An internal standard is a chemical substance added in a fixed, known amount to a sample, blank, and calibration solution to enable accurate quantitative measurements of an analyte. This compound acts as a reference point for precise chemical analysis, particularly in separation techniques like chromatography and instrumental methods such as mass spectrometry and spectroscopy. By using this reference, analysts achieve a higher degree of measurement accuracy.
The Role of Internal Standards in Minimizing Analytical Error
The primary function of an internal standard is to compensate for the various errors inherent in a quantitative analysis procedure. These errors often arise from inconsistent sample handling, which can lead to partial loss of the target analyte during complex preparation steps like extraction, evaporation, or filtration. Since the internal standard is subjected to the same preparation steps, it experiences a proportional loss, ensuring the final ratio remains constant.
Instruments used for analysis can experience fluctuations in response over time due to factors like detector drift, temperature changes, or varying mobile phase flow rates. The internal standard helps normalize the measurement by providing a simultaneous reference signal that is affected by these instrument inconsistencies in the same way as the analyte. Any temporary increase or decrease in the instrument’s sensitivity will equally affect both the analyte and the standard.
Injection Volume Correction
Injection volume inaccuracy is a common issue, especially with manual injection systems. The use of an internal standard corrects for these slight variations because the ratio of the analyte signal to the standard signal remains unchanged, even if the total injected volume is slightly inaccurate.
Mitigating Matrix Effects
The internal standard also helps mitigate the influence of matrix effects. Matrix effects occur when other components in a complex sample, such as biological fluid or environmental extracts, suppress or enhance the analyte’s measured signal. Because the standard is present in the same matrix, it is affected similarly, allowing the calculated ratio to normalize the final result.
Choosing the Right Compound
Selecting an appropriate internal standard is a deliberate process governed by strict chemical and analytical criteria. The compound must not naturally occur in the original sample matrix being analyzed, ensuring that any measured signal is solely from the known, fixed amount added by the analyst. If the compound were already present, it would introduce an unknown variable that would invalidate the quantification method.
The standard must also not chemically react with the analyte or any other components within the sample during the preparation and analysis stages. It must generate a distinct and easily measurable signal, such as a sharp peak in a chromatogram, that is completely separated from all other components. Achieving baseline separation from the analyte and other matrix peaks prevents signal overlap and allows for accurate measurement of its response.
Similarity and Isotopic Labeling
Ideally, the chosen compound should possess physical and chemical characteristics similar to the target analyte. This similarity ensures the standard mimics the analyte’s behavior during extraction efficiency, sample loss, and chromatographic retention time. For the highest accuracy, especially in mass spectrometry, isotopically labeled versions of the analyte—such as a deuterated form—are often used because they behave almost identically but can be distinguished by their mass difference.
Chemical Stability
The selected compound must also be chemically stable under the conditions of the entire analytical process. This includes stability during any heating, solvent exposure, or acidic or basic environments used during sample preparation.
Calculating Results Using the Ratio Method
The core principle for calculating results is measuring the ratio of the analyte’s signal to the standard’s signal, rather than relying on absolute signal intensity. This ratio-based approach compensates for inconsistencies that occur during sample handling and instrument operation. The measured ratio is proportional to the concentration ratio of the two compounds.
A set of calibration solutions is prepared, each containing a different, known concentration of the analyte and a fixed concentration of the internal standard. These solutions are analyzed to determine the signal response, typically the peak area in chromatography. The analyst then calculates the signal ratio (analyte signal divided by internal standard signal) for each calibration solution.
This data is used to construct a calibration curve where the signal ratio is plotted against the corresponding analyte concentration. This curve establishes the proportional relationship between the measured ratio and the true amount of analyte present. When an unknown sample is analyzed, its signal ratio is measured and then used with the calibration curve equation to accurately determine the analyte’s concentration.