Anal. Chem. 1984, 56,625-628
stationary phase, followed by an association with the ligands. Secondly, the plateaus of the selectivity decrease slightly with increasing chain length of the solute molecule. However, the magnitude of this effect is extremely small. This is probably due to the conformational changes resulting from the progressive transition of the solute conformation from a straight chain (small n, values) to a random coil (large n, values).
LITERATURE CITED Howath, Cs.; Melander. W. R.; Molnar, I . J . Chromatogr. 1078, 125, 129-156. Karger, B. L.; Gant, J. R.; Hartkopf, A.; Welner, P. H. J . Chromatogr. 1076, 128, 65-78. Tanaka, N.; Thornton, E. R. J . Am. Chem. SOC. 1077, 9 9 , 7 . 3-0 - 0- 7. 3- 0- 7 Martin, A. J. P. Biochem. SOC. Symp. 1040, No. 3 , 4-10. Melander, W. R.; Horvath, Cs. Chromafographia 1982, 15,86-90, and references contained therein. Berendsen, G. E.; Schoenmakers, P. J.; de Galan, L.; Vigh. G.; VargaPuchony, 2.; Inczedy, J. J . Llq. Chromafogr. 1080, 3, 1669-1686. Krstulovic, A. M.; Colin, H.; Gulochon, G. Anal. Chem. 1082, 5 4 , 2 138-2 143. Colin, H.; Krstulovic, A. M.; Gonnord, M. F.; Gulochon, G.; Yun, 2.; Jandera, P. Chromatographla 1083. 17, 9-16,
-
625
Colin, H.; Yun, 2.; Gulochon, 0 . ; Jandera, P.; Dlez-Masa, J. C. J . Chromatogr. Sci., in press. Grushka, E.; Colin, H.; Gulochon, G. J . Chromatogr. 1082, 248, 325-341. Colin, H.; Guiochon, G.; Dlez-Masa, J. C. Anal. Chem. 1981, 5 3 , 146- 155. Berendsen, G. E.; de Galan, L. J . Chromatogr. ISSO, 196, 21-37. Karch, K.; Sebestian, I.; Halasz, I . J . Chromatogr. 1078, 122, 3-16. Colin, H.; Guiochon, G. J . Chromatogr. 1078, 158, 183-205. Vlgh, Jy.; Varga-Puchony, 2. J . Chromatogr. 1980, 196, 1-9. Lochmuller, C . H.; Hangac, H. H.; Wilder, D. R. J . Chromatogr. Sci. 1981, 19, 130-136. Sleight, R. B. J . Chromatogr. 1974, 83, 31-36. Rehak, V.; Smolkova, E. J . Chromatogr. 1080, 191, 71-79. Rledo, F.; Kovats, E. Sz.; Czencz, M.; Liardon, 0. Helv. Chlm. Acta 1081, 61, 1912-1941. Steudel, R.; Mausle, H. J.; Rosenbauer, D.; Mockel, H.; Freyholdt, T. Angew. Chem., I n t . Ed. Engl. 1081, 2 0 , 394-395. Baker, J.; Ma, C. Y. J . Chromatogr. 1070, 169, 107-115. Schoenmakers, P. J., private communication. Engelhardt, H.; Ahr, G. Chromafographia 1081, 1 4 , 227-233. Slaats, E. H.; Kraak, J. C.; Brugman, W . J. T.; Poppe, H. J . ChromafOgr. 1078, 149, 255-270. van de Venne, J. L. M. Thesis, Elndhoven, 1979. Tchapla, A.; Fabre, C. Tetrahedron 1982, 38, 2147-2155.
RECEIVED for review April 18,1983. Resubmitted December 21, 1983. Accepted December 21,1983.
Oxygen Removal in Liquid Chromatography with a Zinc Oxygen-Scrubber Column W. A. MacCrehan* and W. E. May
National Bureau of Standards, Organic Analytical Research Section, Center for Analytical Chemistry, Washington, D.C. 20234
A slmple and effectlve method has been developed for oxygen removal from llquld chromatographic eluents, based on a zinc scrubber column. The mechanism of the oxygen reductlon has been verlfled by differential pulse polarography. The scrubber column has been applled to remove the oxygen Interference In two llquld chromatographlc detectlon systems, reductive amperometry and molecular fluorescence, and Its advantages are demonstrated In the detectlon of nitro polynuclear aromatlc hydrocarbons.
The removal of atmospheric oxygen from liquid chromatographic (LC) mobile phases may be desirable or necessary for several analytical reasons. Most frequently, solvents are degassed to avoid the physical problem of ”outgassing” on the low pressure end of the analytical column. Molecular fluorescence detection of certain compounds is also affected by the dissolved oxygen. The presence of oxygen can cause intersystem energy transfer from the excited state of the fluorophore resulting in “quenching”of the fluorescence signal through this nonradiative decay pathway (1). Oxygen removal to very low levels is an absolute requirement for most reductive electrochemical detection (2-4) since the oxygen reduction current contributes deleteriously to residual current and detector noise. Finally, some analytes are sensitive to oxygen oxidation during the chromatographic process [i.e., ascorbate (5)] and either oxygen removal or the addition of antioxidants to the mobile phase is necessary to prevent decomposition. Several approaches have been used to remove oxygen from LC mobile phases. Coarse degassing is routinely performed by either evacuation, warming, or purging the solvent reservoir
with helium. Tejada and co-workers (6) have developed an on-line method for elimination of oxygen quenching in fluorescence detection based on catalytic reduction with a packed bed of crushed “three-way automotive catalyst”. More rigorous deoxygenation is required for reductive electrochemical detection and consequently several approaches have emerged. The most simple approach involves continuous purging of the solvent reservoir with high-purity argon or nitrogen. Kissinger and co-workers (3,7)recommend purging at elevated temperature with continuous reflux of the solvent. One very effective approach (4)uses alternate evacuation and purging in a sealed mobile phase reservoir. Electrochemical reduction of the oxygen with a special high-pressure coulometric cell has also been proposed (8) to remove oxygen “on-line” between the pump and injector, although conventional purging of the solvent reservoir was still required, due to less than 100% oxygen reduction. Recently, two similar “on line” approaches have been reported for oxygen removal from mobile phases. A postcolumn deoxygenator has been developed based on the diffusion of oxygen through gas permeable tubing under the influence of a vacuum (9). However, the device has an internal volume of 205 FL which would contribute to chromatographic band broadening. Another membrane oxygen-scrubber described by Warner et al. (IO)used a Cr(I1) solution as a trap to create the concentration gradient through the permeable tubing. However, the internal volume of the tubing required for complete oxygen removal would be too large to be suitable for LC use. All of these deoxygenation procedures either are quite laborious or require additional specialized equipment to be added to the LC system. This work presents a simple and effective chemical method for oxygen removal based on the
This article not subject to U.S. Copyright. Published 1984 by the American Chemical Society
626
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
use of a column packed with zinc particles. EXPERIMENTAL SECTION Oxygen-Scrubber Column. Zinc particles were dry packed with vibration into a 14 x 0.4 cm stainless steel column, using 2-pm end frits. For fluorescence detection, low-purity 100-mesh zinc was found to be adequate to eliminate oxygen quenching. However, for reductive electrochemical detection, high-purity 200-mesh zinc provided lower (by a factor of 2) residual currents. An in-line, 2-rm particle filter was placed in the system following the scrubber column to ensure that no Zn particles could enter. The scrubber column and filter were then inserted in the LC between the pump and injector. The zinc oxygen-scrubber columns were found to have finite useful lifetimes, since the zinc is being consumed by the oxygen. Adequate removal of oxygen to reduce fluorescence quenching could be maintained for 2-3 weeks of continuous operation. However, for the more demanding reductive electrochemical detection, residual currents began to increase after only 3 days of use of the zinc oxygen-scrubber column. The loss in scrubbing efficiency occurs long before most of the zinc in the columns is consumed. However, the mechanism of the decay in performance seems to be a result of dissolution of the zinc, forming channels in the packed bed. Since the surface area exposed to the flowing solvent is decreased with use, the columns eventually fail. They may be easily repacked however, with the old zinc being removed with a drill bit. There is no conditioning required for optimum performance of the zinc oxygen-scrubber column (conditioning is, however, required for the analytical column, vide infra). Downtime, due to scrubber failure, may be avoided by having several zinc-packed columns on hand and replacing them as needed. Supporting Electrolyte. Some ionic electrolyte was necessary to achieve high reduction efficiency of oxygen. In these experiments we used 0.025 mol/L NH40Ac pH 5.4. It was sometimes necessary, depending on the solvent delivery systems used, to coarsely degas the solvent by heating to 50 OC, followed by sonic agitation or evacuation to avoid formation of microbubbles (consisting most probably of hydrogen and nitrogen) at the detector end of the analytical column. Fluorescence Detection. A commercially available LC spectrofluorimetric detector with time-programmable excitation and emission wavelengths was used. A spectral band-pass of 2.5 nm was employed for both emission and excitation monochromators. To eliminate the oxygen quenching of fluorescence, several column volumes of electrolyte were pumped through the scrubber/analytical system before any measurements were made. This step was necessary to allow removal of oxygen already on the surface of the particles packed in the analytical columns. Reductive Electrochemical Detection. A 0.05 mol/L NH40Ac pH 5.4 electrolyte was used to avoid serious iR drop in the amperometric detector cell. Also a stainless steel connector was used to connect the column outlet to the detector. All samples were purged with solvent-saturated argon for at least 5 min prior to sampling using a syringe/loop injection. A commercially available thin-layer amperometric detector with a 3.0 mm diameter gold/mercury film working electrode was used. Applied potentials were measured against a Ag/AgCl, 3 mol/L KCl reference electrode. In order to make high-sensitivitymeasurements in the reductive mode of detection, it was necessary to flow the mobile phase through the scrubber/analytical system with electrolyte for some time. This rigorous requirement was needed to allow oxygen that was entrained within the pores of the silica packing of the analytical column to diffuse out. The most convenient way to accomplish this was to allow the system to operate overnight at 0.05 mL/min flow rate prior to high sensitivity measurements. Use of this preconditioning treatment of the analytical column makes the difference between a residual current of 200-300 nA (after several columns volumes) and 7-10 nA after overnight conditioning, at a flow rate of 1.0 mL/min and an applied potential of -0.7 v. RESULTS AND DISCUSSION We discovered a simple but very effective approach to oxygen removal while developing a method for on-line chem-
90% CH,CN
2 N Flu
9 N nt
-U L
i
I 10
c
I 20
I 30
Minutes Flgure 1. Fluorescence detection of N-PAH after on-line reduction to the amines: column, octadecylsilane bonded-phase silica, 5-pm particles, 25 X 0.46 cm; solvent, CH,CN/H,O as marked, 0.025 mol/L NH,OAc, pH 5.4; flow rate, 1.5 mL/min; zinc oxygen-scrubber, 14 X 0.4 cm, 100-mesh particles; zinc reducer column 4 X 0.3 cm, packed with Zn/silica; sample, 2 pL of a mixture of 10.0 pg/g 2-nitronaphthalene (2N Nap), 10 pg/g 2-nitrofluorene (2N Flu), 11.7 pglg 9-nitroanthacene (9 N Ant), 11.0 pg/g 1-nitropyrene (1N Pyr), 8.0 pg/g g-nitrobenzo[a ] pyrene (6N B(a)P).
_-
100 -
~
0 , 0
p 200
T--+h 400
600
3000
mL of Eluent
Flgure 2. Reduction efficiency as a function of reducer column use:
open circleskoiid line, without zinc oxygen-scrubber;squaresIdashed line, with zinc oxygen-scrubber. Conditions were the same as those given in Figure 1 except that the analyte was I-nitropyrene. ical reduction of nitro polynuclear aromatic hydrocarbons (N-PAH) using small columns packed with zinc metal particles (11). These small %educein columns are inserted between the analytical column and detector to convert the nitro group quantitatively to the amine for fluorescence detection; see Figure 1. However, the small amount of zinc (about 200 mg) in the short reducer column was found to be consumed via dissociation into the mobile phase after only a few hundred milliliters were eluted, resulting in a decreased conversion efficiency from the nitro to the amino form (see Figure 2). In order to prevent the rapid deterioration of the reducer column, we tried inserting a larger volume column (0.4 X 14 cm) be-
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
627
Table I. Fluorescence Signal Enhancement with a Zinc Oxygen-Scrubber Column compound LoIL anthracene fluoranthene pyrene 1-aminopyrene benz[a]anthracene benzo [alpyrene phenanthrene benzo[ blfluoranthene benzo[h Ifluoranthene chrysene dibenz[a, h ]anthracene benzo[ghi Jperylene indeno[ 3,2,3-cd]pyrene I
-0.5
I
I
-0.7
-0.9
-1.3
Flgure 3. Differential pulse analysls of zinc scrubbed eluent: sample, eluent diluted 1:lO with 0.1 mol/L phosphate buffer pH 7.0; electrode, S M M (medium drop); reference electrode, Ag/AgCI 3 mol/L KCI; scan rate, 5 mvls; A, scrubbed eluent alone; B, addition of 4 X mol/L mol/L H,O,. Na,Edta; C, addltion of 1.3 X
tween the pump and injector packed with 100 mesh zinc particles as a sacrificial guard. As seen in Figure 2, the guard column extends the life of the reducer column by over a factor of 25. The mechanism of the guard function of this column was found to be the removal of the oxygen, which was consuming the small amount of zinc of the reducer column. The following sequence of reactions is proposed to explain the oxygen-scrubbing action of the zinc guard column:
-
+ O2 + 2H+ Zn + Hz02+ 2H+
1.3 3.0 2.9 2.7 1.9 1.4 2.1
3.3 6.4 1.2
1
-1.1
Applied Potential in V
Zn
1.2 1.7 9.0
Zn2+
-
+ HzOz
Zn2+
+ 2Hz0
(1) (2)
We felt it was crucial to investigate whether reactions 1and 2 went to completion under the conditions of our flow experiment. We found that oxygen-induced quenching of fluorescence w a ~eliminated when the zinc column was inserted in a LC system (vide infra). Also when a reductive electrochemical flow cell was attached to the outlet of the zinc oxygen scrubber, very low residual currents were found up to an applied potential of -0.8 V (vide infra). Thus, we were certain that reaction 1was essentially complete. However, to test the progress of reaction 2, we performed some “off-line” bulk electrochemical studies of scrubbed eluant. A sample was collected, diluted with pH 7 buffer, and subjected to differential pulse polarographic analysis. Figure 3A gives the result, with a single signal with Ellz= -1.05 V. This peak strongly resembles the response for Zn2+in this electrolyte. However, since HzOzhas its reduction wave at a potential very close to that of Zn2+(Ellz= -0.99 V), it would be impossible to resolve the two waves under these circumstances (owing to the relatively poor resolving power of differential pulse polarography). However, it was possible to “shift” the reduction wave for Zn2+ to more negative potentials by complexation with EDTA (curve B, Figure 3). The resulting curve strongly resembles the “base line” curve obtained in the buffer alone. No evidence for a residual HzOz wave can be seen. To verify that the EDTA was not consuming the HzOzthrough a redox process, a spike of H202 was added in an amount that would correspond to only 30% progress of reaction 2. A single wave, with no time dependence, was observed (see Figure 3C). Thus reaction 2 must approach 99% completion under these conditions. This is a particularly important point since residual HzO2 in the scrubber LC eluent would be undesirable, where it could initiate redox reactions with the analyte and cause
2o
3 0
t I
1
’I -0.1
/
APPLIED POTENTIAL IN V
Flgure 4. Resldual current for reductive detection: zinc oxygenscrubber column 14 X 0.4 cm packed with 200 mesh high-purity zinc; solvent, 70% CH,CN/H,O, 0.05 mol/L NH40AcpH 5.4; flow rate, 1.0 mL/min; detector, thin-layer amperometry with 3 mm diameter Au(Hg) electrode.
column deterioration. No such effects have been observed in our experiments. We have found the oxygen-scrubber column to have two other useful applications described below. Eliminating Oxygen Fluorescence Quenching. The molecular fluorescence signals of several PAH compounds show sensitivity to the quenching effects of oxygen. For example, pyrene shows a significant decrease in signal when air-saturated instead of oxygen-free mobile phases are used (1). The relative fluorescence signal enhancement Lo/L,using the zinc oxygen-scrubber column to eliminate oxygen quenching, may be expressed as fluorescence signal in the absence of oxygen
Lo’L = fluorescence signal in air-saturated solution (3) The “fluorescence enhancement” obtained when using the scrubber is given in Table I for several PAH compounds. Reductive Electrochemical Detection. The removal of oxygen by the zinc scrubber is sufficiently quantitative to allow high sensitivity detection in the reductive mode. Figure 4 shows the current/potential curve for the residual current of a gold/mercury film electrode at a flow rate of 1.0 mL/min. This result compares very favorably to the residual current curves obtained by using the continuous-purge (12)and the gas-permeation postcolumn deoxygenator (9) approaches. The magnitude of the residual current density of the three oxygen removal techniques at a gold-mercury electrode in a thin-layer “flow by” cell are approximately 1nA/mm2 for the zinc ox-
628
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984 90% CH3CN
- .. - - - - - -
/I
1N Cor
--T - - - - - * 1-
'
16
8
24
32
Minutes
Flgure 5. Reductive electrochemical detection of some N-PAH by gradient elution: conditions as in Figure 4 except % CH&N as noted; flow rate, 2.0 mL/min; detector potential, -0.7 V; 20 pL Injection of a sample mixture of 100 ng/g 2N Nap, 107 ng/g 2N Flu, 117 ng/g 9N Ant, 110 ng/g 1N Pyr, 80 ng/g 6N B(a)P, and approximately 100 ng/g 1N Cor.
f
ll\o
a t potentials more negative than -0.80 V. This current rise probably results from the reduction of Zn2+produced in the scrubber column. It may be possible to eliminate the zinc ion interference by employing a cation-exchange column following the scrubber in the LC system. However, for our application to the detection of the easily reduced nitroaromatic compounds (with E,,,,, r -0.75 V), the limitation of the potential range to only -0.8 V does not pose a problem. The residual currents are sufficiently low when the zinc oxygen-scrubber column is used that gradient-elution solvent programs may be used at sensitivities necessary for the detection of N-PAH a t the 100 ng/g level, as shown in Figure 5. This ability to employ gradient elution at these low concentration levels in conjunction with reductive electrochemical detection is an important advantage, since we are interested in simultaneously determining N-PAH containing from twoto-five aromatic rings in extracts of complex matrices, such as diesel soot ( I l ) , in a single chromatographic run. Figure 6 shows low-level detection of 1-nitropyrene in the isocratic mode of operation. The limit of detection (defined as 3 X peak-to-peak noise using a 5-s time constant) for 1-nitropyrene using the scrubber column is about 1 ng/g (20 pg absolute). Registry No. Zn, 7440-66-6;02, 7782-44-7;Ant, 120-12-7;9 N Ant, 602-60-8;Flu, 206-44-0;2 N Flu, 607-57-8;Pyr, 129-00-0; 1 N Pyr, 5522-43-0;B(a)P, 50-32-8; 6 N B(a)P, 63041-90-7;2 N Nap, 581-89-5; 1 N Cor, 81316-84-9; 1-aminopyrene, 1606-67-3; benz[a]anthracene, 56-55-3; phenanthrene, 85-01-8; benzo[/3]fluoranthene, 205-99-2; benzo[k]fluoranthene,207-08-9;chrysene, 218-01-9; dibenz[a,h]anthracene, 53-70-3; benzo[ghi]perylene, 191-24-2;indeno[l,2,3-cd]pyrene, 193-39-5.
0 1 p4
I
Minutes
Figure 8. Isocratic LC detection of 1-nitropyrene. Conditions were the same as those given in Figure 4 except that the sample was 1-nitropyrene (1N Pyr) 2.8 nglg.
ygen-scrubber, 3 nA/mm3 for the continuous purge method (IZ),and 15 nA/mm2 for the gas permeation deoxygenator (9) a t applied potentials of -0.75 V, -0.75 V, and -1.00 V vs. the Ag/AgCl reference, respectively. However, with the zinc oxygen-scrubber the residual current increases substantially
LITERATURE CITED (1) Fox, M. A,; Staley, S. W. Anal. Chem. 1978, 4 8 , 992. (2) MacCrehan, W. A,; Durst, R. A. Anal. Chem. 1978, 50, 2108. (3) Bratln, K.; Kissinger, P. T. J . Liq. Chromafogr. 1981, 3 (Suppl. 2), 321. (4) Senftleber, J.; Bowling, D.; Stabr, M. S.Anal. Chem. 1983, 55. 810. (5) Helllger, F. C. Cum. Sep. 1980, 2, 5-6. (6) Tejada, S. B.; Zweidinger, R. B.; Slgsby, J. E. Paper 820775 presented at the SAE Passenger Car Meeting, Troy, MI, June 7-10, 1982. (7) Shoup, R. E.; Brunlett, C. S.; Jacobs, W. A. "Installation/Operations Manual for LC-4A Amperometric Controller"; Bioanalytical Systems Press, West Lafayette, IN, 1981. (8) Hanekamp, H. B.; Voogt, W. H.; Bos, P.; Frei, R. W. Anal. Chim. Acta 1980, 118, 81. (9) Reim, R. E. Anal. Chem. 1983, 55, 1188.(IO) Rollie, M. E.;Ho, C.-N.; Warner, I. M. Anal. Chem. 1983, 5 5 , 2445. (11) MacCrehan, W. A,: May, W. E. "Proceedings of the 8th International Syrnposlum on Polynuclear Aromatic Hydrocarbons"; Dennis, A. J.. Cooke, M., Eds.; Batelle Press: Columbus, OH; in press. (12) Durst, R. A,; Blubaugh, E. A,; Bunding, K. A,; Fultz, M. L.; MacCrehan, W. A,; Yap, W. T. Clin. Chem. (Winston-Salem, N.C.) 1982, 28, 1922.
RECEIVED for review October 18, 1983. Accepted January 9, 1984.
