How to Use HPLC?
High performance liquid chromatography (HPLC) is a widely used lab technique that allows the analysis of test samples to determine the presence of one or more specific chemicals (the "analytes") as well as the concentrations of those analytes. An HPLC instrument consists of solvents in reservoirs (the "mobile phase"), an injection port, a solvent pump, a column filled with a porous separating agent, and a detector, all connected by tubing. Samples are injected into a stream of solvent being pumped through the column. While passing through the column, the analytes separate from one another. When they exit the column, a detector produces a signal proportional to the amount of each analyte.
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Things You'll Need
- High performance liquid chromatograph instrumentation with data acquisition computer
- Solvents
- Standard sample of analyte chemical
- Sample to be analyzed
- Volumetric flasks
- Pipettes
- Weigh scale
Instructions
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Setting Instrument Parameters
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1
Determine the HPLC parameters that you are going to use to analyze your sample. You will need to know the type of mobile phase solvent, flow rate, type of column packing, type of detector and detector setting. These can often be found in the form of an official test method. For example, Environmental Protection Agency method 8316 gives detailed instructions for analyzing samples for acrylamide compounds using HPLC. If the analysis you are doing does not have an existing method, you will have to develop your parameters by trial and error. Common mobile phase solvents that can be used are acetonitrile, methanol, tetrahydrofuran and water, or a mixture of any of these. A good all-purpose column packing is C18 (octadecyl silica gel). Flow rates are normally in the range of one to two ml per minute. Ultraviolet detectors absorbing at 254 nanometers are very common.
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2
Connect the instrument parts together, power up the various components and start the solvent pump. Prepare a series of 5 to 10 calibration standards by portioning out -- via pipette or a weigh scale -- increasing quantities of the chemical you wish to test. Dissolve these portions in solvent, using volumetric flasks; it normally works well to use the same solvent as your mobile phase. The concentration of your analyte in these standards is typically at the high parts-per-million level.
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3
Inject the calibration standards onto the HPLC for analysis. The HPLC detector will output a real-time response plot on the associated computer; when the analyte exits the column, the response will produce a peak shape. The area of this peak will be proportional to the amount of the analyte and the time taken for the peak to appear (the elution time). It will be unique to that analyte.
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4
Plot the peak area for each calibration standard against the concentration of the analyte in that standard to obtain a linear calibration equation of the form peak area = (M)(concentration) + B where M and B will be numerical values. Use data acquisition software.
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5
Dissolve a known quantity of the sample to be tested in the same solvent as used for the calibration standards. Inject this onto the HPLC instrument and observe the detector response plot. If a peak appears at the same approximate elution time as the calibration standard peak, you can reasonably conclude that your sample contains the analyte.
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6
Record the peak area for the analyte in your sample. Using the calibration equation, find the analyte concentration in your sample.
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Tips & Warnings
An HPLC method can typically analyze for multiple analytes in a single run, if necessary.
It is possible for more than one chemical to have the same elution time, so false positives can happen.
The analyte concentration range of the calibration standards should always exceed the highest analyte concentration expected to be found in samples.
A column oven can be added for increased reproducability.
An autosampler can be added to make automatic injections onto the HPLC.
Use caution when working with mobile phase solvents. Most common solvents, such as methanol or acetonitrile, are toxic by ingestion and inhalation.
References
Resources
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