1500
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978
Since relatively small square wave amplitudes must be used in order to keep the peak width a t a reasonable value, this mode does not do as well as the differential pulse modes. As is the case for the alternate drop modes of Christie et al., potential-dependent adsorption of trace organics of any kind can invalidate the assumption that the capacity current is independent of the previous history of the waveform. In the case of adsorption, the capacity background may be incompletely compensated by these techniques. U p until recently, all electroanalytical techniques a t the dropping mercury electrode had a charging current contribution due to the growing drop. With the advent of the techniques proposed here and the ones developed by Christie et al. (1-3), this charging current background has been eliminated. Not all the problems with trace analysis a t the DME have been dealt with, but a t least headway is being made. I
eo0
I
100
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1
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ACKNOWLEDGMENT The homemade potentiostat and I-E converter used for some of this work was built by Chaim Yarnitzky.
-eo
(mv)
Figure 9. Theoretical currents for regular and alternate drop rapid scan square wave. (A) Difference current (-) ordinary, alternate drop. (B) Individual forward and reverse currents for atternate drop. Conditions: n A E = 5 mV; nEsw= 30 mV; CT = 0.33 (.e.)
LITERATURE CITED (1) J. H.Christie, W.D. Thesis, Colorado State University, Fori Collins, Colo., 1974. ( 2 ) J. H.Christie, L. L. Jackson, and R. A . Osteryoung, Anal. Chem., 48, 242 (1976). (3) J. H Christie, L. L. Jackson, and R. A. Osteryoung, Anal. Chem., 48, 561 (1976). (4) J. H.Christie and R. A. Osteryoung, J . Nectrwnal. Chem., 49, 301 (1974). (5) J. H.Christie, J. A. Turner, and R. A. Osteryoung, Anal. Chem., 49, 1899 (1977). (6) , . J. A. Turner. J. H.Christie. M. Vukovic. and R. A Ostervouna. Anal. Chem.. 49, 1904 (1977). (71 J. A. Turner, Ph.D. Thesis, Colorado State University, Fort Collins, Colo., 1977. (8) T: Kambara, Bull. Chem. SOC. Jpn., 27, 523 (1954). (9) S. C. Rifkin and D. H. Evans, Anal. Chem.. 48, 1616 (1976). (10) G. C. Barker and A . W. Gardner, AERE Harwell, C/R 2297 (1958). (11) G. C. Barker. Anal. Chim. Acta, 18, 118 (1958).
of symmetry relationship gives the largest response, it also gives the broadest peaks. The two peaks, although larger, are too far apart to give a reasonable response. The best symmetry relationship is one where one current is large and the other is fairly small. T h e choice of square wave amplitude is also important as large square amplitudes show considerable broadening. Figure 9 shows the theoretical forward, reverse, and difference currents for the alternate drop mode as well as the difference current for regular square wave. The alternate drop mode shows a marked broadening of the peak and is diminished. T h e reason for the broadening and diminution arises from the same problem that DPR mode has, mainly that the reserve current has been shifted toward the initial potential by 2 E,, and this is sufficient to invalidate the additive nature of the difference measurement.
I
-
RECEI~ED for review August 12, 1977. Accepted .June 9, 1978. This work was supported by the National Science Foundation under Grant CHE-75-00332, and by the Office of Naval Research under Contract N00014-77-C-0004.
Polymer-Bound Thiol for Detection of Disulfides in Liquid Chromatography Eluates J. F. Studebaker” and S. A. Slocum IBM-
Thomas J. Watson Research Center, Yorktown Heights, New York 10598
E. L. Lewis Mount Holyoke College, South Hadley, Massachusetts
limits the applicability of both HPLC and ambient pressure liquid chromatography is the lack of a detection technique which is sensitive and specific for the compound or class of compounds of interest. Frequently, the problem has been solved by mixing the effluent from the chromatography column with one or more reagents which give rise to some change, usually in optical absorption at a certain wavelength, which is proportional to the concentration of compounds having a specific functional group. This technique has been used successfully in amino acid analyzers and in liquid chromatography detection methods which are based on the Technicon AutoAnalyzer. There are some drawbacks to this technique, however. Each reagent added to the eluate makes
A short column packed with thiol-Sepharose is used in an apparatus for detecting disulfides in liquid chromatography eluates. After a thiol-disulfide interchange reaction with the polymer bound thiol occurs, one part of the disulfide leaves in the eluate stream as a thiol, which may then be detected with a mixing manifold and an absorbance detector. The response of this system to cystine has been found to be linear for Injections of 0.15 pg to 2.5 pg.
High pressure liquid chromatography (HPLC) has proved to be a powerful technique for quantitative determination of compounds in complex mixtures. One problem which often 0003-2700/78/0350-1500$01 O O / O
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1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978 FROH LC COLUMN
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Figure 1. Diagram of the apparatus assembled for detection of disulfides. RSSR represents the disulfide in the eluate, P-S- stands for the polymer-bound thiol, and DSSD represents DTNB The reactions which
take place in the apparatus are indicated the detection apparatus more complex mechanically and dilutes the sample further, and stream splitting must be used if the presence of the detection reagents is not to complicate further analysis of the separated fractions. Some of the detection techniques which involve post-column reactions might be easier to carry out with some of the many "solid phase reagents" which have been developed in recent years ( I ) . These reagents would be packed into short columns placed just after the analytical column, an arrangement which would certainly be simpler mechanically. Peaks would be broadened to some extent as they passed through the short column, but it should be possible to reduce the broadening to a level a t which it would be a lesser problem than the dilution which occurs when soluble reagents are added in a mixing tee. Furthermore, fewer detection reagents are mixed with the sample in the solid phase approach. In the present work, we have developed a method, based on a polymer-bound thiol, for detecting disulfides in liquid chromatography eluates. We needed the method in order to use HPLC rather than peptide mapping (2, 3) or diagonal paper electrophoresis (4) for determining which peptides in a proteolytic digest of a protein contained disulfide bonds, but the method should also be applicable to nonprotein disulfides like those used in pesticides or in polymerization processes. The polymer-bound thiol reduces disulfides in the effluent from the analytical column to thiols, which may then be detected by mixing with a solution of 5,5'-dithiobis(2-nitrobenzoic acid) ("DTNB", Ellman's reagent), a reagent which generates an anion with an absorption maximum a t 412 nm when it reacts with a thiol ( 5 ) (Figure 1). The thiols on the polymer are gradually used u p in the process, but they may be regenerated a t any time by washing the column with a reducing agent like /3mercaptoethanol ("8-me"). In methods used previously with peptide mapping (2, 3) and with ambient pressure columns (6), the soluble reagent added to reduce the disulfides was itself capable of producing the D T N B anion. In order to eliminate the resulting interference, it was necessary to add one or two more reagents which reacted selectively with the reducing agent. With the polymer-bound thiol, disulfides are reduced without the addition of a soluble reducing agent to the sample, and the effluent from the thiol-polymer column may be mixed directly with the D T N B stream. Once the fractions containing disulfides are located, the two halves of each disulfide may be identified by reducing it, making specific derivatives (7,8) of the thiols, and separating the derivatives
1501
by chromatography. This procedure is easier when fewer reagents have been added in the detection apparatus. The flow rate and the p H of the effluent from the analytical column may have considerable influence on the sensitivity of the method, so we have investigated the effects of varying those two parameters. If the time necessary for the reduction to go to completion is comparable to or greater than the residence time of a disulfide in the short column used in the solid phase method, the sensitivity would be lower at higher flow rates. In fact, when a polymer-bound disulfide was used in batch procedures for isolating thiol-containing peptides and proteins, the times allowed for the reactions were much longer than the residence times of the eluates in our polymer-thiol column (9). The importance of pH: is a consequence of the p H dependence of the disulfide interchange reaction (10). Though it is unlikely that cyclic disulfides and protein fragments bound together by more than one disulfide bond could be detected with the apparatus, a method based on a polymer-bound dithiol should prove to be suitable for these compounds.
EXPERIMENTAL Apparatus. The liquid chromatograph consisted of a M6000 pump (Waters), an U6K injector (Waters), and a flow-through absorbance detector operated at 405 nm, which is near the 412-nm maximum of the DTNB anion. An ISCO UA-5 detector was used for some of the experiments and a Waters 440 detector for the others. A 30-cm length of 0.020-in. i.d. stainless steel tubing was used in place of a column for all experiments but those shown in Figure 2, in which a 2 mm X 1 m column of Pellionex SAX was used, and Figure 3, in which a 4 mm X 10 cm column of DE 52 cellulose anion exchanger (Whatnian) was used. Prior to packing, the fines were removed from the DE 52 by five cycles of settling and decantation. The thiol--Sepharose (Affi-Gel401, Bio-Rad) was slurry packed in a 4-mm i.d. by 5.0-cm stainless steel column. The capacity of this col.umn was determined by pumping a solution of DTNB through a fresh column until it was saturated and measuring the absorbance of the effluent at 412 nm. On that basis, a column of this size has a capacity of approximately 0.4 bmol thiol at both 0.7 niL/min and 0.3 mL/min. Between periods of use, the column was stored at 5 "C in a solution of 3-me (1% in 0.1 M tris(acetate), 1 mM EDTA, pH 8.0), and it was washed with additional @-mesolution and then buffer just prior to use. When the thiol-Sepharose column had been put into the apparatus and the output from the detector had ceased declining, the column was judged to be free of @-me. From the thiol-Sepharose column, the eluate was directed to a three-port mixing detector (Durrum) ,with ISCO stainless steel fittings. A freshly prepared solution of DTNB ( 5 mg/ 100 mL buffer) was pumped into the opposite port with a second M6000 pump at 0.3 mL/min, and the resulting solution was directed to the optical detector. For integration, the output of the detector was fed to a V/F converter (Dymec 2210),and the frequency output of the converter was counted on a Beckman 6127 Eput meter. The integrator was activated by manually opening the gate just after the injection, and counting was generally discontinued after the voltage reading had returned to the noise level. In those cases where counting was terminated earlier, a cut-off error of approximately 1% was incurred. The baseline detector output was maintained at an average of zero with a digital voltmeter, and the linearity of the integration system was confirmed with artificial step functions in the voltage. Materials. Cystine, DTNB, oxidized glutathione, and dithiothreitol (DTT; Cleland's Reagent) were obtained from Sigma, cystamine hydrochloride and 2,2'-dithiodibenzoic acid were obtained from Aldrich, and solutions of a11 were used within one day of preparation. The concentration of cystine was measured with DTT by the method of Iyer and Klee ( I I ) , using the 0-0.2 absorbance scale of an Aminco DW-2 spectrophotometer. Buffers were either tris(acetate)-EDTA, prepared by adding EDTA (1 mhl) to tris base of the appropriate molarity and titrating to the desired pH with glacial acetic acid, or 0.1 M sodium citrate, pH 7 . 5 . The buffers were generally filtered, heated t o 90 "C and
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978
Table I. Response of the Detection Apparatus to Cystine cystine injected, Crg counts x 0.15 0.30 0.45 0.60 0.75 1.20 2.25
lo3
12.8 26.3 40.3
51.9 68.2 108.6 201
a The eluant was 0.01 M tris(acetate) buffer, 1 mM EDTA, pH 8.0, flowing at 0.7 mL/min. Each entry is the average of duplicate measurements.
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Figure 3. Effect of the detection apparatus on peak width is illustrated here. A sample of DTNB (peak 2), 5,5'-dithiobenzoic acid (peak 3) and an unidentified compound (peak 1) present in the dithiobenzoic acid sample were separated on a 4 mm X 10 cm column of DE 52 anion-exchange cellulose with 0.1 M sodium citrate, pH 7.5, as the eluant. The flow rate was 0.6 mL/minute. The effluent from the column was monitored at 254 nm, (top) without passing through the detection apparatus and (bottom)after passing through the thiol column and mixing with buffer, flowing at 0.3 mL/min, in the mixing tee
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Figure 2. Detection of cystine (-0.45 pg) and oxidized glutathione (- 1.5 pg) after separation on a Pellionex SAX anion-exchange column. The eluant was 1.5 X M tris(acetate), pH 8.4 and the flow rate 0.7 mL/min. (Top)With the thiol-Sepharose column in place, (Bottom) without the thioCSepharose column. "I" denotes the point of injection degassed, then cooled to room temperature for use. For the experiment on the pH dependence of the disulfide interchange reaction, DTNB was prepared in 0.2 M tris(acetate1, pH 8.0, and the eluants were 0.01 M in tridacetate). Experiments on mixing the DTNB solution and the eluant in the ratio of the flow rates showed that the pH of the mixed solution was 8.0 f 0.05 for all the values of pH tested. R E S U L T S AND D I S C U S S I O N The linearity of the detector response to cystine injections is shown in Table I. Though the smallest amount injected in the experiment for Table I was 0.15 p g , it appears that the lower limit of sensitivity is significantly lower than that. It may be possible to increase the Sensitivity still further through the use of a fluorescent thiol reagent (7). We have also injected oxidized glutathione and cystamine into the detection apparatus, and it is about equally sensitive to these compounds and to cystine. Figure 2 demonstrates the detection of cystine and glutathione with the apparatus after they have separated on a n anion-exchange column. It is worth pointing out that the entire experiment shown in Table I was done on a single column, without 8-me regeneration of the thiol column during the experiment. Thus it appears that, with injections of these amounts, there is not a rapid falloff in the yield of the reaction as the thiol sites on the polymer are used up. Figure 2 demonstrates one of the problems in using thiol-Sepharose and similar materials in high performance liquid chromatography. The peaks are considerably broader than one would expect for compounds with retention times of approximately 4 min on the Pellionex SAX column used here. The broadening becomes less noticeable as the peaks become wider, and the experiment shown in Figure 3 dem-
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Figure 4. Effect of eluant flow rate on the detector response. The reciprocal of the flow rate is plotted against peak area. The points marked ( 0 )and those marked (W) represent two different experiments, between which the thiol-Sepharose column was regenerated with P-me onstraks that on a thiol-Sepharose column of 0.6-mL volume, a peak with a half-width equivalent to a volume of 0.84 mL is broadened approximately 14%, but peaks of half-widths 1.8 mL and 5.8 mL are not broadened significantly. It seems reasonable that similar ratios of peak volumes to column volume should give similar results. Thus thiol-Sepharose is suitable for applications in which the peaks have half-widths which are at least three times the short column volume, but a material of higher performance may be necessary for applications in which relatively narrow peaks are expected. The peak broadening effects of the apparatus may also be reduced by using a polymer-thiol column with a smaller volume, but the sensitivity may suffer if the half-life of the exchange reaction is comparable to the residence time in that column. The relationship between these times was investigated by varying the flow rate and determining the resulting variation in peak area. If the reaction is complete a t all flow rates, the plot of peak area vs. the reciprocal of the flow rate, for a constant injection, should be a straight line. The variation in integrated area then reflects only the longer residence time of the DTNB anion within the detector cell at lower flow rates. As Figure 4 indicates, there is a slight upward curvature in the curve of integrated area vs. the
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978
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Figure 5. Effect of the eluant pH on the thiol-disulfide exchange, with the reaction of the thiol and DTNB occurring at essentially constant pH. The points at pH values of 6.0 through 8.0 were taken in triplicate (0.45 Fg cystine injected), and the column was regenerated with @-me for the determinations at 8.5 and 9.0
reciprocal of the flow rate, as would be expected if the reaction were not always complete. Furthermore, the least-squares straight line fit to the points does not pass through zero. The integrated peak area a t 0.7 mL/min falls 17% below the straight line passing through zero and the point a t 0.3 mL/ min, an indication that the reaction is about that far short of completion at 0.7 mL/min. Measurements of the residence times indicate that the residence time of a compound in the thiol-Sepharose column is approximately 1.4 min a t a flow rate of 0.3 mL/min, and approximately 0.5 min at 0.7 m L / min. T h e p H dependence of the disulfide interchange reaction in the short column was also investigated, since it may restrict the pH range of the eluants which may be used for separations with this detection method. The disulfide interchange reaction is best carried out a t alkaline pH, where thiols, which ionize in this region, are in the active anionic form to a significant extent (IO). In the experiment summarized in Figure 3, identical amounts of cystine were injected into the thiolSepharose column a t various values of pH, and the yield of thiol a t each p H was measured with DTNB at a constant pH. As might be expected, there is a significant drop in the yield as the pH is lowered. T h e detection method is clearly still quite sensitive even a t p H 6.0, the lowest pH investigated in t h e present work. Experiments with mixtures of water and methanol or water and dimethylformamide indicated that the activity of thiol-Sepharose declines rapidly when these solvents are used as eluants. This result may be due to the effects of the organic solvents on the structure of the Sepharose backbone. T h e problems of broadening and incompatibility with organic solvents make it desirable to use another material as the support for the covalently bound thiol groups. Controlled pore glass (CPG) would be particularly attractive, because it causes substantially less broadening in a column of the size used in this study. It is a rigid material, which allows operation a t flow rates that are too high for the easily compressed Sepharose. The major difficulty likely to be encountered with CPG is slow hydrolysis of the silica when the detector is operated a t alkaline pH. I t may also be possible to use styrene-based materials like Merrifield resin (12) as the polymeric support. Satisfactory wetting of the material may be a problem with aqueous solvents, however, and the response may change during the course of gradient elutions as the result of changes in the degree of polymer swelling. The latter difficulty has hampered attempts to carry out solid phase peptide synthesis in columns (13). One problem of particular importance to the potential use of this detection method with peptides is the behavior of protein fragments bound together by more than one disulfide
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Figure 6. (a) Reaction of insulin with thiol-Sepharose. (b) Reaction of insulin with a polymer-bound dithiol, giving insulin with two free sulfhydryls and a polymer-bound, cyclic disulfide
bond and of cyclic disulfides, like 1.ipoic acid. As Figure 6 indicates, no part of a protein-like insulin would leave the column, assuming that the polymer backbone is sufficiently rigid to prevent attack of the polymer-insulin disulfide by another polymer-bound thiol. As work on site isolation in co(po1ystyrene-divinylbenzene) polymers has indicated (11, though, the assumption of rigidity in a. polymer like Sepharose is not a good one. Since attack by the second thiol is likely to be slow, a prolonged bleeding of the insulin from the column would probably occur. Experiments with the cyclic disulfide lipoic acid indicate virtually no detectable response with that compound. It should be possible to bind a dithiol to a polymer backbone, thereby creating a solid phase reagent which should react at a rapid rate with disulfides like those of insulin (Figure 6b). It would be necessary that the !,tructure of the dithiol be such that formation of its disulfide would be energetically favored with respect to protein disulfides, but not so highly favored that regeneration of the dithiol form would be difficult. Solid phase reagents or catalysts may be applicable to detection of other groups of compounds in liquid chromatography, making it attactive to use this separation method for some analyses in which its lack of sensitivity or specificity presently makes other methods preferable Enzymes with rather broad specificities may be particularly useful in such methods.
ACKNOWLEDGMENT We thank W. A. Wilson for typing part of the manuscript and V. Coniglione, of Waters Associates, for a three-week loan of a 440 detector.
LITERATURE CITED (1) J. I. Crowley and H. Rapoport, A c c . Chem. Res., 9, 135-44 (1976) and references thereln. (2) H. Maeda, C. B. Glaser, and J. Meienhofer, Eiochem. Eiophys. Res. Commun., 39, 1211-16 (1970). (3) S.S. Ristow, and D. B. Wetlaufer, Eiochem. Eiophys. Res. Commun., 50, 544-50 (1973). (4) J. R. Brown and E. S. Hartiey, Eiochem. J . , 101, 214 (1966). (5) G.Ellman, Arch. Eiochem. Siophys., 82, 70 (1959). (6) K. A. Walsh, R. M. McDonald, and R. A. Bradshaw, Anal. Biochem., 35, 193-202 (1970). (7) T. Sekine, K. Ando, M. Machida, and Y . Kanaoka, Anal Biochem., 48, 557-68 (1972). ( 8 ) A. Witter and H. Tuppy, Eiochim. Eiophys. Acta. 45, 429-42 (1960). (9) T. A. Egorov, A. Svenson, L. Ryden, and J. Carlsson, Proc. NaN. Acad. Sci. U . S . A . , 72, 3029-33 (1975), and references 1-3 therein. (10) R. A. Bradshaw, L. Kanarek, and R. L. Hill, J Bioi. Chem., 242, 3784-98 (1967). (11) K. S. Iyer and W. A. Klee, J , Biol. Chem., 248 (2), 707-10, (1973). (12) R. 6.Merrifield, J . A m . Chem. Soc., 85. 2149-2154, (1963). (13) E. F. Gisin, Heiv. Chim. Acta, 56, 1476-'1482, (1973)
RECEIVED for review March 22, 1977. Resubmitted June 22, 1978. Accepted June 22, 1978.
