Question

Thoroughly explain the separation principles of GC and HPLC

Answer 1

‘Chromatography’ is an analytical technique commonly used for separating a mixture of chemical substances into its individual components so that the individual components can be thoroughly analyzed.

common terms and their definitions used in chromatography

Mobile phase or carrier   solvent moving through the column

Stationary phase or adsorbent   substance that stays fixed inside the column

Eluent   fluid entering the column

Eluate   fluid exiting the column (that is collected in flasks)

Elution      the process of washing out a compound through a column using a suitable solvent

Analyte   mixture whose individual components have to be separated and analyzed

there are around 12 types of chromatography and GC and HPLC are one of them

Gas Chromatography

It is a type of chromatography in which the mobile phase is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen, and the stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside glass or metal tubing, called a column. The instrument used to perform gas chromato­graphic separations is called a gas chromatograph

A gas chromatograph is a chemical analysis instrument for separating chemicals in a complex sample. A gas chromatograph uses a flow-through narrow tube known as the column, through which differ­ent chemical constituents of a sam­ple pass in a gas stream (carrier gas, mobile phase) at different rates depending on their vari­ous chemical and physical properties and their inter­action with a specific column filling, called the station­ary phase.

As the chemicals exit the end of the col­umn, they are detected and identified electronically. The function of the stationary phase in the column is to separate different components, causing each one to exit the column at a different time (retention time). Other parameters that can be used to alter the order or time of retention are the carrier gas flow rate and the temperature.

In a GC analysis, a known volume of gaseous or liquid analyte is injected into the “entrance” (head) of the column, usually using a micro syringe (or, solid phase micro-extraction fibers, or a gas source switching system). As the carrier gas sweeps the analyte molecules through the column, this motion is inhibited by the adsorption of the analyte molecules either onto the column walls or onto packing materials in the column.

The rate at which the molecules progress along the column depends on the strength of adsorption, which in turn depends on the type of molecule and on the stationary phase materials. Since each type of molecule has a different rate of progression, the various components of the analyte mixture are separated as they progress along the column and reach the end of the column at different times (retention time).

A detector is used to monitor the outlet stream from the column; thus, the time at which each component reaches the outlet and the amount of that component can be determined. Generally, substances are identified (qualitatively) by the order in which they emerge (elute) from the column and by the retention time of the analyte in the column.

High-Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry. It is also sometimes referred to as high-pressure liquid chromatography. HPLC is used to separate components of a mixture by using a variety of chemical interactions between the substance being analyzed (analyte) and the chromatography column

In isocratic HPLC, the analyte is forced through a column of the stationary phase (usually a tube packed with small round particles with a certain surface chemistry) by pumping a liquid (mobile phase) at high pressure through the column. The sample to be analyzed is introduced in a small volume to the stream of mobile phase and is re­tarded by specific chemical or physical interactions with the stationary phase as it traverses the length of the column.

The amount of retardation de­pends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time and is considered a reasonably unique identifying character­istic of a given analyte. The use of pressure increases the linear velocity (speed) giving the compo­nents less time to diffuse within the column, leading to improved resolution in the resulting chromatogram.

Common solvents used include any miscible combinations of water or various organic liquids (the most common are methanol and acetonitrile). Water may contain buffers or salts to assist in the separation of the analyte components, or compounds such as Trifluoroacetic acid which acts as an ion pairing agent.

A further refinement to HPLC has been to vary the mobile phase composition during the analysis; this is known as gradient elution. A normal gradient for reverse phase chromatography might start at 5% methanol and progress linearly to 50% methanol over 25 minutes, depending on how hydrophobic the analyte is.

The gradient separates the analyte mixtures as a function of the affinity of the analyte for the current mobile phase composition relative to the stationary phase. This partitioning process is similar to that which occurs during a liquid-liquid extraction but is continuous, not step-wise. In this example, using a water/methanol gradient, the more hydrophobic components will elute (come off the column) under conditions of relatively high methanol; whereas the more hydrophilic compounds will elute under conditions of relatively low methanol.

The choice of solvents, additives and gradient, depends on the nature of the stationary phase and the analyte. Often a series of tests are performed on the analyte and a number of generic runs may be proc­essed in order to find the optimum HPLC method for the analyte — the method which gives the best separation of peaks

types of HPLC

1. Normal phase chromatography

2. Reversed phase chromatography

3. Size exclusion chromatography

4. Ion exchange chromatography

5. Bio-affinity chromatography