166
Anal. Chem. 1986, 58,166-170
66-99-9; 2-naphthol, 135-19-3;9-anthraldehyde, 642-31-9.
Table I. Trends in Chromatographic Retention in Supercritical Xenon and Carbon Dioxide solute phenol 2,6-di-tert-butylphenol 2-naphthaldehyde 2-naphthol 9-anthraldehyde
LITERATURE CITED Novotny, M.; Sprlngston, S. R.; Peaden, P. A,; Fjeldsted, J. C.; Lee, M. L. Anal. Chem. 1981, 53,407A-414A. Fjeldsted, J. C.; Lee, M. L. Anal. Chem. 1984, 56,A618-A628. Smith, R. D.; Felix, W. D.; Fjeldsted, J. C.; Lee, M. L. Anal. Chem.
relative retention, a2ln in xenonb in carbon dioxide 2.60 1.94 4.39 9.58 12.39
1.52 3.59 4.79 7.11
17.30
= ( t ~ -2 t N ) / ( t R 1 - t N ) , where t N = retention time of a nonretained peak (solvent front) and tR1 = retention time of benzaldehyde. bConditions are as follows for xenon and COP, respecaa21
tively: inlet pressure, 1059 psi and 1300 psi; column temperature, O C ; density, 1.21 g/cm3 and 0.51 g/cm3; and linear velocity, 3.5 cm/s and 4.7 cm/s.
27 "C and 40
are likely to invite further explorations in coupling the chromatographic systems to other spectroscopic techniques and, possibly, ionization detectors.
ACKNOWLEDGMENT Thanks are due to Robert Brownlee (Brownlee Laboratories, Santa Clara, CA) for the generous gift of the micropump and to Ken Mahler (SGE, Inc., Austin, TX) for the inlet splitter. The continuous interest of Sharon Smith and Dennis Gerson (IBM Instruments, Danbury, CT) in our work is greatly appreciated. This study was supported by Grant No. CHE 82-00034 from the National Science Foundation and Grant No. N14-82-K-0561 from the Office of Naval Research. Registry No. Xe, 7440-63-3;benzaldehyde, 100-52-7;2,6-ditert-butylphenol, 128-39-2;phenol, 108-95-2;2-naphthaldehyde,
1982,5 4 , 18a3-ia85. Smith, R. D.; Kailnoski, H. T.; Udseth, H. R.; Wright, 6. W. Anal. Chem. 1984, 56, 2476-2480. Fjeldsted, J. C.; Kong, R . C.; Lee, M. L. J . Chromatogr. 1983, 279, 449-455. Chester, T. L. J . Chromatogr. 1984, 299,424-431. Shafer, K. H.; Grifflths, P. R. Anal. Chem. 1983, 55,1939-1942. Olesik, S.V.; French, S. B.; Novotny, M. Chromatographla 1984, 78, 489-495. Krukonis, V. J.; Hugh, M. A.; Seckner, A. J. J . Phys. Chem. 1984, 88,2687-2689. Everett, D. H.; Stageman, J. F. faraday Discuss. Chem. Soc. 1978, 65,230-241. Rentzepls, P. M.; Douglas, D. C. Nature (London) 1981, 293, 165-166. Marshall, D. B.; Strohbusch, F.; Eyring, E. M. J . Chem. Eng. Data 1981,26, 333-334. Rlchter, 6.E.; Kuei, J. C.; Park, N. J.; Crowley, S. J.; Bradshaw, J. S.; Lee, M. L. HRC CC,J . Hlgh Resolut. Chromatogr. Chromatogr. Commun. 1883,4,371-374. Giddings, J. C.; Seager, S. L. J . Chem. Phys. 1960,33, 1579-1580. Peaden, P. A.; Fjeldsted, J. C.; Lee, M. L.; Springston, S.R.; Novotny, M. Anal. Chem. 1982, 5 4 , 1090-1093. Springston, S. R.; Novotny, M. Anal. Chem. 1984, 56, 1762-1766. Lauer, H. H.; McManlgill, D.; Board, R . D. Anal. Chem. 1983, 55, 1370-1375. Sprlngston, S. R. Doctoral Thesis, Department of Chemistry, Indiana University, Bloomington, IN, 1984. Giddings, J. C.; Myers, M. N.; McLaren, L.; Keller, R. A. Science 1968, 762,67-73. Chester, T. L., Procter & Gamble Co., Cincinnati, OH, personal communication, June 1985.
RECEIVED for review July 25, 1985. Accepted September 4, 1985.
Capillary Zone Electrophoresis of Proteins in Untreated Fused Silica Tubing Henk H. Lauer* and Douglass McManigill Hewlett-Packard Laboratories, Chemical Systems Department, 1651 Page Mill Road, Palo Alto, California 94304
Capillary zone electrophoresis (CZE) of proteins In untreated fused silica caplllarles Is possible If Coulombic repulsion between proteins and the caplllary wail can overcome adsorptlon tendencies. Thls Couiomblc repulsion can be achleved elther by ralslng the pH of the buffer solution above the isoelectric polnt values of the sample protelns or by dynamlcally modlfylng the lnterfaclal double layer between the wall and the bulk sdutlon wtth selected ions. Separations of model protelns In the pH range 8-11 are shown and discussed. The low dispersion of sample zones In CZE is demonstrated with a theoretlcal plate number approaching 1 X 10'. A pH step gradlent and a slowdown of the ubiquttous electroosmotic flow are observed to Improve peak resolutlon.
Amino acids and small peptides are well-separated by CZE in untreated glass or fused silica capillaries (1, 2). When applied to large biomolecules like proteins, however, serious adsorption problems can prohibit proper separations (2).
These can be eliminated if the capillary wall is properly coated with a hydrophilic, nonionic phase, such as glycerol propylsilyl ( 1 , 2 ) . Although chemical deactivation of glass and fused silica walls has shown major breakthroughs in capillary gas chromatography, appropriate surface deactivations of capillaries for protein separations by liquid chromatography or CZE are still in an investigative phase. Two other methods to avoid wall adsorption of proteins in solution are based on Coulombic repulsions of species and surface. One utilizes the variation of solution pH relative to the isoelectric point (PI) of a protein ( 3 ) to change its net charge. The other method tries to change the charge characteristics of the wall by coating it dynamically with an ionizable phase ( 4 ) . In the first method a change in solution pH does not necessarily change the sign of charge on the wall. Silica gel in contact with aqueous solutions has a negative charge at pH values above 2 (5-7). A protein bears a net negative charge if the pH of its solution is higher than its isoelectric point. It is therefore possible to adjust the pH of solutions to values
0 1985 American Chemical Society 0003-2700/86/0358-0166$01.50/0
ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986
where both proteins and the wall of the fused silica capillary have net negative charges. Under these conditions proteins should be repelled from the wall and their surface adsorption avoided or strongly diminished thereby increasing the chances for proper separation by CZE. As the bulk (-75%) of reported proteins have PI values above 4 @-IO), it seems reasonable to expect that CZE in untreated fused silica is able to address their separation. Large pH-PI differences, however, may cause structural changes in the protein or even hydrolysis. The second method is based on preferential adsorption of specific ions to the capillary surface, changing its net charge or even its sign. This will consequently cause a change in the zeta potential of the interfacial double layer, which will result in a different electroosmotic flow (11). The success of this method largely depends on how these specific ions interact with the sample. This study describes examples of protein separations by applying the outlined repulsion principle and briefly discusses the phenomenon of electroosmotic flow, its magnitude, and possible control.
EXPERIMENTAL SECTION Apparatus. The apparatus closely resembles that described by Jorgenson (2). A straight length of fused silica capillary (SGE, Austin, TX), approximately 100 cm long with a 52 pm i.d. and 320 wm o.d., connected the anodic reservoir with the electrically grounded cathodic reservoir. A high-voltage dc power supply (0-60 kV, Hipotronics, Brewster, NY) was used to drive the electrophoretic process. Current through the system was measured over a 1-MQresistor in the return circuit of the power supply. Oncolumn UV detection was carried out with a modified ISCO V4 variable-wavelength HPLC detedor (Lincoln,NE)at 230 nm. The output signal was digitized at 10 Hz by a 670 series interface (Nelson Analytical, Cupertino, CA) and transferred via an HP37203 A HP-IB extender to an HP9000 computer (HewlettPackard, Palo Alto, CA). The detector output signal was not calibrated. Therefore, detector response changes with concentration, as shown in the figures, should be considered arbitrary. Electropherograms were replotted with an HP9872T plotter. Injection Procedure. On the high-voltage side, two platinum electrodes were separately immersed in two 2-mL glass vials, one containing sample dissolved in buffer and the other containing buffer only. Samples were electroosmotically introduced into the capillary by inserting the tubing into the sample reservoir and applying a constant voltage during a certain time. After the voltage was switched off, the capillary was placed into the buffer reservoir, and the electrophoretic voltage was applied. Cleaning Procedure. Prior to filling a new capillary with buffer a 20-min flush with 1 M KOH was followed by 45-min flushes with 0.1 M KOH and H20,respectively. After experiments were finished the capillary was flushed and filled with 0.1 M KOH, which remained in the capillary overnight. Before new experiments were started, a 15-min flush with H20 followed by a 15-min flush with the buffer of choice was carried out. All solutions were flushed through the capillary to the 25-mL cathodic reservoir by vacuum. This vacuum was applied with a small, manually driven vacuum pump (Mityvac, Nalge Co., Rochester, NY). This cleaning procedure kept standard deviation in the retention times of consecutive sample runs (n 1 10) virtually below 1%and dayto-day retention time accuracies below 4%. Measurements. In most experiments a nonionized molecule, 4-methyl-3-penten-2-one(mesityl oxide), was used as the electroosmotic flow marker. Only in those cases where this mo'ecule would coelute with a protein of the sample was it omitted. Plate numbers were manually calculated from the peak widths at 0.607 of their heights. The average column diameter was calculated from resistance measurements over a known length, when filled with mercury, as described by Knecht et al. (12). The average value so obtained was 52 f 1 pm, Reagents. All proteins were purchased from Sigma (St. Louis, MO). Those used in this study were selected so as to cover a wide range of isoelectric points and molecular weights (see Table I). Buffers (see Table 11) and other reagents were of AR grade or comparable quality. Buffer solutions were prepared with water
167
Table I. Molecular Weight and PI Values of Model Proteins" protein (origin)
PIb
M.W.
14 000 lysozyme (chicken egg white) 11 cytochrome c (horse heart) 9.4 (10.2) (13000) 9.3 13500 ribonuclease A (bovine pancreas) 17500 (17000) myoglobin (whale skeletal 8.1 (8.2) muscle) myoglobin (equine skeletal 7.3, 7.5 (7.4) 17 500 muscle) 17 500 myoglobin (horse heart) 7.3 myoglobin (dog heart) myoglobin (dog skeletal muscle) 77 000 conalbumin (chicken egg white) 6.6 31 000 6.2 (6.1) carbonic anhydrase (bovine erythrocytes) 35 000 @LactoglobulinB (bovine milk) 5.2 (5.5) 35 000 P-Lactoglobulin A (bovine milk) 5.1 (5.4) 4.7 (4.8) 43 500 (45000) ovalbumin (chicken egg)
" References 3, 8, 9, and 10. Values in parentheses were from a bioproducts catalog, Marine Colloids Division, 1984 (Rockland, ME). Table 11. Buffer Constituents and Dissocation Constants" buffer (cyclohexy1amino)propanesulfonicacid (cyclohexy1amino)ethanesulfonic acid
pK,b 10.4OC
9.55c
glycylglycine
8.40
N-Tris(hydroxymethy1)methylglycine
borate decahydrate
8.15 9.22
N-(2-acetamido)-2-aminoethanesulfonic acid
6.88
abbreviation CAPS CHES GlyGly Tricine ACES
"References 14 and 15. b A t 20 "C. cFrom "Comments", United States Biochem. Corp., summer 1984. of HPLC grade, and no bacterial growth inhibitors were added. All buffer solutions were adjusted to the appropriate pH with 1 M KOH or 1M HC1. Proteins were dissolved in the appropriate buffer solution at concentrations of approximately 1 mg mL-'. If necessary, mesityl oxide was added to the sample solutions at a concentration of approximately 0.01 % (v/v).
RESULTS AND DISCUSSION Jorgenson (2)pointed out that separation of model proteins (see Table I) such as cytochrome c (Cyt. c), lysozyme (Lys.), and ribonuclease A (RNA) in untreated fused silica capillaries with a phosphate buffer a t pH 7 was accompanied by strong tailing, possibly caused by Coulombic interactions of the positively charged proteins and the negatively charged capillary wall. To provide both the proteins and the wall with the same sign (-) of charge, the pH of the buffer solution was increased to pH 12 with 0.025 M Na3P04. In this buffer the electropherogram of these model proteins a t 30 kV showed sharp peaks. However, during consecutive runs these peaks deteriorated in shoulder formations. It was not clear if this degradation was caused by denaturation due to the high pH, Joule heating, or interference of the phosphate buffer with these proteins. In this experimental setup a single run was completed within 8 min, but the current strongly increased from 60 to 100 PA. Currents of this magnitude can raise the average temperature of the bulk fluid to approximately 45-60 "C ( 1 , 13). Low conductivities of bulk fluids can be achieved, however, with the buffer systems described in Table 11, in which zwitterions control the pH. Their possible interaction with the sample proteins are virtually negligible (14,15),and their low conductivities enable separate control of p H and conductivity by adding amounts of electrolyte, such as KCl, to
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986
4.0
8.0
6.0
(mid Flgure 1. Electropherogram of model proteins at pH 11.O: elution order Lys., mesityl oxide, RNA, Cyt. c , myoglobin (wsm); buffer, CAPSIKCI 20110 mM; capillary, length to detector 63 cm; voltage, 16 k V and 5 s for injection, 30 k V and 30 MA for electrophoresis. Detector response Is arbitrary; see Experimental Sectlon.
I
1
I
4.0
6.0
8.0
(mid Flgure 3. Electropherogram of model proteins at pH 8.25: elution order myoglobin (wsrn), myoglobin (hh), CAB, CAA, BLB, BLA; buffer, borate 20 mM; capillary, overall length 101 cm, length to detector 55 cm; voltage, 6 kV and 7 s for injection, 30 kV and 38 pA for electrophoresk.
I
I
I
4.0
6.0
8.0
lL 1
(mid Flgure 2. Electropherogram of model proteins at pH 9.22: elution order mesityl oxide, myoglobin (wsm), conalbumin, BLA, ovalbumin; buffer, CHESlKCl 20110 mM; electrophoresis at 30 kV and 20 MA. Other conditions are the same as in Figure 1.
the system and sample. The necessity to control the conductivity of the bulk fluid and sample was derived from previous experiments with dansylated amino acids. In these experiments sufficient ratios (>200) of buffer-to-sample concentrations appeared necessary to avoid peak deformations possibly due to local perturbations of the electrical field (16). An example of this approach is shown in Figure 1. Here the pH of the buffer was lowered to 11, as no zwitterionic buffer above this value was available, and additional proteins were probed. Lys. elutes before mesityl oxide and shows severe broadening and tailing. This is probably due to a slightly positive net charge on the molecule in this system. Myoglobin (wsm) shows a strong shoulder formation, which disappeared when a fresh sample was prepared and directly electrophoresed, and is interpreted as denaturation of the myoglobin under the high pH conditions. This stability effect was also observed with carbonic anhydrase (CA), ovalbumin, and plactoglobulin A (BLA). Lys., Cyt. c , and RNA, however, showed stable electropherograms for at least 8 h. Decreasing the pH further improved the stability and peak shape of the deteriorating proteins. Examples are shown in Figures 2 and 3. Lys., Cyt. c , and RNA could not be eluted with these buffer systems due to wall adsorption (PIS> pH). Although strongly suppressed, adsorption is probably still responsible for the broad and tailing appearance of conalbumin in Figure 2. This protein (PI 6.6) could not be eluted in a buffer with pH 6.8 (ACES/KCl, 20/10 mM), leading to the conclusion that even before the PI of a protein in solution is reached, its adsorption to the wall may still occur. Similar results were obtained by Kopaciewicz et al. (17).They concluded that proteins having zero net charge at their PI are still able to interfere with other charged species or surfaces due
-
M
I
I
I
5.0
10.0
15.0
(mid Figure 4. Optimum in separation of model proteins at pH 8.22: buffer, Tricine/KCI 10120 mM, pH 8.22; voltage, 2 kV and 6 s for injection, 20 kV and 14 pA for electrophoresis. Other conditions are the same as in Figure 3.
to different charge distributions over the molecule. The separation power and speed of this form of CZE is particularly expressed in Figure 3. This electropherogram shows how three pairs of proteins with different molecular weights, but with equal molecular weights within a pair, can be separated into their separate constituents. This example also shows that a common buffer like borate can be used if precautions are taken to avoid excessive Joule heating. In the examples given so far peak broadening was discussed in general terms. The theoretical plate number (N) can be used, however, as a figure of merit to rate the separation efficiency. For peaks with acceptable shape in Figures 1and 2, N fluctuates from 50000 to 100000, depending on the species and buffer. Peak shape has been optimized in Figure 3, which yields an average number of 174 000 and 200 000 for the specific component carbonic anhydrase B (CAB). The higher plate number in Figure 3 is mainly caused by a smaller injection volume (-5 nL instead of -10 nL for the electroosmotic marker). If this experimental system is stretched to its limits in terms of injection (- 1.5 nL) and detection, its optimum separation efficiency is reached and is depicted in
ANALYTICAL
Figure 4. This electropherogram was generated in the same capillary that was used to produce Figure 3, but 4 months later. The sample input was -3.3 times lower, but the noise level increased -3 times during that time interval, which increased the detection limit from -130 to -500 pg for myoglobin (wsm). The average plate number is 516000 and 836000 for CAB. Though almost 1 X lo6plates are produced, the number is still short of the diffusion limited theoretical plate number of approximately 2.8 X lo6. The latter was calculated from the tube length (L = 55 cm), the estimated diffusion coefficient (D = lo* cm2 s-l), and the experimental dwell time ( t D = 9 min) of CAB with N = L2/2DtD. An improvement in the experimental apparatus (injection, detection, and cooling) is possible but it seems necessary to track other zone-broadening factors not yet accounted for. The effect of pH on the elution order of the constituents in a sample appeared to be small but useful. For example, in Tricine/KCl (10/20 mM), pH 8.22, myoglobin (wsm), myoglobin (hh), and CA eluted at 30 kV and 24 p A within 6 min in this order. When the buffer solution was replaced with CHES/KCl (10/20 mM), pH 9.48, CA and myoglobin (hh) changed positions in the electropherogram. This example shows that development of pH and other possible gradients (temperature, reagents) in this technique should be considered in the future. All previous examples showed that a strong electroosmotic flow is always present. Though very helpful in transporting negatively charged species through the detector, its speed will prevent the development of complete separation if more species are present. This is easily derived from the resolution (R) as defined by IUPAC for chromatography
R = -At = 48,
4(u,, + a,)
where At is the difference in retention times of two adjacent peaks and st is the mean standard deviation of the elution profiles; ueo,ti,, and Aue are the electroosmotic, mean electrophoretic, and difference in electrophoretic velocities, respectively (2). Lowering u,, will therefore increase the resolution, if the other factors remain constant. Electroosmotic velocity (u,) in capillaries has been studied intensively (18-21) and is described as
-€{E
u,, = t
(2)
where e, 9, {, and E are the permittivity of the fluid, its viscosity, the zeta potential, and the electrical field strength, respectively. Apart from the sign, eq 2 is also applicable to the electrophoretic velocity (u,) of a charged particle in a fluid (18). It is assumed, however, that throughout the double layer region both e and 7 retain their normal bulk values and that the double layers are thin with respect to capillary radius or particle size. The linear dependence of the electroosmotic flow on electrical field strength, as predicted by eq 2, was demonstrated by all buffer systems explored in this study for a field strength below 200 V cm-l. To control u,, selectively, that is without changing u, of the species to be separated, manipulation of the parameter { seems to be the only possibility, since a change in the bulk variables e, 7,and the field strength ( E ) will equally affect both u,, and u,. The zeta potential describes electrostatic forces in the interfacial double layer between two phases and is, among others, a function of differential adsorption of ions. Experiments with organic cations, aimed at preventing wall adsorption of positively charged proteins a t pH 7, revealed that 1,4-diaminobutane (putrescine) was rather effective, although not good enough to eliminate tailing of Lys. and Cyt.
CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986
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b
I
I
1
I
4.0
6.0
10.0
15.0
(mid Figure 5. Effect of modifier on retention and resolution of five myoglobins: (a) buffer, Tricine/KCI 10/20mM at pH 8.22; voltage, 16 kV and 7 s for injection, 30 k V and 24 PA for electrophoresis and (b) buffer, same as (a) but 5 mM putrescine added, pH 8.20; voltage, 30 kV and 3 1 pA for electrophoresis. Other conditions are the same as
in (a) and Figure 3.
c during CZE under those conditions. However, large nonlinear changes in the electroosmotic flow, caused by small amounts (0-5 mM) of this divalent organic cation, indicated that the interfacial double layer had been modified and therefore so had its zeta potential. It was also observed that monovalent cations, such as ammonium and trimethyl ammonium, had virtually no effect on u,,. Experiments with different solutions in which the diamine also behaved as twice protonated (7 < pH < 8.5) showed that the electroosmotic flow was still affected. Applying these conditions to the previously discussed example (two myoglobins and CA in Tricine/KCl, 10/20 mM, pH 8.22), but with the addition of 2 mM putrescine, showed a reduction in electroosmotic flow of almost 50% and an increase in resolution between myoglobin (wsrn) and CA from 8.2 to 22.3 (eq 1). A more complex sample, containing five myoglobins (see Table I), was electrophoresed in the same buffer with and without the addition of 5 mM putrescine, as is shown in Figure 5. The electroosmotic flow dropped by 60%, but three myoglobins (esm, dh, and dsm) appeared unresolved between the first and last eluting myoglobin, although the resolution between the latter increased from 5.8 to 23.5 (Figure 5b). Some interference of putrescine with myoglobin (wsrn) showed up in minor peak changes during consecutive runs. The double layer modification only addresses the separation of one type of charged species, positive or negative. The ideal situation would be that where both types of species can be separated in one run. This means that the development of chemically bonded wall deactivators remains an important topic. The remaining problems in CZE, such as very specific wall adsorption, conductivity gradients within the sample zones, and adequate detection, still have to be solved before attempts to improve instrument design can be successful.
ACKNOWLEDGMENT The authors thank Jim Jorgenson and Tomas Hirschfeld for their helpful discussions and Miles Spellman for his assistance with the collection and processing of the experimental data. Registry No. Lysozyme, 9001-63-2;ribonuclease, 9001-99-4; cytochrome c, 9007-43-6; carbonic anhydrase, 9001-03-0; pu-
170
Anal. Chem. 1986, 58, 170-175
trescine, 110-60-1;silica, 7631-86-9.
LITERATURE CITED (1) Lukacs, K. D., Thesis, University of North Carolina, Chapel Hill, 1983. (2) Jorgenson, J. W.; Lukacs, K. D. Science (Washington, D.C.,7883-) 1983, 222,266-272. (3) Kopaciewicz, W.; Regnler, F. E. Anal. Biochem. 1982, 726, 8-16, (4) Green, J. S.;Jorgenson, J. W. Pittsburgh Conference, New Orleans, 1985;paper 892. (5) Bolt, G. H. J . Phys. Chem. 1957, 6 1 , 1166-1169. (6) Stigter, D. “Physical Chemistry: Enriching Topics from Colloid and Surface Science”; van Olphen, H., Mysels, K. J., Eds.; Theorex: La Jolla, CA, 1975;p 304. (7) Seng, H. P. Text. Prax. Int. 1984, 39, 795-798. (8) Rlghetti, P. G.;Caravaggio, T. J . Chromatogr. 1976, 127, 1-28. (9) Malamud, D.; Drysdale. J. W. Anal. Blochem. 1978, 86, 620-647. (IO) Righettl, P. G.; Tudor, G.; Ek, K. J . Chromatogr. 1981, 220, 115-194. (11) Reijenga, J. C.; Aben, G. V. A.; Verheggen, Th. P. E. M.; Everaerts, F. M. J . Chromatogr. 1983, 260, 241-254.
(12) Knecht, L. A.; Guthrie, E. J.; Jorgenson, J. W. Anal. Chem. 1984, 56,
479-482. (13) McManigill, D.; Lauer, H. H., unpublished work. (14) Good, N. E.; Winget, G. D.; Winter, W.; Conolly, T. N.;Jzawa, S.; Singh, R. M. M. Biochemistry 1966, 5 , 467-477. (15) Good, N. E.; Jzawa, S. Methods fnzymol. 1972, 2 4 , 53-68. (16) Thormann, W. flectrophoresls (Weinheim, Fed. Repub. Ger.) 1983, 4 , 383-390. (17) Kopaciewicz, W.; Rounds, M. A,; Fausnaugh, J.; Regnier, F. E.; J . Chromatogr. 1983, 266, 3-21. (18) Hunter, R. J. “Zeta Potential in Colloid Science”; Academic Press: London, 1981. (19) Martln, M.; Guiochon, G. Anal. Chem. 1984, 5 6 , 614-620. (20) Martin, M.; Guiochon, G.; Walbroehl, Y.; Jorgenson, J. W. Anal. Chem. 1985, 5 7 , 559-561. (21) Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1985, 5 7 , 834-841.
RECEIVED for review June 10,1985.Accepted August 19,1985.
Reversed-Phase High-Performance Liquid Chromatographic Determination of Nitroorganics in Munitions Wastewater Thomas F. Jenkins* and Daniel C. Leggett
U S . Army Cold Regions Research and Engineering Laboratory, 72 Lyme Road, Hanover, New Hampshire 03755 Clarence L. Grant and Christopher F. Bauer
Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824
Concentrations of HMX, RDX, TNT, and 2,4-DNT are determlned In munltlons wastewater. Aqueous samples are diluted wlth an equal volume of 76/24 (v/v) methanol-acetonltrlle, flltered through a 0 . 4 - ~ mpolycarbonate membrane, and analyzed by reversed-phase HPLC uslng an LC-8 column wlth 50/38/12 (v/v/v) water-methanol-acetonltrlle. The method provlded linear callbratlon curves to at least several hundred mlcrograrns per liter. Detectlon llmlts were conservatively estimated to be 26, 22, 14, and 10 pg/L for HMX, RDX, TNT, and 2,4-DNT, respectlvely, wlth corresponding standard devlatlons of f3.4, 3.3, 4.4, and 4.6 pg/L up to concentrations of 250 pg/L. At hlgher concentratlons, the percent relative standard devlatlon values were approxlmately f 2 % for HMX and RDX and f4% for TNT and DNT. A ruggedness test Involving the major manlpulatlve steps in the procedure Indicated that conslstent results required glass sample contalners, precondltlonlngof filters, and careful malntenance of sample-to-organlc solvent ratio. The method was tested wlth munition wastewater from several Army ammunltlon plants and found to perform adequately for load and pack wastewaters, wastewater from HYX/RDX manufacture, and contaminated groundwater.
One of the Army’s most serious water pollution problems is the disposal of wash waters used to clean equipment and interior surfaces at munition manufacturing and demilitarization facilities. It has been estimated that up to 2 X lo6 L of this type of wastewater is generated daily from a single production line (1). Current practice is to collect wash water from these processing operations in a holding tank and periodically pump it through a carbon adsorption column prior to discharge to
surface streams. These point discharges are subject to state and federal NPDES permits that generally limit the acceptable concentrations of T N T and RDX (2,4,6-trinitroAlthough toluene and hexahydro-1,3,5-trinitro-l,.3,5-triazine). carbon adsorption technology can reduce TNT and RDX to low part per billion levels, which meet present discharge limitations, these carbon columns have finite lifetimes. Eventually breakthrough occurs and regeneration or replacement is necessary. T o satisfy permit requirements and to check on system performance, daily monitoring of effluent from the carbon adsorption columns is necessary during manufacturing operations. Current monitoring requires separate determinations of TNT and RDX, the two most common explosives used by the U.S. Army. Additionally, monitoring for HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine), also an Army explosive and a common impurity in RDX, and DNT (2,4dinitrotoluene), a low-level impurity in TNT, may also be required in the near future. At present no standard analytical method is available for TNT, RDX, or HMX. Hence, individual Army installations have developed their own procedures, which differ widely in detection limits, specificity, and precision. This work was undertaken to develop a suitable method for analysis of munitions wastewaters at U.S. Army facilities. The major requirements for the method were (a) it must have detection limits sufficiently low to satisfy monitoring requirements, (b) it must be rapid to enable quick action if levels are in excess of discharge limits, (c) it should be free of interferences from common impurities and decomposition products in munitions waste streams such as TNB (trinitrobenzene), SEX (l-acetyloctahydro-3,5,7-trinitro-1,3,5,7-tetrazocine), TAX (l-acetylhexahydro-3,5-dinitro-1,3,5-triazine), 2,6-DNT (2,6-dinitrotoluene), 2Am-DNT (2-amino-4,6-dinitrotoluene), 4Am-DNT (4-amino-2,6-dinitrotoluene), 2,6-
0003-2700/86/0358-0170$0 1.50/0 0 1985 American Chemical Society
