trialkyl barbiturates. NMR spectra on the total products of reaction of 5,5-diethylbarbituric acid and of 5-ethyl-5methylbarbituric acid with excess ethereal diazomethane do show very small and variable quantities of two 0-methylated species (OCH, proton signals at 4.08 and 4.02 ppm) to be present. The identity of these components is being more fully investigated and will be reported later, together with the structural identity of the various products (with OCHB proton signals at 3.86 and 4.00 ppm) arising from the action of excess diazomethane on 1,5-dialkylbarbituric acids.
ACKNOWLEDGMENT The author is grateful to H. W. Avdovich for obtaining the NMR spectra, to G . L. Belec for performing the GLC analyses, and to K. Genest for helpful discussions. The author is particularly grateful to B. C. Flann for making reference samples available for this study. RECEIVED for review September 11, 1969. Accepted December 22,1969.
Quantitative Thin-Layer Chromatography Using a Flame Ionization Detector J. J. Szakasits, P. V. Peurifoy, and L. A. Woods Shell Oil Company, Houston Research Laboratory, P . 0 . Box 100, Deer Park, Texas 77536 A flame ionization detector has been adapted to scan thin-layer chromatographic strips directly and produce a series of signals proportional to the amount of material present for each of the separated organic components. Thin-layer chromatographic separations are carried out on a metal-backed adsorbent strip that is passed directly between the nozzles of a dual-jet flame ionization detector. The signal from the detector is fed to an electrometer, recorder, and digital integrator. The strip is scanned at sufficiently high temperature (300-450 "C) so that all of the sample is removed from the adsorbent by a single scan. Observed background electronic noise is very low and the high detector sensitivity permits use of very small samples. The apparatus is sturdy and easily constructed.
THIN-LAYER CHROMATOGRAPHY (TLC) has many advantages over other chromatographic techniques, but quantitative evaluation of TLC plates has been a difficult task. Quantitative analysis can be made in some cases by densitometry or spotarea measurement, but these techniques require calibration and the relationships are frequently nonlinear. A wide variation in sensitivity is often observed with these techniques. Cotgreave and Lynes ( I ) developed a technique in which a traveling furnace was passed over a TLC plate and the cracked products were swept into a flame ionization detector (FID) by a stream of gas. F. B. Padley (2)described a method whereby a thin quartz rod (0.5-mm diameter) covered with adsorbent (on which TLC separation had been previously made) was passed over the burner of a FID. In our work, a metal-backed TLC adsorbent strip (on which TLC separation has been made previously) is passed directly between the nozzles of a dual-jet FID. The metal strip provides good physical support for the adsorbent, and it is easier to handle than a coated quartz rod. EXPERIMENTAL Apparatus. The main components of the apparatus are the frame with traveling stage, a reversible motor, and a dual-jet FID, as shown in Figure 1, and the TLC-strip assembly as shown in Figure 2. (1) T. Cotgreave and A. Lynes, J. Chrornntog., 30, 117 (1967). (2) F. B. Padley, ibid.,39, 37 (1969).
The frame and traveling-stage portion of the scanner are constructed to provide for the horizontal movement of the TLC-strip through the FID. The traveling stage (holding the TLC-strip with spring-actuated pressure pads) slides on two parallel rails moved by a synchronous reversible motor. The choice of speed of the traveling stage is governed by several parameters of equal importance. These are the detector temperature, which is a compromise of the highest FID temperature at the lowest background noise level; the adsorbent thickness; the type of material used for TLCstrip backing; and the boiling range of the sample. At optimum traveling-stage speed, a sufficiently sharp heatfront exists to give good resolution of closely spaced zones without selective losses of low boiling-point components from the sample. For our work, the major portion of the investigation has been conducted at stage speeds of 9 in./min and a minor portion at 6 in./min. Speeds lower than 6 in./ min have also been tested but show poor resolution and some (2-3 %) light-end losses. The traveling-stage speed of the scanner can be varied by replacing the drive pulley or changing the matched gear-set in the drive mechanism. Limit switches reverse the movement of the traveling stage in the automatic mode. Manual switches are provided to override the automatic reverse. The traveling stage is fabricated of Micarta in order to insulate the TLC-strip electrically from the frame which is maintained at ground potential. The FID is constructed to provide sufficiently high belt temperatures (300-450 "C) so that all the sample present on the TLC-strip is removed and ionized by a single scan, with low background noise. Pure hydrogen is fed through two individually adjustable platinum-tip nozzles (0.010 inch). The early work on this project was carried out using a FID, as shown in Figure 3, in which air entered the detector chamber through a number of small holes drilled in a circular plate. The resulting air supply consisted of a group of jets of relatively high velocity. This design was modified (Figure 4) so that air entered through a sintered, stainless steel membrane, giving many more openings to lower the gas velocity. The aim in modifying the air inlet was to avoid losing volatilized sample by venting from the FID chamber. Also, the FID chamber was enlarged to accommodate a larger platinum-collector electrode (2.2-cm diameter). The larger collector is supported at two points to give greater dimensional stability. The ions formed in the flame are collected between the tips of the burners and TLC-strip, both polarized ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
351
SWITCH
FLPlME IONIZATION H
NlSM
FRAME AN0 SLIDES
TLC
ELlNG STAGE A N 0 TLI? STRIP HOLDER IDLER PULLEYS LIMIT SWITCH
Figure 1. Apparatus for quantitative TLC using a dual-jet name ionization detector
HI JETS
TO ELECTROMETER-
Figure 3. Small-cavity FID
POSITION OF TLC-STI
Figure4. Laree-cavity modified air inlet FID positively, and the collector, which is connected to thecelectrometer circuit operated at ground potential. The metal strip is maintained under spring tension in a stainless steel frame (Figure 2) to avoid any sagging in the flame due to thermal expansion. The strip, 3-mm wide, 100 microns thick (80% Cr and 20% Ni alloy), and 21.5 cm long, is spot-welded to the frame at the supporting arms. To eliminate the need for readjustment of the detector, jets, or the TLGstrip holder, it is advisable to construct the TLC-strip assemblies with close tolerances. Borosilicate glass, quartz, and alumina strips and rods have also been investigated for possible adsorbent support, but with little success. When the support dimensions are large--e.g., a 3-mm wide and 1-mm thick quartz strip-n 352
ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
Figure 2. TLC strip and frame assembly during chromatography
excessively high FID temperature is required to remove all the sample by a single scan. A thickness of less than 1 mm increases the difficulties in handling a glass TLC-strip. Procedure. The FID is connected through an amplifier to a recorder and to an electronic integrator-digitizer and printer, in the same manner as for gas-liquid chromatography (GLC). An automatic data-recording system is important because the strip is scanned rapidly (approximately 3 0 4 0 seconds). The scanner is placed under a protective cover purged with filtered air or nitrogen to eliminate the hackground noise caused by dust and organic vapors in the room air. The TLC-strips are prepared by an even application of the silica gel slurry (0.75 g Research Specialties Silica Gel-G and 2.0 ml distilled water) with an eye dropper to the 3-mm wide strip, forming an adsorbent layer with a thickness of approximately 300400 microns. Following application of the gel, the TLGstrip is dried in a nitrogen-purged oven for 3-5 minutes at 80 "C. Then the strip is run through the FID several times until a flat base line is obtained. The operating conditions for the detector are given in Table I. During the first scan a strong signal is observed, probably due to organic substances in the gel, but this signal rapidly vanishes after several scans. Immediately after the adsorbent is activated, the sample to be analyzed is charged 4 cm from the end of the TLCstrip with a microliter syringe. Normally, 0.5 pl of solvent, such as benzene, containing -5-10 pg of sample is placed on the strip. A sample this small prevents excesSive spreading of the sample on the TLC-strip. The TLGstrip is then placed in the developer (Figure 2). The strips are generally developed to a distance of 10 or 15 cm. In routine application, several strips may be developed simultaneously.
Table I. Detector Operating Conditions Hydrogen Row rate to FID: 75 cc/min Top nozzle Bottom nozzle 10 cc/min Air Row rate to FID: 400 cc/min Position of TLC-strip: -3 mm from top jet -2 m m from bottom jet Electrometer setting: 1 X 10-0 A scale
t
I
I
I
5
IO
15 20 TIME, SECONDS
I
!
25
30
I 0 25
0.75
IO0
Figure 7. Chromatogram of phenothiazene, 2,2 '-dipyridylamine, and n-docosane
PHENOTHIAZINE
I
1
0.25
J
I
0.75
0.50
Figure 8. Separation of a heavy gas oil fraction
Figure 6. Chromatogram of phenothiazine, 2,2'-dipyridylamine, and diphenylamine
Table 11. Analysis of Docosane-Anthracene Blend Anthracene, Run No. K 2 2 , w Dev. %w Dev. 1 2 3 4 5
Known
49.6 48.0 49.4 51.5 49.7 50.0 49.7
0.1 1.7 0.3 1.8 0.0 0.8
TIME, MINUTES
IO0
TIME, MINUTES
Av.
3.50 TIME, MINUTES
Figure 5. TLC separation of anthracene and n-docosane with flame ionization detector scanner
t
'
50.4 52.0 50.6 48.5 50.3 50.0 50.3
0.1 1.7 0.3 1.8 0.0 0.8
After separation, the developing solvent is evaporated in the nitrogen-purged oven (80 "C) for 5 minutes. The TLCstrip is removed from the oven and scanned promptly with the FID. If the adsorbent-covered strip is exposed to room air currents for a prolonged time, a strong background signal is created, due to the adsorption of organic compounds from room air. RESULTS AND DISCUSSION
The value of the TLC-FID scanner lies in the fact that it can be applied in both qualitative and quantitative analysis. Several synthetic blends were prepared to test the quantitative aspects of the detector. A typical separation obtained with a blend of n-docosane and anthracene is shown in Figure 5, and some values obtained from electronic integration are given in
Table 111. Comparison of TLC and Liquid Elution Chromatography of a Heavy Gas Oil Fraction TLC Analvsis. Liquid chromatography,____ w %w 2.0 A Resins A 3.1 B Di- and triaromatics 41 .O B 43.3 C Monoaromatics 4.5 C 2.8 D Saturates 52.5 D 50.2 -
I
Table 11. About 4 pg of each component was applied to the strip and developed with cyclohexane-benzene (35 :2) to a distance of 10 cm. Paraffins as low in molecular weight as n-hexadecane have been chromatographed and scanned successfully. Typical separations of other blends are shown in Figures 6, 7, and 8. Figure 6 shows the separation of three nonhydrocarbons obtained with a developer of cyclohexane-benzene (35 :12). The separation of two nitrogen-containing compounds and one paraffin is shown in Figure 7. This blend was developed with cyclohexane-benzene (21 :4), The chromatographic adsorbent was Research Specialties Silica Gel-G. The separation of a heavy gas oil fraction is shown in Figure 8. This sample had been analyzed previously by large-scale liquid elution chromatography. The TLC technique gives a reasonably close approximation of saturate, aromatic, and resin-type separation as shown in Table 111. In Figure 9, a comparison is shown between a FID scan and a densitometer scan of the TLC of a light gas oil fraction. For ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
353
Table IV. Comparison of Densitometer and FID Scan Data Sample: Gas Oil
Densitometer Area Run 1 2 3 4 5
TIME MINUTES DENSITOMETER SCAN
Run I
"
"
10
1 2 3
I
20 30 40 TIME SECONDS FID SCAN
50
2
3
4
TIME MINUTES DENSITO~ETER SCAN
ni
"j,l q 1
i p 8 B
2 3
[L
IO
20
4
30
40
50
60
TIME SECONDS FID SCAN
Figure 10. Chromatograms of a heavy petroleum fraction the FID scan, the developer was cyclohexane-benzene (105 :6) and the sample size was 0.5 p1 (1 :20 dilution of sample with benzene). The plate used for the densitometer scan was coated with Silica Gel-G (Analtech, 250-p). A 3-pl sample was applied to the plate and developed with cyclohexane-benzene-ethyl acetate (105 : 3 : 3). There are certain similarities between the two traces, but it is obvious that the saturates response (the large peak to the right) of the densitometer scan-
354
2 2.0 9.3 9.0 48.4 31.3
3 3.6 6.5 10.1 45.9 33.9
1
2
3
4 2.8 10.0 9.2 45.2 32.8 FID 4
0.1 43.6 56.3
0.2 43.8 56.0
0.1 43.2 56.7
0.2 42.5 57.3
Average 2.9 8.7 9.7 46.0 32.7
Average 0.2 43.3 56.5
ner is much lower than that of the FID scanner, whereas the aromatics response of the densitometer scanner is exaggerated. The FID response is a direct function of w carbon and should be more representative of the actual composition of the sample. Figure 10 shows another comparison of the FID and densitometer scanners for the separation of a heavy petroleum fraction. Repeatability data for both scanners with the above sample are given in Table IV. Differences between the two scanners and TLC media are again evident in Figure 10. If the heavy material at or near the starting point is of prime interest and represents a small proportion of the total sample, it would be desirable to make two runs with the FID. The first run could be made to analyze the bulk of the material and a second run made with a larger sample to magnify the small components near the starting point. It should be pointed out that different developers were used to obtain the densitometer and FID data shown in Table IV. If the same developers were used, the separation obtained on the metal strip would be somewhat different from that seen on a glass plate. Since the FID has high sensitivity, certain polar developing solvents may cause a slight elevation of the base line. It has been observed that the stability of the base line is influenced by the direction in which the TLC-strip passes through the flame. Somewhat greater stability, without any measurable change in sensitivity or response, is obtained when the strip passes from right to left as shown in Figure 4. If the strip passes in the opposite direction, the hot strip heats the collector electrode unevenly, causing base-line noise. The strip still must be passed through the flame several times before chromatography to remove impurities from the silica gel or other adsorbent. The flame temperature can be varied by a change in the hydrogen pressure supplied to the hydrogen jets. For the system described, a hydrogen pressure of 15 psig gave a satisfactory temperature and low noise. If a single adsorbentcoated strip is used repeatedly, a very small amount of cokelike material builds up at the sample application point with some types of samples. The buildup of coke on the adsorbent adversely affects subsequent separations. For this reason, the metal strip generally is recoated for each run. To keep the separated spots or zones relatively narrow, the sample volume applied to the strip should be 1 p1 or less, and the adsorbent layer should not be thicker than 400 microns or thinner than 100 microns in order to avoid spalling.
Figure 9. Chromatograms of a light gas oil fraction
I
1 3.1 9.1 10.6 44.6 32.6
RECEIVED for review October 2, 1969. Accepted December 22, 1969.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
