Separation of Amino Acids in Plant Tissue Extracts by Capillary Zone Electrophoresis with Indirect UV Detection Using Aromatic Carboxylates

as Background Electrolytes
Z. L. Chen* / C. R. Warren / M. A. A d a m s Department of Botany, The University of Western Australia, Nedlands, WA 6907, Australia

Key Words
Capillary zone electrophoresis Indirect UV detection Amino acids Plant tissue extract

Summary
Amino acids in extracts of plant tissue were separated and detected by capillary zone electrophoresis (CZE) with indirect UV detection. Various aromatic carboxylates such as salicylate, benzoate, phthalate and trimellitate were investigated as background electrolytes (BGEs). A BGE of benzoate gave the best resolution and detector response. Amino acids were separated at a highly alkaline pH to charge amino acids negatively. Separataion was achieved by the co-electroosmotic flow (Co-EOF) by the addition o f the cationic surfactant myristyltrimethylammonium bromide (MTAB) to the electrolyte. The condtions affecting the separation of amino acids, including electrolyte pH, concentrations of both benzoate and MTAB, were investigated and optimised. Separation of a mixture of 17 amino acids at pH 11.20 with indirect UV detection at 225 nm was achieved with a BGE of 10mM benzoate containing 1.0mM MTAB at pH of 11.20. Detection limits ranged between 10 and 50#M. The proposed method was demonstrated by the determination of amino acids in extracts of Eucalypt leaves with direct injection of samples.

Introduction
The accumulation of free amino acids is a widespread response of higher plants to drought and salt stress [12]. The determination of free amino acids in plant tissue has been achieved by chromatographic methods, including gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS) and high perfor180 0009-5893/00/02 180-07 $ 03.00/0

mance liquid chromatography (HPLC) [3-5]. GC or GC-MS methods are based on a variety of derivatization procedures [6-9], they offer high sensitivity and resolution. However, such complicated derivatization procedures are time-consuming. Most free amino acids have low UVextinction coefficients, hence detection of amino acids in HPLC requires formation of a coloured or fluorescent derivative by pre-or post-column derivatization. [10-12]. HPLC derivatization methods give high sensitivity and efficient separation, but the procedures of postcolumn derivatization require complex equipment. Moreover, there is the possibility of excess reagent and the reaction medium interfering with the separation, and several derivatives may be produced from one solute in GC and HPLC derivatization methods [12]. Capillary electrophoresis (CE) is an attractive approach for the separation of free amino acids. Detection of amino acids has been achieved by indirect fluorescence [13], direct UV and indirect UV detection [14--15] and amperometric detection [16]. Indirect UV detection is the most promising method since most commercial CE systems are equipped with UV detectors and most amino acids don't absorb UV [17]. There are several studies on indirect UV detection of amino acids in CZE. Foret et al. [ 18] discussed the use of benzoic acid or sorbic acid as the BGEs for the separation of organic anions and some amino acids. Bruin et al. [ 19] reported the use of salicylate as a BGE at pH 11.0 for the indirect UV detection of 7 amino acids in CZE. Lee et al. [15] investigated several BGEs for CZE separation of amino acids with indirect UV detection, their results indicated that p-aminosalicylic and 4-(N,N-dimethyl amino)-benzoic acid with cyclodextrine additives [20] were the most suitable BGEs. More recently, Chen et al. [21 ] reported the use of salicylate and benzoate as the BGEs for indirect UV detection of 14-18 amino acids, and its application to the determination of amino acids in urine samples. In our previous paper [22], a derivatization GC-FID method was developed for the determination of organic acids and amino acids in plant extracts, which offered high separation efficiency and sensitivity. However, procedures of sample purification and derivatization are Original

Chromatographiagol. 51, No. 3/4, February 2000 9 2000 Friedr. Vieweg & Sohn VerlagsgesellschaftmbH

required, hence it is too slow for routine analysis of multiple samples. In the method described here, we studied systematically the CZE separation of free amino acids with indirect UV detection using various BGEs. We tested several aromatic carboxylates as BGEs, in order to match the mobility of solute ion with the BGE, and enable effective separation and high detection sensitivity. Finally, the proposed method was applied to the determination of amino acids in extracts of plant tissue

deionized water for 5 min, followed by BGE for 5 min. Samples were injected in the hydrodynamic mode for 5 seconds. Separation was carried out with the capillary was thermostated at 25 ~ and a constant voltage of 20 kV (counter-EOF) or-15 or -20 kV (Co-EOF mode).

Electrophoretic Determination
The mobility of various BGEs was determined in 20 mM phosphate buffer at pH 11.0 [ 15]. Mobility was determined in a solution containing all BGEs at 0.1 mM each in deionized water and 0.05 % (v/v) benzyl alcohol as a neutral marker. The voltage was set at +20 kV. To identify the optimal wavelength for detection of BGEs the UV detector was scanned between 200 and 300 nm. The electrophoretic mobility of the BGE was calculated using the equation described in a previous report [ 17].

Experimental
Chemicals
All reagents were of analytical grade and dissolved in Milli-Q water without further purification. Standards of the amino acids tested were prepared daily from a 10 mM stock solution in Milli-Q water and diluted to required concentrations before use. BGEs were prepared by dissolution of an appropriate amount of aromatic carboxylate in Milli-Q water, which contained appropriate amounts of myrisityltrimethylammonium bromide (MTAB). BGEs were filtered through a Millipore 0.45 #m membrane filter and degassed in a benchtop microfuge prior to use. Electrolyte pH was adjusted with 0.1 M NaOH.

Results and Discussion

Characteristics of the BGEs
An effective BGE must be provide a high UV absorbance at a wide range of wavelengths, and its ionic mobility should closely match that of the solute. Aromatic carboxylates have been frequently used in CZE as the BGEs [17] for indirect UV detection of anions as their mobilities can be controlled by the pH. In this work, salicylate, benzoate, phthalate and trimellitate were used as the BGEs. A standard mixture of 16 amino acids was separated using the various BGEs at a concentration of 20 mM and a pH of 11.20 (Figure 1). With all four BGEs, Lys and Pro eleuted first since they are only partly dissociated at pH 11,20, which results in a lower effective mobility. The last eluting peaks with the lowest overall mobility were Cys and Glu, which are double negatively charged and migrated with the greatest effective mobility, against the EOE However, all amino acids could not be completely resolved by either of the four BGEs. This may well reflect differences in the mobility of the test BGE and the mobility of all the amino acids since the mobilities of amino acids are in the range of 1.0 to 4.0. (x 10-4 cm 2 V-is -1) [15]. This was evident as peak deformation for amino acids with mobilities greater or less than that of the BGE. Hence, Asp and Glu with electrophoretic mobilities greater than the BGE gave badly tailing peaks. The best resolution and indirect UV response were obtained with benzoate as a BGE, though Leu and His, Phe and Val comigrated. Better resolution with benzoate than the other BGEs reflects the lesser mobility of benzoate, which more closely matches the mobility of most of the test amino acids [ 17, 23]. Furthermore, amino acids gave the largest indirect UV response with benzoate due to its greater molar absorptivity and its mobility approximating that of solute ions, which results in a greater displacement ratio of UVabsorbing to solute ion and thus a greater detection sensitivity [24]. Therefore, benzoate was used as a BGE in the following studies. 181

Sample Extraction
Frozen leaf tissues were ground with liquid N in a mortar with pestle. Approximately 0.2 g (fresh weight) of ground tissue was weighed into a centrifuge tube and cold (0-4 ~ extraction solution (25 % aqueous ethanol) was added (10mL g-1 fresh weight). The sample was placed on ice, thoroughly homogenised at 8000 rpm with a lab blender (Ultra-turrax) and centrifuged. The supernatant was used as a source of amino acids and injected into the CE without any sample cleanup.

Instrumentation
All CZE experiments were preformed using a Bio-Rad 3000 Capillary Electrophoresis system (Bio-Rad, California, USA) controlled by a computer equipped with CE-3000 software (Bio-Rad). Separation was carried out in fused-silica capillaries (50 #m I.D x 75 cm total length, 70.4 cm effective length). Determination of the mobility of BGEs was performed in shorter capillaries (50ktm I.D. x 40cm total length, 36cm effective length). To determine the optimal wavelength for detection, the UV detector was set for rapid scanning of absorbance between 195 and 295 nm.

Electrophoretic Procedures
Prior to use, a new capillary was pretreated by passing with 0.1 M NaOH for 10 min, and a deionized water for 10 min. Before injection, the capillary was preconditioned with the following cycle: 0.1 M NaOH for 3 min, Original

Chromatographia Vol. 51, No. 3/4, February 2000

O4-

8
c a :D -,4

-4

34

~16 12

16

>

-6

-6

-8
-3 l I 11 I l l I l

6+6 7+8
OxlO
I l I l l

-IDxI0

7 8 9 Migration time (min)

I0

II

12

10 12 Migration tlme (mln)

14

16

(a)

Co)

4-

4-

2-

2-

03 4
16

.0 Q =>
o

0-

18

3 4

111; 3

-2
6*6

-2
8+6

-4xlO
I I l I I

~xlO
l I l I l

1O

12 14 16 Migrationtime (min)

18

10

12 14 Migrationtime (min)

16

18

(c)

(d)

Figure 1
Electropherograms of sixteen amino acids obtained from the test background electrolytes (BGEs) with indirect UV detection. (a) salicylate (235 nm), (b) benzoate (225 nm), (c) phthalate (215 nm), and (d) trimelliate (235 nm). Peaks: 1 = Lys., 2 = Pro., 3 = Trp., 4 - OH-pro., 5 = Leu.; 6 = His, 7 = Phe., 8 = Val., 9 = Met., 10 =Gln., 11 = Ala., 12 = Thr., 13 = Ser., 14 =Asn., 15 = Cys., 16 = Glu. Experimental conditions: applied voltage 20 kV; buffer, 20 mM at pH of 11.20.

C o - E O F M o d e Separation of A m i n o Acids
C o - E O F m o d e , is c o m m o n l y used for the separation o f anionic solutes which co-migrate with the EOF, leading to i m p r o v e d resolution and shorter analysis times. The E O F is reduced or reversed to the direction o f the E O F b y the addition o f cationic surfactant to the B G E [25, 26]. Cationic surfactants such as c e t y l t r i m e t h y l a m m o n i u m b r o m i d e (CTAB) [27] and tetradecyltrimethyla m m o n i u m b r o m i d e (TTAB) [28] have frequently been used to reverse the direction o f the E O E We tested a separation with a B G E o f 20 m M benzoate solution and 0.5 m M m y r i s t y l t r i m e t h y l a m m o n i u m bromide (MTAB) 182

at a p H o f 11.20. Separation was p e r f o r m e d with a reversed polarity o f - 2 0 kV. Seventeen amino acid p e a k s were detected (Figure 2). All p e a k s were well resolved, with the exception o f O H - p r o L e u and His, Phe and Val which co-migrated. The migration order o f all the a m i n o acids was reversed completely with Figure l(b). The first eluting peaks were Asp and Glu, which are double negatively charged and thus m i g r a t e d with the highest effective mobility with the E O E Peak shapes were close to s y m m e t r i c a l for all amino acids, with no bad tailing or fronting (compare Figures lb and 2).

Chromatographia Vol. 51, No. 3/4, February 2000

Original

O-

-2
3 2

> "= -6

1
16

,;,21
7+8

-8
4-6 -3

Ox10

10 Migration time

12

14

(min)

Figure 2
Electropherogram of seventeen amino acids obtained from Co-EOF mode. Peaks: 1 = Lys., 2 = Pro., 3 = Trp., 4 = OH-pro., 5 = Leu.; 6 = His., 7 = Phe., 8 = Val., 9 = Met., 10 =Gln., 11 = Ala., 12 = Thr., 13 = Set., 14 =Asn., 15 - Cys., 16 = Glu., 17 = Asp. Experimental conditions: applied voltage -20 kV; buffer, 20 mM benzoate + 1 mM MTAB at pH of 11.20. Indirect UV detection at 225 nm.

+ +

1
2 3 4 5 A--4 [] 3 1 2 3 4 5

6-

.......................... :>

5-

I'
~=~

[]
........ "" ' ..... ".... "-.X 3

5 6

0
O

6
7 8 9 lO

8
9

2 7-----1

10

0
0

10

15

20

25

3O

11

0 10.6

35
12

10.8

11

11.2

11.4

11.6

BGE concentration (mM)

pH

Figure 3
The effect of BGE concentration on the mobility of amino acids Other conditions as described in Figure 2.

Figure 4
The effect ofBGE pH on the mobility of amino acids Other conditions as described in Figure 2.

Separation Optimization
In C Z E w i t h indirect U V detection, the c o n c e n t r a t i o n o f B G E a n d its pH, as well as the c o n c e n t r a t i o n o f E O F m o d i f i e r d e t e r m i n e the separation e f f i c i e n c y and sensitivity. T h e effect o f b e n z o a t e c o n c e n t r a t i o n on the sep a r a t i o n w a s investigated u s i n g a c o n c e n t r a t i o n o f 1 m M M T A B in the B G E at a p H o f 11.20. T h e effective mobilities o f a m i n o acids d e c r e a s e d as the c o n c e n t r a tion o f b e n z o a t e increased (Figure 3). This is due to a r e d u c t i o n in m o b i l i t y o f the solute as a result o f the i n c r e a s e d total ionic strength o f the electrolyte [17]. This w a s a c c o m p a n i e d b y a c o r r e s p o n d i n g increase in m i g r a t i o n time, h e n c e resolution o f a m i n o acids was better at greater c o n c e n t r a t i o n s o f b e n z o a t e . However, Original detector r e s p o n s e d e c r e a s e d as the c o n c e n t r a t i o n o f b e n z o a t e in the electrolyte increased. This can be attributed to a r e d u c e d d i s p l a c e m e n t ratio o f U V absorbing ion to solute ion [24]. T h u s we m u s t strike a c o m p r o m i s e b e t w e e n sensitivity a n d resolution. Therefore, in this study, 1 0 m M b e n z o a t e w a s c h o s e n as the best c o m p r o m i s e b e t w e e n sensitivity and resolution. The m o b i l i t y and detection r e s p o n s e are p H d e p e n d e n t b e c a u s e the ionization o f solutes and B G E s are affected b y B G E p H [ 19]. The effective m o b i l i t y o f a m i n o acids increased as the B G E p H i n c r e a s e d f r o m 10.80 to 11.40, and a c o r r e s p o n d i n g r e d u c t i o n in m i g r a t i o n time was o b s e r v e d (Figure 4). Peaks o f O H - p r o and Leu, His and Phe, and Val a n d M e t o v e r l a p p e d w h e n the B G E p H was 183

Chromatographia gol. 51, No. 3/4, February 2000

___<>..._ ---O---A ~3
w .. ~ll..dl.~.. v , .....

_o]
.e

2 3 4 5 6

g
7

8 9

--,@.I I I I I

10

-5x10
11

-3

I 12

5
12

I 13

I 14 Migration

I 15

I 16

17

I 18

(a)

time (rain)

MTAB (raM)
Figure 5

O-

The effect of MTAB concentration on the mobility of amino acids Other conditions as described in Figure 2.

-2
0-

-E m 9

-2

16

131211

5+6 7+3

-6x10

-3
I I I f I I

12

13

14
Migration

15

16
time (min)

17

18

(b)
I 18 I 20 I 22

OxlO

I B

I 10

I 12

I I 14 16 Migration time (min)

Figure 7

Electropherograms obtained from plant extracts. (a) Corymbia calophylla, (b) Pinus pinaster Experimental conditions as described in Figure 6.

Figure 6

The elctropherogram of seventeen amino acids obtained under optimum conditions. Experimental conditions: applied voltage - 15 kV; buffer, 10 mM benzoate + 1 mM MTAB at pH of 11.20. Indirect UV detection at 225 nm

equal to or less than 10.8 or greater than 11.40. This reflects greater mobility o f amino acids at greater pH, which led to a reduction in resolution [29].The best resolution and p e a k shape was obtained at a B G E p H o f 11.20. In addition, increasing electrolyte p H increased detection response. This was because elevating electrolyte p H enhanced the displacement ratio [ 19,29]. Hence, optimtma separation, p e a k shapes and detection sensitivity was obtained at a B G E p H o f 11.20. The effect o f M T A B concentration in the B G E on separation was e x a m i n e d over the range o f 0.25-5 m M (Figure 5). Effective mobilities o f amino acids increased as the concentration o f M T A B increased, e.g., the mi184

gration time o f Lys decreased f r o m 9.10 to 8.28 m i n w h e n the concentration o f M T A B increased f r o m 0.25 to 5 m M . A m i n o acids with a m e d i u m mobility such as O H - p r o and Leu, His and Phe, Met and Gln did not resolve well w h e n the concentration o f MTAB was greater than 5 m M , whereas w h e n the concentration o f M T A B was less than 1 m M all a m i n o acids were well resolved. Indicating that resolution can be controlled by altering concentrations o f surfactant. However, increasing the concentration o f M T A B in the B G E led to a reduction in detector response b e c a u s e the added coanion in M T A B c o m p e t e d with solute ions for the disp l a c e m e n t o f the B G E , and thus reduced the displacem e n t ratio [19, 30]. Hence, M T A B concentrations between 0.5 and 1 m M gave the highest resolution and detection sensitivity. The o p t i m i z e d s e p a r a t i o n o f a m i n o acids b y C Z E with indirect U V detection was a c h i e v e d with a B G E o f Original

Chromatographia Vol. 51, No. 3/4, February 2000

Table I. Mobilities, molecular weigth (Mt) structure andpKa of the BGEs and solutes
Mobilty (104cm2/Vs) Benzoic Salicylic Phthalic Trimellic Lys Pro Trp OH-pro Leu His Phe Val Met Gln Ala Thr Set Ash Cys Glu Asp 2.99 3.53 4.94 5.73 0.51 0.69 0.76 1.24 1.35 1.36 1.47 1.48 1.53 1.57 1.62 1.83 2.27 2.68 3.13 3.51 3.86 Mt pKa R.S. D % (migartion time) Detection limit (#M)

121.1 138.1 166.1 210.1 146.2 115 131.1 131.1 155.2 117.2 149.2 146.2 89.09 119.1 105.1 132.1 121.2 147.1 133.1

4.19 2.79 2.98, 5.51 2.42,3.71,5.01. 2.18, 8.95 1.99, 10.60 NA 1.92, 9.73 2.36, 9.60 1.82, 9.17 NA 2.32, 9.62 2.28, 9.21 2.17, 9.13 2.34, 9.69 2.09,9.10 2.21, 9.15 2.02, 8.80 1.96, 10.28 2.19, 9.67 1.88, 9.60

2.4 2.1 1.7 1.3 1.6 1.6 1.4 1.4 0.9 1.1 1.9 1.8 1.6 1.8 1.7 1.5 1.8

45 25 30 20 15 15 10 10 18 45 18 18 17 50 40 20 25

The conditions as in Figure 6. NA. information not available.

Table II. The concentration of amino acids in the plan extracts

determined by the proposed method Solute Corymbia calophylla (mM) 1.02 0.10 n.d. 0.05 Pinus pinaster (mM) 1.52 0.38 0.19 0.57

Analysis of Real Samples

The p r o p o s e d m e t h o d was used to determine amino acids in extracts o f l e a f tissues f r o m plants. Typical electropherograms are presented in Figure 7. Leaves o f C o r y m b i a and Pinus contained four amino acids in detectable concentrations, Pro, OH-Pro, L e u and Met, their identification was verified b y spiking samples with k n o w n a m i n o acids. Concentrations o f amino acids in these samples are listed in Table II. Interference f r o m other anions was evident in a n u m b e r o f samples. A clean-up protocol b a s e d on solid phase extraction (SPE) is being developed to address this problem. These results indicate that the p r o p o s e d m e t h o d is useful for the analysis o f amino acids in extracts o f plant tissues without the n e e d for lengthy derivatization procedures.

Pro OH-pro Leu Met

Conditions as for Figure 6. n. *d-not detected.

10 m M benzoate, 1 m M M T A B at a p H o f 11.20 (Figure 6). The a m i n o acids were generally well separated with sharp s y m m e t r i c a l peaks; however, L e u and His, Phe and Val co-migrated. A m i n o acids m i g r a t e d in the order: Asp, Glu, Cys, Asn, Ser, Thr, Ala, Gln, Met, Val, Phe, His, Leu, OH-Pro, Trp, Pro, Lys. The migration order o f amino acids reflects differences in their charge and mass. For example, Asp and Glu which are double negatively charged, m i g r a t e d faster than Lys and Pro which are only partially dissociated at p H 11.20. Calibration plots were obtained by plotting peak area vs the concentration o f the test acids. The relationship was linear in the concentration range o f 0.02-2.0 mM. Correlation coefficients (r 2) were in the range o f 0.9992 to 0.9999. The detection limits (S/N=3) ranged 10-50 # M and the reproducibility o f the migration time (relative standard derivation, n = 5) from injecting a 1 m M standard mixture ranged between 0 . 7 - 2 . 4 % . Analytical characteristics o f the test solutes using the proposed method, together with theirpKa [31 ], are listed in Table I. Original

Conclusion
C Z E separation o f 15-17 amino acids can be p e r f o r m e d by capillary electrophoresis using C o - E O F m o d e with indirect U V detection. The type o f B G E its concentration and pH, as well as E O F modifiers significantly affect separation and detection sensitivity. The best resolution and detection sensitivity was obtained with a B G E o f 10 m M benzoate containing 1 m M MTAB at p H 11.20. The proposed m e t h o d was demonstrated to be useful for the analysis o f amino acids in extracts o f plant tissues with fast separation and v e r y simple sample preparation. 185

Chromatographia Vol. 51, No. 3/4, February 2000

Acknowledgments
T h e authors thank the Australian R e s e a r c h Council for financial support. Charles W a r r e n is supported b y a D e p a r t m e n t o f C A L M / U W A postgraduate scholarship. We also thank the a n o n y m o u s reviewers for their c o m ments in the revision o f this paper.

References
[1] A.D. Hanson, W.D. Hitz, Ann. Rev. Plant Physiol. 33, 163 (1982). [2] H. Greenway, R. Munns, Annu. Rev. Plant Physiol. 31, 149 (1980). [3] A. Gzik, Environmental & Experimental Botany, 36, 29 (1996). [4] A. Ortiz, V. Martinez, A. Cerdd, Physiologia Plantarum 91, 468 (1994). [5] T. W-M.Fan, T.D. Colmer, A. N. Lane, R. M. Higasahi, Anal. Biochem., 214,260 (1993). [6] J Chauhan, A. Darbre, R. E Catlyle, J. Chromatogr., 227, 305 (1982). [7] H. Kataoka, S. Matsumura, H. KoizumL M.Makita, J. Chromatogr A., 758, 167 (1997). [8] C.-H. Oh, J.-H. Kim, K.-R. Kim, D. M. Brownson, T. J. Mabry,. J.Cbzomatogr., 669, 125 (1994). [9] P Husek, J. Chromatogr., 669, 352 (1995). [10] Y. Tsuruta, H.Inoue, Anal. Chem., 265, 15 (1998). [11] J. E Bowyer, P Clausing, C. D.Newport, J. Chromatogr. B., 666, 241 (1995). [12] E. Heffmann, Chromatography: Fundamentals and application of chromatography and related differential migration method. Amsterdam, (1992).

[13] W. G. Huh~ E. S. Yeung, Anal. Chem., 63,417 (1988). [14] M . J Tornton, J S. Fritz, C. W. Klampfl, J.High Resol. Chromatogr., 20, 647 (1997). [15] Y.H. Lee, T.L Lin, J. Chromatogr. A. 680, 287 (1994). [16] T.M..Olefirowicz, A. G. Ewing, J. Chromatogr.499, 713 (1991). [17] J P Landers, Handbook of Capillary Electrophresis, 2nd, CRC press, Boca Taton, 1997. [18] E Foret, S. Fanali, L. Ossicini, P. Bocek, J. Chromatogr. 470, 299 (1989). [19] G. M. Bruin, A. C. van Asten, X. Xu, H. Poppe, J. Chromatogr. 608, 97 (1992). [20] Y.H. Lee, T.L Lin, J.Chromatogr. A., 716, 335 (1995). [21] H. Chen, Y. Xu, M. P I p , J. Liq. Chrom. & Rel. Technol. 20, 2475 (1997). [22] Z Chen, P Landman, T. D. Colmer, M.A. Adams, Anal. Biochem., 259, 203 (1998). [23] T. Soga, G. A. Ross, J. Chromatogr.A. 767, 223 (1997). [24] S. M. Cousins, P R. Haddad, W.Buehberger, J. Chromatogr. A. 671,397 (1994). [25] K.K.C. Yeung, C. A. Lucy, J. Chromatogr. A. 804, 319 (1998). [26] D. Volgger, A. Zemann, G. Bonn, J. High Resol. Chromatogr., 21, 3 (1998). [27] K. Altria, C Simpson, Anal. Proc. 23,453 (1986). [28] X. Huang, J A. Gordon, M. J Gordon, R. Z. Zare, Anal. Chem. 61,766 (1989). [29] Y. Ma, R. Zhang, J. Chromatogr. 625, 341 (1992). [30] M. W.ENeilen, J. Chromatogr. 588,321 (1991). [31] D. R. Lide, H P R. Frederikse, Eds., Handbook of Chemistry and Physics, CRC Press, Boca Raton, FL, 75 th ed~ 1994. Received: Jun 1, 1999 Revised manuscript received: Aug 25, 1999 Accepted: Sep 16, 1999

186

Chromatographia Vol. 51, No. 3/4, February 2000

Original