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CHROMATOGRAPHY

CHROMATOGRAPHY

A laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary adsorbing medium through which they pass.

CHROMATOGRAPHY

Introduced first by the Russian botanist Mikhail Semenovich Tswett.

Mixtures of solutes dissolved in a common solvent are separated from one another by a differential distribution of the solutes between two phases.

CHROMATOGRAPHY

Two phases in chromatography are: mobile phase is part of the

chromatographic system which carries the solutes through the stationary phase.

stationary phase is the part which the mobile phase flows where the distribution of the solutes between the phases occurs.

PRINCIPLE

Fractionalism of mixtures of substances

In the operation of the chromatogram, a mobile gaseous or liquid phase is use to wash the substances to be separated through a column of a porous material.

PRINCIPLE

Capillary Action – the movement of liquid within the spaces of a material due to the forces of adhesion, cohesion, and surface tension.

PRINCIPLE

The rate of migration of the solute depends upon the rate of interaction of the solute with the two phases, one being the mobile phases and the other stationary phase as the compounds travel through the supporting medium.

PRINCIPLE

The rate of migration of the solute depends upon the rate of interaction of the solute with the two phases, one being the mobile phases and the other stationary phase as the compounds travel through the supporting medium.

• Based on the interactions of solutes with mobile and stationary phases.

MECHANISMS OF SEPARATION IN CHROMATOGRAPHY

ADSORPTION (LIQUID-SOLID) CHROMATOGRAPHY

Based on the competition between the sample and the mobile phase for binding sites of the solid (stationary) phase. Molecules that are soluble in the mobile phase move fastest.

ADSORPTION (LIQUID-SOLID) CHROMATOGRAPHY

Advantages An extensive separation

literature is available on thin layer chromatography methods that are readily transferable to adsorption

The flexibility, speed, and low cost of TLC allow its use in experimental development.

ADSORPTION (LIQUID-SOLID) CHROMATOGRAPHY

Advantages TLC has great value for use

in the preliminary investigation of samples of unknown constituents

Adsorption chromatography, particularly with silica gel, has been widely used for the separation of drugs in both the HPLC and TLC modes.

PARTITION (LIQUID-LIQUID) CHROMATOGRAPHY

separates molecules on the basis of sample volatility.

Depends on the solubility of the solute in nonpolar (organic) or polar (aqueous) solvents

PARTITION (LIQUID-LIQUID) CHROMATOGRAPHY

Advantage: the stationary phase does not leave the solid support and bleed into the detector, and a uniform monomolecular layer of the stationary phase is obtained through the bonding procedure.

Since the chemical influence of the solid support may be largely ignored, the adhering film behaves essentially like a liquid stationary phase.

PARTITION (LIQUID-LIQUID) CHROMATOGRAPHY

ION-EXCHANGE CHROMATOGRAPHY

∞ Based on the net charge of molecules

∞ It is one of the common types of separation mechanism which depends on the nature of the stationary phase.

∞ It has 2 prinicipal types of ion- exchanger is cationic and anionic.

ION-EXCHANGE CHROMATOGRAPHY

∞ Ion exchange matrices can be further categorized as either strong or weak.

∞ It separates amino acid by electric charges based on their respective changes

ION-EXCHANGE CHROMATOGRAPHY

∞ Usually performed in columns

∞ Uses a charged stationary phase to separate charged compounds including anions, cations, amino acids, peptides and proteins.

ION-EXCHANGE CHROMATOGRAPHY

∞PRINCIPLE:∞ Relies on charge

to charge interactions between proteins in the sample and the charges immobilized on the resin.

ION-EXCHANGE CHROMATOGRAPHY

∞PRINCIPLE:∞ Once the solutes are bound,

the column is washed to equilibrate it in the starting buffer,which should be of low ionic strength

∞ Then the bound molecules are eluted off using a gradient be of low ionic strength

ION-EXCHANGE CHROMATOGRAPHY

∞ TWO PRINCIPAL TYPES:

∞ Anion exchange∞ Cation exchange

ION-EXCHANGE CHROMATOGRAPHY

Factors to be considered in Ion Exchange Chromatography:

⓭ Buffers – use anionic buffers for cation exchange and cationic buffers for anion

exchange to avoid difficulty.⓭ pH- influence the charge on the

macromoloecules in solution.

ION-EXCHANGE CHROMATOGRAPHY

Factors to be considered in Ion Exchange Chromatography:

⓭ Salts to use for elutionIons of the eluting salt must displace

other molecules from the charged groups on the stationary phase with either a gradient or step in the 0 to 1.0 range.

ION-EXCHANGE CHROMATOGRAPHY

ION-EXCHANGE CHROMATOGRAPHY

Most molecules have a net charge within a pH range of 2 to 10. When the pH is altered, the net charge on molecules can change drastically.

In this experiment, a mixture of two chemicals is absorbed onto a solid support ion-exchange column and separated during elution under conditions that influence their net charge.

ION-EXCHANGE CHROMATOGRAPHY

InstrumentsEluent GeneratorWater Separation Column

Sample InjectorElectrolytic Eluent Suppressor

DetectorComputer

ION-EXCHANGE CHROMATOGRAPHY

APPLICATIONS:SOFTENING OF WATERDEMINERALIZATION OF WATERPURIFICATION OF SOLUTIONS FREE

FROM IONIC IMPURITIESSEPARATION OF INORGANIC IONSSEPARATION OF SUGARS,AMINO

ACIDS

ION-EXCHANGE CHROMATOGRAPHYADVANTAGES: DISADVANTAGES: Long Life of Resins Cheap maintenance Environmental friendly

because it deals only with substances occurring in water.

Nature and properties of ion exchange resins

Nature of exchanging ions

There are substances (such as organic matter or Fe3+ occurring in some water which can foul the resin.

ION-EXCHANGE CHROMATOGRAPHY

FACTORS AFFECTING THE INSTRUMENTALIZATION1. Column Packing2. Detectors3. Flow Rate4. Sample Size5. Solvent and Temperature

Applications:

Serves as liquid chromatography detectors and as quality control monitors in drug manufactures

Also occurs in air and water quality, medical and clinical laboratories and industrial laboratories

TYPES OF CHROMATOGRAPHY

A.By Chromatographic bed shape

B. By Physical State of Mobile Phase

A. Column ChromatographyBy chromatographic bed shape

• A separation technique in which the stationary bed is within a tube• It works on a much larger scale by packing the same materials into a vertical glass column.

A. Column ChromatographyBy chromatographic bed shape

B. Plane ChromatographyBy chromatographic bed shape

• A separation that takes place on a flat surface or a plane

Example:• Paper Chromatography• Thin Layer Chromatography

Paper chromatography  Based on nature of

solvent, solubility of solute and rate of diffusion.

Uses paper as the stationary phase and a solvent as the mobile phase.

Paper chromatography  Solvent moves

through the paper by a capillary action

Separation depends on the solubility of solute and solvents, the polarity of solvent, and polarity of solutes in the sample.

Paper chromatography  Visualization of the

separated sample occurs by chemical reaction, which produces a color change.

Paper chromatography  Considered to be the

simplest and the most widely used of the chromatographic techniques because its APPLICABILITY TO THE FOLLOWING:

ISOLATION IDENTIFICATION AND QUANTITATIVE

DETERMINATION OF ORGANIC AND INORGANIC COMPOUNDS

Instrumentationof Paper chromatography

1) Lid2) Paper3) Solvent Front4) Solvent

THIN-LAYER CHROMATOGRAPHY

Used as a semi-quantitative screening test screening test

Uses as thin layer of silica gel, alumina gel, polyacrylamide gel, or starch gel attached to glass plate as stationary phase and the mobile phase is liquid solvent.

THIN-LAYER CHROMATOGRAPHY

Fractions in the sample are generally quite soluble in the solvent and move with it up the stationary phase by capillary action.

Separated fractions are also developed in TLC by applying a chemical reaction with the separated fractions to produce color changes

THIN-LAYER CHROMATOGRAPHY Sample movement is compared with the

standard, and fractions are calculated using retention factor (Rf), which is unique for special compounds

THIN-LAYER CHROMATOGRAPHY

ADVANTAGE: DISADVANTAGE: Simple and

economical Easy to perform since

it only involves spotting the stationary phase with the sample & placing one edge of the stationary phase plate in the mobile phase reservoir.

Spots are often faint

TLC is difficult to reproduce

Not typically automated

INSTRUMENTATION

• It can separate nanograms or pictograms of volatile substances.

• It is principally a method for the separation and quantitative determination of gases and volatile liquids and substances.

By Physical State of Mobile Phase

Gas Chromatography

Gas Chromatography

Volatile compounds can be separated in a gas chromatograph, in which the mobile phase is usually a relatively unreactive carrier gas such as helium, nitrogen or hydrogen.

Separations can be carried out in the vapor phase, most parts of a gas chromatograph are temperature controlled; selection of temperature is based on the composition of the sample.

Gas Chromatography

It uses a special detector according to the different kinds of compound and the most widely used are:A. Mass spectrophotometerB. Thermal ConductivityC. Flame Ionization DetectorD. Electron Capture Detector

GAS CHROMATOGRAPHY

Applications: Most effectively used for analyses of

organic compounds, space related, complex mixtures of volatile substances at column temperature of less than -40 °C to greater than 550° C.

Geochemical research projects such as determination of various environmental pollutants at extremely low concentrations.

GAS CHROMATOGRAPHY

ADVANTAGES: DISADVANTAGES: Ability to provide

qualitative information and quantitative information

FAST ANALYSIS Efficient, providing

high resolution Sensitive Nondestructive Requires small

samples Inexpensive

LIMITED to volatile samples

Not suitable for thermally labile samples

Fairly difficult for large preparative samples

Requires spectroscopy usually mass spectroscopy for confirmation of peak identity

COMPONENTS Autosampler- provides the means to

introduce a sample automatically into the inlets. Automatic insertion provides better reproducibility and time-optimization.

Column inlet (or injector)- provides the means to introduce a sample into a continuous flow of carrier gas. The inlet is a piece of hardware attached to the column head.

COMPONENTS

Carrier Gas (mobile phase) - must be chemically inert, include helium, hydrogen and nitrogen. It should be of high purity, and the flow must be tightly controlled to ensure optimum column efficiency and reproducibility of test results.

Detector

GAS CHROMATOGRAPHYGAS-LIQUID

CHROMATOGRAPHYGAS-SOLID

CHROMATOGRAPHY Separates molecules

on the basis of sample volatility

Mobile phase is a gas such as helium and the stationary phase is a high boiling point liquid absorbed onto a solid.

Uses a solid material as an absorbent

Based upon a solid stationary phase on which retention of analytes is the consequence of physical adsorption

Relies upon a large granular surface to aid in the separation of the substances.

GAS CHROMATOGRAPHY

Interferences Volatility of compound Polarity of compounds Column temperature Column packing polarity Flow rate of the gas Length of the column

Application

Use in biomedical research, routine clinical determination and drug researching programs

LIQUID CHROMATOGRAPHY

The mobile phase is percolated through the column by means of either gravity , under pressure generated by a suitable pump or centrifugal force.

SIZE EXCLUSION CHROMATOGRAPHY

Particles of different size will elute (filter) through a stationary phase at different rates. This results in the separation of a solution of particles based on size. Provided, that all the particles are loaded simultaneously or near – simultaneously of the same size should elute together.

SIZE EXCLUSION CHROMATOGRAPHY The support material has certain

range of pore sizes. As solutes travel through, the small molecules can enter the pores, whereas the larger ones cannot and will elute first from column.

The determination of molecular weight, e.g. , of enzymes, and estimation of equilibrium constants can be achieved with relative ease

SIZE EXCLUSION CHROMATOGRAPHY

ADVANTAGES DISADVANTAGES RAPID ROUTINE ANALYSIS IDENTIFYING HIGH MASS

COMPONENTS EVEN IN LOW CONCENTRATION

CAN ANALYZE POLYDISPERSED SAMPLES,BRANCHING STUDIES CAN BE DONE, ABSOLUTE MOLECULAR WEIGHTS CAN BE OBTAINED.

FILTRATIONS MUST BE PERFORMED BEFORE USING THE INSTRUMENT BAD RESPONSE FOR VERY SMALL MOLECULAR WEIGHTS

STANDARDS ARE NEEDED SENSITIVE FOR FLOW RATE

VARIATION. INTERNAL STANDARD SHOULD BE USED WHENEVER POSSIBLE

HIGH INVESTMENT COST

SIZE EXCLUSION CHROMATOGRAPHY

InstrumentsEluentDegasserPumpAuto InjectorSize Exclusion Column

Computer

Instrumentation

High-performance liquid chromatography (HPLC) Uses a pressure for the pumping of

aqueous or organic solution through a column.

The mobile phase is forced under pressure through a long, narrow column, yielding an excellent separation in a relatively short time.

Highly sensitive and specific.

LIQUID CHROMATOGRAPHY

High-performance liquid chromatography (HPLC) Become the primary means of

monitoring the use of drugs and of detecting drug abuse.

Also used to separate the compounds contributing to the fragrance of the flowers.

LIQUID CHROMATOGRAPHY

LIQUID CHROMATOGRAPHY

ADVANTAGES: DISADVANTAGES:An automated process that

takes only a few minutes to produce results.

Uses gravity instead of high speed pump to force compounds through the densely packed tubing.

Results are of high resolution and are easy to read.

Can be reproduce easily via automated process.

Difficult to detect coelution, which may lead to inacurrate compound categorization.

High cost for equipment needed to conduct HPLC.

Operation is complex, requiring a trained technician to operate.

Equipment has low sensitivity to some compounds because of the speed of the process.

High-Performance Liquid Chromatography

Application

Use in biomedical research, routine clinical determination and drug researching programs

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Instruments Fraction Collector Auto Sampler Pumping systems Columns & Packing Detectors Control Data & Processing

Instrumentation

Application

Use in monitoring the use of therapeutic drugs and detecting drug abuse.

also use to separate compounds contributing to the fragrance of flowers