1016
Anal. Chem. 1983, 55, 1016-1019
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RECEIVED for review December 6, 1982. Accepted March 1, 1983.
Amperometric Detection of Reducing Carbohydrates in Liquid Chromatography Noriyukl Watanabe" and Michiro Inoue Department of Industrial Chemistry, Faculty of Engineering, The University of Tokyo, Hongo 7-3- 1, Bunkyo-ku, Tokyo 1 13, Japan
The detectlon of reduclng sugars wlth an amperometrlc detector in high-performance liquid chromatography has been developed. The redox reaction of copper bis( phenanthroline) is coupled with the reducing ability of sugars in alkaline soiutlon at high temperature on postcolumn, enabling glucose to be determlned at levels down to 1 pmol (0.2 ng). The method Is not only sensltlve but also selective owlng to an allowance of the applled potential to the working electrode to be as low as possible. The reagent used Is quite stable as well as noncorrosive. Quantltatlve analysls of sugars can be achieved over a range of 2 to 3 orders of magnltude. Appllcatlons to urine and serum samples are demonstrated.
Detection methods of carbohydrates commonly used in high-performance liquid chromatography may be divided into three categories: (1)refractive index detector (1-5); (2) colorimetric methods using orcinol/sulfuric acid (6-8), tetrazolium blue (9, IO), bicinchonine-copper (11-13), etc.; (3) fluorimetric methods using cerate (14,15),2-cyanoacetamide (16),ethylenediamine (17), etc. The detection limits reported therein range widely from 1pg by refractive index detector to 2 ng by fluorometry using 2-cyanoacetamide. In addition to these, a few trials of coulometric (18)and recent amperometric detection (19) as electrochemical means were reported but their sensitivities are fairly judged as not sufficient. In spite of these wide variety of detections, an easily attainable and more sensitive method is still being pursued. We describe highly sensitive detection of reducing sugars by use of an amperometric detector. A coupling of the redox reaction of metal complex as a mediator with the reducing ability of sugars offers an essential scheme of detection as follows: reducing sugars
Cu(phen)22+ chemically, OH;
A*
Cu(phen)z+
electrochemically*
Cu (phen)22+ where copper bis(phenanthro1ine) complex (CBP) was employed as the mediator. Reducing sugars reduce divalent complex of CBP to monovalent in alkaline solution a t high
temperature in the reaction coil placed after the column. Thus the monovalent CBP formed is reoxidized and the results are measured with an amperometric detector, permitting highly sensitive detection. The method is not only sensitive but also selective and easily performed without any problems such as hazardous corrosion or instability of reagent.
EXPERIMENTAL SECTION Materials. All chemicals were reagent grade commercially available unless otherwise stated. Copper bis(phenanthro1ine) (CBP) was prepared according to the literature (20). It was once recrystallizedfrom distilled water. Reagent solution was prepared by dissolving CBP into the solution containing Na2HP04 as supporting electrolyte. pH of the reagent solution was adjusted to a desired value by addition of 2 M NaOH. The reagent solution was quite stable even for several months. All solutions and eluent were made from singly distilled water. Apparatus. The high-performanceliquid chromatography was equipped with a single plunger piston pump NMP-1U (Nihon Seimitsu Co., Tokyo, Japan) and bellows damper NBD-3 (Nihon Seimitsu Co., Japan), cation exchange column LS212 (Toyo Soda Co., Tokyo, Japan) or anion exchange column IEX220 (Toyo Soda Co., Japan), sample injector with syringe loading loop, Model-7120 (Rheodyne, USA), and an amperometric detector VMD-101 (Yanaco, Kyoto, Japan). The reagent solution containing CBP was delivered by a constant flow rate pump TRI ROTAR-I1 (Japan Spectroscopic Co., Tokyo, Japan) and mixed with the column eluate by means of a simple stainless tee. PTFE tube (0.5 mm id., 5 m length) was used as the reaction coil which was immersed in a water bath, temperature controlled to within *O.l "C. The reaction mixture was passed through the cooling coil (0.25 mm i.d., 30 cm length), dipped in water before reaching to the detector. In all experiments, 20 p L of sample was injected. All optimization studies were carried out under the full HPLC setup, that is, cation exchange column (7.5 mm id., 60 cm length) and water as eluent. RESULTS AND DISCUSSION Cyclic Voltammogram. The cyclic voltammogram for CBP reagent solution was examined with a separate stationary electrochemical cell having glassy carbon as a working electrode. The quasi-reversible wave corresponding to the redox couple of CBP2+/+was obtained. The potential of oxidative peak current was ca. -80 mV vs. AgIAgC1. The peak separation of oxidative and reductive waves was ca. 100 mV.
0003-2700/83/0355-1016$01.50/00 1963 American Chemical Society
ANALYTICAL. CHEMISTRY, VOL. 55, NO. 7, JUNE 1983
1017
Table I. Response Factors Relative to Glucose ( l O O % ) Q re1 response re1 response per wt per wt glucose fructose sorbose xylose fucose mannose arabinose galactose ribose
I
T
'
-80
'
-40
~
~
,
'
0 +40 +80 rnV vs lq/AgCI
"
'
,
Flgure 1. The dependence of peak response and background current peak response, (A) background current on applied potential: (0) reagent; 3 mM CE3P, 0.2 M Na2HPO4,pH 11.2; sample, 5 ppm glucose; reaction bath, 95 O C , 5 m coil.
g
J--l
a
maltose lactose ascorbic acid uric acid methanol ethanol acetaldehyde phenol
100
136 112 109 104 87 83 74 72
69 64 49 42
