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Study Notes: About the HPLC

The theory behind the operation of the High Performance Liquid Chromatograph (HPLC) is similar to that of thin layer chromatography except that this time the stationary phase is packed into a steel or plastic column and the mobile phase (solvent) is moved through the stationary phase under high pressure. The HPLC is very sensitive and will detect very low levels of some compounds.

How Compounds are Separated
The HPLC allows extremely fine adjustments to be made to the mobile phase using dual pumps connected to two different solvents. A ratio in the composition of the mobile phase from:

100% solvent A:   0% solvent B to
0% solvent A:100% solvent B

can be achieved over a HPLC run - the ratio does not need to be linear but can be hyperbolic, stepped or held at various levels for a plateau effect. This technique is called gradient elution and is used to control when chemical compounds in the sample being analysed actually elute (move) off the column.

There are many different elution profiles with the most common being: isocratic (steady state), linear, stepped, convex, concave, and combinations - called complex. Examples of various profiles are illustrated in the following. Remember that you are looking at the way the concentration of solvent B varies over the HPLC run - this is not a trace of the chemical compounds in a particular sample.

Graphs of elution profiles

Start of gradient: 0% solvent B = 100% solvent A
End of gradient: 100% solvent B = 0% solvent A

As you increase the amount of solvent B different molecules of the chemical compounds in the sample elute off the column (apart from the isocratic conditions where solvent B is static).

Gradient elution relies on the relative affinities of the chemical compounds in the sample for the material in the column. This is NOT a solubility effect but rather the effect that at 0% solvent B the chemical compounds attach strongly to the column material and at 100% solvent B none of the compounds in the particular sample will be able to attach to the column material.

Different compounds have different affinities for the column material and hence will elute at different concentrations of solvent B. This is the essential process of the separation by HPLC.

Gradient elution is often developed to suit the particular sample so that each of the different compounds elute in a distinct and well-separated peak. Hence there are a large number of different elution combinations especially when stepped or complex elution is used.

Using the HPLC
While the mobile phase is pumping through the HPLC a quantity of sample is injected into the column using a special injection port (all piping and the column are under high pressure!). From here the sample goes through two short columns (guard column and pre-column) to clean it up prior to reaching the main column where separation occurs.

High Performance Liquid Chromatograph (HPLC)

There are many types of stationary phase materials that can be used in HPLC columns. One type commonly chosen for samples containing proteins (including enzymes) is called C-18.

The C-18 material is very sensitive to particular chemical species that may irreversibly attach to the C-18 and 'poison' the column. Particle contamination of the sample to be tested in the HPLC is also a problem as particles may cause the column to block up, resulting in high pressures and poor performance.

The chemical components eluting from the column are detected using a UV-Vis detector and a trace of the run is automatically generated using a chart recorder. The trace shows a series of peaks, each corresponding to the various compounds that have been separated out of the sample.

The wavelength of the detector must be set correctly prior to the commencement of a run and depends on the sample type (the wavelength is set so that the separated compounds absorb light, which enables them to be detected).

The UV-Vis detector converts the signal from the sample peak into electrical activity that is translated by the chart printer as peak height and width. The paper in the chart recorder runs through the recorder at a specified speed and the ink pen moves to trace out each peak as it comes off the column.

The final result is a trace of the entire sample comprised of a number of peaks of varying peak height and width. This trace is often very consistent for a particular sample type and will act as a characteristic 'signature' under the conditions employed for the HPLC run. Any extra peaks in the signature will immediately raise suspicion of some kind of contamination whether that is bacterial overgrowth, herbicides, pesticides or other contamination.

Sample trace


After passing through the detector the mobile phase and separated sample compounds are run into a waste container for collection.

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