Instrumentation of Column and Gas Chromatography

Contents• Column chromatography• Principle • Instrumentation• Applications • Procedure • Gas chromatography • Instrumentation• Application• references

Column chromatography

•Column chromatography is basically a type of adsorption chromatography techniques.

•Here the separation of components depends upon the extent of adsorption to stationary phase.

•Here the stationary phase is a solid material packed in a vertical column made of glass or metal.

Principle

•When a mixture of mobile phase and sample to be separated are introduced from top of the column, the individual components of mixture move with different rates.

• Those with lower affinity and adsorption to stationary phase move faster and eluted out first while those with greater adsorption affinity move or travel slower and get eluted out last.

Instrumentation

Column chromatography consists of •A packed, three-dimensional stationary

phase inside a glass, plastic, or metal column and can be used for both preparative and analytical purposes.

Procedure:• The stationary phase material is suitably

moistened with mobile phase and packed sufficiently in the column with a cotton or asbestos pad at the bottom.

• The extract material or sample to be separated is placed on the top of packed stationary phase with a second cotton or asbestos pad in between.

• The mobile phase is poured into the column over the sample. A collecting beaker is placed at the bottom of column near the end to collect the elute.

Classification

Column chromatography is therefore classified according to the type of fluid flow system used:

•Gravity chromatography•Low-pressure chromatography•Medium-pressure chromatography

(including fast protein liquid chromatography)

•High-pressure/high-performance liquid chromatography (HPLC)

Gravity Chromatography

•Gravity chromatography uses gravity to pass sample and buffers across the column resin.

•Small-volume columns (~1.5 ml) designed for quick flowthrough by spinning in a microcentrifuge provide a convenient method for rapidly purifying many small samples.

Applications:

•small-scale, rapid preparative chromatography of nucleic acids, peptides, and proteins.

Low-Pressure Chromatography

•Low-pressure chromatography systems are operated at less than 50 psi (0.35 MPa).

• These systems require a sample pump and are often equipped with fraction collectors, gradient capabilities, and detectors to monitor column elution.

Applications:

•Medium-scale preparative chromatography of native or recombinant proteins.

Medium-Pressure Chromatography•Medium-pressure chromatography is

conducted at operating pressures that are actually rather high, up to 3,500 psi (24 MPa).

• Medium-pressure chromatography systems often include additional capabilities such as column switching valves, advanced gradient capabilities, and multi-wavelength detectors.

Applications:

• Preparative and analytical chromatography of a wide variety of molecules, ranging from nonvolatile organics to nucleic acids, peptides, and proteins.

High-Pressure Liquid Chromatography (HPLC)•Conducted at very high pressures — up to

5,000 psi (34 MPa). •HPLC is a powerful analytical tool

providing high resolution and sensitivity, with the ability to detect concentrations down to parts per trillion while having very small sample requirements.

Instrumentation• Solvent Reservoir : Solvent must be deaerated

prior to use either by boiling or by applying a partial vacuum to the solvent reservoir.

• Pump : The pump used in HPLC is pulseless (to avoid pressure changes) with adjustable flow-rate. They may pump solvents from more than one reservoir.

• Pre-column : It contains same stationary phase as that in separating column. It is used to remove any impurities present in the mobile phase, which may contaminate stationary phase in separating column.

• Injector : The sample to be separated is introduced into injector by the help of a syringe. It contains a valve which in one position allows mobile phase to directly enter the separating column. While, in the other position, it passes mobile phase through a loop containing sample mixture. Mobile phase flushes sample mixture into separating column.

•Separating Column : It is made up of stainless steel with length ranging from 10-100 cm and diameter ranging from 2-6 mm. Micro bore columns, often glass-lined, with diameter of 1 mm or less and length of 25 cm are also used. The column can be placed in a thermo stated oven.

•Detector : After exiting the column, the eluate enters a flow-through detector, where it is continuously monitored.

•Amplifier : The electrical signal obtained from the detector is amplified and routed to recorder by amplifier.

•Recorder : The recorder records the developed chromatogram.

Applications:

•Preparative and analytical chromatography of a wide variety of molecules, ranging from nonvolatile organics to nucleic acids, peptides, and proteins.

•Bio-Rad does not manufacture HPLC systems, but we do carry a full selection of HPLC columns.

Precautions:

1. Keep the column in a clean and dust free place.

2. Do not disturb the column till the separation is complete.

3. Avoid gaps within the stationary phase packing.

Applications

•Column chromatography is best suited to separate active principle from plant materials.

• In separation of compounds after organic synthesis to obtain desired molecule.

•To separate or purify natural compound mixtures like alkaloids, glycosides.

instrumentation of Gas chromatography

Gas Chromatography

•In gas chromatography, the components of a sample are dissolved in a solvent and vaporized in order to separate the analytes by distributing the sample between two phases:

• a stationary phase and •a mobile phase.

• The mobile phase is a chemically inert gas that serves to carry the molecules of the analyte through the heated column.

• The stationary phase is either a solid adsorbant, termed gas-solid chromatography (GSC), or a liquid on an inert support, termed gas-liquid chromatography (GLC).

Instrumentation

Consist of •Sample Injection•Carrier Gas cylinder with pressure

regulator•Column Oven•Open Tubular Columns and Packed

Columns•Detection Systems

Sample Injection

•A sample port is necessary for introducing the sample at the head of the column.

•A calibrated microsyringe is used to deliver a sample volume in the range of a few microliters through a rubber septum and into the vaporization chamber.

Carrier Gas•The carrier gas plays an important

role, and varies in the GC used.• Carrier gas must be dry, free of oxygen

and chemically inert mobile-phase employed in gas chromatography.

•Helium is most commonly used because it is safer than, but comprable to hydrogen in efficiency, has a larger range of flow rates and is compatible with many detectors.

Gas cylinder with pressure regulator

•A pressure regulator is used to control the amount of gas to be passed to column.

Column Oven• The thermostated oven serves to control the temperature

of the column within a few tenths of a degree to conduct precise work. 

• The oven can be operated in two manners: • isothermal programming Or

temperature programming.  • In isothermal programming, the temperature of the

column is held constant throughout the entire separation. • The optimum column temperature for isothermal

operation is about the middle point of the boiling range of the sample. 

Open Tubular Columns and Packed Columns•Open tubular columns, which are also

known as capillary columns, come in two basic forms. 

• The first is a wall-coated open tubular (WCOT) column and

• The second type is a support-coated open tubular (SCOT) column. 

Detection Systems•The detector is the device located at the

end of the column which provides a quantitative measurement of the components of the mixture as they elute in combination with the carrier gas. 

Types of Gas Chromatography DetectorsNon-selective• Responds to all compounds present

in carrier gas stream except the carrier gas itself

Selective• Responds to range of compounds with a

common physical or chemical characteristic

Specific• Responds to a single specific compound

only

•Detectors can also be grouped into concentration or mass flow detectors

•Concentration Dependent•The response of such Gas

Chromatography detectors is proportional to the concentration of the solute in the detector such as TCD. Dilution of sample with makeup gas will lower detector response.

•Mass Flow Dependent•Signal is dependent on the rate at which

solute molecules enter the detector such as FID. Response of such detectors is not affected by makeup gas flow rate changes.

Desirable characteristics of detectors •Reproducible response to changes in

eluent composition in carrier gas stream• High sensitivity •  Large linear dynamic range •  Low noise •  Small volume to avoid peak broadening

and resultant loss of resolution •  Preferably non – destructive

Common Gas Chromatography detectors

Flame Ionization Detector (FID)

• Mass sensitive detector •  Response depends on

conducting power of ions or electrons produced on burning of organic compounds in the flame 

•  Selective detector but sample detected must be combustible 

•  Large linear dynamic range (107)

•  No response to inorganic and permanent gases such as CO, CO2, NH3, CS2, N2, etc.

• It is the most widely used detector in Gas Chromatography 

Thermal Conductivity Detector (TCD)• Non-destructive universal detector •  Response depends on the thermal

conductivity difference between the carrier gas and the eluted components 

•  Wide dynamic range (107 – % to ppm levels) 

•  Responds also to inorganic gases such as CO, CO2, NH3, CS2, N2, etc.

Advantages of TCD

•Sample is not wasted •Easy to operate

Applications • Applications• Gas chromatography is a physical separation method in

where volatile mixtures are separated. • It can be used in many different fields such as

pharmaceuticals, cosmetics and even environmental toxins. • Since the samples have to be volatile, human breathe,

blood, saliva and other secretions containing large amounts of organic volatiles can be easily analyzed using GC.

• Knowing the amount of which compound is in a given sample gives a huge advantage in studying the effects of human health and of the environment as well. 

• Air samples can be analyzed using GC.• GC/MS is also another useful method which can determine

the components of a given mixture using the retention times and the abundance of the samples.

REFERENCES• www.chemwiki.ucdavis.edu.com• www.lab-training.com• www.bio-rad.com• www.bheem.hubpages.com• Modern Practice of Gas Chromatography

edited by Robert L. Grob, PhD, Eugene F. Barry, PhD page no 37 

• Liquid Column Chromatography: A Survey of Modern Techniques and Applications

 edited by K. Macek, Z. Deyl, J. Janák page no 57