Internal Standard Method Calculations Auswertung mit Hilfe des inneren Standards

Calculs employ6s dans la methode de dosage avec 6talon interne

D. E. Willis Research Specialist, Monsanto Company, 800 N. Lindbergh Boulevard, St. Louis, Missouri 63166, USA

Quantitation of samples containing non-volatile components (e. g. resins or tars) or components to which the detector is insensitive (e. g. fixed gases or water with flame ionization detector) is a problem continually facing many chemists using gas chromatographic methods. Two methods can be used to solve this problem: a) calibration on an absolute basis by injection of the same amount of sample for each analysis, or b) use of the internal standard method. The first method involves the preparation of standards bracketing the range in which the component(s) of interest will lie and requires considerable skill to precisely inject the same weight of sample each time. Application of this method to multicomponent mixtures can hardly be recommended, except for gas samples, e. g. CI-C4 hydrocarbons, as is commonly done with process analyzers. The internal standard method, on the other hand, can accommodate samples containing a multiplicity of peaks and does not require the precise injection of the sample. The internal standard method is only briefly described in most texts on gas chromatography, despite its high potential utility [2, 6]. One discussion [2] implies that a series of standards must be prepared with component concentrations bracketing the anticipated concentrations of the analyzed substances and a plot of peak ratio versus concentration prepared. Another discussion [ 1 ] does not adequately define area correction factors and gives an equation including "the percentage of internal standard added to the sample" without defining how this percentage is calculated (see equations (2) and (3) below). The use of this equation with the percent internal standard as defined by equation (2) below does not give the correct results. The internal standard is not even mentioned in one discussion of the quantitative interpretation of gas chromatographic data [4]. This note is being written with the hope of correcting some misconceptions about the use and calculations involved in the internal standard method. 42 Chromatographia 5, 1972 Short Communication

The internal standard method involves the addition of a known amount of a pure substance to a known weight of sample. The requirements for a substance used as an internal standard are: a) b) c) d) e) must yield a completely resolved peak, should elute close to the component(s) being measured, should not be present in the original sample, must not react chemically with the sample, and should be present in about same concentration as components measured.

Requirement (a) is to insure a unique and unambiguous area for the peak; (b) is to minimize any change in detector response due to changes in operating conditions (flow, temperature, etc.); (c) and (d) insure that the weight of internal standard is accurately known; and (e) is to minimize the effect of non-linear response of the detector to varying amounts of material. If the component used as internal standard is originally present in the sample, the calculations must be done by the techniques of the standard addition method [7]. Two (or more) internal standards may be used if the components being measured elute at widely different retention times and/or are present at very different concentration levels [5]. In addition to the accurately known weights of sample and standards, it is necessary to experimentally determine the response of each component of interest relative to the internal standard. The relative response factor is given by

F R (i) =

(counts/gram)standard/(Counts/gram)component i (1)

in which integrator counts are a measure of peak areas. The relative response factor thus defmed takes into account the difference in sensitivity of the detector to the standard and the various components. It must be experimentally deter-

mined under the established analytical conditions by the analysis of known blends containing the internal standard. F R indicates the ratio of counts which would be obtained for detection of equal amounts of the component and standard. For example, a relative response factor o f 1.05 indicates that the internal standard will yield 1.05 times as many counts as the component for equal amounts of the two materials. Hence the response of the component must be multiplied by 1.05 to yield equal counts for equal amounts o f material. A response factor may be defined which is divided into the observed area to obtain a corrected area; this is the reciprocal of the factor as defined above. E t t r e [2] has tabulated response data for paraffins, cycloparaffins, and aromatics on two ionization detectors. The preceding points have been well established and are easily understood. The calculations involved in the method have been overlooked in most cases, which creates some existing confusion. Although the actual percent of internal standard (I. S.) in the mixture is given by Wt % I. S. in mixture = weight of standard x 100 weight of standard + weight of sample

Wt % I. S. in mixture = As x 100 As + Ae + An

Wt Std x 100 Wt Std + Wt sample

(5)

where As is the area of the internal standard, Ac is the total corrected area for all observed components (= ~ F R (i) x Ai) and An is the theoretical area of those components which do not elute or are not detected. The percent of a component in the original mixture is % a = Fr(a) x A a x 100/Ac + A n (6)

Combining equations (5) and (6) to eliminate An gives equation (4). The recovery for a sample (percent eluted and/or detected) is given by % Recovery = weight of standard x Z [FR(i ) x Area(i)] x 100 weight of sample x area of standard

(7) (2)
Literature
[1] Bennett, C. E.. Dal Nogare. S. and Safranski. L. W. in "Treatise on Analytical Chemistry," Kolthoff. L M. and Elving. P. J., editors, Part I, Volume 3, p. 1712-13, Interscience Publishers, New York 11961). 12j Dal Nogare. S. and Juvet, R. S.. Jr., "'Gas-Liquid Chromatography," Interscience Publishers, New York 11962). [3] Ettre, L. S., in "Gas Chromatography," Brenner. N., Callen. J, E.. and Weiss, M. D. (editors), Academic Press, New York 11962). [4] Johnson, 14. W. in "'Advances in Chromatography," Giddings. J'. C. and Keller. R. A., editors, Volume 5, Marcel Dekker; New York 11968). [5] Lee. E. H. and Oliver. G. D., Anal. Chem. 31, 1925 (1959). [61 Littlewood, A. B,. "Gas Chromatography," Academic Press, New York 11962). 17] Novak, J. and Novak, Y.. J. Chromatog. 28,392-395 11967). Received: Accepted: June 17,1971 Nov. 7, 1971

this calculation is unnecessary. What is important is that if the sample had contained the weight of standard added, the concentration would be Wt % I. S. (as % of sample) = weight o f standard x 100 weight of sample

(3)
The concentration of any component is then the ratio o f the corrected area of the component to the corrected area of the standard times the weight ratio above, i. e. weight o f standard x area o f A x F R (A) x 100 %A = weight o f sample x area o f standard

(4)
An alternate approach to the derivation of this equation is to use the internal standard to determine the theoretical area of the non-eluted or non-detected components.

Chromatographia 5, 1972

Short Communication

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