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Guide to the Requirements of an HPLC Method
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Overview of an HPLC Method and the choices to be made

In order to use an HPLC System to carry out analysis, you will need an HPLC Method. This will include the following:

Mobile Phase:

Which solvents are required and in what ratio, e.g. it could be methanol and water in the ratio 35:65. Occasionally it may be just pure solvent, more commonly it is a binary mixture (as above) and it can include a third or fourth solvent, a buffer salt (check the concentration and pH) or an ion-pair reagent.

Isocratic means the same solvent is pumped continuously and an isocratic pump will suffice. Gradient means a composition of the mobile phase changes during the run and a gradient pump is required. For a gradient method, you will need a gradient profile (i.e. what changes and over what time period). Temperature:

If the separation should be carried out at elevated or reduced temperature, a column oven or chiller is required. If you do not use the temperature specified in a method, at best the retention times will be different, and at worst, resolution of the sample components will not be achieved.

Detector Type and Settings:

UV-Vis: What wavelength is to be used. If in the visible region (400-800nm) check that the detector has a tungsten lamp or one is available. Wavelength changes may sometimes need to be programmed into a run, and if so, a programmable detector is required. Detection at more than one wavelength may be required to detect all the components.

Refractive Index (RI): No settings are required. Just flush and go!

Electrochemical: Electrode type and Applied potential. too low and you detect nothing, too high and you oxidise everything in sight and get horrendous noise.

Fluorescence: Emission and excitation wavelengths. Any derivatisation required?

Conductivity: Anions or cations, with or without chemical suppression (dual column technique).

Injection Volume:

For a fixed volume autosampler or manual valve, this is set by changing the loop. For a variable volume autosampler, it is set in the autosampler method.

Column Type:

Column packing material, particle size, length, diameter and manufacturer are all important. More than one column type may work, but an HPLC method must specify a column.

Flow rate:

Usually around 1 ml/minute for an analytical column, slower if using polymer based columns.

Run time:

Usually from 2-60 minutes. This is a little longer than it takes for the last peak to elute (note that this is the last peak, not the last peak of interest!). For a gradient run you have to allow for the system to return to the starting conditions after the run is over and then re-equilibrate before the next injection can be made.

Data System Settings:

Data collection. Analogue or digital, input voltage (usually 1 volt unranged), sampling frequency (usually 1-10Hz).

Integration. Integration parameters such as peak width and peak threshold will have to be set, along with other settings appropriate to your samples.

Quantitation: A peak table will have to be set up, where the peaks are listed by their retention times and assigned names and peak time windows so they can be identified by the system.

Calibration. Once you have run calibration standards at appropriate levels, a calibration table is set up for each peak. You will need to select a best fit curve (or straight line!) that will be used by the system to convert peak areas into amounts for each peak

 
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