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An Electrochemical Detector for

Liquid Chromatography with Picogram
Sensitivity
a b a a a
P. T. Kissinger , C. Refshauge , R. Dreiling & R. N. Adams
a
Department of Chemistry , University of Kansas , Lawrence,
Kansas, 66044
b
Department of Chemistry , Michigan State University , East
Lansing, Michigan, 48823
Published online: 05 Dec 2006.

To cite this article: P. T. Kissinger , C. Refshauge , R. Dreiling & R. N. Adams (1973) An

Electrochemical Detector for Liquid Chromatography with Picogram Sensitivity, Analytical Letters,
6:5, 465-477, DOI: 10.1080/00032717308058694

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 ANALYTICAL LETTERS, 6 ( 5 ) , 465-477 (1973)

AN ELECTROCHEMICAL DETECTOR FOR LIQUID

CHROMATOGRAPHY WITH PICOGRAM SENSITIVITY

KEY WORDS: liquid chromatography electrochemical detector,

biogenic amines, thin layer electrochemistry
*
P.T. Kissinger , C. Refshauge, R. Dreiling and R.N. Adams
Downloaded by [Michigan State University] at 19:38 05 February 2015

Department of Chemistry
University of Kansas
Lawrence, Kansas 6 6 0 4 4

ABSTRACT
A simple liquid chromatograph with an extremely sensitive
electrochemical detector is described. The detector, which can
be constructed for less than $5.00 (excluding electronics) , has
an actual dead volume of <1 p 1 and operates well in the 50-100
picogram region. Routine quantitative work at the 5-10 nanogram
level is described. Potential applications to the analysis of
biogenic amines are discussed.

INTRODUCTION
major limitation in the application of modern liquid
A

chromatography (LC) is the sensitivity of detector systems. 1

A few uv detectors have been described which are usable at
low nanogram levels; however, these involve special operating
conditions or compounds with high molar absorptivities.1 1 2 1 3
With ordinary uv absorbing species, quantitative LC is usually
limited to a few tenths of micrograms. We wish to report a

*
Present address: Department of Chemistry, Michigan State
University, East Lansing, Michigan 4 8 8 2 3

465
Copyright @ 1973 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced
or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and
recording, or b y any information storage and retrieval system, without permission in writing from the publisher.
 KISSINGEH ET AL.

s i m p l e and i n e x p e n s i v e l i q u i d c h r o m a t o g r a p h w i t h a n u n u s u a l l y

s e n s i t i v e electrochemical detector. This d e t e c t o r operates

w e l l i n t h e 50-100 picogram r e g i o n f o r c a t e c h o l a m i n e s a n d
c l e a r l y c a n b e made more s e n s i t i v e i f d e s i r e d . Routine

q u a n t i t a t i v e o p e r a t i o n a t t h e 5-10 nanogram l e v e l i s d e s c r i b e d .

E l e c t r o c h e m i c a l d e t e c t o r s i n c h r o m a t o g r a p h y a r e n o t new, and

e x t e n s i v e a p p l i c a t k m s were made by Kemula a n d co-workers in

t h e 1 9 5 0 I s i n t h e i r s t u d i e s o f " c h r o m a t o p o l a r o g r a p h y . ,I 4
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S e v e r a l o t h e r g r o u p s have employed d r o p p i n g mercury a n d

other electrodes, " b u t few o p e r a t e i n t h e s e n s i t i v i t y r a n g e
described herein.

DESIGN

Our i n t e r e s t s l i e p r i m a r i l y i n r a p i d , p r a c t i c a l a n a l y s e s

o f b i o g e n i c amines and r e l a t e d s u b s t a n c e s i n b r a i n t i s s u e ,

c e r e b r a l s p i n a l f l u i d , u r i n e , etc. The l i q u i d c h r o m a t o g r a p h
employed w a s n o t s o p h i s t i c a t e d , b u t r a t h e r w a s d e s i g n e d f o r

routine analyses. The column ( 2 mm i . d . g l a s s , l e n g t h s o f

1 5 , 30 and 50 c m ) , septum i n j e c t i o n p o r t , and a l l f i t t i n g s

were e a s i l y a s s e m b l e d from c o m m e r c i a l l y a v a i l a b l e components

(Chromatronix, I n c . , Berkeley, C a l i f o r n i a ) . The column was

d r y packed w i t h a p e l l i c u l a r c a t i o n e x c h a n g e r e s i n , SCX

(DuPont). A Milton-Roy pump ( I n s t r u m e n t Mini-Pump) w a s con-

n e c t e d t o a c o i l ( c a . 80 f t . ) o f 1 / 1 6 " 0 . d . T e f l o n t u b i n g t o

p a r t i a l l y dampen p u l s a t i o n s . The maximum o p e r a t i n g p r e s s u r e

.
o f t h e s y s t e m was a b o u t 400-500 lbs/inL. F l u i d flows w e r e

0.5-0.8 ml/min. The e l e c t r o c h e m i c a l d e t e c t o r was a t t a c h e d

v i a a s t a n d a r d C h r o m a t r o n i x f i t t i n g t o t h e o u t p u t end o f t h e
column. The d e t e c t o r i s n o t s p e c i f i c t o t h i s p a r t i c u l a r

466
 L I Q U I D CHROMATOGRAPHY

i n s t r u m e n t and c a n be e a s i l y a t t a c h e d t o any l i q u i d chromato-

graph.

The d e t e c t o r i s a working e l e c t r o d e , s e t i n t h e f l o w

stream emerging from t h e column. The e l e c t r o d e i s h e l d a t

a fixed, predetermined p o t e n t i a l t o o x i d i z e (or reduce)

e l e c t r o a c t i v e components p a s s i n g by. A conventional poten-

t i o s t a t i c s y s t e m i s used w i t h a n a u x i l i a r y and reference

electrode. W e have i n v e s t i g a t e d s e v e r a l e l e c t r o d e c o n f i g u r a -
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t i o n s , i n c l u d i n g o n e s where t h e working e l e c t r o d e p r o j e c t e d

i n t o t h e f l o w s t r e a m a t r i g h t a n g l e s and t h e o t h e r two

electrodes w e r e s l i g h t l y downstream.
While by no means optimum i n d e s i g n , t h e a r r a n g e m e n t

shown i n F i g u r e 1 g i v e s e l e g a n t r e s u l t s . This configuration

d o e s n o t r e q u i r e u l t r a - p r e c i s i o n machining a n d c a n b e con-

s t r u c t e d f o r less t h a n $5.00. As shown i n F i g u r e 1,A, t h e

d e t e c t o r c o n s i s t s o f two Jfi i n . t h i c k , 1%i n . s q u a r e L u c i t e

b l o c k s ( t h e s e d i m e n s i o n s were c o n v e n i e n t a n d a r e n o t a t a l l

critical). The t w o b l o c k s are s e p a r a t e d by a t h i n ( 0 . 0 0 5 i n . )

T e f l o n s p a c e r , S , c u t from commercial s h e e t T e f l o n . The

s p a c e r , shown s e p a r a t e l y i n F i g u r e 1 , C , h a s a s l i t , 2 nun wide

and 75 mm l o n g , c u t i n it. One o f t h e b l o c k s (the l e f t s i d e

i n F i g u r e 1,A) h a s two d r i l l b o r e s c u t a t t h e a p p r o x i m a t e

i n d i c a t e d a n g l e s t o provide an i n l e t ( I n ) and o u t l e t ( O u t )

flow stream from t h e column t h r o u g h t h e T e f l o n s l i t r e g i o n .

These h o l e s a r e d r i l l e d s l i g h t l y o v e r s i z e d a n d s t a n d a r d 1 / 1 6
i n . 0.d. Teflon tubing is i n s e r t e d . (These were s l i c e d f l u s h

a t t h e i n n e r f a c e of t h e b l o c k a n d s e a l e d w i t h p a r a f f i n wax.

The e x c e s s wax w a s trimmed smooth w i t h a r a z o r b l a d e a n d , i f

467
 S

A
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C D
Figure 1

necessary, the tubes reamed clear. The upper end of the inlet
tube is connected to a standard Chromatronix 1/16 in. fitting
which is recessed and secured with epoxy cement to the top of
the block.
The opposite block (right side of Figure 1,A) contains
a recessed well whose diameter is about 0.10 in. ( X 3 7 drill)
and 4 in. deep. Again, the well dimensions are not critical.
It should, however, be slightly greater in diameter than the
spacer slit width and positioned slightly downstream of the
point where the inlet tube emerges from the opposite block.

468
 LIQUID CHROMATOGRAPHY

E n t e r i n g t h e s i d e n e a r t h e bottom o f t h e w e l l i s a d r i l l h o l e

i n t o which a copper w i r e c o n t a c t i s s e a l e d ( t h e c o p p e r w i r e

i s shown a s a s o l i d b l a c k d o t emerging p e r p e n d i c u l a r t o t h e

page i n F i g u r e 1 , A ) . The w e l l i s packed f l u s h t o t h e r i m w i t h

carbon p a s t e ( d e s c r i b e d below) which forms t h e working elec-

trode surface.

I f t h e two b l o c k s and T e f l o n s p a c e r o f F i g u r e l , A a r e

p r e s s e d t o g e t h e r and r o t a t e d c o u n t e r - c l o c k w i s e 90°, they

Downloaded by [Michigan State University] at 19:38 05 February 2015

a p p e a r as i n d i c a t e d i n F i g u r e 1,B. Four d r i l l h o l e s i n t h e

corners o f t h e b l o c k s and s p a c e r a l l o w t h e u n i t t o b e a l i g n e d
and t i g h t l y "sandwiched" t o g e t h e r w i t h 1% i n . machine screws.

The d e t e c t o r i s t h e n a t t a c h e d t o t h e end o f t h e column v i a

t h e s t a n d a r d f i t t i n g on t o p . The f l o w p a t t e r n c a n now b e

visualized easily - l i q u i d from t h e column p a s s e s t h r o u g h t h e

i n l e t t u b e a c r o s s t h e working e l e c t r o d e s u r f a c e v i a t h e t h i n

c h a n n e l provided by t h e s p a c e r and e x i t s by t h e o u t l e t t u b e .

The dead volume between t h e column and d e t e c t o r e l e c t r o d e i s

a b o u t 8 p 1 and t h e a c t i v e volume o f t h e d e t e c t o r p e r s e i s

only about 0.7 p l . T h i s l a s t f i g u r e i s an o r d e r o f magnitude

s m a l l e r t h a n t h a t f o r t h e b e s t commercial uv d e t e c t o r .

F i g u r e l , D shows t h e o u t l e t t u b e c o n n e c t i o n t o a s i m p l e

overflow v e s s e l . The a u x i l i a r y e l e c t r o d e (a p l a t i n u m w i r e )

and t h e r e f e r e n c e e l e c t r o d e (a Ag/AgCl w i r e s e t i n a s m a l l

c a p i l l a r y s a l t b r i d g e of 3 M N a C 1 ) d i p i n t o t h e o v e r f l o w

volume and p r o v i d e t h e t h r e e - e l e c t r o d e c e l l .

The c a r b o n p a s t e e l e c t r o d e i s s i m i l a r t o t h o s e used f o r

many y e a r s i n o u r l a b o r a t o r y . The c a r b o n p a s t e i s , however,

s u p e r i o r w i t h r e g a r d t o low l e v e l r e s i d u a l c u r r e n t by v i r t u e

469
 KISSINGER ET AL.

of t h e t y p e o f g r a p h i t e used. I t i s made by t h o r o u q h l y mixing

3 g o f UPG Powder ( U l t r a Carbon Corp., Bay C i t y , Mich.) w i t h

2 m l of Nujol. Thorough mixing r e q u i r e s some t i m e , b u t a

s i n g l e b a t c h s u f f i c e s f o r many e l e c t r o d e s . The p a s t e i s

packed i n t h e w e l l and t h e s u r f a c e smoothed by r u b b i n g t h e

block on a f i n i s h e d paper s u r f a c e (computer c a r d s work v e r y

well). The same e l e c t r o d e s u r f a c e may be used f o r weeks o r

l o n g e r , b u t o c c a s i o n a l l y t r a p p e d b u b b l e s may d i s t o r t t h e s u r -
Downloaded by [Michigan State University] at 19:38 05 February 2015

f a c e and r e q u i r e an e l e c t r o d e change. The r e s u l t i n g s u r f a c e

a r e a v a r i a t i o n may c a u s e a change i n s e n s i t i v i t y o f a few

per c ent . S i n c e w e always employ an i n t e r n a l s t a n d a r d i n t h e

LC o p e r a t i o n , t h e s e s e n s i t i v i t y s h i f t s a r e no problem.

The d e t e c t o r measures t h e l i m i t i n g c u r r e n t ( a t f i x e d

p o t e n t i a l ) a t t h e c a r b o n p a s t e e l e c t r o d e under f o r c e d convec-

tion conditions. T h i s i s one of t h e s i m p l e s t and most s e n s i -

t i v e o f v o l t a m m e t r i c measurements - i.e., o r d i n a r y amperometry.

The c u r r e n t s c o u l d b e measuredwith a s i m p l e v o l t a g e d i v i d e r

and microammeter ( t w o - e l e c t r o d e system of c o n v e n t i o n a l ampero-

metric t i t r a t i o n s ) i f t h e y were l a r g e r . However, f o r t h e

c u r r e n t s e n c o u n t e r e d i n t h i s a p p l i c a t i o n (picoamps t o low
nanoamps), a p o t e n t i o s t a t w i t h low l e v e l c u r r e n t measuring

c a p a b i l i t i e s i s necessary. F i g u r e 2 shows a s i m p l e , h i g h l y

satisfactorycircuit for t h i s application. The o p e r a t i o n a l

a m p l i f i e r s marked OA-1 and OA-2 a r e AD-503JH (Analog D e v i c e s )

and OA-3 i s a F a i r c h i l d 7 4 1 . A l l t h r e e a m p l i f i e r s a r e pro-

vided w i t h 10K trimmers t o n u l l i f y t h e o f f s e t v o l t a g e s .

R e s i s t o r s marked w i t h a n a s t e r i s k i n F i g u r e 3 a r e 1%t o l e r -

ance. The p o t e n t i o s t a t i s housed i n a s m a l l aluminum m i n i

Downloaded by [Michigan State University] at 19:38 05 February 2015

L I Q U I D CHROMATOGRAPHY
 KISSINGER ET AL.

box and can be easily and inexpensively constructed. (Detailed

instructions and circuit boards are available from P.T.K.).
The output is fed typically to a Varian G-14A strip chart
recorder. Some additional filtering at the recorder input
was provided by a 2 vf capacitor across the recorder terminals
and a 100 K series resistor (RC = 0.2 sec). The applied
potential is preset to oxidize (or reduce) the electroactive
components emerging from the column. This obviously pre-
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supposes some knowledge of the electrochemical behavior of the

components. If unknown, this is obtained by ordinary voltam-
metry.
RESULTS
Figure 3,A illustrates the response obtained from a
4 p 1 sample injection containing 6.8 nanograms of norepineph-
rine (NE) and 12.2 nanograms of dopamine (DA). The sample
was eluted from the SCX column using 0.1 M perchloric acid and
the applied potential was +0.8 v. vs. Ag/AgCl. Figure 3 , A is
an actual chart recording and represents typical, routine
operation of the detector. To illustrate reproducibility and
time stability, eleven 4 p 1 samples, each containing 15.3
nanograms of DA, were injected over a 42 minute period. These
results showed a standard deviation of 1.9% in peak height
measurement. This is clearly within the reproducibility
limits of micro injection techniques. The noise level is low
and can be seen (Figure 3,A) to be about 2-3% of the total
peak height at this level of operation. The separation of NE
and DA described above has become the basis of a rapid, routine
determination of the catecholamines in mouse and rat brain
7
tissue which will be described in detail elsewhere.

472
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TVDiCal

473
Figure 3
LC Chromatoarams
L I Q U I D CHROMATOGRAPHY
 KISSINGEE ET AL.

Figure 3 , B shows the response for the same separation

of NE and DA (all conditions as described above) using 5 p1

injections containing 84.6 and 153 picograms of NE and DA,

respectively. It can be noted that, at this sensitivity, the

LC-electrochemical detection compares favorably with the most
sensitive technique used for biogenic amine analyses, the gas

chromatographic-mass fragmentography method. 8 r 9 The latter,

of course, requires quite expensive and complex instrumentation,
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as well as derivatization of the amines.

We believe the performance indicated in Figure 3,B is
about the practical quantitative limit of the present system.
Samples containing about 10 picograms each of NE and DA are
easily detected (signals ca. 4 to 5 X that of noise level),
but quantitative work will require shielding from extraneous
electrical noise and further refinement of the present equip-
ment. Interference from trace impurities in the reagents and,
more importantly, from the practical brain samples becomes a
serious detriment at this level.
Careful calibration of the current output of the poten-
tiostat and recorder allows a calculation of the electrolysis
efficiency of the detector (i.e., equivalents detected/mole
injected). Using about 15 nanogram DA injections, it was
found that the system was electrolysing 4.5% of the total
sample injected. This could obviously be improved by elec-
trode design if desired.lo We have not, as yet, investigated
the full "dynamic range" of the detector since we are not much
interested in levels above 100 nanograms. However, excellent
linear response between 5-200 nanograms of DA ( 5 p l injections)

has been amply demonstrated.

474
 L I Q U I D CKROMATOGRAPHY

In addition to the above NE and DA analyses, we have

injected and detected the compounds listed in Table 1. The
list is presently limited to some compounds of neuropharma-
cological interest to us. Obviously, all compounds which
are oxidizable in the aqueous range of 0.0 to about +1.0 v.
are detectable. A typical survey which includes most aromatic
11
amines, phenolics and various heterocyclics has been compiled.

Residual currents with carbon paste electrodes may interfere

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at cathodic potentials and we have not investigated the reduc-

tion capabilities except €or the easily reduced quinones
listed in Table 1.

TABLE 1
TYPICAL COMPOUNDS EXAMINED WITH LC ELECTROCHEMICAL DETECTOR^
Dopamine 6-Aminodopamine

Dopamine-2-quinone L-Dopa
Norepinephrine 5-Hydroxytryptamine
Norepinephrine-2-quinone 5-Hydroxyindole acetic acid
6-Hydroxydopamine 5,6-Dihydroxytryptamine
6-Hydroxydopamine--Pquinone 5-Hydroxytryptophan
Ascorbic acid

aThe quinones listed were detected at E = +0.3 v. For all

aPP
the other compounds, E = +0.8 v. as for NE and DA.
aPP

The limitations of this type of detector are fairly

obvious. First, it can only sense electroactive components.
(This may be turned to an advantage by "fishing out" some
components among electro-inactive species.) Secondly, the

475
 KISSINGER ET AL.

e l u a n t must be e l e c t r i c a l l y conducting. The l a t t e r i s n o t

v e r y r e s t r i c t i v e s i n c e most a q u e o u s e l e c t r o l y t e s or b u f f e r s

w i l l suffice. F o r carbon paste electrodes t h e c h o i c e of

s o l v e n t s w i l l be l i m i t e d t o a q u e o u s media, or a t most, 20-30%

non-aqueous mixtures. Similar t h i n layer configurations

u t i l i z i n g metal f i l m s o n g l a s s ,l2 p y r o l y t i c g r a p h i t e , and

m e r c u r y electrodes a r e u n d e r a c t i v e i n v e s t i g a t i o n and show

p r o m i s e f o r work i n t o t a l l y n o n - a q u e o u s media.
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ACKNOWLEDGMENTS

The s u p p o r t o f t h i s work b y t h e N a t i o n a l S c i e n c e Founda-

t i o n t h r o u g h G r a n t 32846X i s g r a t e f u l l y a c n o w l e d g e d . The

s u p p o r t of R . D r e i l i n g t h r o u g h t h e NSF-Undergraduate R e s e a r c h

P a r t i c i p a t i o n p r o g r a m i s also a c k n o w l e d g e d . W e are i n d e b t e d t o

C. Duver, G. B o g a t i e a n d P . G r i g s b y for v a l u a b l e a s s i s t a n c e .

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B i o c h e m i c a l P h a r m a c o l o g y , e d s . E. C o s t a , L . L . I v e r s e n
and R. P a o l e t t i , Raven P r e s s , New York, 1 9 7 2 , p. 37.
 L I Q U I D CHROMATOGRAPHY

10. D u r i n g t h e c o u r s e of t h i s work a h i g h e f f i c i e n c y p a c k e d
bed electrode detector for LC o f i n o r g a n i c i o n s w a s
briefly described - D. C. J o h n s o n , L. R. T a y l o r , J . H.
L a r o c h e l l e a n d W. S. Wong, A b s t r a c t 1 2 1 , A m e r i c a n C h e m i c a l
S o c i e t y M i d w e s t R e g i o n a l M e e t i n g , Columbia , M i s s o u r i ,
November , 1 9 7 2 .
11. R. N . A d a m s , E l e c t r o c h e m i s t r y a t S o l i d E l e c t r o d e s ,
Marcel D e k k e r , N e w York, 1 9 6 9 .
12. P. T. K i s s i n g e r a n d C . N. R e i l l e y , A n a l . Chem., 42,
1 2 (1970).
Received M a r c h 2 6 1973
Accepted A p r i l 12 1973
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