tion to be assayed. These anions were checked under the assay conditions described (oxalate ions, pyrophosphate ions, sulfite ions, thiosulfate ions, cupric ions, and dichromate ions) and were found to cause interferences by either forming a more stable complex with pyrithione, by reducing the ferric to ferrous, or by their ability to form a colorless compound with iron, thus reducing the intensity of the ferric pyrithione complex if sufficient excess iron were not present ( 4 ) . It should be mentioned that although sodium 2-pyridinethiol 1-oxide is not stable for long periods of time in acid solution, the ferric pyrithione complex showed neglible loss after 1hour which is more than sufficient time for numerable 0) was assays of this kind. The condition of low pH (pH chosen because of the solubility of the ferric complex. T o ensure that sufficient ferric ion was added, a spectrophotometric titration was performed using a known aliquot of sodium 2-pyridinethiol 1-oxide and varying the concentration of ferric ion. The amount of 1 ml of a 2% solution is -+

0.020 g of Fe(NH4)(S04)2-12 HzO which ensures that the reaction goes to completion instead of the theoretical alhount of 0.00215 g (based on Equation 1).

ACKNOWLEDGMENT The authors express their appreciation to John Wedig of Olin Chemicals for donating pure samples of the degradation and reaction products as well as impurities of sodium 2-pyridinethiol 1-oxide. Special thanks also goes to Sally Fraser for her drawings of the graphs and figures.

LITERATURE CITED (1) Olin Technical Bulletins, Olin Corporation, Rochester, N.Y. (2) R. T. Brooks and P. 0. Sternglanz, Anal. Chem., 31, 561 (1959). (3) A. F. Krivis, E. S.Gazan, G. R . Supp, and M. A. Robinson, Anal. Chern.,35, 966 (1963). (4) J. P. Mehlig, Anal. Chem. 10, 136 (1938).

RECEIVEDfor review October 20, 1975. Accepted February 17, 1976.

Evaluation of Peroxyoxalate Chemiluminescence for Determination of Enzyme Generated Peroxide David C. Williams

Glenn F. Huff, and W. Rudolf Seitz*

Department of Chemistry, University of Georgia, Athens, Ga. 30602

The chemiluminescence (CL) generating reaction of bis(2,4,6-trichlorophenyi) oxalate (TCPO) with hydrogen peroxide In the presence of perylene has been evaluated as a means of determining hydrogen peroxide. in a mixed ethyiacetatemethanol-aqueous buffer solvent system, performlng the reaction in a flow system, CL intensity is linearly proportlonai to peroxide from 7 X M, the detection limit, up to M, the highest concentration tested. The CL intensity is increased both by adding small amounts of triethylamine and by decreasing the concentration of the aqueous buffer. The pH of the aqueous buffer affects CL intensity; however, sensitive analysis for peroxide is possible from pH 4 to 10. Using immobilized glucose oxidase to convert glucose to hydrogen peroxide, the CL reaction of TCPO can be used to determine glucose in urine without interference from uric acid.

Recently, the chemiluminescence of 5-amino-2,3-dihydrophthalazine (luminol) in the presence of ferricyanide has been used for the determination of low concentrations of peroxide (1-4). The utility of this reaction was extended by coupling the enzymatic generation of peroxide from glucose to the CL from luminol(1-4). The luminol system is sensitive to concentrations of peroxide as low as 8-10-9M and is linear over four orders of magnitude. The glucose oxidase (E.C. 1.1.3.4) coupled CL method for glucose is extremely sensitive and specific. In spite of these advantages, two problems, a high background level of light emission and interference problems, have been encountered with luminol-CL analyses. The background light emission arises from the reaction of luminol-ferricyanide with oxygen. As a result of this high background it is necessary that the luminol-ferricyanide mix well, before entering the CL flow cell (I).If this requirement is not met, then the del Present address, Clinical Labarory, H a r t f o r d Hospital, Hartford, Conn.

tection limit is diminished because of a noisy background. Furthermore, with a high level of background light emission, achieving minimum detection limits requires constant flow. If a peristaltic pump is used with the luminol system, the limit of detection is diminished to M H202 because of surges in the flow system. The interference problem was observed when attempting to apply the CL method to the determination of glucose in urine. Uric acid, which may be present in urine a t concentrations as high as 75 mgldl, readily reduces peroxide and oxygen at the basic pH’s required for luminol CL. This causes a decrease of backgroundlpeak height and interferes with quantitation of urine glucose. We have reported a method for removing this interference involving the addition of equimolar portions of Ba(OH)2 and ZnSOI in order to determine urine glucose ( 5 ) .The resulting filtrate, the Somogi filtrate, is free of reducing substances other than glucose (6) and is, therefore, useful for glucose analyses. However, this pretreatment is time consuming. In order to overcome this problem, efforts were made to alter the pH to a value less favorable to the oxidation of uric acid. Since the CL of luminol occurs only in basic solutions a t pH’s between 9 and 12, it was, therefore, decided to replace luminol with another CL reaction Peroxyoxalate CL has been investigated by several workers (7-9). A simplified version of the proposed chemistry for peroxyoxalate CL is given by the reaction sequence shown in Figure 1 (8). Reaction 3 is an energy transfer from the strained ring to a fluorescer. Any fluorescer having a first excited singlet with an appropriate energy level can be excited by the reaction. The aromatic oxalate that was used in this work is bis(2,4,6-trichlorophenyl) oxalate (TCPO). TCPO gives efficient CL, is stable as a solid and in solution and is easy to prepare (7). Perylene was chosen as the fluorescer because it is stable and fluoresces efficiently over a wavelength range where our photomultiplier tube responds sensitively. In addition to the goal of eliminating the interference by ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976

1003

0 0

0 0

I1 I1 Pr-0-C-C-OOH

I1 I1 Pr-O-C-C--PrtClpOp

0 0 I1

I1

Pr-o-C-C-OOH

r

10 F*

1

0-0

-

1

oJ F

30 t ArOH

- [ogj -

rI

f

+ArOY

t Fluorescer

2o

r

F * t 2COp

t hv

Flgure 1. Proposed reaction sequence leading to peroxyoxalate chemiluminescence

Table I. Optimum Concentrations of Reagents for Quantitation of H202 Solvent/Solutes Ethyl acetate TCPO Perylene Methanol triethylamine Water buffer (phosphate)

- l o g Et3N CONCENTRATION ( M I

600 mg/l. 50 mg/l.

50 gl/l. 10-3 M

Figure 2. Effect of triethylamine concentration on CL intensity and background noise

- -

- -

CL intensity 0 0 0 ;Peak-to-peaknoise A A A; Signal to twice the noise 0 - 0 0. The ordinate units refer to the signal-to-noise ratio. CL intensity and noise are in relative units with the noise multiplied by a factor of 20. The peroxide concentration is 1 X lo-' M

-

R

altering the pH, another reason for investigating the peroxyoxalate system is that this reaction was expected to have a lower background because TCPO reacts only with peroxide (8). In this paper, we report the conditions that were found t o be most suitable for the determination of H202 with the peroxyoxalate reaction. The system was then coupled to the enzyme, glucose oxidase, in order to determine glucose concentrations in urine, without interference from uric acid.

EXPERIMENTAL Apparatus. In this study, the flow system was the same as that used by Bostick and Hercules ( 1 ) .An infusion pump drives three syringes containing TCPO and perylene in ethyl acetate, methanol, and aqueous buffer, respectively. Ethyl acetate (EtOAc) was the most suitable solvent investigated for TCPO. Methanol is necessary to render EtOAc and water miscible. The EtOAc and methanol flow lines are joined before entering the CL cell. The buffer flows through a sampling valve, then through the enzyme column, and finally into the CL cell. A sample is introduced to the flow system by drawing the sample into a 1-ml loop and changing the position of the sampling valve. The buffer then pushes the sample out of the loop and into the CL cell. When analyzing for glucose, a column of glucose oxidase immobilized on controlled pore glass is introduced to the flow system between the valve and the CL cell. In the cell, mixing is accomplished with Nz bubbling, and any peroxide in the sample reacts with TCPO to give CL. The light from the CL cell is detected by a photomultiplier, amplified, and recorded. The flow rate was 2.25 ml/min/syringe. For more detail refer to Ref. 1. Reagents. TCPO was prepared by the method of Mohan and Turro (7). After preparation, no special precautions were taken for the storage of TCPO, and problems of stability were not encountered either with the reagent or solutions of the reagent in EtOAc. The TCPO was found to be less stable in dimethoxyethane (glyme) and dioxane. For the enzyme-coupled work, glucose oxidase was immobilized on controlled pore glass by an azo linkage ( 5 ) . Foreign material accumulated in the enzyme column and caused an increasing resistance to flow, but the enzyme remained active. One column was used for nine months before the accumulation of foreign material partially occluded the column, thereby increasing the back pressure and making it necessary to replace the enzyme. One gram of glass to which 1000 units of glucose oxidase had been added was enough to pack the column two times. When the immobilized enzyme was not in use, it was stored at 5 "C. The conditions that were found to be most suitable for quantitative work are shown in Table I. In all but the optimization experiments, these concentrations were used. 1004

ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976

50y 1

Oi 4

,

5

6

,

I

7

8

9

IO

PH

Figure 3. A plot of '/*the signal-to-noise ratio obtained for 2.5 X M peroxide vs. the pH of the peroxide solutions.

The buffers were M acetate for pH 4 and 5. M borax for 8,9.and 10 7,and

M

phosphate for 6 and

RESULTS AND DISCUSSION Solvent System. The first problem with the peroxyoxalate system was t o determine a suitable solvent system for use in analysis. Since efficient peroxyoxalate CL has been observed in dimethoxyethane and dioxane solvents that are miscible with water they were tried first. However, it was found that the TCPO was not stable in these solvents, and there was a high level of background light emission from peroxides formed in the solvents. These problems were attributed t o the tendency of the solvents t o form peroxides. The next solvent system to be investigated was EtOAc. When EtOAc containing TCPO and perylene was introduced t o the CL cell along with peroxide samples, a noisy, irreproducible, signal was observed. This was thought to result from the immisibility of EtOAc with aqueous samples. T o promote mixing, methanol was added to the EtOAc. The signal continually decreased with time when the TCPO and perylene were dissolved in this solvent mixture and introduced t o the

CL cell with H202 samples indicating that the TCPO is unstable in methanol. T o overcome the problem, a third syringe was added to the flow system, and the methanol was mixed with the EtOAc solution directly in the flow system. Catalyst. In the absence of base, it takes several minutes for oxalate ester CL to reach maximum intensity (8).Small amounts of base accelerate and intensify CL. Therefore, by adding base, more CL should occur while the reagents are in the flow cell (The residence time in the flow cell is about 15 seconds). Figure 2 shows intensity as a function of added triethylamine (Et3N). Figure 2 also includes a plot of noise as a function of added Et3N and a combined plot of the ratio of signal to twice the peak-to-peak noise. Both intensity and peak-to-peak noise increase with Et3N; however, above M Et3N, the noise increases more rapidly than the signal. M Et3N was used in subsequent work Therefore, 3.5 X since it gave the optimum signal-to-noise ratio. At low Et3N levels, photomultiplier dark current is the main source of noise. The noise a t higher Et3N levels arises primarily from fluctuations in background CL as reagents flow through the cell in the absence of added peroxide. Since the noise level was proportional to the magnitude of background CL, variations in noise re,flect the background intensity. The nature of the background CL is not presently known. One possible approach to improving signal-to-noise ratios would be to mix the C1 reagents before entering the flow cell and measure intensity at some time after mixing. This was not tried. pH. The next parameter to be investigated was the effect of the pH of the aqueous solution on CL intensity. Figure 3 is a plot of the signal-to-noise ratios-observed as a function of pH. Optimum response is observed a t pH 8; however, sensitive peroxide analysis is possible from pH 4 to 10. Table I1 lists the relative values of signal and noise obtained for the various buffers used to encompass the pH range from 4 to 10. It can be seen that the buffer appears to have an effect other than just a pH effect. If noise and/or signal is plotted vs. pH, discontinuities occur when a shift is made to another buffer system, particularly when the buffer is changed from phosphate to borax. These discontinuities may partly reflect differences in ionic strengths among the various buffers. It should be emphasized that the pH's refer to the aqueous solution. The pH in the CL cell will be higher because of the presence of EtSN. B u f f e r Concentration. The next parameter to be investigated was the effect of buffer concentration on the signalto-noise ratio. For this study, phosphate buffers at pH 6.0 were made up a t various concentrations, and s t h e signal-to-noise ratio for 1 X M peroxide in these buffers was determined. Figure 4 is a plot of 1hthe signal-to-noise ratios vs. the logarithm of the phosphate concentration. The signal-to-noise ratio increases as the buffer strength decreases. The main effect is a decrease in CL intensity with very little change in background noise. Since a minimal buffer concentration is desirable, 1 X M buffer was used in subsequent work. R e a g e n t Concentration. The final parameters to be investigated were the concentrations of TCPO and perylene. In the absence of perylene, there is no measurable CL when M peroxide samples are added to the reagents. Concentrations M caused an equivalent increase in of perylene above both the signal and the noise. A concentration of 2 X 10-4 M (50 mg/l.) was found to give a suitable signal; and, at higher concentrations, there was no gain in the signal-to-noise ratio. The signal-to-noise ratio increased slightly with the increasing concentration of TCPO. In order to conserve the TCPO and speed up the preparation of the solution, 1.3 X 10-3 M TCPO (600 mg/l.) was employed. Higher concentrations of the reagent give a more intense signal and a higher signalto-noise ratio, but several hours of mixing were required to get

Table 11. CL Intensity and Twice the Peak-to-Peak Noise as a Function of pHa

pH

CL intensity Peak-to-peak noise X 2

Buffer

4 5 6

Acetate 34 2.0 Acetate 146 2.5 Phosphate 525 4.5 7 Phosphate 1010 6 8 Borax 645 3 9 Borax 2400 30 10 Borax 1920 30 a Both intensity and noise are given in self-consistent relative units.

150

r

IO0

z

ri \

m 50

01

log

1

I

-3

-2

~

0

~

1

-I

CONCENTRATION 3 -

Figure 4. Effect of phosphate buffer concentrations on the signal-to-

noise ratio '/*the signal-to-noise ratio is plotted vs. the logarithm of the phosphate concentration. The peroxide concentration was 1 X M, and the pH was 6.0

TCPO to dissolve at a concentration of 5.5 X M. The 600 mg/l. dissolves in a few minutes. Response to H202. A plot of the logarithm of peroxide concentration vs. the logarithm of CL intensity was linear over at least 4 orders of magnitude (from to M H202). Higher concentrations of peroxide were not investigated. The limit of detection, having a signal-to-noise ratio of 2, was found to be 7 X M peroxide. This limit of detection is not as good as that of the luminol system (8 X M peroxide),but the peroxyoxalate system has the advantages of a lower background and greater usable pH range which make this sacrifice in sensitivity desirable. Another project that we are working on, involves the use of a peristaltic pump for analyses. In this project, it was found that the high background of the luminol-ferricyanide CL system when combined with the pulsating which originates from the peristaltic pump causes a high noise level. The luminol reaction in the pulsating system has a limit of detection of 1 X M peroxide, but the TCPO system can be used at lower concentrations of peroxide (2 X lo-' M peroxide detection limit). U r i c Acid Interference. T h e performance of the peroxyoxalate system was next checked in the presence of uric acid to see if the interference with the glucose analysis is a problem at neutral pH's. Standard additions of urate were made to 1.0 X M peroxide in M phosphate buffer at pH 6.0 (near optimum for glucose oxidase). Up to concentrations of urate which would be obtained on a suitable dilution of urine Samples which are saturated with uric acid, there was only a 5% decrease in CL intensity (Le., a 200-fold dilution of urine ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976

1005

a

35r

/

GLUCOSE DETERMINED WITHOUT PRECIPITATION ( m g / d l )

Figure 5. A plot of t h e glucose determined in urine samples after precipitation with equimolar portions of Ba(OH), and ZnSO,, to remove possible interferences vs. the glucose determined without cleaning up the samples

samples gives glucose concentrations which are convenient to work with). Furthermore, the decrease in CL intensity increased with time. Since the peroxide in samples for glucose analysis is generated in the flow system, it was anticipated that the rate at which urate reduces peroxide would be sufficiently slow that the analysis for glucose could be accomplished in the presence of uric acid. Response to Glucose. Glucose was determined in 9 urine samples in the presence and absence of uric acid to see if the interference problem had, in fact, been overcome. To remove the uric acid, 1 ml each of 0.1 M Ba(OH)2 and 0.1 M ZnSO4 were added to 0.25 ml of urine. A 0.25-ml aliquot of the re-

sulting filtrates was diluted with 5.0 ml of buffer. The resulting solutions were uric acid free ( 5 ) .Samples and standards were treated in the same manner and the glucose concentrations were determined from a standard curve. To determine glucose in the presence of uric acid, 0.050 ml of urine was diluted to 10 ml with buffer. Again standards were treated the same as the samples, and the glucose concentrations were determined from standard curves. Figure 5 is a plot of the glucose which was determined in the 9 urine samples in the absence of uric acid vs. the glucose determined in the presence of uric acid. The correlation coefficient which was determined was 0.991. It therefore, appears that uric acid does not interfere with the glucose oxidase-CL method when TCPO is used a t a pH of 6.0 instead of luminol at pH 11. This means that the determination of glucose in urine is simplified by using the peroxyoxalate system.

LITERATURE CITED (1) D. T. Bostick and D. M. Hercules, Anal. Chem., 47, 447 (1975). (2) D. T. Bostlck and D. M. Hercules, Anal. Lett.,7, 347 (1974). (3) D. T. Bostick, M.S. thesis, University of Georgia, Athens, Ga., 1974. (4) J. P. Auses, S.L. Cook, and J. T. Maloy. Anal. Chem., 47, 244 (1975). (5) D. C. Williams, G. F. Huff, and W. R . Seitz, Clin. Chem., 22, 372 (1976). (6) S.A. Levlnson and R. P. MacFate, "Clinical Laboratory Diagnosis", Lea and Febiger, Philadelphia, 1969, 359 pp. (7) A. G. Mohan and N. J. Turro, J. Chem. Educ., 51, 526 (1974). (8) M. M. Rauhut, Acc. Chem. Res., 2, 80 (1969). (9) M. M. Rauhut, L. J. Bollyky, B. G. Roberts, M. Loy, R. H. Whltrnan, A. V. lannotta,A. M. Sernsel. and R. A. Clarke, J. Am. Chem. SOC.,89,6515 (1967).

RECEIVEDfor review October 6,1975. Accepted February 17, 1976. This work was supported in part by funds provided to the University of Georgia by PHS, NIH Research Grant No. 17913-01. G.F.H. was supported by NSF Grant No. E P P 750-4665.

Determination of Copper and Iron in Microliter Samples by Flame Atomic Emission Spectrometry with a Tantalum Filament Vaporizer M. R. McCuilough' and T. J. Vickers* Department of Chemistry, Florida State University, Tallahassee, Florida 32306

A simple microsampling attachment for conventional slot burners is described. The device was used for the determination of Cu and Fe by flame atomic emission spectrometry with a nitrous oxide/acetyiene flame. Ten-microliter sampies were used throughout the study. Signals were shown to be insensitive to filament aging up to approximately 50 heating cycles for a single filament and insensitive to the presence of chloride, nitrate, or sulfate as the counterion. For aqueous solutions the absolute detection limits were found to be 0.65 ng for Cu and 0.88 ng for Fe. At the 1 pg/mi level relative standard deviations (ten measurements) were 3.6% for Cu and 2.8% for iron. Measurements made with a control serum sample indicated adequate accuracy and precision for the determination of Cu and Fe at normal levels in human serum using either a standard additions or an analytical curve procedure.

Present address: B-2406, Central Laboratories, Dow Chemical Co., Freeport, Tex. 77541. 1006

ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976

A recent paper (1) described a simple and inexpensive attachment, a tantalum filament vaporizer, to provide for microsampling with a conventional flame spectrometer, and reported the application of this device to the determination of lithium in human serum samples by flame atomic emission spectrometry with an air-acetylene flame. The present report describes a further modification of the tantalum filament vaporizer and extension of the technique to the determination of copper and iron at normal levels in human serum by flame atomic emission spectrometry with a nitrous oxide-acetylene flame.

EXPERIMENTAL Apparatus. A block diagram of the measurement system is shown in Figure 1. Except for the tantalum filament vaporizer and the oscilloscope the arrangement of components is that which is

common to conventional flame atomic emission spectrometry. The instrumentation used, except for the changes noted below, is identical with that described previously ( I ). A Varian-Techtron AB50 nitrous oxide slot burner is used in place of the Alkemade-type burner, and a 10-cm focal length silica lens is used to form an ap-