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HPLC

HPLC is simply liquid chromatography just like you might do it with a jam-jar and a piece of blotting paper, but done in a tube with a high-pressure pump. The resolution is therefore generally better than a jam-jar would provide.

We have two Waters alliance HPLC units, both equipped with diode array detectors, one ("Clyde") also with fluorescence detection, the other ("Bonnie") also with a light-scattering detector.

If these machines are unsuitable for a method, JIC as a whole has a variety of other HPLC-systems in various laboratories.

Stand-alone HPLC chromatography is a classic method that is likely to be with us for years to come. Chemicals are identified largely by retention time, which is very sensitive to minor changes in method. Combined with a mass-spec detector (LC-MS) you can be much more certain about what a peak is.

Absorbance remains a very good way to quantify a peak, provided it's pure. In fact, it is much easier to get accurate quantification from absorbance than it is from mass spectroscopy, because a certain concentration of "X" will generally have the same absorbance from one run to another. However, ionization in the mass spec is more sensitive to small changes in conditions, so mass spec signal may vary from run to run.

If you are developing an HPLC method and have any intention of moving on to do MS, please consult our hints on adaptation to LC-MS.

Mass spec detectors are likely to become increasingly standard on HPLC systems as MS technology improves. The following table sums up the advantages of different sorts of detection methods:

Absorbance	Easy to measure. Most HPLC systems now use diode array detectors, which measure a whole absorbance spectrum simultaneously. They separate out the light into a spectrum using a diffraction grating, and then monitor this with an array of light-sensitive diodes. But this spectrum contains little information (most chemicals have very broad absorbance peaks. That's why we get away with having eyes that operate like a diode-array detector with poor focussing and only 3 diodes!). Nevertheless, the optical spectrum can give you a clue as to whether your peak is pure. Many chemicals absorb only slightly in the visible and near UV ranges, and must be monitored at 250nm or less. This can begin to present problems as solvents may also absorb in this region.
Fluorescence	Much more sensitive than absorbance, and more specific (depends on both excitation wavelength and wavelength of re-emission of light). But not suitable for all chemicals. Especially popular for derivatised amino acids.
Light Scattering	Evaporative light scattering detectors dry off the solvent stream and look at the metabolites scattering light in what's left. This method is billed as the universal detection method since it doesn't rely on the metabolite absorbing at any particular wavelength; in fact, because it is a physical method of measuring things, it is very independant of metabolite chemistry.
Refractive Index	A method worth bearing in mind for things like saccharides and sugars, which absorb little, but refract a good bit. Sensitive, but expensive and specialist.
Mass Spec	Increasingly standard as an HPLC detector, and possibly the best for selectivity. You can enter the mass of your chemical of interest, and it will produce a chromatogram of that chemical. But it relies on the chemical ionizing.