1450
Anal. Chem. 1982, 5 4 , 1450-1452
periodic acid. On the basis of the assumption that periodic acid interfered with detection of acetaldehyde with the reagents, reaction mixtures after cleavage were neutralized with 40% NaOH and treated with arsenite, but neither procedure was successful. Finally, we destroyed the excess periodate in tests by adding 2 drops of ethylene glycol. This procedure was successful with the method of Barker and Summerson (9),which employs p-hydroxydiphenol as reagent, but it was unsatisfactory with the other methods. Possibly the formaldehyde formed upon addition of ethylene glycol interfered with determinations of acetaldehyde employing 3-methyl-2-benzothiazolone hydrozone or N-hydroxybenzenesulfonamide as reagent. Interfering Materials. Barker and Summerson (9) noted that glucose interfers with the analysis, an important point that is not included in two monographs on methods for analyzing fermentation mixtures (5,13). Acetoin, the precursor of 2,3-butanediol (14),would be cleaved to acetaldehyde and acetic acid. Acetoin can be determined separately, and corrections can be made. Diacetyl would yield two molecules of acetic acid and not interfere. Threonine would yield acetaldehyde. Some microorganisms produce acetaldehyde simultaneously with 2,3-butanediol (15). We recommend separation of 2,3-butanediol from acetoin and other interfering
compounds by salting-out chromatography ( 4 ) prior to determination of 2,3-butanediol. LITERATURE CITED Desnuelle, P.; Naudet, M. Bull. SOC.Chlm. Fr. 1945, 72, 871-875. Johnson, M. J. Ind. Eng. Chem., Anal. Ed. 1944, 76, 626-627. Happold, F. C.; Spencer, C. P. Biochim. Biophys. Acta 1952, 8 , 18. Speckman, R. A.; Collins, E. B. Anal. Biochem. 1968, 22, 154-160. Nelsh, A. C. "Analytical Methods for Bacterial Fermentations": Nelsh, A. C., Ed.; Prarie Regional Lab.: Saskatoon, Canada, 1950. Stewart, R. "Oxidation Mechanisms"; W. A. Benjamin: New York. 1964. West, D. M.; Skoog, D. A. J. Am. Chem. SOC.1960, 82,280. Westerfeld, W. W. J. Bioi. Chem. 1945, 761, 495-502. Barker, J. 6.; Summerson, W. H. J . Bo/. Chem. 1941, 738, 535-554. Sawlcki, E.: Hauser. T. R.; Stanley. T. W.; Elbert. W. Anal. Chem. 1961, 33, 93. Lindsay, R. C.: Day, E. A. J. Dairy Sci. 1965, 4 8 , 665-669. Ismall, M. A.; Wolford, R. W. J. Food Sci. 1970, 35, 300-301. Chayken, S. "Biochemistry Laboratory Techniques"; Wiiey: New York, 1966: DO 89-91. rr - JuAlE.; Heym, G. A. J. &Cter/O/. 1956, 7 7 , 425-432. Wood, W. A. "The Bacteria"; Academic Press: New York, 1961; Vol. 2, pp 59-150.
RECEIVED for review February 12, 1982. Accepted April 8, 1982. This investigation was supported by a grant from the Dairy Council of California.
Determination of Trace Amounts of Sulfur in Organic Solvents Erik Klssa Jackson Laboratory-Chemicals
and Pigments Department, E.
I. du Pont de Nemours and Company, Wilmington, Delaware
Sulfur is a known poison of precious metal hydrogenation catalysts. The detection of sulfur in organic liquids to be hydrogenated is therefore of practical interest. Trace amounts of sulfur have been determined by reduction with h e y nickel to nickel sulfide. An addition of acid liberates hydrogen sulfide which is titrated with mercuric acetate (1) or determined spectrophotometrically after conversion with N,N-dimethyl-p-phenylenediamine to methylene blue (2). The elapsed time of the analysis is 3-4 h and the accuracy is only fair. Microcoulometric methods for the determination of sulfur after reduction or oxidation (3-6) are rapid but not applicable to samples containing large amounts of nitrogen or halides. Drushel (7) described a method for the determination of trace sulfur in petroleum fractions involving noncatalytic hydrogenolysis to form hydrogen sulfide determined by the Houston-Atlas H2S analyzer. It was of interest to find out whether the method is applicable to liquids other than hydrocarbons and to sulfur compounds not investigated by Drushel. EXPERIMENTAL SECTION Apparatus. A Houston Atlas Model 856 total sulfur hydrogenator with a ceramic tube was used for hydrogenolysis. The furnace temperature (center zone) was 1300 "C, turned down to 900 "C when the instrument was not in use. The hydrogenolysis tube was positioned so that its end protruded 70 mm from the furnace (measured from the end wall of the furnace to the end of the "Teflon" TFE fluorocarbon resin union holding the septum). A Model 1002 Micro-jector syringe drive (Houston-Atlas, Inc.) was equipped with a 1OO-pLor 10-pL Precision Sampling Co. CGV syringe. The exit end of the hydrogenolysis tube was originally connected to a microfiber filter (Balston, Inc., Catalog No. DFU 9933-05, grade Q, size in. X 11/4in.) with a Teflon tubing. Since carbon deposits in the tubing restricted flow, we connected the filter directly to the hydrogenolysis tube by means of a Teflon 0003-2700/82/0354-1450$0 1.25/0
19898
union. The filtered gases were passed through a gas wash bottle containing 5% acetic acid to the sample chamber of the hydrogen sulfide analyzer (Houston-Atlas, Inc., Model 825R-d). Chemicals. The hydrogen was obtained from Matheson Co., ultrahigh purity grade. The sulfur compounds, obtained either from Eastman Kodak Co. or Aldrich Chemical Co., were analyzed by the Schoniger method for sulfur. Diphenyl sulfone was purified by sublimation. Procedure. Before the furnace temperature was raised to the operating temperature of 1300 "C, the hydrogenolysis tube was cleaned by heating both of its ends which normally protrude from the furnace. With helium flowing through the tube, the exit end was disconnected from the filter and the entrance end pushed into the furnace. After 2-3 min, the exit end of the tube was pulled into the furnace and heated until smoke was no longer visible. The tube was then placed into its operating position and the filter connected to its exit. We found this procedure to be sufficient. For the removal of refractory coke deposits, both ends of the hydrogenolysis tube may have to be opened to allow air to enter the tube and oxidize the accumulated coke (7). While the furnace was heated to the operating temperature, helium was replaced with hydrogen (flow rate 400 mL/min). The syringe was filled with the sample and positioned on the syringe drive so that its barrel was flush with the septum. The syringe drive was operated at speeds 0.25-4.43 pL/min with a 10or 100-pL syringe. When the signal recorded approached its maximum value, the lead acetate paper drive was advanced and the signal recorded again. The second recording representing a steady state was used for the calculation. RESULTS AND DISCUSSION The Houston-Atlas sulfur analyzer is shown schematically in Figure 1. A sulfur-containing sample is fed continuously into the ceramic tube of the pyrolyzer where a t 1250-1300 "C in the presence of hydrogen the analyte is thermally cracked and sulfur converted to hydrogen sulfide. The exit gases are 0 1962 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 8, JULY 1982
1451
Table I. Calibration o f the Sulfur Analyzer
standardizing solution dibutyl sulfide in toluene
thiophene in cyclosol
range, mg/L
syringe 1iolume, pL
0-1.5
100 100
0-5 0-50
100 10
0-1.5
100 100 10 100
0-6 0-50 0-1.5
dibutyl disulfide in rnonochlorobenzene
Table 11. Effect of Sallvent on Response of the Houston-Atlas Sulfur Analyzer
syringe drive setting, amplifier pL/min attenuation 4.43 4.43 2.54 0.25 4.43 2.54 0.25 4.43
calibration curve slope
linear correlation
2.91 8.25 1.22 0.161 3.19 1.15 0.132 2.98
0.9994 0.9996 0.9994 0.9998 0.9989 0.998 0.998 0.9989
700 900 500 500 700 5 00 500 700
LEAD ACETATE P A P E R DRIVE+$
--
V T:
'TEFLON' U N I O N )1
~.----
'TEFLON'
PYROLYZE
FILTER
solvent toluene o-nitrotoluene o-dichlorobenzene chlorobenzene 1-butanol N,N-dimethylformamide ethyl acetate
dibenzyl disulfide instruconcn, ment response mg/L S response per ppm S 2.00 2.00
2.66 2.65
1.33 1.33
2.01
2.75
1.37
2.02 2.03 2.00
2.35 2.40 2.50
1.16 1.18 1.25
2.01
2.50
1.24
ounrn.
,
E U
WASH BOTTLE W I T H ACETIC ACID .."I..
BOTTLE WITH WATER
H?
He
Figure 1. Diagram of the Houston-Atlas sulfur analyzer. 40)
BEGIN PAPER
P
DIBENZYL DISULFIDE I N 0-NITROTOLUENE (2 0 mg S / L )
DFSFT
INJECTION
mean 1.266 std dev u = 0.08 or 6.3% re1
BACKGROUND RESPONSE
ii
I
Y
filtered and passed through acetic acid. The flow of the H2S-containing gas is then directed over the surface of lead acetate paper. The rate of lead sulfide formation is converted to a readout (Figure 2), linearly propoirtional to the sulfur content of the analyte. Since the lead acetate paper and the differential amplifier can handle only a limited amount of H2S (7), the amount of the sample fed into the pyrolyzer has to be adjusted according to the sulfur content. The analyzer was therefore calibrated for three concentration ranges: 0-1.5, 0-6, and 0-50 mg of S/L. The linearity of rlesponse is shown !with calibration data in Table I. The response factor indicated by the slope of the calibration curve is affected only islightly by the chemical composition of the organic liquid containing trace amounts of sulfur. This is shown also with solvents containing C, H, N, 0, or C1 in Table 11. The low sensitivity of the response to the chemical composition of the solvent is of advantage when analyzing solvents of unkown or undefined composition.
I
O
1
2
3
4 5 TIME (MINI
I
I
I
1
6
7
0
9
Figure 2. Typical response curve (dibenzyl disulfide (2.0 mg/L S) in
o-nitrotoluene). Since the structure of sulfur compounds in the organic liquids analyzed is usually unknown, it is important that the analyzer can correctly determine sulfur regardless of the chemical nature of the sulfur compound. Data presented in Table I11 show that sulfur in compounds with R-S-R, R-SS-R, R-SH, R-S(0)-R, R-S(OJ-R, HSRCOOH, and heterocyclic R-S-R functions can be accurately analyzed without a considerable change of the response. Sulfur in sulfonic acid esters, e.g., methyl p-toluenesulfonate, can also be determined. Sulfur in alkali metal salts of sulfonic acids cannot be detected. The precision of the method, determined by five analyses of dibenzyl sulfide in toluene (2.0 mg of S/L) and expressed as the standard deviation, is 2.7% relative. This is in accord with the precision values reported by Drushel(7). The pre-
Table 111. Analysis of Sulfur Compounds in Toluene 0-1.5 mg/L range
sulfur compound
calcd
found
di-n-butyl sulfide
0.51
0.50
dibenzyl disulfide
0.50
0.51
thiophene 1-dodecanethiol dimethyl sulfoxide
0.51
0.54
0.59
0.64
diphenyl sulfoxide diphenyl sulfone 2-mercap topnopionic acid
mg/L sulfur 0-6 mg/L range calcd found 2.0 5.1 2.0 5 .O 5.0 2.0 2.3 5.9 2.0 2.0 2.2
2.0 5 .O 2.0 5.1 5.1 2.0 2.2 5.9 1.8 2.1 2.2
0-50 mg/L range
calcd
found
20.2
20.5
19.9 20.4 23.4
19.8 20.0 23.9
19.9 22.0
20.3 22.9
Anal. Chem. 1982, 5 4 , 1452-1454
1452
cision is not better than 2% of the maximum concentration of each concentration range. This means that an error of 0.02 mg/L S in the 0-1 mg/L S range corresponds to an error of 1mg/L S in the 0-50 mg/L S range. For optimum precision, the response of the analyzer should be kept large by selecting the syringe size according to the sulfur content of the analyte and maintaining a reasonably rapid syringe drive speed. ACKNOWLEDGMENT The author thanks Ward R. Gibson for his skillful assistance in experimental work and D. H. Gehring and C. J. Hensler for their helpful comments.
LITERATURE CITED Granatelli, L. Anal. Chem. 1959 31, 434. Pitt, E. E. H.; Rupprecht, W. E. Fuel lg84, 43, 417. Drushel. H. V. Anal. Lett. 1970, 3, 353. Wallace, L. D. Anal. Chem. 1070, 4 2 , 387. Carter, J. M. Analyst (London) 1972, 97, 929. (6) Killer, F. C. A. In "Recent Analytical Developments in the Petroleum Industry"; Hodges, D. R., Ed., Halsted Press: Wiiey: New York, 1974. (7) Drushel, H. V. Anal. Chem. 1978, 50, 76.
(1) (2) (3) (4) (5)
RECEIVED for review January 13,1982. Accepted March 29, 1982. This is Research and Development Division Publication No. 588.
Electrochemical Assay for Brain Ascorbate with Ascorbate Oxldase James 0. Schenk, Ellen Miller, and Ralph N. Adams" Department of Chemistty, University of Kansas, Lawrence, Kansas 66045
A major research effort in our laboratory for some time has been the development of electrochemical techniques for in vivo monitoring of neurotransmitter systems in small animal brains. Typical experiments utilize very small glass-sheathed graphite-epoxy electrodes implanted in specific areas of brain tissue. Chronoamperometric experiments are conducted at potentials sufficient to oxidize and thus detect catecholamines in the extracellular fluid (ECF). Unfortunately, with most of the electrodes currently available, ascorbic acid (AA) is oxidized at potentials almost identical with that of the catecholamines norepinephrine (NE) and dopamine (DA). Differentiating chemical analysis of the ECF immediately surrounding the electrode tip would be ideal, but sampling technologies for this particular type of in situ measurement have yet to be developed. Due to the complexities of the in vivo experiment, we have turned to the brain slice as a simple model for investigating electrochemical responses in brain tissue. This preparation allows one complete access to the area surrounding the electrode tip. Both electrochemical experiments and subsequent chemical assays on the region immediately adjacent to the electrode tip can be accomplished readily, enabling one to sort out the electroactive species giving rise to the response. Since AA is present in very high concentrations (ca. millimolar) in all neuronal tissue, knowledge of its exact local ECF concentration is very important in interpreting results from in vivo or brain slice electrochemical measurements. It is in the above context that we summarize herein a rapid, specific assay for AA that can be used for any small brain tissue sample. Its use requires no devices not already available to experimenters working with in vivo electrochemistry. It obviates the need to constantly maintain a satisfactorily performing liquid chromatography system for AA analyses which, at best, has proven to be difficult and tedious. It requires only the purchase of the enzyme ascorbic acid oxidase (L-ascorbate:oxygen oxidoreductase, EC 1.10.3.31, which is stable under stored refrigerated conditions. Ascorbic acid oxidase (AAO) catalyzes the reaction
-
L-AA + 1/202 dehydroascorbic acid
+ H20
The reaction is extremely rapid and specific and, under the conditions used, can be shown to cause negligible oxidation of the catecholamines. Following conventional tissue sampling and homogenization, the assay consists of three simple steps: (1) A chronoamperometric measurement is made on the homogenized sample in buffer solution. This becomes a 0003-2700/82/0354-1452$01.25/0
measure of all electroactive species oxidized at that potential. (2) A standard addition of a few microliters of standard AA solution is made and a second chronoamperometric run calibrates the electrode under actual assay conditions. (3) A fixed amount of AAO is added and a third amperometric response detects the almost instantaneous removal of all AA. Since each of the electrochemical measurements requires only 1 s or less, the entire assay for an individual sample is completed in a few minutes and a simple calculation gives the AA content. Since air oxidation of AA samples is a serious source of error, the minimal handling and rapidity of the proposed assay are particularly advantageous. EXPERIMENTAL SECTION Reagents and Solutions. All chemicals for buffer solutions, etc. were reagent grade and were used as received. AAO was purchased from Boehringer-Mannheim (Indianapolis). Enzyme solutions having a concentration of 1 mg/100 pL were prepared in 0.05 M phosphate buffer, pH 5.6, fresh before each set of assays. When not in use they may be kept at 0 "C and are stable for at least 2 weeks (longest time tested) with no deterioration. The AA stock solutions were made in doubled distilled water and were 1.00-1.50 mM. These solutions, stored in a refrigerator, were stable for at least 2 h. Two buffer media were evaluated for the assays. For routine tissue samples 0.05 M phosphate/0.001 M EDTA (ethylenediaminetetraacetic acid), pH 7.0, can be used. If samples are to be evaluated from brain slices or used in pharmacological or physiological experiments, the bicarbonate buffer for maintaining the functioning tissue works equally well. This has the composition: 124 mM NaCl, 5 mM KC1, 1.24 mM KHZP04, 1.3 mM MgS04,2.5 mM CaCl,, 26 mM NaHC03,and 10 mM glucose. This buffer is saturated with a 95% 02/5% C 0 2 gas mixture while in use. Apparatus. A small electrochemicalcell of approximately 100 pL volume (Figure 1)was constructed by cutting off the end of an Eppendorf plastic pipet tip and force-fitting the piece over the end of a glassy carbon electrode (BioanalyticalSystems,Model GCE). This gives an easily removable but watertight seal. When the electrode is used in the inverted position, it forms an extremely convenient cell, A small platinum wire auxiliary electrode and a Ag/AgCl wire reference electrode are positioned with a micromanipulator (or any convenient holder) as seen in Figure l. This arrangement provides complete access for solution additions, etc. Miniature electochemical cells of this general design have been reported ( I ) . The potentiostat for applying the potential in the chronoamperometric measurements was a PAR 174A (Princeton Applied Research Corp.) and the current-time response was displayed on 0 1982 American Chemical Society
