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Anal. Chem. 1984. 56. 1507-1514
turn converted into current-loop pulses for reliable distant transmission by U5, transistor 2N3906, and optoisolator 4N33. The remaining BCD digits of xinare then processed. STEP goes high, so that SELECT goes high and U3 is reset. U7 then selects yin,and its three and one-half digits are processed as for xin. Except for the last BCD digit of yin, each nibble coming from pin 20 to 23 of U8 is attached to a special nibble of 0011 on the most significant side of the 8-bit byte input of UART U9. Each whole-digit byte thus becomes a hex code 30-39 (which corresponds to an ASCII of “O”-‘9”). It also makes each half-digit byte into a hex code from 3A to 3F (ASCII, u.n-u?n . . ). The specifications of chip U8 provide details for reconstruction of xin and yinat the computer. Pin 6 of U14 goes low only for the last whole digit of yin, changing the above-mentioned special nibble into 0010. The acquisition program thereby monitors this digit according to lines 1090-1100 so that acquisition always begins with xin. Lines 1190-1200 restore the special nibble for this digit to 0011 for further processing. Following analog-to-digital conversion of yin,STEP goes high, making END low and CLKEN high. Conversion and transmission then halt until arrival of the next pulse from the sampling rate generator at pin 11 of U4.
The parameters of UART to be specified by the computer RS-232 interface are as follows: 1200 baud; 8 data bits; 1stop bit; no parity. +V is connected to +5 V of the RS-232 interface, and pin 3 and 7 of the DB25 connector of the interface are connected to the transistor leads of 4N33 as shown. Registry No. Cytochrome c, 9007-43-6; ribonuclease A, 9001-99-4.
LITERATURE CITED Privalov, P. L.; Khechinashvili, N. N. J . Mol. Biol. 1974, 8 6 , 665-684. Privalov, P. L.; Filimonov, V. V.; Venkstern, I. V.; Vayev, A. A. J . Mol. Biol. 1975, 9 7 , 279-288. Schott, F. J.; Grubert, M.; Wangler, W.;Ackermann, Th. Biophys. Chem. 1981, 14, 25-30. Bevington, P. R. ”Data Reduction and Error Analysis for the Physical Sciences”; McGraw-Hill: New York, 1969; pp 235-242. Savitzky, A.; Golay, M. J. E. Anal. Chem. 1964, 3 6 , 1627-1639. Bevington, P. R. “Data Reduction and Error Analysis for the Physical Sciences”; McGraw-Hill: New York, 1969; pp 265-271. Privalov, P. L.; Khechinashvili, N. N.; Atanasov, B. P. Biopo~mers 1971, 10, 1865-1890. Jackson, W. M.; Brandts, J. F. Biochemistry W70, 9 , 2294-2301. Krishnan, K. S.; Brandts, J. F. Methods Enzymol. 1978, 4 9 , 3-14.
RECEIVED for review December 14,1983. Accepted March 22, 1984. This work was supported by a grant to A.G.M. from the U.S.A. Public Health Service (NIH 1R01 GM-29274-03).
Postcolumn Addition of Buffer for Thermospray Liquid ChromatographylMass Spectrometry Identification of Pesticides Robert D. Voyksner,* Joan T. Bursey, and Edo D. Pellizzari Analytical and Chemical Sciences, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709
Coaxlai and right-angle tees were evaluated as methods of postcoiumn buffer addition for thermospray LC/MS analysis. The coaxial tee, which showed dlghtiy better total ion current stability, was optimized to produce the best sensitivity. This tee was used together with a gradient LC separation to obtain thermospray LC/MS spectra for 15 carbamate and urea pesticides. The detection limits for these pesticides are also reported.
Carbamate pesticides have high usage in the United States by virtue of their high effectiveness and low mammalian toxicity ( I , 2). The ability to separate and identify these pesticides is important because of their increasing presence in the environment, along with their degradation and metabolic products. Analytical procedures for determination of these pesticides are rather limited. The pesticides are thermally labile which prevents direct analysis by gas chromatography. Spectrometric methods lack specificity or sensitivity (3-5). Although high-performance liquid chromatography (HPLC) is ideally suited for carbamate separation, mass spectrometry is appropriate for their detection. Liquid chromatography/mass spectrometry (LC/MS) has developed several approaches to overcome the problems as-
sociated with coupling the effluent of the LC to the source of a MS, including moving belt (6),direct liquid introduction (9, thermospray (€9,atmospheric pressure ionization (9),and semipermeable membrane (10). Although the moving-belt and direct liquid introduction (DLI) interfaces have been used in the analysis of carbamates (11-16), these techniques have been hampered by either lack of sensitivity or thermal degradation of the sample. The DLI interface usually requires a 1/100 split, and the moving belt requires the sample to be thermally desorbed from a surface and sometimes requires a split depending on the solvent system used. On the other hand, thermospray LC/MS does not require splitting the LC effluent or thermal desorption. Thermospray (TSP) is also an ionization technique, the exact nature of which is still being investigated (17, 18). In qualitative appearance, the spectra resemble field desorption and ammonia chemical ionization spectra (17). The ionization process requires that a volatile buffer be present in the LC effluent. The most commonly used buffer has been ammonium acetate; however, others have been reported (18). Most LC separations are developed before consideration of the use of mass spectrometry as a detector. This situation poses a problem for TSP LC/MS analysis since a separation scheme which has already been developed cannot be used unless ammonium acetate or an equivalent buffer is present for ion formation. If a buffer is added to accommodate the
0003-2700/84/0356-1507$01.50/00 1984 American Chemical Society
1508
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984 Asulam
A t
Fluometuron
Right angle tee
0.015-cm Stainless Steel Tubing
HPLC In 0.8 to 2 mLimin Figure 1. Schematic of thermospray interface: (A) vaporizer; (B) vaporizer thermocouple; (C) jet chamber: (D) source thermocouple; (E)ion exit cone; (F)aerosol thermocouple; (G) lenses; (H) quadrupole assembly; (I) liquid nitrogen trap and forepump; (J) source block heater; (K) vaporizer heater.
Coaxial tee
Right Angle Tee SSI 01.0165 Zero Dead Volume Tee
I I
i
1
0 inj.
2.5
5
Time (min) Flgure 3. UV traces used to evaluate coaxial tee and right-angle tee.
0.0625" Tubing
I
l j I
I
:
,
J
Conditions: 60% MeOH in H20 at a flow of 1.2 mL/min through a Zorbax CN column, UV 254 nm, with 0.5 M buffer added at a flow of 0.4 mL/min into the tee.
addition of the buffer solution and have evaluated TSP LC/MS for the analysis of carbamate and urea pesticides. YBuffer Addition
Coaxial Tee
Column
0.875"
-----
t
Buffer Addition
Flgure 2. Schematic of the two tees evaluated for postcolumn addition.
TSP interface, the chromatographic resolution may suffer. Other problems arise in performing gradient elution analysis. Many buffers are not soluble in one of the solvents, resulting in a variation of the buffer concentration during the analysis. This variation in buffer concentration can produce changes in sensitivity due to dependence of the operation of the TSP on buffer concentration (19). These problems would be irrelevant if a method to add the buffer postcolumn could be developed. Previously established LC conditions then could easily be translated to TSP LC/MS with no buffer concentration variance. In this paper we have explored methods for postcolumn
EXPERIMENTAL SECTION Equipment. The LC consisted of two 6000A pumps (Waters Assoc., Milford, MA) controlled by a 720 system controller (Waters ASSOC.,Milford, MA) and a Model 709 pump (Milton Ray Co., St. Petersburg, FL) used for the postcolumn addition of buffer. The samples were injected with a U6K injector (Waters ASSOC., Milford, MA) and separated on a Zorbax CN column, 25 cm X 4.6 mm (Du Pont Inst., Wilmington, DE). The mixing tees evaluated for postcolumn addition were located between the column and UV detector. A 440 UV detector (Waters Assoc., Milford, MA) set at 254 nm and a 2-wm filter (Rainin Inst., Woburn, MA) were in line before the TSP interface. The TSP interface (Figure 1) (Finnigan MAT Co., San Jose, CA) was installed on a Finnigan 4500 quadrupole mass spectrometer. The interface included a temperature controller and readout, The temperature zones monitored were the vaporizer, source jet, and aerosol (just past the ion exit cone). Electrical cartridge heaters were used in the source and vaporizer assembly. This interface did not require any splitting of the LC effluent. The large volume of solvent was pumped out of the source with a liquid nitrogen cold trap prior to a mechanical rough pump. The mass spectrometer was operated with an ion entrance voltage of -15 V, extractor voltage of -30 V, lens voltage of 0 V, and a quadrupole entrance of -48 V in the positive ion mode. The instrument was scanned from mlz 150 to 550 at a rate of 2 s per scan. The presence of numerous solvent-buffer cluster ions limited the lower mass range available to the technique (20). The data were collected and processed with an INCOS data system (Finnigan MAT Co., San Jose, CA).
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
-
No tee __
Table I. Dependence of Sensitivity on the Percentage of Water for the TSP Analysis of Identical Injections of Four Pesticides absolute areab ion 5% 50% 95% compd obsvda H20 H*0 H2O 248 3500 316000 500000 asulam 418000 993000 208 8800 aldicarb 516 000 741 000 233 7600 fluometuron 113000 500000 270 1500 alachlor
Asulam Aldicarb Fluometuron Alachlor
a The base peak of the compound which was used for the absolute intensity measurements. Pesticides analyzed through a sample loop individually in a water and methanol mobile phase containing 0.1 M ammonium acetate. The amounts injected and instrumental conditions were the same for the three different water solutions used.
The pesticides were obtained from the EPA repository (Research Triangle Park, NC) and ranged in purity from 99.9 to 95%. Standards were diluted with methanol or acetonitrile in water. Instrumental Operation. The mass calibration of the quadrupole was verified daily with poly(propy1ene glycol) (mol wt 3000). The separation scheme for the carbamates and ureas previously developed was a linear gradient of 50% methanol in water to 70% methanol in water in 15 min. The 70% methanol in water solvent composition is held for 5 min before cycling to the initial 50% methanol composition. The TSP interface was optimized by watching for the best stability and intensity of the solvent-buffer ion in the mass range of 30 to 150 on the oscilloscope. Previous experiments showed that the analyte signal correlated very closely with the solvent signal (21). By recording the optimal conditions at the two extremes of the gradient, the temperatures could be varied in sequence with the change in solvent composition. The optimal temperatures for 50% methanol in water were vaporizer 210 O C , areosol 165 "C, and jet 190 "C for a flow of 1.6 mL/min. At 70% methanol in water these temperatures were 208 "C, 145 "C, and 182 "C, respectively. During gradient analysis, the vaporizer and jet-source heaters were manually reduced in power to linearly reduce the aerosol temperature from 165 to 145 "C. Even at optimal settings,the solvent ion current fluctuated as much as 20%. For all samples analyzed, both UV and mass spectrometric detection were used.
1509
Right angle tee Aldicarb
I
Fluometuron
Aldicarb
h
Coaxial tee
0
Fluometuron
I
I
2.5
5
Time (min)
RESULTS AND DISCUSSION
Flgure 4. TIC traces for no tee and two different tees at constant conditions of 1.6 mL/min into the mass spectrometer, gain, sample, and column. For the no-tee situation, 0.1 M ammonium acetate was added through the column. I n the right-angle and coaxial tee, buffer was added postcolumn so final concentration is 0.1 M.
Two methods of postcolumn buffer addition were examined, involving the use of a right-angle tee or a coaxial tee (Figure 2), respectively. Both tees were located between the UV detector and LC column. A 0.5 M ammonium acetate solution added through the tee a t a rate of 0.4 mL/min resulting in a 0.01 M final concentration (ammonium acetate) when a column flow of 1.2 mL/min was used. Each tee was evaluated in the analysis of four pesticides by using isocratic LC conditions. Figure 3 shows that there was no loss of resolution when either tee was incoporated. In fact, the chromatographic peak width decreased, due to the increased flow through the UV cell, giving the appearance of increased resolution. The coaxial tee appeared to be the better choice since it appeared
to produce less base-line noise than the right-angle tee. The total ion current traces from the HPLC/MS analyses of the pesticides show little difference between the two tees (Figure 4). The coaxial tee appeared to produce less noise and was ultimately chosen over the right-angle tee for postcolumn buffer addition. Figure 4 also shows the degradation of the established pesticide separation when ammonium acetate is added in a precolumn fashion, demonstrating the necessity of developing a method of postcolumn buffer addition. Another advantage of having postcolumn buffer addition was the increased MS intensity for the four pesticides compounds compared to precolumn additions of the buffer. Ion intensities were 3-5 times greater with the postcolumn buffer
Table 11. Effect of Buffer Flow into the Coaxial Tee on Sensitivity column flow,= buffer flow, mL/min mL/min 1.2 1.1 1.0 0.9 0.8 60/40 methanol/water.
0.4 0.5 0.6 0.7 0.8
f~
aldicarb m/z 208 24 000 48 300 59 000 46 500 50 000
absolute intensity for base peak asulam fluometuron m/z 248 m/z 250 3 800 15 400 27 000 20 000 29 000
0.5 M ammonium acetate in water added coaxiallv.
12 300 18 700 2 1 000 19 000 18 000
alachlor m/z 287 3900 5500 4600 3800 3100
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ANALYTICAL CHEMISTRY. VOL. 56, NO. 8, JULY 1984
LC Chromatogram Using U V -ion
Inj.
I
I
I
I
I
0
5
10
15
20
Time (min)
Total Ion Chromatogrnm
3:20
6:40
1o:oo Time (min)
1320
16:40
2o:oo
and total ion chromatogram for the analysis of 15 pesticides, 600 ng injected. Condiions: 50% methanol in water to 70% methanol in 15-min linear gradient, at a flow of 1.2 mL/min. 0.5 M ammonium acetate added postcolumn at a flow of 0.5 mL/min. Figure 5. UV
addition and were primarily due to the increased volume of water (Table I). Coaxial Tee Optimization. The postcolumn addition of buffer through the coaxial tee was optimized to produce the highest possible sensitivity and resolution (peak shape). As
previously shown, the postcolumn addition of water and buffer increased sensitivity. However, if more water was added postcolumn to take advantage of this enhanced sensitivity, the sample concentration was decreased. Thus, an optimal column/buffer flow rate was expeded to exist which provided
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
Table 111. Structure, Mass,Intensity, and Proposed Ion Composition for the Pesticides Analyzed by HPLC/MS compd and structure asulam H2N
0
230
190 231 248
100
191 2 08
100
[M [M
+ HI' + ",I+
232
233 250
100 54
[M [M
+ HI' + NH,]'
24 8
249 266
100
300
185 241 301 318
100 35 2 59
3 66 383
100
190
R
YH3
proposed composition a
ions
S02NHCOOCH3
aldicarb
relative intensity
MW
37 8
14
---
[M + HI' [M + NH,]'
CH3S-Z -7=N-O-C-Fi]-CH3 H3 H
H
fluometuron 0
" P A linuron
5)
1
N-C-N,
,CH3 O-CH3
phenmedipham
CH3-0-C-N-H
70
IN + HI'
[M
+ ",I+
___ ___ IN + HI+ [M
+ ",I+
CHI
benzoylprop ethyl
365
28
[M + H]+ [M + ",I+
CI H3 C-F-$-O-C2 Hg H O
carbofuran
m
(
c
H
221
3
i
222 239
67 100
[M [M
+ HI+ + ",I+
2
0-C-N-CH3 I1 I O H
diuron
C
232
l
[M + HI+ [M t ",I+ [M + (",),I'
233 2 50 278
100
182 192 301 318
12 44
-__
1 100
[M
209
210 227 4 36
69 100 4
[M + HI+ [M + ",I+ [M, + ",I+
201
202 219
100
420
2
30 6
bH &-NICH~IZ
desrnedipham
300
-__
+ HIt
[M + ",I+
N-C-0-C2 Hg
propoxur
??
0-C-N-CHs
carbaryl or
0-C-N-CH,
12
+ HI+ [M, + NH]+ [M
[M+ ",I+
1511
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
Table I11 (Continued) compd and structure BPMC
MW
ions
relative intensity
207
208 225
100
0
17
proposed compositiona [M + HI+ [M + NH,]'
0-C-NH-CH3
alachlor
269
CHzCH3
propachlor
211
0
CH(CH312
kJ-l-CH,C!
a
238 270 287
212 224 423 440
37 100
54
100
48 2
