+I
E
I-
H
W
I
Table I. Precision of Analysis of Dimethylnitrosamine from Aqueous and Aqueous Algal Suspensiona Standard Sample, deviation of Coefficient ng/ml Recovery, peak height of variation Aqueous solution
z
+
10 25 50 100 500
d
0.26 0.59 1.54 0.89 1.32
2.04 1.62 2.07 0.62 0.16
0.29 1.OO 2.08 3.19
2.35 2.77 2.79 2.35
Algal culturea 10 25 50 100 a
96.9 100.0 100.0 95.6
Algal suspensions contain approximately 3 x
lo7
cells/ml
Chlorella ellipsoidea.
Aqueous solution A Algalculture
min and a column temperature of 210 “C allows recovery of DPN and DBN with about the accuracy described above for DMN and a coefficient of variation less than 3 S 7 J for each compound. The combination of the Chromosorb 103 column system, the Ascarite precolumn in the injection port, and the FID results in a sensitive qualitative and quantitative method for the direct analysis of aqueous biological systems for aliphatic Nnitrosamines. The system is about 100 times more sensitive than methods of N-nitrosamine analysis previously reported.
solutions is shown in Table I. The accuracy and precision of the analysis of DMN in algal culture were determined from two injections of six replicate algal solutions spiked with each of the various quantities of DMN. Both the recovery of the added DMN and the precision of the method were very good (Table I, Figure 2). To minimize band broadening so that the quantitative analysis of DPN and DBN can be performed with this system, the carrier gas flow and column temperature must be increased over the conditions listed above. A He flow rate of 140 ml/
RECEIVED for review August 14, 1972. Accepted October 16, 1972. Contribution from the Agricultural Research Service, USDA, in cooperation with Colorado State University Experiment Station, Scientific Journal Series No. 1769. This research was supported in part through the financial assistance of the Environmental Protection Agency. Trade names and company names are included as a matter of convenience to the reader, and such inclusion does not constitute any preferential endorsement by the U. S. Department of Agriculture of products named over similar products available on the market.
QUANTITY OF DMN
(ng/ml) Figure 2. Relationship of concentration of DMN to peak height and recovery of various amounts of DMN added to algal suspensions 0
Flow Programming in Combined Gas Chromatography-Mass Spectrometry Michael A. Grayson, Ram L. Levy, and Clarence J. Wolf Mc Donne11 Douglas Research Laboratories, McDonnell Douglas Corp., S t . Louis, Mo. 63166
IN THE TECHNIQUE of flow programmed gas chromatography (FPGC), the carrier gas pressure at the column inlet is increased in a uniform fashion (linearly, logarithmically, etc.), thereby causing the flow rate through the column to increase with time. FPGC was first described in 1962 (1, 2), and several reviews of this technique have been published ( 3 , 4 ) . M. Morgantini, Boll. Sub. Chim. Procinciali (Bologna), 13, 545 (1962). (2) J. H. Purnell, “Gas Chromatography,” Wiley, New York, N.Y., 1962, p 387. (3) R. P. W. Scott, “Progiess in Gas Chromatography,” J. H. Purnell, Ed., Interscience, New Yolk, N.Y., 1968, p 271. (4) C. Costa Neto, J. 7’.Koffer, and J. W. DeAlencar, J. Chromatogr., 15, 301 (1964). (1)
The major effect of FPGC on a chromatographic analysis is to shorten the time required for the separation of a given mixture in a manner analogous to the more popular technique of temperature programmed gas chromatography (TPGC). However, in certain cases, FPGC is more desirable than TPGC when a reduced analysis time is required ( 3 , 5 ) . For instance, thermally labile compounds which decompose at the upper temperature limit required to elute them using TPGC can be eluted well below their decomposition temperature using FPGC. In addition, the boiling point range of compounds which can be chromatographed on a given column can be ( 5 ) C. J. Wolf and J. Q. Walker, Amer. Lab., 3 ( 5 ) , 10 (1971).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
373
Column outlet flow greater than amount taken by GC-separator
I
GC
+
Separator
-W/+-
MS
Vent taken by
GC.separator GC
GC-separator
Separator MS Separator
MS
Figure 2. Vent connection of column outlet to molecular effusion separator (low flow rate separator)
10
20
30
40
50
GC flow rate at column outlet. cm3 atm/min
Figure 4. Ratio of GC peak area as seen by the TIM and FID plotted against column outlet flow. The GC-SEP restriction accepts 27 cm3 atm/min
Vent
restriction GC
restriction Separator
MS
An Analabs flow programmer (Model FP2078) was used to program the flow rate from 20 to 80 cm3/min. The ion source pressure was monitored during the entire FPGC/MS experiment with a Bayard Alpert ionization gauge. RESULTS AND DISCUSSION
extended by use of FPGC to include those which, if eluted by TPGC, would cause excessive “column bleed,” or even degradation of the liquid phase (5). In combined gas chromatography/mass spectrometry (GC/MS) the sensitivity is limited by the extent of “column bleeding” and only liquid phases that do not mask spectral patterns of the sample compounds can be used (6, 7). In TPGC/MS, the continually changing rate of column bleeding causes a continual change in the intensity and pattern of the background spectra which limits the sensitivity and complicates the extraction of net spectra (6). Wolf andWalker (5)point out that the amount of stationary phase eluated as “column bleed” increases exponentially with temperature but only linearly with flow rate. Hence, less “column bleed’ would result during FPGC. The disadvantages of TPGC in GC/MS have been discussed by Levy et al. (6). The specific advantages of FPGC are, therefore, of special interest in GCjMS where the deleterious effects of the column bleed are pronounced. Despite these apparent advantages, the use of FPGC/MS has received only passing reference in the literature (8). EXPERIMENTAL
The FPGC/MS system consisted of a Bendix time-of-flight mass spectrometer (Model 12-101) coupled to a Beckman gas chromatograph (Model GC-4) with a molecular effusion type separator. The active surface area of the separator was 150 mm2 of porous silver membrane (pore size 0.8 pm). Complete details of the separator design appear elsewhere (8). (6) R. L. Levy. H. Gesser, T. S . Herman, and F. W. Hougen. ANAL.CHEM., 41, 1480 (1969). (7) R. Teranishi. R. G. Buttery, W. H. McFadden, R. T. Mon, and J. Wasserman, ibid.,36, 1509 (1964). (8) M. A. Grayson and C . J. Wolf, ibid., 42, 426 (1970). 374
One of the major problems associated with the use of FPGC together with GC/MS is the development of a molecular separator that operates efficiently over a broad range of carrier gas flow rates. Unfortunately, most molecular separators are optimized to operate at one flow rate. However, several flow path arrangements can be made to circumvent this difficulty. The principal configurations consist of three different approaches: The outlet of the G C column is directly connected to the molecular separator and the entire column effluent is introduced to the separator; the outlet of the G C column is connected to a “flow splitter,” thus allowing only a fixed flow to enter the separator while the excess flow is vented; the separator is optimized for the highest flow rate encountered in the program. In the latter case, an external source of carrier gas is added at the G C outlet to complement the column flow rate. In the first arrangement (Figure l), the increase in flow rate during FPGC produces a pressure increase in the separator, thereby yielding a pressure increase in the ion source of the mass spectrometer. At high flow rates the pressure in the separator exceeds that required for molecular flow through the orifices of the porous frit, and, as a result, the separator functions as a splitter rather than as an enrichment device. In addition, the difference in the column outlet pressure may result in some differences in retention times of the FPGC and FPGC/MS chromatograms. The second approach for connecting the components of the system for FPGC/MS is illustrated in Figure 2 . In this arrangement, the pressure inside the separator remains constant as the flow rate through the column increases. The gas flow in excess of that entering theGC-SEP restriction is vented to the atmosphere. Consequently, the amount of sample lost through the vent increases as the flow rate increases during FPGC analysis. This system functions as a variable splitter and is an unsatisfactory approach because the majority of the sample is vented during the high pressure portion of the flow program.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
0 w
C
E
A
L
3
Y
C
E
L
3
100
L 1
140
180 i 9 6
-
Temperature (OC)
-
196
-
0
20
30
40
-
-
40
Flow rate (cm’/min)
9 5 10 15 20
0
Time (min)
Figure 5. Comparison of elution of n-octane with flow programming ( A ) and temperature programming ( B )
However, this experimental arrangement is ideal when performing isorheic (constant pressure) chromatography since the chromatograms obtained during combined GC/MS will be identical to those obtained during regular GC. The GCSEP restriction is chosen such that the carrier gas flow rate into the separator is slightly less than the lowest carrier gas flow rate of the flow program. An atmospheric vent is provided by a T in the connecting line between the column outlet and the GC-SEP restriction. A small piece of wide bore tubing (-1-mm i.d.) is connected to the sidearm of the T to minimize back-diffusion of air into the separator. If the GC-SEP restriction is designed so that -95% of the carrier gas exiting the column enters the separator, the column outlet is at atmospheric pressure. The third procedure for conducting FPGC/MS is illustrated in Figure 3. An auxiliary helium supply is attached to the venting sidearm of the T. In addition, the GC-SEP restriction and associated separator are chosen so that they will accept the highest carrier gas flow rate encountered in the flow program. The pressure of auxiliary gas supply is adjusted to provide a flow rate slightly greater than the difference between the minimum and maximum flows used during the flow program. The total effluent from the column enters the separator regardless of the flow exiting the column during the flow program. The only gas vented is that of the auxiliary supply. During the initial portion of the flow program, i.e., at low flow rates, the concentration of eluate in the carrier gas stream is diluted by the addition of gas from the auxiliary supply. During the latter stages of the flow program, i.e., at high flow rates, very little dilution occurs since most of the auxiliary gas is vented to atmosphere.
The yield as a function of carrier gas flow rate was determined for a system similar to that shown in Figure 3. An aliquot of sample was injected and the area of the eluting peak was measured by the flame ionization detector (FID) of the G C and the total ion monitor (TIM) of the MS. The same procedure was repeated for several different fixed flow rates. Some flow rates greater than the flow the GC-SEP restriction can accept were used to check the reliability of the system. Ideally, the ratio of the areas of the peaks detected by the TIM and the FID will be constant for all flow rates up to the maximum useable flow rate permitted by the design of the GC-SEP restriction. When the flow from the G C exceeds the flow rate, the peak area ratio decreases. Various fixed helium carrier gas flow rates ranging from 10 to 43 cm3 atm/min were used with a GC-SEP restrictor designed to accommodate 27cm3atm/min. The ratios of the FID to TIM areas as a function of trow rate are shown in Figure 4. The results indicate that the yield is essentially constant for flow rates equal to or less than the design limit of 27 ,ma atmpnin. Thus, flow programming with GCiMS produces no change in yield during the analysis provided that the G C is connected to the MS in a manner similar to that shown in Figure 3. By the appropriate use of vents and an auxiliary gas supply between the G C column outlet and the GC-SEP restrictor, molecular effusion separators can be operated so that the G C column outlet is maintained at atmospheric pressure during an analysis. The major advantages of FPGC/MS can be illustrated in an experiment in which the total-ion-monitor (TIM) chromatograms obtained during FPGC are compared to those observed in TPGC. A single column (5-m by 1.4-mm i.d. packed with 10% SE 30 on 60/80 mesh Chromasorb G) was used and the
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
375
experimental conditions were chosen so that in both FPGC and TPGC, n-octane eluted in 11.5 min. The TIM chromatograms are illustrated in Figure 5. During the flow programmed mode (Figure 5 4 , the column was held isothermally at 100 "C and the flow increased from 6 to 40 cm3atm/min at a rate of 2.8 cm3atmhnin*. During the temperature programmed mode (Figure 5B),the column was heated from 100 to 196 "C at a rate of 8 "C/min with a helium flow of 6 cm3 atm/min. The small peaks before and after the main n-octane peak are isooctane and octene, respectively. The relatively low background current observed in the FPGC analysis illustrates the utility of this method to extend
the useful range of a chromatographic column while minimizing the effect of the "column bleed." The major ions in the background spectra occur at m/e of 133, 147, 189, 191, 205, 265, and 279. The intensity of these peaks in TPGC during the elution of n-octane is more than 10 times larger than in FPGC indicating the advantage of FPGC-MS in obtaining the net mass spectrum.
RECEIVED for review August 14, 1972. Accepted October 19, 1972. This research was conducted under the McDonnell Douglas Independent Research and Development Program.
Chromatographic and Spectral Analysis of Terpene and n-Alkyl Alcohol Carbamates R . C . Gueldner, F . Y . Hutto,' A . C . Thompson, and P . A ; Hedin Entomology Research Dicision, Agricultural Research Sercice, U S D A , State College, Miss. 39762 IN THE COURSE of investigations of the essential oil of the cotton plant, Gossypium hirsutum L., Minyard ( I ) found several tertiary alcohols that were difficult to separate from other oxygenated classes by column chromatography and that were dehydrated during GLC. Hedin, Gueldner, and Thompson (2), therefore, developed a procedure based on the work of Goodlett (3) to prepare the carbamates of several terpene alcohols by reaction of trichloroacetyl isocyanate (TCAIC) with the alcohols and then hydrolysis of the TCAIC esters to the carbamates with aqueous methanolic KOH. This procedure was used to isolate the terpene alcohols from the other oxygenated compounds present in the essential oil ofthe cotton plant. The present paper describes the preparation of the carbamates of several terpene alcohols and other alcohols by the method of Hedin, Gueldner, and Thompson ( 2 ) and their spectral characterization and the conditions for the separation of the carbamates by TLC and GLC.
2500
4000
1OOC
'
'
1500
1300
900
1100
'
0
2
4000
8
6
4
2333
1500
1300
10
12
WO
1100
1
100
EXPERIMENTAL
Apparatus. All infrared spectra were obtained with a Perkin-Elmer Model 521 infrared spectrophotometer. Proton magnetic resonance spectra were obtained with a Varian A-60 analytical NMR spectrometer. GLC separations were performed on a Varian Aerograph 90-P TC instrument with the following columns and conditions: Column A, 0.0064- x 0.610-m aluminum column packed with 16 DEGS on 60/80 mesh Gas Chrom P treated with HMDS. Carrier gas flow He at 200 ml/min, column temperature 160 "C, injector 180 "C; Column B, 0.0064- X 1 Present address, Department of Agronomy, Mississippi State University; State College. Miss. 39762.
(1) J . P. Minyard, Jr., "Constituents of the Cotton Plant: The Volatile Fractions," University .Microfilms, Ann Arbor. Mich.? NO,67-13,768 (1967). (2) P. A. Hedin. R . C. Gueldner, and A. C. Thompson, ANAL. CHEW.42,403 (1970). ( 3 ) V. W. Goodlet; ibid.. 37,431 (1965). 376
I
0
2
4
6
8
10
12
Figure 1. Infrared spectra of ( A ) (-)-isopulegol, and ( B ) (-))-isopulegol carbamate plotted as % transmission cs. cm-I
1.524-1-11 aluminum column packed with 20% Apiezon L on 60/80 mesh Gas Chrom P treated with HMDS. Carrier gas flow, He at 160 ml/min; column temperature 180 "C, injector 205 "C, detector 210 "C. Reagents. Trichloroacetyl isocyanate was obtained from Eastman Organic Chemicals (reagent 3937) and used without purification. Procedure. PREPARATION OF CARBAMATES. The alcohol (0.0172 mole) was dissolved in 30 ml of CC1,. After a slight molar excess (0.023 mole or 2.2 ml) of TCAIC was added, the mixture was allowed to stand in an ice bath for 5-10 min, and then it was extracted with 50 ml of 5 % KOH in 80% aqueous methanol. The aqueous basic methanol phase was
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
