Microdetermination of Acetone in Biological Fluids M. U. TSAO, G. H. LOWREY, and E. J. GRAHAM Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Mich.
b The method is based on the diffusion of acetone from the sample on a strip of filter paper into 2,4-dinitrophenylhydrazine reagent in a photometer tube. The color of the resultant acetone 2,4-dinitrophenylhydrazone in alcoholic alkali is developed and read in the tube. From 0.5 to 8 y of acetone may be determined without prior dilution of the sample. At a concentration of 50 mg. acetone in the sample, the limits of error a t the 9570 confidence interval are 1.2 and 1.5% for urine and blood, respectively. A large number of samples may be run simultaneously. This method was designed for research purposes, but may be used as a routine clinical procedure.
yo
F
of the methods n o ~ vavailable for acetone determination in biological fluids are suitalde for micro scale adapt'ation. The methods based on prrcipitation of acetone with mercurio sulfate f o l l o n d b!. gravimetric ( 1 6 ) , t>itrinietric (1, 16, I?), or colorinietric (4) procedures are subject, to serious u r o r whni minute amounts of precipitate arc to bc frcxed from contaminants. Methods utilizing the red color from the reaction between acetone and salicylaldc~hytleare relatively spccific and sensit,ivc, (2. 3, 6 4 , 10, 13, 1 4 ) . However, they have serious deficiencirs (I), as described by Barnes and K i c k ; furthermore, the high volatility of salicylaldehyde can be troublesonit1 i n a laboratory where reagents for carbonyl compounds, such as 2,4diiiitropl-ienylhydrazine, are used. The product of the reaction between acetonr and 2,4-dinitrophenylhydrazine g i w s a reddish bronm with alcoholic alkali; this is the basis of several sensitive methods for acetone (5, 9, 11, 12). Where extraction of the reaction product n-ith carbon tetrachloride is employed (5, 9, 12), the recovery is low (12). TVhere extraction is not nwded, a distillation necessarily precctles t'he reaction (12). A method has been described which involves distillation of acetone into a n excessive amount of iodine and back-titration with thiosulfate (11). This is not specific for acetone in biological fluids, even though high sensitivity for carbonyl compounds can be obtained. I n a study using small animals, a procedure for the microdetermination of acetone in blood and urine samples EW
Figure 1. Diffusion rod with filter paper strip
was necessary. Furthermore, the samples contained significant amounts of a nonvolatile carbonyl compound, which eliminated the method which involved the extraction of 2,4-dinitrophenylhydrazone of acetone 11ith carbon tetrachloride (6). The method described here uses the high volatility of acetone for its isolation by diffusion and the relative color smsitivity of the 2,4dinitrophenylhydrazone. Because all steps are carried out in the same photometer tube, a high degree of precision has been achieved. The feasibility of siniultaneouq analysis of multiple samples \vas niadc possible by the modification of a unique apparatus described previously for the transfer of material by diffusion (1.5). APPARATUS
DIFFUSION ROD. Diffusion rod, suspend the filter paper strip onto ivhicli the sample has been pipetted for diffusion of acetone from the paper into the reagent in the photometer tube. The glass diffusion rod is 100 mm. long, with a diameter of 3 mni. (Figure 1). It has a flattened segment 20 mm. from the lower end, t o which a stainless steel wire, such as one from stainless steel nire gauze, can be securely fastened. A part of the \\-ire forms a loop around the rod. as a guide for sliding the filter paper with the sample on it into the yhotometer tube (Klett tube, Klett hlfg. Co., Ken- York, X. Y.), thus avoiding contact between the filter paper and the wall of the tube. The filter paper strip is cut to form a collar around the diffusion
Figure 2. bath
Diffusion rack and ice
rod. It is kept from falling off by flattening the lower end of the rod so that the paper collar may be slipped past the disklike end but cannot slip do\T n of its own accord. The upper end of the diffusion rod is set into a one-hole S o . 0 rubber stopper which fits into a Klett tube. The diffusion rod ma;\- be flattened a t these two points by heating a glass rod red-hot and pinching lightly with glass-blowing forceps. DIFFUSIOS RACKAND ICEBITH. A desiccator of 21-cm. diametrr is used as an ice bath and as a support for the diffusion rack. The rack used in this study has 24 p1:ices for holding Klett tubes (Figure 2). It consists of three flat stainless steel rings fastened together with four vertical rods. The bottom ring or plate supports the Klett tubes and has no holes. The center ring is mainly the support of the rack and rests on the rim of the desiccator. The top and center rings have 24 holes of 16.5mm. diameter to accommodate the Klett tubes. The three rings are spaced so that the Klett tubes rest securely in the rack with 10.5 cni. of the tube above the center ring, and 2.0 cm. below it. When the desiccator. D , is filled with an ice-water niivture to t h r brim, the Klett tubes in the diffusion rack have the lower 2.0 cm. immersed in ice water. Because the diffusion rack promotes diffusion of acetone from the paper strip on the diffusion rod into the reagent in the bottom of Klett tube, mild heating of the paper and chilling of the reagent VOL. 31,
NO. 2, FEBRUARY 1959
31 1
hasten the transfer of acetone. A heata reagent blank and an acetone standard ing element, -4 (Zoalite infrared heating of comparable concentration with the element SZ S35, Burdick Corp., Milton, unknowns all in triplicate. M7is., or equivalent) is suspended in the COLORDEVELOPMENT. Add 0.3 nil. of center of the rack. A thermoswitch, Bl 9-V sodium hydroxide with a syringe is placed in a hole in the rack t o maintain pipet to the tube. Mix and let stand exthe temperature reading from a thermactly 30 seconds, and add 3 ml. of 50% istor-type thermometer, C, in a Xlett ethyl alcohol with a syringe pipet, Proctube at 37' to 39" C. when the top ring ess each tube in this manner. Read the has its center area covered mith asbestos tubes in the colorimeter against the reshields; the thermosn-itch operates a agent blank a t 15 minutes or some other variable transformer which supplies curfixed time interval after the addition of rent to the heating element a t approxithe ethyl alcohol. mately 40 bolts. ' 4 metal shield 18 em. CALCULATION AXD CBLIBRATIOS. high (not shown), the inside surface of The absorbance reading of the sample which is highly polished, encircles the tubes; this acts as a reflector and facilitube corresponds to the acetone contates even heating of the tubes. ii centration of the sample. From a stainless steel disk (not shonm) of a calibration curve constructed with abdiameter just small enough to pass sorbance readings and their correthrough the opening of the top ring of sponding acetone standards of the conthe rack and yet large enough to rest on centrations in milligrams per cent, the the center ring, supports an asbestos disk acetone concentration of the unknown to insulate the ice bath from the heating. can easily be read. The acetone standAn asbestos roof on top of the diffusion ard run with the samples checks any rack cuts doiTn the air convection currents. possible error in the procedure and KLETT-SUMMERSON PHOTOELECTRIC should give the same colorimeter readCOLORIMETER, with filter 54. ings from day to day. PIPETS.Micropipets of lo-, 20-, and 150-p1, capacities. Automatic syringe EXPERIMENTAL pipets: one 0.5-ml. tuberculin syringe adjusted to deliver 0.3 ml. and one 10Absorption Maxima of Acetone 2,4ml. syringe adjusted to deliver 3 nil. Dini?rophenylhydrazone. The absorption spectra of t h e hydrazone in REAGENTS alkaline solution was determined on a solution of a purified sample in alco2,&DISITROPHENYLHYDRAZOKE. holic sodium hydroxide (0.25.V in 70% mixture of 10 ml. (alcoholic sulfuric acid prepared from 50 nil. of 95% ethyl alcoethyl alcohol) solution containing 5 hol] and 0.25 ml. of concentrated sulfuric mg. per ml. of hydrazone. A Beckman acid) and 10 mg. of 2,4-dinitrophenylspectrophotometer was used. A sharp hydrazine is heated in a glass-stoppered absorption maximum was noted a t centrifuge tube to about 50" in warm 430 mp and a broad one a t 540 mp. water with constant shaking until most The same maxima were observed when of the solid dissolves. The undissolved an acetone solution was allowed to react material is centrifuged down but allowed with a solution of 2,4-dinitrophenylt o remain in the tube. This reagent hydrazine hydrochloride in 50% ethyl must be prepared fresh daily and kept in the refrigerator when not in use. alcohol and the mixture then made 9N SoDIuhf HYDROXIDE, prepared by alkaline with sodium hydroxide as demixing equal volumes of water and satscribed in procedure. Although the urated and sedimented sodium hydrox430 mp maximum not only is a sharper ide in water. absorption peak but also gives a greater 50% ethyl alcohol. extinction coefficient than the 540 mp maximum, the reagent blank at PROCEDURE that Ivave length is, unfortunately, much too high. Consequently a 54 DIFFUSION.Pipet 150 pl. of the 2,4dinitrophenylhydrazine reagent into the filter was used in the Klett-Summerson bottom of the Klett tubes. S o t c h filter photoelectric colorimeter for reading paper strips (8 X 16 mm.) a t the ends the color of acetone 2,4-dinitrophenylso t h a t a paper collar can be formed by hydrazone in alcoholic alkaline solution hooking the ends together. P u t the in all the absorbance determinations. paper on the diffusion rod and place the 2,4-Dinitrophenylhydrazine Rearod part-way into the Klett tube so that gent. T h e preparation of this reagent the rod can be quickly slid into place was based on the relative stability of and the tube stoppered with minimum 2,4-dinitrophenylhydrazine in acid opportunity for the acetone on the paper solution and t h e desirability of a high to escape. Pipet 10 or 20 bl. of sample onto the paper and immediately stopper concentration of t h e reagent in the the tube. Place the tube in the diffusion reaction mixture to ensure completion rack which has been thermally equiliof hydrazone formation by acetone. brated t o maintain the thermometer When a mineral acid was not included in reading a t 37" t o 39" over the ice bath the reagent, an extremely high reagent filled with crushed ice and water. Alblank n-as found. Sulfuric acid is betlow the diffusion process t o proceed for ter than hydrochloric in that the reagent 1.5 hours. At the end of this period, has less tendency to creep up the wall remove the tubes and let them stand a t of the colorimeter tube during diffusion room temperature for 30 minutes. Run 312
ANALYTICAL CHEMISTRY
of the acetone. If the hydrazone mas allowed to precipitate out of the reaction mixture, there was no linearity betneen the final color in terms of absorbance and the concentration of acetone; therefore, 95% ethyl alcohol was used in the reagent to keep the acetone 2,4-dinitrophenylhydrazonein solution during and after the diffusion period. The reagent blank ri>eq steadily with time even when the reagent is kept a t 5 " ; consequently, it is necessary to prepare a fresh reagent daily. The amount of reagent used is sufficient to react with up to 8 y of acetone so far as the linearity of absorbance z's. concentration is concerned. With less than 150 pl. of reagent. there occasionally was not enough reagent to cover the bottom of the colorimeter tube; consequently the diffusion process was hindered by the diminished absorbing surface. Diffusion Process. The temperature for t h e diffusion process was determined by the limits between t h e niavinium temperatures above n-hich there might be u n t o m r d production of volatile carbonyl compound from t h e biological fluid, and the minimum temperatures below n-hich the vapor pressure of acetone and water will be so loiv as to render the diffusion process impractical. Although the temperature reading in the control tube was maintained a t 37" to 39O, the actual temperature of the filter paper on n hich sample was placed might be somewhat higher n hen radiant heat was absorbed by the blood sample. It cannot be assumed, holyever, that metabolic processes do not go on in the blood sample, a t least during the initial phase of the diffusion process, unless inhibitors are added to the blood sample. At the end of the diffusion process the dry appearance of the filter paper indicates that practically all of the water, as well as the acetone, in the sample has been transferred from the filter paper into the reagent in the colorimeter tube by diffusion. Hence, there is no likelihood that any residual acetone might remain on the filter paper because of its solubility in water. The rate of transfer of acetone from the filter paper into the ZJ4-dinitrophenylhydrazine reagent x a s studied with acetone standards (50 nig. % solution), blood, and blood or urine with the equivalent of 25 and 50 mg. % acetone added. The conditions used were as described in the procedure, except that the length of the diffusion period TTas varied and quadruplicate determinations mere made. The results are expressed in terms of absorbance readings in the Klett photoelectric colorimeter ( x lo3) and tabulated in Table I. At the end of 1 hour of diffusion the filter paper strips do not alvvays appear dry; therefore the dif-
fusion study started with the 1-hour period. The results indicate that there is no significant difference between the results from 1.5-, 2-, and 2.5-hour diffusion periods. Color Development. At the end of the diffusion period, the colorimeter tubes were permitted to stand for 30 minutes t o reach room temperature before sodium hydroxide !vas added. The effect of the period of standing on the results )!as studied by adding acetone standard to the reagent in colorimeter tubes and noting the development of color after l 5 , 2 0 , 30, and 45 minutes of standing. The means of the quadruplicate runs showed no significant difference; therefore, the reaction between acetone and the reagent is rapid and 30-minute standing is ample for the tubes to reach room temperature and complete the hydrazone formation. The amount of sodium hydroxide added did not seem to be critical as long as an excess over that required t o neutralize the sulfuric acid in the 2,4-dinitrophenylhydrazinewas present. To keep both the sodium sulfate and the hydrazone from causing turbidity in the final solution, a solvent meeting this requirement was found50% ethyl alcohol in water. The color of the final solution was not too stable; a decrease of 2% in absorbance readings in 15 minutes after the color development has been observed. Useful Range. A stock standard of acetone wis prepared by weighing acetone into water in a volumetric flask and making up to volume with nater. From this standard, solutions containing 5, 10, 20, 40, 60, 80, and 100 mg. % acetone mere prepared and used shortly afterwards. Applying the procedure as described to these standards, the results of the means from triplicate analyses, expressed in terms of absorbance, were plotted. The procedure yields linear relationship betn-een the concentration of acetone in the sample and the absorbance of the final solution from 5 to 80 mg. yo. This range spans the limits of normal and pathological blood acetone values. There has been no occasion, as yet, to dilute the sample to bring the concentration of acetone into the effective range of the procedure. For urine samples of low acetone content-containing less than 5 mg. %-it might be necessary to double the sample size so that a reliable colorimeter reading could be obtained. However, the extra amount of water in the larger volume of sample could lower the results; thus corrections must be made, or a separate calibration curve set up for the larger volume. Reproducibility. An acetone solution was added to blood or urine samples so t h a t the acetone concentration of the samples was increased by
~~
Table 1.
Rate of Transfer of Acetone
Sample PI.
25 mg.yo std. 50 mg.Cc std. Blood Blood 25 mg.% acetone Blood 50 mg.% acetone Vrine 25 mg.yo acetone
++ +
10 10 10 10 10 10
20
50 mg.YO. On each of the four blood and three urine samples simultaneous runs of 11 tubes were made. The standard deviations, in terms of per cent of absolute value, were 1.6, 1.0, and 1.6 for blood and 1.6, 1.2, and 0.7 for urine. Greater precision is obtained \Then samples are analyzed in triplicate. Recovery of Added Acetone from Blood and Urine. Stock acetone standard was added to a fresh venous blood sample and to water, so t h a t the increase in acetone concentration in both was 50 nig.Yo. The acetone levels of the blood and water with acetone added and t h a t of the original blood were analyzed in triplicate. The mean values from the triplicates were used for calculation of recovery of acetone added to blood. The recovery of 5 y of acetone from blood samples was calculated (with absorbance as units) by the following equation:
70recovery
=
blood with acetone added - blood water with acetone added
x 100
~
by Diffusion
Diffusion Periods, Hours 1.5 2 Transfer Rate -I I ii i8 128 129 131 27 28 31 .. 103 103 .. 164 159 79 79 i8 173 168 168 1
2.5 81 131 27 104
161 80
170
reading the absorbance immediately after color development, and subsequent to 30-minute incubation a t 37". Other conditions may be used to simplify the calculation of results. Preservation of Sample. Blood samples sealed in a syringe and urine samples kept in a closed vessel may be preserved in the refrigerator for 24 hours without any change in acetone level. Because the metabolic processes of the blood may continue after the placement of blood sample on the filter paper for analysis, acetone other than the preformed acetone might be included in the result. To test whether this is the case, metabolic inhibitors nere added to blood samples with and without the addition of acetone and the results were compared with the same blood samples without the inhibitors. Sodium fluoride, sodium azide, mercuric chloride, dinitrophenol, malonic acid, or citric acid a t 1mJI concentration showed no significant effect on the acetone values of blood. DISCUSSION
The results with four blood samples were: 99, 86, 92, and 94% recovery. The same method was applied to recovery of acetone added to three urine samples. The results were: 102, 102, and 99% recovery. Interfering Substances. The isolation of acetone from biological samples by diffusion eliminated the majority of substances t h a t would interfere with procedures where diffusion or distillation is not employed. Acetaldehyde, being the most likely interfering substance, was studied for its effect on the acetone determination. Immediately after color development, it yielded about the same absorbance as that of acetone of equal weight. However, the absorbance due to acetone drops only 5% after 30 minutes a t 37", while that of acetaldehyde reduces by 75y0 under the same conditions. I n situations where the concentration of acetaldehyde might be significantly high in relation to that of the acetone, the quantities of both the acetone and acetaldehyde mag be determined by
There are several possible sources of error. I n the preparation of 2,4dinitrophenylhydrazine reagent, either excessive and/or prolonged heating invariably causes a high reagent blank Lvhich otherwise remains fairly constant from one preparation to another. Because of the high volatility of acetone, the standard solutions for the construction of the calibration curve must be prepared from fresh stock standard shortly before use. Also, despite the high volatility of acetone, any glass\yare dried with acetone is highly contaminated and other means of drying glassware should be used. During the transfer of the sample from the pipet onto the filter paper strip, care should be taken not to blow the last trace of blood into a bubble which would spatter onto the inside wall of the Klett tube; poor results follow such a seemingly slight error. While the assembled Klett tube is being heated in the diffusion rack, the temperature control within the specified VOL. 31, NO. 2, FEBRUARY 1959
313
range is critical; allouing the teniperature to escecd the limits causes low reproducibility and overheating brings about high reagent blanks as nell. This method has been used routinely in this laboratory for the past 6 months on clinical blood and urine samples with satisfactory results. ACKNOWLEDGMENT
This work was aided by a grant from the Michigan hlemorial Phoenix Project. The interest and encouragement of James L. Wilson are deeply appreciated.
LITERATURE CITED
(1) Barnes. R . H., Wick, il. \I7,)J . Biol. Cheni. 131, 413 (1939). (2) Behre, J. Ah.> Ibid., 136, 25 (1940). 1 3 ) Behre. J. A.. Benedict. S. R.. Ibid.. 70.
487 (19’6).
’
Crandall. L. -i.!Jr., Ibzd., 133, 539 (1940). (5, Greenberg, L. A,, Lester, E)., Ibid., 154, 177 (1944). (6) Klendshoj, S . C., Feldstein, Milton, Can. J . X e d . Technol. 17, 74 (1985). (71 \ , Lauersen. Fritz. Klin. Wochschr. 15. 339 (1936): 18) RIarenzi. A. D.. Braemer, E. S.. Pubis. inst. invest. &ropuiK Linin. naci, litoral (Rosario, Ary.) 17, 140 (1953). (9) ;2licharls, G. D., Margen, Sheldon, 14)
~,
Liebert, Gei:tld, Kinsell, L. \I-,, J . Clin. Invest. 30, 1483 (1951). 110) Nadeau. GUT,,Can. Med. .-lssoc. J . 67, 158 (1952). ” ’ (11) Ra paport, F., Baner, B., J . Lab. Clin. s e d . 2 8 , 1770 (1943). (12) Rutman, J. Z., Ibid.,*41, 648 (1953). (13) Schvivenkenbacher, 1% ., Z . klin. X e d . 134, 325 (1938). (14) Sobotka, Harry, Carr, J. J., “Anriual Review of Medicine,” Vol. 6 , p. 260, -4nnual Review, Stanford, Calif., 1955. (15) Tsao, 11.E., Baumann, bl. L., Wark! Shirley, . ~ X A L .CHEM.24, 722 (1952). (16) Jan Slyke, D. D., J . Biol. Chem. 32, 453 (1917). (17) Weichselbaum, T. E., Ibid., 140, 5 (1941). RECEIVED for review March 26. 1958. .iccepted September 12, 1958. ~
Colorimetric Determination of Alkyl Nitrites SIR: The Griess Ilosvay method has been used in air pollution work to determine nitrogen dioxide in laboratory and field operations and has been adapted for use in automatic instrunientation ( 5 ) . I n this latter investigation ( 5 ) it was shown that alkyl nitrites also react n i ‘ h the reagent in the automatic nitrogen dioxide analyzer used. It appeared of interest to determine the reactivity of the alkyl nitrites directly without having to consider absorption efficiencies. The amount of reaction of n-butyl nitrite, krt-butyl nitrite, n-amyl nitrite, and isoamyl nitrite on a micromole basis was determined for comparison a i t h the reactivity of sodium nitrite and nitrogen dioxide (8). The concentrations ranged between 0.07 and 0.7 micromole per 10 ml. of Table 1.
Colorimetric Results with Alkyl Nitrites
llethod
Absorbance Units/ pmole Alkyl Xitritea Alkyl Prepara- .hbsorbance Units/ tion pmole KaNOs Nitrite n-Butyl A 0 64 zk 0 04 B 0 79 =!= 0 05 C 0 79 zk 0 0 7 tert-Butyl B 0 70 i 0 03” n-Amyl B 0 81 i 0 OAC C 0 91 i o 04 Isoamyl A 0 77 i 0 06 B 0 74 i 0 04 C 0 79 0 03 A = untreated, B = atmospheiic distillation, C = vacuum distillation. a Standard deviation given after average values. * Average of three determinations. c Average of two determinations. of
314
ANALYTICAL CHEMISTRY
solution. The reproducibility of the results ranged from =!=5 to &lo% for a given sample of an individual alkyl nitrite (Table I). Saltzman has reported that the reactivity of nitrogen dioxide compared nith inorganic nitrite is in the ratio of 0.72 to 1. Patty and Petty found this ratio to be 57 Z!C 2.6% ( 1 ) . Thomas (4) found it to be 0.9, using the automatic analyzer (@, for which the ratio drifted upnard towards 0.9 over a period of months. Changes in the gas-liquid contactor column with time may account for this behavior. The difference in the two former values may be due to the use of S-(1-naphthyl)ethylenediamine dihydrochloride rather than 1-naphthylamine by Saltzman and a higher concentration of sulfanilic acid. Saltzman’s data ( 2 ) indicate an increase in the ratio with increasing sulfanilic acid concentration. Table I shons that the alkyl nitrites react to about the same extent as nitrogen dioxide. Distillation, particularly vacuum distillation, increases reactivity through improved purity of the alkyl nitrites. Undoubtedly, a portion of the diffcrence in the observed ratios from unity is the rtsult of appreciably less than 100% purity in the alkyl nitrites. High purity is difficult to achieve and maintain, bccause of the instability of the alkyl nitrites to heat and light. It has been suggested that the ratio for nitrogen dioxide and alkyl nitrites may involve a reverse oxidation-reduction system (4). A comprehensive study of the kinetics of the reactions involved would be extremely useful. The results obtained in this inyestigation and elsewhere indicate that the method u s d for nitrogrn dioxide is not
specific but responsive to all compounds, inorganic and organic, containing the 0-N-0 group. These compounds include the acyl peroxy nitrite found in the Los Angeles type atmospheres by infrared investigations (3). Furthermore, the complex reaction products formed by the reaction of nitric oxide and nitrogen dioxide with olefins often contain reactive O--h’-O groups. Consequently, methods are needed to differentiate nitrogen dioxide from organic nitrites. EXPERIMENTAL DETAILS
The alkyl nitrites included n-butyl nitrite, tert-butyl nitrite, n-amyl nitrite, and isoamyl nitrite from commercial sources. These nitrites were subjected to atmospheric and vacuum distillations during various stages of the investigation. A number of the lots of alkyl nitrites were dried over calcium chloride. The solutions for the colorinictric determinations Fvere prepared by dissolving 1 ml. of the alkyl nitrite in 75 ml. of glacial acetic acid and diluting to 250 ml. with distilled water. One milliliter of this solution was diluted to 100 ml. with distilled ivater to make the necessary solutions in the microgram range. An aliquot of test solution n a s added to 10 nil. of a reagent prepared by dissolving 5 grams of sulfanilic acid in nearly a liter of tvater containing 140 ml. of glacial acetic acid, adding 20 ml. of O.lyo -Y-(l-naphthyl)-ethylenediamine dihydrochloride solution. and diluting to 1 liter ( 2 ) . After 10 minutes the color wab read in a Beckman Model D r spectrophotometer a t 550 nip. The stability of 0.4% tert-butyl nitrite in acetic acid-water solution n as followed by means of its spectruni in the 3000- to 4000-A. region using a Cary Model 11 spectrophotometer.
