Anal. Chem. 1983, 5 5 , 1013-1016
1013
Donnan Dialysis Preconcentration for Ion Chromatography James E. DINuinzlo" and Michael Jubara Department of Chiamistry, Wright State Universi@, Dayton, Ohio 45435
Donnan dlalysls techniques are used for the preconcentratlon of Cu, NI, Co, Ctl, and Mn prlor to Separation by Ion chromatography. The problems associated with the separation of low levels of analyte! Ions In a hlgh lonlc strength receiver electrolyte and the conslderatlons necessary to properly select a compatlble receiver electrolyte and moblle phase combination to allow the determlnatlon of trace analyte Ions In a hlgh Ionic strength recelveir electrolyte are dlscussed. Enrlchment factors of 80-fold are obtalned for a 1 h preconcentration and over 100-fold for longer time periods. The preclslon of the method Is about 12% at the parts-per-bllllon level.
The development of ion chromatography as a technique for the simultaneous determination of ionic species has sparked an enormous amount of research in this area. At present the major limitation with the technique lies with the detection systems currently available. A number of detection systems have been used for ion chromatography. These include conductometric ( 1 4 , electrochemical ( 4 ) ,and various reaction detection systems (5-7). None of these systems, however, is sensitive enough to allow truly trace analysis to be performed. In general, the absolute detection limit for ion chromatography is around 1 ng ( 4 , 8), but because of the small sample sizes normally used, the concentration detection limit is not coniparably small. Because of limited sensitivity associated with ion chromatography, preconcentration techniques will play an important role in determining the applicability of the method for trace analysis. Unfortunately, this is one aspect of ion chromatography which has received almost no attention. The on-columm preconcentration method has been used almost exclusively in ion chromatography (9-12). In this procedure the sample is passed through the column, or through a precolumn connected to the top of the analytical column, and the ions in the sample are collected a t the top of the column. After they are rinsed, the analyte ions are eluted. Although the on-column preconcentration technique is capable of very high enrichments (13),there are a number of disadvantages associated with this method. First, this method is time-consuming and expensive. This is particularly true when the chromatograph is used for both the preconcentration and analysis stepis. Furthermore, because of the nature of the method, careful washing and reequilibration of the column are necessary a t the end of the separation to ensure efficient preconcentration of the following sample. Some of these problems can be minimized by use of an off-line pumping system with precolumn collection. This alternative suffers from problems associated with the need to connect the precolumn to the chromatographic systems as well as the expense of the off-line pumping system. A second and potentially more serious problem with the on-column preconcentration method deals with the sample matrix. In general samples of high ion strength cannot be preconcentrated by the on-column method. In ion chromatography the total exchange capacity of the system is usually
very small (14). While on-column preconcentration of samples containing essentially only trace levels of ions is possible, the presence of ions in high concentration will drastically interfere with the technique. This interference results because the bulk ions in the sample will quickly exhaust the system exchange capacity. Once this occurs analyte ions can no longer be collected on tho column and/or precolumn. This can result in a negative error for the analysis and low recoveries of analyte ions. Donnan dialysis is a technique which has been used for preconcentration of both anions (15) and cations (16). The method, which AS itself based on an ion-exchange process, has been shown to be simple, inexpensive, and precise. This technique has been used for preconcentration of trace ions from samples of both high and low ionic strength without difficulty. The purpose of this paper is to describe the use of Donnan dialysis preconcentration for the determination of metals by ion chromatography.
EXPERIMENTAL SECTION Procedure. Metal preconcentrations were performed in a manner similar to that previously described ( 1 7 ) . In all cases the diameter of the membrane in the dialysis cell was 3 cm and 200-mL aliquots of sample were preconcentrated into the receiver electrolyte. For the chromatography studies 0.5 mL of receiver electrolyte was used. For determinations by atomic absorption spectrophotometry 4.0 mL of receiver electrolyte was used. The preconcentration time was 1 h unless otherwise stated. Determination of the preconcentrated metals was accomplished by analysis of the receiver electrolyte. For measurement by atomic absorption spectrophotometry the electrolyte was aspirated directly into an air-acetylene flame. For separation and determination by chromatography 20 pL of the electrolyte was injected onto the column. The metals were eluted by use of a tartrate or oxalate buffer gradient. The mobile phase flow rate was 1 mL/min. Eluted metals were detected with a spectrophotometric reaction detection system based on the formation of colored metal complexes with 4-(2.pyridylazo)resorcinol (PAR). The metal-PAR complexes were monitored at 525 nm. The reaction detection system consisted of a peristaltic pump which delivered the PAR solution at a flow rate of 0.1 mL/min through 3/32 in. i.d. Tygon tubing to a zero dead volume Kel-F tee fitting. The column eluent entered the tee through 0.042 in. i.d. Teflon tubing at a 180' angle from the PAR solution fitting. The PAR and eluent then exibed the tee into a 0.5-m mixing coil composed of 0.042 in. i.d. Teflon tubing. The end of the coil was joined to the detector inlet. Apparatus. An Altex Model 420 liquid chromatograph (Altex Scientific Inc., Berkely, CA) with a Partisil 10 SCX l o p 4.6 mm X 25 cm cation-exchange column was used for the metal ion separations. The reaction detection system consisted of a Schoeffel Instruments Spectroflow Monitor SF770 variable wavelength detector (Schoeffel Instrument Corp., Westwood, NJ) and a Buchler polystaltic pump (Fisher Scientific). A Varian Techtron Model AA-6 atomic absorption spectrophotometer with a Model BC-9 simultaneous background corrector was used for the determination of metals by atomic absorption. The cation-exchange membranes were type P-1010 (RAI Research Corp., Hauppauge, Long Island, NY). The membranes were pretreated in a manner previously described (18) and were stored in water when not in use. Prior to preconcentration tlhe membranes were equilibrated with at least three fresh portions of receiver electrolyte over at least 1 h before use.
0003-2700/83/0355-1013$01.50/00 1983 Amerlcan Chemical Society
1014
ANALYTICAL CHEMISTRY, VOL. 55, NO. 7, JUNE 1983
“1 \
I e I
9 h
I O
~
t
n
0 2 i
I
- 05
01
Ionic
0:
IO
I
50
IO 2 0
Strenglh
Figure 2. Effect of ionic strength of injected sample on chromatographic performance: (0)relative peak area for 0.4 p g of NI, ( 0 ) height of void volume peak: sample volume, 20 WL; ionic strength adjusted with NaCI.
-
0
30 Time
(rr
n )
O21
Flgure 1. Deterioration of separation efficiency due to high salt sample
matrix: sample matrix, (top) deionized H,O, (bottom) 0.1 M MgS04; sample volume, 20 pLL; (1) 12 ppm Cu, (2)10 ppm Ni, (3) 20 ppm Zn, (4)12 ppm Co, (5) 185 ppm Cd; eluent, 1.5 mF-2.5 mF oxalate buffer at pH 3.0.
The PAR solution used in the reaction detection system consisted of 5 X M sodium 4-(2-pyridylazo)resorcinol(Aldrich Chemical Co. Milwaukee, WI) in a pH 9 ammonium hydroxide/ ammonium acetate buffer (6). This solution was stored in polyethylene and prepared fresh every 3-5 days. All chemicals used were analytical reagent grade. Water was doubly distilled and deionized. All sample and mobile phase solutions were filtered through 0.45-pm filters prior to use.
RESULTS AND DISCUSSION For preconcentration to occur by Donnan dialysis, the receiver electrolyte must be of higher ionic strength than the sample. To make the method generally applicable, it is necessary to have an ionic strength of the receiver electrolyte of at least 0.1 M ( 1 7 ) . I t has been previously demonstrated that a receiver electrolyte consisting of a mixture of MgS04 is ideal for preconcentrating cations by Donnan and A12(S04)3 dialysis (16,17,19). However, serious difficulties result when trying to apply Donnan dialysis preconcentration to ion chromatography because of the high salt content of the receiver electrolyte. The difficulty associated with the separation of trace levels of ions in a high ionic strength sample matrix is illustrated by the chromatograms in Figure 1. When the sample contains bulk ions, the chromatographic efficiency of the system is greatly diminished. When only low levels of analyte ions are present in the sample, the separation of the test ions can be achieved relatively easily. In the presence of large amounts of Mg only slight separation is possible. The phenomenon observed in Figure 1 appears to result from overloading of the ion exchange column due to the presence of the bulk ion. Figure 2 shows the data obtained in a more detailed study of this problem. The data show the effect of sample ionic strength on the relative chromatographic peak area of Ni and on the height of the peak which occurs a t the void volume. For dilute solutions the peak area remains constant and there is no peak observed at the column void volume. As the ionic strength of the sample increases, the peak area of Ni begins to decrease. Associated with this is the appearance of and an increase in the height of the void volume peak. This indicates that the metal is being washed off the column at the void volume and that at least part of the metal is unretained. At very high ionic strengths the measured peak area is very small while the void volume peak is quite large. The reduc-
A
0
30 Time ( m i n )
Flgure 3. Separation of preconcentrated sample: sample matrix, 0.1
M A12(S0,), at pH 0.5; sample volume, 20 wL; (1) 50 ppb Cu, (2) 25 ppb Ni, (3) 25 ppb Co, (4)125 ppb Cd, (5) 50 ppb Mn; eluent, 9 mF-16 mF tartrate buffer at pH 4.0.
tions in both resolution and quantitative recovery represent major difficulties with attempting to determine trace ions in high salt solutions by ion chromatography. In order to overcome the difficulties encountered with injection of high ionic strength samples, yet at the same time have a receiver electrolyte of sufficient ionic strength to allow Donnan dialysis, it was necessary to design the preconcentration and separation steps together. Proper selection of a receiver electrolyte and mobile phase eluent was required to allow successful application of Donnan dialysis preconcentration. The receiver electrolyte chosen was a solution of A1,(S04)3adjusted to pH 0.5 with H2S04. This was used in conjunction with an approximately 0.01 F tartrate buffer a t pH 4.0. Figure 3 shows the separation of five metals obtained by using the A12(S04)3/tartratesystem. Separation of all metals was achieved with gradient elution. This system was successful because the eluent acted to essentially eliminate the receiver electrolyte in the injected sample. Although the sample injected onto the column had a high proton concentration, the buffer capacity of the tartrate eluent reduced it to a much smaller value. Likewise, since Al(II1) forms a very strong anionic complex with tartrate, it is effectively eliminated from interacting with the cation-exchange column. The net result is that the sample appeared to be free of receiver electrolyte.
ANALYTICAL CHEMISTRY, VOL. 55, NO. 7, JUNE 1983
1
&I
1
Table I. Calibration of Various Metals with Donnan Dialysis Preconcentration linear regression data for enrichmenta detection limit,b slope intercept correlation metal PPb 2 1.7 0.995 68.0 cu
20
E FIO
i
Ni
1.73.6 3.57.9 38.9 76.0
co Cd Mn -4
0
-2
log F12(S04)3T
Flgure 4. Effecl: of aluminum sulfate concentration on enrlchment factor: all receiver electrolytes adjusted to pH 0.5 with H2S04;samples analyzed by atomic absorption spectrophotometry; preconcentratlon time, 1 h; receiver volume, 4.0 mL; enrichment factor, the ratio of the concentratlon of analyte in the receiver electrolyte after preconcentration divided by the original concentration in the sample.
standard solutions
10
RSD preconcentrated solutions
7
12
Ki
6
13
Cd
4 2
10
10
a
-
100
25
Mn 6 13 Data obtained a t a concentration for each metal approximately 20 times above the detection limit. Peak areas are measured manually,
1 50
0.9
Oo
c0
/
1.0
2!
--__
cU
E F
1.
0.996 0.999 0.995 0.999
Table 11. Influence of Donnan Dialysis Preconcentration on Reproducibility'
metal
-
1.4 1.5
receiver electrolyte: 0.1 M Al,(SO,), at pH 0.5 receiver volume: 0.5 mL enrichment time: 1 h a Chromatographic peak areas used for regression analysis. Based on analysis of 20 VL of receiver electrolvte.
150-1
100
11015
I50
200
T i r e (rntn)
Flgure 5. Time dependence on preconcentration: receiver electrolyte, 0.1 M AI,(SO,), aLt pH 0.5; receiver volume, 0.5 mL; sample, 50 ppb Go. The presence of Al(II1) has been shown to be critical if rapid preconcentration is desired (17,20). Figure 4 shows the effect of the A12(S04)3concentration on preconcentration into the Al(III)/H+ recieiver electrolyte. The enrichment factor increases as the amount of Al(II1) in the electrolyte increases. At Al(II1) concentrations greater than M the enrichment factor is approximately constant. On the basis of these data a receiver electrolyte consisting of 0.1 M Al,(SO& adjusted to pH 0.5 with H2S04was used in this study. This receiver electrolyte possesses an ionic strength high enough to allow it to be used with a wide variety of samples and allow high enrichments to be achieved in a reasonable time. It is worth noting that receiver electrolytes containing lower concentrations of A12(S0J3 could be used in many cases. The limiting factor for their use would be the ionic strength of the sample solution. Figure 5 shows the relationship between time and enrichment factor for preconcentration by Donnan dialysis prior to ion-chromatographic determination. Enrichments on the order of 80-fold can be achieved for 1-h preconcentration and well over 100-folldfor longer preconcentrations. The difference in enrichment factors obtained for the data in Figures 4 and 5 is due to the fact that a much larger volume of receiver electrolyte was used in the former than in the latter. A number of iatudies were performed in order to characterize the method for analysis. Table I shows the linear regression
.-
data obtained from calibration curves form 1 h preconcentrations of standard aqueous solutions containing Cu, Ni, Co(II), Cd, and Mn(I1). The calibration curves were linear over 2 orders of magnitude for all of the metals and the detection limits ranged from l ppb for Ni to 25 ppb for Cd. A number of variables have been investigated to determine their influence on the precision of Donnan dialysis preconcentration. These include temperature and stirring rate ( 2 7 ) , sample ionic strength (16),and the presence of phosphate (20), surfactants, arAdcomplexing agents (21) in the sample. These factors have been examined in detail and were not investigated here. In this study the precision of the method was investigated by examining the reproducibility of the system with and without Donnan dialysis preconcentration. These data are shown in Table 11. The average precision for a series of standard solutions analyzed on a single day is about 5%. The average precision for a series of preconcentrated samples analyzed on a single day is about 12%. The difference between these data can be attributed to the Donnan dialysis preconcentration. This is in agreement with the previously published data on the precision of Donnan dialysis. Donnan dialysis is a viable alternative to on-column techniques for preconcentration of trace ions prior to ion-chromatographic analysis. The technique and principle of matching the eluent with the receiver electrolyte should be applicable to the preconcentration of anions as well as cations. The advantages of simplicity, low cost, and high enrichment factors more than compensate for the somewhat reduced precision of the method and make Donnan dialysis ideal for routine analysis of large numbers of samples. Registry No. Cu, 7440-50-8; Xi, 7440-02-0; Co, 7440-48-4; Cd, '7440-43-9; Mn, '7439-96-5.
LITERATURE CITED (1)
Small, H.; Steven, T. S.;Bauman. W. C. Anal. Chem. 1975, 4 7 , 1801.
1016
Anal. Chem. 1983, 55, 1016-1019
(2) Maugh, T. H. Science 1980, 208, 164. (3) Nordmeyer, F. R.; Hansen, C. D.; Eatough, D. J.; Rollins. D. K.; Lamb, J. D. Anal. Chem. 1980, 52, 853. (4) Bond, A. M.; Wallace, G. G. Anal. Chem. 1982, 54, 1206. (5) Fritz, J. S.;Story, J. N. Anal. Chem. 197a, 4 6 , 825. (6) Jezorek, J. R.; Frelser, H. Anal. Chem. 1979, 57, 373. (7) Krull, I. S.;Lankmayr, E. P. Am. Lab. (FairfieM,Conn.) 1982, 74,18. (8) Molnar, I.; Knauer, H.; Wilk, D. J. Chromatogr. 1980, 207, 225. (9) Wetzel, R. A.; Anderson, C. A.; Schleicher, W.; Crook, G. D. Anal. Chem. 1979, 57, 1532. (10) Cassldy, R. M.; Elchuk, S. J. Chromafogr. Sci. 1980, 18,212. (11) Leyden, D. E.; Wegschelder, W. Anal. Chem. 1981, 53, 1059A. (12) Pensenstadler, D. F.; Fulmer, M. A. Anal. Chem. 1981, 53, 859A. (13) Cassidy, R. M.; Elchuk, S. J. Chromafogr. Scl. 1981, 19, 503.
(14) Smith, F. C., Jr.; Chang, R. C. CRC Crit. Rev. Anal. Chem. 1980, 9 , 197. (15) Cox, J. A.; Cheng, K. U. Anal. Chem. 1978, 50,601. (16) Wilson, R. L.; DINunzlo, J. E. Anal. Chem. 1981, 53, 692. (17) Cox, J. A.; DINunzlo, J. E. Anal. Chem. 1977, 4 9 , 1272. (18) Blaedel, W. J.; Klssel, T. R. Anal. Chem. 1972, 4 4 , 2109. (19) Cox, J. A.; Twardowski, 2 . Anal. Chem. 1980, 52, 1503. (20) DiNunzlo, J. E.; Wilson, R. L.; Gatchell, F. P. Talanta 1983, 30, 5 7 . (21) Cox, J. A.; Twardowski, 2 . Anal. Chim. Acta 1980, 719,39.
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
