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Trinitrobenzenesulfonic acid: a chromophore, electrophore and pre-column derivatizing agent for high-performance liquid chromatography of alkylamines
Analytica
Chimica
Eisevier Scientific
141
Acta,
Publishing
(1982)
269-278
Company,
Amsterdam
-
Printed
in The Netherlands
TRINITROBENZENESULFONIC ACID: A CHROMOPHORE, ELECTROPHORE AND PRE-COLUMN DERIVATIZING AGENT FOR HIGHPERFORMANCE LIQUID CHROMATOGRAPHY OF ALKYLAMINES
W. LOWRY CAUDILL Department (Received
and R. MARK
of Chemistry, 8th March
Indiana
WGH’I’MAN*
University,
Bloomington,
IN 4 7405
(U.S.A.)
1982)
SUMMARY Trinitrobenzenesulfonic acid is shown to be an ideal pre-column derivatizing agent for high-performance liquid chromatography of alkylamines. The reaction is quantitative and the trinitrophenyl derivatives are amenable to ultra-violet and electrochemical detection_ Electrochemical detection with either a glassy carbon or pressure-annealed pyrolytic graphite working electrode provides lower detection limits than ultraviolet detection and thus is preferable
for trace determinations.
Trinitrobenzenesulfonic acid (TNBS) was introduced as a derivatizing agent for amino acids in 1959 by Okuyama and Satake [l] _ The trinitrophenyl (TNP) derivatives of aliphatic amines that can be formed in alkaline
solutions have many properties that facilitate their use in quantitative work. Direct spectrophotometric determination is possible because TNP derivatives have an absorbance maximum at approximately 350 nm, a wavelength where TNBS is essentially transparent. The derivatives can be isolated in crystalline form in high yield so that authentic standards are easily obtained [ 2,3]. In this report, it is shown that TNP derivatives are also advantageous for electrochemical detection because they are reduced at easily accessible pctentials. There has been, however, little use of TNBS for trace determinations or as a pre-column reagent for the derivatization of compounds separated by liquid chromatography_ It was recently shown that TNBS can be used quantitatively to derivatize y-aminobutyric acid, a neurotransmitter, in whole rat brain homogenates using liquid chromatography with electrochemical detection (1.c.e.c.) [4]. Trinitrobenzenesulfonic acid is of interest as a derivatizing agent for use as an electrophore for 1.c.e.c. to take advantage of the low detection limits of this method. The low detection limits of 1.c.e.c. have been demonstrated for easily oxidized compounds where picogram measurements are routine [5, 63. Carbon electrodes at fixed potential are used primarily in this application Properly prepared electrodes are stable, and thus do not require electrode resurfacing or other forms of treatment for several months. However, carbon electrodes are not reliable when operated at very negative potentials. 0003-267O/SZ/OOOO-0000/$02_75
o
1982
Elsevier Scientific
Publishing
Company
270
Various interferences, either in the mobile phase or in the sample, cause the electrode response to deteriorate [7, 83. Chief among these interferences is oxygen, the presence of which can lead to a large baseline offset and increased faradaic noise. To minimize these problems,- detectors for reducible compounds
have employed
renewable
mercury
surfaces (dropping
mercury elec-
trodes or metal amalgams [ 9-151). Another successful approach has been to use a dualelectrode system in which a generator electrode is used to form an oxidizable product [S] . However, electroreduction of trinitro compounds occurs at sufficiently low potentials that a single carbon electrode can be employed for trace level detection. For routine determinations, this approach offers mode.
many
of
the advantages
demonstrated
by
1.c.e.c.
in the oxidative
EXPERIMENTAL
Reagents _%ll aqueous solutions were prepared in doubly-distilled water with reagent
grade chemicals as described previously [ 41. -y-Aminobutyric acid, ~alanine, L-alanine, glycine, a-aminobutyric acid, P-aminoisobutyric acid, 6 -aminovaleric acid, 2,4-dinitrophenyl-y-aminobutyric acid, and 2,4,6-trinitrobenzene-sulfonic acid (TNBS) were obtained from Sigma Chemical Company (St. Louis, MO). Crystalline forms of 2,4,6trinitrophenyl7aminobutyric acid, 2,4,6-trinitrophenyl+&~.nine, 2,4,6-trinitrophenyl-L-alanine, 2,4,6trinitrophenyld aminovaleric acid, 2,4,6-trinitrophenylglycine, 2,4,6-trinitrophenyl-ac-aminobutyric acid, and 2,4,6-trinitrophenylQ-aminoisobutyric acid were prepared for use as standards with a procedure similar to that of Okuyama and Satake [ 11. !k?icroreac tion A sample (200 ~1) containing the above-specified amino acids (except for
6 aminovaleric acid which served as internal standard) at pH 9.2 with 0.2 M potassium tetraborate is dispensed into an Eppendorf polypropylene test tube (1.5 ml, Brinkmann Instruments, Westbury, NY). The derivatizing agent, T-NBS, (20 ~1, 0.1 M) is added and the reaction is allowed to progress for 30 min. The reaction is terminated by the addition of perchloric acid (2.0 M, 180 ,ul) containing the internal standard, 2,4,6-trinitrophenyl-&amincvaleric acid (2.2 X 10m6M). The final pH is 0.2. The sample is then extracted twice with 400-~1 aliquots of toluene. A portion of the combined toluene fraction (500 ~1) is back-extracted with pH 9.0 buffer (250 ~1, Fisher, Cincinnati, OH). The aqueous phase, which contains the derivatized amino acids and the internal standard, is placed in a vial (V vial, 1.0 ml, Anspec Co., Ann Arbor, MI). The vial is sealed with a serum cap and the aqueous phase is deosygenated by nitrogen bubbling for 2 min. The deoxygenated aqueous solution is then injected onto the column.
271
Apparatus The liquid chromatographic apparatus was described previously [ 41. A constant, reduced level of oxygen in the mobile phase was accomplished by deoxygenation of the mobile phase with nitrogen and the utilization of stainless steel tubing throughout the chromatograph. The columns were either 4.6-mm i.d. X 25 cm Biophase (&pm particle size) reversed-phase columns (Bioanalytical Systems, West Lafayette, IN) or g-mm i-d. X 50 cm Zipax (30-pm particle size) strong anion-exchange columns (SAX, DuPont, Wilmington, DE). The mobile phase for the reversed-phase separation contained citric acid (0.098 M), disodium hydrogenphosphate (0.016 M), acetic acid (0.43 M) (pH 2.5) and 35% methanol (HPLC Grade, Fisher, Cincinnati, OH). The SAX mobile phase consisted of potassium hydrogenphthalate (0.037 M), sodium acetate (0.039 M) and disodium ethylenedinitrilotetraacetate (0.001 M). The flow rate for the reversed-phase separation was 1.5 ml min-’ and for the SAX system was 0.8 ml min-‘. the working electrode was made of glassy For amperometric detection, New York, NY) or the oxidized basal carbon (Tokai GC-20, Atomergic, plane of pressure-annealed pyrolytic graphite (Union Carbide Co., Parma, OH). The pyrolytic graphite electrode was pretreated with anodic oxidation as described elsewhere [16] _ The glassy carbon was polished to a mirror finish with use of 600~grit sandpaper, followed by polishing on a felt cloth with 5+m, 0_3+m, and 0_05+m alumina (Fisher, Cincinnati, OH), successively_ The flow cell, identical for both electrode materials, was a modification of that previously described [16] . The auxiliary electrode was made of stainless steel which served as the upper block of the cell. _4 precut spacer (51+m thick: Tefzel, DuPont, 1Vilmington, DE) was laminated onto the auxiliary electrode_ Lamination was achieved in 1 h at 260°C. The entire assembly was then clamped together with 4 bolts. The working electrode area was 0.33 cm’ and the cell volume was 1.7 ~1. The working electrode potentials were -0.6 V and -0.8 V vs. a saturated calomel electrode (SCE) for the reversed-phase and S-4X systems, respectively_ For ultraviolet (u-v_) detection, the optical unit of an Altex Model 300 isocratic liquid chromatograph (lo-mm path length, 8+1 cell volume, Altex Scientific, Berkeley, CA) was used with single wavelength monitoring at 254 nm. Voltammetric studies employed a PARC Model 174A. Measurements were made with a disk-shaped glassy carbon electrode (0.05Scm’ surface area). RESULTS
AND
DISCUSSION
Properties of trinitrophenyl derivatives Gram-scale quantities of the TNP derivatives of each of the amino acids were prepared and each possessed a sharp melting point (see Table 1). Ring substitution by the amino group is expected for these derivatives [ 17,181 and was verified for the glycine derivative by mass spectrometry (parent ion m/z = 264). All of the TNP derivatives possess a high molar absorptivity at
272 TABLE
1
Recovery of the trinitrophenyl derivatives of the compounds cited after extraction and deoxygenation (Melting pcints of.the sepvately prepared derivatives are also given) Compound
Recover,-
<‘Fop
L-.L\latIiIIe yXminobutwic acid P-,\minoisobutpric acid 8-hminomleric acid
100 2 0.886 101 f. 0.613 101 i 0.993 -
nr.P.&j
Compound
144-145 149-151 143-1-14 114-115
a-Aminobutwic &Alanine Glycine
Recovery acid
94.3 + 2 26 94.2 f 2.44 91.2 f 2.60
I&p.
CC>
119-120 189-189.5 166-167
“Mean recoveries determined with 1 X 1Oa Prl solutions of the amino acids. The 98% confidence limits (n = 9) were obtained from a 2-sided Student’s t-test.
254 nm which makes them ideal for U.V. detection following liquid chromatographic separation (e.g., for the r-aminobutyric acid derivative, e = 2.2 X 10’ I mol-’ cm-‘). The electrochemistry of the y-aminobutyric acid derivative was characterized at carbon electrodes to determine the parameters for amperometric detection. The response of the amperometric detector is directly proportional to the number of electrons per mole transferred in the electrolysis, which
suggests
that
TNP
derivatives
are ideal because
at very negative
poten-
they are reduced in an overall 18-electron process. This process is evidenced by polarography in aqueous solutions as three 4electron, 2-proton, irreversible waves at moderate potentials and as a 6-electron wave at potentials that are near the negative limit for a mercury electrode. The 4electron, Z-proton waves correspond to the reduction of each nitro group to the corresponding hydrosylamine [19-223. _At a carbon electrode in the buffer used for reversed-phase chromatography, only the first three reduction waves are apparent in the potential range available (Fig. 1). The peak potentials for these waves are shifted negatively with respect to those obtained at mercury electrodes_ The peak potentials move in a negative direction with an increase in pH from 2 to 6 (43.7 mV/pH unit, 45.5 mV/pH unit, and 66.1 mV/pH unit for the first, second and third waves, respectively). The oxidation wave observed on the reverse scan suggests that the aromatic multi-hydroxylamine is the product of these reductions. Figure 1 also shows a cyclic voltammogram for 2,4&nitrophenyi-yaminobutyric acid. Dinitrophenyl derivatives can be easily formed from the more common derivatizing agent for amines, 2,4_dinitrofluorobenzene. The dinitrophenyl derivatives are reduced at more negative potentials than the corresponding TNP derivatives and only two voltammetric waves are observed_ Therefore, the TNP derivatives are superior for amperometric detection with carbon electrodes, because they are easier to reduce and more electrons per mole of derivative are transferred at any given potential. tials
, $7 ,
’
\
.
.
273
-3
I
!_‘I
_
_“_-..-
c
_____.___..
_.a-.
/’
. . . . .-.
:
I
_-
-.--
-CT-...--
_.... , s-.-“-
.-.
------&
, to.6
t -0.2
- 0.2
- 0.6
-i.O
EC: !
Fig. 1. Cyclic voltammograms at a glassy CL..bon electrode in the deoxygenated mobile phase used with reversed-phase columns_ Scan rate, 200 mV s-l. (-) 2,4,6-Trinitrophenyl-y-arninobutyric acid, 1 x 10“ M; (---) 2,4-dinitrophenyl-y-aminobutyric acid, 1 x lo* M; (---) background.
L.c.e.c.
of TNP derivatives
The TNP derivatives are readily separated by the reversed-phase column and are detected amperometrically with carbon electrodes (Fig. 2). An unexpected problem is that csygen in solution is retained on reversed-phase columns. The retention time of the oxygen peak is virtual!y independent of the mobile phase composition, but is a function of flow rate. Because the concentration of oxygen in aqueous solutions is greater than 10e4 M at standard temperature and pressure, oxygen can be a significant interference for trace analysis. To remove oxygen, all samples were purged with nitrogen for 2 min in a sealed micro vial. As shown in Fig. 2, this procedure does not result in an alteration of the concentrations of trinitrophenyl derivatives even from volumes as small as 200 r_tl_ With amperometric detection, the noise depends on the magnitude of the residual current. For a faradaic species in the mobile phase, it has been shown that the value of the amperometric noise is dependent on the degree of fluctuations in the flow rate [13, 23, 243 _ Thus, with amperometric detection at negative potentials, such as are required here, it is imperative to reduce the amount of oxygen in the mobile phase to trace levels unless totally pulse-free pumping systems are employed. For example, for a glassy carbon detector operated at -0.6 V in a deoxygenated mobile phase, a residual current of 53 n-4 was observed with a 0.6-n.4 peak-to-peak noise level. When the flow of nitrogen in the mobile phase was turned off, the residual current rapidly increased, and, at a residual current level of 380 nA, the peak-to-peak noise had increased 3-fold. Furthermore, oxygen in the buffer also leads to gradual fluctuations in the background signal as has been previously shown [S] . This may arise from outgassing of the solvent by the pump and it certainly precludes trace measurements_
274
A
a
102
I
I
10 l-l.4
’;
I: 2
2
s
U
i
I I
, 0
i0
2c)
0 TIME
(mtn)
10
20
30
- 0.1
-c.3
-0.5
-0.7
-0.9
-1.1
E(V’
Fig. 2. Reversed-phase chromatograms with amperometric detection of TNP derivatives of amino acids (50 pmol of each derivative): deoxygenation; (B) with sample deoxygenation. Pezk assignments: oxygen; (I) glycine; (2) L-alanine; (3) o-alanine; (4) y-aminobutyric butyric acid; (6) a-aminobutyric acid; and (7) 6 -aminovaleric acid.
of a standard
solution
(A) without sample S, solvent front; 02, acid; (5) p-aminoiso-
Fig. 3. Hydrodynamic voltammograms at a glassy carbon electrode obtained following acid derivative reversed-phase separation. Fiow rate, 1.5 ml min-‘. (0) y-Aminobutyric (1 x lo* M); (0) O1 in sampleat room temperature and atmosphericpressure;(----)
residualcurrent.
The selection of the potential for detection of TNP derivatives involves a series of tradeoffs. This is illustrated in the hydrodynamic voltammograms for oxygen, the TNP derivatives, and the residual currents that were obtained by recording chromatograms at increasingly negative potentials (Fig. 3). The voltammograms of the other derivatives are very similar to that for the y-aminobutyric acid derivative. The stepwise reduction that is apparent in Fig. 1 is hardly observed in the hydrodynamic voltammogram, possibly because the electrode surface had deteriorated after extended use_ The composite waves are shifted to negative potentials as expected [ 251. At potentials beyond -0.9 V, the current decreases suggesting either that electrogenerated products are reacting with the y-aminobutyric acid derivative or that the electrode response is deteriorating. The dissimilarity in shape of the curve for injected oxygen to that for the background indicates that oxygen levels are effectively reduced. It can be seen that at -0.6 V, only partial electrolysis of oxygen occurs, and yet, as described above, this is of sufficient magnitude
275
to cause serious interferences unless removed. The magnitude of the residual current and the current for oxygen are considerably larger at the potential for the 12electron reduction of the ~eminobutyric acid derivative than at the potential for the s-electron reduction (-0.6 V) and thus this potential was employed_
Micrcmanipulation of sample The reaction of TNBS with primary amines has been shown to be a nucleophilic aromatic substitution with the rate-limiting step being the formation of a tetra-substituted ring carbon intermediate [ 1’7, 18]_ The second-order rate constants for y-aminobutyric acid, L-alanine, fl-alanine and glycine have been shown to be 2-3 orders of magnitude greater than that of hydroxide ion [lS] _ The optimum pH for the reaction which will minimize side products, such as picric acid, and which will provide for 100% derivatization at room temperature, has been determined to be 9.0 [ 31. Under the conditions described in the experimental section, the microreaction proceeds to 100% derivatization of the amine. The efficiency of the reaction was determined by comparing the amount of derivative formed under the conditions given in the experimental section with a known amount of authentic TNP derivative following separation on an anion-exchange column (Fig. 4A and B). Direct injection of the derivatized sample onto the reversed-phase column is not possible because TNBS remains in high concentration (-lo-’ M) in the reaction mixtures_ Trinitrobenzenesulfonic acid elutes very quickly on the reversed phase column and hinders trace determination of the TNP derivatives. High concentrations of TNBS also cause a considerable decrease in column lifetime. To alleviate these problems, an extraction procedure with toluene was employed_ The recoveries obtained following the extraction procedure for deoxygenated samples are given in
Fig. 4. Chromatograms obtained with the SAX column with amperometric detection. (A) Chromatogram of standard 2,4,6-trinitrophenylr-aminobutyric acid (500 pmol, peak 1); (B) chromatogram of microderivatized -y-aminobutyric acid (500 pmol, peak 1); (C) chromatogram of microderivatizedand extracted r-aminobutyric acid (800 pmol, peak 1).
276
Table 1. The extraction procedure, which is based on the relative acidity of TNBS vs. the derivatives, is obviously very efficient for the compounds listed. Trinitrobenzenesulfonic acid is almost totally removed as are most other polar, unidentified reaction side products (Fig. 4C). However, this extraction procedure is not universal; more polar compounds such as TNP glutamate and TNP aspartate are only recovered at less than 50% yield. If larger sample volumes are available, pre-column cleanup with an anionexchange resin would be preferable. Because errors may arise during the manipulation of the small volumes as a resnlt of dispensing errors or sample evaporation, the addition of an internal standard is essential 1261. For the compounds in this work, the TNP derivative of 6 aminovaleric acid is ideal for this role. Analysis of the working curves obtained from the 1.c.e.c. chromatograms using the internal standard after microreaction and extraction shows that linear working curves for l-500 pmol are found for all of the compounds employed_ Unextracted, electroactive reaction side products co-elute with the C(-alanineand glycine derivatives, but these are u.v.-transparent. Comparison
of detection
methods
The U.V. detector and both types of electrochemical detectors are suitable for chromatographic detection of the TNP derivatives_ Figure 5A shows a typical chromatogram of a micro-derivatized mixture of amino acids (corresponding to 25 pmol of each derivative) that was obtained with u-v. detection. All of the TNP derivatives are completely resolved. One undesirable feature is the appearance of a toluene peak at 42 min which increases the time of determination with respect to electrochemical detection.
Fig. 5. Reversed-phase chromatograms of a microderivatized mixture of amino acids. (A) U.V. detection of 25 pmol of each derivative; (B) amperometric detection with glassy carbon (1 pmol of each derivative); (C) amperometric detection with the pyrolytic graphite (1 pmol of each derivative). Peak assignments: (1) glycine; (2) L&mine; (3) p-alanine; (4) y-aminobutyric acid; (5) p-aminoisobutyric acid; (6) cr-aminobutyric acid; (7) the internal standard, 6-aminovaleric acid (50 pmol); (8) toluene.
277
Figures 5B and 5C show chromatograms of micro-derivatfzed mixtures of amino acids (corresponding to 1 pmol of each derivative) that were obtained with the glassy carbon and the pyrolytic graphite detectors, respectively. The signal-to-noise (peak-to-peak) ratios of the derivatized y-aminobutyric acid peaks for the three modes of detection were found to be: U.V. detection (25 pmol of the derivative), S/N = 5.4; glassy carbon (1 pmol of the derivative), S/N = 8.0; and pyrolytic graphite (1 pmol of the derivative), S/N = 14. Comparison of these ratios for each of these detectors indicates that the pyrolytic graphite detector has detection limits ca. 2 X lower than the glassy carbon detector and ca. 66 X lower than the U.V. detector_ The difference in the signal-to-noise ratios of the two electrochemical detectors is consistently observed, and may arise because only a portion of the surface-oxidized basal plane of pyrolytic graphite is available for electrolysis, resulting in “islands” of carbon where elect.rolysis occurs. These isolated sites would be expected to be less dependent on flow rate fluctuations [23], and their presence is implied because the absolute amperometric current at these pyrolytic graphite electrodes is less than that of glassy carbon electrodes of identical geometric area. Freshly oxidized pressure-annealed pyrolytic graphite shows the theoretically expected dependence of current on geometric area [27, 281, but this response decays to a stable value after prolonged use [IS], with the observed, improved signal-to-noise ratios. Although the differences in signal-to-noise ratios for the two electrochemical detectors are not significant for routine applications, these observations may be important for planning strategies to improve detection limits with electrochemical detectors. Despite the very high molar absorptivity of these compounds, the signal-to-noise ratios do demonstrate that the electrochemical detectors are preferable to u-v. detection for trace detection of TNP derivatives_ All of these data suggest that TNBS is an ideal derivatizing agent for the trace detection of alkylamines. The reaction is quantitative and the reagent can easily be removed before chromatographic separation of the derivatives because it is such a strong acid. The derivatives are well suited for both u.v. and electrochemical detection with the latter method preferred for trace determination. The gifts of samples of pressure-annealed pyrolytic graphite from Union Carbide are gratefully acknowledged_ This work was supported by NSF Research Grant BNS 81-00044. R.M.W. is an Alfred P. Sloan Fellow and the recipient of an NIH Research Career Deveiopment Award (K04 NS 00356-04). REFERENCES 1 T. Okuyama and K. Satake, J. Biochem. (Tokyo), 47 (1960) 454. 2 K. Satake, T. Okuyama, h3. Ohashi and T. Shinoda, J. Biochem. (Tokyo), 654. 3 L. C. Mockrasch, Anal. Biochem., 1s (1967)
64.
47
(1960)
278
4 W. L. Caudill, G. P. Houck and R. M. Wightman, J. Chromatogr., Biomed. Appl., 227’ (19S2) 331. 5 P. T. Kissinger, C. S. Bruntlett and R. E. Shoup, Life Sci., 28 (1981) 455. 6 I_ N. Mefford, J. Neuroxi. Me’&., 3 (1981) 207. 7 P. T. Kissinger, Anal. Chem., 49 (1977) 447A. 8 W. A. MacCrehan and R. A_ Durst, Anal. Chem., 53 (1981) 1700. 9 K. Bratin, P. T. Kissinger, R. C. Briner and C. S. Bruntlett, Anal. Chim. Acta, 130 (1981) 295. 10 R. Samuelson, J. O’Dea and J. Osteryoung, Anal. Chem., 52 (1980) 2215. 11 D. L. Rabenstein and R. Saetre, Anal. Chem., 49 (1977) 1036. 12 W. A. MacCrehan and R. A. Durst, Anal. Chem., 50 (1978) 2108. 13 W. A. MacCrehan, Anal. Chem., 53 (1961) 74. l-1 R. C. Buchta and L. J. Papa, J. Chromatogr. Sci., 14 (1976) 213. 15 S. K. Vohra and G. W. Harrington, J. Chromatogr. Sci., 18 (1980) 379. 16 R. M. Wightman, E. C. Paik, S. Borman and M. A. Dayton, Anal. Chem., 50 (19SG) 1410. 17 A_ R. Goldfarb, Biochemistry, 5 (1966) 2570. 1s G. E. Means, W. I. Congdon and M. L. Bender, Biochemistry, ll(l972) 3564. 19 II. B. Ehlers and J. W. Sease, Anal. Chem., 31 (1959) 16. 20 S. F. Dennis, A. S. Powell and M_ J. Astel. J. Am. Chem. Sot., 71(1949) 1484. 21 G. W. Jackson and J. S. Dereska, J. Electrochem. Sot., 112 (1965) 1218. 22 J. Pearson, Trans. Faraday SOC., 44 (1948) 683. 23 D. E. Weisshaar, D. E. Taliman and J. L. Anderson, Anal. Chem., 53 (1981) 1809. 24 D. G. Swartzfager, Anal. Chem., 48 (1976) 2189. 25 J. L. Anderson, D. E. Weisshaar and D. E. Tailman, Anal. Chem., 53 (1981) 906. 26 L. R. Snyder and Sj. van der Wal. Anal. Chem., 53 (1981) 877. 27 S. G. Weber and W. C. Purdy, Anal. Chim. Acta, 100 (1978) 531. 28 Y. Hirata, P. T. Lin, M. Novotny and R. M_ Wightman, J. Chromatogr., Biomed. Appi., 181 (1980) 287.
Chimica
Eisevier Scientific
141
Acta,
Publishing
(1982)
269-278
Company,
Amsterdam
-
Printed
in The Netherlands
TRINITROBENZENESULFONIC ACID: A CHROMOPHORE, ELECTROPHORE AND PRE-COLUMN DERIVATIZING AGENT FOR HIGHPERFORMANCE LIQUID CHROMATOGRAPHY OF ALKYLAMINES
W. LOWRY CAUDILL Department (Received
and R. MARK
of Chemistry, 8th March
Indiana
WGH’I’MAN*
University,
Bloomington,
IN 4 7405
(U.S.A.)
1982)
SUMMARY Trinitrobenzenesulfonic acid is shown to be an ideal pre-column derivatizing agent for high-performance liquid chromatography of alkylamines. The reaction is quantitative and the trinitrophenyl derivatives are amenable to ultra-violet and electrochemical detection_ Electrochemical detection with either a glassy carbon or pressure-annealed pyrolytic graphite working electrode provides lower detection limits than ultraviolet detection and thus is preferable
for trace determinations.
Trinitrobenzenesulfonic acid (TNBS) was introduced as a derivatizing agent for amino acids in 1959 by Okuyama and Satake [l] _ The trinitrophenyl (TNP) derivatives of aliphatic amines that can be formed in alkaline
solutions have many properties that facilitate their use in quantitative work. Direct spectrophotometric determination is possible because TNP derivatives have an absorbance maximum at approximately 350 nm, a wavelength where TNBS is essentially transparent. The derivatives can be isolated in crystalline form in high yield so that authentic standards are easily obtained [ 2,3]. In this report, it is shown that TNP derivatives are also advantageous for electrochemical detection because they are reduced at easily accessible pctentials. There has been, however, little use of TNBS for trace determinations or as a pre-column reagent for the derivatization of compounds separated by liquid chromatography_ It was recently shown that TNBS can be used quantitatively to derivatize y-aminobutyric acid, a neurotransmitter, in whole rat brain homogenates using liquid chromatography with electrochemical detection (1.c.e.c.) [4]. Trinitrobenzenesulfonic acid is of interest as a derivatizing agent for use as an electrophore for 1.c.e.c. to take advantage of the low detection limits of this method. The low detection limits of 1.c.e.c. have been demonstrated for easily oxidized compounds where picogram measurements are routine [5, 63. Carbon electrodes at fixed potential are used primarily in this application Properly prepared electrodes are stable, and thus do not require electrode resurfacing or other forms of treatment for several months. However, carbon electrodes are not reliable when operated at very negative potentials. 0003-267O/SZ/OOOO-0000/$02_75
o
1982
Elsevier Scientific
Publishing
Company
270
Various interferences, either in the mobile phase or in the sample, cause the electrode response to deteriorate [7, 83. Chief among these interferences is oxygen, the presence of which can lead to a large baseline offset and increased faradaic noise. To minimize these problems,- detectors for reducible compounds
have employed
renewable
mercury
surfaces (dropping
mercury elec-
trodes or metal amalgams [ 9-151). Another successful approach has been to use a dualelectrode system in which a generator electrode is used to form an oxidizable product [S] . However, electroreduction of trinitro compounds occurs at sufficiently low potentials that a single carbon electrode can be employed for trace level detection. For routine determinations, this approach offers mode.
many
of
the advantages
demonstrated
by
1.c.e.c.
in the oxidative
EXPERIMENTAL
Reagents _%ll aqueous solutions were prepared in doubly-distilled water with reagent
grade chemicals as described previously [ 41. -y-Aminobutyric acid, ~alanine, L-alanine, glycine, a-aminobutyric acid, P-aminoisobutyric acid, 6 -aminovaleric acid, 2,4-dinitrophenyl-y-aminobutyric acid, and 2,4,6-trinitrobenzene-sulfonic acid (TNBS) were obtained from Sigma Chemical Company (St. Louis, MO). Crystalline forms of 2,4,6trinitrophenyl7aminobutyric acid, 2,4,6-trinitrophenyl+&~.nine, 2,4,6-trinitrophenyl-L-alanine, 2,4,6trinitrophenyld aminovaleric acid, 2,4,6-trinitrophenylglycine, 2,4,6-trinitrophenyl-ac-aminobutyric acid, and 2,4,6-trinitrophenylQ-aminoisobutyric acid were prepared for use as standards with a procedure similar to that of Okuyama and Satake [ 11. !k?icroreac tion A sample (200 ~1) containing the above-specified amino acids (except for
6 aminovaleric acid which served as internal standard) at pH 9.2 with 0.2 M potassium tetraborate is dispensed into an Eppendorf polypropylene test tube (1.5 ml, Brinkmann Instruments, Westbury, NY). The derivatizing agent, T-NBS, (20 ~1, 0.1 M) is added and the reaction is allowed to progress for 30 min. The reaction is terminated by the addition of perchloric acid (2.0 M, 180 ,ul) containing the internal standard, 2,4,6-trinitrophenyl-&amincvaleric acid (2.2 X 10m6M). The final pH is 0.2. The sample is then extracted twice with 400-~1 aliquots of toluene. A portion of the combined toluene fraction (500 ~1) is back-extracted with pH 9.0 buffer (250 ~1, Fisher, Cincinnati, OH). The aqueous phase, which contains the derivatized amino acids and the internal standard, is placed in a vial (V vial, 1.0 ml, Anspec Co., Ann Arbor, MI). The vial is sealed with a serum cap and the aqueous phase is deosygenated by nitrogen bubbling for 2 min. The deoxygenated aqueous solution is then injected onto the column.
271
Apparatus The liquid chromatographic apparatus was described previously [ 41. A constant, reduced level of oxygen in the mobile phase was accomplished by deoxygenation of the mobile phase with nitrogen and the utilization of stainless steel tubing throughout the chromatograph. The columns were either 4.6-mm i.d. X 25 cm Biophase (&pm particle size) reversed-phase columns (Bioanalytical Systems, West Lafayette, IN) or g-mm i-d. X 50 cm Zipax (30-pm particle size) strong anion-exchange columns (SAX, DuPont, Wilmington, DE). The mobile phase for the reversed-phase separation contained citric acid (0.098 M), disodium hydrogenphosphate (0.016 M), acetic acid (0.43 M) (pH 2.5) and 35% methanol (HPLC Grade, Fisher, Cincinnati, OH). The SAX mobile phase consisted of potassium hydrogenphthalate (0.037 M), sodium acetate (0.039 M) and disodium ethylenedinitrilotetraacetate (0.001 M). The flow rate for the reversed-phase separation was 1.5 ml min-’ and for the SAX system was 0.8 ml min-‘. the working electrode was made of glassy For amperometric detection, New York, NY) or the oxidized basal carbon (Tokai GC-20, Atomergic, plane of pressure-annealed pyrolytic graphite (Union Carbide Co., Parma, OH). The pyrolytic graphite electrode was pretreated with anodic oxidation as described elsewhere [16] _ The glassy carbon was polished to a mirror finish with use of 600~grit sandpaper, followed by polishing on a felt cloth with 5+m, 0_3+m, and 0_05+m alumina (Fisher, Cincinnati, OH), successively_ The flow cell, identical for both electrode materials, was a modification of that previously described [16] . The auxiliary electrode was made of stainless steel which served as the upper block of the cell. _4 precut spacer (51+m thick: Tefzel, DuPont, 1Vilmington, DE) was laminated onto the auxiliary electrode_ Lamination was achieved in 1 h at 260°C. The entire assembly was then clamped together with 4 bolts. The working electrode area was 0.33 cm’ and the cell volume was 1.7 ~1. The working electrode potentials were -0.6 V and -0.8 V vs. a saturated calomel electrode (SCE) for the reversed-phase and S-4X systems, respectively_ For ultraviolet (u-v_) detection, the optical unit of an Altex Model 300 isocratic liquid chromatograph (lo-mm path length, 8+1 cell volume, Altex Scientific, Berkeley, CA) was used with single wavelength monitoring at 254 nm. Voltammetric studies employed a PARC Model 174A. Measurements were made with a disk-shaped glassy carbon electrode (0.05Scm’ surface area). RESULTS
AND
DISCUSSION
Properties of trinitrophenyl derivatives Gram-scale quantities of the TNP derivatives of each of the amino acids were prepared and each possessed a sharp melting point (see Table 1). Ring substitution by the amino group is expected for these derivatives [ 17,181 and was verified for the glycine derivative by mass spectrometry (parent ion m/z = 264). All of the TNP derivatives possess a high molar absorptivity at
272 TABLE
1
Recovery of the trinitrophenyl derivatives of the compounds cited after extraction and deoxygenation (Melting pcints of.the sepvately prepared derivatives are also given) Compound
Recover,-
<‘Fop
L-.L\latIiIIe yXminobutwic acid P-,\minoisobutpric acid 8-hminomleric acid
100 2 0.886 101 f. 0.613 101 i 0.993 -
nr.P.&j
Compound
144-145 149-151 143-1-14 114-115
a-Aminobutwic &Alanine Glycine
Recovery acid
94.3 + 2 26 94.2 f 2.44 91.2 f 2.60
I&p.
CC>
119-120 189-189.5 166-167
“Mean recoveries determined with 1 X 1Oa Prl solutions of the amino acids. The 98% confidence limits (n = 9) were obtained from a 2-sided Student’s t-test.
254 nm which makes them ideal for U.V. detection following liquid chromatographic separation (e.g., for the r-aminobutyric acid derivative, e = 2.2 X 10’ I mol-’ cm-‘). The electrochemistry of the y-aminobutyric acid derivative was characterized at carbon electrodes to determine the parameters for amperometric detection. The response of the amperometric detector is directly proportional to the number of electrons per mole transferred in the electrolysis, which
suggests
that
TNP
derivatives
are ideal because
at very negative
poten-
they are reduced in an overall 18-electron process. This process is evidenced by polarography in aqueous solutions as three 4electron, 2-proton, irreversible waves at moderate potentials and as a 6-electron wave at potentials that are near the negative limit for a mercury electrode. The 4electron, Z-proton waves correspond to the reduction of each nitro group to the corresponding hydrosylamine [19-223. _At a carbon electrode in the buffer used for reversed-phase chromatography, only the first three reduction waves are apparent in the potential range available (Fig. 1). The peak potentials for these waves are shifted negatively with respect to those obtained at mercury electrodes_ The peak potentials move in a negative direction with an increase in pH from 2 to 6 (43.7 mV/pH unit, 45.5 mV/pH unit, and 66.1 mV/pH unit for the first, second and third waves, respectively). The oxidation wave observed on the reverse scan suggests that the aromatic multi-hydroxylamine is the product of these reductions. Figure 1 also shows a cyclic voltammogram for 2,4&nitrophenyi-yaminobutyric acid. Dinitrophenyl derivatives can be easily formed from the more common derivatizing agent for amines, 2,4_dinitrofluorobenzene. The dinitrophenyl derivatives are reduced at more negative potentials than the corresponding TNP derivatives and only two voltammetric waves are observed_ Therefore, the TNP derivatives are superior for amperometric detection with carbon electrodes, because they are easier to reduce and more electrons per mole of derivative are transferred at any given potential. tials
, $7 ,
’
\
.
.
273
-3
I
!_‘I
_
_“_-..-
c
_____.___..
_.a-.
/’
. . . . .-.
:
I
_-
-.--
-CT-...--
_.... , s-.-“-
.-.
------&
, to.6
t -0.2
- 0.2
- 0.6
-i.O
EC: !
Fig. 1. Cyclic voltammograms at a glassy CL..bon electrode in the deoxygenated mobile phase used with reversed-phase columns_ Scan rate, 200 mV s-l. (-) 2,4,6-Trinitrophenyl-y-arninobutyric acid, 1 x 10“ M; (---) 2,4-dinitrophenyl-y-aminobutyric acid, 1 x lo* M; (---) background.
L.c.e.c.
of TNP derivatives
The TNP derivatives are readily separated by the reversed-phase column and are detected amperometrically with carbon electrodes (Fig. 2). An unexpected problem is that csygen in solution is retained on reversed-phase columns. The retention time of the oxygen peak is virtual!y independent of the mobile phase composition, but is a function of flow rate. Because the concentration of oxygen in aqueous solutions is greater than 10e4 M at standard temperature and pressure, oxygen can be a significant interference for trace analysis. To remove oxygen, all samples were purged with nitrogen for 2 min in a sealed micro vial. As shown in Fig. 2, this procedure does not result in an alteration of the concentrations of trinitrophenyl derivatives even from volumes as small as 200 r_tl_ With amperometric detection, the noise depends on the magnitude of the residual current. For a faradaic species in the mobile phase, it has been shown that the value of the amperometric noise is dependent on the degree of fluctuations in the flow rate [13, 23, 243 _ Thus, with amperometric detection at negative potentials, such as are required here, it is imperative to reduce the amount of oxygen in the mobile phase to trace levels unless totally pulse-free pumping systems are employed. For example, for a glassy carbon detector operated at -0.6 V in a deoxygenated mobile phase, a residual current of 53 n-4 was observed with a 0.6-n.4 peak-to-peak noise level. When the flow of nitrogen in the mobile phase was turned off, the residual current rapidly increased, and, at a residual current level of 380 nA, the peak-to-peak noise had increased 3-fold. Furthermore, oxygen in the buffer also leads to gradual fluctuations in the background signal as has been previously shown [S] . This may arise from outgassing of the solvent by the pump and it certainly precludes trace measurements_
274
A
a
102
I
I
10 l-l.4
’;
I: 2
2
s
U
i
I I
, 0
i0
2c)
0 TIME
(mtn)
10
20
30
- 0.1
-c.3
-0.5
-0.7
-0.9
-1.1
E(V’
Fig. 2. Reversed-phase chromatograms with amperometric detection of TNP derivatives of amino acids (50 pmol of each derivative): deoxygenation; (B) with sample deoxygenation. Pezk assignments: oxygen; (I) glycine; (2) L-alanine; (3) o-alanine; (4) y-aminobutyric butyric acid; (6) a-aminobutyric acid; and (7) 6 -aminovaleric acid.
of a standard
solution
(A) without sample S, solvent front; 02, acid; (5) p-aminoiso-
Fig. 3. Hydrodynamic voltammograms at a glassy carbon electrode obtained following acid derivative reversed-phase separation. Fiow rate, 1.5 ml min-‘. (0) y-Aminobutyric (1 x lo* M); (0) O1 in sampleat room temperature and atmosphericpressure;(----)
residualcurrent.
The selection of the potential for detection of TNP derivatives involves a series of tradeoffs. This is illustrated in the hydrodynamic voltammograms for oxygen, the TNP derivatives, and the residual currents that were obtained by recording chromatograms at increasingly negative potentials (Fig. 3). The voltammograms of the other derivatives are very similar to that for the y-aminobutyric acid derivative. The stepwise reduction that is apparent in Fig. 1 is hardly observed in the hydrodynamic voltammogram, possibly because the electrode surface had deteriorated after extended use_ The composite waves are shifted to negative potentials as expected [ 251. At potentials beyond -0.9 V, the current decreases suggesting either that electrogenerated products are reacting with the y-aminobutyric acid derivative or that the electrode response is deteriorating. The dissimilarity in shape of the curve for injected oxygen to that for the background indicates that oxygen levels are effectively reduced. It can be seen that at -0.6 V, only partial electrolysis of oxygen occurs, and yet, as described above, this is of sufficient magnitude
275
to cause serious interferences unless removed. The magnitude of the residual current and the current for oxygen are considerably larger at the potential for the 12electron reduction of the ~eminobutyric acid derivative than at the potential for the s-electron reduction (-0.6 V) and thus this potential was employed_
Micrcmanipulation of sample The reaction of TNBS with primary amines has been shown to be a nucleophilic aromatic substitution with the rate-limiting step being the formation of a tetra-substituted ring carbon intermediate [ 1’7, 18]_ The second-order rate constants for y-aminobutyric acid, L-alanine, fl-alanine and glycine have been shown to be 2-3 orders of magnitude greater than that of hydroxide ion [lS] _ The optimum pH for the reaction which will minimize side products, such as picric acid, and which will provide for 100% derivatization at room temperature, has been determined to be 9.0 [ 31. Under the conditions described in the experimental section, the microreaction proceeds to 100% derivatization of the amine. The efficiency of the reaction was determined by comparing the amount of derivative formed under the conditions given in the experimental section with a known amount of authentic TNP derivative following separation on an anion-exchange column (Fig. 4A and B). Direct injection of the derivatized sample onto the reversed-phase column is not possible because TNBS remains in high concentration (-lo-’ M) in the reaction mixtures_ Trinitrobenzenesulfonic acid elutes very quickly on the reversed phase column and hinders trace determination of the TNP derivatives. High concentrations of TNBS also cause a considerable decrease in column lifetime. To alleviate these problems, an extraction procedure with toluene was employed_ The recoveries obtained following the extraction procedure for deoxygenated samples are given in
Fig. 4. Chromatograms obtained with the SAX column with amperometric detection. (A) Chromatogram of standard 2,4,6-trinitrophenylr-aminobutyric acid (500 pmol, peak 1); (B) chromatogram of microderivatized -y-aminobutyric acid (500 pmol, peak 1); (C) chromatogram of microderivatizedand extracted r-aminobutyric acid (800 pmol, peak 1).
276
Table 1. The extraction procedure, which is based on the relative acidity of TNBS vs. the derivatives, is obviously very efficient for the compounds listed. Trinitrobenzenesulfonic acid is almost totally removed as are most other polar, unidentified reaction side products (Fig. 4C). However, this extraction procedure is not universal; more polar compounds such as TNP glutamate and TNP aspartate are only recovered at less than 50% yield. If larger sample volumes are available, pre-column cleanup with an anionexchange resin would be preferable. Because errors may arise during the manipulation of the small volumes as a resnlt of dispensing errors or sample evaporation, the addition of an internal standard is essential 1261. For the compounds in this work, the TNP derivative of 6 aminovaleric acid is ideal for this role. Analysis of the working curves obtained from the 1.c.e.c. chromatograms using the internal standard after microreaction and extraction shows that linear working curves for l-500 pmol are found for all of the compounds employed_ Unextracted, electroactive reaction side products co-elute with the C(-alanineand glycine derivatives, but these are u.v.-transparent. Comparison
of detection
methods
The U.V. detector and both types of electrochemical detectors are suitable for chromatographic detection of the TNP derivatives_ Figure 5A shows a typical chromatogram of a micro-derivatized mixture of amino acids (corresponding to 25 pmol of each derivative) that was obtained with u-v. detection. All of the TNP derivatives are completely resolved. One undesirable feature is the appearance of a toluene peak at 42 min which increases the time of determination with respect to electrochemical detection.
Fig. 5. Reversed-phase chromatograms of a microderivatized mixture of amino acids. (A) U.V. detection of 25 pmol of each derivative; (B) amperometric detection with glassy carbon (1 pmol of each derivative); (C) amperometric detection with the pyrolytic graphite (1 pmol of each derivative). Peak assignments: (1) glycine; (2) L&mine; (3) p-alanine; (4) y-aminobutyric acid; (5) p-aminoisobutyric acid; (6) cr-aminobutyric acid; (7) the internal standard, 6-aminovaleric acid (50 pmol); (8) toluene.
277
Figures 5B and 5C show chromatograms of micro-derivatfzed mixtures of amino acids (corresponding to 1 pmol of each derivative) that were obtained with the glassy carbon and the pyrolytic graphite detectors, respectively. The signal-to-noise (peak-to-peak) ratios of the derivatized y-aminobutyric acid peaks for the three modes of detection were found to be: U.V. detection (25 pmol of the derivative), S/N = 5.4; glassy carbon (1 pmol of the derivative), S/N = 8.0; and pyrolytic graphite (1 pmol of the derivative), S/N = 14. Comparison of these ratios for each of these detectors indicates that the pyrolytic graphite detector has detection limits ca. 2 X lower than the glassy carbon detector and ca. 66 X lower than the U.V. detector_ The difference in the signal-to-noise ratios of the two electrochemical detectors is consistently observed, and may arise because only a portion of the surface-oxidized basal plane of pyrolytic graphite is available for electrolysis, resulting in “islands” of carbon where elect.rolysis occurs. These isolated sites would be expected to be less dependent on flow rate fluctuations [23], and their presence is implied because the absolute amperometric current at these pyrolytic graphite electrodes is less than that of glassy carbon electrodes of identical geometric area. Freshly oxidized pressure-annealed pyrolytic graphite shows the theoretically expected dependence of current on geometric area [27, 281, but this response decays to a stable value after prolonged use [IS], with the observed, improved signal-to-noise ratios. Although the differences in signal-to-noise ratios for the two electrochemical detectors are not significant for routine applications, these observations may be important for planning strategies to improve detection limits with electrochemical detectors. Despite the very high molar absorptivity of these compounds, the signal-to-noise ratios do demonstrate that the electrochemical detectors are preferable to u-v. detection for trace detection of TNP derivatives_ All of these data suggest that TNBS is an ideal derivatizing agent for the trace detection of alkylamines. The reaction is quantitative and the reagent can easily be removed before chromatographic separation of the derivatives because it is such a strong acid. The derivatives are well suited for both u.v. and electrochemical detection with the latter method preferred for trace determination. The gifts of samples of pressure-annealed pyrolytic graphite from Union Carbide are gratefully acknowledged_ This work was supported by NSF Research Grant BNS 81-00044. R.M.W. is an Alfred P. Sloan Fellow and the recipient of an NIH Research Career Deveiopment Award (K04 NS 00356-04). REFERENCES 1 T. Okuyama and K. Satake, J. Biochem. (Tokyo), 47 (1960) 454. 2 K. Satake, T. Okuyama, h3. Ohashi and T. Shinoda, J. Biochem. (Tokyo), 654. 3 L. C. Mockrasch, Anal. Biochem., 1s (1967)
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