~~~
Table IV.
Amt. of osmium added, pg. 80
80 80 80 80
80 80 161 161 161 161 161
Blank Blank Blank
Determination of Osmium in Iron-Copper-Nickel Buttons Amt. of osmium Amt. of osmium Osmium
found in residue, p g . 35 48 47 47
found i n filtrate, p g . 42 27 25 25 28 27 27 35
50
48 48 117 101 87 122 139
52
69 30
...
16 ...
...
...
Total osmium found, pg. 77 75 72 72 78 75 75 152 153 106 152 155 0 0 1
lost, pg.
3 5
8 8 2
5 5
9 8 5
9 6 ...
which is carried over in the vapors can be recovered by perchloric acid distillation. Test of Procedure. Buttons containing various amounts of osmium were analyzed for osmium by the proposed procedure. The results are given in Table IV. General Scheme of Analysis for Six Platinum Metals. The above method was developed to allow the integration of osmium with a general analytical scheme for the six platinum metals. The scheme is outlined in Figure 2.
... LITERATURE CITED
(1) Marks, A. G., Beamish, F. E., ANAL.
CHEY.30, 1464 (1958).
if the evaporated cake from the button parting was dissolved and subjected to ion exchange procedures immediately after the evaporation of the base metal bolution. Presumably this copper leak was caused by evaporating the parting acid under nitrogen, which resulted in a cake containing copper in the lower valence state. As indicated above, the presence of large amounts of copper in the effluent resulted in excessive losses of osmium during the subsequent evaporation. This difficulty can be prevented by allowing the residue from the evaporation, moist-
ened with 20 ml. of 1 : l hydrochloric acid, to stand open to the atmosphere overnight a$ indicated in the procedure. During this period copper is converted to a form readily removed by a cation exchange resin Osmium Loss during Evaporation of Effluent. Small losses of osmium undoubtedly result during the evaporation of the large volumes of effluent, even after saturation with sulfur dioxide. For the most precise work the effluent can be evaporated in small portions in the apparatus shown in Figure 1. T h e osmium
( 2 ) Plummer, 11.E. V., Beamish, F. E., Ibid.. 31. 1141 11959).
(3) Tertipis, G. ~ G . ,'Beamish, F. E., Tahnta 10, 1139 (1963). ( 4 ) Thiers, It., Graydon, W.,Beamish, F. E., AXAL.CHEM.20, 831 (1948). ( 5 ) \-an Loon, J. C., Beamish, F. E., Ibad., 36, 872 (1964). (6) Ibzd., p. l i i l . ( 7 ) Westland. -4.D.. Beamish. F. E.. ' Ibtd., 26, 739 (1954). (8) Westland, A. D., Beamish, F. E., Microchim.Scfa. 5 , 40 (1957). ( 9 ) Zachariasen, H., Beamish, F. E., AXAL.CHEM.34, 964 (1962). RECEIVED for review June 24, 1964. Accepted October 9, 1964.
Carbon and Hydrogen Analyses by CouIo metric ElectroIysis of Water H.
S. HABER,
D. A. BUDE, R.
P.
BUCK, and K. W. GARDINER
Bell & Howell Research Center, Pasadena, Calif.
b A coulometric method for the simultaneous determination of carbon and hydrogen in organic compounds employs a rapid combustion in a modified quartz tube, using nitrogen carrier gas. A weighed sample ( 2 to 10 mg.) i s covered with cobaltocobaltic oxide for the initial combustion and a downstream copper oxide packing i s used to assure complete oxidation. Additional combustion tube packings of copper and silver oxyvanadate are employed to remove the oxidation products of nitrogen, sulfur, and the halogens. The hydrogen content of the sample i s determined coulometrically as water in a specially developed high capacity Keidel-type electrolytic cell containing rhodium electrodes. The carbon content of the sample i s also determined as water in a second electrolytic cell after the carbon dioxide from the combustion has been converted to an 116
e
ANALYTICAL CHEMISTRY
equivalent amount of water with a specially prepared charge of lithium hydroxide. Electrolysis currents are automctically integrated by calibrated voltage-to-frequency converters and pulse counters, and the coulombs measured are related to milligrams of carbon and hydrogen by Faraday's law. The absolute standard deviations (CT) were i0.3% for carbon and f 0.03% for hydrogen.
A
determination of carbon and hydrogen in organic compounds has received a great deal of attention in recent years. While functional group determination5 and instrumental methods for structure analysis, such as infrared, are certainly important, elemental determinations do provide the basic means for diagnostic and confirmatory testing in organic syntheses and proof of structure work. UTOMATED
Gnfortunately, the? highly accurate classical t,echniques for elemental analyses tend to be both laborious and As a consequence, time-consuming. much effort has been directed toward supplementing or replacing some of the widely used classical procedures, such as the Pregl niet,hod, with automated microanalytical techniques. In a recently published article (S), Francis reviews some of the earlier work and most of the more recent, developments involving the automated instrumental determination of carbon, hydrogen, and nitrogen, and describes several commercially available instruments. The developnient of an automatic coulometric analyzer for the determination of carbon and hydrogen that, is capable of providing the accuracy of the classical method will be described here. The method is an expansion of previously published work (4)and only the initial sample weight and straightfor~
i'
RI
zoofi
MOVING H E A T E R
I
I
-9 5 0'
C.-,--
CELL
RI
zoon
STATIONARY HEATERS
8 00 ' C.-,
-\y I 5"
P t PLUG
I-
CELL-
C u 4"
-6 0 0 e,-.C
IAp,(VOd
4'"
Ap WOOLf
5 8 5 m m ( 9 m m 1.0. X llmrnO.0. 9 U A R T Z ) -
Figure 1 ,
Combustion system Figure 3.
ward absolute electrical measurements are required. The earlier work (4) established the feasilility of converting carbon dioxide to water with lithium hydroxide (LiOH) and the electrolytic meaiurement of this water. Unfortunately, the integral of electrolysis current with time had to be related to sample composition by a calibration curve as the absolute coulometric concept had not yet been established. hlso, the problem of handling interfering species had not been solved with the earlier system. EXPERIMENTAL
Apparatus. Gas Purification Train. This is a two-piece module consisting of a copper oxide preheater and chemical absorber which removes the hydrocarbons, water, or carbon dioxide generally .3resent in trace amounts in commercial cylinder gases. Combustion S F t e m . Combustion Tube. =\s shown in Figure 1,a 585-mm. length of 911-mm. quartz tubing with a 6-mm. side arm inlet. The downstream end of the tube is ground square and sized on a lathe with a carbon mandrel to accommodate a special 0-ring-to--Swagelok adapter. The upstream end is sealed with a X o . 000 silicone rubber st'opper. Heaters. The heaters for the combustion tube were designed to operate directly from the Il~O-voltline to eliminate the need for c'ontrol transformers. Opt'imal operating temperatures are attained after 2 to 3 minutes from startup. The heaters 'were fabricated by winding lengths of Hoskins 875 wire on hexagonal mandrels and preoxidizing the wound elements in a muffle furnace a t 1000" C. for 8 hours. Sections of the oxidized heat,ing elements were then fitted over 16-rnm. quartz sleeving. Transite end plates were machined to receive the quartz; sleeving and the heater assemblies were enclosed within a 2-inch stainless steel shell (0.016-inch wall). The inside of the shell is insulated with aluminum-backed Fiberfrax. The moving heater is 3 inches in length and attains a temperature of 950" C. The two stationary heaters are combined into a single unit 10 inches long with a Transite disk separating each 5-inch section. The upstream heater section maintains the copper oxide/copper packing a t 800" C. and the downstream section attains a temperature of 600" C. for the silver/silver vanadate packing.
Figure 2.
LiOH converter
Electrolytic Cells. The electrolytic cells used in this system are based on the Keidel-type ( 7 ) cell but are of special design and are not, as yet, commercially available. The active electrode elements in each cell are 5-mil (0.005-inch) diameter rhodium wire. These are bifilar wound on a 1/i6-inch (0.063-inch) stainless steel mandrel and encapsulated in borosilicate glass tubing which is bent into hairpin configuration. A micarta end plate and Teflon ferrules are used to seal the ends of the element into a Teflon end-cap. The assembled cells are internally coated with a solution of phosphoric acid in acetone. Excess coating solution is aspirated off and the cells are electrolyzed to dryness before use. Power Supply. The electrolytic cells are operated in parallel a t 75 volts with a Power Designs, Inc., transistorized power supply, Xodel 105 T h . Lithium Hydroxide Converter. As shown in Figure 2, this module consists of a 7 - X 9-mm. borosilicate tube approximately 200-mm. in length charged
Integrator circuit
with 125- to 130-mm. of specially prepared LiOH. The charged tube is inserted into a 1-inch square aluminum heat sink, 140-mm. in length and maintained at constant temperature, 210" i 10" C., with a Variac-controlled 100-watt cartridge heater. Special 0ring-to-Swagelok adapters are used t o connect the tube to the system. Integration and Readout. Electrolysis cell currents are integrated (as voltages) with Vidar Model 240 voltageto-frequency converters, as indicated in Figure 3, and Computer Measurements Co. Model 308B totalizing counters are employed for readout purposes. Reagents. Cobalto-Cobaltic Oxide. This reagent may be prepared b y the method of Vecera, Snobl, and Synek (IO). The prepared material must be kept in a desiccator. Silver Vanadate. Prepared by the method of Ebeling and Malter ( 2 ) . Store in a desiccator after preparation. Silver Wool. Available from the American Platinum and Silver Division of Engelhard Industries, Inc., 231 S e w Jersey Railroad Ave., Kewark 5, X. J. Lithium Hydroxide. Granlox anhydrous LiOH, Maywood Chemical Works, 100 W.Hunter Ave., Maywood, N. J., is ground to a fine powder ( - 100mesh) with a mortar and pestle in a dry box and must be stored in a desiccator until ready to use. Procedure. T h e combined system used for the simultaneous determination of carbon and hydrogen in organic compounds is shown in Figure 4. Nitrogen carrier gai, regulated a t 10 ml./minute, is purified by passing it over hot copper ovide to remove hydrocarbons, and the traces of water
A
P COPPER O X I D E 8 ASCARIT€/ M G ( C 1 0 4 ) 2 c FLOWRATOR N2CARRlER D SOLENOID VALVE
E F PCOPPER i PLUG O X I D E G COPPER
Figure 4.
H AG,IVO,) / A G WOOL I SPECIAL E L E C T R O L Y T I C C E L L J LiOH COVERTER K S P E C I A L ELECTROLYTIC C E L L
M L FVLTO O WFR A INTEGRATORS TOR N,BLEED
N TOTALIZING COUNTERS
'
I
p+
Carbon-hydrogen analyzer system VOL. 37,
NO. 1 , JANUARY 1965
117
Table I.
Typical Analytical Data of NBS Anisic Acid (CHBO. CsHsCOOH) Theory: C = 63.15%; H = 5.307,
Sample wt. 6,385 7.203 4.825 5.310 3.389 4,832 3.713 3,632 4.621 3.205 Mean
%C 62,94 63.33 63.07 63.07 63.21 63.43 63,21 63.22 63.25 63.09 63,18
4 % from theory -0.21 + O . 18 -0.08
A,
Table 11.
%
from theory +0.02 + O . 05
%H 5.32 5.37 5.30 -0.08 5.30 +0.06 5.30 +0.28 5.31 +0.06 5.30 +0.07 5.32 + O . 10 5.30 -0.06 5.27 Mean 5.31
Typical Analytical Data of NBS Acetanilide (CH3CONHCeHJ Theory: C = 71,09%; H = 6.7170
Sample wt. 5.547 3.471 3,310 4.374
0.00 0.00 0.00
70 C 70.87 70.79 70.91 70.85
70.82 3.749 70.87 70.85 3.211 3,836 70.72 5,033 70. 83 3.985 70.79 Mean 70,86 4.238
+0.01
0.00
+0.02
0.00
-0.03
a t 800" C. and following the copper oxide packing, is used to reduce any oxides of nitrogen to elemental nitrogen. Other potentially interfering species, such as the oxides of sulfur and the halogens (except fluorine), are removed by a downstream packing of silver vanadate and silver wool maintained a t 600" C. The hydrogen and carbon contents are then measured coulometrically as described in the Discussion. Analysis time is on the order of 15 to 30 minutes.
and carbon dioxide generated are absorbed by the combined hscarite and magnesium perchlorate trap. A sample in the range of 2 to 10 mg. is weighed in a nickel boat using a conventional microbalance, covered with cobaltocobaltic oxide (Co,04), and introduced into the combustion tube in front of the copper oxide filling, while backflushing with nitrogen. The moving heater is turned on and started forward after about 2 minutes are allowed for it to attain its optimal temperature of 950" C. The heater travels a t a rate of 1 inch per minute and slowly distills the sample, partially oxidized, out of the boat and into the heated copper oxide section of the combustion tube maintained a t 800" C., where oxidation of the sample is completed. d zone of metallic copper turnings, maintained
RESULTS
The accuracy of the developed system was established by the combustion of samples of Sational Bureau of Standards benzoic acid, anisic acid, acetanilide, and iodobenzoic acid, as
Table 111.
4 70 from theory -0.22 -0.30 -0.18 -0.24 -0.27 -0.22 -0.24 -0.27 -0.26 -0.30
Benzoic acid CsHsCOOH Anisic acid CHaO CsHbCOOH Acetanilide CH&O?iHCsH6
samples 22
Interference
10
...
p-Nitrophenylacetonitrile
NO2CeH4 CH&N Iodobenzoic acid C6HsCOOHI 2-Bromonapthalene CIOH7Br Sulfanilamide NII,C6H,SO2NH, 6-E thoxy-2-mercapto benzothiazole
SoH 6.68 6.78 6.81 6.76 6.76 6.78
6.78 6.75 6.75 6.75 Mean 6 , 7 6
5%
from t,heory -0.03 t-0.07
+o. 10 +0.05 +0.05 +0.07 $0.07
$0.04 +0.04 +0.04
well as by other purified samples containing potentially interfering elements, both alone and in combination. Results on two typical compounds are given in Table I and Table 11. summary of the results of 101 consecutive analyses from 11 different compounds containing the elements carbon, hydrogen, oxygen, nitrogen, sulfur, bromine, and iodine is presented in Table 111. The standard deviation ( u ) calculated from even the worst group of data was 0.3y0for carbon and 0.O3yGfor hydrogen. These standard deviations were calculated from X, where n-1
Summary of Analytical Data
% Carbon No. of
.A,
(x)
Theory 68.85
Found 68.80
63.15
yo Hydrogen Std. dev.
Std. dev.
0.30
Theory 4.95
Found(X) 4.96
63.18
0.14
5.30
5.31
0.025
(U )
(u)
0.025
10
N
71.09
70.86
0.06
6.71
6.76
0.034
5
N
59.26
59.23
0.11
3.73
3.76
0.022
14
I
33.90
33.85
0.12
2.03
2.03
0.010
5
Br
58.00
57.96
0.09
3.40
3.41
0.012
10
S, N
41.85
41.72
0.05
4.68
4.71
0.023
4
s, N
51,16
50.97
0.13
4.29
4.29
0.000
5
c1, s, N
31.87
31.93
0.06
2.23
2.21
0.022
s, N
40.46
40,39
0.09
4.24
4.25
0.016
74,47
74.52
0.19
4.86
4.86
0.011
CgHQONS2 A-, .V-Dirhlorobenzene
sulfonamide CsHs02SSC12 Acetamide, 2,P-dichloro-N[P-hydroxy-a-(hgdroxympthyl)-p(methylsulfonyl) phenethyll-D +-threo CI:HIsOs XSCL 8-Hydros yquinoline HO.CgHsS
11 8
e
ANALYTICAL CHEMISTRY
11
C1,
5
N
The Co304 oxidant used in the combustion boat has a finite blank for both water and carbon dioxide and this blank correction has been used in treating the data presented above. The correction in integrator counts per weight of C0304 is essentially constant when the CoaOl supply is kept in a desiccator. The blank for hydrogen is appreciable, being of the order of 60 to 70 integrator counts per milligram of Co304. The carbon blank is generally quite small, and can be eliminated entirely by taking adequate precautionary measures when handling the reagent. DISCUSSION
Combustion Tube Packings. I n general, the oxidation of organic substances t o water and carbon dioxide presents little difficulty to the analyst. T h e selection of specific combustion tube fillings is largely a matter of one's own experiences. I n the developed system discussed here, the copper oxide packing has proved to be completely reliable for completing the oxidation of hydrocarbon fragments when Co304is used a!; the added internal oxidant. The silver vanadate packing (2, 5 , 8 ) is very efficient for retaining sulfur and halogen oxidation products and is especially important when using nitrogen carrier gas because silver wool alone is not as effective unless used in an oxygen stream where a film of silver oxide can be formed. Hot metallic copper is completely satisfactory for removing oxides of nitrogen (1, 6). Our studies confirmed the work of Mendoza (9) in regard to the use of manganese dioxide (MnOJ packing in the downstream end of the combustion tube for the purpoi,e of retaining the oxides of nitrogen. We found that M n 0 2 absorbs water strongly, which is desorbed very slowly even at 160" C. Lithium Hydroxide Converter. T h e reaction of LiOH with carbon dioxide to produce water is straightforward and well established.
2LiOH
+ COZ-*
Lid203
+ H20
Determination of the optimum conditions of temperature, residence time, length of charge, particle size, etc. constituted a major portion of the actual study. The best LiOH to use as a converter charge was found to be commercial anhydrous LiOH (see Reagents). This material contains about o.5yO water and it must, therefore, be equilibrated in the converter a t operating temperature by paising dry carrier gas over it until a constant background level (approximately 500 pa.) of liberated water is attained. This treatment requires approximately 1.5 to 2 hours and, in practice, an auxiliary pre-
treated LiOH converter module is maintained on a stand-by basis. The optimum temperature and flow rate conditions for a LiOH charge were established by the combustion of pure graphite samples and conversion of the generated carbon dioxide to water with a LiOH charge. The water released was absorbed and weighed in Pregl tubes. These studies established a converter temperature of 205" to 225' C. and a flow rate of 10-15 ml./minute to be near optimum. Our studies showed that the release of water from a converter becomes slower and slower as the charge becomes exhausted. Because of this tailing effect, the charge should be changed after 10 to 15 analyses involving samples of about 5-mg. in size. Special Electrolytic Cells. Keideltype electrolytic cells ( 7 ) which are currently available commercially are normally used in applications where peak currents do not exceed 20 to 25 ma. Peak current levels in the carbonhydrogen system frequently exceed 50 ma. and positive errors are observed when standard cells are used. These positive errors have been attributed to the catalytic recombination of the hydrogen and oxygen generated by electrolysis of water within the cell. Consequently, an electrolytic cell was designed specifically to handle higher current loads and, if possible, to eliminate the recombination error. Essentially, the special cell is a scaledup version of a standard moisture cell, as used in the CEC Type 26-304 Xoisture Monitor, with several major modifications. The inner diameter of the cell tubing was increased from 0.025 inch to 0.063 inch. The special cells are not potted in epoxy resin as are the standard cells. Five-mil (0.005-inch) rhodium electrode wire is used in place of 5-mil platinum electrode wire. In addition to these changes, the special cells are mounted in an aluminum case with the inlet end of the cell aligned with a cooling port. T o minimize further recombination errors in the carbon determination, it is also necessary to apply a controlled bleed of dry nitrogen upstream of the electrolytic cell. An optimum bleed rate of nitrogen of approximately 40 ml. /minute was consistent with complete electrolysis of water, yet provided minimum residence time to achieve the least recombination. It should be made clear that such a bleed cannot be applied to the upstream (hydrogen measuring) electrolytic cell, as the subsequent flow rate through the LiOH converter and the second electrolytic cell would be too great for efficient operation. One feature of the recombination effect found in the course of this investigation was the occurrence of a
progressively increasing positive error during any given series of analyses especially when using platinum electrodes. This phenomenon was thought to be due to gradual catalytic activation of the cell electrodes by prolonged passage of high current densities. The rhodium electrode cell is not immune to activation under all operating conditions, but rhodium electrodes will not activate so fast nor to so great an extent as platinum electrodes. Errors caused by recombination effects can indeed be nearly eliminated provided the cell temperature is decreased by the application of external cooling, the current density is decreased, and electrolysis products are diluted and rapidly removed with a controlled nitrogen bleed. Peak currents must be kept below 100 ma. and this is readily accomplished by strict adherence to the designated carrier gas flow rate and sample weight. Special electrolytic cells have been operated intermittently at peak currents of 200 ma. for 1 hour without damage. Voltage-to-Frequency Converter Integrators. Electrolysis cell currents are measured automatically by applying the voltage drop generated across thermally stable resistors to calibrated voltage-to-frequency converter integrators with totalizing counters. A bucking circuit is employed to provide a small variable biasing voltage to the voltage-to-frequency converter so that the steady state background signal can be cancelled out. h simplified schematic of the integrating circuit is presented in Figure 3. Resistor R1 is used to limit the current to the electrolysis cell. Resistor RZ is used to convert electrolysis current to a potential and the high wattage rating is designed to minimize resistance changes due to heating effects. The integrators are calibrated coulometrically by substituting a resistance decade box in place of the electrolysis cell, passing a simulated electrolysis current through a precision resistor (10.0570) and measuring the iR drop on a precision L &: K potentiometer for an exact time interval. Time intervals are measured with a Standard Electric Time Co. electric stopclock, precise to +0.01 second. I n this manner, the number of counts per coulomb is established for each integrator over the desired range of current (voltage) input levels. The number of counts per coulomb is related to the number of coulombs per milligram of carbon and hydrogen as established by Faraday's law and, thus, each integrator is calibrated in terms of counts per milligram of carbon and hydrogen. The voltageto-frequency converters have a linearity of =!=o.05~0, and the established calibration factors are reproducible to better than *O.1Y0 over a range of 1 to 100 ma. VOL. 37, NO. 1, JANUARY 1965
119
LITERATURE CITED
ACKNOWLEDGMENT
The authors gratefully acknowledge the cooperation of the Upjohn Co. which supplied several purified organic samples for this investigation, and the technical assistance provided by E. E Martin and R. W. Eldridge in fabricating the equipment.
(1) Bennet, A., Analyst 74, 188 (1949). (2) Ebeling, M., Malter, L., Mikrochem. J. 1 . 169 (1963). (3) Fiancis,' H. J., Jr., ANAL. CHEM. 36, No. 7, 31A (1964). (4) Haber, H. S., Gardiner, K. W., Microchem. J. 6, 83 (1962). (5) Ingram, G., J . SOC. Chem. Ind. 62, 175 (1943). (6) Kainz. G.. Mikrochemie 35, 569 (1950). ' '
(7) Keidel, F. A., ANAL.CHEM.31, 2043 (1959). (8) Lebedera, A. I., Nikolaeva, N. A., Orestova, V. A., Izv. Akad. Nauk SSSR, Otd. Khim. Nauk. 1961, p. 1350; C . A . 55, 26828 (1961). (9) Mendoza, M. P., Fuel 37 (1958). (10) Vecera, 'M., M., Snobl. D.. ' Svnek.-' L.. --' Mikrochik. Acta 28, 9'(1958)." RECEIVEDfor review August 24, 1964. Accepted October 5, 1964. Division of Analytical Chemistry, ACS, 148th Meeting, Chicago, Ill., August 1964.
Resorufin Butyrate and Indoxyl Acetate as Fhorogenic Substrates for Cholinesterase GEORGE G. GUILBAULT and DAVID N. KRAMER Defensive Research Division, Chemical Research and Development laboratories, Edgewocd Arsenal, Md.
b Two new fluorogenic substrates are described for the determination of cholinesterase: resorufin butyrate and indoxyl acetate. Both are nonfluorescent compounds which are hydrolyzed by cholinesterase to highly fluorescent materials. The rate of change in the fluorescence of the solution due to production of resorufin (Aex = 540-570 mp, A,, 580 mp) = and indoxyl (Aex = 395 mp, A,, 470 mp), AF/At, is measured and correlated with enzyme activity. The preparation of the substrate solutions is relatively simple, and complete analysis requires only 2 to 5 minutes. Employing the methods described, 0.00030 to 0.060 unit per milliliter of horse serum cholinesterase may b e determined with a relative standard deviation of about f 1.0%. In addition to cholinesterase, acylase, acid phosphatase, and chymotrypsin hydrolyzed the substrates in varying degrees.
N
METHODS have been described for the determination of cholinesterase, based on enzymatic hydrolysis. Most of these involve either the measurement of the rate of acid production by p H change (6) or by the manometric determination of carbon dioxide liberation (IO),or utilize colorimetric (1, 2) or electrochemical (6) techniques. Since fluorogenic substrates are generally several orders of magnitude more sensitive to measurement than chromogenic ones (@, it was hoped that a fluorometric method could be developed for measurement of extremely small concentrations of enzyme and substrate. I n a recent publication ( d ) , a simple, rapid procedure was described for the assay of lipase activity in the presence of other esterases. The method was based on the hydrolysis of fluorescein
120
UMEROUS
ANALYTICAL CHEMISTRY
esters catalyzed by lipase. Recently we announced that resorufin acetate was hydrolyzed by horse serum cholinesterase to produce the highly fluorescent compound resorufin ( 8 ) . Various authors (11) have reported the colorimetric assay of cholinesterase, based on the formation of indigo blue from indoxyl acetate. Gehauf (3) and Kramer ( 7 ) have reported that indoxyl is a highly fluorescent compound, which is easily air oxidized to indigo blue. The present study discusses the development of a simple fluorometric assay for extremely small quantities of cholinesterase, based on the hydrolysis of indoxyl acetate to a stable fluorescent compound, indigo white. The hydrolysis of these substrates by other enzymes is discussed. EXPERIMENTAL
Reagents. Enzymes. Cholinesterase, horse serum (Armour Industrial Chemical Co.), specific activity 1.80 units-one unit represents one pmole of acetylcholine hydrolyzed per milligram of enzyme per minute. Solutions were prepared by dissolving the material, purified by the Strelitz procedure (6), in 0.1M tris buffer, p H 7.40. Acet'ylcholinesterase, bovine erythrocytes (Winthrop Labs), specific activity
1-90 units-one unit equals one pmole of acetylcholine hydrolyzed per milligram of enzyme per minute. Acetylcholinesterase, eel (these labs), specific activity 6.0 units (ACh). Lipase, porcine pancreas, and steapsin, a- and y-chymotrypsin were as described previously (4). p - Chymotrypsin, bovine pancreas (Calbiochem. Co.), activity 6000 units per mg. (ATEE). Acid phosphatase, potato (Nutritional Biochem. Co.), activity 100 units per mg. (Kornberg). Acylase, (Armour Research Co.), activity 1.0 unit per mg. (Armour). Tris Buffers. Tris(hydroxymethy1) aminomethane, p H 7.4 and 8.0, 0.01 and 0.1M, was prepared by dissolving t h e appropriate amount of Sigma 7-9 buffer (Sigma Chemical Co.) in distilled water. HC1, O.lM, was added to adjust the p H . Substrates, Indoxyl acetate(1). A stock 0.083M solution of this substrate was prepared by dissolving 145 mg. of the compound (Mann Research Labs) in 10 ml. of dioxane. Resorufin esters. A series of resorufin esters was prepared for testing as possible substrates for cholinesterase : resorufin acetate, propionate, butyrate, and benzoate, These esters of resorufin(1V) were prepared by treating resorufin (Eastman Organics, Practical Grade', with the appropriate acid an-
Table I. Resorufin Esters: Preparation and Properties A, Recryst. solvent Esters M.P. hsx, mr 450 1 . 4 2 x 104 Ethanol 223" da Resorufin acetate 450 1.30 x 104 Ethanol 1770b Resorufin propionate 440 1 . 2 4 x 104 Benzene 130'" Resorufin butyrate 470 8 . 0 x 103 Benzene 2030d Resoruh benzoate Literature value 223' d * Calcd. for C15Hl~04N:C, 66.91; H, 4.12; 0, 23.77 Found : C, 66.7; H. 4.2: 0 . 24.0. c Calcd. for CllHlS04N: C, 67.84; H: 4.63; 0 ; 22.59 Found : C, 68.2; H, 4.9; 0, 22.4. d Calcd. for ClsH1104N: C, 71.93; H, 3.49; 0, 20.17 Found: C, 70.3; HI 3.9; 0, 20.0. 5
