Anal. Chem. 1902, 5 4 , 2368-2372
2360
ference and was inert to most substances. The angle of incidence of the beam with the SS tip which yielded the highest ion abundance was found to be 0 = 60°,at least in our instrument. Furthermore, the ion yield for the sample is related to the probe size. A plateau in sensitivity was noted with the 2.5 mm diameter tips. The ion beam spread caused by the neutral beam/sample/tip surface interaction is a factor in the absolute ion sensitivity. The wide range of gases studied support the assumption that, of the commonly used gases, xenon is the most efficient neutral beam for the ionization of the sample on the probe tip. The most important aspect of the FABMS technique is sample preparation because a number of contaminants have the property of suppressing ionization of the sample, sometimes completely. This is especially critical for those samples that have been derivatized, exposed to buffers, or treated with enzymes. The sample cleanup procedure which we have found most effective consists of repeated dilution followed by lyophilzation and the use of Sep-PAK or separation by HPLC depending on the nature of the impurity. The most efficient method for the preparation of homogeneous solutions consists of first dissolving the sample in a solvent and then adding the matrix. Furthermore, the addition of acids or bases to the sample matrix is often required to produce a sample spectrum observable above the glycerol background. LITERATURE CITED (1) Barber, M.; Bordoli, R. S.; Sedgwlck, R. D.; Tyler, A. N. J . Chem. Soc., Chem. Commun. 1981, 7, 325-327. (2) Surman, D. J.; Vickerman, J. C. J . Chem. SOC., Chem. Commun. 1981, 7 , 324-325.
Devienne, F. M.; Roustan, J . 4 . C.R. Hebd. Seances Acad. Sci., Ser. B 1976, 2 8 3 , 397-399. Bennlnghoven, A. Surf. Sci. 1973, 35, 427-457. Bennlnghoven, A.; Sichtermann, W. Org. Mass Spectrom. 1977, 12, 595-597. Benninghoven, A.; Sichtermann, W. K. Anal. Chem. 1978, 50, 1180-1 184. Barber, M.; Bordoli, R. S.; Garner, G. V.; Gordon, D. 6.; Sedgwick, R. D.; Tetler, L. W.; Tyler, A. N. Biochem. J . 1981, 197, 401-404. Barber, M.; Bordoll, R. S . ; Sedgwick, R. D.; Tyler, A. N. Nature (London) 1981, 2 9 3 , 270-275. Morrls, H. R.; Panico, M.; Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, N. Blochem. Biophys. Res. Commun. 1981, 101, 623-631. Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N.; Bycroft, B. W. Biochem. Biophys Res. Commun. 1981, 10 I , 632-638. Dell, A.; Morris, H. R.; Levin, M. D.; Hecht, S. M. Biochem. Biophys. Res. Commun. 1981, 102, 730-738. Rinehart, K. L., Jr.; Gaudioso, L. A.; Moore, M. L.; Pandey, R. C.; Cook, J. C., Jr.; Barber, M.; Sedgwick, R. D.; Bordoli, R. S.; Tyler, A. N.; Green, B. N. J . Am. Chem. SOC. 1981, 103, 6517-6520. Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N.; Green, B. N.; Parr, V. C.; Gower, J. L. Blomed. Mass Spectrom. 1982, 9 , 11-17. Wllliams, D. H.; Bradley, C. V.; Santikarn, S.; Bojessen, G. Biochem. J . 1982, 201, 105-117. Beynon, J. H.; Cameron, D.; Todd, J. F. J. Anal. Chem. 1982, 5 4 , 679A. Ratz, R.; Schroeder, H.; Ulrich, H.; Kober, E.; Grundmann, C. J . Am. Chem. SOC. 1962, 8 4 , 551-555. Franks, J.; Ghander, A. M. Vacuum 1974, 2 4 , 489-491. Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tetler, L. W. Org. Mass Spectrom. 1981, 16, 256-260. McNeal, C. J. Anal. Chem. 1982, 5 4 , 43A-50A. Olson, K. L.; Rinehart, K. L., Jr.; Cook, J. C., Jr. Biomed. Mass Spectrom. 1977, 4 , 284-290.
.
RECEIVED for review June 7,1982. Accepted August 23,1982. This work was supported by the National Institutes of Health (Grant No. RR00317 from the NIH Division of Research Resources) and from the Office of Naval Research (Grant No. ND0014-78-C-0421).
Exponential Dilution Chambers for Scale Expansion in Flow Injection Analysis Kent K. Stewart"' and A. Gregory Rosenfeld* Nutrient Composition Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, Science and Education, Unifed States Department of Agriculture, Beltsville, Maryland 20705
The use of a small mlxlng chamber In flow injection analysis systems is a unlque and useful method to extend the range of detectlon for these systems. Colorimetric, fluorometric, conductometric, and flame emlssion detectors demonstrate the usefulness of this system. The scale expansion system Is useful In those methods both requiring and not requiring a reagent to produce a detectable specles. I t Is thought that this system will be useful In a number of flow injection appllcatlons.
Flow injection analysis (FIA) and its automated counterpart, automated multiple flow injection analysis (AMFIA), show considerable promise as tools for automated analyses. Several Present address: V i r g i n i a Polytechnic I n s t i t u t e a n d State University, Department of F o o d Science & Technology, Blacksburg,
VA 24061.
Present address: U n i v e r s i t y of M a r y l a n d School of Medicine,
655 West B a l t i m o r e St., Baltimore, MD 21201.
reviews (1-4) and one textbook ( 5 ) have recently been published on this subject. Like many techniques, FIA and AMFIA systems have limited concentration ranges of usefulness. These limitations are attributed to the restricted dynamic range of the detector and to the concentration of a detectable species. This limited concentration range of usefulness is determined by: (1)the sensitivity of the detector to low concentrations of a detectable species, (2) the maximum limit of the detector and the possible nonlinearity of the detector at high concentrations of the analyte, and (3) the limiting concentrations of the reagents used to produce a detectable species when a chemical reaction is necessary. In this paper we describe a technique for scale expansion in FIA and AMFIA and demonstrate its usefulness by application to several different types of determinations. BACKGROUND Traditional FIA systems use the measurement of peak area or peak height to quantitate the concentration of the injected analyte. Ruzicka and Hansen (5) use peak width as a means of quantitating the concentration of an analyte in a FIA system
This article not subject to US. Copyright. Published 1982 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 13, NOVEMBER 1982
Table I. List of Symbols parameters or variable
gymbols
determinant concn (mol L-' ) initial ',C C,, time dependent in gradient chamber volume (ml) gradient chamber sample
parameter of variable
symbols
flow rate (mL s - ' )
f
time for all sample to enter gradient chamber
t2
V,
vs
that performs analyses tdmilar to classical titrations-herein termed FIA-pseudotitrations. Pardue and Fields (6) define titration as the reaction between equivalent amounts of substances. In FIA-pseudotitrations, the equivalence point occurs when the reacting substances are equal in concentration. The differences between FIA-pseudotitrations and classical titrations can have important implications, particularly when dealing with their physical-chemical characteristics. In FIA-pseudotitrationi~,an injected sample bolus is transported into an exponential dilution chamber where it is mixed with the carrier stream. The resulting chamber effluent is combined with an unsegmented stream of titrant and the appropriate species is eventually detected downstream. AAternatively, the titrant solution can be used as a carrier stream and diluant, eliminating the necessity for a postdilution chamber reagent addition. Quantification of the detected species is accomplished by peak width measurements. The peak width is measured as the time (or volume) the detector signal remains above (or below) a preset level-this quantity is termed the equivalence time. Ruzicka and Hansen indicate that the equivalence time for these types of FIA-pseudotitrations is proportional to the logarithm of the ratio of concentration of the originall analyte to the concentration of the titrant and demonstrate ithe usefulness of this method for both a calcium-EDTA titration and an acid--base titration (7). Several other workers have done FIA-pseudotitrations using similar approaches (8-15). We wish to point out that FIA-pseudotitration systems are part of a more general situation in unsegmented flowing systems whereby exponential dilution chambers and time measurements can be used for the measurement of analyte concentrations in flow injection analysis under a wide variety of situations. Our independent studies reported here are, in part, corroborated by the theoretical work of Pardue and Fields (6, 16). We are in substantial agreement with these workers where our work: overlaps. THEORY When a sample boluci is introduced into an exponential dilution chamber, and the analyte concentration profile monitored a t the dilution chamber exit, an exponential rise and fall with time is observed. Pardue and Fields (6, 16) described the time course and maximum concentration pro. duced by such a concentration profile, with eq 1 and 2, respectively. Pungor et al. (17) also discuss the theory of
[AJgmax= Cas0[l- exp[-ft2/v,]] FIA-pseudotitrations. Equation 2 provides the theoretical basis for the measurement of peak height when a mixing chamber is used in FIA, ias was done by Nagy et al. (18). We have employed the notation used by Pardue and Fields (6)-as illustrated in Table I. In addition, Pardue and Fields (6) relate the equivalence time to the original concentration of the injected sample. This
2369
relationship is shown in eq 3. The practical implications of eq 3 are that exponential dilution chambers can be used as a scale expansion system for many FIA and AMFIA systems when equivalence time is used as a measure of sample concentration.
At,, = (ug/f) In [exp(u,/v,) - llCaso - ( u g / f ) In [AI,"' (3) We have tested this scale expansion system by using standard chemistries and several flow-through detectors. Colorimetric sugar analyses, colorimetric base determinations, colorimetric phosphate determinations, flame emission sodium determinations, fluorescent fluorescein and leucine determinations, and conductance salt determinations are among those investigated thus far. Linear plots of the logarithm of the original concentration vs. the equivalence time were obtained in all cases. EXPERIMENTAL SECTION The principal components of the apparatus are shown in Figure 1,as previously described (19). A microprocessor system was used with the AMFIA system to control the operation of the system and to make the time measurements. The second pump (P2)and the mixing "TEE" were not used for those systems requiring no prior chemistry to form the measured species. Several three-pump systems using one sample solvent (Pl) and two reagent pumps (P2 and P3) were also used. Depulsed positive displacement pumps (20) were used in all system configurations. In addition, the entire system was connected by size 26 AWG (0.46 mm i.d.) Teflon tubing and Cheminert fittings, unless otherwise noted. The equivalence time was measured as the time (or volume) the detector signal remained above (or below) a preset level. One-Pump Systems. Several one-pump systems were investigated over the course of this study. Each system resembled Figure 1;however, only pump P1 was used and, therefore, points B and E were directly connected with Teflon tubing. 1. Colorimetric Hydroxide Determination. The carrier stream consisted of a phenolphthalein solution (0.05 g in 50 mL of ethanol and 50 mL of water) diluted to 0.3% with water-the flow rate was 9 mL/min. A colorimeter equipped with a 570-nm interference filter and a PO-pL flow cell was used. A Valco pneumatically activated sample-injection valve with a 200-pL sample volume and a 200-pL exponential dilution chamber was also used. 2. Conductometric Sodium Chloride Determination. This system was similar to that described above, except that the carrier stream was 1.0 X 10" M NaCl, the sample volume was 20 pL, the flow rate was 8.0 mL/min, and a Wescan conductivity meter with a nuqber 900 flow-through conductivity cell was used. 3. Flame Emission Sodium Determination (21). The system was similar to the ones described above except that the carrier stream of deionized water was pumped at 2.8 mL/min, the sample volume was 50 pL, and the detector was a PerkinElmer 603 atomic absorption spectrophotometer equipped with a sodium hollow cathode lamp. 4. Fluorescent Fluorescein Determination. The carrier stream of deionized water was pumped at 8.2 mL/min. The system's components included: a 100-pL sample loop, a 100-pL exponential dilution chamber, and a reaction coil which consisted of 25 ft of 0.46 mm i.d. Teflon tubing thermostated at 30 "C. Ah additional 10 f t qf this tubing, thermostated at 20 "C, was used as a cooling coil-the total reaction time was 15 s. For this system, the reaction and cooling coils would be located at point F on Figure 1. The detector was an Aminco "Fluoromonitor" equipped with a 0.1 mm i.d. flow cell (20 pL), a Corning 7-54 primary filter, and a Wratten 2A secondary filter. Two-Pump System. The two-pump system resembled Figure 1, however, only the sample solvent and reagent no. 1 solutions-pumps P1 and P2, respectively-were required. Here, points C and E on Figure 1were directly connected with Teflon tubing. Fluorescence Leucine Determination. The carrier streap consisted of 0.1 M phosphate buffer, pH 7.0, adjusted to a flow
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ANALYTICAL CHEMISTRY, VOL. 54, NO 13, NOVEMBER 1982
--
-
- - - - -- - - _ _ _
PULSE SUPPRESSOR
PJLSESUPPRESSOR
SCiVENT
REAWN
I/
-
BUBBLE TRAP
S A N P L I SOLIEN- PUhw
REAGENT PUMP
EXPMjfNTIrZL DILUTION CHAMBER
F
4
/I
COMPJTER
DETECTOR
SAMPLER
PERISTALTIC PUMP
\' TO WASTE
1 4I RECORDER
Figure 1. Schematic diagram of the princlpai apparatus components. Dashed lines surrounding pump configurations P2 and P3 indicate the variability
in their use. The letters A-E are used to clarify different pump systems and are explained throughout the text. rate of 3.0 mL/min. The reagent stream was o-phthalaldehyde (400 mg of o-phthaladehyde and 0.2 mL of 2-mercaptoethanol per liter of 0.1 M borate buffer, pH 10.0) pumped at 3.0 mL/min. The remaining components of this system were the same as those described for the fluorescein assay. Three-Pump Systems. Two three-pump systems were investigated here. These systems are illustrated in Figure 1. 1. Total Sugar Determination. This method will be described in detail elsewhere. The chemistry used for the detection of the sugars was a modification of that used in the automated sugar analysis of Mundie et al. (22) and Hudson et al. (23) and was similar to that of Vratny and Ouhrabkova (24) and Blakeny and Mutton (25). Briefly, 100-pL samples were inserted into a carrier stream, PI, of distilled water (0.75 mL/min), mixed with 1 N sulfuric acid, P2 (0.75 mL/min), hydrolyzed for 15 s at 100 "C (10 f t of 0.51 mm i.d. Teflon tubing connected from points C to D), subsequently mixed with alkaline p-hydroxybenzoic acid hydrazide, P3 (5% (w/v) in 0.5 N hydrochloric acid, diluted with 1 N sodium hydroxide to a final concentration of 7.5 mM) (3.0 mL/min), and heated at 100 O C for 43 s in a 25 ft reaction coil (0.51 mm i.d. Teflon tubing located between points E and F). The yellow reaction mixture was cooled in a 15 O C water bath and passed through a 200-pL flow cell and the absorbance measured at 400 nm. The dilution chamber was placed either just after the sample insertion valve (as shown in Figure 1)or just before the detector (points A and B are connected directly with tubing and the dilution chamber is placed after point F). 2. Phosphate Determination. This method was based on a procedure initially described by Kraml (26)and later modified by Howe and Beecher (27). The sample loop and the dilution vial were both 100 pL. The samples were inserted into a carrier stream of deionized water pumped at 4.0 mL/min, P1, mixed with 1.0% ammonium molybdate in 1.25 N sulfuric acid pumped at 2.0 mL/min, P2 (the reaction coil was 35 cm long and placed
between points C and D), and then mixed with a solution of stannous chloride dihydrate (200 mg/L) in 1 N sulfuric acid containing 0.2% hydrazine sulfate, P3. The resulting solution was heated in 8 m of reaction coil (0.46 mm i.d. Teflon tubing) at 60 "C, cooled in 2 m of reaction coil at 30 "C (both reaction coils were located between points E and F), and detected with a colorimeter equipped with a 700-nm filter and a Schoeffel flow cell. The dilution chamber was placed just after the sample insertion valve. RESULTS Plots of the logarithm of the analyte concentration vs. the equivalence time were made for each determination. The concentration range through which each plot remains linear (denoted the linear range) and a ratio of the maximum to minimum concentration delimiting this range (denoted max/min ratio) were manually determined. These data are shown in Table 11. In all cases, the exponential dilution chamber significantly increased the linear range of the system under study. The smallest increase was seen in conductance measurements, where the max/min ratio changed from 20 to 54, whereas fluorescent leucine and fluorescein determinations showed ratio changes from 30 to approximately 1000. Ratios of 1000 were common when the exponential dilution chamber was used. Furthermore, the data obtained from the total sugar determination indicated that the position of the dilution chamber was important. Here, the max/min ratio changed from 100 when the dilution chamber was placed just after the sample value to 50 when it was placed just before the detector. Dilution chamber performance was studied by varying the flow rates of solutions passing through the chamber and by varying the volume of the chamber itself. These experiments
ANALYTICAL CHEMISTRY, VOL. 54, NO. 13, NOVEMBER 1982
2371
Table 11. Linear Ranges with and without Dilution Vial analyte sodium chloride fluorescein leucine sodium chloride fluorescein leucine sodium hydroxide inorganic phosphate sucrose before reaction before detector
detector
linear range, M
A. No Dilution Vial Peak Area Measurements conductance 4.4 X 10-'-8.8 X fluorescence 1 x 10-5-3 x fluorescence 9.2 X 10-'-2.4 X
lo-'
B. Dilution Vial-Peak Width Measurements conductance 1.4 X 10-'-7.5 X lo-' fluorescence 1 x 10-5-1 x 10-2 fluorescence 7.6 X 10-*-7.6X flame emission 4.3 X 10-'-4.3 X colorimetric 0.01-1.00 colorimetric 1.1 x io-*-i.i x 10-5
max/min ratio of linear range 20 30 26
54 1000 1000 1000 100 1000
colormetric
showed that the flow rate has little affect on the linear range, or upon the max/min ratio of the particular system under study. However, the fllow rate had a direct affect on the analysis time. Slower flow rates increased the equivalence time, leading to a decrea~3ein the number of samples analyzed per hour. The dilution chamber volume had a direct affect on the linear range. The linear range decreased as the ratio of the exponential dilution chamber volume to sample volume increased. However, the maximin ratio was not significantly altered with changes in dilution chamber volume.
DISCUSSION These results demoncitrate that the insertion of an exponential dilution chamber into a FIA, M I A , or similar system provides a simple means of extending the linear ranges of these systems. Although there is some loss in sensitivity, this loss is often of little practical importance to many laboratoriesmost assay systems have more than adequate sensitivity and a slight loss in sensitivity can be compensated for by modifying the reaction conditions. The advantages gained by an increased linear range lessen the need for sample dilution and often far outweigh the disadvantages of decreased sensitivity. If necessary, the dilution effect and subsequent sensitivity loss can be substantially reduced by using a small dilution chamber (i.e., 100 p L volume). A substantial decrease in analysis time is seen when using an exponential dilution system rather than a linear dilution system. This is brought about by the disproportionate increase in equivalence time as the sample concentration is increased-the equivalence time is proportional to the logarithm of the sample concentration. If a system is designed for the determination of the most dilute sample, the equivalence time doubles when analyte concentrations are 100-fold greater than the most dilute sample. The analysis of samples with widely varying concentrations is simplified by using a microprocessor controlled feed back system coupled to an AMFIA system (19,28). Fields (6, 16), as mentioned earlier, laid the theoretical basis for estimating sample concentrations by measuring peak height or peak width. The work reported here c o n f i s their findings for peak width measurements and demonstrates the broad applicability of peak width measurements to different assays and to various methods of detection. Nagy et al. (18)noted that peak height measurements could be used in FIA systems with dilution chambers. It seems likely that the exponential dilution chamber can be used for systems where both peak width and peak height measurements are desirable. It should be a simple task to develop a system in which both measurements are used. With the data at hand, we predict that peak height measurements would be more precise and sensitive to subtle changes in concentration while peak width measurements would allow analysis of
2.9 X 10-6-2.9X 1.46 X 10-$-7.3 X
100 50
samples over a wide range of concentrations. Investigations must be undertaken to determine the effects of several parameters on the sensitivity, precision, and linear range of FIA, AMFIA, and similar systems that use peak width measurements. Such parameters which must be studied include: sample size, flow rates, dilution chamber volume, reaction tube length and diameter, and detector and data acquisition system reliability. These studies will be published elsewhere. The use of exponential dilution chambers in AMFIA and similar systems has interesting implications for the development of detectors, data systems, and reagent concentrations. A detector is needed only as a trigger to turn on and off a clock or a counter. For any particular assay, a detector must be accurate and precise at the points where the counter is either turned on or off. Therefore, it is necessary for the analyst to calibrate the analytical system with a series of standards under normal operating conditions in order to ensure accuracy and precision of the detector at the trigger points. Once this is accomplished, neither a linear detector response nor an accurate and precise detector signal between the trigger points is required. In light of these findings, the cost of detectors with these characteristics could be dramatically reduced. Equally significant in its modification of detectors, the use of exponential dilution chambers has implications for the development of data collection systems. Since the primary measurement is a time interval, the data acquisition system could be a rather simple, inexpensive digital counter or clock. In addition, the calculation of results, requiring exponentiation and multiplication, could be done with an inexpensive microprocessor. Reagent concentration is yet another parameter of FIA, AMFIA, and similar systems that is affected by exponential dilution chambers. When a reagent is necessary to produce a detectable species, it must be a t a sufficient level to completely react with an analyte present a t a concentration corresponding to the trigger points. Since these analyte concentrations are low, it is not required that the reagent be present at those concentrations necessary to produce a linear response throughout the entire sample bolus. Thus, less concentrated reagents can be used for assays requiring a chemical reaction. The use of an exponential dilution chamber in FIA, AMFIA, and similar systems, therefore, offers several possibilities for the development of low cost detectors and data systems, as well as limiting the use of chemical reagents. Some mention should be made as to the best method for introducing an exponential gradient into FIA, AMFIA, and similar systems. Ruzicka and his co-workers (7)originally used three types of gradient systems: the long narrow tube, the short wide tube, and the stirred chamber. More recently, this group suggests that short narrow tubes may also be used (13).
2372
Anal. Chem. 1982, 5 4 , 2372-2375
In our work (14), we find that each of these sytems will give exponential gradients. Our studies indicate that stirred chambers give exponential behavior over a wider range of concentrations than do gradient systems using the various sizes and lengths of tubing. Many of the objections that others have to stirred chambers may be overcome by using small (100 pL) dilution chambers. ACKNOWLEDGMENT The skillful technical assistance of Darla Higgs is gratefully acknowledged. LITERATURE CITED (1) (2) (3) (4)
Betteridge, D. Anal. Chem. 1978, 5 0 , 832A-846A. Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1980, 114, 19-44. Ranger, C. B. Anal. Chem. 1981, 53, 20A-32A. Stewart, K. K. Talanta 1981,,,28, 789-797. ( 5 ) Ruzicka, J.; Hansen, E. H. Flow Injection Analysis"; Wiley: New York, 1981. (6) Pardue, H. L.; Fields, B. Anal. Chlm. Acta 1981, 124, 39-63. (7) Ruzicka, J.; Hansen, E. H.; Mosbaek, H. Anal. Chim. Acta 1977, 92, 235-249. (8) Nagy, G.; Toth, K.; Pungor, E. Anal. Chem. 1975, 4 7 , 1460-1462. (9) Nagy, G.;Feher, Z.; Toth, K.; Pungor, E. Anal. Chim. Acta 1977, 9 1 , 87-96. (IO) Nagy, G.;Feher, 2.; Toth, K.; Pungor, E. Anal. Chlm. Acta 1977, 9 1 , 97-106. (11) Nagy, G.;Feher, Z.; Toth, K.; Pungor, E. Anal. Chlm. Acta 1978, 100, 181-191. (12) Astrom, 0.Anal. Chim. Acta 1979, 105, 67-75. (13) Ramsing, A. U.; Ruzicka, J.; Hansen, E. H. Anal. Chlm. Acta 1981, 129,1-17. (14) Stewart, K. K.; Rosenfeld, A. G. J . Autom. Chem. 1981, 3 , 30-32.
(15) Horvai, G.;Toth, K.; Pungor, E. Anal. Chim. Acta 1976, 82, 45-54. (16) Pardue, H. L.; Fields, B. Anal. Chlm. Acta 1981, 124, 65-79. (17) Pungor, E.; Feher, 2.; Nagy, G.; Toth, K.; Horvai, G.; Gratzl, M. Anal. Chim. Acta 1979, 109, 1-24. (18) Nagy, G.;Feher, 2.; Pungor, E. Anal. Chim. Acta 1970, 52,47-54. (19) Brown, J. F.; Stewart, K. K.; Higgs, D. J . Autom. Chem. 1981, 3 , 182-186. (20) Stewart, K. K. Anal. Chem. 1977, 4 9 , 2125. (21) Wolf, W. R.; Stewart, K. K. Anal. Chem. 1979, 5 1 , 1201-1205. (22) Mundie, C. M.; Cheshlre, M. V.; Anderson, H. A.; Inkson, R. H. E. Anal. Biochem. 1978, 7 1 , 604-607. (23) Hudson, G.J.; John, P. M. V.; Bailey, B. S.; Southgate, D. A. T. J . Sci. Food Agric. 1976, 2 7 , 681-687. (24) Vratny, P.; Ouhrabkova. J. J . Chromatogr. 1980, 191, 313-317. (25) Blakeney, A. B.; Mutton, L. L. J . Scl. FoodAgrlc. 1980, 3 1 , 889-897. (26) Kraml, M. Clln. Chlm. Acta 1966, 13, 442-448. (27) Howe, J. C.; Beecher, G. R. J . Nutr. 1981, 111, 708-720. (28) Stewart, K. K.; Brown, J. F.; Golden, 6. M. Anal. Chim. Acta 1980, 114. 119-127.
RECEIVED for review February 17, 1982. Resubmitted and accepted August 23, 1982. A preliminary report of this work was presented at the 1981 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 9-13, Atlantic City, NJ, by K. K. Stewart and A. G. Rosenfeld, paper number 824. This work was supported in part by an Interagency Reimbursable Agreement No. 2Y01-HB60041-05 from the National Heart, Lung, and Blood Institute, NIH. Mention of trademark or proprietary products does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply their approval to the exclusion of other products that may also be suitable.
CORRESPONDENCE Soft Negative Ionization of Nonvolatile Molecules by Introduction of Liquid Solutions into a Chemical Ionization Mass Spectrometer Sir: Over the last 6 years our efforts have been directed toward interfacing a high-performance liquid chromatograph (HPLC) to a quadrupole mass spectrometer by nebulizing a constant fraction of the liquid effluent from the chromatograph into a high-pressure chemical ionization (CI) source. This method has been called direct liquid introduction (DLI) (1). Initial efforts to develop the analytical potential of the DLI method were restrained by experimental difficulties such as instabilities of the vacuum (2),clogging of the interface (3), or improper HPLC packings and solvents ( 4 ) . These inconveniences being now less commonplace, it becomes easier to test the performance of the DLI method by applying it to the separation and detection of known nonvolatile organic molecules as also currently done in other laboratories (5-9). The performance characteristics of this combined liquid chromatography/mass spectrometry (LC/MS) interface are significantly improved when the droplets from the nebulizer are allowed to drift through a heated zone (8-10), sometimes referred to as the desolvation chamber, prior to introduction into the CI source. Since solute ions may arise in part from the liquid solution (11-16), a complete desolvation to dryness of the liquid droplets within the desolvation chamber should be avoided. By use of an appropriate geometry for the desolvation chamber, the droplets are accelerated to sonic velocities. This approach is comparable to that followed by Vestal et al. (12,13,16) who have shown that rapid thermal vaporization of high speed droplets assists the ionization of
nonvolatile molecules when neutral solutions are nebulized in the presence of an external ionization source or produces gas-phase solute ions directly when electrolytic solutions are nebulized and vaporized. The operating principle of our high-speed DLI device and preliminary results obtained for different fragile molecules have been previously reported (10). This correspondence describes in detail the device used in these experiments. Also included are the mass spectra obtained for vitamin B12and the antibiotic erythromycin A. They show the capability of handling polar molecules of molecular weight over m / z 1000 and the occurrence of electron capture chemical ionization (17) under DLI LC/MS conditions. EXPERIMENTAL SECTION General Equipment. A Waters Associates, Inc. (Milford,MA), Model 6000A solvent delivery system and Model U6K injector were used with a Merck (Darmstadt, GFR) reversed-phase column (4.6 mm X 25 cm) packed with 10-gm Lichrosorb RP 18. Mass spectra were recorded on a Nermag Model R-10-10-C quadrupole mass spectrometer, equipped with a Nermag LC/MS interface and Model SIDAR l l l B data processing system. No liquid nitrogen cryopump ( 2 ) was used in this study. The instrument has an operable mass range up to 1500 amu and is fitted with a conversion dynode electron multiplier detector for recording of negative ions. Primary ionization of the solvent vapors was accomplished by a 70-eV beam of electrons from a heated rhenium ribbon. The standard metallic cage around the rhenium ribbon
0003-2700/82/0354-2372$01.25/00 1982 American Chemical Society
