How the Double Internal Standard Method Revolutionizes Chemical Analysis
Explore the MethodImagine trying to find specific individuals in a huge city where millions of residents are constantly moving. This is similar to the challenge faced by analytical chemists when trying to determine the exact amount of specific substances in complex mixtures—from environmental monitoring to pharmaceutical quality control.
Traditional analysis methods often face the problem of component loss at various stages of sample preparation, which significantly reduces measurement accuracy.
The revolutionary method of double internal standard, developed by Russian scientists I.G. Zenkevich and K.M. Korolev, achieves incredible measurement accuracy even in the most challenging conditions .
Chromatography is a powerful physicochemical method for separating and analyzing complex mixtures, based on the different distribution of components between two phases—stationary and mobile. The method is so effective that modern pharmaceuticals, forensics, or environmental monitoring would be impossible without it.
However, precise quantitative measurements in chromatography face a serious problem—analyte losses (determinable substances) during sample preparation stages: extraction, purification, and concentration. These losses can reach 30-40%, which is completely unacceptable when high measurement accuracy is required.
Traditionally, to compensate for these losses, the internal standard method is used—adding a known amount of a substance with properties similar to the determined analyte to the sample. But this approach has limitations—it's not accurate enough when different components are lost to varying degrees.
Chromatography was first developed in 1900 by Russian botanist Mikhail Tsvet to separate plant pigments.
The innovative idea of Zenkevich and Korolev was to use not one, but two internal standards—specially selected homologs (chemical "relatives") of the determined compounds. This approach allows for mathematically precise accounting of losses at all stages of analysis, since the change in the ratio of the two standards reflects the degree of loss of the determined substances .
The method is based on a fundamental principle: in homologous series (series of chemically similar compounds differing from each other by a specific structural unit), there is a regular change in physicochemical properties. This means we can predict how a particular substance will behave during analysis if we know the behavior of its "relatives" .
To demonstrate the method's effectiveness, researchers conducted an experiment with model samples that simulated real complex matrices with sorption properties.
The experiment consisted of several clearly organized stages:
The polar sorbent Silipor 75 (silica gel with a specific surface area of 75 m²/g) was saturated with polar alkanecarboxylic acids of various chain lengths.
Two preselected homologs of the determined acids with known concentrations were added to the initial samples.
Acids were extracted from the sorbent using a suitable organic solvent.
For gas chromatographic analysis, polar carboxylic acids were converted to their ethyl esters—less polar and more volatile compounds.
Separation and quantitative determination of the obtained esters was performed on a chromatograph with a flame ionization detector .
The experimental results were impressive: the relative error of determinations was only (-1)-(-8)%, which is 2-4 times more accurate than traditional methods of quantitative chromatographic analysis.
| Method | Error Range (%) | Advantages |
|---|---|---|
| Double Internal Standard | (-1)-(-8) | High accuracy, loss compensation |
| Absolute Calibration | (-15)-(-25) | Simplicity |
| Single Internal Standard | (-10)-(-20) | Partial loss compensation |
| Standard Additions | (-5)-(-15) | Accounts for matrix effects |
| Acid (Carbon Atoms) | Initial Amount (mg) | Found Amount (mg) | Error (%) |
|---|---|---|---|
| Acetic (C2) | 10.0 | 9.8 | -2.0 |
| Butyric (C4) | 10.0 | 9.9 | -1.0 |
| Caproic (C6) | 10.0 | 9.5 | -5.0 |
| Caprylic (C8) | 10.0 | 9.3 | -7.0 |
| Capric (C10) | 10.0 | 9.2 | -8.0 |
The method is applicable even when homologs of the determined analytes are already present in the original samples and can be extended to any number of carbon atoms in the molecules of such standards .
Successful application of the double internal standard method requires a set of specific reagents and materials:
| Reagent/Material | Function in Analysis | Special Requirements |
|---|---|---|
| Two homologous internal standards | Compensate for analyte losses at all preparation stages | Must be chemical "relatives" of the determined analytes |
| Polar sorbent Silipor 75 | Model complex matrices with sorption properties | Specific surface area 75 m²/g |
| Extraction solvents | Extract analytes from matrix | High purity, selected for specific analytes |
| Derivatization reagents | Convert analytes to derivatives suitable for chromatographic analysis | For acids: esterifying reagents (e.g., ethanol with catalyst) |
| Gas chromatograph | Separate and quantitatively determine mixture components | Preferably with various detector types (FID, MS) |
| Iristectorigenin A | 39012-01-6 | C17H14O7 |
| Isoamyl salicylate | 87-20-7 | C12H16O3 |
| Isobutyl decanoate | 30673-38-2 | C14H28O2 |
| Isopropyl benzoate | 939-48-0 | C10H12O2 |
| N-Boc-PEG8-alcohol | 1345337-22-5 | C21H43NO10 |
The double internal standard method finds application in various fields of analytical chemistry. It is particularly useful for:
Analysis of natural objects (water, soil, plant and animal tissues)
Quality control of medicinal products
Determining contaminants in the environment
Food quality and safety testing
Recent work by Zenkevich and colleagues demonstrates the possibility of combining the double internal standard method with high-performance liquid chromatography-mass spectrometry (HPLC-MS) for determining such complex analytes as monoethanolamine in aqueous solutions .
The double internal standard method developed by I.G. Zenkevich and K.M. Korolev represents an elegant solution to the complex problem of quantitative chromatographic analysis under conditions of possible analyte losses at sample preparation stages.
This approach combines a fundamental scientific basis (patterns of property changes in homologous series) with practical applicability in various fields of analytical chemistry. The accuracy and reliability of the method open new possibilities for analysts working with the most complex samples—from medical diagnostic kits to environmental monitoring.
As chemical science continues to face increasingly complex analytical challenges, methods like the double internal standard will play an increasingly important role in obtaining reliable and accurate results, upon which crucial decisions in medicine, ecology, and industry are based .