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Internal Standards #1: How Does It Work?

In this first in the series of blogs about internal standards, we look at how they work in HPLC and LC-MS applications.
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Written byJohn Dolan
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In this technical article you'll learn how an internal standard is used in quantitative analysis.

An internal standard is used to aid quantification of an analyte, especially when volumetric loss of the sample is probable, as with a multi-step sample preparation scheme. In concept it is quite simple: you add a constant amount of the internal standard to every sample, and instead of keeping track of absolute peak area or height for calibration purposes, you use the ratio of the area or height of the analyte and internal standard. For example, consider the blue chromatogram inset in the figure. The normal sample is shown as the peaks comprising lines. To this sample, a known amount of another compound (represented by the solid peak) is added as an internal standard. The same process is used to prepare the calibration samples: calibrators at different concentrations each have the same amount of internal standard added to them. When the chromatogram is integrated, the peak areas of the analyte and the internal standard are measured. The ratio of these areas is then used in the calibration process.

By reading the full article you will learn how internal standards can be used to help correct for volumetric recovery errors in sample preparation.

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Meet the Author(s):

  • John Dolan

    John Dolan is considered to be one of the world’s top experts in HPLC. He has written more than 300 user-oriented articles on HPLC troubleshooting over the last 30 years, in addition to more than 100 peer-reviewed technical articles on HPLC and related techniques. His three books (co-authored with Lloyd Snyder), Troubleshooting HPLC Systems, Introduction to Modern Liquid Chromatography (3rd edn), and High-Performance Gradient Elution, are standard references on thousands of desks around the world. He has taught HPLC training classes around the world to more than 10,000 students.

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