Anal. Chem. 1981, 53, 1380-1383
1380
Table V. Results of Analyses of Human Urines with CN- and SCN- Recovery Testa amt added, -wg/mL CN' SCN0.00 1.00
2.00 3.00 4.00
0.0 5.0 10.0 15.0 20.0
amt found, d m L CNSCN-
__l__l_-
0.00
0.94 1.81 3.3lC 3.98
3.1 7.9 13.6 16.8 21.5
recovery, % CNSCN94 91 110 100
96 105 91 92
Mean of five a The sample was no. 3 in Table IV. Mean of replicate analyses, standard deviation = 0.1. five replicate analyses, standard deviation = 0.31.
(4) Deshmukh, G. S.; Tatwawadl, S.V. J . Sci. Ind. Res., Secf B 1980, 19, 195-198. (5) Pettlgrew, A. R.; Fell, G. S . Clin. Chem. (Winston-Salem, N.C.)1972, 78, 996-1000.
(6) "Standard Methods for the Examination of Water and Wastewater", 14th ed.; American Public Health Associatlon, American Water Works Associatlon,and Water Pollution Control Federation: New York, 1975; Pp 367-372, pp 383-386. (7) Epstein, J. Anal. Chem. 1949, 19, 292-274. (8) Aldridge, W. N. Analyst (London) 1944, 69, 262-265; 1945, 70,
__
474-475. . . ..
(9) (IO) (11) (12) (13) (14)
(15)
ACKNOWLEDGMENT Authors thank Y. Tsugita, Y. Matsushita, and H. Iida of
(16)
the Environmental Protection Research Center (Nishi-ku, Osaka, Japan) for their assistance to colrect wastewater Samples.
(18)
LITERATURE CITED
(20)
(1) Bark, L. S.; Hiysori, H. G. Analyst(London) 1963, 88,751-760. (2) Guilbault G. G.; Kramer, D. N. Anal. Cfiem. 1965, 37, 1395-1399; 1968. 38. 834-836. (3) Darr,'R. W.: Capson, T. Hifernan, F. D. Anal. Chem. 1980, 52, 1379- 1380. L.;
(17)
(19)
Butts, W. C.; Kuehneman, M.; Widdowson, G. M. Clin.
Chem. (Winsfon-Salem. N.C.) 1974. 20. 1344-1348. Danchik, R.'S,; Bok, D. F. Anal. Chem. 1968, 40, 2215-2216. Ahuja, S. d . Pharm. Sci. 1976, 65, 163-182. Dorozd, ,I, J. Chromatogr., 1976, 113, 303-356. Knapp, D. R. "Handbook of Analytical Derivatizatlon Reactions";Wlley: New York, 1979. Blau, K., Klng, C;. S., Eds. "Handbook of Derlvatlves for Chromatography"; Heyden: London, 1978. Funazo, K.; Tanaka, M.; Shono, T. Chem. Lett. 1979, 309-310; Anal. Chem. 1980, 52, 1222-1224. Funazo, K.; Kusano, K.; Tanaka, M.; Shono, T. Anal. Len. 1980, 73, 751-757. Nota, G.;Palomtari, R. J . Chromatogr. 1973, 84, 37-41; 1978, 123, 411-413. Vafentour, J. C.: Aggarwel, V ; Sunshine, I. Anal. Chem. 1974, 46, 924-925. De Brabanaer, H. F ; Verbeke, R. J. Chromatogr. 1977, 738, 13'1-142. fhomson, I.; AnUerson, R. A. J . Cbrornatagr. 1980, 188, 357-362.
R E C E Nfor ~ review March 12,1981. Accepted May 13,1981. This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education of Japan.
Reversed-Phase Liquid Chromatographic Resolution of Amino ers by Mixed Chelate Complexation Moriyuki Nimura, Tsukasa Sumuki, Yoko Kasahara, and Toshio Kinoshlta * School of Pharmaceutical Sciences, Kitasato University, 9- I Shirokane-5, Minato-ku, Tokyo
Separation of enantbmers of arnlno acids by reversed-phase liquid chromatography has been accomplished by using the copper( X I ) complex of optically active N-(p-toluenesunonyl)-L-phen~lalan~ne(TosPhe) as the mobile phase. The stereoselectivity is ascribed to the differences In the stability of the ternary complex of enantiomerlc amino acids with the TssPhe-Cu(II) reagent. The column eluent is monitored for fluorescence alter reaction with a-phthaladehyde. Amino acids are detectabie up to picomole range.
The many attempts to resolve the enantiomeric amino acids by high-performance liquid chromatography (HPLC) have followed two general approaches: precolumn derivatization converting amino acids into diastereomers ( I ) or the use of a chiral stationary phase (2)or mobile phase containing chiral additive(s) ( 3 ) . The chiral derivatization methods for the resolution of enantiomeric amino acids by reversed-phase HPLC have been described in a previous report ( 4 ) . Resolution of enantiomeric Dns or DNP derivatives of amino acids have also been developed by using a chiral stationary phase (5) or chiral eluant (6-8), but the procedures for precolumn derivatization such as dansylation can be tedious. In contrast, various metal chelate additives to the mobile phase have been used for the separation of underivatized
108,Japan
amino acid enantiomers on cornnierically available stationary phases. Recently, Grushka et al. (9) have separated underivatized D,L-amino acids on a reversed-phase column containing the chiral reagent L-aspartylcyclohexylamidecopper(I1). However, conventional postcolunin reaction for the highly sensitive detection of amino acids is not applicable to their system since L-aspartylcyclohexylilrnide has a free amino group. Gil-Av et al. have reported the methods for the resolution of free amino acid enantiomers on an ion-exchange column (3) and reversed-phase colunin (IO) using a chiral eluant containing a copper-proline complex accompanied by sensitive fluorometric detection using o-phthalaldehyde (QPTA) reagent. The present paper describes a method for separating free D-and L-amino acids on a reversed-phase column using mobile phases containing the chiral reagent N-(p-toluenesulfony)-I,phenylalaninecopper(I1) [TosPhe-Cu(II)]. TosPhe is readily prepared by the reaction of L-phenylalanine with p-toluenesulfonyl chloride in alkaline media. The resolution was accomplished on a short column 10 cm long filled with a noctylsilyl bonded silica gel, and amino acids were monitored by fluorescence postcolumn reaction using OPTA reagent.
EXPERIMENTAL SECTION Reagent. Amino acids and other reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan) and Tokyo
0003-2700/81/0353-1380$01.25/0 0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981
1381
*---y -1
!
1.0-
0
&--
I _ _ 10 025 05 mMOf TosPhe (TosPhe Cu = 2 I )
Flgure 1. Influence of TosPhe-Cu(I1) concentration on separation factor ( a ,solid line) and resolution value (Rs, dashed line): mobile phase, aqueous solution containing TosPheCu(I1)in concentration as given in the figure; pH 6.0. TIME(min)
Flgure 3. Separation of o,L-amino acids with TosPhe-Cu(I1)eluent: mobile phase, 10% acetonitrile in an aqueous solution containing 1 mM TosPhe and 0.5 mM CuS04.5H20;pH 6.0. Approximately 0.5 nmol of each amino acid was injected.
1
0
10
20 30 TIME(rnln)
40
50
Flgure 2. Separation of o,L-amino acids with TosPhe-Cu(I1)eluent: mobile phase, aqueous solution containing 1 mM TosPhe and 0.5 mM CuSO4*5H20; pH 6.0. Approximately 0.5 nmol of each amino acid was injected.
Chemical Industry Co. (Tokyo, Japan). Chemically bonded noctyhilyl silica gel Develosil C8 (particle size; 5 km) was obtained from Nomura Chemical (Seto-shi, Japan), and its unreacted accessible silanol groups were capped with trimethylsilane (11) in our laboratory. Water and acetonitrile were distilled by using glass apparatus before use. TosPhe was prepared as described by McChensney and Swann (12).
The mobile phase consisted of acetonitrile-water containing TosPhe and CuS04.5H20in 2:l molar ratio. The pH of the mobile phase was adjusted with 5% aqueous sodium carbonate solution. The OPTA reagent containing EDTA.2Na (2.5 g/L) for postcolumn derivatization was prepared as described by Hare and Gil-Av (3, 13). Chromatographic System. Both mobile phase and postcolumn reagent were delivered at a constant flow rate of 1.0 m.L/min using a double plunger pump (SanukiIndustry Co., Tokyo, Japan). Trimethylsilyl-treated Develosil C8 was packed in the stainless steel column tube (10 cm x 4.0 mm i.d.) in our laboratory by the conventional slurry packing technique. The column was operated a t 30 “C utilizing a Taiyo thermo unit C-600 (Taiyo Scientific Industry Co., Tokyo, Japan). The column eluate was mixed with the OPTA reagent in a mixing T-piece and a PTFE-tubing reaction coil (50 cm X 0.5 mm i.d.). Fluorescence intensity of the effluent was measured at 455 nm; excitation of fluorescence was achieved at 340 nm, using an RF-500 LCA spectrofluoromonitor (Shimadzu Seisakusho Ltd.,Kyoto, Japan) equipped with a xenon discharge lamp.
0
10
20
30
40
TIME(min)
Flgure 4. Separation of o,L-amino acids with TosPhe-Cu(I1)eluent: mobile phase, 15% acetonitrile in an aqueous solution containing 1 mM TosPhe and 0.5 mM CuS0,.5H20; pH 6.0. Approximately 0.5 nmd of each amino acid was injected.
RESULTS AND DISCUSSION In the following discussion, the symbols k ’, a , and Rs represent capacity ratio, separation factor, and resolution value, respectively. These values changed with alteration of the pH and the concentrations of TosPhe-Cu(I1) complex and acetonitrile in the mobile phase. k’and a increased with the increase in the pH in the range from pH 5 to p H 7, so most amino acids can be resolved in this pH range. However, the racemic pairs of alanine, aspartic acid, glutamic acid, and asparagine were resolved a t pH 6 or above. Moreover, the Rs values markedly decreased a t p H 7 due to the tailing of chromatographic peaks (Table I). Therefore, the pH of the mobile phase was adjusted to 6. The dependence of a and Rs on the concentration of TosPhe-Cu(I1) is shown in Figure 1. Both a and Ra increased as the concentration of TosPhe-Cu(I1) increased. The resolution of glutamic acid enantiomers could be achieved a t 1
1382
ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981
Table I. Capacity Ratio ( k ' ) , Separation Factor ( a ) ,and Resolution Value (Rs) as a Function of pHa pH 5.0 amino acid glutamicacid
D
alanine
L L D
valine
a
Rs
1.60 1.60 2'oo 2.00
l.oo
o.oo
1.00
0.00
1.27
1.38
3'00
3.80
k' 2.20
2.63
i:;: lg:iE iy:;:
pH 7.0
a
Rs
1.20
0.80
1.35
1.87
2.05
6.85
1.30 2.52 2.08 8.25 8.38 Mobile phase: aqueous solution containing 1 mM TosPhe and 0.5 mM CuSO;5H20. tyrosine
a
L D
PH 6.0
____
k'
L D
6'44
01
Rs
3.40 2'70
1.26
0.48
3'60 5.00
1.39
1.65
2.08
2.45
2.24
3.89
k'
ii:;: ::k
-
Table 11. Capacity Ratio ( k ' ) ,Separation Factor (a),and Resolution Value (Rs) as a Function of Acetonitrile Concentration Acetonitrile Concentration" 10% acetonitrile
0% acetonitrile
amino acid aspartic acid glutamic acid asparagine
k' D L D L D L
glutamine alanine valine tyrosine
D L L D
L D L
norvaline
D L D
phenylglycine
L
isoleucine leucine norleucine phenylalanine tryptophan
D L D L D L D L D I, D
1.44 2.00 2.20 2.63 2.70 2.93 3.52 3.52 2.40 3.24 8.25 16.90 10.51 21.86 12.00 25.00 22.81 52.00
a
Rs
k'
1.39
1.10
0.10
1.20
0.80
1.09
0.39
1.00
0.00
1.35
1.87
2.05
6.85
2.08
8.25
2.08
7.35
2.28
10.85
b b b b
0.29 0.28 0.58 0.60 0.60 0.68 0.68 0.78 1.10 3.70 6.40 4.50 8.00 5.20 9.24 9.20 21.80 9.40 19.74 16.00 26.60 16.80 31.50 37.40 71.20
b
a
15% acetonitrile Rs
2.90
0.64
2.07
0.90
1.00
0.00
1.00
0.00
1.41
1.21
1.73
4.32
1.78
5.56
1.78
4.75
2.37
8.57
2.10
7.39
1.66
4.53
1.88
5.88
1.90
6.04
b
k'
0.05 0.05 0.10 0.10 0.18 0.18 0.20 0.20 0.12 0.12 1.00
1.54 1.20 1.90 1.22 1.96 2.60 5.00 2.60 4.40 3.82 5.64 4.10 6.60 9.50 14.70 13.20 20.80
a
Rs
1.00
0.00
1.00
0.00
1.00
0.00
1.00
0.00
1.00
0.00
1.54
1.71
1.58
2.21
1.61
2.06
1.92
5.45
1.69
3.33
1.48
3.16
1.61
3.79
1.55
4.30
1.58
4.14
a Mobile phase: acetonitrile in an aqueous solution containing 1 mM TosPhe and 0.5 mM CuS0;5H20, pH 6.0. t,, = 2.0 min. Not measured as the amino acid was retained over 1 h.
m M TosPhe and 0.5 mM Cu(I1) (Figure 1). The separation of the amino acids was dependent on acetonitrile concentration (Table 11). When acetonitrile was omitted, leucine, phenylalanine, and tryptophan were retained on the column for over an hour. Addition of acetonitrile a t a concentration from 10% to 15% enabled rapid separation of a several amino acid isomers (Table 11, Figures 2-4). TosPhe does not interfere with the detection of amino acids with OPTA which reacts with primary but not with secondary amines and amides. Precipitation of copper ions by the alkaline OPTA reagent is prevented by adding EDTA in the reagent (3). Consequently, picomole levels of amino acids are detected. In the mobile phase, TosPhe and copper(I1) ion can be assumed to form a binary complex [ (To~Phe)~CuI1] as shown in Figure 5. Recently, Karmen et al. (8) have reported a method for separating D- and L-Dns derivatives of amino acids by adding Cu(I1) complexes of proline and arginine. Their study suggests that N-sulfonylamino acid, free amino acid, and copper(I1) form a ternary complex. Accordingly free amino acid, TosPhe,
r
'1
Flgure 5. Proposed structure of binary complex of TosPhe with cop-
per(I1).
l o r P h ~ - C ~ l I I ) - D - ~a cm~~d ~ ~ T o s P h e - C ~ ( l l I-L.Arnino
atld
Flgure 6. Proposed structure of ternary complexes formed from D
and L-amino acid with TosPhe and copper(I1). and copper(I1) appear to form a ternary complex. Enantiomeric pairs of amino acids may form complexes of different conformation. The ternary complex of D-amino acid may assume trans conformation around the Cu(I1) ion and that of L-isomer cis conformation (Figure 6). These molecular
Anal. Chem. 1981, 53, 1383-1386
models support the fact that L-amino acid was eluted before the D-isomer except for acidic amino acids since the trans isomer is thermodynamically more stable than the cis one (13). The difference in mobility among amino acid appears to be based on different hydrophobic and steric interaction between the alkyl substituent on the a-carbon of the amino acid and n-octyl residue on the bonded phase silica gels. Consequently, amino acids which have higher hydrophobic alkyl substituents were retained longer than those having lower hydrophobic moiety. For isomers with an equal number of carbon atoms, those pairs with the branched side chain such as valine and leucine are eluted faster than those with a linear side chain such as norvaline and norleucine. These results agreed with the results of Grushka et al. (9) and Karmen et al. (7). A preliminary test showed that TosPhe does not react with ninhydrin. This fact suggests that proline may also be detected after separation by the present system with slight modifications although it is not detectable when copperproline complex is used as the mobile phase (3, 10).
1383
LITERATURE CITED Tamegal, T.; Ohme, M.; Kawabe, K.; Tomoeda, M. J. L/quMChromtoor. 1979. 2. 1229-1250. D&ankov, V. A,;Rogozhin, S. V. J. Chromatcgr. 1971, 60, 260-283. Hare, P. E.; GiCAv, E. Sclence 1979, 204, 1226-1228. Nimura, N.; Ogura, H.; Kinoshtta, T. J. Chromatcgr. 1980, 202, 375-380. Pirkle, W. H.; House, D. W.; Finn, J. M. J. Chromatcgr. 1980, 192, 143-158. Lindner, W.; LePage, J. N.; Davies, G.; Seitz, D. E.; Karger, B. L. J. ChrOmatOgr. 1979, 185, 323-344. LePage, J. N.;Lindner, W.; Davles, G.; Seitz, D. E.; Karger, B. L. Am/. Chem. 1979, 51, 433-435. Lam, S.; Chow, F.; Karmen, A. J. Chromatcgr. 1980, 199. 295-305. Gilon, C.; Leshem, R.; Grushka, E. Anal. Ch8m. 1980, 52. 1206- 1209. GICAv, E.; Tishbee, A.; Hare, P. E. J. Am. Chem. Soc. 1980, 102, 5115-5117 - . . . - . .. . Evans, M. B.; Dale, A. D.; Little, C. J. Chromatographis 1980, 13, 5-10. McChensney, E. W.; Swann, W. K., Jr. J. Am. Chem. Soc. 193’1, 59, 1116-1 118. JozefonvMz, J.; Mulier, D.; Petit, M. A. J. Chem. Soc., Dalton Trans. 1980, 76-79.
REZEIVED for review February 6,1981. Accepted April 6,1981.
Spurious Peaks in Rapid-Scan Square-Wave Polarography Adina Lavy Feder and Chaim Yarnitrky Department of Chemistry, Technion-Israel Institute of Technology, Haifa, Israel
John J. O’Dea and Janet Osteryoung” Department of Chemistry, State University of
New York at Buffalo, Buffalo, New York 14214
Square-wave polarography can exhibR spurlous peaks due to a resonance between the frequency of the applied potential and the mechanical frequency of oscillation of the dropping mercury electrode. The peak hbights can correspond to signals arising from the faradaic response of solution in the concentration range 10-T-lOd M. The resonant frequency is a function of the drop mass and can be estimated from a semiempirical equation, f = (1/27r)( a / M b/M”3)‘’2 where a = 1.51 X 10’ mg and b = -2.64 X lo5 rng1l3.
+
Square-wave voltammetry at high scan rates provides peak-shaped signals which appear well-suited to both physical chemical investigation and analysis (1-5). We have designed a One-Drop Square-Wave Analyzer (ODSWA) for the special purpose of using the rapid-scan square-wave waveform at the dropping mercury electrode (DME) for analysis (6). Work with this instrument has shown that the sensitivity is typically greater than that for differential pulse polarography for normal operating conditions, and of course the scan of potential is accomplished in much shorter time for square-wave polarography (7). In addition, the cost of constructing commercially an instrument with parameters similar to those of the one-drop square-wave analyzer should be comparable to that of other routine instrumentation for pulse voltammetry (e.g., the PARC Model 174 polarographic analyzer). Therefore there is interest in defining carefully the chhracteristics of this technique, especially those which might arise from instrumental artifacts or from physical phenomena neglected in the development of theory. Here we report on a phenomenon not previously seen, a spurious peak which arises from a resonance between the
frequency of the applied potential and the mechanical frequency of oscillation of the growing mercury drop of the DME. This peak could be misinterpreted as arising from an unidentified analyte or could interfere with measurement of the peak current for an analyte which is present. Thus although the phenomenon itself is a laboratory curiosity, it can have important practical consequences for the user of square-wave voltammetry.
EXPERIMENTAL SECTION The potential-time waveform applied in square-wave voltammetry consists of a staircase with tread width equal to the square-wave period on which is superimposed a symmetrical square wave. In the first half of each period, the square-wave pulse is in the same direction as the riser of the staircase; this is the forward pulse, and the current measured during this pulse is the forward current. Correspondinglyduring the second half period the square-wave pulse is in the opposite or reverse direction and the current is the reverse current. The usual signal output is the forward current minus the reverse current and is referred to as the net current. The parameters of this technique are the square-wave frequency, f , the step height of the base staircase, AE, the amplitude of the square-wavepulse, E,, and the delay time, td, at the initial potential, Ei. Two different kinds of instrumentation were used in these experiments. The first is the one-drop square-wave analyzer already described (6). This instrument was operated at a fixed frequency of 25 Hz, and the current was sampled and integrated over the last 10 ms of each pulse. (Note that the version described in ref 6 is designed for mains frequency of 60 Hz and therefore operates at 30 Hz.) The second is based on a Digital Equipment Corp. PDP 8/e minicomputer with homemade interface and analog electronics and is described more fully elsewhere (8). The current was sampled and integrated over the last one-third of each pulse.
0003-2700/81/0353-1383$01.25/00 1981 American Chemical Society
