Anal. Chem. 1990, 62, 2599-2606
products, plasma, and environmental samples such as river water and particulate extracts. A number of enhancements to the system are planned, specifically in the method of peak recognition where the authors plan to implement multidetector approaches to confirm the presence of the analyte. Improvements are also to be made to the routines determining the times and duration of the heart cuts. The amalgamation of the cybernetic software and the Midas system, although needing complex reprogramming, would lead to a significant decrease in the complexity of the various software subsections. For this application the phenyl column and UV detection were not appropriate and hence were not utilized by the system. As the aim is to produce a system capable of analysis without prior knowledge of the sample type, flexibility in separation modes and detection schemes is of upmost importance. Future work will include the development of a coupled size exclusion/reverse-phase system to better fulfil the need for flexibility.
CONCLUSIONS The use of a column-switching procedure for the analysis of complex matrices can result in the rapid and accurate analysis of low levels of substances that would otherwise prove difficult to analyze. The use of a computer system based on adaptive intelligent software significantly reduces the analyst’s workload by allowing the automated analysis of such samples. Other advantages including minimized sample loss and increased operator safety are also realized. It is expected that simultaneous development of both the software and the chromatographic apparatus will lead to a system capable of
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automatically developing analytical methods for a wide range of analytes in many different matrices. ACKNOWLEDGMENT We gratefully acknowledge the support of A.J.P. who is in receipt of a Science and Engineering Research Council CASE award, cosponsored by ESSO Petroleum Co. Ltd. (ESSO Research Centre, Abingdon, U.K.). We are also indebted to Technic01 Ltd. for the supply of columns used in this study and to Comus Instruments Ltd. for the loan of the Midas chromatographic data station and the adapted software. Registry No. Benzo[a]pyrene, 50-32-8.
LITERATURE CITED (1) Sonnefeld, W. J.; Zoller, W. H.: May. W. E.: Wise, S.A. Anal. Chem. 1982, 5 4 , 723. (2) Karger, 6 . L.; Giese, R. W.; Synder, L. R. TrAC, Trends Anal. Chem. 1983, 2, 106. (3) Williams, R. A,; Macrea, R.; Sheperd, M. J. J . Chromatogr. 1989, 477. . - . 315 (4) Ramsteiner, K. A.; Bohm, K. H. J . Chromatogr. 1983, 260, 33. (5) Wiegand, M. D.; Wiegand, P. M.; Crouch, S. R. Anal. Chim. Acta 1988. 167, 241. (6) Willmott, P. W.; Mackenzie, I.; Dolphin, R. J. J . Chromatogr. 1987, 167, 31. (7) Harvey, R. D., Ed. Polycyclic Aromatic Hydrocarbons and Carcinogenesis; ACS Monograph Series No. 283; American Chemical Society: Washington, DC, 1985. (8) Hoffman, D.; Wynder, C. E. Chemical Carcinogens; ACS Monograph Series No. 173, American Chemical Society: Washington, DC, 1976. (9) Lee, M. L.: Novotny. M. V.; Bartle, K. D. Analytical Chemistfy of Polycyclic Aromatic Compounds; Academic Press: New York, 1981. (IO) Fielden, P. R.; Packham, A. J. J , Chromatogr. 1989, 497, 277. (11) Fieiden, P. R.; Packham, A. J. J . Chromatogr. 1990, 516, 355.
.
RECEIVED for review March 20, 1990. Revised manuscript received August 6, 1990. Accepted August 23, 1990.
Determination of Aromatic and Sulfur-Containing Amino Acids, Peptides, and Proteins Using High-Performance Liquid Chromatography with Photolytic Electrochemical Detection Lin Dou and Ira S. Krull* Department of Chemistry, T h e Barnett Institute (341MU), Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115
Aromatk amlno acids, sutfw-contalnlng amino acids, peptldes contalnlng such constltuents, and protelns can now be detected In hlgh-performance liquid chromatography by the use of on-line, postcolumn, continuous photolytk derivatizatlon with electrochemical (HPLC-hv-EC) detection. The overall approach is a very simple, reproduclble, rapid, and fully automatable approach for the determination of certain amino aclds, peptides, and protelns wlth excellent selectlvlty, sensltlvtty, and llnearltles of response. Dual-electrode response ratlos, IamponAamp-off behavior, and chromatographk capacity factors all contrlbute to the enhanced selectivity of the overall HPLC-h V-EC determlnatlon for these particular classes of Moorganks and biopolymers. The analytical figures of merit, chromatography detection, and method validation approaches have all been opthnally derlved and demonstrated reproducible. Applications of the basic methodology to real-world samples are demonstrated and valldated. *To whom correspondence and reprint requests should be addressed.
INTRODUCTION The analyses of amino acids, peptides, and proteins are very important in biological and medical research. Since the first use of a chromatographic method for the qualitative and quantitative analysis of amino acids done by Stanford Moore, using ion-exchange (IE) chromatography in the mid-1950s ( I , 2 ) , more rapid, reproducible high-performance liquid chromatography (HPLC) methods have been developed for the determinations of amino acids and peptides with relatively simple and inexpensive instrumentation. Depending on the method being used in HPLC, the analysis of amino acids can be classified into two main approaches. The first method uses nonderivatization detection, in which the amino acids are chromatographed from a column and detected by a UV detector at 200-210-nm wavelength (3). The method is straightforward, nondestructive, useful, detects both primary and secondary ambo acids and their derivatives directly, and needs no pre/postcolumn derivatization. However, the sensitivity and selectivity of this detection method are poor. The low UV extinction coefficients of most amino acids limit the use of direct UV detection. The current detection methods
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for amino acids and peptides have to rely on the second approach, derivatization detection, mainly via the reactions of the primary amino group. The use of a reversed-phase separation model and pre/postcolumn chemical derivatizations, to convert amino acids into a mixture which can be easily separated and detected by commoniy used UV and fluorescence (FL) detectors, has provided a very sensitive means for peptide mapping and protein sequencing. In addition, it has provided rapid separations (within 1h normally) and relatively inexpensive instrumentation. Pre/postcolumn derivatization dramatically changes both detectability and chromatographic behavior of amino acids and peptides, so that high separation resolution and high detection sensitivity can be achieved. The most commonly used sensitive derivatization detection for amino acid analysis now is pre/postcolumn modification using o-phthalaldehyde (OPA) reagent, which gives strong fluorescent derivatives for final detection (4-6). The disadvantages of this method are the instability of the fluorescent products and the reproducibility of sensitivity due to fluorescence quenching. Other pre/postcolumn chemical derivatization methods, using different reagents, such as dansyl chloride and 9-fluorenylmethyloxycarbonyl chloride (FMOC), also suffer some limitations (7-10). The advantages of a photolytic derivatization method for liquid chromatography-electrochemical (LCEC)detection have been described (11). The use of light as a derivatization "reagent" greatly simplifies the derivatization procedures and avoids some drawbacks in chemical derivatizations. The method can be used on-line, without any mixing of chemical reagents and analytes, and postcolumn, without significant band broadening from the reactor, by using a knitted open tubular design, with excellent hydrodynamic performance (12). Due to the selectivity of the photochemical properties of different analytes, the lamp-off/lamp-on response differences, and the high sensitivity of electrochemical detection, the method is highly selective and sensitive. This method has been used for the analysis of many classes of compounds, with many unique advantages (13-16). The use of this method for the analysis of aromatic and sulfur-containing amino acids, peptides, and proteins seemed highly possible and would provide a new means for the determination of biological mixtures. In this paper, we describe the use of postcolumn, on-line photolytic derivatizations in liquid chromatography with electrochemical detection for the determinations of these amino acids, peptides containing these amino acids, and proteins. Typical chromatograms, optimized separation detection conditions, dual-electrode response ratios, minimum detection limits, calibration plots and linear response ranges, cyclic and hydrodynamic voltammograms, lamp-on/lamp-off responses as a function of working potentials, method validation, and other analytical figures of merit have been derived. The method is selective and sensitive. The detection limits for amino acids and peptides studied are low picomole levels. The method can be used for the detection of these biological compounds but not for peptide mapping derivatization, since peptides that contain neither aromatic amino acids nor sulfur-containing amino acids have no EC or hv-EC responses.
EXPERIMENTAL SECTION Apparatus. As described in a previous paper (13),the instrumentation consisted of three parts: the HPLC system, the photolysis unit, and the electrochemical detector. The HPLC system was composed of a Model 590 solvent delivery system (Waters Corp., Milford, MA), a LiChroma Damp I11 pulse dampener (Handy and Harmon Tube Co., Norristown, PA), a Rheodyne Model 7010 injector with a 20-kL sample loop (Rheodyne, Inc., Cotati, CA), and reversed-phase columns [E. Merck GmbH., Darmstadt, FRG, Supelco, Inc., Supelco Park, Bellefonte, PA, and YMC, Inc., Morris Plains, NJ (through Eicon Scientific, Inc., Medway, MA)]. Electrochemical detection was
performed by using Model LC-4B dual amperometric controllers, a dual glassy-carbonworking electrode half-cellused in the parallel mode, a stainless steel auxiliary electrode half-cell,and a Ag/AgCl reference electrode (RE-lB), all obtained from Bioanalytical Systems, Inc., (West Lafayette, IN). The postcolumn, on-line photolysis unit was made of a low-pressure mercury lamp as the UV light source (Model 816 W batch irradiator) (Photronix Corp., Medway, MA), a knitted open tubular (KOT) reactor knitted in 0.5 mm i.d. X 1.6 mm 0.d. Teflon tubing (Alltech Associates, Deerfield, IL)wrapped on the W light source, and a power supply accompanyingthe Photronix batch irradiator. At times, a Zn lamp accompanied with its power supply (BHK, Inc., Monrovia, CA), with a self-made quartz coil as ptcolumn photolysis reactor, was used. Both reactors were bathed in ice water (0-5 "C) when used. Cyclic voltammetry (CV) was carried out by using a CV-1B cyclic voltammograph (Bioanalytical Systems, Inc.), a glassy-carbon working electrode, a Ag/AgCl reference electrode, and a Pt auxiliary electrode. Upchurch fingertight fittings (Alltech Associates, Deerfield, IL) were used to connect the column, irradiation unit, and EC detector. A Brown, Boveri and Co. (BBC) Model SE 120 dual-pen chart recorder (Brown, Boveri and Co., Goerz/Metrawatt Division, Vienna, Austria) or an OmniScribe dual-pen chart recorder (Houston Instruments, Austin, TX), and a Hitachi Model D-2000 chromatographic integrator (EM Science, Inc., Cherry Hill, NJ) were used for recording of the chromatograms. Chemicals, Reagents, and Solvents. Reagent grade sodium hydrogen phosphates (monobasic and dibasic) were obtained from Fisher Scientific Co. (Fair Lawn, NJ). HPLC grade methanol and 2-propanol used in the mobile phases were obtained from EM Science, Inc. (Cherry Hill, NJ), as the Omnisolv grade. Tetrabutylammonium hydrogen sulfate (TBAHS) was bought from Fluka Chemical Corp. (Ronkonkoma, NY). ACS grade sodium chloride was bought from Sigma Chemical Co. (St. Louis, MO). All amino acids, peptides, and proteins were obtained from Sigma Chemical Co. as Sigma grade and used as standards. Deionized water was prepared in our laboratory by using a Barnstead water purification system (Sybron Corp., Boston, MA). Chromatographic and Electrochemical Detection Conditions. At most times, the mobile phase used for isocratic elution was a 1:s(v/v) Me0H:phosphate buffer solution. The phosphate buffer was prepared by dissolving 3.0 g of Na2HP04and 3.0 g of NaH2P04in 1 L of water (pH 6.8). At times, slightly different mobile phases were also used as indicated in the text or tables/figures. The gradient elution and ion-pair chromatography separation conditions will also be given in the text or tables/ figures. All HPLC solvents were filtered through a Millipore 0.45-pm filter (Millipore Corp., Bedford, MA) and degassed under vacuum with stirring before use. In the case of protein separation and detection, the mobile phase was on-line, continuously degassed with helium gas. The flow rate for the determination was 1.3or 1.0 mL/min, otherwise as given in the text or figures/tables. The applied potentials for the determinations were +0.65 and +OB0 V for the dual glassy-carbon working electrodes, except for the potentials of the hydrodynamic voltammetry of Phe, which varied from +0.30 to +1.20 V. At times, some other applied potentials were used. The conditions for CV are shown in the figures.
RESULTS AND DISCUSSION Electrochemical Properties of Amino Acids Studied. The study of electrochemical activities of some aromatic amino acids under the conditions used for the separation were carried out by performing cyclic voltammetry (CV) and hydrodynamic voltammetry (HDV) using glassy-carbon working electrodes. The cyclic voltammograms of Phe, Tyr, and Trp were recorded both with and without photolysis. Phe was not inherently electrochemically active, while Tyr and Trp did show EC signals even with no irradiation. However, Phe did show an EC oxidative current after photolysis. At the same time, the CV currents for Tyr and Trp also increased if photolysis was carried out. It was also observed that the CV currents changed with irradiation time, but they did not show an increase for all three amino acids when 5-min or longer irradiation times
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
t
1
0 bmpon
I
2601
t-lnmfI
1,2 and 3
E c 0.1 V
(a) lamp on
1 2 3
0.2
0.4
0.6
0.8
1.0
1.2
l
. 20ppmTyr . 25 ppm Pha . 26 ppm Trp
Y
2 (b) h m p o n
3
Is-
1.4
POTENTIAL (V VI. Ag/AgCI)
Figure 1. Hydrodynamic voltammograms of Trp. Conditions: C,8 reversed-phase column, 4.0 i.d. X 125 mm; 1:99 Me0H:buffer; buffer, 3 g of NaH,PO, in 1 L of water: flow rate, 1.3 3 g of Na,HPO, mL/min; KOT postcolumn reactor; glassy-carbon working electrode; AglAgCl reference electrode.
+
were employed. These CV studies for the aromatic amino acids suggested the use of photolysis in LCEC determination of amino acids, peptides, or proteins containing such constituents. The HD voltammograms for three aromatic amino acids have been obtained under both lamp-off and lamp-on conditions. All HD voltammograms were recorded by plotting the EC current vs applied potential with the analytical column and postcolumn photolysis unit on-line. In Figure 1,for Trp, when the lamp was off, there was no EC response until the potential reached +0.65 V, with a half-wave potential at +0.84 V. The similar HD voltammogram for Tyr showed that the EC response started from +0.70 V and had a half-wave potential at +0.82 V. Both HD voltammograms reached a response plateau at +LOO v. As shown by HD voltammograms (17), Phe had no inherent oxidative EC activity. The EC detector showed no signal for Phe for applied potentials up to +1.20 V (the maximum tolerated potential of the glassycarbon electrode is +1.30 V) when the lamp was off. On the other hand, HD voltammograms for the same amino acids obtained under lamp-on conditions were different from the ones obtained under lamp-off conditions. Trp had an EC response starting from +0.20 V after on-line photolysis and had a response plateau at +0.65 V (Figure 1). Tyr started to have an EC response at +0.30 V when irradiated and had a half-wave potential of +0.62 V. The HD voltammogram of Phe when photolyzed had a EC response and a plateau, as shown in ref 17. One thing we could notice was that all HD voltammograms obtained under lamp-on conditions showed a second oxidation at potentials higher than +1.10 V. Selectivity of Detection for Amino Acids in HPLCh u-EC Determination. On-line photolytic derivatizations for the electrochemical detection of aromatic amino acids, peptides, and proteins were carried out by using a postcolumn reactor (KOT or quartz coil) with UV light irradiation. In Figure 2, under lamp-off conditions, the chromatograms obtained at different applied potentials (+0.60 and +OB0 V) for the mixture of Tyr, Phe, and Trp are shown. Although both Tyr and Trp showed inherent EC response at the potential of +OB0 V without photolysis, they showed no EC responses at +0.60 V. However, when the lamp was turned on, both Tyr and Trp showed sensitive EC responses even at +0.60 V. This provided very high selectivity in terms of lamp-on and lamp-off response differences as well as the selection of applied potentials. The peaks for both amino acids obtained at +0.80 V, lamp-on conditions, were 2 or 3 times as high as the ones obtained under lamp-off conditions. If higher potentials (e.g. +0.90 V) had been used, it is expected that the EC response
EC 0.8V
i
2
II
r
l
l
l
.
.
,
I
0
4
8
12
10
20
24
28
0
TIME (minutes)
I 4
I 8
I 12
I 16
I 20
, 24
TIME (minuter)
Figure 2. RPLC-hv€C chromatograms of (1) 20 ppm Tyr, (2) 25 ppm Phe, and (3) 25 ppm Trp. Conditions: CI8 reversed-phase column, 4.0 i.d. X 125 mm; 1:99 Me0H:buffer; buffer, 3 g of Na,HPO, 3g of NaH,PO, in 1 L of water; flow rate, 1.3 mLlmin; KOT postcolumn reactor, 2.5 ml; dual glassycarbon working electrode, +0.6 and 4-0.8 V; Ag/AgCI reference electrode; injection volume, 20 pL.
+
differences between lamp-off and lamp-on conditions would be larger. This simply suggested the increase in detection sensitivity by photolysis, even for inherently electrochemically active amino acids. Phe was not inherently electrochemically active, and did not show any EC response when the lamp was off, which agreed with CV results. As a matter of fact, as shown in HDV performed on Phe under lamp-off conditions (In,there were no EC signals for Phe without irradiation, even when the applied potential reached +1.20 V. However, when irradiated, Phe showed sensitive EC signals at both +0.80 and +0.60 V. The peptides containing these aromatic amino acid residues also displayed this unique selective EC response. Thus, in addition to the selectivity provided by HPLC column separations, two other selectivity modes could be used for the determination: (1)the selectivity provided by the choice of applied potential and (2) using or not using irradiation. The dual electrode ratios can also be used to identify analytes. Another two aromatic amino acids, o-Tyr and m-Tyr, were also studied by using this HPLC-hu-EC method. Both o-Tyr and m-Tyr showed similar hv-EC properties as Tyr. When the lamp was on, both could be seen by an EC detector under +0.70 V, +0.60 V, or even lower potentials. When the lamp was turned off, however, these two peaks could be seen only at potentials of +0.70 V or above (very small peaks at a potential +0.70 V) and there were no responses at +0.60 V. Optimization of Separation and Detection Conditions. The optimization of the hu-EC detection of amino acids mainly included the choice of applied potential (from HD voltammograms), the selection of irradiation time (residence time of the analyte in the postcolumn reactor), and the choice of the postcolumn photolysis reactor. The residence time study for Phe was obtained by plotting the EC response vs the
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
Table I. Ratios of Peak Width, Peak Area, and Peak Height for Two Reactionsa
lamp on lamp off analyte W1/W2b A1/A2 H1/H2 W1/W2 A1/A2 H1/H2 Tyr Phe Trp
0.53 c
0.79
3.58 19.45 2.28
7.19 22.65 2.96
0.47 d 0.71
1.16 d 1.02
2.17 d 1.35
See Figure 2 for the chromatographic and EC conditions. W1, peak width in KOT/Hg; W2, peak width in quartz coil/Zn; Al, peak area in KOT/Hg; A2, peak area in quartz coil/Zn; H1, peak height in KOT/Hg; H2, peak height in quartz coil/Zn. Peak was too small in quartz coil/Zn lamp system to be measured. dThere were no peaks for Phe when lamps were off. a
Table 111. Relative Standard Deviation of EC Response Measurement in the Linearity Studyasb
Phe PPm
TrP RSD, %
PPm
RSD, %
2.4 1.0 1.3 2.5 4.6
0.2 2.0 20.0 200.0
6.9 2.0 3.5 4.8
1.2
3.5 35.0 175.0 350.0
a n = 3; the EC responses were measured in peak height. See Figure 2 for conditions. Table IV. Summary of Dual-Electrode Responses and Their Ratio for Phea
Table 11. Linear Range of Response for Amino Acids and Peptides by HPLC-hv-EC"Detection
analyte
linear response range, orders of magnitude
3.5 TYr Phe 3.3 3.7 TrP peptide no. 1 2.4 peptide no. 3 3.3 a See Figures 2 and 5 for conditions.
peak height f SD PPm
LOD, ppb or ng/mL 70 100 50 500 100
residence time. Experimentally, the residence time change was accomplished by changing the volume of the KOT reactors, not the flow rate, because the flow rate itself can change the EC response, even when other parameters remain constant, which would complicate the selection of optimal irradiation time. By use of a constant flow rate and KOT reactors with different volumes, EC response changes would be a direct result of the change of irradiation time. The optimal residence time for the hu-EC detection of Phe was 1.8 min. A longer irradiation time (larger KOT reactor volume) would give no better EC responses, but rather more band broadening. This agreed with the results of the CV studies, which suggested that no more than 2-min irradiation should be used for all three amino acids studied. Tyr, Trp, and Phe all have UV absorption at 254 nm, although they all also have stronger absorption at 200-220 nm. Both a Zn lamp (having a main irradiation line at 210 nm) and a mercury lamp (having a maximum irradiation a t 254 nm) should be able to be used for the photolysis. Unfortunately, the Zn lamp can only be used with a quartz coil reactor (the Teflon KOT reactor is not transparent a t 210 nm), which has a large postcolumn volume giving a broadened peak. Therefore, we would realize lower separation efficiency, as well as higher detection limits. On the other hand, the lower absorbed UV light as 254 nm, by use of a mercury lamp with the KOT reactor (lower photolytic derivatization efficiency), may be balanced out by the excellent hydrodynamic performance of the KOT reactor, leading to minimum band broadening. In fact, a comparison was made in terms of the band broadening and photolytic efficiency for two postcolumn reactors (KOT with Hg lamp and quartz coil with Zn lamp). The KOT reactor with a mercury lamp was superior to the quartz coil reactor with a Zn lamp. The band broadening of analytes was more pronounced for the quartz coil reactor. When a KOT reactor was used, the band broadening due to the reactor was greatly reduced because of the excellent hydrodynamic properties of the reactor (12). Also, the higher photolytic efficiency of the KOT reactor was clear from its higher peak height and larger peak area. The comparisons of peak width, peak height, and peak area under the same
1.2
3.5 175.0 350.0
0.9
v
20.5 f 0.5 59.3 f 0.6 2465.0 f 60.6 3833.0 f 175.6
0.7 V
response ratio (SD)
7.0 f 0.1 21.9 f 0.2 890.0 f 17.3 1616.7 f 16.1
2.93 f 0.07 2.70 f 0.03 2.77 f 0.07 2.37 f 0.11
a n = 3; EC responses were measured in peak heights. Chromatographic conditions: CIS reversed-phse column, 4 i.d. X 125 mm; mobile phase, 0.5:99.5 Me0H:buffer; buffer, 3 g of NazHPOl + 3 g of NaH2P04in 1 L of water; flow rate, 1.0 mL/min; injection volume, 20 pL; KOT postcolumn reactor, 1.8 mL. Electrochemical detection conditions: glassy-carbon working electrode; applied potentials of 0.7 and 0.9 V vs AgIAgC1 reference electrode.
experimental conditions for these two reactors are given in Table I. Linearity of Response Range and Detection Limits. In Table 11,the linearity of response ranges is indicated for Tyr, Trp, and Phe. For Tyr and Trp, the linear ranges of responses were from detection limits up to a few hundred ppm (pg/mL), 3-4 orders of magnitude. For Phe, the linear range of response was 3.3 orders or magnitude, and the calibration plot became nonlinear when the concentration of Phe was higher than 200 ppm (pg/mL). The r2 for all plots was 0.999 or better within the linear ranges of response. Besides the high selectivity provided by different modes, the method was highly sensitive. Detection limits for the three aromatic amino acids are given in Table 11, obtained by using a 20-pL loop injector, which were down to low picomol levels. The overall sensitivity of this method was comparable to that of FL derivatization detection or better than that of UV detection at 210 nm (28). Reproducibility of the Detection and Dual-Electrode Response Ratio. All the experimental results for hv-EC detection were repeated three times. The three injections were reproducible and stable. The standard deviations (SD) were measured, and the relative standard deviations (RSD) were within 8% for the residence time, linearity, dual-electrode ratio studies, etc. In Table 111, the RSDs are listed for Phe and Trp for the linearity studies. The good reproducibility of this method is one of the advantages over FL derivatization detection. The dual-electrode response ratio (RR),offering another mode for selectivity, is unique for each analyte separated by chromatography and can be used for the identification of analytes. The RR values for Phe were obtained by using this hv-EC method and are given in Table IV. The RR values remained constant over the concentration range studied (the differences were within experimental error) except the one at 350 ppm (pg/mL), which was beyond the linear response range. Sulfur-Containing Amino Acid Determinations Using HPLC-hv-ECDetection. Although sulfur-containing amino acids, methionine, cysteine, and cystine, are inherently elec-
ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
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Table V. List of Peptides Used
2
no. 1 Phe-Gly-Gly-Phe no. 2 Phe-Leu-Glu-Glu-Leu no. 3 Trp-Leu
no. 4 Tyr-Ala no. 5 Lys-Cys-Thr-Cys-Cys-Ala no. 6 Tyr-Tyr-Tyr-Tyr-Tyr-Tyr
1
L ' ,
I
I
I
5
I
(
1
I
I
a
O L
I
I
I
I
I
I
5
10
I
I
# , I 10
TIME (minulei)
Figue 3. RPLGhu-EC detection of (1) 80 ppm cystine and (2) 50 ppm Met: (a) lamp on: (b) lamp off. Conditions: YMC AQ-300 reversed-phase column 4.6 X 250 mm; phosphate buffer, 3 g of Na,HPO, 3 g of NaH2P0, in 1 L of water: flow rate: 1.0 mL/min; KOT postcolumn reactor, 2.5 ml; glassycarbon working electrode at +0.65 V vs Ag/AgCI.
+
trochemically active, the direct LCEC detection of these amino acids is difficult with most common glassy-carbon electrodes (cysteine can be directly detected by using a glassy-carbon electrode). Iriyama et al. reported on the use of a preanodized glassy-carbon electrode for the determination of methionine in human serum by LCEC detection (19). Without this preanodization step, methionine could not be determined at any potential within the working-potential range of a glassy-carbon electrode. The reason is that a very high potential, +1.70 V vs Ag/AgCl, is needed for methionine to be oxidized, which is far beyond the working-potential range. Mercury or Au/Hg amalgam electrodes have been successfully used for LCEC detection of thiols (20-23). Although carbon electrodes can be used for the direct oxidation of thiols, the potential needed is much higher than the potential needed for a mercury electrode. The oxidized form of cysteine, cystine, can be reduced by using electrochemical reduction but requires very high reduction potentials, such as -1.00 V, and therefore strict deoxgenation of the mobile phase. The use of on-line, postcolumn photolysis followed by electrochemical detection seemed to provide another way for the determination of these amino acids, even for some other thiols and disulfides. As shown in Figure 3, the separation followed by hv-EC detection of methionine and cystine was achieved by using a YMC aqueous solution reversed-phase column and a hv-EC detector. Both amino acids had no oxidative EC responses under mild detection conditions, +0.65 V vs Ag/AgCl, without irradiation, and could be detected at the same potential after photolysis. Again, as in the case of aromatic amino acids, the detection selectivity provided by the lamp-on/lamp-off response differences was obtained for methionine and cystine, and it will be useful for the determination of these amino acids in complex mixtures. On the other hand, cysteine behaved differently. It was inherently electrochemically active and could be detected without being photolyzed, but the peak significantly decreased with on-line, postcolumn irradiation. It seemed that the photooxidation of cysteine was taking place during the irradiation, forming a disulfide that could not be detected oxidatively. The reverse process, the photocleavage of the sulfmulfur bond of cystine, giving an electroactive thiol group, may be considered the main mechanism of hv-EC detection of cystine. It was not clear at this time what products of irradiation of methionine were responsible for the oxidative EC response. Low picomol levels of methionine and cystine could be detected using this HPLC-hv-EC method.
- 2
\
6
12
6
12
TIME (minutes) Flgure 4. Peptide separation and detection using RPLC-hu-EC: (1) sample no. 6 (see text), Tyr-Tyr-Tyr-Tyr-Tyr-Tyr; (2) sample no. 2,
Phe-GlyGly-Phe: (a) lamp on; (b) lamp off. Conditions: C,8 reversed-phase column, 4 X 125 mm; 40:60 Me0H:buffer; buffer; 3 g of Na,HPO, 3 g of NaH,PO, in 1 L of water: flow rate: 1.0 mL/min; KOT postcolumn reactor, 1.8 mL; glassy-carbon working electrode: applied potential, 0.8 V vs Ag/AgCI reference electrode.
+
Peptide Determinations by Isocratic RPLC-I,u-EC Detection. The sensitive and selective determination of aromatic and sulfur-containingamino acids could be extended to peptides containing such constituents. HPLC separation of such peptides, combined with hu-EC detections, showed the same detection properties as for the corresponding amino acids. Several separation modes were used for the separation of peptides, including isocratic reversed-phase, ion-pair, and gradient reversed-phase elution. The mobile phases used in these separations modes were all compatible with hu-EC detection. The peptides used all contained either aromatic amino acids (Trp, Tyr, and Phe) or Cys and are listed in Table V. The retention times for peptides nos. 1 and 3, Table V, had only small differences under isocratic elution condition and could have been changed if different mobile phases had been used. The EC response decreased or disappeared when the lamp was turned off depending on the applied potential and analyte. At +0.65 V, both nos. 1 and 3 showed no peak when the lamp was off, but no. 3 did show a peak at the applied potential of +0.90 V, with a peak height reduced to 1/3 when compared to the same peak obtained under lamp-on conditions. These lamp-on/ lamp-off EC behaviors were similar to the ones of Phe and Trp reported above. These results suggested that only the aromatic amino acids in these peptides were responsible for the hv-EC response (no. 1contained Phe, and no. 3 contained Trp). The separation of nos. 2 and 6 under isocratic HPLC and hv-EC detection is shown in Figure 4. When the lamp was on, both nos. 2 and 6 could be detected (Figure 4a). However, when the lamp was off, the no. 6 peak reduced to and no. 2 peak disappeared (at the same potential) (Figure 4b). As in the case above, an EC detection at +0.60 V showed no peaks for both nos. 2 and 6 when the lamp was off. Again, the
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3 r
1
4
5
10
15
TIME (minutes) Figure 5. Ion-pair RPLC-hU+C chromatogram of peptides: (1) sample no. 5; (2) sample no. 1; (3) sample no. 3. Conditions: C,, reversedphase column, 4 X 125 mm; 3 8 6 2 MeOH:H,O with 2.0 g of Na,HPO,, 2.0 g of NaH,PO,, and 1.0 g of TBAHS in 1 L of the mobile phase; flow rate, 1.0 mL/min; KOT postcolumn reactor, 1.8 mL; glassy-carbon working electrode; applied potential; 0.8 V; Ag/AgCI reference electrode.
Table VI. Dual-Electrode Response Ratio for Peptides"** ppm
peptide no. 1
3 10 30 100
1.93 1.96 1.88
resDonse ratio RSD, '70 peptide no. 3 3.21 2.85 1.28
RSD, %
1.81
2.49
1.79
1.24
The ratio of EC responses at poa See Figure 5 for conditions. tentials of +0.85 and +0.70 V: n = 3.
lamp-on/lamp-off EC behaviors of these two peptides corresponded to Phe and Tyr (no. 2 contained Phe, and no. 6 contained Tyr). This selectivity provided by lamp-on/ lamp-off behavior and different applied potentials was useful in the determination of these peptides, as in the case of amino acid determinations. Ion-Pair Chromatography with Photolytic Electrochemical Detection for Peptides. Generally, the separation of peptides by isocratic elution in the reversed phase is not ideal, because of the wide range of polarity of the samples. Ion-pair reversed-phase chromatography has been successfully used for peptide separations (24). The addition of ion-pair reagents to the mobile phase significantly neutralizes the electrical charges of peptide samples and therefore improves the separation. In our previous studies, the compatibility of mobile phases of ion-pair reversed-phase liquid chromatography with hv-EC (RPLC-hu-EC) detection was shown for the ion-pair RPLC-hv-EC detection of inorganic species (16). The separation of peptides containing aromatic amino acids using ion-pair reversed-phase chromatography followed by photolytic electrochemical detection, as indicated in Figure 5, shows a more powerful resolution of peptides than reversed-phase chromatography. The linear response ranges of the detections were 2.4 and 3.3 orders of magnitude for peptides nos. 1 and 3, Table 11,with detection limits of 500 and 100 ppb (ng/mL), respectively. The dual-electrode response ratios for these peptides are shown in Table VI. The ratios a t different sample concentrations were almost the same for each peptide, and the RSDs (n = 3) for each measurement were within 4%. Cys-containing peptide, sample no. 5, Phe-containing peptide, no. 1,and Trp-containing peptide, no. 3, were well separated from each other, although they could not be resolved in the reversed phase. The EC response of sample no. 5 decreased
~~
0
4
8
12
16
20
24
TIME (minutes) Figure 6. Gradient RPLC-huIC chromatogram of pepttdes: (1) sample no. 4; (2) sample no. 6; (3) sample no. 3; (4) sample no. 2. Conditions: C,, reversed-phase column, 4 X 125 mm; mobile-phase A = 8 % MeOH, 2 % i-PrOH, 90% H 2 0 , 0.05% TFA, 2.8 g of NaCi in 1 L of mobile-phase A; mobile-phase B = 60% MeOH, 10% i-PrOH, 3 0 % H,O, 0.05% TFA, 4.2 g of NaCl in 1 L of mobile-phase B; gradient, 0-20 min from 0% B to 100% E; glassy-carbon working electrode at 0.8 V vs Ag/AgCI; KOT postcolumn reactor (1.8 mL), lamp on.
when photolyzed for the reason mentioned above for cysteine. The inherent advantages of high selectivity and sensitivity of hu-EC detection were also gained in these detections. Gradient Elution and Photolytic Electrochemical Detection for Peptides. Another approach to improve the resolution of peptide separations is to use gradient elution. To exploit the full advantages of photolytic electrochemical detection in peptide determinations, the combination of reversed-phase gradient elution with hv-EC was carried out. In the case of a MeOH/i-PrOH/H20/TFA (NaC1 as electrolyte) mobile phase used for the gradient elution, a close examination of baseline change was performed. There was no baseline drift for the mobile phase changing from 5 to 90% MeOH (v/v). Normally, the use of high-percentage organic solvents in the mobile phase damaged the postcolumn reactor made from Teflon, but MeOH is an exception. 90% MeOH in water could be used in the mobile phase without dissolving the reactor. High percentages of i-PrOH in the mobile phase, however, could shorten the life of the reactor. A gradient from 0 to 100% B for a peptide separation with hv-EC detection is shown in Figure 6. The separation was complete within 20 min and the detection was sensitive and selective,as discussed. Protein Determination Using Photolytic Electrochemical Detection. UV and FL detection, relying on the absorption of light of aromatic amino acid residues, are the most commonly used detection methods in protein separation. The major concerns in these detections are selectivity and sensitivity (especially the concentratin detection limit). Electrochemical detection was generally thought not to be useful in protein separations, because the diffusion of larger biomolecules to the electrode surface, where the electron transfer takes place, was very slow. Also, some electrochemically active residues in the protein may be folded in the inside of the molecule, so that is could not be seen by the electrodes. The postcolumn, on-line photolysis of proteins followed by
ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
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TIME (minuter)
Flgure 8. Chromatograms of insulin samples using RPLC-hv-EC: (a) insulin standard, lamp on; (b) insulin formulation, lamp on; (c) insulin
3
standard, lamp off. Conditions: Supelco C, wide-pore (300 A) reversed-phase column, 5 cm X 4.6 mm i.d.; mobile-phase A = 1 % i-PrOH, 4 % MeOH, 95% water with 2.8 g of NaCl in 1 L of mobilephase A; mobile-phase B = 10% i-PrOH, 60% MeOH, 30% water with 4.2 g of NaCl in 1 L of mobile-phase B; gradient, 0-5 min from 55% to 70% B; flow rate, 1.0 mL/min; KO1 postcolumn reactor, 2.0 mL; EC detection: glassy-carbon electrode at 0.8 V vs Ag/AgCI. Table VII. Spiked Amino Acid and Protein Determinations
Using HPLC-JIV-ECDetection analyte I
I
10
1
1
20
30
TIME (minutes)
Figure 7. RPLC-h V-ECdetection of proteins: (1, 2) ribonuclease A; (3) lysozyme : (4) b-lactoglobulin A; (a) lamp off (b) lamp on. Conditions: Supelco C, wide-pore (300 A) reversed-phase column, 5 cm X 4.6 mm i.d.; mobile-phase A = 2 % i-PrOH,8 % MeOH, 90% water, 0.05% TFA with 2.8 g of NaCl in 1 L of mobile-phase A; mobile-phase B = 10% I-PrOH, 70% MeOH, 2 0 % water, 0.05% TFA with 4.2 g of NaCl In 1 L of mobile-phase B; gradlent, 0-20 min from 5 % to 100% B; flow rate, 1.5 mL/mln; KOT postcolumn reactor, lamp on; EC detection, glassy-carbon electrode at 0.8 V vs Ag/AgCI.
EC detection, however, provides ways to overcome these difficulties in EC detection in biopolymers. The degradation of a biopolymer's backbone by photolytic cleavage may be able to produce lower molecular weight photoproducts having their own EC properties. This was true as shown by the results in Figure 7. In Figure l a , the EC detection of three standard proteins showed no signals when no photolysis was performed. In Figure 7b, the chromatogram was taken under the same conditions except with photolysis. The chromatogram showed three well-separated proteins by a wide-pore reversed-phase column using a MeOH/i-PrOH/H20/TFA mobile phase in gradient elution. Triplicate injections were made, and the chromatograms were reproducible in terms of retention time, peak height, and peak shape (RSD < 3% for retention time measurements,
