AN ASSIGNMENT ON

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

SUBMITTED BY AHANA.P-B080460CH ALEN ALEX NINAN-B080567CH AMITHA ASOKKUMAR-B080770CH ANURAG KUMAR SHASTRI-B080777CH APARNA M-B080541CH

INTRODUCTION
High-performance liquid chromatography (or high-pressure liquid chromatography, HPLC) is a chromatographic technique that can separate a mixture of compounds, and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture. HPLC utilizes different types of stationary phase (typically, hydrophobic saturated carbon chains), a pump that moves the mobile phase(s) and analyte through the column, and a detector that provides a characteristic retention time for the analyte. The detector may also provide other characteristic information (i.e. UV/Vis spectroscopic data for analyte if so equipped). High performance liquid chromatography is basically a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster. It also allows you to use a very much smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture. The other major improvement over column chromatography concerns the detection methods which can be used. These methods are highly automated and extremely sensitive.

LOOKING AT THE WHOLE PROCESS

INSTRUMENTATION:
Main components in an HPLC system include the solvent reservoir, or multiple reservoirs, a high-pressure pump, a column, injector system and the detector.

The reservoir holds the solvent, which is referred to as the mobile phase because it moves. There are usually a minimum of two reservoirs in a system, with each holding up to 1000 cc of solvent and usually fitted with a gas diffuser through which helium can be bubbled. A pump is used to generate a specified flow of the mobile phase. Although manual injection of samples is still possible, most HPLCs are now fully automated and controlled by computer. The injector, or auto sampler, introduces the solvent into a phase stream that carries the sample into the high pressure (up to 400 bar) column, which contains specific packing material needed to effect separation. The packing material is referred to as the stationary phase because it is held in place by the column hardware. A detector is needed to see the separated compound bands as they elute from the high pressure column. The information is sent from the detector to a computer which generates the chromatogram. The mobile phase exits the detector and is either sent to a waste, or collected, as desired. With HPLC, a high purity helium is to degasse the mobile phase. Helium as well as Nitrogen is also used as a Nebulization gas.

WORKING MECHANISM
Injection of the sample Injection of the sample is entirely automated, and you wouldn't be expected to know how this is done at this introductory level. Because of the pressures involved, it is not the same as in gas chromatography (if you have already studied that).

Retention time The time taken for a particular compound to travel through the column to the detector is known as its retention time. This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound. Different compounds have different retention times. For a particular compound, the retention time will vary depending on:

the pressure used (because that affects the flow rate of the solvent) the nature of the stationary phase (not only what material it is made of, but also particle size) the exact composition of the solvent the temperature of the column

That means that conditions have to be carefully controlled if you are using retention times as a way of identifying compounds.

The detector There are several ways of detecting when a substance has passed through the column. A common method which is easy to explain uses ultra-violet absorption. Many organic compounds absorb UV light of various wavelengths. If you have a beam of UV light shining through the stream of liquid coming out of the column, and a UV detector on the opposite side of the stream, you can get a direct reading of how much of the light is absorbed. The amount of light absorbed will depend on the amount of a particular compound that is passing through the beam at the time.

Different compounds absorb most strongly in different parts of the UV spectrum. Methanol, for example, absorbs at wavelengths below 205 nm, and water below 190 nm. If you were using a methanol-water mixture as the solvent, you would therefore have to use a wavelength greater than 205 nm to avoid false readings from the solvent.

TYPES OF DETECTORS

UV detector

The light source is a D2 lamp. This detector is used mainly to detect components having an absorption wavelength of 400 nm or less in the ultraviolet region. A D2 lamp and a W lamp are used as the light source. This detector is effective in the detection of coloring components such as dyes and stains because of coverage of the visible light region. Data on the spectrum from the ultraviolet to visible light range is also collected. Fluorescent substances can be detected specifically with high sensitivity. Change in the refractive index is detected. Components absorbing no ultraviolet light can also be detected despite low sensitivity. Mainly inorganic ions are detected by monitoring the conductivity.

UV-VIS detector

Diode array detector (DAD) Fluorescence (FL) detector Differential refractive index (RI) detector Conductivity detector

Coupling HPLC to a mass spectrometer When the detector is showing a peak, some of what is passing through the detector at that time can be diverted to a mass spectrometer. There it will give a fragmentation pattern which can be compared against a computer database of known patterns. That means that the identity of a huge range of compounds can be found without having to know their retention times.