, (B) Chromatographic development chamber, (C)Detection & Visualization.

COLUMN CHROMATOGRAPHY In column chromatography, the stationary

phase is held in a narrow tube through which the mobile phase is forced under pressure.

COLUMN CHROMATOGRAPHY

In this method, the mixture to be separated is dissolved in suitable solvent and allowed to pass through tube containing adsorbent. The components which has greater adsorbing power is adsorbed in upper pert of the column. The next component is adsorbed in lower portion of column which has lesser absorbing power than the first component.

HPLC HPLC was referred as High Pressure Liquid

Chromatography but nowadays the term is referred as High Performance Liquid Chromatography.

Types of HPLC :Based on modes of chromatography1. Normal phase mode2. Reverse phase mode

Based on principle of separation3. Adsorption chromatography4. Ion exchange chromatography5. Partition chromatography6. Size exclusion

Conti…

Based on elution technique1. Isocratic separation2. Gradient separation

Based on type of analysis3. Qualitative analysis 4. Quantitative analysis

HPLC(HIGH PERFORMANCE LIQUID CHROMATOGRAPHY)

HPLC (HIGH PERFORMANCE LIQUID CHROMATOGRAPHY) A solvent reservoir A high pressure pump Sample injection port A column A detector and recording unit

SOLVENT RESERVOIR

Mobile phase contents are contained in a glass reservoir. The mobile phase , or solvent, in HPTLC is usually a mixture of polar and non- polar liquid components whose respective concentrations are varied depending on the composition of the sample.

Isocratic separationGradient separation

PUMP

A pump aspirates the mobile phase from the solvent reservoir and forces it through the system’s column and detector. Depending on the particle size of stationary phase, the flow rate and the composition of the mobile phase, operating pressures are generated.

SAMPLE INJECTOR

The injection can be single or an automated injection. An injector for an HPLC system should provide injection of the liquid sample within the range of 0.1-100 ml of volume with high reproducibility and under high pressure.

COLUMN

Columns are usually made up of polished stainless steel, are between 50 and 300 mm long and have an internal diameter between 2- 5 mm. Ideally the temperature of column and mobile phase should be constant during an analysis.

Column packing◦Microporous◦Pellicular◦Bonded Phase

DETECTOR

The HPLC detectors are located at the end of the column, detect the analytes as they elute from the chromatographic column. Commonly used detectors are UV – spectroscope, fluorescence, electrochemical and mass spectrometric detectors.

DATA COLLECTION DEVICES

Signals from detector may be collected on chart recorders or electronic integrators that vary in complexity and in their ability to process, store and reprocess chromatographic data. The computer integrates the response of the detector to each component and placed it into a chromatograph that is easy to read and interpret.

Applications of HPLC

Pharmaceutical applicationsPharmaceutical quality controlTablet dissolution study of pharmaceutical dosages form.Environmental applicationsDetection of phenolic compounds in drinking water.Bio monitoring of pollutants.Food and flavorMeasurement of quality of soft drinks and water.Sugar analysis in fruit juices.Analysis of adulterants in food materials.Preservative analysis.

Applications of HPTLC

Clinical applicationsUrine analysis,antibiotic analysis in blood.Analysis of bilirubin, and other drugs in hepatic

disorders.Detection of endogenous neuropeptides in

extracellular fluid of brain,etc.

Identification and quantification of drugs in biological samples.

Identification of steroids in blood.Analysis of dyes.Determination of cocaine and other drugs of

abuse in blood, urine etc.

FORENSIC APPLICATION

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