Improved Resolution in High-Sensitivity Dual-Label Gas Radiochromatography Paul J. Thomas and H. J. Dutton Northern Regional Research Laboratory, Peoria, Ill. 61604 The simultaneous and independent measurement of carbon-14and tritium isotopes in compounds separated by gas-liquid chromatography can be performed by automatic serial collection of fractions in vials with subsequent scintillation counting. This paper describes the application of digital computer techniques to speed the analysis of counting data, to improve the sensitivity and resolution of the analysis, and to plot the individual isotopic curves. This improvement is obtained by combining and averaging the results of two to four replicate analyses of a given sample into a single plot of radioactivity vs. chromatographic retention time. With these techniques, samples having an activity of around 1-10 nanocuries/mg can be easily analyzed. The profile of radioactivity vs. chromatographic retention time is superior in its signal-to-noise-ratio, in its resolution of closely spaced peaks, and in its sensitivity to that obtainable with any continuous flow system. THEQUANTITATIVE ANALYSIS of either 14C or tritium isotopes in compounds separated by gas-liquid chromatography (GLC) has been accomplished by several methods. These include serial collection of fractions for scintillation counting (1-3) ; cumulative collection for continuous integral counting (3, 4); continuous ion-chamber monitoring of the effluent vapor (5-8); and continuous proportional counting of the effluent, with or without combustion (9, IO). Some of these methods are employed in commercially available instruments. Simultaneous analysis for 14C and tritium in dual-labeled compounds has required separate combustion and reaction trains for each isotope. Automatic collection of serial fractions for scintillation counting as described by Dutton ( I , 2) gives the highest sensitivity for compounds of low specific activity-Le., 0.5 to 100 nanocuries/mg-because the time of counting is not limited to residence time, as in a differential detector. High sensitivity is primarily caused by the increased time available for counting collected fractions (up to 100 minutes or more) and the resulting improved statistical accuracy. High resolution in this present method can be provided by the close spacing of fractions and the constant time interval of collection. This high resolution has now been improved significantly by the application of modern data-processing techniques available with a high-speed digital computer. These techniques enable us to combine and average the results of two to four repeated analyses on the same sample and then to approximate a true (1) H. J. Dutton in "Advances in Tracer Methodology," Vol. S. Rothchild, Ed., Plenum Press, New York, 1962, pp 147-152. (2) H. J. Dutton, J. Amer. Oil Chem. Soc., 38, 631 (1961).
1,
(3) A. Karrnen and H. R. Tritch, Nature, 186, 150 (1960). (4) G. Popjlk, A. E. Lowe, D. Moore, L. Brown, and F. A. Smith, J. Lipid Res., 1, 29 (1959). ( 5 ) P. Riesz and K. E. Wilzbach, J . Phys. Chem., 62, 6 (1958). (6) L. H. Mason, H. J. Dutton, and L. R. Bair, J. Chromatogr., 2, 322 (1959). (7) F. Cacace and Inam-U1-Haq, Science, 131, 732 (1960). (8) H. E. Dobbs, J . Chromatogr., 5, 32 (1961). (9) R. Wolfgang and F. S . Rowland, ANAL.CHEM.,30, 903 (1958). (10) A. T. James and E. A . Piper, J . Chromatogr., 5, 265 (1961).
I\
Pump
Figure 1. Modified automatic system for condensation of effluent in scintillation solvent differential curve of the distribution of radioactivity in the column effluent. The shape and resolution of overlapping peaks for radioactivity are nearly as good as those of the thermal conductivity curve. A complete description of this technique and some examples of the results are given below. Simultaneous analysis of dual-labeled compounds is impossible with ion-chamber monitoring, and it can be performed by proportional counting only after chemical separation of 14CO2 and 3Hz from the combustion products of the effluent. Because tritium and 14C isotopes can be easily resolved by scintillation spectrometry, our procedure is particularly useful for the analysis of dual-labeled compounds in GLC effluents. EXPERIMENTAL Apparatus. The system for automatic serial collection of fractions was slightly altered from that described by Dutton (132). An Aerograph Model A-90 gas-chromatographic instrument was modified by the addition of an auxiliary stainless-steel collection tube, A (3-inches X 1/dnch 0.d. and SJls-inch i.d.), enclosed in an aluminum block placed at the top of the detector oven (Figure 1). The aluminum block was maintained at 250" to 260 "C by a 75-W cartridge heater and controller. The effluent streams from both the sample side and the reference side of the thermal conductivity detector cell were VOL. 41, NO. 4, APRIL 1969
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brought together through a tee into the interior end of the collector tube. A heavy-walled aluminum tube, B (21/,-inches x s/,,-inch 0.d. and l/16-inchid,), was inserted to a depth of 11/2 inches into the exterior end of the stainless steel tube, and sealed to it with a washer made of Teflon (Dupont) and a stainless steel packing nut. The outer end (ca. 1 inch) of the aluminum tube was covered with a sleeve made of Teflon (Dupont), C, machined to a standard male taper. The 3/16-in~hbore of the sleeve was reduced to ‘/I6 inch at the tip, to cover and insulate the front face of the aluminum tube. A small glass tee, D , with a 7/15 standard female taper on the short arm was placed over the sleeve made of Teflon (Dupont). The two sides of the long arm of the tee were connected to l/*-inch 0.d. Polyflo tubing. A toluene-base liquid scintillation mixture was introduced through the lower tubing (with a metering pump); liquid and gas flow together out the upper tubing to the fraction collector. An air-filled damping reservoir, E, was provided at the pump to smooth the flow. The fraction collector was operated on a time basis by a 0 to 60 second interval timer. Fractions were collected directly in 20-ml scintillation counting vials, F. A solenoid-operated glass valve, G, connected to the interval timer interrupted the flow of solvent at the collector between fractions. An event marker on the thermal conductivity recorder chart was also connected to the interval timer. MATERIALS 1 4C-Labeled methyl esters of palmitic, stearic, oleic, and linoleic acids with specific activities of 70 to 100 microcuries/ mg were purchased from New England Nuclear Corp. Tritiumlabeled methyl stearate with a specific activity of cu. 8.3 microcuries/mg was a remnant from earlier experiments of Jones et al. (ZZ) in which tritium gas was added to methyl oleate. Small amounts of tritium-labeled methyl esters can also be conveniently prepared by the procedure of Mounts and Dutton (12). A standard sample was made by mixing trace amounts of these labeled esters with soybean methyl esters. The final activity was 9 nanocuries of 14C and 40 nanocuries tritium/mg of esters. The composition of the mixture by weight was not detectably different from soybean methyl esters. Procedure. For the separation of fatty methyl esters, a 1/4-inch 0.d. x 10-ft stainless steel column was packed with 15 Hi-Eff IBP (diethylene glycol succinate polyester) on SO/lOO mesh Gas Chrom P. (The packing was obtained, precoated and pretested, from Applied Science Laboratories.) The column was maintained at 195” to 200 “C with helium flowing at 100 ml/min. Under these conditions methyl stearate has a retention of 20 to 25 minutes. The interval timer was generally set for about 45 to 50 seconds, and the metering pump was set at about 20 ml/min. Methyl stearate generally fell between fraction numbers 25 and 30; a total of 50 to 80 fractions were collected, depending on the sample. The filled vials from the fraction collector were counted in either a Packard Tri-Carb or a Beckman Model LS-250 scintillation counter. The latter was equipped with automatic external standardization and automatic quench compensation, as well as a teletypewriter printer and paper tape punch. No significant quenching was noted in any sample collected with the system as described. Both counters were provided with at least two voltage-discriminator channels to provide simultaneous data on 14C and tritium isotopes. Data from the scintillation counter were transferred to an IBM 1130 computer either directly from the punched paper tape through an IBM 1134 Tape Reader or from hand-punched data cards through an IBM 1440 Card Read-Punch. Interpretation of
z
(11) E. P. Jones, L. H. Mason, H. J. Dutton, and R. F. Nystrom, J . Org. Chem., 25, 1413 (1960). (12) T. L. Mounts and H. J. Dutton, J. Label. Compounds, 3, 343 (1967). 658
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the teletypewriter character codes required a machine-language subroutine. Each fraction collected was counted in duplicate and the duplicate counts were compared. If a significant deviation was noted, an error message was printed identifying the fraction number. Dual-labeled samples were resolved by computer programs making use of standard procedures (13, 14) as follows: (1) The voltage-discriminator “windows” of the scintillation spectrometer were adjusted to achieve a balance between high efficiency and low “spill-over.’’ Counting efficiency for each isotope in each channel was determined as a function of the external standard ratio by counting a series of quenched standards. The function was approximated by a second degree polynomial “quench curve.” (2) The efficiency of each sample counted was determined from its external standard ratio by reference to the quench curves. With our samples in the Beckman Model LS-250, the routinely observed efficiencies were: Tritium, 40z in channel A, 0.5% in channel B; 14C, 9 % in channel A, 67z in channel B. The absolute disintegration rate for each isotope was then determined by solving the following two simultaneous equations : net cpm chn. A = 14C eff. chn. A X 14C dpm
+ 3H eff. chn. A X 3H dpm net cpm chn. B = 14C eff. chn. B X 14C dpm + 3H eff. chn. B X 3H dpm If a single analysis was done, data from the scintillation counter were reduced to a plot of disintegrations per minute in each fraction us. the relative retention time at the beginning of the fraction; the points were connected by straight lines, and the resulting graph, complete with grid lines, was produced by an IBM 1627 x-y plotter. Two or more repetitive analyses of the same sample were combined, as follows : (1) The absolute disintegration rate for each isotope in each fraction of each chromatographic “run” was stored. The maximum count and the minimum count were selected by the program for each run. The minimum count was subtracted from all fractions as a background or baseline correction. (2) The exact position of the methyl stearate peak, determined by inspection of the recorder chart, was specified to the program in terms of the fraction numbers. Each fraction was then assigned an x value corresponding to its position relative to methyl stearate. (3) A scale of relative retention values from 0 to 2.5 times that of methyl stearate was divided into 1250 increments. The computer, beginning at 0, calculated a y value at each increment for each of the separate runs by straight-line interpolation between the data points on either side. The calculated values for each separate run were added and divided by the maximum count to give a “per cent of full scale” figure that was recorded by the plotter as a point on the curve. A new point was calculated at every ljl00 inch interval along the x axis, and the plotter connected these points by straight lines. The resulting graph of radioactivity cs. relative retention time is, in essence, a radiochromatogram. Integration of the “peaks” was obtained by summing the y values for each increment between specified values of the relative retention time. The area under each peak as a per cent of the total was then calculated and printed out. A complete triplicate analysis of a single sample required (13) F. A. Blanchard, Mary R. Wagner, and I. T. Takahashi in “Advances in Tracer Methodology,” Vol. 4, S. Rothchild, Ed., Plenum Press, New York, 1968, p 133. (14) M. I. Krichevsky, S. A. Zaveler, and J. Bulkeley, Anal. E o chern., 22, 442 (1968).
Rilativi Ritontion Tim
0.5
1.o 1.5 Relative Retention Time
2.0
1
5 Relative Retention Time
Figure 2. Computer-plotted profile of radioactivity in the effluent of a gas-liquid chromatograph, averaged from three repeated analyses as described in the text.
Figure 3. Profile of radioactivity in the effluent of a gas-liquid chromatograph, obtained as in Figure 2.
The sample contained 14C-labeled methyl palmitate (C), methyl stearate (E), methyl oleate (F),and methyl linoleate (C), diluted with soybean oil methyl esters to a final specific activity of 9 nanocuries/mg. Tritium-labeled methyl stearate was added to a final specific activity of 40 nanocuries/mg. Methyl stearate is assigned a relative retention time of 1.0. The thermal conductivity analysis is shown in the insert. H is methyl linolenate
The sample consisted of methyl esters prepared from the lipids of immature soybean seeds incubated in sodium acetate-l-14C for 15 minutes. The actual measured activity of the total sample before analysis was 845 cpm/mg-i.e., less than 0.5 nanocurie/mg. Methyl stearate is assigned a relative retention time of 1.0. Peaks are identified as follows: methyl laurate ( A ) , methyl myristate (B), methyl palmitoleate (D); others as in Figure 2. The small peaks after 1.7 are not reproducible and must be assigned to background fluctuations
3 to 5 hours for gas chromatography and collection of fractions; 24 to 48 hours for liquid scintillation counting (unattended); and less than 15 minutes for data processing and output, starting from the punched tape. Dual-labeled samples were handled in essentially the same length of time. The plotting routine was simply run twice by using the appropriate data for each isotope. The plots were superimposed on the same chart by using different colored inks. An accurate determination of the isotope ratio could be obtained by plotting 14C dpm os. 3H dpm for all the fractions collected in a particular peak. RESULTS AND DISCUSSION
The important features of the collection system are: (1) A high temperature is maintained in the gas stream up to the point of its contact with the scintillation solvent. (2) The holdup volume at all points of the liquid stream is kept to a minimum to eliminate peak tailing. This is especially important in the glass tee, D,and the collector valve, G. (3) The joining of the sample and reference streams from the exit of the thermal conductivity cell eliminated about 80z of the noise introduced by gas pressure changes within the cell. This noise was further reduced by the air reservoir at the pump. The radiochromatogram computed from triplicate analyses of 1- to 2-mg aliquots of the standard sample is shown in Figure 2. This example illustrates the high resolution achieved by the system we describe. In addition to this standard sample, we have made triplicate analyses of nearly 50 samples, which were isolated from algae and higher plants grown in the presence of carbon dioxide-IC, and from seeds incubated in acetate-l-14C. Figure 3 shows that useful results can be obtained with specific activities as low as 0.5 nanocurie/mg in a mixture of methyl esters. In selecting conditions such as fraction spacing, there is a certain degree of “trade-off” between resolution and sensitivity; that is, sensitivity will be increased by collecting fewer fractions with longer intervals; resolution will be correspondingly decreased. The practice of collecting only a single
fraction for each peak observed gives very poor resolution and can lead to serious errors of interpretation, because a small amount of material with high specific activity may be mistakenly identified if it overlaps another compound of low specific activity. When a single run is graphed by plotting the count in each fraction DS. its retention time, as we have described, the results are deceptive. The top of each peak will generally fall in a single fraction and will appear as a sharp point. While each point will be plotted with a precision of =k0.002 relative retention units, its position is significant only to about 1 0 . 0 3 units, and compounds differing in relative retention by that amount could not be distinguished. Moreover, with compounds of very low specific activity, the total count in each fraction may be close to the background level. Thus, a noticeable degree of uncertainty may be expected. This uncertainty could be reduced by repeating the analysis and averaging the two (or more) sets of data. Between repetitions, however, there may be slight changes in the conditions of the chromatographic column, leading to variations in the composition of each fraction collected. The data could not, therefore, be averaged by simply summing the count in corresponding vials for each run and dividing by the number of runs. Such a procedure would at best give no increase in resolution of adjacent peaks; in most cases it would drastically reduce resolution. The logical alternative, and the method we adopted, was to scale the data points on a common axis, interpolate at many increments between points, and average the data at corresponding increments for the several runs. This procedure, which was possible only with the aid of a digital computer, successfully reduced the “noise” or random error, in the final plot. It also led to an increase in resolution when the fractions of successive runs did not coincide, because the total number of actual data points was increased from perhaps 60 to 180. Peak positions could now be considered significant to a precision of *0.01 relative retention units, subject to the limitation described below. Calculations of the percentage of radioactivity in each peak showed an average VOL. 41, NO. 4, APRIL 1969
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deviation of 0.4% when calculated from a single run, and only 0.06 when calculated from a triplicate. The ultimate capability of the system is demonstrated by Figure 2, which compares the radioactive analysis of a standard dual-labeled mixture, described above, with the corresponding thermal conductivity analysis. This mixture was employed routinely to test the performance of our system. The data shown in the figure are typical of the results obtained when the system was functioning properly. From measurements of the peak widths, and using the general relation that the peak width is inversely proportional to the square root of the number of theoretical plates for the column, we can estimate the resolution of our radioactivity separation. Our estimation is that this resolution corresponds to about 60% of the number of theoretical plates obtained for the nonradioactive separation. In matching peaks of the thermal conductivity recording with those of the radioactivity profile, it is necessary to make a slight correction for the “time lag.” This lag leads to a small uncertainty in the precise position of the radioactive peak; however, this uncertainty is generally smaller than 1 to 2% of the retention time. If a dual-labeled compound is present, the coincidence of the 14C peak and the tritium peak is visibly apparent. Therefore, if a small amount of tritium-labeled standard is added to a mixture of 1Glabeled compounds, peaks may be matched to a precision of 0.5 of the retention time. On closer examination of dual-labeled compounds, a small separation may be observed due to an isotope effect (15). In the analysis shown in Figure 2, the ratio of 14C to tritium increased from 0.09 in the first half of the methyl stearate peak to 0.11 in the second half. This increase amounts to a difference in retention time for the two isotopic compounds of roughly 1 part in 300. When this very slight isotopic effect is taken into account, the precision of determining isotope ratios with this technique is unsurpassed by any other system. Simultaneous independent measurement of carbon and hydrogen cannot be done with ionization detectors. The analysis can be performed with continuous-flow scintillation counters (16), or with proportional counters, by combustion of the effluent, splitting of the stream, chemical separation of carbon dioxide from water, (15) P. D. Klein, Sep. Sci., 1, 511 (1966). (16) G. Popja’k, A. E. Lowe, and D. Moore, J. Lipid Res., 3, 364 (1962).
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reduction of the water to hydrogen, and subsequent measurement of the radioactivity of the carbon dioxide and hydrogen in separate counters (17). This procedure introduces many uncertainties in the relative amounts of the two isotopes, since there may be variations in the efficiency at the combustion, reduction, trapping, and counting stages. By contrast, the system we describe measures both isotopes on the same collected fractions, and the efficiency of collection must be identical for both. The efficiency of counting the collected fractions is easily determined with modern scintillation counters (13, 14). Dual-label radiochromatography offers many advantages for chromatographic analysis. Tritium-labeled internal standards may be added to 14C-labeled samples, or cice versa, to check the coincidence of retention times, the relative specific activities, and the relative collection efficiency of different compounds. The use of tritium-labeled standards for programmed-temperature gas chromatography of fatty ester ozonolysis products, as outlined by Dutton (18), can be extended by using IC-carboxyl-labeled standards to distinguish aldehydes from aldehyde-esters. Liquid-liquid chromatography should also be amenable to double-labeled isotope analysis with this procedure. The use of computer programs such as we describe greatly shortens the time required for computation and processing of the data and increases the accuracy and credibility of the results. We will provide any or all of these computer programs upon request. ACKNOWLEDGMENT
We are grateful to J. 0. Ernst and R. 0. Butterfield of this Laboratory for helpful advice on computer programming. RECEIVED for review October 16, 1968. Accepted January 16, 1969. Presented at 156th meeting of the American Chemical Society, Atlantic City, N.J., September 1968. The Northern Regional Research Laboratory is headquarters for the Northern Utilization Research and Development Division, Agricultural Research Service, U S . Department of Agriculture. Mention of trade or company names is for identification only and does not imply endorsement by the Department. (17) Leon Swell, Anal. Biuchem., 16, 70 (1966). (18) H. J. Dutton, presented at Pittsburgh Conference onAnalytica1
Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1968.
