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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