In the Laboratory
Internal Standard Calculations in Chromatography Judy A. Magee and Antony C. Herd Department of Applied Science and Technology, Otago Polytechnic, Dunedin, New Zealand
Despite the importance of internal standard methods in quantitative chromatography, analytical chemistry textbooks for undergraduates either skim over the issue or, in some instances, provide confusing references to ill-defined response factors. Although specialist texts (e.g. 1, 2) derive equations for use, they are not particularly suitable as a method of teaching the calculation for this important method to undergraduates. The most versatile quantitative method is one in which different amounts of internal standard are weighed into the standard and reference solutions. This note demonstrates a simple calculation method which students find easy to follow and which can also be applied to simpler scenarios such as area normalization. The method is demonstrated by an example based on an industrial sample. Consider the determination of two active ingredients in an herbicide. A reference solution is prepared by weighing the active ingredients and the internal standard into a vial and dissolving in a suitable solvent. The sample solution is prepared by weighing the sample and internal standard into a second vial and dissolving in approximately the same volume of solvent. After chromatographing both solutions and determining peak areas, the following tables are drawn up. Relative response factors are derived from the reference solution results as shown in Table 1, in which the weight and area data are from experimental data and the response column data are obtained by dividing areas by weights. Relative responses are obtained by dividing responses by the response of the internal standard. The experimentally determined data and the calculated response for the internal standard are shown entered in Table 2a. Responses for the actives can be obtained by multiplying the internal standard response by the appropriate relative response factors, and the weights of the actives are calculated, remembering that response is area/weight. The completed calculation is shown as Table 2b. If the weight of herbicide sample was 0.1588 g the %m/m of actives X and Y are respectively 27.8 and 25.8. The method is easy for students to follow and clearly illustrates the meaning of response and relative response factors. It is instructive to point out that as well as overcoming variations in the injected volume, the internal standard method does not require careful volume measurements in making the solutions. The method can be taught alone as a calculation for either GLC or HPLC, or in the context of a laboratory session. Triazines at nominal levels of 25% in a commercial herbicide using THF as solvent are used in this laboratory course, but many alternative examples should be available from local analytical laboratories.
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Table 1. The Reference Solution Compound
Wt/mg
Area
Response
Relative Response
Active X
39.4
480,105
12,185
1.05
Active Y
40.2
438,002
10,896
0.94
Internal standard
38.8
450,119
11,601
1.00
Table 2a. Measured Data for the Sample Solution Compound
Relative Response
Wt/mg
Area
Response
Active X
–
497,423
–
1.05
Active Y
–
413,266
–
0.94
40.5
435,216
10,746
1.00
Internal standard
Table 2b. Measured and Calculated Data for the Sample Solution Compound
Wt/mg
Area
Response
Relative Response
Active X
44.1
497,423
11,283
1.05
Active Y
40.9
413,266
10,101
0.94
Internal standard
40.5
435,216
10,746
1.00
The method can also be used for analysis of solvent blends when the area normalization method is employed. Although not strictly necessary, it has the advantage of requiring one calculation method. The only modifications are that any compound can be chosen to have a relative response factor of 1.00 and in the sample solution an estimated mass or volume needs to be inserted into the table before calculation and subsequent normalization. Although these calculations are routinely made by chromatographic software packages, we believe that it is instructive for the students to perform the manual version at least once during their training. Literature Cited 1. Modern Practice of Gas Chromatography, 3rd ed.; Grob, R. L., Ed.; Wiley: New York, 1995. 2. Braithwaite, A.; Smith, F. J. Chromatographic Methods, 5th ed.; Blackie: London, 1996.
Journal of Chemical Education • Vol. 76 No. 2 February 1999 • JChemEd.chem.wisc.edu
