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Anal. Chem. 1981, 53, 1963-1965 TO 'VACUUM SYSTEM
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Figure 1. Simplified sample tube cracker: (A) indentation to prevent plug from sliding up tubing; (13) Teflon disks, '/e in. thick with a 1-2 mm diameter hole through the middle; (C) disk cut from 200-mesh screen; (D) size 1817 groundl-glass ball joint; (E) size 18/9 groundglass socket joint; (F) position of scratch on sample tubing; (G) FETFE O-ring; (H)size 7 Ace-Thred connector; (I) nylon bushing from Ace-Thred connector; (J) sarrple ampule made from 6 mm 0.d. glass tubing. The ball and socket jolnt must be held together with a spring-tension clamp; the clamp is not shown.
flexible tubing and requires some machined parts. Although this method is effective, sometimes the tubing shatters if it is not positioned properly within the tube cracker. Also, the cost becomes a significant €actorif multiple tube crackers are needed. Consequently, another method has been developed that consistently results in clean breakage of the glass tubing and utilizes readily available, low-cost materials. The apparatus shown in Figure 1,for cracking 6-mm 0.d. tubing (J),utilizes a size 18/7 ground-glass ball joint (D) and an adaptor consisting of a size 18/9 ground-glass socket (E), attached to a size 7 Ace-thred connector (H) (Ace Glass Inc., Vineland, NJ). The two halves of the ball and socket joint, are greased with
high-vacuum stopcock grease and must be held together with a spring-tension clamp (this is not shown in the figure). Thle tubing to be cracked is scratched about 3 cm from the end and then inserted through the O-ring (G)until the scratch is positioned at the base of the ball joint (F). The bushing (I) is tightened to seal the O-ring aroung the tubing and the system is evacuated. The tubing may then be cracked by slightly flexing the adapter; if the tubing is correctly positioned, little force is needed to crack it. If the tubing doer3 not break easily, it cain be repositioned without opening the system by sliding it up or down through the O-ring seal. Because the scratch can be seen through the glass adapter, correct positioning is easily achieved. A Teflon plug (B) with a small (1-2 mm diameter) center hole may be pressed into the connecting tube of the ball joinit to prevent the upper portion of the tubing from being blown up into the vacuum system when the tubing is cracked or when the bushing (I) is loosened to vent the system. In Figure 1, a small disk of 200-mesh screen (C) has been sandwiched between two center-drilled disks (B) that were cut from a l/s in. thick sheet of Teflon with a stopper-boring tool. The fine mesh screen prevents any small chips of glass from entering the vacuum system. Changing sample tubes is easiest if the bushing (I) is completely removed. The O-ring will come out with the lower portion of the sample tube and allow the upper portion of the sample tube to fall out. Although the upper ball joint may be a size 18/9, the smaller inside diameter of a size 18/7 ball joint results in a tighter fit around the 6-mm tubing and thus less flexure is required to crack the tubing. The use of excessive grease on the ground joints should be avoided to prevent grease from squeezing out at the base of the ball andl causing the upper portion of the cracked tubing to stick behind! when the lower portion is removed. Although there should be no problems with leakage with high-quality ball and socket, joints, if problems do develop, the joints can be reground withi a slurry of grinding compound. The use of O-ring type ball. joints is not recommended as they are more likely to leak when flexed during the fracture operation. This system has been used successfully for cracking both Pyrex and quartz tubing. The method is equally effective for 9-mm 0.d. tubing if a size 11Ace-Thred connector and a size 18/9 ball joint are used. However, the inside diameters of the ground joints vary from one manufacturer to another and, because of the closer tollerances encountered when using 9-mm tubing, joints should be chosen that easily accept the tubing that is to be cracked.
LITERATURE CITED (1) DesMarais, D. J.; Hayes, J. M Anal. Chem. 1978, 48, 1651-1652.
RECEIVED for review April 27, 1981. Accepted July 6, 1981.
Micro Liquid Chrormatography/Mass Spectrometry Diaphragm Probe Interface Jack D. Henion" Dlagnostlc Laboratory, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
Timothy Wachs Department of Chemlstty, Cornell University, Ithaca, New York 14853
Several groups have reported LC/MS results on a variety of compounds (1-4)in the recent past. The referenced work generally utilizes either the moving belt introduced by Scott (5) or the direct liquid introduction (DLI) LC/MS interface
first reported by McLafferty (6). These and other approaches have provided an increasingly viable means of accomplishing LC/MS, but routine sensitivity has not been comparable to that afforded by GC/MS. Many resesarchers involved with
0003-2700/81/0353-1963$01.25/00 1981 American Chemical Soclety
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Flgure 1. (A) micro LC effluentinlet line, (6) water cooling inlet tube, (C) Teflon washer for malntainlng vacuum seal between probe tip/ cooling chamber and probe shaft, (D) throughput tube collet, (E) 0.010 in. i.d. X in. 0.d. stainless steel throughput tube containing the 0.004 In. 1.d. X 0.008 in. 0.d. mlcrobore tubing, (F) water cooling chamber, (G)Kalrez 3-ring, (H) diaphragm containing 5 p m pinhole in center, (I)
removable endcap. environmental and toxicology studies require LC/MS detection limits better than currently available. We report the construction and preliminary results of a modified DLI micro LC/MS interface patterned after the unit recently introduced by Hewlett-Packard Co. (7).This device offers several advantages over existing commercial units which include: (a) increased routine sensitivity approaching that afforded by modern GC/MS, (b) reduced construction and LC solvent costs, and (c) practical add-on to existing GC/MS instruments equipped with a chemical ionization (CI) source. While micro LC/MS has been proposed (8) and reported (9-11), the present apparatus is the first to combine the simplicity of unchanged, commercially available conventional LC pumps and hardware with total effluent introduction into the mass spectrometer (MS). EXPERIMENTAL SECTION In this DLI micro LC/MS probe interface (Figure l),a central, small bore (0.004 in. i.d.1 stainless steel tube transfers total micro LC effluent to the water-cooled probe tip housing a thin, stainless steel diaphragm. The diaphragm (Optimation, Derry, NH) has a precisely centered “one-shot” laser generated pinhole (5 pm diameter) which is held firmly against the exit end of the small bore tubing at the probe tip. The threaded end-cap accomplishes this by pressing the diaphragm against the O-ring and the probe tip pedestal. The metal-tu-metaldiaphragm seal affords zero dead volume at the probe tip while permitting totaltransfer of the micro LC effluent through the 5 pm pinhole into the CI ion source. The rounded end-cap butts against the ion source probe inlet affording the necessary “tightness” for CI. Construction of the probe tip is facilitated by a two-part process. The entire cooling chamber (Figure 1) and diaphragm support pedestal me fabricated separately from the main probe shaft. This allows machining the cooling chamber, soldering the three ‘/I6 in. tubes comprising the throughput and coolant tubing, and smoothing the pedestal surface. A Teflon gasket provides a vacuum and coolant seal when the diaphragm support pedestal is tightened onto the main probe shaft. The central small bore throughput tubing (0.004 in. i.d. X 0.008 in. o.d., Part No. HTX-32, Small Parts, Inc., Miami, FL) is soldered casefully inside a 0.010 in. i.d. X ‘/I6 in. 0.d. stainless tube to afford strength and ease of coupling to the micro LC column exit. The overall length of this throughput tube is kept to a minimum to reduce extra column effects. Both ends are machined smooth and flat for efficient transfer of total micro LC effluent. The micro LC column exit is attached directly to the micro LC/MS probe throughput tube with a symmetrical in-line Valco low dead volume filter union (P. J. Cobert, St. Louis, MO, Part No. 4078). The UV *letectoris eliminated from the system to exclude its large extra column effects. The micro LC column used in this work was a 1.0 mm i.d. X 1.58 mm 0.d. x 15 cm 10 pm HRSM C18column (C-M Laboratories, Nutley, NJ). It had a plate count of 9000 plates/m and was attached directly to a U6K injector (Waters Associates, Milford, MA). The injector was connected by 0.010 in. i d . tubing through a 0.4 pm in-line filter (HewlettPackard Catalog No. 7814-62701,Palo Alto, CA) to a Model 6000 pump driven by an M-660 solvent programmer (Waters Associates, Milford, MA). Micro LC flow can be accomplished with this
Flgure 2. NCI micro LC/MS TICP and EICP for 30-ng levels of dexamethasone and 6-P-hydroxyprednisoioneusing 50 % CH,CN/H20 at 34 pL/min as micro LC/MS eluant/CI reactant gas. The micro LC column was a C,, HRSM connected to an unchanged Waters ALC-202 pump and solvent programmer. X X SFECTRUR D I S P L A \ ‘ / E D I T
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PC‘I LC PlS OF S T E F Q I D S F L U S DES USING 50.4 CH3CN/H20 100 LIL PlIN FLULI 15 CW HRSN C18
Flgure 3. PCI micro LC/MS TICP and EICP for 100-ng levels of the steroids shown in Figure 2 in the presence of trans- and cisdiethylstilbestrol (DES) using 50% CH,CN/H,O at 34 pL/min as micro LC/MS eluant/CI reactant gas. The trace 13-min component represents 20 ng of DES.
equipment by setting the pump at 0 mL/min eluant flow while the M-660 solvent programmer is set at 0.1 mL/min B with the percent B set at 50%. This combination of settings on the unchanged hardware yields a nominal eluant flow rate of 50 pL/min but a measured flow rate of 35 pL/min. This system provided reliable micro flow under these conditions. When the above conditions had been established, the micro LC/MS probe produced the steady, 1in. long “jet” of total micro LC effluent (CH,CN/H,O) necessary for optimum micro LC/MS performance as determined by previous experiments. When the “jetting” micro LC/MS probe was inserted into the standard solid probe inlet of a Hewlett-Packard 5985B MS, a stable ion current base line from m / z 100 to 500 was maintained over 4-h time periods. The ion source temperature was maintained at 250 “C and the liquid nitrogen cryropump on the MS was operated in the normal manner. The MS was operated in either the NCI or PCI mode in these experiments using the micro LC eluant as the CI reactant gas (6). The compounds studied in this work display comparable micro
ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981
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Flgure 4. (A, upper) Micro LC UV trace for a TLC scrape of an unknown sample confiscated from a race track. The flow rate was 34 pL/min 50% CH,CN/H,O on a C,* HRSM mlcro LC column and UV detection was 239 nrn. (B, lower) NCI micro LC/MS TICP and EICP for the sample described in 4A. The major component observed at 2.8 min retention time was shown to be dexamethasone.
LC/MS sensitivity response for both NCI and PCI modes, although certain molecules containing more electronegative hetero atoms can reveal enhanced sensitivity in the NCI mode (I). In the micro LC/MS results described below, the MS was scanned repetitively from m/z 100 to 400 in 3 s at an ion source pressure of 0.8 torr measured at the GC/MS transfer line 6 in. from the ion source. Data acquisition commenced at sample injection.
RESULTS AND DISCUSSION The micro LC/MS extiracted ion current profiles (EICP) (12) of the NCI mass spectral base peaks for 30 ng each of 6-P-hydroxyprednisolone and dexamethasone using 50% CH3CN/H20 as micro LC/MS eluant/CI reactant gas are shown in Figure 2. These steroids are easily resolved by the C18 HRSM micro LC column used in this work but have not been adequately separated by micro LC equipment reported earlier (10). Thus, the improved separation and comparable sensitivity of this system offer increased versatility for inicro LC/MS analysis of mixtuire components that are difficult to separate chromatographically. When a mixture of 100 ng levels of the above mentioned steroids and diethylstilbestrol (DES) was subjected to PCI micro LC/MS under the same micro LC conditions, the EICPs shown in Figure 3 were obtained. These components can be found at trace levels in variious biological tissues and are often difficult to differentiate from endogenous materials. The EICPs of the (M f 1)’ ions for trans- and cis-DES isomers are readily observed at 7.8 and 12.8 min, respectively, in Figure 3. The cis component represents 20 ng of material detected under these micro EC/MS conditions. Selected ion moni-
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toring (SIM) micro LC/MS techniques could offer an alternative to identifying these materials at low levels in biologicid matrices (13). The PCI micro LC/MS base peaks for 6-13hydroxyprednisolone ( m / z 317.3) and dexamethasone (mlz 333.3) are also readily detected and their PCI micro LC/MS full scan ( m / z 125-4010) mass spectra (not shown here) easily differentiate these important steroids. Figure 4 shows the micro LC UV trace and NCI micro LC/MS total ion current profile (TICP), respectively, for a thin-layer chromatography (TLC) scrape of an unknown sample confiscated from a race track. The micro LC UV trace shown in Figure 4A reveals just one component following a known solvent impurity at 2.0 min retention time. However, in the NCI micro LC/ MS EICP shown in Figure 4B, the major component at 2.8 min retention time is joined by two smaller components at 1.6 and 4.4 min retention time that were not observed in the UV trace. The m / z 297.3 and 390.3 ions at 2.8 rnin retention time represent the NCI base peak and (M - 2)- for dexamethasone. The m/z 100-450 NCI full scan mais spectrum of this comiponent was identical with that of auithentic dexamethasone obtained under the same conditionn. The unknown compoinent at 4.4 min has a base peak at m/z 127 in its NCI micro LC/MS spectrum with an apparent molecular weight of 366. This unknown material appears to be unrelated to the dexamethasone identified as the major component at the 2.8-min retention time. It is interesting to note that this 7-min micro LC/MS experiment identified dexamethasone in the suspect TLC scraped “spot” and detected at least two other components in the TLC scrape thait had previously gone unnoticed. This phenomenon is not uncommon with LC/MS due to the unique specificity and sensitivity of an MS as a detector.
ACKNOWLEDGMENT The authors wish to express their sincere thanks to R. Jenkins and E. Cramer for their assistance with the design and construction of the micro LC/MS interface and P. J. Henton for preparing the TLC scraped sample.
LITERATURE CITED (1) McFadden, W. H. J . Chromatogr. Scl. 1980, 18, 97-115. (2) Henion, J. D.; Mayiin, 0. A. Blomed. Mass Specfrom. 1980, 7 , 115-1 2 1. (3) Karger, B. L.; Kirby, D. P.; Vouros, P.; Foltz, R. L.; Hidy, B. Anal. Chem. 1979. 51. 2324-2328. (4) Arplno, P. J.; Guiochon, G. Anal. Chem. 1979, 51, 682A-701A. (5) Scott, R. P. W.; Scott, C. G.; Munroe, M.; Hess, J., Jr. J . Chromatcgr. 1974, 99, 395-399. (6) Baidwln, M. A.; McLBfferty, F. W. Org. Mass Spectrom. 1979, 7 , 1355-1354. (7) A. Melera, Paper No, 085, 30th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, OH, March 5-9. 1979. (8) McLafferty, F. W.; Baldwin, M. A. U.S.Patent 3 997 298, 1976. (9) Tsuge, S.; Hirata, Y.; Takeuchi, T. Anal. Chem. 1979, 51, 166-169. (10) Henion, J. D. J. Chromatogr. Scl. 1981, 19, 57-64. (11) Schafer, K. H.; Levsen, K. J . Chromatogr. 1981, 206, 245-252. (12) Watson, J. T.; Falkner, F. C.; Sweetman, B. J. Blomed. Mass Spec.. trom. 1974. 1 . 156.
(13) Day, Ei-W.,’ J;. Vanatta, L. E.; Sleck, R. F. J . Assoc. Off. Anal. Chem. 1975, 58, 520-523.
RECEIVED for review June 5, 1981. Accepted July 23, 1981. This paper is based on ,work supported by the New York State Racing and Wagering Board and the Harry M. Zweig Memorial Fund for Equine Research and was first reported at the 32nd Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Paper No. 753, Atlantic City, NJ, March 9-14, 1981.
