1019
Anal. Chem. 1982, 54, 1019-1021
Retention Index Scheme and Calibration Method for Gas Chromatography with Electron Capture Detection Frank Pacholec and Colin F. Poole” Department of Chemistty, Wayne State University, Detroit, Michlgan 48202 The electron-capture detector remains the most sensitive of the standard gus chromatographic detectors in common use. This deceptively simple device has been variously described as “the most misunderstood, finest performing, poorest performing, most inconsistent in response detector in use”, by many chromatogiraphers who felt that such descriptions were appropriate based on their experiences ( I ) . The general evolution in detect,or design, represented by the method of electron collection, namely, dc operation, pulsed modulated, and most recently constant-current variable frequency operation in conjunction with the availability of new fl sources of high thermal stability, have done much to make the analysts life an easier one. Yet still, nearly all the improvements in detector operation in the last 2 decades have been associated with the developiment of new materials for construction and advances in electronics and signal processing (2). Very little attention has been given to the chemical aspects of its use or to the area of response standardization. Two areas where improvement is needed are the accurate measurement of column retention and a general method of detector calibration. In this paper we ?wishto describe a novel calibration marker scheme which sirriultaneously provides a retention index scale and internal calibration curve for each sample analyzed. Many methods of constructing retention index scales have been described in the literature (3). The most widely used scheme is the Kovats retention index scale having fixed point9 defined by the adjusted retention volume of the normal hydrocarbons. However, the normal hydrocarbons are virtually insensitive to the (electron-capturedetector and are unsuitable for use with it. The mathematical simplicity of the Kovats scheme has much to recommend it, and we propose to use a similar scheme i n which the fixed points are the normal bromoalkanes which have an adequate electron-capture detector response. Provided that the logarithm of the adjusted retention times is a linear function of the carbon number of the n-bromoalkanes on the stationary phase used in the analysis, then identical formulas to those described by Kovats may be used to calculate retention index values. On the n-bromoalkane scale, the physical meaning of the retention index value remains the same except that it is defined with respect to a different series of fixed points. Several methods are used to calibrate the response of the detector to the compounds of interest. The most common method is to construct a calibration curve using several different concentrations of each substance to be determined. An alternative method makes use of an internal standard of known concentration which is serially added to each sample to be analyzed. The internal standard is able to correct to some extent for changes in the detector response and injection volume between rums. Intuitively, the accuracy of the internal standard calibratilnn method should be the greatest when its structure, concentration, and retention time match closely those of the substance to be measured. The approach may be imagined to be less accurate if several components in a complex mixture me to be determined as it is unlikely that the internal. standard is either close in retention time, concentration, or structure to all the substances to be measured. The new calibraticin method proposed here makes use of two features of the response of the electron-capture detector to
XI
XP
Flgure 1. Plot of log detector response against log concentration for the “caiibratlon method”: Y , = response for bromobenzene on 17bromoalkane calibration curve 1; X , = concentration of an imaginary n-bromoalkane corresponding to Y , ; X , = concentration of bromobenzene having a response Y , . ESTABLISHEC
PLOT
I
SAMPLE
RUN
I
Figure 2. Plot of log detector response against log concentration for the “error reduction method”: Y , = response for bromobenzene on n-bromoalkane calibration curve 3;X 3 = uncorrected concentration of bromobenzene on calibration curve 2; Y,’ = response for bromo-
benzene on n-bromoalkane calibration curve 1. a homologous series of internal standards. In general the response of the detector is concentration dependent and, for a homologous series of n-bromoalkanes, is proportional to the concentration of bromide ions generated on electron capture and independent of the length of the alkyl chain (possible exceptions might be the first few members of the homologous series). As the n-bromoalkanes are used to define the fixed points on the retention index scale and also to provide the basis for the detector calibration curve, we will term them “calibration markers” to indicate their dual role. Two methods of calibration have been evaluated. These are termed “the calibration method” and the “error reduction method”. The procedures will be described with the aid of Figures 1and 2 using bromobenzene as the test substance and the n-bromoalkanes Csthrough C8 as calibration markers. For the calibration method, it is necessary to establish the relationship between the calibration curve for the calibration markers (Figure 1,curve 1)and bromobenzene (Figure 1,curve 2). This information is stored in the computing integrator and forms the data base for all subsequent work. Each poinit on the calibration curve for bromobenzene was generated from the average of five runs for each concentration. Since ti calibration marker plot is obtained with each run, each point on the n-bromoalkane standard plot is an average of 25 runs. The n-bromoalkane calibration plot is generated by arranging: for the different calibration markers to be present in stepwise 0 1982 American Chemical Society
1020
ANALYTICAL CHEMISTRY, VOL. 54, NO. 6, MAY 1982
Table I
actual moles of bromobenzene 2.43 x
1.22 x l o - “
6.09
3.04
X
X
lo-”
lo-’’
1.52 X 10‘’
calibration using n-bromoheptane as internal std moles % error
std calibration using curve 2 moles % error 2.30 2.19 2.80 1.98 2.68 3.15 2.57 2.39 2.51 f 0.37 1.47 1.27 1.34 1.44 1.48 1.57 1.43 1.43 1.42 i 0.09 6.78 6.73 6.75 6.99 8.00 7.09 7.35 7.27 7.12 i 0.43 3.40 3.26 3.21 3.72 3.58 3.49 3.71 3.48 t 0.2 1.48 1.43 1.47 1.58 1.52 1.49 1.54 1.60 1.51 i: 0.06
5.7 10.2 14.8 18.9 9.8 29.0 5.3 2.0 12.0% 20.5 4.1 9.8 18.0 22.0 28.7 17.2 17.2 17.2% 11.3 10.5 10.8 14.8 33.0 16.4 20.7 19.4 17.1% 11.5 6.9 5.2 22.0 17.8 14.8 22.0 14.3% 2.9 5.9 3.3 3.9 0.0
2.0 1.3 5.3 3.1%
2.11 2.10 2.71 1.88 2.27 2.54 1.95 1.71 2.15 i: 0.34 1.32 1.25 1.28 1.29 1.29 1.28 1.11 1.09 1.23 * 0.08 6.51 6.38 6.60 6.53 6.28 6.00 6.08 5.99 6.30 i 0.25 3.13 2.92 3.17 3.42 3.15 3.18 3.11
3.15 f 0.15 1.37 1.46 1.39 1.19 1.29 1.34 1.29 1.19 1.31 i: 0.09
increasing concentration to cover the concentration range of bromobenzene to be investigated. In this work, a concentration range of about 1000,within the linear operating range of the detector was used. The two calibration curves can be represented by the equation of a straight line. By use of simple algebra the detector response to bromobenzene can be related to a concentration term obtained with respect to the calibration marker curve by eq 1.
for calibration marker, curve 1,y1 = M I X l + C1 for bromobenzene, curve 2, y2 = M2X2
+ Cz
X = concentration Expressed in words, the calibration method works as follows. The sample of bromobenzene is coinjected with a mixture of calibration markers. The chromatogram obtained after normal data handling reproduces the calibration curve for the cali-
13.5 13.1 11.5 23.4 6.6 4.5 19.6 29.5 15.2% 8.2 2.5 4.9 5.7 5.7 4.9 9.0 10.7 6.5% 6.9 4.8 8.4 7.4 3.1 1.5 0.2 1.6 4.2% 2.6 3.8 4.1 12.3 3.4 4.4 2.1 4.7% 9.9 3.9 8.6 21.7 15.1 11.8
15.1 21.7 13.5%
calibration method moles % error 1.99 1.80 2.76 1.91 2.27 2.67 1.95 1.80 2.14 i 0.38 1.30 1.25 1.22 1.30 1.29 1.35 1.16 1.16 1.25 f 0.07 6.33 6.56 6.40 6.65 6.09 6.98 5.73 6.37 6.38 * 0.37 3.21 3.22 3.18 3.18 3.25 3.09 3.10 3.18 f 0.06 1.44 1.50 1.42 1.26 1.32 1.41 1.36 1.26 1.37 t 0.09
18.4 26.2 13.1 21.3 7.0 9.4 19.2 26.2 17.6% 6.6 2.0 0.0
6.6 5.7 10.7 4.9 4.9 5.2% 4.0 7.7 6.1 9.2 0.0
14.6 5.9 4.6 6.3% 5.4 5.7 4.4 4.4 6.7 1.4 1.8 4.3% 5.3 1.3 6.6 17.1 13.1 7.2 10.5 17.1 9.8%
error reduction method moles % error 2.03 1.95 2.13 1.89 2.28 2.66 1.98 1.77 2.16 * 0.36 1.31 1.24 1.24 1.31 1.29 1.31 1.14 1.24 1.26 i 0.06 6.35 6.53 6.41 6.59
6.21 5.59 6.02 6.27 6.24 f 0.32 3.22 3.21 3.31 3.38 3.23 3.24 3.14 3.25 i 0.08 1.45 1.50 1.43 1.50 1.32 1.41 1.36 1.26 1.40 i: 0.09
16.6 20.0 11.9 22.2 6.6 9.0 18.9 27.0 16.5% 7.3 1.6 1.6 8.3 5.7 8.3 6.6 1.6 5.1% 4.2 7.2 5.2 8.2 2.0 8.2 1.1
2.1 4.8% 5.6 5.4 8.5 10.8 6.3 6.6 3.3 6.6%
4.6 1.3 5.9 1.3 13.2 7.2 10.5 17.1 7.6%
bration markers (Figure 1,curve 1)and provides a retention index value and the area response of the detector for bromobenzene. The detector response for bromobenzene is treated as if it was an imaginary bromoalkane of concentration XI.This value for XI is then used to calculate the true concentration of bromobenzene X 2 using eq 1 and the established plots stored in the integrator memory as shown in Figure 1. In the “error reduction method, the observed response for bromobenzene is used to calculate its uncorrected concentration X 3 from calibration curve 2. If the bromobenzene had been an imaginary n-bromoalkane of concentration XB,it would generate a response Y3on curve 3, the sample run, and Y3/ on curve 1,the established plot. The ratio Y,’/Y3 is used to correct for the response-concentration difference between the two calibration curves 1and 3. The corrected value for the response Y3”, not shown in Figure 2, is then used to calculate a corrected concentration X4for bromobenzene using calibration curve 2 as described previously. The error reduction method assumes that the small change in the detector response characteristics represented by the nonsuperimpos-
ANALYTICAL CHEMISTRY, VOL. 54, NO. 6, MAY 1982
ability of curves 1 and 3 faithfully mirror the expected change in the detector response to the sample, in this case, bromobenzene. For small changes in the detector operating conditions, this is a reasonable assumption. This may not always be the case, as the nature of the detector response is a function of the mollecular structure of the test solute and the thermal energy of the captured electron.
EXPERIMENTAL SECTION The C6to CISn-bromoalkanesand bromobenzene were obtained from Aldrich Chemical Co. (Milwaukee,WI). Stock concentrations were made up on ti weight to weight basis using pesticide residue grade n-hexane (J. T. Baker, Phillipsburg, NJ) as the dilution solvent. For chromatography, a Varian 3700 gas chromatograph with a 8-m Ci pulse-modulated constant-current electron-capture detector was used. The column was 3 f t X 0.25 in. o.d. packed with 5% O'V-101 on Gas Chrom Q, 100-120 mesh. The column temperature, detector temperature, and injection temperature were 55, 400, andl 210 "C, respectively. The carrier gas was argon-methane (955) at a flow rate of 30 mL min-l. The carrier gas was purified by passage through a Go-Getter (Alltech Associates, Deerfield, L)oxysorb trap and a molecular sieve trap. For data handling a Spectra Physics SP4100 computing integrator and a Texas Instruments 59 programmable calculator were used. RESULTS AND DISCUSSION Initial experiments were performed to establish the three assumptions macle in the introduction to this paper. It was confirmed that the log of the adjusted retention time of the n-bromoalkanes was a linear function of their carbon number. Retention index values could then be calculated in a manner analogous to that described by Kovats ( 3 ) . The electroncapture detector was shown to produce an identical response for equal molar concentrations of each of the n-bromoalkanes from C6 through CU. By variation of the concentration of n-bromoalkanes in a stepwise fashion, a linear calibration curve for detector response against the molar concentration of n-bromoalkanes was obtained. By use of accurately prepared standards of bromobenzene as a test solute, four methods of detector calibration were evaluated a t five different concentration levels. Each measurement was repeated seven or eight times and the mean value of the bromobenzene concentration measured and the average relative percentage error in its determination calculated. Tho raw data and the statistical interpretation are collated in Table t. Several conclusions can be drawn from this information. The conventional method of calibration using an accurately prepared calibration curve produces a relative percentage error in the range 5-30%. At the highest concentration level investigated, the standard calibration curve method gave an average relative percentage error of 3.1%" This result is atypical of the method and of data obtained in preliminary studies but is recorded here so as not to bias the data set. An average relative percentage error of 12-20% is typical for the results normally obtained with the conventional calibration curve imethod. It should be noted that this error is consiberably higher than can be associated with an injection volume effect. This usually averages about 3% with a 4-@L injection volume a3 determined in an independent experiment. For the brornobemene concentration of 2.43 X mol, which
0
1021
is very close to the detection limit, the average relative percentage error of 12-189'0 obtained by the four calibration methods would be acceptable for this concentration level. At the next three higher concentration levels, both the accuracy and the precision of the internal standard method, calibration method, and error reduction method are all significantly better than the conventional calibration curve method. There is no evidence that the error reduction method produces a significant increase in either accuracy or precision compared to the calibration method. It does modify the absolute value of the concentration of bromobenzene obtained, but statistically this value is no closer to the true value than the value obtained by the calibration method. The method of internal standard calibration using n-bromoheptane and the calibration method are similar in accuracy and precision. The data set presented for bromobenzene is typical of several other test substances investigated. These include the isomers of dichlorobenzene and the dialkyl phthalate estera. The "calibration m e t h o d described in this report has several advantages. First of all, it provides a retention index value to aid compound identification. Simultaneously it enables an internal calibration curve to be generated which can be used as a universal calibration method. The technique is compatible with the use of modern chromatographic data stations which after preliminary programming and calibration are capable of performing all calculations and library searchcs with the minimum of operator intervention. The method would be most useful and time saving when multiple cornponent mixtures are to be analyzed on capillary columns. Under these circumstances a single substance internal standard is not ideal as an accurate calibration and retention marker. The n-bromoalkanes or some similar homologous series of compounds could provide a general method of defining the detector response to a compound. The sensitivity of any substance could be determined as if it was an imaginary bromoalkane. If the response of one of the bromoalkanes is calculated or defined, then the response obtained for any other electron-capturing compound could be established with respect to this value. As the detector responses of all bromoalkanes are simply related to each other, a suitable internal calibratioin standard would be available for any compound to be determined. This has the advantage that it places detection sensitivity on a uniform chemical scale and removes some of the arbitrariness associated with the operating conditions of the detector from the measurement of such quantities.
LITERATURE CITED (1) David, D. J. "Gas Chromatographic Detectors"; Wiley: New York, 1974: on 76-1 13. (2) %tkis:k., Poole,.C. F., Eds. "Electron-Capture. Theory and Practice in Chromatography"; Elsevler: Amsterdam, 1981. (3) Haken, J. K. Adv. Chromatogr. lg76, 74, 387-407.
RECEIVED for review January 18, 1982. Accepted February 10,1982. Work in the authors' laboratory is supported by the donors of the Petroleum Research Fund, administered by the American Chemical Society, and the Camille and Henry Dreyfus Foundation.
