Unrecognized Systematic Errors in Quantitative Analysis by Gas-Liquid Chromatography Adam Shatkay” and Soiange Fiavian Israel Institute for Biological Research, Ness-Ziona, Israel
The errors inherent in the accepted methods of quantitative gas chromatographic analysis are discussed. I t Is shown that in addition to the usual parameters affecting the measurements (wch as temperature and gas flow), it is necessary to consider also the volume of llquid InJected, and the concentration of the standards. This applies not only to the “direct” measurements using peak area vs. quantity for the calibration curve, but also to the “internal standards” technique, which Is tacitly assumed to depend only on relative data, considered to be independent of quantity and concentration of the samples. Our argument is supported by experimental results obtained using various combinations of analyles and standards, various chromatographs, various columns, various detectors, and various integrators.
Gas-liquid chromatography (GLC) has become a very popular tool in quantitative analysis. In fact, the very informative and comprehensive text by Littlewood ( I ) states t h a t gas chromatography is mainly used for quantitative analysis, as the interpretation of the data is simple, and the apparatus does not require skilled personnel. This claim appears to us rather dangerous. We shall attempt to point out that the interpretation of the GLC data is not very straight forward, and that unless the quantitative methods are tested in ways which have not been in common use up to now, the results of an analysis can easily be in error by *lo%, or more in some cases, without the investigator being aware of any deviation from accuracy. Two methods are claimed to give reasonably accurate quantitative results in GLC analysis. One is based on the direct calibration of the quantity of pure analyte vs. the peak area, when the instrument parameters (such as temperature, gas flow, etc.) are kept constant. The other method is based on the use of internal standard or standards-it employs a calibration curve where the abscissa is the ratio of the quantity of the analyte to the quantity of the standard, while the ordinate is the corresponding ratio of the peak area of the analyte to the peak area of the standard. These two methods have been considered a t length in the literature (1-5), and we shall summarize here only the salient features, in order to point out the weaknesses of these methods, which have been either tacitly ignored or not realized before. In the “direct” method, a volume of solution ( u ) containing a quantity of the analyte ( q d y t egrams or moles) is introduced into the column. A calibration curve of the peak areas (Amd*) corresponding to qanalyteis prepared; it need not be linear. When an unknown sample is analyzed, its peak area is determined, and the quantity of the analyte is read off the calibration curve. As the volume injected is known, the can also be calculated. concentration (Canaigte) The above argument assumes that the volume of the analyte solution is adequately known. In the common quantitative analysis, the 10-pL syringe is most popular; it is graduated in 0.1-pL divisions and can be assumed accurate to within f0.1 2222
ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
pL. When ca. 2 pL of liquid are introduced, this corresponds to the range of *570. When the quantity injected is less than 2 pL, the possible error increases. On introduction of samples greater than ca. 4 wL, the reproducibility decreases considerably, as can be seen in Figures 7 and 8. This appears to be due to the difficulty in transferring quantitatively all the volume of the liquid from the syringe to the column. When a large volume is introduced, only a small part of the piston serves as a seal, and the liquid tends to evaporate through the top of the syringe. Next, the assumption that all the instrument parameters are constant, both during the establishment of the calibration curve and during the analysis, is not always justified. Thus a shift might occur in the temperatures of the various instrument components, or in the various gas flows. Such factors would introduce systematic errors in any continuous analysis: the duplicate samples would show only small fluctuations, so that the precision might be high, while the accuracy might be low. This is illustrated in Figure 7 and 8, where 100 measurements taken in 15 independent runs are plotted together. As a solution to the above two problems associated with the “direct” method, the “internal standard” method is supposed to be independent of the exact volume introduced into the column, and if the effects of temperature and gas flow on the sensitivity of the instrument to analyte and standard are similar, then fluctuations in these parameters should not affect the applicability of the calibration curve, which depends only on the ratios between the analyte and the standard. We shall attempt to show that such trust in the “internal standard” method might be misleading. Furthermore, we shall show that even in the “direct” method, in addition to the limitations listed above, the concentration itself is a parameter which cannot be neglected. We have started our investigations in order to develop an analytic method which would assist the research carried out in our Institute on the use of 31Pin NMR studies. For this purpose we employed some esters of phosphoric acid. In the course of the investigation, we have extended our measurements to cover quite dissimilar substances, such as halogen derivatives of benzene, or simple hydrocarbons like dodecane. The effects described below were found in all the materials tested by us. However, for brevity, only representative experiments will be described in detail, without quoting the data for all the substances used.
EXPERIMENTAL Materials. For analytes and standards, we have used a number of esters of phosphoric acid (trimethyl phosphate, triethyl phosphate, dimethyl methylphosphonate), 1-bromo-4-chlorobenzene, undecane, and dodecane. All these materials were commercially obtained (from Merck, B.D.H. and Aldrich);all were of analytical grade and were used without any further purification. Instruments. (a) The following gas chromatographs were used: ( a l ) Packard, Oven model 804, Temp. control model 873, Electrometer model 842, H.V. supply model 838. (a2) Packard, Oven model 805, Temp. control model 873, Electrometer model 844, H.V. supply model 834. (a3) Packard, Oven model 802, Temp. control model 871, Electrometer model 840, H.V. supply model
871. (a4) Pye Unicam Series 802. (b) The following detectors were used: (bl) Flame ionization detector, Packard model 881. (b2) Flame ionization detector, Pye model 802. (b3) Thermal conductivity detector, Packard model 839. (c) The following integrators were used: (cl) Infotronics digital integrator model CRS-100. (c2) Honeywell Disc-chart Integrator model 201-B. (c3) Weighing of areas plotted on Leeds & Northrop Ltd. Speedomax W Recorder. (c4) Home-made capacitor-integrator, combined with Radiometer PHM-64 potentiometer. (c5) Home-made integrator based on Hewlett-Packard model 9810 Desk calculator. (d) The following columns were used (length 2 m, diameter 5 mm): ( d l ) 15% Silicone oil DC 550 on Chromosorb W, DMCS 60/80 mesh. (d2) 5% Silicone oil SE 30 on Chromosorb W, DMCS 80/100 mesh. (d3) 10% Silicone oil DC 550 + 5% Polyethylene glycol succinate on Chromosorb W, DMCS 80/100 mesh. Most of the results described below were obtained on using gas chromatograph a1 with detector b l and integrator cl. On use of the other instruments, similar effects were observed. Syringes. We have used the 10-pL Hamilton syringes. The reproducibility and the linearity of the volumes delivered was checked by using a number of syringes, and by extracting the same volumes from different regions of the syringe (e.g., 1 pL between the marks 1-2 or 3-4 pL). Working Conditions. Usually we used intake temperature of 150 "C, isothermal column temperature of 110 "C, and detector temperature of 150 "C. The effects of changes in these temperatures by 50-100 "C were tested in some experiments, to study the influence of variations in temperature. While the retention times and peak shapes and areas were altered by such changes, the effects described in the following discussion were present at all the temperatures tested. The gas flows were usually: H2 50 cm3/min, N2 30 cm3/min, air 500 cm3/min. These also were altered in some experiments, with results similar to those stated for the temperature changes.
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Flgure 1. Calibration curve of the ratio of areas vs. ratio of weights, for dimethyl methylphosphonate and undecane. (0):experimental results for the concentration of undecane ca. 3 X g/cm3. (A): experimental results for the concentration of undecane ca. 1 X g/cm3. 0:experimental result for the concentration of undecane ca. 2 x g/cm3 .a,
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