Anal. Chem. 1988, 6 0 , 284-286
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TECHNICAL NOTES Catalytic Oxygen Scrubber for Liquid Chromatography W.A. MacCrehan* and S. D. Yang’ Organic Analytical Research Division, Center for Analytical Chemistry, National Bureau of Standards, Gaithersburg, Maryland 20899
B. A. Benner, Jr. Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742 Often in liquid chromatography it is desirable or necessary to remove dissolved oxygen, since it quenches fluorescence (1-3), produces a large residual current in reductive electrochemical detection (3-5), or causes oxidation of sensitive analytes (6,7).Sparging with inert gas, either by direct means (4)or assisted by heating (7)or vacuum (5, B), is probably the most frequently used method for oxygen removal. However, for routine LC work this approach is quite cumbersome. Several ”on-line” methods for mobile phase oxygen removal have thus been developed, including a high-pressure electrochemical cell (9) and the use of gas-permeable tubing in either vacuum ( 1 0 , I I ) or chemical oxygen reducing solution (2). One alternative recently developed was the use of a “chemical” zinc oxygen scrubber (3), where the zinc metal reduces the oxygen in the mobile phase directly. However, in routine application, this approach has shown many disadvantages. After 3-5 working days of use, the efficiency of the scrubber column declines rapidly (and without warning) or shows a dramatic increase in mobile phase back pressure. For reductive electrochemical detection, the zinc ions produced in the reduction reaction limit the useful current-potential range. Also, since the zinc metal particles of the scrubber column are consumed by the reduction of mobile phase oxygen, it is necessary to employ a small-particle (0.5-pm), high-capacity filter following the scrubber to prevent zinc particles from accumulating on the inlet frit and column packing. Even so, particle accumulation in the LC system may still be troublesome with the zinc oxygen scrubber. One elegant solution to the oxygen reduction problem has been developed by Tejada et al. (12,13) based on the use of catalytic reduction of mobile phase oxygen by alcohols added to the mobile phase. We tried a sample of the “crushed three-way automotive catalyst” (13) (a gift from Dr. Tejada) but found that a t room temperature the catalyst did not reduce oxygen levels sufficiently for the use with reductive electrochemical detection a t high sensitivity. For use with fluorescence detection, Tejada (13) found that an elevated temperature (80 “C) was required for optimum catalytic activity. This requirement is a nuisance for routine LC work and prompted our further investigations into other catalyst materials. In this paper we describe an oxygen scrubber for use in liquid chromatography based on the reduction of oxygen by methanol added to the mobile phase. A short column, packed with platinum-on-alumina catalyst, provides the active surface for this reaction and is inserted in the chromatographic system just before the sample injection valve. Oxygen in the mobile phase is substantiallly reduced by the addition of as little as 1% methanol. The effectiveness of the platinum oxygen Current address: National Center for Clinical Laboratory, Bei-
jing Hospital, Beijing, People’s Republic of China.
scrubber is demonstrated both for high-sensitivity reductive electrochemical detection and for the elimination of oxygen quenching in fluorescence detection. EXPERIMENTAL SECTION Chromatographic Apparatus. The liquid chromatographic apparatus consisted of conventional, commercially available equipment. For the fluorescence detection, a double monochromator instrument with wavelength programming of both excitation and emission wavelengths was used. “HPLC” grade methanol, water, and acetonitrile were used throughout. Buffers were prepared by neutralizing monochloroacetic acid with ammonium hydroxide, as monitored with a glass pH electrode. The effectivenessof the oxygen scrubber for use in reductive electrochemical detection was tested with a mixture of nitro polynuclear aromatic hydrocarbons (NPAH),prepared by dilution of National Bureau of Standards’ standard reference material (SRM) 1587 with methanol. A monomeric, octadecylsilane modified silica (C-18) column (25 X 0.46 cm, 5-pm particles) was used with a solvent gradient from 9% (v/v) acetonitrile, 91% (admixture of 64% methanol, 5% 1-propanol, 0.2 mol/L ammonium chloroacetate pH 3.2) to 40% acetonitrile, 60% of the admixture. Detection was at a gold/mercury thin-film electrode (1.2 mm diameter) held at a constant potential of -600 mV versus a silver/silver chloride reference electrode. Samples were sparged with argon for 5 min before injection of a 20-pL volume. Measurements of the residual current at various potentials and solvent compositions were made by choosing the desired parameters and allowing at least 30 min for the system to equilibrate (at a flow rate of 1.5 mL/min) before the current values were recorded. For the catalysis of the reaction of methanol with oxygen in flowing streams, we then evaluated several commercially available, precious metal catalysts coated on solid supports. Platinum, rhodium, and palladium, present as 5% coatings on carbon (Alfa Products, Danvers, MA), were dry packed into a 15 X 0.46 cm stainless steel column. For the comparison, we used a mobile phase consisting of a 50% methanol/electrolyte mixture and monitored the residual current at the gold/mercury thin-film electrode at an applied potential of -700 mV. Rhodium provided little or no catalysis, palladium provided good reduction for only about 30 min of use, but platinum provided very efficient reduction of oxygen. Subsequent experiments have shown that commercially available 10% platinum-on-alumina (Alfa Products, Danvers, MA) is an excellent catalyst for oxygen scrubbing of LC eluents. This material produces less mobile phase back pressure (generally 200-600 psi) than the similar carbon support (looCr2000 psi) in 60-70% methanol solvents at 1.5 mL/min and provides excellent catalysis of the methanol oxygen reduction. Oxygen Scrubber Column. The scrubber consists of an empty 15 X 0.46 cm LC column dry packed (with vibration) with the 10% platinum-on-alumina catalyst. The scrubber is placed between the solvent delivery pump and the injector. Before the final connection is made to the system, it is necessary t o pump solvent through the scrubber for 30 min to waste, allowing fines to be eluted. It is necessary to use a high-capacity,small-particle (0.5-fim)filter following the scrubber to prevent any residual fines
This article not subject to US. Copyright. Published 1988 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 3, FEBRUARY 1, 1988
0
/ -0 5
0 0
Applied Potential in V
40 60 80 % MeOH in Mobile Phase
20
0
-1.0
100
Figure 2. Residual current as a function of methanol content at -600
Figure 1. Residual current with the platinum oxygen scrubber.
mV.
from plugging the LC column. We have found the useful lifetime of the scrubber column to be several months of continuous use. Repacking of the column is only necessary when the back pressure exceeds 1000 psi at normal flow rates. Caution: Care should be taken in the handling and disposal of the catalyst material, since even small quantities may cause oxidizable liquids, as well as damp paper, to burst into flame in the open air.
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l 1000
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RESULTS AND DISCUSSION
-
The mechanism of the oxygen removal by the alcoholic solvent at the platinum scrubber is catalytic, since the material is not consumed in the reaction. The oxygen reduction reaction may follow two pathways:
-
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+ CHBOH
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platinum catalyst
platinum catalyst
+
200 100 -
HzO + CHzO
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0
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285
+ 3H20
The formation of formaldehyde as the oxidation product from the methanol oxidation was confirmed by reacting a solution of dinitrophenylhydrazine with scrubbed mobile phase containing 5% methanol. The dinitrophenylhydrazone derivative may be separated from the unreacted reagent by reverse-phase HPLC and detected at 365 nm (14). A single, well-defined formaldehyde derivative was found for the platinum oxygen scrubber product using this procedure. On the basis of a formaldehyde standard, the concentration of aldehyde formed was approximately 0.53 mmol/L. Formation of formic acid in the reduction of oxygen by the methanol is another active pathway for the reaction. By measurement of the pH of the mobile phase before and after the scrubber, combined with the known p K a , the amount of formic acid formed was estimated to be 0.13 mmol/L. Thus, at this concentration of methanol in unbuffered media, the first reduction to the aldehyde predominates, but some acid is also formed. In a single experiment scrubbing a 5% 1-butanol mobile phase, three aldehyde adducts of the dinitrophenylhydrazine were found, including formaldehyde. The total concentration of aldehydes formed was approximately 0.47 mmol/L. The acidity also increased, but analysis of the acid concentration formed by the reaction is confounded by the possible formation of formic and other lower carbon acids in addition to 1-butanoic acid (which has a relatively higher pKJ. Because of the formation of unwanted aldehydes and acids, some caution should be exercised in the use of the oxygen scrubber. For some analytes, reaction with the aldehyde products might be possible during the chromatographic separation, making use of the scrubber ill-advised for such systems. The acidity resulting from the scrubber will always be less than millimolar and thus the use of a mobile phase buffer of at least 10 mM would be advisable for pH-sensitive systems.
I
I
20
I
I
1
1
1
1
40 60 80 % MeOH in Mobile Phase
1
1
100
Figure 3. Residual current as a function of methanol content at -1000 mV.
The completeness of the oxygen reduction is evidenced by the low residual current as a function of the applied potential at the gold/mercury electrode (see Figure 1). This is the mobile phase composition used in our laboratory for the reductive electrochemical detection of nitro polycyclic aromatic hydrocarbons (15) and contains 54% methanol. The residual current under these conditions is quite low and comparable to that previously achieved with a zinc oxygen scrubber (3). To investigate the amount of methanol required for oxygen reduction, we examined the residual current at two different applied potentials as a function of the methanol content of the mobile phase. At a potential of -600 mV (Figure 2), on the diffusion plateau of the first oxygen reduction wave, as little as 1% methanol provides substantial reduction of oxygen and 3% methanol provides excellent reduction. At -lo00 mV (Figure 3), on the diffusion plateau of the second oxygen reduction wave, 1-3 % methanol provides substantial reduction of the oxygen, but about 50% methanol is required to provide the most complete reduction. One operational advantage of the use of the platinum oxygen scrubber is the elimination of the long initial delay for oxygen removal from the chromatographic system at the beginning of a working day. At the end of each day’s experiments, the chromatographic column is flushed with pure methanol. This not only cleans the system of contaminants and buffers but, with the oxygen scrubber, assures low oxygen levels for the next day’s use. As long as the chromatographic system is not disassembled, the oxygen levels in the closed system do not return to atmospheric levels for at least 72 h. Thus, after elution of only 10-15 column volumes of solvent, the oxygen levels in the chromatographic system are quite low,
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nitro polycyclic aromatic hydrocarbons to the fluorescent amine ( 1 4 , 1 5 ) . By use of the platinum oxygen scrubber, the working life of the reducer column has been extended to more than 10 days before repacking with fresh zinc is required. The platinum oxygen scrubber is a simple and convenient method of oxygen removal in chromatographic systems. Substantial reduction of oxygen occurs with the addition of as little as 1% methanol to the mobile phase. Thus, this approach to oxygen scrubbing should be compatible with many LC separations. I (I
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1 0
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40
1
Flgure 4. Reductive electrochemical LC detection of nitro polycyclic aromatic hydrocarbons using the platinum oxygen scrubber: peak identity (concentration In Fg/mL), (1) oxygen, (2) 2-nitrofluorene (1.5), (3) 9-nitroanthracene (0.79), (4) perdeuterio-1-nitropyrene (1.4), (5) 1-nitropyrene (1.4), (6) 3-nitrofluoranthene (1.5), (7) 7-nitrobenz[a 1anthracene (1.5), (8) 6-nitrochrysene (1.3), (9) 6-nRrobenzo[a]pyrene (1.O).
__-Table I. Fluorescence Signal Enhancement with Oxygen __ Scrubbers
compound naphthalene acenaphthene fluorene phenanthrene anthracene fluoranthene pyrene benz[a]anthracene chrysene benzo[b]fluoranthene benzo[k]fluoranthene benzo[a]pyrene indeno[l,2,3-cd]pyrene
platinum ( X , n = 3) 2.4 2.0 1.3 2.5 1.2
1.8
zinc (ref 3)
2.7 1.2 1.7
10.4
9.0
2.6 2.7 1.9 1.5
3.0 2.7 1.9
3.0
2.9
1.3
1.2
1.4
and the system may be used to make sensitive measurements. This contrasts sharply against the bulk purging approach, where system startup may require elution of more than 100 column volumes to reach the lowest oxygen levels. The application of the platinum oxygen scrubber to high sensitivity, reductive electrochemical detection of nitro polyclic aromatic hydrocarbons is shown in Figure 4. Although a gradient-elution solvent program is used for this chromatogram, the residual current change with the oxygen scrubber is relatively small. This scrubber is also quite suitable for use in LC fluorescence detection, to eliminate the quenching caused by dissolved molecular oxygen. Table I shows the "fluorescence signal enhancement" (1-3) for several polycyclic aromatic hydrocarbons using the platinum oxygen scrubber as compared to measurements with air-saturated mobile phases. Another function for oxygen scrubbers that has been important in our laboratory has been to extend the lifetime of the zinc reducer column, used for the "on-line" reduction of
ACKNOWLEDGMENT The authors thank Willie E. May of the National Bureau of Standards for suggesting identification of formaldehyde by dinitrophenylhydrazine derivatization and for other helpful discussions. Registry No. Naphthalene, 91-20-3;acenaphthene, 83-32-9; fluorene, 86-73-7;phenanthrene, 85-01-8; anthracene, 120-12-7; fluoranthene, 206-44-0; pyrene, 129-00-0; benz[a]anthracene, 56-55-3; chrysene, 218-01-9; benzo[b]fluoranthene, 205-99-2; benzo[k]fluoranthene, 207-08-9; benzo[a]pyrene, 50-32-8; indeno[l,2,3-cd]pyrene, 193-39-5; oxygen, 7782-44-7; platinum, 7440-06-4; alumina, 1344-28-1; methanol, 67-56-1. LITERATURE CITED Fox, M. A.; Staley, S.W. Anal. Chem. 1978, 4 8 , 992-995. Rollie, M. E..; Ho. C.-N.; Warner, 1. M. Anal. Chem. 1983, 5 5 , 2445-2448. MacCrehan, W. A.; May, W. E. Anal. Chem. 1984, 5 6 , 625-628. Wightman, R. M.; Paik, E. C.; Borman, S.; Dayton, M. A. Anal. Chem. 1978, 5 0 , 1410-1414. Senftleber, F.; Bowling, D.; Stahr, M. S. Anal. Chem. 1983, 5 5 , 8 10-812. Margolis, S. A.; Black, I. J. Assoc. Off. Anal. Chem. 1987, 2 0 , 806-809. Heiliger, F. C. Curr. Sep. 1980, 2 , 5-6. Michael, L.; Zatka, A. Anal. Chim. Acta 1979, 705, 109-117. Hanekamp, H. B.; Voogt, W. H.; Bos, P.; Frel, R. W. Anal. Chim. Acta 1980, 118, 81-86, Trojanek, A.; Holub, K. Anal. Chim. Acta 1980, 121, 23-28, Relm, R. E. Anal. Chem. 1983, 5 5 , 1188-1199. Tejada, S.6.;Zweidinger, R. B.; Slgsby. J. E. Paper 820775 Presented at the Passenger Car Meeting, Troy, MI, June 7-10, 1982. Tejada, S.B.; Zweidinger, R. B.; Sigsby, J. E. Anal. Chem. 1988, 5 8 , 1827-1834. Levin, J.-0.; Andersson, K.; Lindahl. R.; Nllsson, C.-A. Anal. Chem. 1985, 5 7 , 1032-1035. MacCrehan, W. A.; May, W. E. Proceedings of the 8th International Symposium on Polynuclear Aromatic Hydrocarbons : Dennis, A. J., Cooke, M., Eds.; Battelle Press: Columbus OH, 1964; pp 857-869. MacCrehan, W. A.; May, W. E.; Benner, B. A,; Yang, S. D. Anal. Chem. 1988, 6 0 , 194-199.
RECEIVED for review February 24,1987. Accepted September 23, 1987. Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. Work related to the fluorescence enhancement by the oxygen scrubber was in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of Maryland (B.A.B.).
