ANALYTICAL CHEMISTRY
902 cence). The interference by formaldehyde is more pronounced on account of the greater rate of dye formation with this substance. Attempts to eliminate this interference by initial reaction of pyruvaldehyde samples containing formaldehyde with dimedone were unsuccessful. Apparently pyruvaldehyde also undergoes a rapid reaction with dimedone. The presence of reducing sugars a t fairly high concentrations may result in either high or low values, depending on the mode of degradation of the carbohydrate under these conditions. DXylose causes high values, which increase with time of standing] because of appreciable conversion to pyruvaldehyde (18). DGlucose appears to undergo sufficient degradation to diacetyl or formaldehyde (11, fb) to cause low results, although in view of the greater interference by the lower concentration of this substance, as shown in Table VII, it seems that formation of pyruvaldehyde is in this case a competing reaction which may be more concentration dependent. The following substances did not interfere a t a weight ratio of substance tested to pyruvaldehyde of 1000 to 1: furfural, acetol, methyl ethyl ketone, pyruvic acid, lactic acid, levulinic acid, acetic acid, propionic acid, acetone, acetaldehyde, isobutyraldehyde, methanol, ethanol, and 1-butanol. PRECISION
The precision of the method indicated by the data in Table VI11
is slightly outside the limits of reproducibility by the fluorometer. However, the handling of this number of samples is a rather severe test of precision, owing to the considerable elapsed time
between the first addition of sulfuric acid to the first sample and the placing of the six samples in the 50' bath. In a number of applications of the method where development for only two or three samples has been carried out at one time the precision has been generally better than that indicated in the table. LITERATURE CITED
(1)Aryama, N., J. Bid. Chem., 77,359 (1928). (2) Baer, E.,Arbeitsphysiol., 1, 130 (1928). (3) Barrensheen, H., and Dregnus, M., Bwchem. Z., 233,296 (1931). (4) Dische, Z.,and Robbins, S., Zbid., 271,304 (1934). (5) Eegriwe, E.,Z . anal. Chem., 110,22 (1937). (6) Fischler, F.,and Boettner, R., Zbid.,77,359 (1928). (7) Friedmann, F.,J . Biol. Chem., 73,133 (1927). (8) Knopfer, G.,Monatsh., 32,765 (1911). (9) Levene, P. A., and Walti, A., "Organic Syntheses,'' Coll. Vol. 11,p. 5,New York, John Wiley & Sons, 1943. (10) Simon, E., and Neuberg, C., Biochem. Z.,232,479 (1931). (11) Speck, J. C.,ANAL.CHEM.,20, 647 (1948). (12) Speck, J. C.,unpublished work. (13) Spoehr, H. A,, and Strain, H. H., J. BioZ. Chem., 89,503 (1930) (14) Witzemann, E.J., Evans, W. L., Hass, H., and Schroeder,E. F., "Organic Syntheses," Coll. Vol. 11, p. 305, New York, John Wiley & Sons, 1943. RECEIVED March 27, 1948. From a thesis submitted by Barbara J. Thornton to the Graduate School of Michigan State College for the M.S. degree. This paper reports research undertaken in cooperation with the Quartermaster Food and Container Institute for the Armed Forces, and has been assigned number 164 in the series of papers approved for publication. The views or conclusions contained in this report are those of the authors. They are not to be construed aa necessarily reflecting the views or indorsement of the Department of the Army.
Fluorometric Determination of Acetol ARLINGTON A. FORIST AND JOHN C. SPECK, JR. Michigan State College, East Lansing, Mich. The fluorescence of 3-hydroxyquinaldine, which is formed in the reaction of o-aminobenzaldehyde with acetol, has been applied in a method for determining acetol. The effects of the following variables have been determined: concentration of sodium hydroxide, reagent concentration, acetol concentration, time, and final pH. The proposed method is sufficiently sensitive to permit determination of acetol at concentrations of 0.3 mg. per liter. Ite usefulness is limited by pronounced interference of furfural, diacetyl, and formaldehyde.
R""""
TTwork in this laboratory involving investigation of producta from reaction of amino acids with reducing sugars has led to a semch for a method for determining acetol a t low concentrations in the presence of other carbonyl compounds. The proposed analytical procedure] which has been developed in an attempt to meet these requirements] is based upon a reaction first described by Baudisch (1, 2) in which acet,ol combines with o-aminobenaaldehyde in alkaline solution to form 3-hydroxyquinaldine (Equation 1). Upon irradiation with ultraviolet light, 3-hydroxyquinaldine gives a strong blue fluorescence.
aldehyde and sodium hydroxide solutions to 1 ml. of distilled water. The flasks are immersed in a bath of boiling water and the stoppers are inserted. Heating is continued for a total of 30 minutes, after which the flasks are transferred to a cold water bath and rapid1 brought to room temperature. To each solution are then a d d e i 2 ml. of 0.5 N hydrochloric acid and 5 ml. of pH 6.6 McIlvaine buffer (3). The solutions are diluted to the mark with distilled water, and the fluorescence of the unknown is determined in a fluorometer equipped with standard filters used in the vitamin BI determination. The instrument is calibrated by determining the fluorescence obtained by the above procedure with solutions of known acetol content. Two points are sufficient to establish a curve, as it is perfectly linear over the recommended concentration range. Temperature control is recommended during fluorescence measurements. EXPERIMENTAL
RECOMMENDED PROCEDURE
One milliliter of the unknown solution, containing 0.30 to 6 micrograms of acetol, is placed in a 25-ml. volumetric flask equipped with a lass stopper. To this is then added 1 ml. of t6e eaminobenaalde%yde reagent (0.2 mg. of o-aminobenaaldeh de per ml.) followed by 5 ml. of 0.2 N sodium hydcoxide. A blanc is also prepared by adding the same amounts of the o-aminobena-
Materials. Acetol used in these ex eriments wm obtained from Bios Laboratories and was purifieB by ordinary distillation a t reduced pressure. The fraction collected boiled a t 42" to 45" C. a t 15 mm. and was stored in a desiccator over Drierite. For preparation of acetol stock solutions, approximately 100 mg. of such a fraction were accurately wei hed into a 50-ml. volumetric flask and diluted to the mark wit% redistilled water. The purity of the acetol was checked by oxidation of aliquots of these stock solutions with periodic acid. In a typical determination,
903
V O L U M E 22, NO. 7, J U L Y 1 9 5 0 Table I.
Effect of Time of Heating
Time, Minutes
c3
2 60
33.0 54.0 63.5 75.0 76.5
10 15 30
W
45
a a W
I
Fluorometer Reading
5
2
Table 11.
50
0
Mixture
K
Acetol Furfural-acetol
0 3
....
0.2
04 0.5 CONCENTRATION OF O-AMINOBENZ-
0.6
0.3
> 100
100:1 10:1 1000:1 100:1 10:1 1000:1 100:1 1000:1
Diacetyl-acetol Formaldehyde-acetol
0.I
Fluorometer Reading 68.0
Weight Ratio
Pyruvic acid-acetol
40
Interferences
Acetaldehyde-acetol
>
1OO:l
84.0 60.5 65.0 5.0 100 5 38.5 57.0
ALDEHYDE REAGENT, MG*/ML*
Figure 1.
4
Effect of o-Aminobenzaldehyde Concentration
Effect of Reaction Conditions. In order to test the effect of variation of reaction conditions on the formation of the fluorescent compound, sets of identical samples were treated according to the recommended procedure, with the exception of the condition being studied which was varied from sample to sample. In all cases separate blanks were prepared for each sample, and a quinine sulfate solution (0.3 mg. of quinine sulfate per liter, 0.1 AT in sulfuric acid) was employed for maintaining a reference point (100 on the scale of the instrument). The results of variation in the time of heating are shown in Table I. The relationship between fluorescence and the concentrations of o-aminobenzaldehyde and of sodium hydroxide is shown in Figures 1 and 2, respectively.
(I
60
W
a a
W b
sodium hydroxide solution, cooled during the neutralization, and diluted to 250 ml. witkdistilled water.
50
I
0
a
s
2 40 1
I
I
0.1
0.2
0.3
I
I
0.4 0.5 NORMALITY OF SODIUM HYDROXIDE
Figure 2. Effect of Sodium Hydroxide Concentration Used in Condensation upon Final Reading
the acetol concentration found by periodic acid oxidation was 2.080 grams liter, as compared with 2.076 grams per liter actually weig ed. o-Aminobenzaldeh de was purchased from Bios Laboratories and was not purifiej before use. Diacetyl was obtained from Forest Products Chemical Com any and was fractionally distilled. Other compounds t e s t e f as possible interferences were either Eastman Kodak best grade or C.P. chemicals. Hydrochloric acid, sulfuric acid, sodium hydroxide, citric acid, and diRodium phos hate used in this work were all C.P. quality. Mallinckrodt U.1.P. quinine sulfate was used to prepare reference standards. Apparatus. A Model 12B Coleman electronic photofluorometer, equip ed with a B-1 priinary filter and a PC-1 secondary filter, an$ the standard cuvettes supplied for this instrument were employed for fluorescence measurements. A Beckman pH meter with glass electrode was used for pH determinations.
R"'
Preparation of o-Aminobenzaldehyde Reagent. Solid o-aminobenzaldehyde undergoes polymerization upon standing, with the formation of water-insoluble material. This reaction may be reversed by solution in concentrated hydrochloric acid, and, providing neutralization is carricd out immediately after solution is efiected, the resulting solutions contain only a negligible amount of insoluble material and remain clear. Accordingly, the following procedure was adopted for preparation of solutions of this compound : Fifty milligrams of o-aminobenzaldehyde were dissolved in 25 ml. of concentrated hydrochloric acid (specific The solution was then adjusted to pH 7.0 (glass e?::t%ie)l%k
5.5
6.0
64
7.0
7.5
PH Figure 3.
Effect of pH on Fluorescence
Effect of pH on Fluorescence. The fluorescence obtained with acetol-o-aminobenzaldehyde reaction mixtures is dependent upon the final acidity of the solution. Data showing this variation of fluorescence with pH (Figure 3) were obtained by carrying out the reaction of ident,ical samples of acetol with o-aminobenzaldehyde according to the recommended procedure and varying the pH of the buffer solution added after neutralization. Seprtrate blanks were again prepared, and the quinine sulfate solution was used as the reference. Interferences. The data in Table 11, which indicate interference by several 'compounds, were obtained with solutions containing the same aliquot of acetol standard stock solution and the compound to be examined for interference in the weight ratio indicated. The most pronounced interference is that of diacetyl.
ANALYTICAL CHEMISTRY
904 Table 111. Precision Acetol Weighed, Mg./L. 0.82 1.64 2.40 3.28 4.09 4.91 5.73
Found, Mg./L. 0.8,O.g 1.6, 1.6 2.5,2.5 3.2,3.3 4.2,4.2 4.8,4.9 5.7,5.8 6.6,6.5
6.56
Formaldehyde, in large excess, forms colored solutions which fluoresce beyond the range measurable by the fluorometer. .4t lower concentrations of formaldehyde, in which case little coloration develops, low values are obtained, possibly due to reaction of formaldehyde with acetol. The presence of furfural results in high values. Acetaldehyde interferes only when present in very large excess, whereas the interference by pyruvic acid is small. The following substances do not interfere when present in a weight ratio of 1000 to 1 : methanol, ethanol, acetone, lactic acid, and levulinic acid. DISCUSSION
This quantitative application of Baudisch’s qualitative test for acetol gives good results in the absence of the interfering sub-
stances mentioned above. As shown in Table 111, the precision of the method appears to be nearly within the limits of the fluorometer. The fluorescence is dependent on temperature. However, the fluorescent compound is stable, exhibiting no decay, in the absence of strong illumination, during 2 hours. A single determination requires approximately one hour, and several can be run simultaneously. As yet, no means has heed found for reducing the effect of any of the interfering substances. This is unfortunate, for the interferences of diacetyl and furfural limit the usefulness of the method in application to certain problems in carbohydrate chemistry. LITERATURE CITED (1) Baudisch, O., Biochem. Z.,89, 279 (1918). (2) Baudisch, O., and Deuel, H. J., J . Am. Chem. Soc., 44, 1585 (1922). (3) McIlvaine, T. C., J . Biol. Chem., 49, 183 (1921).
RECEIVED December 27, 1949. This paper reports research undertaken in cooperation with the Quartermaster Food and Container Institute for the Armed Forces, and has been assigned number 283 in the series of papers approved for publication. The views or conclusions contained in this report ar0 those of the authors. They ale not to be construed as necessarily retlecting the views or endorsement of the Department of the h r m y
Spectrochemical Analysis of Brines R. G . RUSSELL, Gulf Research & Development Company, Pittsburgh, Pa. A spectrographic procedure for the analysis of most of the cations in brine solutions utilizes an alternating current spark form of excitation on 0.5-inch briquets combined with a series of synthetic standards. A relative standard method is used to obtain the concentrations of the major constituents. The shortcomings of the method as well as its advantages are discussed. An error of *lo% is claimed for the procedure.
T
HE complete quantitative chemical analysis of brines has always been a difficult and tedious task. In the determination of the cations this has been occasioned both by the difficult chemical separations as, for instance, small amounts of potassium from large amounts of sodium, or small quantities of barium and strontium from larger amounts of calcium, and the de termination of very small amounts of some constituents. In addition, there is difficulty in determining the anions in the form in which they occur in the original sample. Contact of the brine solutions with the air continually changes the form of the anion-i.e., bicarbonate changes to carbonate. As the result of these chemical difficulties, shortened and approximate methods of brine analysis have been developed in many laboratories, All alkalies are reported as sodium. Barium and strontium are not determined, but are partially precipitated with and determined as calcium. Aluminum and iron are precipitated as the hydroxides, along with any other materials that would be precipitated a t this point, and reported as RzO~. Many of the elements such as boron, manganese, and lithium, present only in small amounts, are not detected in a sample of the size usually taken for chemical analysis. The investigation reported here has been carried out to develop a more rapid method for the quantitative analysis of brines. In addition, more information can be provided by the spectroscopic method than by the usual chemical analysis. QUALITATIVE ANALYSIS
Before any attempt is made to analyze a sample quantitatively, a so-called qualitative analysis is made. The sample is evapo-
rated to dryness over an open flame in the presence of a little hydrochloric acid. Three 10-mg. portions of the dried residue are weighed and arced, using 10-ampere direct current and a large Littrow quartz prism spectrograph. The purpose of using an exact weight of sample is to enable the operator to estimate more accurately the quantities of elementa present. These qualitative results become of value to the quantitative analysis only as the operator learns to place them in the proper percentage groupings. On the basis of many other samples that have been analyzed chemically, the operator is able to place the elementa in their correct groupings by remembering the densities of linea
Table I.
Qualitative Analysis of a Typical Brine Residue (% refen, to cations detected) %
>
10
NS
1-10
CS
Mg
0.1-1
K
Sr Si B
0.01-0.1
Fe
< 0.01
Pb
Li A1
cu
Ba Ti
2
