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Preconcentration of an analyte dialysate in a flow-injection system
ANALYTICA
CHIMICA ACTA Analytica Chimica Acta 308 (1995) 214-221
Preconcentration
of an analyte dialysate in a flow-injection system J.F. van Staden *, C.J. Hattingh
Department of Chemistry, Unioersity of Pretoria, Pretoria 0002 South Africa
Received 3 June 1994; revised manuscript received 16 September 1994
Abstract Preconcentration of an analyte dialysate was studied in a flow-injection system where hyphenation of the dialysis/preconcentration was done on-line. The dependence of the combined dialysis/preconcentration process on a number of variables was evaluated and optimised. As timing played a very important role in the whole system, a time regulated system from a computer was employed to control various functions of the system. The system was applied to the determination of copper in food supplements containing vitamin-mineral conditioners for dogs and cats. Suitable results were obtained from a
calibration curve between 0.5 and 5 mg/l of soluble copper with a detection limit of 0.295 mg/l. The values obtained by the hyphenated dialysis/preconcentration/FIA system compared favourably with the standard manual atomic absorption spectrometric method and the value of 0.05% (m/m> given by the producers. A relative standard deviation of 5.74% was obtained. Keywords: Flow injection; Preconcentration; Dialysis
1. Introduction
The use of dialysis membranes in flow-through dialysers as part of flow-injection systems is extremely useful to remove suspended solids and macromolecules in samples. Dialysers, however, not only remove interferences but also serve as an automated diluter of the analyte. The nature of the membrane determines the features of the mass transfer across the membrane [l-4] and, therefore, the nature and size of the molecules and ions that pass through it. The classification of the dialysis process as passive or active (Donnan) also depends on the nature of the membrane [l].
* Corresponding
author.
0003.2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0003-2670(94)00479-X
In active (Donnan) dialysis, ions with a given charge are transferred across an ion-exchange membrane. Active or Donnan dialysis is a promising technique for the preconcentration, recovery and speciation of ionic species 15-161. There are, however, only a few publications available where Donnan dialysis as a preconcentrator were combined with flow-injection analysis (FIA). Koropchak and Allen [17] employed flow-injection Donnan dialysis as preconcentrator of cations for flame atomic absorption spectrophotometry (AAS). By using a similar set-up [17] Kasthurikrishnan and Koropchak [18] used flow-injection Donnan dialysis with inductively coupled plasma emission spectrometry. Berggren [ 191 used Donnan dialysis for the speciation of cadmium(H) in combination with differential-pulse anodic stripping voltammetry. The author developed
J.F. mn Staden, C.J. Hattingh/Analytica
a flow-injection/Donnan dialysis/differential pulse anodic stripping voltammetric system for the determination of free cadmium ions in solutions containing negatively charged and neutral organic cadmium(H) complexes. It is, however, not always possible to use Donnan dialysis. An alternative is to use passive dialysis. In the passive process a neutral membrane differentiates between separations on a molecular scale and with a preciseness of molecular order allowing species within a given range of molecular mass to diffuse across a neutral membrane. Passive dialysis is a highly efficient molecular filter, but is inefficient in terms of solute diffusion rates across a semi-permeable filter with an efficiency of only about O-7% [4]. Passive dialysis is, therefore, very suitable for controllable dilution. In some samples the extent of interferences, like undesired suspended solids and the presence of unwanted macromolecules (like for example protein), is of such a nature that the samples cannot be analyzed directly on a flow-injection system. Passive dialysis membranes offer a solution to this problem as it is ideally suitable for employment in an automated sample preparation and processing procedure before a final processed sample is delivered in a well-defined and interference-free condition for detection obviating manual operations which are time consuming, tedious, expensive and operator intensive when used in routine laboratories. There is, however, one main problem that may arise from this, and that is that in some samples the content of analyte may fall in a concentration range where the concentration of analyte in the dialysate is below the detection limit of a dialyser/FIA system. To circumvent this problem one must find a way to concentrate the analyte in the dialysate again after the dilution effect of the dialyser in the flow system. This is possible by applying a hyphenated dialysis/preconcentration technique in a dialysis/preconcentration/FIA system where the analyte zone in the dialysate is preconcentrated before processing it for detection. Dialysis was used as a sample pretreatment system and intermediate separation process for liquid chromatography (LC). The sample is dialysed and the dialysate may then be treated further in three different ways: (i) injected directly into the chromatograph; (ii) undergo derivatization or (iii) preconcentration before injection. Turnell and Cooper
Chimica Acta 308 (1995) 214-221
215
[20,21] selected the last two options in two pioneering papers in this regard. The group [20] introduced dialysis as an automated pretreatment procedure for amino acid analysis of biological samples prior to LC obviating manual tedious deproteinization of serum samples. The concept initiated by Turnell and Cooper [20,21] was used by the authors [22] as basis to automate the system further in a trademark for the automatic sequential trace enrichment of dialysates (ASTED). In doing so, elimination of high-molecular-weight components, using dialysis, maintains the performance of the chromatographic column whilst trace enrichment presents the diffusible analytes in a concentration form suitable for trace analysis. In doing so Turnell and Cooper [22] succeeded in providing an ASTED system as an effective sample preparation method for LC that is rapid, inexpensive and simple to operate. There are a number of methods available whereby samples may be preconcentrated before detection in a FIA system. Kuban et al. [23] used solvent extraction to preconcentrate copper in a flow-injection manifold before detection with flame AAS. Ion-exchange resins are generally used for on-line preconcentration of an analyte in FIA [24]. Carbonell et al. [2.5] compared a number of cation-exchange resins that were commercially available. So far dialysis was not used on-line as an intermediate in a FIA system where preconcentration of the analyte in the dialysate forms part of the same manifold system. In the FIA system described by Fernandez et al. [26] dialysis solutions were concentrated, but the dialyser did not form part of the flow-injection manifold. This paper reports on the study of preconcentration of an analyte dialysate where hyphenation of the dialysis/preconcentration was done on-line in an FIA system and the system applied for the determination of copper in food supplements for dogs and cats containing vitamin-mineral conditioners.
2. Experimental 2.1. Reagents
and solutions
All reagents were prepared from analytical-reagent grade chemicals unless specified otherwise. All aqueous solutions were prepared with doubly distilled, deionised water.
216
J.F. van Staden, C.J. Hattingh/Analytica
A stock standard copper(U) solution containing 1000 mg/l of copper ion was prepared by dissolving 7.859 g copper sulphate pentahydrate and diluting to 2 1 with water. Working standard copper(I1) solutions containing copper(U) ions in the range 0.5-20 mg/l were prepared by suitable dilution of the stock solution. The eluent consists of a 1.0 mol/l HCI, 1.0 mol/l HNO, and 0.1 mol/l NaCl solution. The buffer solution was prepared by dissolving 33.55 g ammonium acetate in 1500 ml deionised water. A 25% (v/v) ammonia solution was used to adjust the pH to 9.2. The solution was diluted to 2 1 with deionized water. The pH of the final solution was 8.7.
Pl Sample Donor --b
.g “’
*
Ll..
.I
’ W;ste
Fig. 1. Flow diagram tion/FIA system.
Dialyser unit A slightly modified version of a 12-in. Technicon AutoAnalyzer II unit was used for the dialysis process. The dialyser consisted of a 160 X 30 X 25 mm module (laboratory made as modified versions of 12-in. Technicon dialysers). The donor and acceptor channels of the dialyser unit consisted of semi-tubular grooves with an id. of 0.5 mm (i.e., 0.5 mm wide and 0.5 mm deep) and with a distance (width) of 15 mm between the entrance and exit sides of a channel. The pathlengths of the dialyser were 300 mm for both the donor and acceptor channels. The dialysis unit was equipped with a Technicon pre-mount dialysis membrane Type C. Ion-exchange column The ion-exchange column consisted of a 12 mm X 1.4 mm i.d. tube packed with a 16% crosslinked, styrene based sulphonic acid resin used in Dionex OnGuard-H pretreatment cartridges. This resin has a high affinity for multivalent cations and transition metals.
specmultiof 10 ray in a 0.5 flame
Waste
P2 1wm Buffer :..__ _____ _‘I., ,., _____ ‘d “4 ’ z. &““’ mn JI- 12Wrnrn 8 ID-0.635mm I _“, waste
cn \Di, I I
of the hyphenated
was used for samples zinc.
2.2. Instrumentation
Detector A Varian AA-1275 flame atomic absorption trometer was used as detector. A Varian 103 element hollow-cathode lamp, with a current mA, was used to give a monochromatic light the detector. A wavelength of 324.6 nm and mm slit width was used. An N,O-acetylene
Chimica Acta 308 (1995) 214-221
“2 “3 “4 Di
= valve2 = valve3 =vaIw4 =dlalyser
dialysis/preconcentra-
with a high concentration
of
2.3. Flow system A schematic diagram of the flow system used is given in Fig. 1. The manifold consisted of Tygon tubing (inside diameters given in Fig. 1) cut into the required lengths and wound around glass tubes with an o.d. of 10 mm. The dialyser and ion-exchange column were incorporated in succession in a hyphenated mode into the FIA manifold for dialysing and preconcentrating the sample before final detection. The following equipment also formed part of the FIA system: Cenco- and Gilson-Minipuls peristaltic pumps (operating at 10 rpm) were used to supply the different streams and VICI Valco lo-port multi-functional valves were used to channel the analyte in different forms into the various channels. The valves, peristaltic pumps and the AAS detector were coupled to a computer. The whole FIA system was controlled from the computer with a FlowTEK program [27]. The dashed line in Fig. 1 presents the flow in the FIA system when pump 2 (P2) is in operation and pump 3 (P3) is stopped. There was no instance where both pumps P2 and P3 were in operation at the same time. 2.4. Procedure The configuration of the FIA system is shown in Fig. 1. The pumps and valves operated as described below.
J.F. uan Staden, C.J. Hattingh/Analytica
and V4 were arranged in such a way that each one switched between two channels; valve V3 between the ion-exchange column and detector and valve V4 between the ion-exchange column and waste. Valves V3 and V4 were also time regulated with other parts of the system. The timing diagram for treating samples is outlined in Fig. 2. The system was switched on and allowed to run for about 5 min in order to equilibrate the flow dynamics of all parts throughout the whole system. The computer was then actuated and the timing sequence started in the load position which was only used in the beginning of a run. The following timing sequences were then followed with time regulated from the computer by the FlowTEK [27] program. 6) Starting of system Pumps: Pumps P2 and P3 were switched off. Pump Pl was actuated by the computer and was in operation. Valves: Valves Vl and V2 were in load position. Operation: Pump Pl was actuated by the computer for 354 s and the loop of
Operation of pumps and ualues. Pumps were either in operation or switched off. Two types of valves were used: valves for sampling purposes and valves to channel the analyte zone to different streams. A VICI Valco 10 port multi-functional injection valve (Vl in Fig. 1) was arranged in such a way that it serves as a sampling valve with a sample loop having a volume of 22 ml. In the load position, sample was aspirated through the loop to waste filling the whole 22 ml volume with sample. On switching to the inject position (which is time regulated with pumps Pl and P3) the donor stream was interrupted and part of the contents of the loop placed into the donor stream (with a time allocation of 510 s regulated via pump P3). A VICI Valco 10 port multi-functional injection valve (V2 in Fig. 1) was also arranged in such a way that it serves as a sampling valve with a sample loop having a volume of 1.000 ml. This valve was used to place 1.000 ml eluent in the buffer stream at certain time intervals to sweep and strip out the preconcentrated analyte from the column instantly and regenerate the column at the same time. The valve was time regulated with pumps P2 and P3 and valves V3 and Vi Valves V3
i
Sample 1
I
1Load
PI
;
P2
z
Vl v2 v3 v4
_I
217
Chimica Acta 308 (1995) 214-221
r-
Dialyrls\Preconcentration
/Delection( ._
I
Sample 2
1 Dlalyslo\Preconcentration
etectb
I
Inject Load
Inject Load Column Delector Waste Column
j!,
:: :
i:
‘I 0
1;
W w
384
:
v vv v v W 874 s3.7 1019 1099 ,228 Tzle 12%
v vv v v 1748 ,808 109.3 ,033 1973
/s
Fig. 2. Timing diagram of the hyphenated
dialysis/preconcentration/FIA
system.
218
(ii)
(iii)
(iv)
J.F.
valve Vl filled completely with sample aspirated at a flow rate of 3.9 ml/mm by the peristaltic pump through the valve. Valve V3 was channelled to the detector and valve V4 to waste. Dialysis/preconcentration At 354 s pump Pl was switched off, pump P3 was actuated and valve V3 channelled to the ion-exchange column in order to get the dialyser/preconcentration part to reach equilibrium. 10 s was allowed for this before the sampling valve Vl switched at 364 s to the inject position for a period of 510 s placing precisely 21.250 ml of the contents of the loop into the donor stream. This amount of analyte was allowed to be dialysed and preconcentrated for a period of 570 s. In the mean time at 874 s pump Pl was actuated to operate and valve Vl switched to the load position. Detection At 934 s pump P3 was switched off and pump P2 actuated to operate. Valve V3 was channelled to the detector and valve V4 to the ion-exchange column. A time of 25 s was allowed with both pump P3 in operation and valve V2 in the load position in order to fill the l.OOO-ml loop with eluent. At 959 s the sampling valve V2 was switched to the inject position for a period of 60 s placing precisely 1.000 ml of the eluent as a zone into the buffer stream transporting it to the ion-exchange column to strip backwards the preconcentrated analyte instantly to the detector and at the same time regenerating the ion-exchange column. At 1019 s injection valve V2 was switched to the load position. Completion of a run At 1099 s the sequence for sample 1 was completed when pump P2
Chimica Acta 308 (1995) 214-221
(v)
was switched off and valve V4 channelled to waste. At this stage sampling of the second sample was already in operation. The system proceeded with the timing program as illustrated in Fig. 2 for the next samples following the same sequences as outlined above.
3. Results and discussion The combined dialysis/preconcentration/FIA unit offers certain advantages over systems where the hyphenated dialysis/preconcentration components are split into more than one manifold using two separate FIA systems. The main advantage of the proposed system lies in the power of the combined features inherent to the system and as such the system optimization must be done where the dialysis and preconcentration components are both inclusive in one single FIA system. The evaluation and optimization of the dialysis part had been dealt with before [3,28] and are not repeated here. There are however a number of variables which influence the successful eluting of copper(U) dialysate from the preconcentrating column in the FIA manifold and this was studied. Fast stripping of the preconcentrated copper(U) ions from the column into the flow injection stream in a single small analyte bolus was very important in order to keep the contribution of this variable to dispersion to the absolute minimum. It was therefore necessary to study the type and concentration of eluent to be used. Different concentrations of HCl and HNO,, as well as the ratio between the two acids were compared. Peak height and peak width are two parameters which played an important role in the system in order to obtain accurate and precise results, peak height for sensitivity and peak width for sample throughput rate. The results are given in Table 1. It is clear from Table 1 that an eluent with a final concentration of 1 mol/l for both acids gave the best results with a relative peak height of 62 mm and a relative peak width of 6 mm. A higher concentration of acid decreased the reproducibility of the system and will damage the valve in the long run. It was
J.F. can Staden, C.J. Hattingh/Analytica Table 1 Determination
of the optimum eluent composition
[HCII: [HNO,]
Relative peak height (mm)
Relative peak width (mm)
l:o 0.7:o 1:0.5 1:l OS:1 0.1:O.l
40 32 58 62 54 11
14 21 8 6 9 45
therefore decided to use an eluent where the concentration of both acids were 1 mol/l. Most samples also contain sodium chloride as one of the additives. Although results show that the influence of NaCl was minimal, a solution of 0.1 mol/l NaCl was used in the final eluent to obtain optimum working conditions. The flow rate of the eluent, dimensions of the column and flow direction of the buffer streams in the preconcentrating and eluting mode also contribute to the efficiency of an ion-exchange column. In the work done by Kuban et al. [29], Fang and Welz [30] and Purohit and Devi [31] the authors concentrated on evaluation of the flow rate of the eluent and dimensions of the column. Smith and Chang [32] recommend columns with an internal diameter ranging from 1 to 3 mm. The authors also found that peak height increased with an increase in column length and a decrease in internal diameter of the column. A smaller internal diameter tended to decrease the dispersion. We found, however, that long columns with a small diameter tended to cause back pressure on the FIA system with a sharp decrease in accuracy and precision. With the resin in the Dionex OnGuard pretreatment cartridges a 12 mm X 1.4 mm i.d. column gave optimum results. Smith and Chang [32] also found that the flow rate of the eluent stream did not have a great effect on the effectiveness of the system. In the proposed combined dialysis/preconcentration FIA system the various parameters did not only have an influence on the effectiveness of the ion-exchange column, but also on the effectiveness of the dialyser. The influence of flow rate of the donor and acceptor stream, sample volume and time of dialysis were then investigated. A preliminary investigation showed that the above-mentioned parameters mutu-
Chimica Acta 308 (1995) 214-221
219
ally influenced each other and this had an overall effect on the performance of the whole system. At this stage the sample volume was optimised, fixed at exactly 22 ml and the time of dialysis chosen in such a way that the whole sample volume was first dialysed, the dialysate preconcentrated and then eluted. As seen later this did not give the best performance. The flow rate of the acceptor stream influences both the mass transfer across the membrane [3,28] and the preconcentration and elution process on the ion-exchange column and had to satisfy the prerequisites of both techniques at the same time. A flow rate of 1.4 ml/min gave the best results for both the dialyser and preconcentration/elution systems and was chosen for further work. The flow rate of the donor stream was varied between 0.6 and 3.9 ml/min and investigated. We found that the amount of copper(H) ions that dialysed, increased with a decrease in flow rate of the donor stream. The precision, however, decreased at the same time especially with the lower flow rates. A flow rate of 2.50 ml/min for the donor stream was chosen as the best condition for both the amount of copper(H) ions to be dialysed and precision, It was clear at this stage that timing also plays a very important role in the whole system and that a time regulated system from the computer could improve the performance. It was found previously [3,28] that the majority of mass transfer across a dialysis membrane of an analyte bolus took place at the front head part where the sample plug was more concentrated and that the rear part tended to give a tailing effect. The tailing effect influenced the preconcentration and elution steps of the combined system leading to a decrease in precision. We found that the precision decreased with longer dialysis times which confirmed the results obtained in the previous investigations [3,28]. As the system was very complex and the different parts and parameters were interdependent, these parts and parameters were optimised in a time-regulated system from the computer and after evaluation we found that the final timing sequences as outlined in the timing diagram in Fig. 2 gave the best results. It was found that the timing sequence for pumps Pl and P2 had to be arranged in such a way that the sampling loop of exactly 22 ml had to be filled completely, but that only 21.250 ml of the sample
220
J.F. can &den,
C.J. Hattingh /Anaiytica
should be placed into the donor stream in order to get the best precision and accuracy. A period of 570 s gave the optimum time for dialysing and preconcentrating the sampling zone. The time allocated for stripping and regenerating of the ion-exchange column together with the zone of 1.000 ml of eluent containing 1 mol/l HCl, 1 mol/l HNO, and 0.1 mol/l NaCl for this purpose was sufficient to obtain reliable precision and accuracy and for optimum performance of the whole system. The direction of preconcentration and elution through the ion-exchange column was also an important factor to consider in sample dispersion and peak width in the signal output obtained. A drastic increase in precision of results was found when the flow direction of the buffer streams for preconcentration and elution opposed each other. Copper ions from the diaiysate are preconcentrated at the entrance of the column. If the flow direction of the buffer stream is the same for the elution step, the copper(I1) ions in the column will be spread through the column before elution. This increases the dispersion. If the flow direction for elution is changed to oppose the direction of preconcentration, the copper(I1) ions will be stripped from the concentrated side in a more concentrated plug and fed to the detector. The complexity of the system also had a major influence on the linearity of the calibration curve. A non-linear calibration curve for copper(I1) ion solutions between 0.5 and 20 mg/l was obtained under optimum experimental conditions as outlined in Fig. 3. There are two regions where the calibration graph tended to give linearity, although it was still non-linear. These were between 5-20 mg/l and 0.5-5 mg/l (Fig. 3). The linear regression equation for 0.5-5 mg/l, obtained by the method described by McCormick and Roach [33], was y = 0.24791~ +
Table 2 Performance supplements
Chimica Acta 308 (199s) 214-221
002-
Fig. 3. Calibration
graph for copper(H) between 0.5 and 20 mg/l.
0.75935; r = 0.9989 (n = 3), with y = peak height and x = copper(I1) ion concentration in mg/l. The system was then applied to the determination of copper in food supplements containing vitaminmineral conditioners for dogs and cats. About 2-g samples of food supplements were mixed with water to obtain a slurry and shaken for 30 min. The slurry was diluted with buffer solution to 1000 ml to give a solution with insoluble particles suspended through the whole mixture. The solution mixtures were left overnight before analyses. The sample solutions were injected directly into the system. The samples were also analyzed directly by AAS after centrifugation and filtration several times manually which is very operator intensive. The performance of the proposed method is given in Table 2. The values obtained by the proposed hyphenated dialysis/preconcentration/ FIA system compares favourably with the standard manual AAS method as can be seen from Table 2. It is also in good agreement with the value of 0.05% (m/m) given by the manufacturer. The precision of the method was determined through 15 repet-
and reproducibility of the hyphenated dialysis/preconcentration/FIA containing vitamin-mineral conditioners for dogs and cats
system
for the determination
of copper
Sample No.
Standard AAS method (%, m/m)
FIA method (%, m/m)
R.S.D. (%) a
1 2 3 4
0.047 0.045 0.046 0.046
0.045 0.043 0.046 0.044
5.74 5.41 5.22 5.01
a n = 15 for the proposed
FIA method.
in food
J.F. L~CIII Staden, C.J. Hattingh/Analytica
itive analyses of a number of samples as given in Table 2. The method provided satisfactory precision as deduced from the calculated standard deviation values. The detection limit was calculated as 0.295 mg/l for the calibration graph (Fig. 3) using the methods given by Miller and Miller [34] giving a detection limit of 0.015% (m/m) on a 2-g dog food sample treated as above.
4. Conclusions In a study of preconcentration in a flow-injection system where hyphenation of the dialysis/preconcentration was done on-line, the dependence of the combined dialysis process on a number of variables was evaluated and optimised. As timing played a very important role in the whole system, a time-regulated system from a computer was employed to control the various functions of the system. The proposed system is suitable for the determination of copper in food supplements containing vitaminmineral conditioners for dogs and cats, and satisfactory results were obtained between 0.5 and 5 mg/l of soluble copper in the injected samples. The detection limit was 0.295 mg/l (0.015% (m/m) in the food sample).
References [II M. Valcarcel
and M.D. Luque de Castro, Non-chromatographic Continuous Separation Techniques, Royal Society of Chemistry, London, 1991. I21 J.K. Foreman and P.B. Stockwell, Automatic Chemical Analysis, Ellis Horwood, Chichester, 1975. t31 J.F. van Staden and A. van Renshurg, Analyst, 115 (1990) 1049. 141 J.F. van Staden, Fresenius’ J. Anal. Chem., (1994) in press. [51 R.M. Wallace, Ind. Eng. Chem. Proc. Design Div., 6 (1967) 423. l61 J.A. Cox and J.E. DiNunzio, Anal. Chem., 49 (1977) 1272. (71 J.A. Cox and 2. Twardowski, Anal. Chem., 52 (1980) 1503.
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221
[S] J.A. Cox and Z. Twardowski, Anal. Chim. Acta, 119 (1980) 39. [9] R.L. Wilson and J.E. Di Nunzio, Anal. Chem., 53 (1980) 692. [lo] J.A. Cox, E. Olbrych and K. Brajter, Anal. Chem., 53 (1981) 1308. [ll] J.A. Cox, T. Gray, K.S. Yoon, Y. Kim and Z. Twardowski, Analyst, 109 (1984) 1603. [12] J.A. Cox, K. Slonawska, D.K. Gatchell and A.G. Hiebert, Anal. Chem., 56 (1984) 650. [13] J.A. Cox, K. Slonawska and K. Brajter, Anal. Chem., 57 (1985) 2403. [14] J.A. Cox and N. Tanaka, Anal. Chem., 57 (1985) 2370. [15] K. Brajter and K. Slonawska, Mikrochim. Acta, 1 (1986) 231. [16] T.C. Chuang, Y.K. Lin and C.Y. Chen, J. Membrane Sci., 37 (1988) 131. [17] J.A. Koropchak and L. Allen, Anal. Chem., 61 (1989) 1410. [18] N. Kasthurikrishnan and J.A. Koropchak, Anal. Chem., 65 (1993) 857. [19] D. Berggren, Int. J. Environ. Anal. Chem., 41 (1990) 133. [20] D.C. Turnell and J.D.H. Cooper, J. Autom. Chem., 7 (1985) 177. [21] J.D.H. Cooper and DC. Turnell, J. Autom. Chem. 7 (1985) 181. 1221 D.C. Turnell and J.D.H. Cooper, J. Autom. Chem., 8 (1986) 151. 1231 V. Kuban, J. Komarek and D. Cajkova, Collect. Czech. Chem. Commun., 54 (1989) 2683. [24] Z. Fang, Flow Injection Separation and Preconcentration, VCH, Weinheim, 1993. [25] V. Carbonell, A. Salvador and M. de la Guardia, Fresenius’ J. Anal Chem., 342 (1992) 529. [26] P. Fernandez, C. Peres Conde, A. Gutierrez and C. Camara, Talanta, 38 (1991) 1387. [27] G.D. Marshall and J.F. van Staden, Anal. Instrum., 20 (1992) 79. [28] J.F. van Staden and C.J. Hattingh, Anal. Chim. Acta, submitted for publication. 1291 V. Kuban, J. Komarek and Z. Zdrahal, Collect. Czech. Chem. Commun., 54 (1989) 1785. [30] Z. Fang and B. Welz, J. Anal. At. Spectrosc., 4 (1989) 543. 1311 R. Purohit and S. Devi, Talanta, 38 (1991) 753. [32] F.C. Smith and R.C. Chang, The Practice of Ion Chromatog raphy, Wiley, New York, 1983. [33] D. McCormick and A. Roach, Measurement, Statistics and Computation: Analytical Chemistry by Open Learning, Wiley, Chichester, 1987. [34] J.C. Miller and J.N. Miller, Statistics for Analytical Chemistry, 2nd edn, Ellis Horwood, Chichester, 1988.
CHIMICA ACTA Analytica Chimica Acta 308 (1995) 214-221
Preconcentration
of an analyte dialysate in a flow-injection system J.F. van Staden *, C.J. Hattingh
Department of Chemistry, Unioersity of Pretoria, Pretoria 0002 South Africa
Received 3 June 1994; revised manuscript received 16 September 1994
Abstract Preconcentration of an analyte dialysate was studied in a flow-injection system where hyphenation of the dialysis/preconcentration was done on-line. The dependence of the combined dialysis/preconcentration process on a number of variables was evaluated and optimised. As timing played a very important role in the whole system, a time regulated system from a computer was employed to control various functions of the system. The system was applied to the determination of copper in food supplements containing vitamin-mineral conditioners for dogs and cats. Suitable results were obtained from a
calibration curve between 0.5 and 5 mg/l of soluble copper with a detection limit of 0.295 mg/l. The values obtained by the hyphenated dialysis/preconcentration/FIA system compared favourably with the standard manual atomic absorption spectrometric method and the value of 0.05% (m/m> given by the producers. A relative standard deviation of 5.74% was obtained. Keywords: Flow injection; Preconcentration; Dialysis
1. Introduction
The use of dialysis membranes in flow-through dialysers as part of flow-injection systems is extremely useful to remove suspended solids and macromolecules in samples. Dialysers, however, not only remove interferences but also serve as an automated diluter of the analyte. The nature of the membrane determines the features of the mass transfer across the membrane [l-4] and, therefore, the nature and size of the molecules and ions that pass through it. The classification of the dialysis process as passive or active (Donnan) also depends on the nature of the membrane [l].
* Corresponding
author.
0003.2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0003-2670(94)00479-X
In active (Donnan) dialysis, ions with a given charge are transferred across an ion-exchange membrane. Active or Donnan dialysis is a promising technique for the preconcentration, recovery and speciation of ionic species 15-161. There are, however, only a few publications available where Donnan dialysis as a preconcentrator were combined with flow-injection analysis (FIA). Koropchak and Allen [17] employed flow-injection Donnan dialysis as preconcentrator of cations for flame atomic absorption spectrophotometry (AAS). By using a similar set-up [17] Kasthurikrishnan and Koropchak [18] used flow-injection Donnan dialysis with inductively coupled plasma emission spectrometry. Berggren [ 191 used Donnan dialysis for the speciation of cadmium(H) in combination with differential-pulse anodic stripping voltammetry. The author developed
J.F. mn Staden, C.J. Hattingh/Analytica
a flow-injection/Donnan dialysis/differential pulse anodic stripping voltammetric system for the determination of free cadmium ions in solutions containing negatively charged and neutral organic cadmium(H) complexes. It is, however, not always possible to use Donnan dialysis. An alternative is to use passive dialysis. In the passive process a neutral membrane differentiates between separations on a molecular scale and with a preciseness of molecular order allowing species within a given range of molecular mass to diffuse across a neutral membrane. Passive dialysis is a highly efficient molecular filter, but is inefficient in terms of solute diffusion rates across a semi-permeable filter with an efficiency of only about O-7% [4]. Passive dialysis is, therefore, very suitable for controllable dilution. In some samples the extent of interferences, like undesired suspended solids and the presence of unwanted macromolecules (like for example protein), is of such a nature that the samples cannot be analyzed directly on a flow-injection system. Passive dialysis membranes offer a solution to this problem as it is ideally suitable for employment in an automated sample preparation and processing procedure before a final processed sample is delivered in a well-defined and interference-free condition for detection obviating manual operations which are time consuming, tedious, expensive and operator intensive when used in routine laboratories. There is, however, one main problem that may arise from this, and that is that in some samples the content of analyte may fall in a concentration range where the concentration of analyte in the dialysate is below the detection limit of a dialyser/FIA system. To circumvent this problem one must find a way to concentrate the analyte in the dialysate again after the dilution effect of the dialyser in the flow system. This is possible by applying a hyphenated dialysis/preconcentration technique in a dialysis/preconcentration/FIA system where the analyte zone in the dialysate is preconcentrated before processing it for detection. Dialysis was used as a sample pretreatment system and intermediate separation process for liquid chromatography (LC). The sample is dialysed and the dialysate may then be treated further in three different ways: (i) injected directly into the chromatograph; (ii) undergo derivatization or (iii) preconcentration before injection. Turnell and Cooper
Chimica Acta 308 (1995) 214-221
215
[20,21] selected the last two options in two pioneering papers in this regard. The group [20] introduced dialysis as an automated pretreatment procedure for amino acid analysis of biological samples prior to LC obviating manual tedious deproteinization of serum samples. The concept initiated by Turnell and Cooper [20,21] was used by the authors [22] as basis to automate the system further in a trademark for the automatic sequential trace enrichment of dialysates (ASTED). In doing so, elimination of high-molecular-weight components, using dialysis, maintains the performance of the chromatographic column whilst trace enrichment presents the diffusible analytes in a concentration form suitable for trace analysis. In doing so Turnell and Cooper [22] succeeded in providing an ASTED system as an effective sample preparation method for LC that is rapid, inexpensive and simple to operate. There are a number of methods available whereby samples may be preconcentrated before detection in a FIA system. Kuban et al. [23] used solvent extraction to preconcentrate copper in a flow-injection manifold before detection with flame AAS. Ion-exchange resins are generally used for on-line preconcentration of an analyte in FIA [24]. Carbonell et al. [2.5] compared a number of cation-exchange resins that were commercially available. So far dialysis was not used on-line as an intermediate in a FIA system where preconcentration of the analyte in the dialysate forms part of the same manifold system. In the FIA system described by Fernandez et al. [26] dialysis solutions were concentrated, but the dialyser did not form part of the flow-injection manifold. This paper reports on the study of preconcentration of an analyte dialysate where hyphenation of the dialysis/preconcentration was done on-line in an FIA system and the system applied for the determination of copper in food supplements for dogs and cats containing vitamin-mineral conditioners.
2. Experimental 2.1. Reagents
and solutions
All reagents were prepared from analytical-reagent grade chemicals unless specified otherwise. All aqueous solutions were prepared with doubly distilled, deionised water.
216
J.F. van Staden, C.J. Hattingh/Analytica
A stock standard copper(U) solution containing 1000 mg/l of copper ion was prepared by dissolving 7.859 g copper sulphate pentahydrate and diluting to 2 1 with water. Working standard copper(I1) solutions containing copper(U) ions in the range 0.5-20 mg/l were prepared by suitable dilution of the stock solution. The eluent consists of a 1.0 mol/l HCI, 1.0 mol/l HNO, and 0.1 mol/l NaCl solution. The buffer solution was prepared by dissolving 33.55 g ammonium acetate in 1500 ml deionised water. A 25% (v/v) ammonia solution was used to adjust the pH to 9.2. The solution was diluted to 2 1 with deionized water. The pH of the final solution was 8.7.
Pl Sample Donor --b
.g “’
*
Ll..
.I
’ W;ste
Fig. 1. Flow diagram tion/FIA system.
Dialyser unit A slightly modified version of a 12-in. Technicon AutoAnalyzer II unit was used for the dialysis process. The dialyser consisted of a 160 X 30 X 25 mm module (laboratory made as modified versions of 12-in. Technicon dialysers). The donor and acceptor channels of the dialyser unit consisted of semi-tubular grooves with an id. of 0.5 mm (i.e., 0.5 mm wide and 0.5 mm deep) and with a distance (width) of 15 mm between the entrance and exit sides of a channel. The pathlengths of the dialyser were 300 mm for both the donor and acceptor channels. The dialysis unit was equipped with a Technicon pre-mount dialysis membrane Type C. Ion-exchange column The ion-exchange column consisted of a 12 mm X 1.4 mm i.d. tube packed with a 16% crosslinked, styrene based sulphonic acid resin used in Dionex OnGuard-H pretreatment cartridges. This resin has a high affinity for multivalent cations and transition metals.
specmultiof 10 ray in a 0.5 flame
Waste
P2 1wm Buffer :..__ _____ _‘I., ,., _____ ‘d “4 ’ z. &““’ mn JI- 12Wrnrn 8 ID-0.635mm I _“, waste
cn \Di, I I
of the hyphenated
was used for samples zinc.
2.2. Instrumentation
Detector A Varian AA-1275 flame atomic absorption trometer was used as detector. A Varian 103 element hollow-cathode lamp, with a current mA, was used to give a monochromatic light the detector. A wavelength of 324.6 nm and mm slit width was used. An N,O-acetylene
Chimica Acta 308 (1995) 214-221
“2 “3 “4 Di
= valve2 = valve3 =vaIw4 =dlalyser
dialysis/preconcentra-
with a high concentration
of
2.3. Flow system A schematic diagram of the flow system used is given in Fig. 1. The manifold consisted of Tygon tubing (inside diameters given in Fig. 1) cut into the required lengths and wound around glass tubes with an o.d. of 10 mm. The dialyser and ion-exchange column were incorporated in succession in a hyphenated mode into the FIA manifold for dialysing and preconcentrating the sample before final detection. The following equipment also formed part of the FIA system: Cenco- and Gilson-Minipuls peristaltic pumps (operating at 10 rpm) were used to supply the different streams and VICI Valco lo-port multi-functional valves were used to channel the analyte in different forms into the various channels. The valves, peristaltic pumps and the AAS detector were coupled to a computer. The whole FIA system was controlled from the computer with a FlowTEK program [27]. The dashed line in Fig. 1 presents the flow in the FIA system when pump 2 (P2) is in operation and pump 3 (P3) is stopped. There was no instance where both pumps P2 and P3 were in operation at the same time. 2.4. Procedure The configuration of the FIA system is shown in Fig. 1. The pumps and valves operated as described below.
J.F. uan Staden, C.J. Hattingh/Analytica
and V4 were arranged in such a way that each one switched between two channels; valve V3 between the ion-exchange column and detector and valve V4 between the ion-exchange column and waste. Valves V3 and V4 were also time regulated with other parts of the system. The timing diagram for treating samples is outlined in Fig. 2. The system was switched on and allowed to run for about 5 min in order to equilibrate the flow dynamics of all parts throughout the whole system. The computer was then actuated and the timing sequence started in the load position which was only used in the beginning of a run. The following timing sequences were then followed with time regulated from the computer by the FlowTEK [27] program. 6) Starting of system Pumps: Pumps P2 and P3 were switched off. Pump Pl was actuated by the computer and was in operation. Valves: Valves Vl and V2 were in load position. Operation: Pump Pl was actuated by the computer for 354 s and the loop of
Operation of pumps and ualues. Pumps were either in operation or switched off. Two types of valves were used: valves for sampling purposes and valves to channel the analyte zone to different streams. A VICI Valco 10 port multi-functional injection valve (Vl in Fig. 1) was arranged in such a way that it serves as a sampling valve with a sample loop having a volume of 22 ml. In the load position, sample was aspirated through the loop to waste filling the whole 22 ml volume with sample. On switching to the inject position (which is time regulated with pumps Pl and P3) the donor stream was interrupted and part of the contents of the loop placed into the donor stream (with a time allocation of 510 s regulated via pump P3). A VICI Valco 10 port multi-functional injection valve (V2 in Fig. 1) was also arranged in such a way that it serves as a sampling valve with a sample loop having a volume of 1.000 ml. This valve was used to place 1.000 ml eluent in the buffer stream at certain time intervals to sweep and strip out the preconcentrated analyte from the column instantly and regenerate the column at the same time. The valve was time regulated with pumps P2 and P3 and valves V3 and Vi Valves V3
i
Sample 1
I
1Load
PI
;
P2
z
Vl v2 v3 v4
_I
217
Chimica Acta 308 (1995) 214-221
r-
Dialyrls\Preconcentration
/Delection( ._
I
Sample 2
1 Dlalyslo\Preconcentration
etectb
I
Inject Load
Inject Load Column Delector Waste Column
j!,
:: :
i:
‘I 0
1;
W w
384
:
v vv v v W 874 s3.7 1019 1099 ,228 Tzle 12%
v vv v v 1748 ,808 109.3 ,033 1973
/s
Fig. 2. Timing diagram of the hyphenated
dialysis/preconcentration/FIA
system.
218
(ii)
(iii)
(iv)
J.F.
valve Vl filled completely with sample aspirated at a flow rate of 3.9 ml/mm by the peristaltic pump through the valve. Valve V3 was channelled to the detector and valve V4 to waste. Dialysis/preconcentration At 354 s pump Pl was switched off, pump P3 was actuated and valve V3 channelled to the ion-exchange column in order to get the dialyser/preconcentration part to reach equilibrium. 10 s was allowed for this before the sampling valve Vl switched at 364 s to the inject position for a period of 510 s placing precisely 21.250 ml of the contents of the loop into the donor stream. This amount of analyte was allowed to be dialysed and preconcentrated for a period of 570 s. In the mean time at 874 s pump Pl was actuated to operate and valve Vl switched to the load position. Detection At 934 s pump P3 was switched off and pump P2 actuated to operate. Valve V3 was channelled to the detector and valve V4 to the ion-exchange column. A time of 25 s was allowed with both pump P3 in operation and valve V2 in the load position in order to fill the l.OOO-ml loop with eluent. At 959 s the sampling valve V2 was switched to the inject position for a period of 60 s placing precisely 1.000 ml of the eluent as a zone into the buffer stream transporting it to the ion-exchange column to strip backwards the preconcentrated analyte instantly to the detector and at the same time regenerating the ion-exchange column. At 1019 s injection valve V2 was switched to the load position. Completion of a run At 1099 s the sequence for sample 1 was completed when pump P2
Chimica Acta 308 (1995) 214-221
(v)
was switched off and valve V4 channelled to waste. At this stage sampling of the second sample was already in operation. The system proceeded with the timing program as illustrated in Fig. 2 for the next samples following the same sequences as outlined above.
3. Results and discussion The combined dialysis/preconcentration/FIA unit offers certain advantages over systems where the hyphenated dialysis/preconcentration components are split into more than one manifold using two separate FIA systems. The main advantage of the proposed system lies in the power of the combined features inherent to the system and as such the system optimization must be done where the dialysis and preconcentration components are both inclusive in one single FIA system. The evaluation and optimization of the dialysis part had been dealt with before [3,28] and are not repeated here. There are however a number of variables which influence the successful eluting of copper(U) dialysate from the preconcentrating column in the FIA manifold and this was studied. Fast stripping of the preconcentrated copper(U) ions from the column into the flow injection stream in a single small analyte bolus was very important in order to keep the contribution of this variable to dispersion to the absolute minimum. It was therefore necessary to study the type and concentration of eluent to be used. Different concentrations of HCl and HNO,, as well as the ratio between the two acids were compared. Peak height and peak width are two parameters which played an important role in the system in order to obtain accurate and precise results, peak height for sensitivity and peak width for sample throughput rate. The results are given in Table 1. It is clear from Table 1 that an eluent with a final concentration of 1 mol/l for both acids gave the best results with a relative peak height of 62 mm and a relative peak width of 6 mm. A higher concentration of acid decreased the reproducibility of the system and will damage the valve in the long run. It was
J.F. can Staden, C.J. Hattingh/Analytica Table 1 Determination
of the optimum eluent composition
[HCII: [HNO,]
Relative peak height (mm)
Relative peak width (mm)
l:o 0.7:o 1:0.5 1:l OS:1 0.1:O.l
40 32 58 62 54 11
14 21 8 6 9 45
therefore decided to use an eluent where the concentration of both acids were 1 mol/l. Most samples also contain sodium chloride as one of the additives. Although results show that the influence of NaCl was minimal, a solution of 0.1 mol/l NaCl was used in the final eluent to obtain optimum working conditions. The flow rate of the eluent, dimensions of the column and flow direction of the buffer streams in the preconcentrating and eluting mode also contribute to the efficiency of an ion-exchange column. In the work done by Kuban et al. [29], Fang and Welz [30] and Purohit and Devi [31] the authors concentrated on evaluation of the flow rate of the eluent and dimensions of the column. Smith and Chang [32] recommend columns with an internal diameter ranging from 1 to 3 mm. The authors also found that peak height increased with an increase in column length and a decrease in internal diameter of the column. A smaller internal diameter tended to decrease the dispersion. We found, however, that long columns with a small diameter tended to cause back pressure on the FIA system with a sharp decrease in accuracy and precision. With the resin in the Dionex OnGuard pretreatment cartridges a 12 mm X 1.4 mm i.d. column gave optimum results. Smith and Chang [32] also found that the flow rate of the eluent stream did not have a great effect on the effectiveness of the system. In the proposed combined dialysis/preconcentration FIA system the various parameters did not only have an influence on the effectiveness of the ion-exchange column, but also on the effectiveness of the dialyser. The influence of flow rate of the donor and acceptor stream, sample volume and time of dialysis were then investigated. A preliminary investigation showed that the above-mentioned parameters mutu-
Chimica Acta 308 (1995) 214-221
219
ally influenced each other and this had an overall effect on the performance of the whole system. At this stage the sample volume was optimised, fixed at exactly 22 ml and the time of dialysis chosen in such a way that the whole sample volume was first dialysed, the dialysate preconcentrated and then eluted. As seen later this did not give the best performance. The flow rate of the acceptor stream influences both the mass transfer across the membrane [3,28] and the preconcentration and elution process on the ion-exchange column and had to satisfy the prerequisites of both techniques at the same time. A flow rate of 1.4 ml/min gave the best results for both the dialyser and preconcentration/elution systems and was chosen for further work. The flow rate of the donor stream was varied between 0.6 and 3.9 ml/min and investigated. We found that the amount of copper(H) ions that dialysed, increased with a decrease in flow rate of the donor stream. The precision, however, decreased at the same time especially with the lower flow rates. A flow rate of 2.50 ml/min for the donor stream was chosen as the best condition for both the amount of copper(H) ions to be dialysed and precision, It was clear at this stage that timing also plays a very important role in the whole system and that a time regulated system from the computer could improve the performance. It was found previously [3,28] that the majority of mass transfer across a dialysis membrane of an analyte bolus took place at the front head part where the sample plug was more concentrated and that the rear part tended to give a tailing effect. The tailing effect influenced the preconcentration and elution steps of the combined system leading to a decrease in precision. We found that the precision decreased with longer dialysis times which confirmed the results obtained in the previous investigations [3,28]. As the system was very complex and the different parts and parameters were interdependent, these parts and parameters were optimised in a time-regulated system from the computer and after evaluation we found that the final timing sequences as outlined in the timing diagram in Fig. 2 gave the best results. It was found that the timing sequence for pumps Pl and P2 had to be arranged in such a way that the sampling loop of exactly 22 ml had to be filled completely, but that only 21.250 ml of the sample
220
J.F. can &den,
C.J. Hattingh /Anaiytica
should be placed into the donor stream in order to get the best precision and accuracy. A period of 570 s gave the optimum time for dialysing and preconcentrating the sampling zone. The time allocated for stripping and regenerating of the ion-exchange column together with the zone of 1.000 ml of eluent containing 1 mol/l HCl, 1 mol/l HNO, and 0.1 mol/l NaCl for this purpose was sufficient to obtain reliable precision and accuracy and for optimum performance of the whole system. The direction of preconcentration and elution through the ion-exchange column was also an important factor to consider in sample dispersion and peak width in the signal output obtained. A drastic increase in precision of results was found when the flow direction of the buffer streams for preconcentration and elution opposed each other. Copper ions from the diaiysate are preconcentrated at the entrance of the column. If the flow direction of the buffer stream is the same for the elution step, the copper(I1) ions in the column will be spread through the column before elution. This increases the dispersion. If the flow direction for elution is changed to oppose the direction of preconcentration, the copper(I1) ions will be stripped from the concentrated side in a more concentrated plug and fed to the detector. The complexity of the system also had a major influence on the linearity of the calibration curve. A non-linear calibration curve for copper(I1) ion solutions between 0.5 and 20 mg/l was obtained under optimum experimental conditions as outlined in Fig. 3. There are two regions where the calibration graph tended to give linearity, although it was still non-linear. These were between 5-20 mg/l and 0.5-5 mg/l (Fig. 3). The linear regression equation for 0.5-5 mg/l, obtained by the method described by McCormick and Roach [33], was y = 0.24791~ +
Table 2 Performance supplements
Chimica Acta 308 (199s) 214-221
002-
Fig. 3. Calibration
graph for copper(H) between 0.5 and 20 mg/l.
0.75935; r = 0.9989 (n = 3), with y = peak height and x = copper(I1) ion concentration in mg/l. The system was then applied to the determination of copper in food supplements containing vitaminmineral conditioners for dogs and cats. About 2-g samples of food supplements were mixed with water to obtain a slurry and shaken for 30 min. The slurry was diluted with buffer solution to 1000 ml to give a solution with insoluble particles suspended through the whole mixture. The solution mixtures were left overnight before analyses. The sample solutions were injected directly into the system. The samples were also analyzed directly by AAS after centrifugation and filtration several times manually which is very operator intensive. The performance of the proposed method is given in Table 2. The values obtained by the proposed hyphenated dialysis/preconcentration/ FIA system compares favourably with the standard manual AAS method as can be seen from Table 2. It is also in good agreement with the value of 0.05% (m/m) given by the manufacturer. The precision of the method was determined through 15 repet-
and reproducibility of the hyphenated dialysis/preconcentration/FIA containing vitamin-mineral conditioners for dogs and cats
system
for the determination
of copper
Sample No.
Standard AAS method (%, m/m)
FIA method (%, m/m)
R.S.D. (%) a
1 2 3 4
0.047 0.045 0.046 0.046
0.045 0.043 0.046 0.044
5.74 5.41 5.22 5.01
a n = 15 for the proposed
FIA method.
in food
J.F. L~CIII Staden, C.J. Hattingh/Analytica
itive analyses of a number of samples as given in Table 2. The method provided satisfactory precision as deduced from the calculated standard deviation values. The detection limit was calculated as 0.295 mg/l for the calibration graph (Fig. 3) using the methods given by Miller and Miller [34] giving a detection limit of 0.015% (m/m) on a 2-g dog food sample treated as above.
4. Conclusions In a study of preconcentration in a flow-injection system where hyphenation of the dialysis/preconcentration was done on-line, the dependence of the combined dialysis process on a number of variables was evaluated and optimised. As timing played a very important role in the whole system, a time-regulated system from a computer was employed to control the various functions of the system. The proposed system is suitable for the determination of copper in food supplements containing vitaminmineral conditioners for dogs and cats, and satisfactory results were obtained between 0.5 and 5 mg/l of soluble copper in the injected samples. The detection limit was 0.295 mg/l (0.015% (m/m) in the food sample).
References [II M. Valcarcel
and M.D. Luque de Castro, Non-chromatographic Continuous Separation Techniques, Royal Society of Chemistry, London, 1991. I21 J.K. Foreman and P.B. Stockwell, Automatic Chemical Analysis, Ellis Horwood, Chichester, 1975. t31 J.F. van Staden and A. van Renshurg, Analyst, 115 (1990) 1049. 141 J.F. van Staden, Fresenius’ J. Anal. Chem., (1994) in press. [51 R.M. Wallace, Ind. Eng. Chem. Proc. Design Div., 6 (1967) 423. l61 J.A. Cox and J.E. DiNunzio, Anal. Chem., 49 (1977) 1272. (71 J.A. Cox and 2. Twardowski, Anal. Chem., 52 (1980) 1503.
Chimica Acta 308 (1995) 214-221
221
[S] J.A. Cox and Z. Twardowski, Anal. Chim. Acta, 119 (1980) 39. [9] R.L. Wilson and J.E. Di Nunzio, Anal. Chem., 53 (1980) 692. [lo] J.A. Cox, E. Olbrych and K. Brajter, Anal. Chem., 53 (1981) 1308. [ll] J.A. Cox, T. Gray, K.S. Yoon, Y. Kim and Z. Twardowski, Analyst, 109 (1984) 1603. [12] J.A. Cox, K. Slonawska, D.K. Gatchell and A.G. Hiebert, Anal. Chem., 56 (1984) 650. [13] J.A. Cox, K. Slonawska and K. Brajter, Anal. Chem., 57 (1985) 2403. [14] J.A. Cox and N. Tanaka, Anal. Chem., 57 (1985) 2370. [15] K. Brajter and K. Slonawska, Mikrochim. Acta, 1 (1986) 231. [16] T.C. Chuang, Y.K. Lin and C.Y. Chen, J. Membrane Sci., 37 (1988) 131. [17] J.A. Koropchak and L. Allen, Anal. Chem., 61 (1989) 1410. [18] N. Kasthurikrishnan and J.A. Koropchak, Anal. Chem., 65 (1993) 857. [19] D. Berggren, Int. J. Environ. Anal. Chem., 41 (1990) 133. [20] D.C. Turnell and J.D.H. Cooper, J. Autom. Chem., 7 (1985) 177. [21] J.D.H. Cooper and DC. Turnell, J. Autom. Chem. 7 (1985) 181. 1221 D.C. Turnell and J.D.H. Cooper, J. Autom. Chem., 8 (1986) 151. 1231 V. Kuban, J. Komarek and D. Cajkova, Collect. Czech. Chem. Commun., 54 (1989) 2683. [24] Z. Fang, Flow Injection Separation and Preconcentration, VCH, Weinheim, 1993. [25] V. Carbonell, A. Salvador and M. de la Guardia, Fresenius’ J. Anal Chem., 342 (1992) 529. [26] P. Fernandez, C. Peres Conde, A. Gutierrez and C. Camara, Talanta, 38 (1991) 1387. [27] G.D. Marshall and J.F. van Staden, Anal. Instrum., 20 (1992) 79. [28] J.F. van Staden and C.J. Hattingh, Anal. Chim. Acta, submitted for publication. 1291 V. Kuban, J. Komarek and Z. Zdrahal, Collect. Czech. Chem. Commun., 54 (1989) 1785. [30] Z. Fang and B. Welz, J. Anal. At. Spectrosc., 4 (1989) 543. 1311 R. Purohit and S. Devi, Talanta, 38 (1991) 753. [32] F.C. Smith and R.C. Chang, The Practice of Ion Chromatog raphy, Wiley, New York, 1983. [33] D. McCormick and A. Roach, Measurement, Statistics and Computation: Analytical Chemistry by Open Learning, Wiley, Chichester, 1987. [34] J.C. Miller and J.N. Miller, Statistics for Analytical Chemistry, 2nd edn, Ellis Horwood, Chichester, 1988.