Anal. Chem. 1905, 57, 2423-2425
ACKNOWLEDGMENT The authors wish to than K. Govindaraju, C.R.P.G., Vandoeuvre-Nancy, France, for providing samples of standard reference materials. Registry No. Fluorine, 7782-41-4;chlorine, 7782-50-5. LITERATURE C I T E D (1) Bennett, H.; Hawley, N. G. “Methods of Silicate Analysis”; Academic Press: London and New York, 1965.
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(2) Bloxam, T. W. Chem. Geol. 1988, 3 , 89-94. (3) Clements, R. L.; Sergeant, 0. A.; Webb, P. J. Analyst (London) 1971, 96,51-54. (4) Farzaneh, A,; Troll, G. Geochem. J . 1977, 11, 177-181. (5) Farzaneh, A,; Troll, G. Z . Anal. Chem. 1978, 292, 293-295. (6) Hetman, J. S. Bull. Cent. Rech. Pau-SNPA 1975, 9 , 183-190. (7) Fuge, R. Chem. Geol. 1978, 17, 37-43. (8) Govindaraju, K. Geostand. Newsl. 1984, 8 , special Issue. (9) Troll, G.; Farzaneh, A. Geostand. Newsl. 1980, 2 , 43-47.
RECEIVED for review March 25,1985. Accepted May 16,1985.
High-speed Microbore Liquid Chromatography with Electrochemical Detection Using 3-pm C18 Packing Material E d w a r d J. Caliguri, Peter Capella, Leo Bottari, and I v a n N. Mefford* Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02167 Much of the interest in microbore HPLC has been a result of increased mass sensitivity and lower solvent consumption (1). In addition, microbore HPLC seems more suited to methods of detection such as mass spectrometry (2) where removal of excess solvent poses a problem. Lowering the detector volume of UV or fluorescence detectors in order to eliminate extracolumn dispersion has generally increased the noise as a result of diffraction and decreased the sensitivity due to a reduction in path length (3, 4). As a result, the theoretical decrease in detection limits is not observed. Although more limited in application, amperometric detectors are readily adapted to microbore HPLC and can be modified to provide signal enhancement beyond the chromatographic increase in peak concentration as a result of increased coulometric yield and lower detector noise, both byproducts of the lower flow rates (5). The use of short (7-10 cm) microbore columns packed with 3-pm material allows one to take advantage of both signal enhancement and high sample throughput. The present work describes the performance of high-speed microbore columns using electrochemical detection, as well as packing procedures and detector modifications. EXPERIMENTAL SECTION Column Preparation. Bulk stainless steel tubing, ‘/I6 in. 0.d. and 1.2 mm i.d. was purchased from a local chromatography supply house. Columns were cut to between 7 and 10 cm in length. The tubing was then rinsed with acetone prior to packing. The exit end of the tubing was seated in a stainless steel auxiliary electrode block (MF 1018, Bioanalytical Systems, Inc., (BAS), West Lafayette, IN) into which a ‘/I6 in., 0.5 pm stainless steel frit had been placed. The ferrule on the inlet end was seated into a Model 7413 Rheodyne injection valve. The column, with ferrules seated was disconnected from the injection valve and then attached to the slurry reservoir. The slurry reservoir consisted of a 25 cm length by 4.6 mm i.d. stainless steel column blank. The exit fitting was drilled out to accommodate a l/ls in. tube. The slurry was prepared by suspending 200 mg of Hypersil C18, 3-wm reverse-phase packing material (Shandon) in 4.5 mL of degassed 2-propanol. This was then placed in the slurry reservoir and attached to the slurry pump (Chemco Model 124A) which had been purged with degassed 2-propanol. Flow was then initiated, using 2-propanol as the packing solvent. Initial flow was at 400 bar and was elevated gradually over a 1-min period to 700 bar (10 OOO psi). Pressure was maintained at 700 bar for 5 h. At that time, the slurry pump was turned off and the column pressure allowed slowly to equilibrate with atmospheric pressure (30 min).
The packed column was then disconnected from the reservoir and placed directly into the column port of the 7413 Rheodyne injector (see Figure 1). Electrochemical Detection. Detection was accomplished at a glassy carbon working electrode (TL-aA, BAS). The detector electrode was attached to the stainless steel auxiliary electrode block, separated by a very thin plastic film, 10 pm in thickness (Saran Wrap). The flow chamber was a 3 mm x 14 mm rectangle cut in the plastic film, giving a total cell volume of 420 nL. As the electrode sits in the middle of the flow chamber, only dispersion in the first half of the cell contributes to band broadening, giving an effective cell volume of 210 nL. An electrode potential of +0.60 V vs. Ag/AgCl reference was maintained and the current monitored with a Model LC-SA amperometric detector (BAS) which had been modified to have a time constant of 0.3 s. Pulseless solvent delivery was maintained with a Model 400-02 solvent metering pump (Applied Chromatography Systems, Inc., State College, PA). Chromatograms were recorded on a strip chart recorder. Retention times were measured manually as were the values of the plate height, H, number of theoretical plates, N , and peak asymmetry. Peak width was measured at half height and peak asymmetry at 10% peak height. Chromatographic resolution was accomplished for a series of catechol compounds: 3,4-dihydroxyphenylglycol,norepinephrine, 3,4-dihydroxyphenylalanine,epinephrine, 3,4-dihydroxyphenylacetic acid, and dopamine (Sigma Chemical Co., St. Louis, MO). The solvent used for separation of these compounds was 0.2 M NaH2P04,30 mg/L sodium octyl sulfate, 50 mg/L EDTA, and 0.6% acetone, adjusted to a final pH of 3.2. RESULTS AND DISCUSSION Figure 2 shows a typical chromatogram obtained for the separation of a mixture of catecholamines and related compounds. Conditions are described in the figure legend. No marked effect on efficiency was observed with injection volumes of 0.5-5 pL. Peak asymmetries for those compounds with k’greater than 1.5 were typically between 1.0 and 1.10. The average peak asymmetry for epinephrine, k ’ = 2.3, was 1.03 A 0.031 (one standard deviation) measured a t 10% peak height. A 10-cm column packed as described typically gave a plate count of 4500-5500 (45000-55000 plates/m) at our normal operating flow rates of 200-250 pL/min with a back pressure of 3000-3500 psi. Columns such as these give relatively high speed separations at approximately 95 plates/s. This can be compared to the work of DiCesare et al. (6) which accomplished separation at 100-150 plates/s using a 4.6 mm i.d. reverse-phase column.
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
A
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Figure 1. Injection port, column, and electrode configuration. Columns are 1.2 mm i.d., while length can vary between 7 and 10 cm.
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Figure 2. High-speed microbore resolution of biogenic amines: peak 1, Dopeg, 1.10 ng; peak 2, NE, 2.06 ng; peak 3, Dopa, 3.31 ng; peak 4, E, 0.79 ng; peak 5, DHBA; peak 6, Dopac, 1.34 ng; peak 7, DA, 0.97 ngl.
Figure 3 shows the flow rate dependence of theoretical plate height. Optimum flow rate is shown here to be between 80 and 100 pL/min. The 10-cm length was found to offer the optimum efficiency and speed of analysis. While this work was in progress, an article appeared describing efficient packing procedures for small particle microbore columns (7). Our slurry concentration is adjusted to be 45 mg of packing material/mL of 2-propanol, oqmpared to 65 mg/mL of 2-propanol used by Meyer and Hartdck (7). We used 2-propanol as the packing solvent at a packing pressure of 10 000 psi (the maximum operating pressure of our commercial slurry packing pump) while Meyer and Hartwick used methanol as the slurry solvent at a packing pressure of 8000 psi. As our interest is in application of this technique to determination of biogenic amines, the column performance was
200
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Flowrate uI/min
Glassy Carbon
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k=64 k=3.3
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'-OM
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OA
A OHBA
Figure 3. Plot of plate height (in micrometers) vs. flow rate for compounds with different k' values: dopamine (DA), 3,4dihydroxybenzylamine (DHBA), epinephrine (E).
tested with catecholamines using ion pairing to effect resolution. A minimum plate height of 16 pm was obtained at an optimum flow rate of 80-100 pL/min. A practical comparison of the efficiency of these columns can be made by comparing their performance with published separations of the same compounds using a conventional width (4.6 mm) X 7.5 cm length column packed with 3-pm reverse-phase material (8). Similar plate height and plate counts per meter are obtained. Similar values for the minimum plate height have been found by Welling et al. (9) for microbore columns using a different packing procedure. Our routinely used flow rates of 200-250 pL/min give a pressure drop of 3000-3500 psi. This corresponds to a flow rate of 2.9-3.7 mL/min for a 10 cm X 4.6 mm i.d. column packed with the same material. Flow rates of 0.2 mL/min or greater do not normally require a special microbore HPLC pump. In addition, injection of 5 pL of sample onto the column causes no observable effect on peak widths or symmetry for peaks with k'> 1. This overcomes one of the primary criticisms of the practical use of microbore HPLC, namely, the extremely small injection volumes. Finally, column lifetime appears to be comparable to commercially available microbore or analytical scale HPLC columns. Columns typically last for 3 months or more with routine use. As each column contains only 150 to 200 mg of packing material, the cost is minimal, less than $4.00 per column. Including the cost of fittings and ferrules the cost per column is around $10.00, The separation achieved in this work is accomplished in approximately 5 min. This will allow routine analysis of six catechol compounds extracted with aluminum oxide simultaneously. This represents a significant decrease in analysis time over conventional separations but does not approach the ulta-high-speedseparations previously described by Scott (10) or DiDesare et al. (6, 11). When coupled with amperometric detection, high speed microbore HPLC offers limits of detection for biogenic amines of 1pg or less. The low cost of these columns and fast analysis times make them very appealing for routine use in the analysis of biogenic amines.
LITERATURE CITED (1) (2) (3) (4)
Scott, R. P. W.; Kucera, P.J . Chromafogr. 1979, 169, 51-52. Krein, P.; Devant, G.; Hardy, M. J . Chromafogr. 1982, 257,129-139. Bruins, A. P.; Drentl, 8. F. H. J . Chromafogr. 198% 271, 71-82. Cook, N. H. C.; Olsen, K.; Archer, B. G. L . C. Mag. 1984, 2, 514-524. (5) Caliguri, E. J.; Mefford, I. N., unpublished observations. (6) DiCesare, J. L.; Dong, M.; Atwood, J. G. J . Chromatogr. 1981, 217, 369-386.
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Anal. Chem. 1985, 57, 2425-2427 (7) Meyer, R. F.; Hartwick, R. A. Anal. Chem. 1984, 56, 2211-2214. (8) Lin, P. Y. T.; Bulawa, M. C.; Wong, P.;Lin, L.; Scott, J.; Blarlk, C. L. J . Liq. Chromafogr. 1984 7 (3). 509-538. (9) Welling, P.; Poppe. H.; Kraak, J. C. J . Chromatogr. 1985, 327, 450-457. (10) Scott, R. P. W. In “Advances in Chromatography”;Giddings, J. C., Grushka, E., Cazes, J., Brown, P. R., E&.; Marcel Dekker: New York, 1983; Vol. 22. pp 247-294.
(11) DiCesare, J. L.; Dong, M. W.; Ettre, L. S. Chromafographia 1981, 74 (5).257-268.
RECEIVED for review February 19, 1985. Accepted June, 11, 1985. This work was supported by a grant from the Scottish Rite Schizophrenia Research Foundation.
Utility of Silver Ion Attachment in Fast Atom Bombardment Mass Spectrometry Brian D. Musselman,* John Allison, and J. Throck Watson
Departments of Biochemistry and Chemistry, Michigan State University, East Lansing, Michigan 48824 The addition of metal salts to the glycerol (G) solvent utilized in fast atom bombardment (FAB) mass spectrometry (MS) results in the generation of adducts between the cation and molecules of both the analyte and solvent. Several investigators have outlined procedures for the addition of a salt such as LiCl to the solvent in order to produce adduct ions such as (M Li)+ (where M = analyte) (1). The presence of these salts leads to additional unwanted ionic species in the mass spectrum. These include adduct ions arising from the combination of the cation with the solvent to form ion clusters Li)+ and ions which may be the product of such as (G, unimolecular dissociation of these clusters (2). Additional problems are apparent when samples of biological origin are analyzed. The presence of sodium and potassium ions in these samples results in the formation of both cationated solvent ions (G, + Na+), cationated analyte (M Na)+, and cluster ions containing one or more cations such as (G, + Na + K - H)+, which further complicate interpretation of the mass spectrum. These experiments detail the use of silver salts dissolved in the FAB solvent for the generation of unique cation-analyte clusters which can be used to identify the molecular weight of unknown components while minimizing the generation of new cluster species. For purposes of illustration, consider the FAB mass spectrum of cholic acid (mol wt 408) in Figure la as an unknown. The three major peaks could represent protonated molecules of three different analytes having even molecular weights or they could represent cluster ions and fragment ions of a single analyte. An alternate interpretation of the spectrum suggests that the peak a t m / z 373 could while the peak represent a protonated molecule (M H)+, at m / z 355 could represent the fragment ion (M H - H20)+ arising from the loss of water from the protonated molecule, a fragmentation frequently observed in mass spectra obtained by FAB ionization of carbohydrates. Both of these interpretations are reasonable, but incorrect in this case, as revealed during subsequent analysis of the analyte in glycerol containing 0.14 M AgN03 which resulted in the generation of an adduct, (M + Ag)+. Figure l b illustrates the results of this analysis. Abundant ionic species which were not apparent in the original mass spectrum were obtained a t m / z 515 and 517. Additional analyses of the sample a t different concentrations indicated that the abundance of these ions could be correlated to sample concentration. The silver ion adducts are easily recognized by virtue of the characteristic peak intensity pattern (51%:49%) of the 1mAg:109Agisotopes. The appearance of (Aganalyte)+adducts is evident even when the protonated analyte molecule is not observed in mass spectra obtained using pure glycerol as a solvent (Figure la). This analysis of cholic acid in two different FAB matrices illustrates the potential of using silver salts to produce (Aganalyte)+ adducts which can be used to
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determine molecular weight information for unknown components. The molecular weight of the analyte in this case is determined to be 408 as obtained by subtracting 107 from the nominal mass of the (Aganalyte)+adduct a t m / z 515. With this verification of the molecular weight by intentional formation of the silver ion adduct, major peaks in the original FAB mass spectrum, Figure la, can be interpreted as labeled in Figure lb. Note that the peak at m / z 409 representing the protonated molecule is not readily distinguishable in either spectrum (Figure l), a feature that would surely contribute to misinterpretation of the FAB spectrum in Figure l a , if it were an unknown. Protonated cholic acid dimer (mlz 817) is also present, but not shown in the partial mass spectra in Figure 1.
EXPERIMENTAL SECTION Solutions of silver salts in glycerol were prepared by first dissolving the salt in water to make a 5% w/w solution; aliquots of this solution were subsequently added to known volumes of glycerol to obtain the desired concentration of silver salt. Mixing was completed with a magnetic stirring apparatus. All FAB analyses were completed using 1pL of a glycerol solution containing the silver salt as delivered from a 0-5-pL micropipet onto the aluminum FAB probe tip. A series of experiments were conducted to determine the relative utility of silver salts (AgC10, and AgN03) dissolved in glycerol. The utility of granulated silver metal (-222, +325 mesh size Cerac, Inc., Milwaukee, WI) suspended in the solvent, was also examined. Optimum concentrations of each silver salt required for generation of silver adducts were determined. Silver metal suspended in the glycerol did not result in the formation of silver adducts with the analyte or the solvent. Solutions of sucrose, acetaminophen sulfate, arachidonic acid, the tripeptide Leu-Gly-Phe, and cholic acid were prepared by dissolving each in H,O/methanol (75:25) to the desired concentration; 1-pL aliquots of this solution were applied to the FAB probe tip upon which a 1-pL aliquot of the silver salt in glycerol solution had been applied previously. Excess water/methanol was evaporated from the probe tip by cautious use of a heat gun. Aliquots of a solution of 1.8 g of sucrose in 10 mL of water were g of AgN03, pipetted into 0.5 g of glycerol containing 9 X mixed by agitation, and analyzed after 5-min intervals. The relative abundances of the protonated molecule and the silver ion adduct of the analyte were monitored as a function of sucrose concentration. A similar experiment was completed using aliquots of 1.0 X lo-’ g of tripeptide dissolved in 5 mL of water/methanol (4;l). Determination of the minimum concentration of silver salt necessary to provide useful analytical data was completed as follows: glycerol (0.5 i 0.03 g) was added to each of ten l/*-dram vials. A stock solution of 0.45 g of AgN03 in 1 mL of distilled water was prepared. Volumes of 5, 10, 15, 20, 25, 30, 40, 50, 7 5 , and 100 pL of the stock solution were pipetted into each of the ten vials. Distilled water was added to each of the first nine vials in order to make a final volume of 100 FL in each vial. Analysis of the tripeptide, Leu-Gly-Phe,was completed by measuring the 0 1985 American Chemical Society
