Anal. Chem. 1984, 56, 217-221
stand weathering effects and provide more specificity, the value of a crude source library is greatly enhanced.
LITERATURE CITED (1) “Annual Book of ASTM Standards”; American Society for Testing and Materials: Philadelphla, PA, 1982; Part 31, D3415-79. (2) Bentz, A. P. Arlai. Chem. 1976, 4 8 , 454A. (3) “Annual Book of ASTM Standards”; American Society for Testing and Materials: Philadelphia, PA, 1982; Part 31, D3327-74T. (4) “Annual Book of ASTM Standards”; American Society for Testing and Materials: Philadelphla, PA, 1982; Part 31, D3414-75T. (5) Brown, C. W.; Lynch, P. F.; Ahmadjian, M. Appi. Spectrosc. Rev. 1875, 9 , 223. (8) “Annual Book of ASTM Standards”; American Society for Testing and Materials: Philadelphla, PA, 1982; Part 31, D3650-78. (7) “Annual Book of ASTM Standards”; American Society for Testing and Materlals: Philadelphia, PA, 1982; Part 31, D3328-78. (8) Bentz, A. P. ”Petroleum in the Marine Environment”; Petrakis, L., Weiss, F. T.; Eds.; American Chemical Society: Washington, DC, 1980; Chapter 3. Bentz, A. P. Anal. Chem. 1878, 5 0 , 655A. Bentz, A. P. ASTM Stand. News 1961, July, 10. Anderson, C. P.; Kllleen, T. J.; Taft, J. 6.; Bentz, A. P. Environ. Sci. Technoi. 1980, October, 1230. Ahmadjian, M.; Baer, C. D.; Lynch, P. F.; Brown, C. W. Environ. Sci. Technoi. 1976, IO,777. Ahmadjian, M.; Baer, C. D.; Brown, C. W.; Westervelt, V. M.; Grant, D. F.; Bentz, A. P. Anal. Chem. 1876, 48, 628. Klileen, T. J.; Eastwood, D.; Hendrick, M. S. Taianta 1981, 2 8 , 1. Baer, C. D.; Brown, C. W. Appi. Spectrosc. 1977, 31, 524. Selfert, W. K.; Moldowan, J. M. Geochim. Cosmochim. Acta 1978, 4 2 , 77.
217
(17) Pym, J. 0.; Ray, J. E.; Smith, G. W.; Whitehead, E. V. Anal. Chem. 1975. 4 7 , 1617. (18) Shen, J., unpublished results, 1982. (19) Saadeh, N. K.; Shen, J., unpublished results, 1982. (20) Engen, R. J., Arabian American Oil Co., Dhahran, Saudi Arabia, unpublished results, 1982. (21) Johnson, J. C.; McAuiiffe, C. D.; Brown, R. A. ASTM Spec. Tech. Pubi. 1978, No. 659, 141. Mackay, D.; Paterson, S.; Boehm, P. D.; Fiest, D. L. I n ”Proceedings, 1981 Oil Spill Conference”, American Petroleum Institue, 1981. Ewald, M.; Lamotle, M.; Garrigues, P.; Rima, J.; Veyres, A,; Lapouyade, R.; Bourgeois, G. I n “Advances in Organic Geochemistry 1981”, Bjoroy, M., et al., Eds.; Wiiey: New York, 1983; pp 705-709. Atlas, R. M.; Boehm, P. D.; Calder, J. A. Estuarine Coastal Sheif Scl. 1981, 72, 589. Seifert, W. K.; Moldowan, J. M. Geochim. Cosmochim. Acta 1879, 43, 111. Shen, J.; Saadeh, N. K., unpublished results, 1983. Volkman, J. K.; Alexander, R.; Kagi, R. I.; Woodhouse, G. W. Geochim. Cosmochirn. Acta 1983, 4 7 , 785. Schmitter, J. M.; Sucrow, W.; Arplno, P. J. Geochim. Cosmochim. Acta 1982, 4 6 , 2345. Phiip, R. P.; Gilbert, T. D.; Friedrich, J. Geochim. Cosmochim. Acta 1981, 4 5 , 1173.
RECEIVED for review July 11, 1983. Accepted September 6, 1983. Thanks are given to the Arabian American Oil Co. for permission to publish this work. Parts of this paper have been first presented at the Annual Meeting of Federation of Analytical Chemistry and Society of Spectroscopy, Philadelphia, PA, Sept 25-30, 1983.
Multiple Peak Formation in Reversed-Phase Liquid Chromatography of Papain’ S. A. Cohen? K. P. Benedek, Shannian Dong, Yitzhak Tapuhi, and B. L. Karger* Institute of Chemical Analysis and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115 This paper presents the reversed-phase ilquld chromatographic behavlor of papaln, a proteolytic enzyme, under acldic conditions with 10 mM phosphoric acid. Wlth an n-butyl bonded phase and 1-propanol as organic modifier, two major peaks widely separated from one another in gradlent eiutlon were observed at pH 2.2 and 5 ‘C. As the column temperature was raised, the later eluting peak grew at the expense of the early eluting peak. Slmllar behavior was observed at a mobile phase of pH 3.0, except that at any given temperature the first peak was relatively larger than at pH 2.2. Flnaiiy, at pH 4.8, only the first peak was observed even at 21 ‘C. Enzymatic assay of the collected fractlons showed the early peak to be active and the late peak to be inactive. Reinjection of the fractions resutted In the appearance of both peaks from the first fraction and only the late eluting peak for the second fraction. These results have been interpreted In terms of the Irreversible denaturatlon of papain on the chromatographic phase, with the first peak being native and the second denatured. Moreover, incubation studies revealed that the denaturationoccurred at a slow rate when the papain was In contact wlth the n-alkyl bonded phase. As a consequence of these studies, papain could be rapidly isolated in an actlve state under reversed-phase chromatographic conditions.
The reversed-phase liquid chromatographic separation of proteins by using n-alkyl bonded phases on small particle * T h i s paper i s dedicated t o t h e m e m o r y of Y i t z h a k T a p u h i . Present address: Waters Associates, M i l f o r d , M A 01757.
porous silica has often been found to yield symmetrical peaks of narrow widths (1-3). Best results have generally been found by using low pH and low ionic strength gradients of water/ acetonitrile or propanol. One concern voiced with this approach is that enzymatic species will often not maintain activity under such elution conditions (4).Nevertheless, some relatively stable enzymes have been isolated in an active state (5,6). Moreover, other enzymes can reversibly recover their activity by change in the elution solution to that of native state conditions. In addition, many applications (e.g., isolation for sequence analysis) do not require chromatographicseparation in active states. Thus, because of its high resolving power and applicability, the reversed-phase procedure is under active study. In order to optimize current phase systems and to find new systems yielding milder elution conditions, it is necessary to understand the phenomena involved in protein retention in reversed-phase chromatography. Valuable empirical studies have been conducted concerning the role of n-alkyl chain length (7), silica type (8), and mobile phase conditions (9) on peak shape and recovery. However, the large size and complexity of proteins have led at times to inconsistent and indeed contradictory results. The problem is in part one of finding model proteins which can reveal underlying retention and general chromatographic behavior as a function of specific variables. Our group has been interested in this problem and in particular has been studying proteins that are relatively stable under acidic conditions and/or in the presence of low molecular weight alcohols. We have found that for certain of these proteins, depending on the mobile phase gradient conditions and column temperature, two or more peaks from a single species can be
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observed. These peaks can be related to native (i.e., active) and denatured (i.e., inactive) forms of the protein. We have previously shown that soybean trypsin inhibitor (STI) produces several chromatographic peaks widely separated from one another a t low pH (10). On the basis of reinjection experiments and analysis of collected peaks (using sodium dodecyl sulfate poly(acry1amide) gel electrophoresis (SDS PAGE) and inhibition assays), we were able to show that the above behavior is caused by irreversible denaturation in the chromatographic column with the early eluting peaks being active and the last peak, widely separated from the early pet& in the gradient, denatured. The extent of denaturation was found to be a function of mobile phase pH, column temperature, and the incubation time that STI spends on the column prior to elution. In this paper, we examine another protein that exhibits similar behavior to STI, namely, the proteolytic enzyme, papain. As with STI, papain in acid media undergoes irreversible denaturation ( 1 1 ) . Two chromatographic peaks, widely separated under gradient elution conditions, are observed, the relative amounts of the two species depending on pH, column temperature, and incubation time. The fist peak will be shown to be native and the second denatured. Indeed, under proper conditions, activg papain can be recovered in a purified state with no appearance of the denatured peak. These studies, in conjunction with those of STI, provide insight into important parameters in the denaturation process. As such, papain and STI are useful model proteins to assess phenomena in the reversed-phase chromatographic process and to suggest procedures to recover species in an active state. In addition, the results reveal that one cannot simply assume that multiple peaks from an injected protein are a consequence of impurities. EXPERIMENTAL SECTION The chromatographicsystem consisted of two M6000A pumps controlled by a Model 660 gradient programmer (Waters Associates, Milford, MA), a Model 7125 injection valve (Rheodyne, Inc., Cotati, CA) and a Schoeffel770 (Kratos, Analytical Instruments, Westwood, NJ) variable wavelength detector. The column packing ma;terialwas LiChrospher SI 500 bonded with butyldimethylchlorosilane and end-capped with a mixture of hexamethyldisilazane and trimethylchlorosilane. The phase was slurry packed in carbon tetrachloride:methanol(9:1) in a stainless steel column 100 X 4.6 mm. In most experiment8 either of two mobile phase gradient systems were employed. In the first, mobile phase A was 10 mM H3P04(pH 2.2) and mobile phase B was 1-propanokwater(45/55, v/v) with an overall H3P04concentration of 10 mM. The second system consisted of 10 mM NaH2P04(pH 4.8) as mobile phase A, and mobile phase fi was similar to the first gradient except 10 mM NaH2P04replaced H,Pd4. In general, a linear gradient from 5 to 85% B in 30 min was died with a flow rate of 1mL/min. Detection was conducted at 210 nm. Column temperature was coptrolled by immersing the injector, 3 m of 0.01 in. i.d. tubing prior to the injector, and the column in a water bath. At temperatures above 20 O C the temperature was controlled within f0.2 "C of that stated, while below 20 "C control was within f0.5 "C. Samples of papain (2-3 mg/mL) were made up in mobile phase A or water, and for anaIytical purposes, an injection volume of 20 pL (40-60 wg of sample) was used. For preparative scale, required for the reinjection, SDS electrophoresisand/or activity measurements, 100 pL of a 20 mg/mL (2 mg) solution of protein was employed. For papain chrorpatographed at pH 4.8, fractions from eluted peaks of preparative runs were collected in chilled water, lyophilized, and theq reconstituted with the appropriate solvent. In the case of preparative scale collections of eluted peaks at pH 2.2, the chilled fraction6 were first neutralized prior to lyophilization. c h e m i c a l s . Silane reagents were purchased from Silar Laboratories, Inc., Scotia, NY,and LiChrospher SI 500 silica was from MCB Reagents, Inc., Gibbstown, NJ. All chemicals for activity
T = 10 "C
T=i.5 ' C
T=26"C
I
0
I
5
I 10
I
f5
I 20
I 25
TIME, minutes
Flgure 1. HPLC chromatographic behavior of papain as a function of temperature: column, C4 bonded phase on 10-pm LiChrospher SI500; mobile phase A, 10 mM H3P04, pH 2.2; mobile phase 6,H,0/1propanol, 55/45 (v/v), in which the total H3P04concentration is 10 mM; linear gradient from 5 to 8 5 % mobile phase B in 30 min; flow rate, 1 mL/min; sample, 20 pL/mL papain in mobile phase A; detection at 210 nm.
Chromatography.
and SDS PAGE measurements were obtained from Sigma, St. Louis, MO. Also, papain, type IV, was from Sigma and used as received. Mobile-phase components, including water (HPLC grade), propanol (HPLC grade), and &PO4 (reagent grade) were obtained from J. T. Baker Chemical Co., Phillipsburg, NJ. Coomassie Brilliant Blue R-250 was obtained from BioRad Laboratories, Richmond, CA. E n z y m e Assay. Activity measurements were made by the spectrophotometricdeterminationof p-nitroanilineformed during hydrothe hydrolysis of (N-a-benzoylarginine)-p-nitroanilide chloride based on the procedure of Erlanger e$ al. (12), as adapted by Arnon (13). Activity was measured by following the change in absorbance at 410 nm with a Spectronic 20 spectrometer (Bausch and Lomb, Rochester, NY). A control sample consisted of 0.1 mL of stock papain solution which was assayed for activity, as described above. Protein quantities were determined by the Coomassie Blue dye binding technique (14). SDS PAGE. SDS PAGE was conducted on a gel electrophoresis system GE 2/4 using a power supply EPS 500/400 (Pharmacia, Sweden). The procedure was based on that of Laemmli (15). RESULTS AND D I S C U S S I O N
The elution profile of papain chromatographed on a reversed-phase column is sensitive to mobile phase pH and column temperature. Figure 1shows the influence of temperature on peak distribution when the pH of the aqueous portion of the gradient is 2.2 (10 mM H3P04). At a column temperature of 5 OC, the bulk of the material is distributed equally between two major peaks with retention times in the gradient of approximately 11.6 and 23.2 min, respectively. (The shoulder on the peak eluting at 11.6 min may represent an impurity.) Increasing the column temperature has a distinct and significant effect on the chromatogram observed. A decrease in peak area of the early peak and the simultaneous increase in Chromatographic Behavior.
ANALYTICAL CHEMISTRY, VOL. 56, NO. 2, FEBRUARY 1984
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h
A.
FRACTION I
h
T =/5 "C
FRACTION D A = 0.01AUFS I
0
10 15 TIME, minutes
5
I
1
20
25
0
i
T.21 "C
I
I
0
5
I
5
10 15 TIME, minutes
20
25
Flgure 3. Reinjection experiments for collected fractions of papain at pH 2.2 and 5 "C; see Figure 1 for other conditions: (top chromato-
gram) 250 pg of papain; (middle chromatogram) reinjection of peak I; (lower chromatogram)reinjection of peak 11.
I
10 15 TIME, minutes
I
I
20
25
chromatographic behavior of papain as a function of temperature at pH 3.0 other conditions similar to Figure 1. (6)HPLC chromatographic behavior of papain at a column temperature of 21 "C and pH 4.8; other conditions similar to Figure 1.
Flgure 2. (A) HPLC
the late eluting peak is seen. Indeed at 21 "C, the early component has essentially disappeared. Further increase in temperature does not markedly influence the chromatogram. It can be also seen that raising column temperature decreases retention for both major peaks (most clearly observed for the late eluting peak). Note also that in the chromatograms of Figure 1,there is substantial material eluting early in the gradient. Papain, a proteolytic enzyme, is degraded into peptide fragments by self-digestion. In freshly prepared samples, injected within 1 min of dissolution, there are no peaks prior to the first major component (e.g., see Figure 2A, 5 "C). As shown in Figure 2A, a similar trend in peak distribution for papain as a function of column temperature occurs in a mobile phase system in which the aqueous pH is 3.0. In this study, however, at any given temperature the early peak is more prominent than at pH 2.2. For example, at 21 "C the early peak still constitutes a significant portion of the mass eluted, whereas at pH 2.2 it has virtually disappeared. Indeed, at pH 4.8 and 21 "C, only the first major peak is found (Figure 2B). At pH 5.5 and room temperature, a chromatogram identical with that in Figure 2B was also observed. Similar trends in changes of the early eluting vs. the late eluting peak as function of pH and column temperature were observed for STI (IO). Chromatographic Peak Examination. In order to ascertain the nature of the first and second peaks, we collected each fraction for electrophoretic and enzymatic activity characterization. Experiments were conducted with samples collected for gradient elution at pH 2.2 and 5 "C under which roughly equal peak areas of the first and second components were found (see Figure 1). Since it is known that papain is
not stable for long periods of time at high acidity (11,13),the samples upon collection were immediately neutralized to pH 7. Next, the samples were subjected to lyophilization and reconstituted in distilled water for subsequent examination. SDS PAGE revealed that both collected fractions had the same molecular weight and that this weight was identical with a standard papain sample. This result supports the contention that the two peaks arise from the same protein and are not a consequence of impurities. The enzymatic activity assay revealed that the first peak was active but that the second peak was inactive. Thus, it can be concluded that the first peak is a native form and the second is denatured. On the basis of the SDS PAGE and enzymatic activity results, it is reasonable to conclude that the phenomena being observed in Figures 1 and 2 are a consequence of the denaturation of native papain, as previously observed for STI (IO). A denatured form of papain would be favored by lower pH and higher temperature (11). It can be seen that the second peak (denatured) grows at the expense of the first (native) with system changes in these directions. Conversely, the native state is stabilized by higher pH and lower temperature. A t pH 4.8 and 21 "C, only the native form appears, and this condition provides a means for isolating active papain by reversed-phase chromatography. Further information concerning the chromatographic denaturation process was obtained by reinjection of collected fractions. In this experiment, the first and second peaks at pH 2.2 and 5 "C were again collected and immediately reinjected into the column (without neutralization or lyophilization). The results are shown in Figure 3. Fraction I upon reinjection produced both the first and second peaks, whereas fraction I1 only produced the second peak. As already noted, at this low pH, papain undergoes irreversible denaturation in solution (11). Since the second fraction does not yield any of the native first peak, irreversible denaturation is also observed in the reinjection experiment. (Of course, irreversibility in the reinjection experiment can only be a relative measure, since reinjection was not studied as a function of the time of standing of the collected fraction.) Note also that the proteolytic impurities disappear upon the reinjection of fractions I and 11.
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Table I. Mass and Activity Recovery of Collected Major Peak of Papain at pH 4.8, 2 1 'C, Using C4 Bonded Phasea recovery (% of original sample) mass activity specific activity
59 85
114
a For chromatographic conditions, see Figure 2B; injected amount was 2 mg. _____l___l
n + d L 15 mm
e
Flgure 4. Chromatographic behavior of papain as a function of incubation time on the column. Conditions were similar to those of Flgure 1 except injection time relative to the start of the gradient Is varied.
It is important to point out that the retention difference between the native and denatured peaks is large, particularly in light of the fact that protein retention is known to be very sensitive to small changes in organic modifier concentration and that gradient elution is used in Figures 1 and 2. Papain is known to be globular in its native state conformation (13). A denatured species would possess a different conformation which would be more unfolded than the globular shape. The size of such a species adsorbing to the bonded phase surface would thus be expected to be larger than in the native state. Therefore, a much stronger binding of the denatured state to the surface would be anticipated. In other words, the number of sites of interaction of a globular native state protein with the stationary phase would be less than that of an unfolded species (16). Hence, the large difference in retention between native and denatured species found for both papain and STI (10) is reasonable. On-ColumnIncubation. Since it is possible to obtain both native and denatured peaks in the same chromatogram, papain can represent a good model protein to probe the factors influencing the unfolding process in reversed-phase chromatography. Of course, one must always be cautious in generalizing from the results with a single protein, particularly given the diversity of properties of proteins. We have nevertheless begun to explore the causes of papain denaturation. In this paper we report on the important role of the contact (incubation) time on unfolding of the protein with the n-alkyl stationary phase. Previously, we have shown that this incubation time influences the relative amounts of native to denatured peaks for STI (IO). Figure 4 illustrates the role of incubation time on the denaturation process of papain by using the common condition of pH 2.2 and 5 OC. In this experiment a solution of papain in mobile phase A was injected on the column from 30 min prior to the start of the gradient to 8 min after the gradient began. In Figure 4, S represents the start of the gradient from the programmer; a delay time of 4 min takes place before the gradient reaches the head of the column. It can be seen that large changes in the relative amounts of native to denatured papain occur, depending on the time of incubation and whether the protein injection is before or after the gradient.
(The difference in the relative amounts of the native to denatured peaks for injection at the start of the gradient in Figure 4 vs. Figure 1 at 5 "C may be a result of the use of two different C4bonded phase columns in the two experiments.) We next allowed papain to incubate in a solution of mobile phase A at 5 "C for 30 min. This sample was then injected into the column, and gradient elution was begun in the normal manner. A chromatogram similar to that of C in Figure 4 was found. Thus, the change in the relative amounts of native to denatured protein observed in Figure 4 can be attributed to denaturation processes on the bonded phase surface and not to that in solution. Slow denaturation of proteins on hydrophobic surfaces has been observed in other adsorption studies (17). Figure 4 and the previous results with STI (10) thus reveal that an important factor in the reversed-phase chromatographic behavior of these proteins is the rate of denaturation or unfolding on the hydrophobic support. In principle, it should be possible to measure this rate by varying the time of injection prior to the start of the gradient. Moreover, it is expected that this measured rate should be dependent on the mobile phase conditions during incubation. Papain Purification. Figure 2B has shown that at pH 4.8 and 21 "C it is possible to elute native state papain from a C4 reversed-phasecolumn. We therefore decided to employ this condition for the rapid purification of commercial papain. We injected 2 mg of native papain onto the C4 bonded phase column at pH 4.8,21 "C, and collected the major peak. After lyophilizing and reconstituting in distilled water, the mass recovery (Coomassie Blue method (14))and activity recovery were determined. Table I shows the results of this experiment, and it can be seen that the specific activity is higher than in the original sample. It is interesting to note that roughly 60% of the protein is recovered in the major peak. Less than full recovery may be due to proteolytic decomposition of the native species, impurities in the original sample, losses in collection, and/or protein remaining on the column. There are very few examples in the literature on the purification of an active enzyme by reversed-phase liquid chromatography (e.g., ref 5,6, and 18). This work and that of STI (10) provide further examples. For certain enzymes, separation of active species on high coverage n-alkyl bonded phases is thus possible. It is clear that mobile phase conditions, e.g., pH, organic modifier, column temperature, can play a significant role on the extent of denaturation. Furthermore, as Figure 4 illustrates, rapid separation can be important. The conditions of sample collection after the column can also influence recovery of active species (18). In the case of papain, collection at room temperature of the native peak eluting at pH 2.2 and a column temperature of 5 "C caused irreversible denaturation, However, collection, followed by immediate neutralization to pH 7 , arrested the denaturation process.
CONCLUSION The results of this paper reveal that for relatively stable proteins, higher pH, lower temperature, and rapid separation favor the elution of such proteins in active form. Indeed, the isolation of active papain at room temperature at pH 4.8 has
Anal. Chem. 1984, 56,221-225
been accomplished. It has also been observed that the rate of denaturation on the bonded phase can play a role in the chromatographic behavior of the protein.
Registry No. Papain, 9001-73-4. LITERATURE CITED (1) Lewis, R. V.; Fallon, A,; Stein, S.;Gibson, K. D.; Udenfriend, S. Anal. Biochem. 1980, 704, 153.
(2) Hearn, M. T. W. In “Advances In Chromatography”; Glddings, J. C., Grushka, E., Cazes, J., Brown, P. R.. Eds.; Marcel Dekker: New York, 1982;Vol. 20. (3) Cooke, N. H. C.; Archer, B. G.; OHare, M. J.; Nice, E. C.; Capp, M. J . Chromatogr. 1983, 2 5 5 , 115. (4) Frlesen, H.J. In “Practical Aspects of Modern High Performance Liquid Chromatography”; Molnar, I., Ed.; de Gruyter: Berlin, 1982. (5) Titanl, K.; Sasagawa, T.; Resing, K.; Walsh, K. A. Anal. Biochem. 1982. 723,408. (6) Berchtold, M. W.; Helzmann, C. W.; Wilson, K. J. Anal. Biochem. 1983, 729, 120. (7) Nice, E. C.; Capp, M. W.; Cooke, N.; O’Hare, M. J. J . Chromatogr. 1981, 278, 569.
221
(8) Pearson, J. D.; Lin, N. T.; Regnier, F. E. Anal. Biochem. 1982, 724, 217. (9) Mahoney, W. C.;Hermodson, M. A. J . Biol. Chem. 1980, 255, 1 1 199. (IO) Cohen, S. A.; Dong, S.; Benedek, K.; Karger, B. L. Symposium Proceedings, Filth International Symposium on Affinity Chromatography and Biological Recognition, Academic Press: New York, in press.
(11) Glazer, A. N.; Smith, E. L. J . Bid. Chem. 1960, 245, PC43. (12) Erlanger, B. F.; Kokowsky, N.; Cohen, W. Arch. Biochem. Biophys. 1961, 9 5 , 271. (13) Arnon, R. Immunochemistry 1985, 2 , 107. (14) Bradford, M. M. Anal. Biochem. 1978, 72, 248. (15) Laemmll, U. K. Nature (London) 1970, 227, 680. (16) Jennlssen, H. P. J . Chromatogr. 1978, 159, 71. (17) Soderquist, M. E.; Walton, A. G. J . Colloid Interface Sci. 1980, 7 5 , 386. (18) Strickler, M. P.; Guski, M. J.; Doctor, B. P. J . Liq. Chromatogr. 1981, 4, 1765.
RECEIVED for review August 19, 1983. Accepted October 7, 1983. The authors gratefully acknowledge the NIH under Grant GM15847 for support of this research. This is Paper No. 133 from the Institute of Chemical Analysis.
Determination of Trace Anions in Water by Multidimensional Ion Chromatography Thomas B. Hoover* and George D. Yager
U S . Environmental Protection Agency, Environmental Research Laboratory, Athens, Georgia 30613
Selenate, selenne, and arsenate Ions were separated from the major anions Chloride, nitrate, and sulfate In drinking water, surface water, and groundwater sources by collecting a selected portlon of the Ion chromatogram, after suppresslon, on a concentrator column and relnjectlng at the orlglnal chromatographlc condltlons. Statlstlcal detectlon llmlts varled from 0.02 to 1.2 pg of trace element dependlng on the mlnor components to be separated and on the water matrix but Independent of lnltlal sample slre from 2 to 10 mL. The maxlmum reliably separated molar ratlo was 1300 for SUIfateiselenate In well water. Carbonate-bicarbonate eluent composltlons were optlmlred for each trace Ion.
Determination of trace components in the presence of major interferences is a common and troublesome problem in many areas of analytical chemistry. Several workers have reported the use of ion chromatography (IC) for the determination of trace anions individually or as peaks well-separated from other components, but no attempts to separate overlapping peaks have been reported. Wetzel et al. (1) showed that part-perbillion levels of individual ions in water could be determined by IC in which the traces were collected on concentrator columns, which were 50-mm lengths containing the same ion exchange resin as the separator column. Bynum et al. (2) reported that the IC response to 100 ppm of sulfate was reduced by the presence of more than 0.4% chloride, although the peaks were well separated. Smee et al. (3)used IC for the determination of fluoride, chloride, nitrate, and sulfate in natural waters. In controlled studies they found that fractional part-per-million levels of F-,C1-, or NO3- gave about 10% greater response when the remaining three ions were added at 20- to 200-fold greater concentrations. Sulfate did not seem to be affected. The authors did not report whether chromatographic peaks overlapped.
This paper is concerned with the extraction of a component that may be completely buried under the peak of a major ion. The approach we used is the elementary one of collecting the portion of the chromatogram suspected to contain the trace ion on a concentrator column and reinjecting a t the original chromatographic conditions. The term “recycle Chromatography” was introduced by Porath and Bennich ( 4 ) for the continuous recycling of eluent in gel chromatography but is used here for the discrete operation that has also been termed “heart-cutting” (5, 6). The procedure, in which no attempt is made to improve resolution through changes of the fixed or mobile phases, obviously is a special case of multidimensional chromatography or column switching (7). We have chosen to illustrate the technique by the speciation of arsenic and selenium in drinking water and water supplies. The National Interim Primary Drinking Water Regulations (8) mandate maximum total concentrations of arsenic and selenium of 50 and 10 ppb, respectively, whereas nitrate nitrogen may be present a t 10 ppm. Chloride and sulfate are not controlled but are typically present a t several parts per million. IC can readily determine arsenate, selenite, and selenate species in water in the absence of interferences (9). At the permissible concentrations, however, they will usually be completely obscured by the major anions. Arsenic and selenium are ordinarily a t their highest oxidation state in surface waters but that is not necessarily the case in groundwaters. When concentrations are great enough to require removal, it is important to know which species are present in designing the treatment technology. The water matrices used in this study-treated municipal drinking water, river water, and water from a shallow well-did not contain detectable amounts of the toxic anions and all experiments were made with known additions. Although the concept of recycling to separate trace components from major interferences is simple, several limitations and conditions are immediately apparent in IC:
This article not subject to U S . Copyright. Published 1984 by the American Chemical Society
