Internal Standards: Strategies From the Frontline

by | Oct 5, 2023 | Articles, Gas Chromatography, Liquid Chromatography

Explore strategies for choosing the ideal internal standard in chromatography techniques amidst changing regulations and rigorous detection limits.

Chromatography techniques, from GC-MS to HPLC, hinge significantly on the suitable choice of internal standards. But in an era of rapidly evolving regulation and increasingly rigorous detection limits, it can be challenging for workflows to keep pace. Engaging in connected communities such as Chromotography Forum (CF) offers a distinct advantage—a platform for collective discussion that leverages the shared insights and expertise of its user base.

Avoiding internal standard interference

When selecting an appropriate internal standard for chromatographic analyses, practitioners often grapple with finding a chemical compound that does not interfere with the peak of interest and does not interact with the testing medium. Ideally, the internal standard should elute near the analyte's time, maintain stability, exhibit good solubility in the working solvent, and be absent from the sample matrix.

Standard addition: An alternative approach?

In certain contexts, such as headspace (HS) analysis, the method of standard addition is sometimes favored over internal standards. This method involves spiking samples with different levels of a standard and demonstrating a consistent linear relationship in the recovery of the substance to its original amount before spiking. This strategy proves especially beneficial when dealing with a matrix that doesn't exactly match the actual sample.

Practical insights into standard addition

CF user Chromatographer1 elucidates the standard addition method with a hypothetical scenario involving naproxen and ethanol. By preparing solutions of ethanol at varying concentrations, adding them to naproxen, and plotting the resultant ethanol areas against the added amounts, the x-axis intercept of the plotted line indicates the ethanol amount in the unspiked solution. While detailed, this method offers a solid approach to identifying the presence of an analyte in a specific sample.

But why the insistence on a gas internal standard when a volatile liquid might suffice?  Discussions among analysts reveals that adherence to specific reference methods and the capabilities of existing lab equipment, like thermal desorption units, significantly influence this choice. The need to follow reference methods and avoid explaining deviations to accrediting bodies highlights the importance of selecting suitable gas phase internal standards.

Internal standards in HS-GC: A detailed methodology

Forum user Peter Apps provides a nuanced methodology for utilizing internal standards in HS-GC. The internal standard, which should be chemically akin to the analyte and separable from it and other volatiles in the sample, is used to spike the sample to give a peak about the same size as the analyte peak. Calibration is set up using the analyte spiked into a matrix that matches the sample matrix, and the ratio of the analyte peak area to internal standard peak area is plotted against the analyte concentration or added analyte concentration to derive the calibration equation.

Conclusion: Navigating through choices

The choice between using an internal standard and alternative methods such as standard addition hinges significantly on the specific context of the analysis and the nature of the samples and analytes involved.

Discover solutions and navigate through critical scientific choices with ease by joining Chromatography Forum—your community where expertise and insights converge!

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