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What Is an Internal Standard?

By Paul Reed
Updated May 23, 2024
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An internal standard is a known amount of chemical added to an analytical sample, which allows technicians to calibrate laboratory instruments. This also allows for the calculation of amounts of unknown chemicals, because the instrument will show the amount of the known standard as a reference. Tests that can benefit from an internal standard include gas and liquid chromatography.

Gas chromatography passes a vaporized sample through a column packed with a solid, very pure form of soil. The sample is moved through the column with an inert, or non-reactive, gas such as nitrogen or argon. Different molecules absorb on the column and then are released based on their molecular structure. The gas then carries the separated sample to a flame to create ions, or molecules with small electrical charges. A detector determines the amount of each ion and plots the results on a chart.

Liquid chromatography also separates samples into different molecules, but uses a liquid solvent to move the sample by gravity or pressure across a stationary material. A chromatography bed material may be a resin, diatomaceous earth, or even another liquid. The sample is loaded at the top, and solvent is added to push the sample down through the column material.

Parts of the sample will pass through the column at different rates based on molecular or ionic characteristics. Small samples of liquid, called aliquots, are taken at regular intervals from the bottom of the column. These samples will contain the different molecules that have separated in the column, and are analyzed to determine their composition.

In both types of chromatography, many variables can affect results. The flow rate of the carrier gas can change slightly, or columns may be at different temperatures for different samples. Adding a calibrated internal standard provides a known result for any sample placed in the test instrument. If some variable in the instrument, such as gas flow rate, changes by a small percentage, the standard will change by the same amount.

Once the instrument has been calibrated by running a sample with the internal standard, an unknown chemical sample can be run through the same instrument, with the standard included. The results will show the chemical makeup of the unknown, plus the internal standard, plotted on the graph created by the instrument. Knowing the amount of internal standard, the relative amount of unknown chemical can be determined, which will allow a calculation of the concentration.

It is important to note that an internal standard is not used to chemically react with any sample. Rather, it is used to compare molecules of any unknown material with the known amount of the standard. This technique is known as setting one variable, because the known standard will always remain the same from sample to sample, regardless of other conditions that may change.

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