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 HPLC :high performance liquid chromatography

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مُساهمةموضوع: HPLC :high performance liquid chromatography   السبت ديسمبر 05, 2009 6:16 pm

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY - HPLC


High performance liquid chromatography is a powerful tool inanalysis. This page looks at how it is carried out and shows how ituses the same principles as in thin layer chromatography and columnchromatography.

Carrying out HPLC

Introduction

Highperformance liquid chromatography is basically a highly improved formof column chromatography. Instead of a solvent being allowed to dripthrough a column under gravity, it is forced through under highpressures of up to 400 atmospheres. That makes it much faster.Italso allows you to use a very much smaller particle size for the columnpacking material which gives a much greater surface area forinteractions between the stationary phase and the molecules flowingpast it. This allows a much better separation of the components of themixture.Theother major improvement over column chromatography concerns thedetection methods which can be used. These
methods are highly automatedand extremely sensitive.

The column and the solvent

Confusingly, there are two variants in use in HPLC depending on the relative
polarity of the solvent and the stationary phase.

Normal phase HPLC

Thisis essentially just the same as you will already have read about inthin layer chromatography or column chromatography. Although it isdescribed as "normal", it isn't the most commonly used form of HPLC.Thecolumn is filled with tiny silica particles, and the solvent isnon-polar - hexane, for example. A typical column has an internaldiameter of 4.6 mm (and may be less than that), and a length of 150 to250 mm.Polarcompounds in the mixture being passed through the column will sticklonger to the polar silica than non-polar compounds will. The non-polarones will therefore pass more quickly through the column.

Reversed phase HPLC

Inthis case, the column size is the same, but the silica is modified tomake it non-polar by attaching long hydrocarbon chains to its surface -typically with either 8 or 18 carbon atoms in them. A polar solvent isused - for example, a mixture of water and an alcohol such as methanol.Inthis case, there will be a strong attraction between the polar solventand polar molecules in the mixture being passed through the column.There won't be as much attraction between the hydrocarbon chainsattached to the silica (the stationary phase) and the polar moleculesin the solution. Polar molecules in the mixture will therefore spendmost of their time moving with the solvent.Non-polarcompounds in the mixture will tend to form attractions with thehydrocarbon groups because of van der Waals dispersion forces. Theywill also be less soluble in the solvent because of the need to breakhydrogen bonds as they squeeze in between the water or methanolmolecules, for example. They therefore spend less time in solution inthe solvent and this will slow them down on their way through thecolumn.That means that now it is the polar molecules that will travel through the column more quickly.Reversed phase HPLC is the most commonly used form of HPLC.

Looking at the whole processA flow scheme for HPLC




Injection of the sample

Injection ofthe sample is entirely automated, and you wouldn't be expected to knowhow this is done at this introductory level. Because of the pressuresinvolved, 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 tothe point at which the display shows a maximum peak height for thatcompound.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 areseveral ways of detecting when a substance has passed through thecolumn. A common method which is easy to explain uses ultra-violetabsorption.Many organiccompounds absorb UV light of various wavelengths. If you have a beam ofUV 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 adirect 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.



You mightwonder why the solvents used don't absorb UV light. They do! Butdifferent compounds absorb most strongly in different parts of the UVspectrum.Methanol, forexample, absorbs at wavelengths below 205 nm, and water below 190 nm.If you were using a methanol-water mixture as the solvent, you wouldtherefore have to use a wavelength greater than 205 nm to avoid falsereadings from the solvent.

Interpreting the output from the detector

The outputwill be recorded as a series of peaks - each one representing acompound in the mixture passing through the detector and absorbing UVlight. As long as you were careful to control the conditions on thecolumn, you could use the retention times to help to identify thecompounds present - provided, of course, that you (or somebody else)had already measured them for pure samples of the various compoundsunder those identical conditions.But you canalso use the peaks as a way of measuring the quantities of thecompounds present. Let's suppose that you are interested in aparticular compound, X.If youinjected a solution containing a known amount of pure X into themachine, not only could you record its retention time, but you couldalso relate the amount of X to the peak that was formed.The areaunder the peak is proportional to the amount of X which has passed thedetector, and this area can be calculated automatically by the computerlinked to the display. The area it would measure is shown in green inthe (very simplified) diagram.



If thesolution of X was less concentrated, the area under the peak would beless - although the retention time will still be the same. For example:


This meansthat it is possible to calibrate the machine so that it can be used tofind how much of a substance is present - even in very small quantities.Be careful,though! If you had two different substances in the mixture (X and Y)could you say anything about their relative amounts? Not if you were using UV absorption as your detection method.




In thediagram, the area under the peak for Y is less than that for X. Thatmay be because there is less Y than X, but it could equally well bebecause Y absorbs UV light at the wavelength you are using less than Xdoes. There might be large quantities of Y present, but if it onlyabsorbed weakly, it would only give a small peak.

Coupling HPLC to a mass spectrometer

This iswhere it gets really clever! When the detector is showing a peak, someof what is passing through the detector at that time can be diverted toa mass spectrometer. There it will give a fragmentation pattern whichcan be compared against a computer database of known patterns. Thatmeans that the identity of a huge range of compounds can be foundwithout having to know their retention times.

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