How to Calibrate an HPLC

HPLC is a high-performance liquid chromatography test that forces solvents through a column under high pressures of up to 400 atmospheres. The high pressure makes this chromatography test perform quicker than other chromatography tests with detection methods for HPLC automated and sensitive. In order to perform HPLC, a series of calibration standards containing the specific compounds in which you are testing need to be prepared using a calibration curve.

Things You'll Need

• HPLC column
• Samples
• HPLC water
• Standards for samples
• Volumetric flasks
• Microsoft Word or Excel

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Instructions

• 1

  Prepare a series of dilutions to prepare calibration standards containing the compounds that are to be analyzed. Make the standards range from 20 micrograms per milliliter to 100 micrograms per milliliter using volumetric flasks. Use HPLC grade water for your dilutions. If you are analyzing caffeine, for instance, you want to obtain a standard for caffeine and dilute that to create a standard curve.

• 2

  Prepare your sample that you want to analyze in the HPLC. For instance, if you want to analyze caffeine, you can choose tea or coffee.

• 3

  Dilute the sample with HPLC grade water so that it is in the range of your samples.

• 4

  Inject the samples into the HPLC column. Follow the specific instructions provided by the manufacturer for your particular column.

• 5

  Make a graph of the data obtained for your standard solutions with peak area versus the sample concentration.

• 6

  Determine the concentration of each component present in your sample from the standard curve you created.