Anal. Chem. 1982, 5 4 , 2387-2388
LITERATURE CITED
2387
(7) Butler, W. L.; Hopkins, D. W.
.-- .--.
fhofochem. fhotobiol. 1970, 12.
ARO-Arin
(1) Clarke, E. G. C. "Isolation and Identification of Drugs"; The Pharmaceutical Press, London, 1969; p 192. (2) Matthew, W. R.; Browne, H. C.; Weber, J. B. J. Assoc. Off. Anal. Chem. 1972, 5 5 , 789-793. (3) Wallace, J. E.; Biggs, J. D.; Ladd, S. L. Anal. Chem. 1968, 4 0 , 2207-2210. (4) Clark, C. R.; Darling, C. M.; Chan, J. L.; Nichols, A. C. Anal. Chem. 1977. 4 9 . 2080-2083. (5) Predmore; D. B.; Christian, G. D. Anal. Chem. 1976, 48, 361-363. (6) Cahili, J. E. Am. Lab. (Fairfield, Conn.) 1979, 7 1 , 79-85.
(8) Butler, W. L.; Hopkins, D. W. fhofochem. fhotobiol. 1970, 12, 451-456. (9) Lawrence, A. H. J. Am. Leather Chem. Assoc. 1980, 7 5 , 403-407. (10) Fell, A. F. R o c . Anal. Div. Chem. Soc. 1978, 15, 260-267. (11) Talsky, G.; Mayring, L.; Kreuzer, H. Angew. Chem., Int. Ed. Engl. 1978, 17, 785-799. (12) O'Haver, T. C.; Green, G. L. Anal. Chem. 1976, 4 8 , 312-318.
RECEIVED for review April 26, 1982. Accepted July 29, 1982.
Nanoliter Cuvette! Cell for Fluorimetry L. Hirschy, B. Smith, IE. Volgtman, and J. D. Wlnefordner" Department of Chemistry, IJniversity of Florida, Gainesville, Florida 326 1 1
A number of investigators have recently introduced microfluorescence systems (1-10). Work in this area is primarily aimed a t developing low-volume, flow-through detectors for HPLC. Diebold and Zare (1)and Voigtman et al. (2) have introduced windlowless flows cells in which the column effluent forms a droplet that acts as the detector cell. Hershberger and co-workers (3) developed a system that sheaths the column effluent with flowing solvent to provide the fluorescence cell. Recently, Folestad et al. ( 4 ) described an HPLC detector in which the cell is formed by a free falling jet stream of effluent. Volumes for these cells range from 6 nL to 20 pL. Another approach to the development of flow cells has been the use of quartz capillary tubes. Several groups have described such systems (4-8). Although most of these have had microliter volumes or greater, Folestad andl co-workers ( 4 ) did describe one detector cell for which the dead volume was only 1 nL. The absolute detection limits for these flowing systems are quite good (as low as the femtogram level), but they all require a larger initial sample that is subsequently diluted in the chromatographic process. Vurek and B o w " (9) and Vurek and Pegram (10) have described the only microfluorimeters useful for discrete samples. Unfortunately, the cells described had fairly high volumes (200 nL-50 pL) and were made from poor optical quality material. This paper describes what we believe to be the first reloadable nanoliter cell u,seful for discrete samples. The cell is made from the small& commercially available Suprasil quartz tubing (0.05 mm i.d.) and has an optical volume of 0.5 nL. It is designed for convenient sample introduction and easy cleaning. EXPERIMENTAL SECTION Apparatus. A schematic diagram of the nanoliter cuvette cell appears in Figure 1. A 100 mm length of Suprasil capillary tubing obtained from Vitro Dynamics, Inc., Rockaway, NJ (0.05 mm i.d. X 0.08 mm o.d.), is inserted into a 50 mm length of '/I6 in. 0.d. stainless steel tubing (0.010 in. i.d.) so that the one end is flush (50 mm of capillary tubing protrudes from one end of the stainless steel tubing). It is held in place with a drop of epoxy at the end of the stainless steel tubing where the capillary protrudes. The cell is connected via Swagelok fittings to a three-way valve system which allows the top of the capillary to be opened to the atmosphere, flushed with argon, or closed. The '/le in. Swagelok fitting holding the tubing in place allows for convenient cell replacement. The cell is suspended at a 30' angle, as suggested by Folestad et al. (4),to minimize scatter problems. Excitation is achieved with 337.1-nm radiation from a Photochemical Research Associiates (London, Ontario, Canada) Ni0003-2700/82/0354-2387$01.25J0
Table I. Fluorescence Limits of Detection by Use of the Nanoliter Cuvette Cell for Several Model Compounds limits of detection M
coumarin-500 rhodamine-6-G cresyl violet quinine anthracene
1.3 X l o - * 1.8 x lo-' 1.1 x 10-7 1.6 x 10-7 1.4X
ndmL 7 10 32 64 170
fg 3 5
16 32 85
tromite N2 laser. The excitation radiation is focused on the capillary cell with a quartz lens. Fluorescence is collected at a 90" angle to the excitation beam by another quartz lens and focused onto the entrance slit of a Jobin Yvon J Y H10 monochromator, with 1-mm slits. Light exiting the monochromator is detected with an RCA 1P28 photomultiplier tube. The signal from the PM tube is amplified by a PAR 161 current-to-voltage converter, which also serves to stretch the pulse. The stretched pulse is then fed into a PAR CW-1 boxcar averager, which is optimized with an oscilloscope (Tektionix 454) to synchronize the boxcar gate with the pulse. The signal from the boxcar is recorded on a potentiometric strip chart recorder. Reagents. Rhodamine-6-Gand cresyl violet are obtained from Exciton and coumarin-500 is obtained from Photochemical Research Associates, anthracene from Eastman, and quinine hydrochloride dihydrate from Aldrich. Solvents used are ethanol (USP reagent quality, Industrial Chemicals Co.) and deionized water. Procedure. Solutions are prepared from the above reagents and solvents. Successive dilution is utilized to prepare dilute solutions of each compound. Samples are introduced into the nanoliter cuvette cell by opening the valve to the atmosphere and allowing capillary action to pull the solution up. Droplets of 1 pL are easily sampled in this manner, although the cell requires only 5 nL of liquid. After fluorescence measurements are made, the cell is flushed with argon and rinsed several times with solvent, and the next sample is introduced. There is no need to change the capillary except when it is broken.
RESULTS AND DISCUSSION The detection limits of five fluorescent compounds are given in Table I. These values are calculated by taking 3 times the standard deviation of the blank noise and dividing by the slope of the calibration curve. The absolute detection limits were calculated by considering only the region of the capillary illuminated by the laser as the sample volume. As mentioned in the Experimental Section, however, 5 nL of sample is actually required to fill the tube. In either case, this system is capable of detecting subpicogram quantities. @ 1982 American Chemical Society
2388
Anal. Chem. 1882. 54, 2388-2390
fluorescence. If this noise could be minimized so that the limiting noise is the shot noise of the PM tube, then the detection limits could possibly be lowered by up to 4 orders of magnitude. This problem has been effectively dealt with by several groups (5-9), through the use of optical fibers. By use of the fiber to collect the analyte fluorescence along the axis of the capillary tube, collection of the quartz fluorescence is minimized. Another way to minimize the unwanted signal is to time resolve one fluorescence signal from the other. Work toward these improvements is progress.
LITERATURE CITED
Flgure 1. Schematic diagram of the nanollter cwene cell
The detection ability of this system is limited by the blank noise. The blank noise was 4 orders of magnitude higher than the shot noise from the dark current of the PM tube and remained unchanged when the empty cuvette was illuminated or when it was filled with solvent and illuminated. The blank signal peaked at 450 nm, which led us to believe that it was not due to scatter from the laser and was more likely quartz
(1) Diebald. 0. J.; &re. R. N. Scknce 1977. 196, 1439-1441. (2) Volglman. E.; Jurgensen. A,; Winetordner. J. D. Anal. Chem. 1981, 53, 1921-1923. (3) Hershberger. L. W.; Galls. J. 8.; Christian. G. D. Anal. chem. 1979, 51, 1444-1446. (4) Folestad, S.; Johnson. L.: Josefsson, 8.; Galle, 8. Anal. Chem. 1982, 54, 925-929. (5) smim. R. M.; Jackm. K. W.; AldOus. K. M. Anal. Chem. 1977. 49, 2051-2053. (6) Vurek. 0. 0. Anal. chem.1982, 54. 840-842. (7) Sepaniak, M. J.; Yeung. E. S. J . Chromatog. 1980, 190. 3 3 7 3 8 3 . (8) Thacker. L. H. J . Ghromatogr. 1877. 136, 213-220. (9) Vurek, G. 0.; Bawman, R. L. Anal. Biochsm. 1969. 29, 238-247. (10) Vurek, 0. 0.; Pegram, S. E. Anal. Bicchem. 1966. 16, 409-419.
RFCEIYEDfor review June 11,1982, Accepted August 9,1982. This work is supported totally by NIH-GM-11373-20.
Coupling of Fused Silica Capillary Gas Chromatographic Columns to Three Mass Spectrometers Trescott E. Jensen, Ray Kaminrky, Bruce D. McVeety, Timothy J. Wozniak, and Ronald A. Hites' School 01 Publk and Environmental A i f a h and L%pamnt 01 Chemlstty, Indiana Univers& Bbomlngton. Indiana 47405
The connection of the effluent of a gas chromatograph with the ion soulce of a mass spectrometer (GC/MS) has provided the analyst of organic compounds with an instrument more powerful than the s u m of its parts. For the last 15 years, this connection has been achieved by devices that separate the organic compounds from the gas chromatographic carrier gas (I, 2). These devices were a very succeasful strategy when used with packed GC columns. The advent of flexible fused silica capillary GC columns, however, has made such GC/MS interface devices obsolete. Unfortunately, few commercially available instruments are designed fo-r such modern GC columns. We h v e . herefoe. m(dified three H w l e n - p a c M ( ~ pGC/MS ) ins&,mente in our labratory that the end of the GC column directly entersthe ion source, hi^ paper will show that this strategy has been very successful, Thus, we will demonstrate that the best GC/MS interface is no interface.
EXPERIMENTAL SECTION Model 5985B Modification. The capillary column isolation valve assembly was removed from the transfer box heated zone and replaced with a length of in. stainless steel tubing running from the GC oven to the transfer line prohe assembly. A in. Swagelok tee was welded into this length of tubing to provide an entry for chemical ionization reagent gases that would sweep past the end of the column and into the ion source. Reagent gas flow was controlled by a flow controller needle valve and a shut off valve between the GC flow monitor and the tee fitting. A in. Swagelok union was welded to the end of the in. tube in the GC oven. By threading the column through the tube in the GC oven to the exit of the GC/MS probe within the connecting
bellows unit to the souce (and clipping off the column end), the end of column can he brought to within 2 cm of the electron beam. AU fittings are sealed with standard graphite vespel ferrules. With a 30 m x 0.25 mm i.d. J&W DB-5 column and He as the carrier gas at 25 cm/s, the source pressure is 4 X lo4 torr when the column is at ambient temperature. We have found this pressure satisfactory and have not experienced any significant difficulty with background from neutrals. Model 5982 Modification. The direct injection prohe assembly was removed at the source manifold flange. An interface prohe with a '/,$in. Swaeelok fitting welded in one end and a seal flange plate welded on th; other pr&dru the vacuum seal and wnnertiun hetween the GC own and column and the ion oowce. To decrease dead volume and allow simple alignment of the column with the ion source, a stainless steel insert fills the space within the prohe and aligns with the ion souce inlet. To ensure an inert surface in contact with the column, we installed a glass-lined tube within the of this insert, Fused silica capillary column installation is directlv from the GC oven through the interface D r o k . the end is positioned next to the source plkger. In our instrument, the source preasure with He flowing at 25 cm/s through a HP 25 m X 0.22 mm i.d. SE-54 column is 5 X torr. Instrument tuning is done hy introduction of perfluorotrihutylamine (PFTBA) controlled by both a metering and a shut off valve through one of the original sample inlet lines. In a similar manner, chemical ionization reagent gas can be introduced to the source through the other original sample inlet line. Model 5995 Modification. To provide a direct path for the introduction of a capillary column into the ion source, we removed the large source isolation valve. This was done by cutting the valve unit off the interface stem of the vacuum manifold on the manifold side of the valve body. A length of 'I8 in. i.d. stainless steel tubing equivalent in length and diameter to the piece re-
0003-2700/82/0354-2388$01.25/0 0 1982 American Chemical SocieIy
