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Miniaturized Voltammetric Detectors
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MINIATURIZED VOLTAMMETRIC DETECTORS MASASHI GOT0 Research Center for Resource and Energy Conservation, Nagoya University, Chikusa-ku, Nagoya, Japan INTRODUCTION Micro high-performance liquid chromatography (micro HPLC) [l] using packed columns of I.D. less than 1 mm and packed or opentubular capillary chromatography (capillary LC) [ 2 , 31 are attractive in that the consumption of the mobile phase and sample is much less than that in conventional HPLC. Flow rates of the mobile phase employed in micro HPLC and capillary LC are typically 0.1 - 30 pl/min. These extremely slow flow rates require small volume detectors and these have been designed for spectrophotometric detection by W [l] and fluorescence methods [ 4 ] . The usefulness of electrochemical detectors has already been recognized [5 - 8 1 . Numerous voltammetric detectors have been described for HPLC. These can be broadly divided into coulometric detectors that use a large surface area working electrode, and amperometric detectors that use a small surface area working electrode. These differ in their methods of detection and estimation, in that the former measures the quantity of electricity flowing when 100% of the target substance is electrolyzed at constant potential, whereas the the latter measures the current flowing when just part of the target substance is electrolyzed at constant potential. Of the various voltammetric detectors utilized for HPLC, the voltammetric detector employing a thin-layer electrolytic cell is considered to be the most suitable for micro HPLC and capillary LC, as the cell volume is easily reduced. The miniaturized voltammetric detectors suitable for micro HPLC and capillary LC and their general features are reviewed here. 1
VOLTAMMETRIC DETECTORS WITH A SINGLE WORKING ELECTRODE Goto et al. [ 9 ] and Hirata et al. [lo] independently designed sub-microliter voltammetric detectors having one working electrode for use in micro HPLC and capillary LC, respectively. Figure 1 shows the cell structure used in the single working electrode detector built by Goto et al. [9]. The thin-layer cavity is constructed of two fluorocarbon resin blocks separated 2
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by a t e f l o n s h e e t 2 0 45 pm t h i c k and 0.5 2 nun wide. A working e l e c t r o d e i s made w i t h a 3-mm d i a m e t e r g l a s s y c a r b o n d i s k c o n t a i n e d i n one of t h e b l o c k s . The r e f e r e n c e e l e c t r o d e , s i l v e r / s i l v e r chloride electrode, is held i n a c y l i n d r i c a l hole i n t h e o t h e r block. A s t a i n l e s s - s t e e l t u b e s e r v e s b o t h a s t h e c o u n t e r e l e c t r o d e and t h e e x i t l i n e . The volume i n t h e s e c e l l s i s a b o u t 0.06 - 0.3 p l . The c e l l was u t i l i z e d f o r t h e
B
M
F i g . 1. Voltammetric d e t e c t o r c e l l w i t h s i n g l e working e l e c t r o d e by Goto e t a l . [ 9 1 . (A) S i d e view o f c e l l , (B) f r o n t view o f s p a c e r . 1 = Working e l e c t r o d e ( g l a s s y c a r b o n ) , 2 = r e f e r e n c e e l e c t r o d e (Ag/AgCl), 3 = c o u n t e r e l e c t r o d e d o u b l i n g as o u t l e t (stainless-steel t u b e ) , 4 = spacer ( t e f l o n s h e e t ) , 5 = micro s e p a r a t i o n column d o u b l i n g a s i n l e t , 6 = s l o t . d e t e r m i n a t i o n o f aminophenol isomers s e p a r a t e d on a micro-column (147 x 0.5 mm I. D . )
packed w i t h silica-ODS o f 1 0 pm p a r t i c l e
chromatograms o f t h e m i x t u r e of s i z e . F i g u r e 2 shows aminophenol isomers a t d i f f e r e n t a p p l i e d p o t e n t i a l s . When t h e a p p l i e d p o t e n t i a l w a s 1.00 V (vs. Ag/AgCl), a l l t h r e e i s o m e r s s e p a r a t e d on t h e micro-column w e r e d e t e c t e d . A t lower a p p l i e d p o t e n t i a l s of 0.75 V o r 0.50 V, o n l y p- and o-aminophenol c o u l d be s e l e c t i v e l y d e t e c t e d . Three i s o m e r s w e r e c o m p l e t e l y e l u t e d w i t h i n 1 0 min a t a f l o w r a t e o f 8 . 3 pl/min u s i n g 0.1 N p e r c h l o r i c a c i d as t h e mobile phase. The l i n e a r dynamic r a n g e between peak c u r r e n t and c o n c e n t r a t i o n w a s a b o u t 1 0 ' (10 pg - 1 0 ng) and t h e
311
2
C
Fig. 2 . applied Applied v, flow
Typical chromatograms of aminophenol isomers at different potentials [9]. Peaks: 1 = p-, 2 = m-, 3 = o-aminophenol. potential (vs. Ag/AgCl): (A) 1.00 V, (B) 0.75 V, (C) 0.50 rate: 8 . 3 pl/min.
minimum detectable amount was about 10 pg for three isomers. The electrolytic efficiency in the cell under the experimental conditions of A in Fig. 2 was quite high, about 60%,and sensitivity was found to be about ten times better than in conventional HPLC. Figure 3 shows the cell structure used in the single working electrode detector built by Hirata et al. [lo]. A pressureannealed pyrolytic graphite is used as the working electrode, a saturated calomel as the reference electrode and a platinum wire as the counter-electrode. The cell is constructed by sandwiching a 50-pm thick polyethylene sheet, containing a channel 1 mm wide and 3 mm long, between a Lucite plate and the graphite electrode plate and designed to hold a volume of 0.15 p l . The cell was applied to the determination of tyrosine and tryptophan metabolites in urine with a 60 m microcapillary column packed with 30 pm porous silica particles. The experiments were conducted at a low flow rate of 1 pl/min using 0.2 M acetate buffer (pH 4 . 0 ) as mobile phase and the metabolites were detected by measuring oxidation current with the potential of the working
312
2 5
I
Fig. 3 . Cell for voltammetric detector with single working electrode by Hirata et al. [lo]. 1 = Working electrode (graphite), 2 = reference electrode (sat. Hg/Hg2C12), 3 = counter electrode (platinum wire), 4 = spacer (polyethylene sheet), 5 = microcapillary separation column, 6 = conductor (copper plate), 7 = teflon tube, 8 = inlet, 9 = outlet. electrode set at 1.00 V vs. sat. Hg/Hg2C12. Figure 4 shows the chromatogram of p-hydroxyphenylacetic acid (PHPAA), 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA) and vanillic acid (VA) in human urine. Because of the reduced dimensions of the cell and the lower flow rates compared to conventional HPLC, the detector had a detection limit approximately ten times higher than a large volume cell. For the compounds investigated here, the electrochemical method was far more sensitive than fluorescence or UV detection. VOLTAMMETRIC DETECTORS WITH TWO WORKING ELECTRODES A new approach to electrochemical detection involves the use of two working electrodes, operated simultaneously at different potentials. Several dual voltammetric detectors for HPLC have recently been developed, which provide for enhanced performance [11 - 231. They can basically be classified into the three types of configuration of the two working electrodes with respect to the flow axis, as shown in Fig. 5 [241. 3
313
I
1
2
I
4
6
I
t (hrl
8
I0
I I2
Fig. 4 . Typical chromatogram of metabolites of tyrosine and tryptophan in human urine [lo]. Peaks: 2 = PHPAA, 3 = 5-HIAA, 4 = HVA, 5 = VA. Applied potential: 1.0 V vs. sat. Hg/Hg2C12, flow rate: 1 pl/min. In the "parallel-adjacent" configuration, the working electrodes are placed adjacent to each other on one side of the rectangular thin-layer channel. In the "series" configuration, the working electrodes are positioned along the flow stream on one side of the channel. In the "parallel-opposed" configuration, the working electrodes are placed opposed to one another on both sides of the channel. All voltammetric detectors with two working electrodes (dual voltammetric detectors) can simultaneously provide two chromatograms of both oxidations or both reductions or oxidation and reduction. Dual voltammetric detector of parallel-adjacent type The parallel-adjacent dual voltammetric detector (PADVD) is analogous to the dual wavelength UV absorbance detector, and can provide useful qualitative information from peak current ratios at different potentials. PADVD has not been used for micro HPLC or capillary LC yet, because the detector of this type requires a relatively large cell volume, compared with those of other types. 3.1
314
B
A
1
1-
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i ]
Parallel- Adjacent
Series
Parallel-Opposed
F i g . 5. Three t y p e s i n c o n f i g u r a t i o n of d u a l v o l t a m m e t r i c d e t e c t o r [ 2 4 ] . (A) S i d e view, ( B ) f r o n t view. W 1 and W2 r e p r e s e n t t h e two working e l e c t r o d e s . The a r r o w s show t h e d i r e c t i o n o f f l o w .
3.2
Dual v o l t a m m e t r i c d e t e c t o r i n series t y p e
The series d u a l v o l t a m m e t r i c d e t e c t o r (SDVD) i s a n a l o g o u s to t h e f l u o r e s c e n c e d e t e c t o r , and t h e p r o d u c t of e l e c t r o d e r e a c t i o n a t t h e upstream working e l e c t r o d e is d e t e c t e d a t downstream working e l e c t r o d e . Goto e t a l . [26, 271 b u i l t a s u b - m i c r o l i t e r t h i n - l a y e r c e l l c o n t a i n i n g two g l a s s y c a r b o n e l e c t r o d e s i n series, as shown i n F i g . 6 t o s e r v e f o r m i c r o HPLC. The t h i n - l a y e r c a v i t y is c o n s t r u c t e d of two f l u o r o c a r b o n r e s i n b l o c k s s e p a r a t e d by a t e f l o n s h e e t 50 pm t h i c k and 2 mm wide. Two working e l e c t r o d e s are made w i t h g l a s s y c a r b o n d i s k s , 3 mm i n d i a m e t e ' , c o n t a i n e d i n one of t h e b l o c k s . The r e f e r e n c e e l e c t r o d e , s i l v e r /
31s
B
Fig. 6. Cell for voltammetric detector with two working electrodes in series [25]. (A) Side view of cell, ( B ) front view of spacer. 1, 2 = Working electrodes (glassy carbon), 3 = reference electrode (Ag/AgCl), 4 = counter electrode doubling as outlet (platinum tube), 5 = spacer (teflon sheet), 6 = micro separation column doubling as inlet, 7 = slot. silver chloride, is held in a cylindrical hole in the other block. A platinum tube serves both as the counter electrode and the exit line. The cell was successfully used for the selective detection of catecholamines, indoleamine and their metabolites in human urine based on their electrochemical reversibility. The principle of selective detection by SDVD is as follows. The upstream and downstream electrodes are set at potentials where the reductant (analyte) is oxidized and its oxidant is reduced, respectively. The reductant, of a reversible or quasi-reversible species, is oxidized at the anode and the product of this electrode reaction is re-reduced at the cathode, whereas the reductant of an irreversible species is not re-reduced at the cathode. Therefore, the reversible and/or quasi-reversible species present in irreversible species can be detected selectively by measuring the re-reducing current. The reversible species can, moreover, be selectively detected from quasi-reversible species by adequately
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selecting both electrode potentials. Care is required in selecting the applied potentials of such voltammetric detectors. To maximize the analytical response and selectivity and minimize background interference, the applied potentials should be held at the minimum potential for oxidation or the maximum potential for reduction, at which currents reach the limiting values for the analyte under investigation. The optimum applied potentials can be determined directly by hydrodynamic voltammetry (HDV), in which peak current is measured against applied potential, point by point, for the analyte injected into the stream flowing through the voltammetric detector under the experimental conditions of micro-HPLC. However, HDV measurements require several hours for completion. Alternatively, the approximate applied potentials for voltammetric detectors can be established,indirectly, much more rapidly by cyclic voltammetry (CV), which may be carried out on a stationary solution (without stirring) in a separate voltammetric cell [261. Cyclic semidifferential voltammetry (CSV) [27 301 provides higher sensitivity and better resolution than ordinary CV. The technique measures the semi-derivative of current with respect to time ( e ) versus applied potential ( E ) under the same experimental conditions as in CV. The instrument for CSV (model VMA-010) is now commercially available from Yanagimoto Co. Kyoto, Japan. A glassy carbon disk, 3 mm in diameter, is used as the working electrode. A
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silver/silver chloride electrode and a platinum wire are used for the reference and the counter electrode, respectively. (i) Catecholamine analysis in human urine [25, 261. The electrochemical behavior of noradrenaline (NA), adrenaline (AD), dopamine (DA) and 1-dopa (LD) on the glassy carbon electrode in the mobile phase used in micro HPLC, the Britton-Robinson (B-R) buffer of pH 1.8 containing 0.5 mM sodium 1-heptane sulphonate (HSA) as the ion-pair reagent and 0.1 mM EDTA (disodium salt) as the masking reagent for ion (11) ion, were studied by CSV [261. Figure 7 shows the cyclic semi-derivative voltammograms of catecholamines at a scan rate of 4 0 mV/sec. Each species investigated showed only one oxidation and one re-reduction peak. Theoretically, the anodic peak potential ( E ) in the cyclic e Pa versus E curves coincides with the cathodic peak potential ( EPC) for the reversible system [28]. It is clear that the catechol
317
-LI
E
f -
-
L-
u
0-4-
ae
05 E( V
02
VS
Ag1AgCI )
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Fig. 7. Cyclic semi-derivative voltammograms of 0.1 mM catecholamines in B-R pH 1.8 buffer containing 0.5 mM HSA and 0.1 mM EDTA at a scan rate of 4 0 mV/sec [ 2 6 ] . form is reversibly oxidized to the quinone form and the electrode reactions of all four catecholamines are reversible in this medium. For selective detection of catecholamines by SDVD, the upstream electrode (anode) and the downstream electrode (cathode) of the twin-electrode cell should be set at the end potentials (Eea and Eec), at which the oxidation and re-reduction wave in the cyclic e versus E curve are complete, respectively. Therefore, the potentials (vs. Ag/AgCl) of 0.60 V and 0.20 V were, respectively, chosen a s the optimum applied potentials of anode and cathode for selective detection of catecholamines in other electroactive species from Fig. 7. The micro-HPLC system with a miniaturized precolm and dual voltamnetric
318 4
i 4.
Fig. 8 . Block diagram of micro HPLC system with a miniaturized preconcentration column and dual voltammetric detector [25, 311. 1 = Micro-feeder, 2 = micro-syringe, 3 = three-way v,alve, 4 = sample injector, 5 = mobile phase, 6 = buffer solution, 7 = water, 8 = six-way valve, 9 = mixing joint, 10 = micro-precolumn, 11 = micro-separation column, 12 = twin-electrode cell, 13 = dual potentiostat, 14 = dual-pen recorder. detector,as shown in Fig. 8, was used for direct injection analysis of catecholamines in human urine. Three micro-feeders, micro-syringes and three-way valves are used to feed the mobile phase, buffersolution and water. A sample injector and six-way valve are used for sample injection and alternative connection of the precolumn with the sample enrichment system and chromatographic system, respectively. A dual potentiostat is employed to control independently the potentials of the two working electrodes and to measure the currents. The anodic and cathodic chromatograms are simultaneously recorded on a dual pen recorder. The micro-separation column for analysis is filled with silica-ODS of 10 Vrn particle size in a teflon tube 150 x 0.5 mm I. D. The micro-precolumn for enrichment is made by packing alumina of 30 um particle size in a teflon tube 20 x 0.5 mm I. D. The mobile phase for analysis is B-R buffer of pH 1.8 containing 0.5 mM HSA and 0.1 mM EDTA. The buffer solution for pretreatment of the micro-precolumn and pH adjustment of the sample is 1 M Tris
319 b u f f e r , PH 8.7,
c o n t a i n i n g 0.25 % EDTA ( d i s o d i u m s a l t ) and 0 . 5 %
sodium hydrogen s u l f i t e f o r s t a b i l i z i n g c a t e c h o l a m i n e s . The a n a l y t i c a l p r o c e d u r e s are as f o l l o w s .
Raw
human u r i n e
i s t a k e n w i t h a m i c r o - s y r i n g e and i n j e c t e d i n t o t h e sample l o o p (100 p l ) o f t h e sample i n j e c t o r t h r o u g h a s o l u t i o n f i l t e r . The sample i s d e l i v e r e d by water a t a f l o w r a t e o f 33 pl/min and mixed w i t h T r i s b u f f e r (pH 8.7) a t t h e same f l o w r a t e i n t h e mixing j o i n t t o a d j u s t t h e sample t o pH 8.6.
The sample i s
injected into the precolumn f o r 1 5 min f o r e n r i c h m e n t w i t h a mixed f l o w of w a t e r and b u f f e r s o l u t i o n and t h e n t h e precolumn i s washed f o r an additional 15 min w i t h a f l o w c o n s i s t i n g of w a t e r a l o n e , w i t h o u t any b u f f e r s o l u t i o n .
By s w i t c h i n g
t h e six-way v a l v e , t h e m o b i l e p h a s e i s i n t r o d u c e d i n t o t h e s e p a r a t i o n column t h r o u g h t h e micro precolumn a t a f l o w r a t e o f 8.3 pl/min.
I n t h i s p r o c e d u r e , t h e a d s o r b e d c a t e c h o l a m i n e s are
e l u t e d from t h e the
precolumn and s i m u l t a n e o u s l y s e p a r a t e d by
s e p a r a t i o n column. The s e p a r a t e d c a t e c h o l a m i n e s a r e
introduced i n t o t h e twin-electrode, t h i n - l a y e r e l e c t r o l y t i c cell i n series c o n f i g u r a t i o n , i n which t h e u p s t r e a m e l e c t r o d e a n d downstream e l e c t r o d e a r e set a t p o t e n t i a l s ( v s . Ag/AgCl)
o f 0.60 V
and 0.20 V, r e s p e c t i v e l y . The c a t e c h o l a m i n e s are s e l e c t i v e l y d e t e c t e d by m o n i t o r i n g t h e r e - r e d u c t i o n
c u r r e n t a t t h e cathode.
Chromatograms of c a t e c h o l a m i n e s i n 1 0 0 p 1 o f human u r i n e i n j e c t e d d i r e c t l y w i t h o u t a n y p r e t r e a t m e n t i n t o t h e micro-HPLC s y s t e m were measured u s i n g SDVD. F i g u r e 9 shows examples o f t h e s e l e c t i v e d e t . e c t i o n of c a t e c h o l a m i n e s i n u r i n e from h e a l t h y humans. P a r t s A and B a r e , r e s p e c t i v e l y , t h e a n o d i c and c a t h o d i c chromatograms. Of p a r t i c u l a r i n t e r e s t i n p a r t s A are t h e p e a k s a p p e a r i n g a s t h e background o f NA and AD i n F i g . 9 a of LD i n F i g . 9b.
and a s t h a t
By r e c o r d i n g t h e r e - r e d u c t i o n c u r r e n t , t h e
i n t e r f e r e n c e s from t h e compounds r e s p o n s i b l e f o r t h e s e p e a k s c o u l d b e removed, a s shown i n p a r t s B i n F i g . 9
on t h e b a s i s o f
t h e i r electrochemical i r r e v e r s i b i l i t y . The c o l l e c t i o n e f f i c i e n c y , t h e magnitude of f r a c t i o n o f u p s t r e a m p r o d u c t s t h a t are c o n v e r t e d a t t h e downstream w o r k i n g e l e c t r o d e , i s i m p o r t a n t f o r s e l e c t i v e d e t e c t i o n i n HPLC. Goto e t
a l . o b t a i n e d t h e c o l l e c t i o n e f f i c i e n c i e s o f 0.68 t o 0.78 f o r c a t e c h o l a m i n e s a t a f l o w r a t e of 8.3 Vl/min
[26].
These v a l u e s
are m u c h h i g h e r t h a n t h o s e of 0.30 t o 0.31 o b t a i n e d i n SDVD a t a f l o w r a t e of 1 . 6 ml/min i n t h e o r d i n a r y HPLC [221.
The l i n e a r
320 b
Fig* 9. Selective detection Of catecholamines in human urine by SDVD i261- ( A ) Anodic response, (B) cathodic response. Sample: 100 1.11 of healthy human urine, applied potentials (vs. Ag/AgC1): anode 0.60 v, cathode 0.20 V, flow rate: 8 . 3 pl/min. dynamic range between peak current and amount injected was about l o 3 and the minimum detectable amount in the system was about 10 pg for catecholamines. (ii) Analysis of biogenic amine metabolites in human urine [ 2 4 ] . By means of CSV, the electrochemical behavior of biogenic amine metabolites was studied in the mobile phase of micro-HPLC, the B-R buffer of pH 3.6 containing 10 % methanol, 50 mM sodium perchlorate and 0.1 mM EDTA (disodium salt). FigUre 10 shows the cyclic semi-derivative voltammograms of 3 , 4-dihydroxyphenylacetic acid (DOPAC),homovanillic acid (HVA) and 5-hydroxyindole-3-acetic acid (5-HIAA) at a scan rate of 100 mV/sec. All the species
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-4 0
v
Fig. 1 0 . C y c l i c s e m i - d e r i v a t i v e voltammograms o f 1 . 0 mM DOPAC, HVA and 5-HIAA i n B-R b u f f e r o f pH 3 . 6 c o n t a i n i n g 1 0 % methanol, 50 mM sodium p e r c h l o r a t e and 0 . 1 mM EDTA a t a scan r a t e o f 1 0 0 mV/ sec [ 2 4 1 . i n v e s t i g a t e d showed o x i d a t i o n and r e - r e d u c t i o n p e a k s . I t i s i n t e r e s t i n g t h a t t h r e e successive o x i d a t i o n s t e p s w e r e observed f o r 5-HIAA, w h i l e one o x i d a t i o n s t e p was o s t e n s i b l y o b s e r v e d f o r DOPAC and HVA. On t h e o t h e r hand, two r e - r e d u c t i o n s t e p s
were observed f o r HVA and 5-HIAA, w h i l e o n l y one r e - r e d u c t i o n s t e p was o b s e r v e d f o r DOPAC. These f a c t s i n d i c a t e t h a t t h e e l e c t r o d e r e a c t i o n o f DOPAC is n e a r l y r e v e r s i b l e , w h i l e t h o s e o f HVA and 5-HIM are q u a s i - r e v e r s i b l e i n t h i s medium. For selective d e t e c t i o n o f b i o g e n i c amine m e t a b o l i t e s , t h e p o t e n t i a l s (vs. Ag/AgCI) o f 0.80 V and
-0.05
V w e r e chosen a s t h e s u i t a b l e p o t e n t i a l s o f t h e
upstream and downstream e l e c t r o d e , r e s p e c t i v e l y , from F i g . 1 0 .
322
11
11
Fig. 11. Block diagram of the micro-HPLC system with series dual voltammetric detector 1241. 1 = Micro-feeder, 2 = micro-syringe, 3 = three-way valve, 4 = mobile phase, 5 = sample injector (0.3 ~ 1 1 , 6 = guard column, 7 = separation column, 8 = twin-electrode cell, 9 = dual potentiostat, 10 = dual pen recorder, 11 = waste. The micro HPLC system used for analysis is shown schematically in Fig. 11. A micro-feeder, a micro-syringe and a three-way valve are used to feed the mobile phase. A sample injector with 0.3 p1 loop is used for sample injection. The guard column and micro-separation column are made by packing silica-ODS of 10 pm particle size in a teflon tube 2 0 mm x 0.5 mm I. D. and 165 x 0 . 5 mm I. D., respectively. The analytical procedures are as follows. Only 0.3 p 1 of supernatant of raw human urine is injected into the micro-HPLC system. The biogenic amine metabolites are separated at the flow rate of 8 . 3 pl/min. Typical chromatograms for the simultaneous determination of DOPAC, 5-HIAA and HVA in 0 . 3 pl of human urine directly injected without any pretreatment in the micro-HPLC system are shown in Fig. 12. Parts A and B are, respectively, the anodic and cathodic chromatograms. Peaks 3 , 4 and 5 are due to DOPAC, 5-HIM and HVA in urine, respectively. These were identified by the retention time and/or the peak current ratio of cathodic to anodic
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F i g . 1 2 . S e l e c t i v e d e t e c t i o n of b i o g e n i c amine m e t a b o l i t e s i n human u r i n e by SDVD 1 2 4 1 . (A) Anodic r e s p o n s e , (B) c a t o d i c r e s p o n s e . Peaks: 3 = DOPAC, 4 = 5-HIAAI 5 = HVA. Sample: 0.3 l.11 of h e a l t h y human u r i n e , p o t e n t i a l s ( v s . Ag/AgCl): anode 0.80 V , c a t h o d e -0.05 V , flow r a t e : 8 . 3 pl/min. by comparison w i t h t h e s t a n d a r d s . Of p a r t i c u l a r i n t e r e s t a r e t h e peaks of X I Y and z i n p a r t s A of F i g . 1 2 . BY r e c o r d i n g t h e c a t h o d i c r e s p o n s e , it was shown t h a t t h e r e were e s s e n t i a l l y no c a t h o d i c peaks c o r r e s p o n d i n g t o t h e a n o d i c peaks o f y and z, s u g g e s t i n g t h a t t h e compound o r compounds p r o d u c i n g t h e a n o d i c peaks a r e i r r e v e r s i b l y o x i d i z e d . S i n c e t h e main compounds r e s p o n s i b l e t o t h e a n o d i c peak o f x were a l s o i r r e v e r s i b l y o x i d i z e d , DOPAC c o u l d s e l e c t i v e l y be d e t e c t e d on t h e c a t h o d i c chromato-
grams. The d e t e c t i o n l i m i t s o f b i o g e n i c amine m e t a b o l i t e s by t h i s system were 1 0 pg f o r DOPAC, 20 pg f o r 5-HIAA and 20 pg f o r HVA,
324
respectively,and t h e range of l i n e a r i t y was about
lo3.
The
c o l l e c t i o n e f f i c i e n c i e s i n t h e system were found t o be 0 . 6 1 f o r 0.20 f o r 5-HIAA and 0.30 f o r HVA, r e s p e c t i v e l y , a t a flow r a t e of 8.3 pl/min. I t should be noted t h a t t h e s e values a r e
DOPAC,
much l a r g e r than those of 0.31 f o r DOPAC, 0.05 f o r 5 - H I M and 0.05 f o r HVA, r e s p e c t i v e l y , obtained i n SDVD a t t h e flow r a t e of 1 . 6 ml/min
[221.
3.3 Dual voltammetric d e t e c t o r of parallel-opposed type The parallel-opposed dual voltammetric d e t e c t o r (PODVD) i s analogous t o t h e photomultiplier tube and t h e product of t h e e l e c t r o d e r e a c t i o n a t one working e l e c t r o d e can d i f f u s e t o t h e o p p o s i t e working e l e c t r o d e where s t a r t i n g m a t e r i a l may be c r e a t e d . Goto e t a l . [31, 321 r e c e n t l y b u i l t a twin-electrode t h i n l a y e r e l e c t r o l y t i c c e l l i n parallel-opposed c o n f i g u r a t i o n f o r micro-HPLC a s shown i n Fig. 1 3 .
The t h i n - l a y e r c a v i t y i s
constructed of two fluorocarbon r e s i n blocks separated by a t e f l o n s h e e t 30
-
50 Um t h i c k and 2 mm wide. Two working e l e c t r o d e s a r e
made with g l a s s y carbon p l a t e s 1 c m long and 2 c m wide contained i n each block. The r e f e r e n c e e l e c t r o d e , s i l v e r / s i l v e r c h l o r i d e ,
i s held i n a c y l i n d r i c a l hole i n one of t h e blocks. A platinum tube serves both a s t h e counter e l e c t r o d e and t h e e x i t l i n e .
(i)Current a m p l i f i c a t i o n i n parallel-opposed dual voltammetric For slower flow r a t e s , c a t a l y t i c a m p l i f i c a t i o n of d e t e c t o r response f o r r e v e r s i b l e and q u a s i - r e v e r s i b l e a n a l y t e s may be achieved by r e c y c l i n g t h e redox couple between t h e two working e l e c t r o d e s i n PODVD. Consider a case where only oxidant is i n i t i a l l y i n t h e flow stream. The two working e l e c t r o d e s of PODVD a r e set a t p o t e n t i a l s where t h e oxidant i s s u f f i c i e n t l y reduced and i t s r e d u c t a n t i s s u f f i c i e n t l y oxidized, r e s p e c t i v e l y . The equations r e l a t i n g c a t h o d i c c u r r e n t (Ic)and anodic c u r r e n t (Ia)t o s y s t e m v a r i a b l e s a r e as follows. Ic/nFC = L D W / b + 0.2711 (1) d e t e c t i o n f o r micro HPLC 1321.
-
-Ia/nFC = L D W / b 0.07U (2) where n i s t h e e l e c t r o n t r a n s f e r number, F t h e Faraday's c o n s t a n t ,
C the bulk concentration of a n a l y t e , L t h e l e n g t h of e l e c t r o d e , W t h e width of channel o r e l e c t r o d e , b t h e h e i g h t of channel, LI t h e s o l v e n t volume flow rate and D t h e s o l u t e d i f f u s i o n c o e f f i c i e n t . The colLection e f f i c i e n c y (Q,) , t h e r a t i o of anodic c u r r e n t to cathodic c u r r e n t i n t h i s c a s e , i s represented as
326
A 6
m I
B
F i g . 13. C e l l f o r v o l t a m m e t r i c d e t e c t o r w i t h t w o working e l e c t r o d e s i n p a r a l l e l - o p p o s e d c o n f i g u r a t i o n [31] (A) S i d e view of c e l l , (B) f r o n t view o f s p a c e r . 1, 2 = Working e l e c t r o d e s ( g l a s s y c a r b o n ) , 3 = r e f e r e n c e e l e c t r o d e (Ag/AgCl), 4 = c o u n t e r e l e c t r o d e doubling a s o u t l e t (platinum t u b e ) , 5 = spacer ( t e f l o n s h e e t ) , 6 = m i c r o - s e p a r a t i o n column d o u b l i n g as i n l e t , 7 = s l o t .
.
-
0.34U/(LDW/b + 0.27U) The c u r r e n t a m p l i f i c a t i o n e f f i c i e n c y ( Q a ) and e f f e c t i v e Qc = 1
(3)
a m p l i f i c a t i o n e f f i c i e n c y ( Q e ) i n PODVD a r e , r e s p e c t i v e l y , d e f i n e d a s t h e r a t i o s o f c a t h o d i c and a n o d i c c u r r e n t t o c o u l o m e t r i c c u r r e n t i n t h i s case a s @a= 0 . 2 7
+
(LDW/b)( 1 / U )
(4)
and
Qe = - 0.07 + ( L D W / b ) (1/U) (5) The c u r r e n t a m p l i f i c a t i o n and c o l l e c t i o n e f f i c i e n c y i n t h e d e t e c t o r a t f l o w r a t e s o f 1 . 4 t o 1 1 . 2 Ul/min were i n v e s t i g a t e d by u s i n g f e r r i c y a n i d e a s a n a l y t e i n B-R b u f f e r o f pH 1.8 c o n t a i n i n g 1 mM HSA, 0 . 1 mM EDTA and 50 mM p e r c h l o r a t e as s o l v e n t . The r e s u l t s are shown i n F i g . 1 4 . The l i n e s r e p r e s e n t t h e o r e t i c a l e x p e c t a t i o n s of eqns. ( 3 - 5 ) , w h i l e t h e p o i n t s r e p r e s e n t e x p e r i m e n t a l d a t a . The c o l l e c t i o n e f f i c i e n c i e s from 0.98 t o 0.84 were o b t a i n e d i n t h e flow rate r a n g e from 1 . 4 t o 1 1 . 2 pl/min, r e s p e c t i v e l y . I t i s i n t e r e s t i n g t h a t t h e s e v a l u e s are m u c h h i g h e r t h a n t h o s e (4J.37 o b t a i n e d i n SDVD a t a f l o w r a t e o f 1 ml/min i n t h e o r d i n a r y HPLC
326
F i g . 1 4 . Dependence of c u r r e n t a m p l i f i c a t i o n e f f i c i e n c i e s and c o l l e c t i o n e f f i c i e n c y i n PODVD on flow r a t e [321. The l i n e s r e p r e s e n t t h e o r e t i c a l e x p e c t a t i o n s of e q n s . ( 3 - 5 ) . The p o i n t s r e p r e s e n t e x p e r i m e n t a l d a t a . Sample: 100 V M f e r r i c y a n i d e i n B-R b u f f e r of pH 1 . 8 , c h a n n e l h e i g h t : 3 0 um. (1) Qc, ( 2 ) Oa, (3)
[ 2 0 1 . The e f f e c t i v e a m p l i f i c a t i o n e f f i c i e n c y v a r i e d c o n s i d e r a b l y ,
from 2.4
t o 1 9 . 5 , when t h e flow r a t e changed from 1 1 . 2 t o 1 . 4 p l /
min, a s t h e t h e o r y p r e d i c t s . I t s h o u l d be n o t e d t h a t t h o s e v a l u e s
are mch larger than those ( 0 . 7 - 0.8) o b t a i n e d f o r c a t e c h o l a m i n e s i n SDVD a t a flow rate o f 8 . 3 pl/min i n micro HPLC 1 2 4 1 . (ii)Catecholamine
a n a l y s i s i n human serum [311. The
same
b a s i c micro-HPLC system.as t h a t shown i n F i g . 8,was used f o r d e t e r m i n a t i o n of c a t e c h o l a m i n e s i n human serum. The d i f f e r e n c e s a r e a s f o l l o w s : t h e 200 ul l o o p i s s e t and PODVD is a p p l i e d a s d e t e c t o r . The a n a l y t i c a l p r o c e d u r e s a r e a s f o l l o w s . The human
321
blood sample i s drawn i n a serum s e p a r a t i o n t u b e by v e n i p u n c t u r e . A f t e r i n c u b a t i o n a t room t e m p e r a t u r e f o r a b o u t 1 0 min t o i n d u c e c o a g u l a t i o n , t h e t u b e i s c e n t r i f u g e d , s e p a r a t i n g t h e serum from
t h e blood c e l l s . Then, 500 1.11 of t h e raw human serum is removed t o a t e s t t u b e , t o which had been added 0.5 mg of s o l i d EDTA (disodium s a l t ) , 0 . 5 mg of s o l i d sodium hydrogen s u l f i t e , and 1 0 ~1 o f 25 pg/ ?.113 , 4-dihydroxybenzylamine hydrobromide (DHBA) s t a n d a r d
s o l u t i o n . The t e s t t u b e
is
sealed
shaken f o r 1 min by hand
and a l a r g e p o r t i o n o f t h e mixed sample s o l u t i o n i s t r a n s f e r r e d t o a n u l t r a f i l t r a t i o n c e l l . The u l t r a f i l t r a t i o n i s c a r r i e d o u t by s t i r r i n g t h e s o l u t i o n w i t h a magnetic s t i r r e r under a n i t r o g e n p r e s s u r e of 2 . 5 kg/cm2 i n a n i c e b a t h . The f i l t r a t e d serum i s t a k e n w i t h a m i c r o - s y r i n g e and i n j e c t e d i n t o t h e sample l o o p (200 1.11) of t h e sample i n j e c t o r . The o t h e r p r o c e d u r e s a r e a l m o s t t h e same as in
u r i n a r y catecholamine
a n a l y s i s . The d i f f e r e n c e s are
a s f o l l o w s : t h e t i m e f o r sample e n r i c h m e n t and t h e t i m e f o r precolumn washing a r e b o t h 1 0 min; t h e mobile p h a s e used i s t h e B-R
b u f f e r a t pH 8 . 7 c o n t a i n i n g 2 mM HSA, 0 . 1 mM EDTA and 50 mM
p e r c h l o r a t e , t h e separated catecholamines a r e introduced i n t o t h e t w i n - e l e c t r o d e c e l l i n p a r a l l e l - o p p o s e d c o n f i g u r a t i o n , i n which t h e lower and upper working e l e c t r o d e a r e s e t a t t h e p o t e n t i a l s ( v s . Ag/AgCl) o f 0.60 V and 0 . 2 0 V, r e s p e c t i v e l y , and t h e c a t e c h o l a m i n e s are s e l e c t i v e l y d e t e c t e d w i t h h i g h s e n s i t i v i t y by m o n i t o r i n g t h e r e - r e d u c t i o n c u r r e n t a t t h e c a t h o d e and d e t e r m i n e d by comparing w i t h t h e r e s p o n s e o f i n t e r n a l s t a n d a r d . F i g u r e 1 5 shows t y p i c a l chromatograms o f c a t e c h o l a m i n e s i n human serum a t a mobile phase f l o w r a t e o f 8 . 3 pl/min o b t a i n e d u s i n g t h e proposed p r o c e d u r e s and t h e micro-HPLC system w i t h precolumn and PODVD. P a r t s A and B a r e , r e s p e c t i v e l y , t h e a n o d i c and c a t h o d i c chromatograms. Of p a r t i c u l a r i n t e r e s t i n p a r t s A
are
t h e peaks a p p e a r i n g , r e s p e c t i v e l y , a s t h e s h o u l d e r o f NA i n F i g . 15b and t h e background of AD i n F i g . 1 5 a . By r e c o r d i n g t h e c a t h o d i c c u r r e n t , t h e i n t e r f e r e n c e s from t h e compounds r e s p o n s i b l e f o r t h e s e peaks c o u l d be removed, as shown i n p a r t s B, on t h e b a s i s o f t h e i r e l e c t r o c h e m i c a l i r r e v e r s i b i l i t y . Both t h e a n o d i c and c a t h o d i c peak h e i g h t r a t i o s o f NA and AD t o DHBA were l i n e a r w i t h e a c h amount of c a t e c h o l a m i n e i n j e c t e d , w i t h a good c o r r e l a t i o n . The l i n e a r dynamic r a n g e w a s a b o u t l o 3 and t h e minimum d e t e c t a b l e amount i n t h e proposed system was a b o u t 3 pg f o r NA and AD a t t h e f l o w r a t e o f 8 . 3 pl/min.
T h i s d e t e c t i o n l i m i t by PODVD i s a b o u t
328
a
?
z
1 (min)
Fig. 15. S e l e c t i v e and s e n s i t i v e d e t e c t i o n of catecholamines i n human serum by PODVD 1311. (A) Anodic response, (B) c a t h o d i c response. Sample: 200 p 1 of human serum spiked with 1 0 0 pg of DHBA, p o t e n t i a l s (vs. Ag/AgCl): anode 0.60 V, cathode 0.20 v, flow rate: 8.3 p l / m i n . t h r e e times b e t t e r than t h a t i n [25] given by SDVD. The chromatographic peak r a t i o s of c a t h o d i c t o anodic, c o l l e c t i o n e f f i c i e n c i e s , f o r a standard s o l u t i o n were 0 . 9 6 , 0.79, 0.68 f o r NA, 0.95, 0.81, 0.66 f o r AD and 0.97, 0.86, 0.70 f o r DHBA a t t h e flow rates (pl/min) of 1.4, 5.6, 1 1 . 2 , r e s p e c t i v e l y . The e l e c t r o l y t i c e f f i c i e n c i e s ( % ) obtained from t h e c a t h o d i c peak a r e a s i n t h i s micro-HPLC system were 1132 f o r NA, 1000 f o r AD and
1324 f o r DHBA a t t h e flow r a t e of 1.4 pl/min, by assuming a = 2. Figure 16 shows t h e chromatograms of catecholamines i n u l t r a f i l t r a t e d human serum (200 p l ) spiked with 100 pg DHBA.directly
329 a
t (hr)
b
t m'n)
Fig. 16. Chromatograms of catecholamines in healthy human serum by PODVD at very slow flow-rates [32]. (A) Anodic response, (B) cathodic response. Peaks: 1 = NA, 2 = AD, 3 = DHBA (internal standard). Sample: 2 0 0 p1 of human serum spiked with 100 pg DHBA, flow rates (pl/min) : (a) 1.4, (b) 5.6. injected to the micro-HPLC system at lower flow-rates. It is clear that the selective and sensitive detection of catecholamines on the basis of electrochemical reversibility can be performed by using the cathodic chromatograms from PODVD at very low flowrates. CONCLUSION Compared with ultraviolet and fluorescence detectors of the same cell volume, voltammetric detectors for micro-HPLC or capillary LC have a much greater detection sensitivity, this being 4
330
true for biogenic mines and their metabolites. Another merit of such detectors is that several substances can be detected selectively by constructing a thin-layer electrolytic cell containing two or more working electrodes and setting the respective electrodes at selected potentials. The SDVD with anode and cathode is a powerful tool for the selective detection of reversible and/or quasi-reversible species for micro-HPLC or capillary LC, because the collection efficiency increases with decreasing the flow-rate of mobile phase. Moreover, the PODVD with anode and cathode can provide an enhancement in sensitivity by recycling oxidation and re-reduction between the two working electrodes at slow flow rates of mobile phase. Thus, the PODVD is the most advantageous type of detector for reversible and/or quasi-reversible species in micro HPLC and capillary LC. The constituent of biological fluids and tissues include many electroactive substances Voltammetric detectors are likely to findmany applicationsin the field of clinical analysis. Typical electroactive species are as follows; phenols, methoxyphenols, catechols, catecholamines, hydroxycoumarins, quinones, estrogens, tocophenols, morphine derivatives, anilines, sulfonamides, indoles, phenotiazines, purines, ascorbic acid, thiols, cyanide, aldehydes, uric acid, nitro compounds, aromatic amines,etc.
.
REFERENCES 1 D. Ishii, K. AsaL, K. Hibi, T. Jonokuchi and M. Nagaya, J. Chromatogr., 144 (1977) 157. 2 D. Ishii and T. Takeuchi, J. Chromatogr. Sci., 18 (1980) 462. 3 M. V. Novotny, J. Chromatogr. Sci., 18 (1980) 473. 4 Y. Hirata and M. V. Novotny, J. Chromatogr., 186 (1979) 521. 5 P. T. Kissinger, Anal. Chem., 49 (1977) 447A. 6 K. Brunt, Pharm. Weekbl., 113 (1978) 689. 7 R. J. Rucki, Talanta, 27 (1980) 147. 8 R. E. Majors, H. G. Barth and C. H. Lochmuller, Anal. Chem., 54 (1982) 323R. 9 M. Goto, Y. Koyanagi and D. Ishii, J. Chromatogr., 208 (1981) 261. 10 Y. Hirata, P. T. Lin, M. V. Novotny and R. M. Wightman, J. Chromatogr., 181 (1980) 287. 11 C. L. Blank, J. Chromatogr., 117 (1976) 35. 12 G. W. Schieffer, Anal. Chem., 52 (1980) 1994. 13 W. A. MacCrehan and R. A. Durst, Anal. Chem., 53 (1981) 1700. 14 D. A. Roston and P. T. Kissinger, Anal. Chem., 53 (1981) 1965. 15 K. B. Bratin and P. T. Kissinger, J. Liq. Chromatogr. Suppl., 4 (1981) 321. 16 D. A. Roston and P. T. Kissinger, Anal. Chem., 54 (1982) 429. 17 T. Kurahashi, The Yanaco News, 11 (1982) 8. 18 R. E. Shoup and G. S. Mayer, Anal. Chem., 54 (1982) 1164. 19 S. G. Weber and W. C. Purdy, Anal. Chem., 54 (1982) 1757.
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20 21 22 23 24 25 26 27 28 29 30 31 32
D. A. Roston and P. T. Kissinger, Anal. Chem., 5 4 (1982) 1798. K. Inoue, T. Otake and K. Kyogoku, Yakugaku Zasshi, 102 (1982) 1041. G. S. Mayer and R. E. Shoup, J. Chromatogr., 255 (1983) 533. L. A. Allison and R. E. Shoup, Anal. Chem., 55 (1983) 8. M. Goto, E. Sakurai and D. Ishii, J. Liq. Chromatogr. Suppl., 6 (1983) 1907. M. Goto, T. Nakamura and D. Ishii, J. Chromatogr., 226 (1981) 33. M. Goto, E. Sakurai and D. Ishii, J. Chromatogr., 238 (1982) 357. M. Goto and D. Ishii, J. Electroanal. Chem., 61 (1975) 361. P. Dalrymple-Alford, M. Goto and K. B. Oldham, J. Electroanal. Chem., 85 (1977) 1. P. Dalrymple-Alford, M. Goto and K. B. Oldham, Anal. Chem., 49 (1977) 1390. M. Goto, M. Kato and D. Ishii, Anal. Chim. Acta, 126 (1981) 95. M. Goto, G . Zou and D. Ishii, J. Chromatogr., 275 (1983) 271. M. Goto, G . Zou and D. Ishii, J. Chromatogr., 268 (1983) 157.
MINIATURIZED VOLTAMMETRIC DETECTORS MASASHI GOT0 Research Center for Resource and Energy Conservation, Nagoya University, Chikusa-ku, Nagoya, Japan INTRODUCTION Micro high-performance liquid chromatography (micro HPLC) [l] using packed columns of I.D. less than 1 mm and packed or opentubular capillary chromatography (capillary LC) [ 2 , 31 are attractive in that the consumption of the mobile phase and sample is much less than that in conventional HPLC. Flow rates of the mobile phase employed in micro HPLC and capillary LC are typically 0.1 - 30 pl/min. These extremely slow flow rates require small volume detectors and these have been designed for spectrophotometric detection by W [l] and fluorescence methods [ 4 ] . The usefulness of electrochemical detectors has already been recognized [5 - 8 1 . Numerous voltammetric detectors have been described for HPLC. These can be broadly divided into coulometric detectors that use a large surface area working electrode, and amperometric detectors that use a small surface area working electrode. These differ in their methods of detection and estimation, in that the former measures the quantity of electricity flowing when 100% of the target substance is electrolyzed at constant potential, whereas the the latter measures the current flowing when just part of the target substance is electrolyzed at constant potential. Of the various voltammetric detectors utilized for HPLC, the voltammetric detector employing a thin-layer electrolytic cell is considered to be the most suitable for micro HPLC and capillary LC, as the cell volume is easily reduced. The miniaturized voltammetric detectors suitable for micro HPLC and capillary LC and their general features are reviewed here. 1
VOLTAMMETRIC DETECTORS WITH A SINGLE WORKING ELECTRODE Goto et al. [ 9 ] and Hirata et al. [lo] independently designed sub-microliter voltammetric detectors having one working electrode for use in micro HPLC and capillary LC, respectively. Figure 1 shows the cell structure used in the single working electrode detector built by Goto et al. [9]. The thin-layer cavity is constructed of two fluorocarbon resin blocks separated 2
310
-
-
by a t e f l o n s h e e t 2 0 45 pm t h i c k and 0.5 2 nun wide. A working e l e c t r o d e i s made w i t h a 3-mm d i a m e t e r g l a s s y c a r b o n d i s k c o n t a i n e d i n one of t h e b l o c k s . The r e f e r e n c e e l e c t r o d e , s i l v e r / s i l v e r chloride electrode, is held i n a c y l i n d r i c a l hole i n t h e o t h e r block. A s t a i n l e s s - s t e e l t u b e s e r v e s b o t h a s t h e c o u n t e r e l e c t r o d e and t h e e x i t l i n e . The volume i n t h e s e c e l l s i s a b o u t 0.06 - 0.3 p l . The c e l l was u t i l i z e d f o r t h e
B
M
F i g . 1. Voltammetric d e t e c t o r c e l l w i t h s i n g l e working e l e c t r o d e by Goto e t a l . [ 9 1 . (A) S i d e view o f c e l l , (B) f r o n t view o f s p a c e r . 1 = Working e l e c t r o d e ( g l a s s y c a r b o n ) , 2 = r e f e r e n c e e l e c t r o d e (Ag/AgCl), 3 = c o u n t e r e l e c t r o d e d o u b l i n g as o u t l e t (stainless-steel t u b e ) , 4 = spacer ( t e f l o n s h e e t ) , 5 = micro s e p a r a t i o n column d o u b l i n g a s i n l e t , 6 = s l o t . d e t e r m i n a t i o n o f aminophenol isomers s e p a r a t e d on a micro-column (147 x 0.5 mm I. D . )
packed w i t h silica-ODS o f 1 0 pm p a r t i c l e
chromatograms o f t h e m i x t u r e of s i z e . F i g u r e 2 shows aminophenol isomers a t d i f f e r e n t a p p l i e d p o t e n t i a l s . When t h e a p p l i e d p o t e n t i a l w a s 1.00 V (vs. Ag/AgCl), a l l t h r e e i s o m e r s s e p a r a t e d on t h e micro-column w e r e d e t e c t e d . A t lower a p p l i e d p o t e n t i a l s of 0.75 V o r 0.50 V, o n l y p- and o-aminophenol c o u l d be s e l e c t i v e l y d e t e c t e d . Three i s o m e r s w e r e c o m p l e t e l y e l u t e d w i t h i n 1 0 min a t a f l o w r a t e o f 8 . 3 pl/min u s i n g 0.1 N p e r c h l o r i c a c i d as t h e mobile phase. The l i n e a r dynamic r a n g e between peak c u r r e n t and c o n c e n t r a t i o n w a s a b o u t 1 0 ' (10 pg - 1 0 ng) and t h e
311
2
C
Fig. 2 . applied Applied v, flow
Typical chromatograms of aminophenol isomers at different potentials [9]. Peaks: 1 = p-, 2 = m-, 3 = o-aminophenol. potential (vs. Ag/AgCl): (A) 1.00 V, (B) 0.75 V, (C) 0.50 rate: 8 . 3 pl/min.
minimum detectable amount was about 10 pg for three isomers. The electrolytic efficiency in the cell under the experimental conditions of A in Fig. 2 was quite high, about 60%,and sensitivity was found to be about ten times better than in conventional HPLC. Figure 3 shows the cell structure used in the single working electrode detector built by Hirata et al. [lo]. A pressureannealed pyrolytic graphite is used as the working electrode, a saturated calomel as the reference electrode and a platinum wire as the counter-electrode. The cell is constructed by sandwiching a 50-pm thick polyethylene sheet, containing a channel 1 mm wide and 3 mm long, between a Lucite plate and the graphite electrode plate and designed to hold a volume of 0.15 p l . The cell was applied to the determination of tyrosine and tryptophan metabolites in urine with a 60 m microcapillary column packed with 30 pm porous silica particles. The experiments were conducted at a low flow rate of 1 pl/min using 0.2 M acetate buffer (pH 4 . 0 ) as mobile phase and the metabolites were detected by measuring oxidation current with the potential of the working
312
2 5
I
Fig. 3 . Cell for voltammetric detector with single working electrode by Hirata et al. [lo]. 1 = Working electrode (graphite), 2 = reference electrode (sat. Hg/Hg2C12), 3 = counter electrode (platinum wire), 4 = spacer (polyethylene sheet), 5 = microcapillary separation column, 6 = conductor (copper plate), 7 = teflon tube, 8 = inlet, 9 = outlet. electrode set at 1.00 V vs. sat. Hg/Hg2C12. Figure 4 shows the chromatogram of p-hydroxyphenylacetic acid (PHPAA), 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA) and vanillic acid (VA) in human urine. Because of the reduced dimensions of the cell and the lower flow rates compared to conventional HPLC, the detector had a detection limit approximately ten times higher than a large volume cell. For the compounds investigated here, the electrochemical method was far more sensitive than fluorescence or UV detection. VOLTAMMETRIC DETECTORS WITH TWO WORKING ELECTRODES A new approach to electrochemical detection involves the use of two working electrodes, operated simultaneously at different potentials. Several dual voltammetric detectors for HPLC have recently been developed, which provide for enhanced performance [11 - 231. They can basically be classified into the three types of configuration of the two working electrodes with respect to the flow axis, as shown in Fig. 5 [241. 3
313
I
1
2
I
4
6
I
t (hrl
8
I0
I I2
Fig. 4 . Typical chromatogram of metabolites of tyrosine and tryptophan in human urine [lo]. Peaks: 2 = PHPAA, 3 = 5-HIAA, 4 = HVA, 5 = VA. Applied potential: 1.0 V vs. sat. Hg/Hg2C12, flow rate: 1 pl/min. In the "parallel-adjacent" configuration, the working electrodes are placed adjacent to each other on one side of the rectangular thin-layer channel. In the "series" configuration, the working electrodes are positioned along the flow stream on one side of the channel. In the "parallel-opposed" configuration, the working electrodes are placed opposed to one another on both sides of the channel. All voltammetric detectors with two working electrodes (dual voltammetric detectors) can simultaneously provide two chromatograms of both oxidations or both reductions or oxidation and reduction. Dual voltammetric detector of parallel-adjacent type The parallel-adjacent dual voltammetric detector (PADVD) is analogous to the dual wavelength UV absorbance detector, and can provide useful qualitative information from peak current ratios at different potentials. PADVD has not been used for micro HPLC or capillary LC yet, because the detector of this type requires a relatively large cell volume, compared with those of other types. 3.1
314
B
A
1
1-
-
i ]
Parallel- Adjacent
Series
Parallel-Opposed
F i g . 5. Three t y p e s i n c o n f i g u r a t i o n of d u a l v o l t a m m e t r i c d e t e c t o r [ 2 4 ] . (A) S i d e view, ( B ) f r o n t view. W 1 and W2 r e p r e s e n t t h e two working e l e c t r o d e s . The a r r o w s show t h e d i r e c t i o n o f f l o w .
3.2
Dual v o l t a m m e t r i c d e t e c t o r i n series t y p e
The series d u a l v o l t a m m e t r i c d e t e c t o r (SDVD) i s a n a l o g o u s to t h e f l u o r e s c e n c e d e t e c t o r , and t h e p r o d u c t of e l e c t r o d e r e a c t i o n a t t h e upstream working e l e c t r o d e is d e t e c t e d a t downstream working e l e c t r o d e . Goto e t a l . [26, 271 b u i l t a s u b - m i c r o l i t e r t h i n - l a y e r c e l l c o n t a i n i n g two g l a s s y c a r b o n e l e c t r o d e s i n series, as shown i n F i g . 6 t o s e r v e f o r m i c r o HPLC. The t h i n - l a y e r c a v i t y is c o n s t r u c t e d of two f l u o r o c a r b o n r e s i n b l o c k s s e p a r a t e d by a t e f l o n s h e e t 50 pm t h i c k and 2 mm wide. Two working e l e c t r o d e s are made w i t h g l a s s y c a r b o n d i s k s , 3 mm i n d i a m e t e ' , c o n t a i n e d i n one of t h e b l o c k s . The r e f e r e n c e e l e c t r o d e , s i l v e r /
31s
B
Fig. 6. Cell for voltammetric detector with two working electrodes in series [25]. (A) Side view of cell, ( B ) front view of spacer. 1, 2 = Working electrodes (glassy carbon), 3 = reference electrode (Ag/AgCl), 4 = counter electrode doubling as outlet (platinum tube), 5 = spacer (teflon sheet), 6 = micro separation column doubling as inlet, 7 = slot. silver chloride, is held in a cylindrical hole in the other block. A platinum tube serves both as the counter electrode and the exit line. The cell was successfully used for the selective detection of catecholamines, indoleamine and their metabolites in human urine based on their electrochemical reversibility. The principle of selective detection by SDVD is as follows. The upstream and downstream electrodes are set at potentials where the reductant (analyte) is oxidized and its oxidant is reduced, respectively. The reductant, of a reversible or quasi-reversible species, is oxidized at the anode and the product of this electrode reaction is re-reduced at the cathode, whereas the reductant of an irreversible species is not re-reduced at the cathode. Therefore, the reversible and/or quasi-reversible species present in irreversible species can be detected selectively by measuring the re-reducing current. The reversible species can, moreover, be selectively detected from quasi-reversible species by adequately
316
selecting both electrode potentials. Care is required in selecting the applied potentials of such voltammetric detectors. To maximize the analytical response and selectivity and minimize background interference, the applied potentials should be held at the minimum potential for oxidation or the maximum potential for reduction, at which currents reach the limiting values for the analyte under investigation. The optimum applied potentials can be determined directly by hydrodynamic voltammetry (HDV), in which peak current is measured against applied potential, point by point, for the analyte injected into the stream flowing through the voltammetric detector under the experimental conditions of micro-HPLC. However, HDV measurements require several hours for completion. Alternatively, the approximate applied potentials for voltammetric detectors can be established,indirectly, much more rapidly by cyclic voltammetry (CV), which may be carried out on a stationary solution (without stirring) in a separate voltammetric cell [261. Cyclic semidifferential voltammetry (CSV) [27 301 provides higher sensitivity and better resolution than ordinary CV. The technique measures the semi-derivative of current with respect to time ( e ) versus applied potential ( E ) under the same experimental conditions as in CV. The instrument for CSV (model VMA-010) is now commercially available from Yanagimoto Co. Kyoto, Japan. A glassy carbon disk, 3 mm in diameter, is used as the working electrode. A
-
silver/silver chloride electrode and a platinum wire are used for the reference and the counter electrode, respectively. (i) Catecholamine analysis in human urine [25, 261. The electrochemical behavior of noradrenaline (NA), adrenaline (AD), dopamine (DA) and 1-dopa (LD) on the glassy carbon electrode in the mobile phase used in micro HPLC, the Britton-Robinson (B-R) buffer of pH 1.8 containing 0.5 mM sodium 1-heptane sulphonate (HSA) as the ion-pair reagent and 0.1 mM EDTA (disodium salt) as the masking reagent for ion (11) ion, were studied by CSV [261. Figure 7 shows the cyclic semi-derivative voltammograms of catecholamines at a scan rate of 4 0 mV/sec. Each species investigated showed only one oxidation and one re-reduction peak. Theoretically, the anodic peak potential ( E ) in the cyclic e Pa versus E curves coincides with the cathodic peak potential ( EPC) for the reversible system [28]. It is clear that the catechol
317
-LI
E
f -
-
L-
u
0-4-
ae
05 E( V
02
VS
Ag1AgCI )
-
Fig. 7. Cyclic semi-derivative voltammograms of 0.1 mM catecholamines in B-R pH 1.8 buffer containing 0.5 mM HSA and 0.1 mM EDTA at a scan rate of 4 0 mV/sec [ 2 6 ] . form is reversibly oxidized to the quinone form and the electrode reactions of all four catecholamines are reversible in this medium. For selective detection of catecholamines by SDVD, the upstream electrode (anode) and the downstream electrode (cathode) of the twin-electrode cell should be set at the end potentials (Eea and Eec), at which the oxidation and re-reduction wave in the cyclic e versus E curve are complete, respectively. Therefore, the potentials (vs. Ag/AgCl) of 0.60 V and 0.20 V were, respectively, chosen a s the optimum applied potentials of anode and cathode for selective detection of catecholamines in other electroactive species from Fig. 7. The micro-HPLC system with a miniaturized precolm and dual voltamnetric
318 4
i 4.
Fig. 8 . Block diagram of micro HPLC system with a miniaturized preconcentration column and dual voltammetric detector [25, 311. 1 = Micro-feeder, 2 = micro-syringe, 3 = three-way v,alve, 4 = sample injector, 5 = mobile phase, 6 = buffer solution, 7 = water, 8 = six-way valve, 9 = mixing joint, 10 = micro-precolumn, 11 = micro-separation column, 12 = twin-electrode cell, 13 = dual potentiostat, 14 = dual-pen recorder. detector,as shown in Fig. 8, was used for direct injection analysis of catecholamines in human urine. Three micro-feeders, micro-syringes and three-way valves are used to feed the mobile phase, buffersolution and water. A sample injector and six-way valve are used for sample injection and alternative connection of the precolumn with the sample enrichment system and chromatographic system, respectively. A dual potentiostat is employed to control independently the potentials of the two working electrodes and to measure the currents. The anodic and cathodic chromatograms are simultaneously recorded on a dual pen recorder. The micro-separation column for analysis is filled with silica-ODS of 10 Vrn particle size in a teflon tube 150 x 0.5 mm I. D. The micro-precolumn for enrichment is made by packing alumina of 30 um particle size in a teflon tube 20 x 0.5 mm I. D. The mobile phase for analysis is B-R buffer of pH 1.8 containing 0.5 mM HSA and 0.1 mM EDTA. The buffer solution for pretreatment of the micro-precolumn and pH adjustment of the sample is 1 M Tris
319 b u f f e r , PH 8.7,
c o n t a i n i n g 0.25 % EDTA ( d i s o d i u m s a l t ) and 0 . 5 %
sodium hydrogen s u l f i t e f o r s t a b i l i z i n g c a t e c h o l a m i n e s . The a n a l y t i c a l p r o c e d u r e s are as f o l l o w s .
Raw
human u r i n e
i s t a k e n w i t h a m i c r o - s y r i n g e and i n j e c t e d i n t o t h e sample l o o p (100 p l ) o f t h e sample i n j e c t o r t h r o u g h a s o l u t i o n f i l t e r . The sample i s d e l i v e r e d by water a t a f l o w r a t e o f 33 pl/min and mixed w i t h T r i s b u f f e r (pH 8.7) a t t h e same f l o w r a t e i n t h e mixing j o i n t t o a d j u s t t h e sample t o pH 8.6.
The sample i s
injected into the precolumn f o r 1 5 min f o r e n r i c h m e n t w i t h a mixed f l o w of w a t e r and b u f f e r s o l u t i o n and t h e n t h e precolumn i s washed f o r an additional 15 min w i t h a f l o w c o n s i s t i n g of w a t e r a l o n e , w i t h o u t any b u f f e r s o l u t i o n .
By s w i t c h i n g
t h e six-way v a l v e , t h e m o b i l e p h a s e i s i n t r o d u c e d i n t o t h e s e p a r a t i o n column t h r o u g h t h e micro precolumn a t a f l o w r a t e o f 8.3 pl/min.
I n t h i s p r o c e d u r e , t h e a d s o r b e d c a t e c h o l a m i n e s are
e l u t e d from t h e the
precolumn and s i m u l t a n e o u s l y s e p a r a t e d by
s e p a r a t i o n column. The s e p a r a t e d c a t e c h o l a m i n e s a r e
introduced i n t o t h e twin-electrode, t h i n - l a y e r e l e c t r o l y t i c cell i n series c o n f i g u r a t i o n , i n which t h e u p s t r e a m e l e c t r o d e a n d downstream e l e c t r o d e a r e set a t p o t e n t i a l s ( v s . Ag/AgCl)
o f 0.60 V
and 0.20 V, r e s p e c t i v e l y . The c a t e c h o l a m i n e s are s e l e c t i v e l y d e t e c t e d by m o n i t o r i n g t h e r e - r e d u c t i o n
c u r r e n t a t t h e cathode.
Chromatograms of c a t e c h o l a m i n e s i n 1 0 0 p 1 o f human u r i n e i n j e c t e d d i r e c t l y w i t h o u t a n y p r e t r e a t m e n t i n t o t h e micro-HPLC s y s t e m were measured u s i n g SDVD. F i g u r e 9 shows examples o f t h e s e l e c t i v e d e t . e c t i o n of c a t e c h o l a m i n e s i n u r i n e from h e a l t h y humans. P a r t s A and B a r e , r e s p e c t i v e l y , t h e a n o d i c and c a t h o d i c chromatograms. Of p a r t i c u l a r i n t e r e s t i n p a r t s A are t h e p e a k s a p p e a r i n g a s t h e background o f NA and AD i n F i g . 9 a of LD i n F i g . 9b.
and a s t h a t
By r e c o r d i n g t h e r e - r e d u c t i o n c u r r e n t , t h e
i n t e r f e r e n c e s from t h e compounds r e s p o n s i b l e f o r t h e s e p e a k s c o u l d b e removed, a s shown i n p a r t s B i n F i g . 9
on t h e b a s i s o f
t h e i r electrochemical i r r e v e r s i b i l i t y . The c o l l e c t i o n e f f i c i e n c y , t h e magnitude of f r a c t i o n o f u p s t r e a m p r o d u c t s t h a t are c o n v e r t e d a t t h e downstream w o r k i n g e l e c t r o d e , i s i m p o r t a n t f o r s e l e c t i v e d e t e c t i o n i n HPLC. Goto e t
a l . o b t a i n e d t h e c o l l e c t i o n e f f i c i e n c i e s o f 0.68 t o 0.78 f o r c a t e c h o l a m i n e s a t a f l o w r a t e of 8.3 Vl/min
[26].
These v a l u e s
are m u c h h i g h e r t h a n t h o s e of 0.30 t o 0.31 o b t a i n e d i n SDVD a t a f l o w r a t e of 1 . 6 ml/min i n t h e o r d i n a r y HPLC [221.
The l i n e a r
320 b
Fig* 9. Selective detection Of catecholamines in human urine by SDVD i261- ( A ) Anodic response, (B) cathodic response. Sample: 100 1.11 of healthy human urine, applied potentials (vs. Ag/AgC1): anode 0.60 v, cathode 0.20 V, flow rate: 8 . 3 pl/min. dynamic range between peak current and amount injected was about l o 3 and the minimum detectable amount in the system was about 10 pg for catecholamines. (ii) Analysis of biogenic amine metabolites in human urine [ 2 4 ] . By means of CSV, the electrochemical behavior of biogenic amine metabolites was studied in the mobile phase of micro-HPLC, the B-R buffer of pH 3.6 containing 10 % methanol, 50 mM sodium perchlorate and 0.1 mM EDTA (disodium salt). FigUre 10 shows the cyclic semi-derivative voltammograms of 3 , 4-dihydroxyphenylacetic acid (DOPAC),homovanillic acid (HVA) and 5-hydroxyindole-3-acetic acid (5-HIAA) at a scan rate of 100 mV/sec. All the species
321
-j./
-4 0
v
Fig. 1 0 . C y c l i c s e m i - d e r i v a t i v e voltammograms o f 1 . 0 mM DOPAC, HVA and 5-HIAA i n B-R b u f f e r o f pH 3 . 6 c o n t a i n i n g 1 0 % methanol, 50 mM sodium p e r c h l o r a t e and 0 . 1 mM EDTA a t a scan r a t e o f 1 0 0 mV/ sec [ 2 4 1 . i n v e s t i g a t e d showed o x i d a t i o n and r e - r e d u c t i o n p e a k s . I t i s i n t e r e s t i n g t h a t t h r e e successive o x i d a t i o n s t e p s w e r e observed f o r 5-HIAA, w h i l e one o x i d a t i o n s t e p was o s t e n s i b l y o b s e r v e d f o r DOPAC and HVA. On t h e o t h e r hand, two r e - r e d u c t i o n s t e p s
were observed f o r HVA and 5-HIAA, w h i l e o n l y one r e - r e d u c t i o n s t e p was o b s e r v e d f o r DOPAC. These f a c t s i n d i c a t e t h a t t h e e l e c t r o d e r e a c t i o n o f DOPAC is n e a r l y r e v e r s i b l e , w h i l e t h o s e o f HVA and 5-HIM are q u a s i - r e v e r s i b l e i n t h i s medium. For selective d e t e c t i o n o f b i o g e n i c amine m e t a b o l i t e s , t h e p o t e n t i a l s (vs. Ag/AgCI) o f 0.80 V and
-0.05
V w e r e chosen a s t h e s u i t a b l e p o t e n t i a l s o f t h e
upstream and downstream e l e c t r o d e , r e s p e c t i v e l y , from F i g . 1 0 .
322
11
11
Fig. 11. Block diagram of the micro-HPLC system with series dual voltammetric detector 1241. 1 = Micro-feeder, 2 = micro-syringe, 3 = three-way valve, 4 = mobile phase, 5 = sample injector (0.3 ~ 1 1 , 6 = guard column, 7 = separation column, 8 = twin-electrode cell, 9 = dual potentiostat, 10 = dual pen recorder, 11 = waste. The micro HPLC system used for analysis is shown schematically in Fig. 11. A micro-feeder, a micro-syringe and a three-way valve are used to feed the mobile phase. A sample injector with 0.3 p1 loop is used for sample injection. The guard column and micro-separation column are made by packing silica-ODS of 10 pm particle size in a teflon tube 2 0 mm x 0.5 mm I. D. and 165 x 0 . 5 mm I. D., respectively. The analytical procedures are as follows. Only 0.3 p 1 of supernatant of raw human urine is injected into the micro-HPLC system. The biogenic amine metabolites are separated at the flow rate of 8 . 3 pl/min. Typical chromatograms for the simultaneous determination of DOPAC, 5-HIAA and HVA in 0 . 3 pl of human urine directly injected without any pretreatment in the micro-HPLC system are shown in Fig. 12. Parts A and B are, respectively, the anodic and cathodic chromatograms. Peaks 3 , 4 and 5 are due to DOPAC, 5-HIM and HVA in urine, respectively. These were identified by the retention time and/or the peak current ratio of cathodic to anodic
323
F i g . 1 2 . S e l e c t i v e d e t e c t i o n of b i o g e n i c amine m e t a b o l i t e s i n human u r i n e by SDVD 1 2 4 1 . (A) Anodic r e s p o n s e , (B) c a t o d i c r e s p o n s e . Peaks: 3 = DOPAC, 4 = 5-HIAAI 5 = HVA. Sample: 0.3 l.11 of h e a l t h y human u r i n e , p o t e n t i a l s ( v s . Ag/AgCl): anode 0.80 V , c a t h o d e -0.05 V , flow r a t e : 8 . 3 pl/min. by comparison w i t h t h e s t a n d a r d s . Of p a r t i c u l a r i n t e r e s t a r e t h e peaks of X I Y and z i n p a r t s A of F i g . 1 2 . BY r e c o r d i n g t h e c a t h o d i c r e s p o n s e , it was shown t h a t t h e r e were e s s e n t i a l l y no c a t h o d i c peaks c o r r e s p o n d i n g t o t h e a n o d i c peaks o f y and z, s u g g e s t i n g t h a t t h e compound o r compounds p r o d u c i n g t h e a n o d i c peaks a r e i r r e v e r s i b l y o x i d i z e d . S i n c e t h e main compounds r e s p o n s i b l e t o t h e a n o d i c peak o f x were a l s o i r r e v e r s i b l y o x i d i z e d , DOPAC c o u l d s e l e c t i v e l y be d e t e c t e d on t h e c a t h o d i c chromato-
grams. The d e t e c t i o n l i m i t s o f b i o g e n i c amine m e t a b o l i t e s by t h i s system were 1 0 pg f o r DOPAC, 20 pg f o r 5-HIAA and 20 pg f o r HVA,
324
respectively,and t h e range of l i n e a r i t y was about
lo3.
The
c o l l e c t i o n e f f i c i e n c i e s i n t h e system were found t o be 0 . 6 1 f o r 0.20 f o r 5-HIAA and 0.30 f o r HVA, r e s p e c t i v e l y , a t a flow r a t e of 8.3 pl/min. I t should be noted t h a t t h e s e values a r e
DOPAC,
much l a r g e r than those of 0.31 f o r DOPAC, 0.05 f o r 5 - H I M and 0.05 f o r HVA, r e s p e c t i v e l y , obtained i n SDVD a t t h e flow r a t e of 1 . 6 ml/min
[221.
3.3 Dual voltammetric d e t e c t o r of parallel-opposed type The parallel-opposed dual voltammetric d e t e c t o r (PODVD) i s analogous t o t h e photomultiplier tube and t h e product of t h e e l e c t r o d e r e a c t i o n a t one working e l e c t r o d e can d i f f u s e t o t h e o p p o s i t e working e l e c t r o d e where s t a r t i n g m a t e r i a l may be c r e a t e d . Goto e t a l . [31, 321 r e c e n t l y b u i l t a twin-electrode t h i n l a y e r e l e c t r o l y t i c c e l l i n parallel-opposed c o n f i g u r a t i o n f o r micro-HPLC a s shown i n Fig. 1 3 .
The t h i n - l a y e r c a v i t y i s
constructed of two fluorocarbon r e s i n blocks separated by a t e f l o n s h e e t 30
-
50 Um t h i c k and 2 mm wide. Two working e l e c t r o d e s a r e
made with g l a s s y carbon p l a t e s 1 c m long and 2 c m wide contained i n each block. The r e f e r e n c e e l e c t r o d e , s i l v e r / s i l v e r c h l o r i d e ,
i s held i n a c y l i n d r i c a l hole i n one of t h e blocks. A platinum tube serves both a s t h e counter e l e c t r o d e and t h e e x i t l i n e .
(i)Current a m p l i f i c a t i o n i n parallel-opposed dual voltammetric For slower flow r a t e s , c a t a l y t i c a m p l i f i c a t i o n of d e t e c t o r response f o r r e v e r s i b l e and q u a s i - r e v e r s i b l e a n a l y t e s may be achieved by r e c y c l i n g t h e redox couple between t h e two working e l e c t r o d e s i n PODVD. Consider a case where only oxidant is i n i t i a l l y i n t h e flow stream. The two working e l e c t r o d e s of PODVD a r e set a t p o t e n t i a l s where t h e oxidant i s s u f f i c i e n t l y reduced and i t s r e d u c t a n t i s s u f f i c i e n t l y oxidized, r e s p e c t i v e l y . The equations r e l a t i n g c a t h o d i c c u r r e n t (Ic)and anodic c u r r e n t (Ia)t o s y s t e m v a r i a b l e s a r e as follows. Ic/nFC = L D W / b + 0.2711 (1) d e t e c t i o n f o r micro HPLC 1321.
-
-Ia/nFC = L D W / b 0.07U (2) where n i s t h e e l e c t r o n t r a n s f e r number, F t h e Faraday's c o n s t a n t ,
C the bulk concentration of a n a l y t e , L t h e l e n g t h of e l e c t r o d e , W t h e width of channel o r e l e c t r o d e , b t h e h e i g h t of channel, LI t h e s o l v e n t volume flow rate and D t h e s o l u t e d i f f u s i o n c o e f f i c i e n t . The colLection e f f i c i e n c y (Q,) , t h e r a t i o of anodic c u r r e n t to cathodic c u r r e n t i n t h i s c a s e , i s represented as
326
A 6
m I
B
F i g . 13. C e l l f o r v o l t a m m e t r i c d e t e c t o r w i t h t w o working e l e c t r o d e s i n p a r a l l e l - o p p o s e d c o n f i g u r a t i o n [31] (A) S i d e view of c e l l , (B) f r o n t view o f s p a c e r . 1, 2 = Working e l e c t r o d e s ( g l a s s y c a r b o n ) , 3 = r e f e r e n c e e l e c t r o d e (Ag/AgCl), 4 = c o u n t e r e l e c t r o d e doubling a s o u t l e t (platinum t u b e ) , 5 = spacer ( t e f l o n s h e e t ) , 6 = m i c r o - s e p a r a t i o n column d o u b l i n g as i n l e t , 7 = s l o t .
.
-
0.34U/(LDW/b + 0.27U) The c u r r e n t a m p l i f i c a t i o n e f f i c i e n c y ( Q a ) and e f f e c t i v e Qc = 1
(3)
a m p l i f i c a t i o n e f f i c i e n c y ( Q e ) i n PODVD a r e , r e s p e c t i v e l y , d e f i n e d a s t h e r a t i o s o f c a t h o d i c and a n o d i c c u r r e n t t o c o u l o m e t r i c c u r r e n t i n t h i s case a s @a= 0 . 2 7
+
(LDW/b)( 1 / U )
(4)
and
Qe = - 0.07 + ( L D W / b ) (1/U) (5) The c u r r e n t a m p l i f i c a t i o n and c o l l e c t i o n e f f i c i e n c y i n t h e d e t e c t o r a t f l o w r a t e s o f 1 . 4 t o 1 1 . 2 Ul/min were i n v e s t i g a t e d by u s i n g f e r r i c y a n i d e a s a n a l y t e i n B-R b u f f e r o f pH 1.8 c o n t a i n i n g 1 mM HSA, 0 . 1 mM EDTA and 50 mM p e r c h l o r a t e as s o l v e n t . The r e s u l t s are shown i n F i g . 1 4 . The l i n e s r e p r e s e n t t h e o r e t i c a l e x p e c t a t i o n s of eqns. ( 3 - 5 ) , w h i l e t h e p o i n t s r e p r e s e n t e x p e r i m e n t a l d a t a . The c o l l e c t i o n e f f i c i e n c i e s from 0.98 t o 0.84 were o b t a i n e d i n t h e flow rate r a n g e from 1 . 4 t o 1 1 . 2 pl/min, r e s p e c t i v e l y . I t i s i n t e r e s t i n g t h a t t h e s e v a l u e s are m u c h h i g h e r t h a n t h o s e (4J.37 o b t a i n e d i n SDVD a t a f l o w r a t e o f 1 ml/min i n t h e o r d i n a r y HPLC
326
F i g . 1 4 . Dependence of c u r r e n t a m p l i f i c a t i o n e f f i c i e n c i e s and c o l l e c t i o n e f f i c i e n c y i n PODVD on flow r a t e [321. The l i n e s r e p r e s e n t t h e o r e t i c a l e x p e c t a t i o n s of e q n s . ( 3 - 5 ) . The p o i n t s r e p r e s e n t e x p e r i m e n t a l d a t a . Sample: 100 V M f e r r i c y a n i d e i n B-R b u f f e r of pH 1 . 8 , c h a n n e l h e i g h t : 3 0 um. (1) Qc, ( 2 ) Oa, (3)
[ 2 0 1 . The e f f e c t i v e a m p l i f i c a t i o n e f f i c i e n c y v a r i e d c o n s i d e r a b l y ,
from 2.4
t o 1 9 . 5 , when t h e flow r a t e changed from 1 1 . 2 t o 1 . 4 p l /
min, a s t h e t h e o r y p r e d i c t s . I t s h o u l d be n o t e d t h a t t h o s e v a l u e s
are mch larger than those ( 0 . 7 - 0.8) o b t a i n e d f o r c a t e c h o l a m i n e s i n SDVD a t a flow rate o f 8 . 3 pl/min i n micro HPLC 1 2 4 1 . (ii)Catecholamine
a n a l y s i s i n human serum [311. The
same
b a s i c micro-HPLC system.as t h a t shown i n F i g . 8,was used f o r d e t e r m i n a t i o n of c a t e c h o l a m i n e s i n human serum. The d i f f e r e n c e s a r e a s f o l l o w s : t h e 200 ul l o o p i s s e t and PODVD is a p p l i e d a s d e t e c t o r . The a n a l y t i c a l p r o c e d u r e s a r e a s f o l l o w s . The human
321
blood sample i s drawn i n a serum s e p a r a t i o n t u b e by v e n i p u n c t u r e . A f t e r i n c u b a t i o n a t room t e m p e r a t u r e f o r a b o u t 1 0 min t o i n d u c e c o a g u l a t i o n , t h e t u b e i s c e n t r i f u g e d , s e p a r a t i n g t h e serum from
t h e blood c e l l s . Then, 500 1.11 of t h e raw human serum is removed t o a t e s t t u b e , t o which had been added 0.5 mg of s o l i d EDTA (disodium s a l t ) , 0 . 5 mg of s o l i d sodium hydrogen s u l f i t e , and 1 0 ~1 o f 25 pg/ ?.113 , 4-dihydroxybenzylamine hydrobromide (DHBA) s t a n d a r d
s o l u t i o n . The t e s t t u b e
is
sealed
shaken f o r 1 min by hand
and a l a r g e p o r t i o n o f t h e mixed sample s o l u t i o n i s t r a n s f e r r e d t o a n u l t r a f i l t r a t i o n c e l l . The u l t r a f i l t r a t i o n i s c a r r i e d o u t by s t i r r i n g t h e s o l u t i o n w i t h a magnetic s t i r r e r under a n i t r o g e n p r e s s u r e of 2 . 5 kg/cm2 i n a n i c e b a t h . The f i l t r a t e d serum i s t a k e n w i t h a m i c r o - s y r i n g e and i n j e c t e d i n t o t h e sample l o o p (200 1.11) of t h e sample i n j e c t o r . The o t h e r p r o c e d u r e s a r e a l m o s t t h e same as in
u r i n a r y catecholamine
a n a l y s i s . The d i f f e r e n c e s are
a s f o l l o w s : t h e t i m e f o r sample e n r i c h m e n t and t h e t i m e f o r precolumn washing a r e b o t h 1 0 min; t h e mobile p h a s e used i s t h e B-R
b u f f e r a t pH 8 . 7 c o n t a i n i n g 2 mM HSA, 0 . 1 mM EDTA and 50 mM
p e r c h l o r a t e , t h e separated catecholamines a r e introduced i n t o t h e t w i n - e l e c t r o d e c e l l i n p a r a l l e l - o p p o s e d c o n f i g u r a t i o n , i n which t h e lower and upper working e l e c t r o d e a r e s e t a t t h e p o t e n t i a l s ( v s . Ag/AgCl) o f 0.60 V and 0 . 2 0 V, r e s p e c t i v e l y , and t h e c a t e c h o l a m i n e s are s e l e c t i v e l y d e t e c t e d w i t h h i g h s e n s i t i v i t y by m o n i t o r i n g t h e r e - r e d u c t i o n c u r r e n t a t t h e c a t h o d e and d e t e r m i n e d by comparing w i t h t h e r e s p o n s e o f i n t e r n a l s t a n d a r d . F i g u r e 1 5 shows t y p i c a l chromatograms o f c a t e c h o l a m i n e s i n human serum a t a mobile phase f l o w r a t e o f 8 . 3 pl/min o b t a i n e d u s i n g t h e proposed p r o c e d u r e s and t h e micro-HPLC system w i t h precolumn and PODVD. P a r t s A and B a r e , r e s p e c t i v e l y , t h e a n o d i c and c a t h o d i c chromatograms. Of p a r t i c u l a r i n t e r e s t i n p a r t s A
are
t h e peaks a p p e a r i n g , r e s p e c t i v e l y , a s t h e s h o u l d e r o f NA i n F i g . 15b and t h e background of AD i n F i g . 1 5 a . By r e c o r d i n g t h e c a t h o d i c c u r r e n t , t h e i n t e r f e r e n c e s from t h e compounds r e s p o n s i b l e f o r t h e s e peaks c o u l d be removed, as shown i n p a r t s B, on t h e b a s i s o f t h e i r e l e c t r o c h e m i c a l i r r e v e r s i b i l i t y . Both t h e a n o d i c and c a t h o d i c peak h e i g h t r a t i o s o f NA and AD t o DHBA were l i n e a r w i t h e a c h amount of c a t e c h o l a m i n e i n j e c t e d , w i t h a good c o r r e l a t i o n . The l i n e a r dynamic r a n g e w a s a b o u t l o 3 and t h e minimum d e t e c t a b l e amount i n t h e proposed system was a b o u t 3 pg f o r NA and AD a t t h e f l o w r a t e o f 8 . 3 pl/min.
T h i s d e t e c t i o n l i m i t by PODVD i s a b o u t
328
a
?
z
1 (min)
Fig. 15. S e l e c t i v e and s e n s i t i v e d e t e c t i o n of catecholamines i n human serum by PODVD 1311. (A) Anodic response, (B) c a t h o d i c response. Sample: 200 p 1 of human serum spiked with 1 0 0 pg of DHBA, p o t e n t i a l s (vs. Ag/AgCl): anode 0.60 V, cathode 0.20 v, flow rate: 8.3 p l / m i n . t h r e e times b e t t e r than t h a t i n [25] given by SDVD. The chromatographic peak r a t i o s of c a t h o d i c t o anodic, c o l l e c t i o n e f f i c i e n c i e s , f o r a standard s o l u t i o n were 0 . 9 6 , 0.79, 0.68 f o r NA, 0.95, 0.81, 0.66 f o r AD and 0.97, 0.86, 0.70 f o r DHBA a t t h e flow rates (pl/min) of 1.4, 5.6, 1 1 . 2 , r e s p e c t i v e l y . The e l e c t r o l y t i c e f f i c i e n c i e s ( % ) obtained from t h e c a t h o d i c peak a r e a s i n t h i s micro-HPLC system were 1132 f o r NA, 1000 f o r AD and
1324 f o r DHBA a t t h e flow r a t e of 1.4 pl/min, by assuming a = 2. Figure 16 shows t h e chromatograms of catecholamines i n u l t r a f i l t r a t e d human serum (200 p l ) spiked with 100 pg DHBA.directly
329 a
t (hr)
b
t m'n)
Fig. 16. Chromatograms of catecholamines in healthy human serum by PODVD at very slow flow-rates [32]. (A) Anodic response, (B) cathodic response. Peaks: 1 = NA, 2 = AD, 3 = DHBA (internal standard). Sample: 2 0 0 p1 of human serum spiked with 100 pg DHBA, flow rates (pl/min) : (a) 1.4, (b) 5.6. injected to the micro-HPLC system at lower flow-rates. It is clear that the selective and sensitive detection of catecholamines on the basis of electrochemical reversibility can be performed by using the cathodic chromatograms from PODVD at very low flowrates. CONCLUSION Compared with ultraviolet and fluorescence detectors of the same cell volume, voltammetric detectors for micro-HPLC or capillary LC have a much greater detection sensitivity, this being 4
330
true for biogenic mines and their metabolites. Another merit of such detectors is that several substances can be detected selectively by constructing a thin-layer electrolytic cell containing two or more working electrodes and setting the respective electrodes at selected potentials. The SDVD with anode and cathode is a powerful tool for the selective detection of reversible and/or quasi-reversible species for micro-HPLC or capillary LC, because the collection efficiency increases with decreasing the flow-rate of mobile phase. Moreover, the PODVD with anode and cathode can provide an enhancement in sensitivity by recycling oxidation and re-reduction between the two working electrodes at slow flow rates of mobile phase. Thus, the PODVD is the most advantageous type of detector for reversible and/or quasi-reversible species in micro HPLC and capillary LC. The constituent of biological fluids and tissues include many electroactive substances Voltammetric detectors are likely to findmany applicationsin the field of clinical analysis. Typical electroactive species are as follows; phenols, methoxyphenols, catechols, catecholamines, hydroxycoumarins, quinones, estrogens, tocophenols, morphine derivatives, anilines, sulfonamides, indoles, phenotiazines, purines, ascorbic acid, thiols, cyanide, aldehydes, uric acid, nitro compounds, aromatic amines,etc.
.
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