882
ANALYTICAL CHEMISTRY
terniine the two dilution ratios and, thus, the concentration of nickel carbon>-lin the calibrating gas. T h e final concentration, X,is given by
n-here p
=
P = F1 = F2 = Fa = I.'? =
vapor pressure of nickel carbonyl a t 0" C . atmospheric pressure f l o rate ~ through first 08 Flonrat,or f l o rate ~ through 01 Flowator flox rate through second 08 Flonrator flow rate through 2L F l o w a t o r
The vapor pressure, p , of nickel carbonyl a t 0" C. is 134 nini., and F, and F , are held constant a t 10,000 cc. per niinute and 23,000 cc. per minute, respectively. If atmospheric pressure, P , is take:i as 760 mm., the eqiintion rednws to
-Y = 0.000iOS F,F:j p.p.m. Carbon monoxide is used in the bubbler and for the first dilution t o prevent premature deconiposition of the nickel carbonyl. Air is used for the second dilution to promote oxidation of the deposit on the hot disk. S o evidence of decomposition of nickel carbonyl in any part of the system except on the hot disk was found. DISCUSSIOS
From the basic principlea of operation, it is seen that the mininiiini concentration of nickel carbonyl that the instrunient can detect depends upon the qu;tritity of deposit that is allowed to build up on the surface of the disk. This, in turn, depends upon the rate of sample flow aiitl npon the rate of rotation of the
disk. Large saniplr floir P, hon e w r , tend to cool the surface and decrease the deposition efhieiicy, so that for given values of temperature and rate of rotation, there exists a n optimum rate of f l o ~ . T h e optimum rate Eo1 the iristrunient described in this paper was found t o be about 500 cc. per n-incite. At this flow rate, the instrument gives full-sc ale deflection a t a concentratioii of approximately 4 p.p.m. T h e sensitivity of the instrunLentcan be increased appreciably by altering the position of the light beam mith respect to the nozzle so t h a t a greater quantitv of material is deposited a t a given point before the reflectance at that point is measured. This, however, introduces a longer t h e lag beta een deposition and measurement. K i t h a &minute time lag (which was not considered excessive) the instrument registered a deflection of 1yc of full scale for 0.2 p.p.m. A time lag of 10 minutes results in 1% of full-scale deflection for 0.05 p.p.ni. The instrument is capable of even greater sensitivity because, with the particular nozzle which was used, the time of deposition a t any point on the periphery of the disk is approximately 40 minutes. Although the instrument described in this paper n as designed for the detection of nickel carbonjl, it is also sensitive t o iron carbonyl and should be easily adapted to the detection of tetraethyllead and other metallo-organic gases and vapors. LITERATURE CITED (1) Arch. I n d . H u g . and Occupational M e d . 9, 531 (1954). (2) Chem. Processing (;lrnerican C o n f e r e n c e of G o v e r n m e n t a l Indus-
trial Hygienists) 15, 134 (1952). (3) G a r r a t t , A. P., Thompson, H. K., J . Chem. SOC.(Lo7ldon) 1934, 1824. R E C E I V Efor D review 11s. 2 6 , 19.53. Accepted .Ianiiar~-28, 1956.
Indicator for Titration of Calcium in Presence of Magnesium Using Disodium Dihydrogen Ethylenediamine Tet raacetate HARVEY DIEHL and JOHN L. ELLINGBOE Department of Chemistry, lowa State College, Ames, lowa
A new indicator, designated calcein, has been prepared for the titration of calcium in the presence of magnesium with disodium clihydrogen ethylenediamine tetraacetate. No preliminary treatment is necessary be>ond dissolving the sample and adjusting the pH to a value of 12. Excessisel3 large amounts of sodium and magnesium cause the results for calciuttl to be slightly low. Interference 1)) copper and iron is obTiated by the addition of c! ankle.
A
SE\T- i.itlicatoi for the titration of calcium with disodium dihydrogen ethylenediamine tetraacetate [the disodium salt of (ethj-1enedinitrilo)tetraacetic acid] in the presence of magnesium has been prepared by condensing irninodiacetic acid w t h fluorescein. This is a procedure analogous t o t h a t employed by Schwarzenbach and others for the preparation of the so-called metal phthaleins (4). In highly alkaline solution t h e indicator is brown and its calciuni complex is a yellow-green. At lower pH values the free indicator is also yellow-green. Magnesium does not form a complex with the indicator. T h e indicator m a y be used for t h e determination of calcium in water, limestone, or other calcium compounds. I t has been given the trivial name calcein. I n the analysis of limestone or water, the total calcium and
niagtiesiuni can be deterinilied b y using disodium dihydrogen ethylenediamine tetraacetate as the titrant with Eriochronie Black T as the indicator (1-3). Either the calcium or magnesium must then be determined separately and the other calculated b y difference. AIagnesium cannot be determined in the presence of calcium because the formation constant of the calcium complex of ethylenediamine tetraacetate is two orders of magnitude greater than that of the magnesiwn complex. T o determine calcium directly using disodium dihydrogen et,hylenediamine tetraacetate as the titrant, the pH is niade siifficiently high so that the magnesium is largely precipitated as the hydroxide and an indicator is used which combines with calcium only. Murexide is such an indicator ( 5 , 6), b u t the end point with it is rather indefinite and is made worse by increasing amounts of magnesium. A sharper end point is obtained with calcein than with niurexide, and larger quantities of magnesium may be present without impairing the end point. T h e niagnesium may exceed the calcium b y a factor of 20 to 30 without interference. Large amounts of sodium salts-2 t o 3 granis of sodium chloride, for exanipledo not affect the titration. Strontium and barium interfere and are titrated along with calcium; the end point with either alone is t h e same as t h a t with calcium. Copper and iron interfere with the end point, b u t such interference is easily obviated b y the addition of cyanide. T h e titration of calcium may be performed in the presence of chloride, nitrate, acetate. and sulfate.
V O L U M E 28, NO. 5, M A Y 1 9 5 6
883
I3elow p H 12, both the indicator and its calcium complex have a yvllow-green color. Above p H 12 the indicator is brown, but tkir d c i u m coniplex has the same yellow-green color. T h e titration is carried out a t a p H above 12 so t h a t the end point is nisrked by a change from yellox-green to brown. T h e indicator can be added either as the solid (one part of indicator mixed with 100 parts of potassium chloride) or as a 27, solution in dilute sodium hydroside. A better end point is obtained if t h e solid indicat,or contains some charcoal (1 part of indicator, 10 parts of charcoal, and 100 parts of pot,assiuni chloride). T h e calcium complex appears much greener xhen this is doqe and, when large amounts of iron are present, the end point is much better. K h e n thc iric!icat,or is used alone or in conjunction with charcoal, it, is completely reversible. T h e end point is better in diffuse light tliaii in illumination of high intensity.
SOLID VTTH CHARCOAL. Grind together 1 gram of the indicat,or, 10 grams of charcoal (Sorite A is satisfactory), and 100 grams of potassium chloride. It is convenient t o measure out eit,her of t.he solid indicators with a small metal scoop t,hat holds about 0.07 gram of the mixture. Calcium in Water. Pipet a 50-ml. sample into a conical flask. Add 1 or 2 drops of 2% indicator solution (or a scoop of a 1% solid mixture, about 0.07 gram) and 5 ml. of 1.W sodium hydroxide containing 1 gram of sodium cyanide per 100 ml. Titrate with 0.02.V disodium dihydrogen ethylenediamine tetraacetate until the color changes from yellow-green to brown. Vigorous stirring is necessary throughout the titration. Do not carry out the titration under a fluorescent lamp or in light of high intensity. Standardize the disodium dihydrogen ethylenediamine tetraacetate solution against Iceland spar or a primary standard grade of calcium carbonate.
Table 11. Analysis of Limestone and Gypsum Table 1. Pwpnratiun"
f> I 0
Analyses on Calcein
Seutralization Equivalrnt, h-aOH in Water
Sitrogen, Iijeldalil
207 187
3 38 4 16
c
Seutralization Equivalent. Bromination
88
h-eutralization Equiralent, HClOi in Acetic Acid
194
T-arioui preparations ohtained from numerous attempts a t purification.
Owing to the high p H at which the titration is performed, some calcium is precipitated as the hydroxide at the beginning. T.igorous stirring is necessary to dissolve the hydroxide as the titration progresses. If the stirring is slow, false end points are ohtained, the color returning after each change as the stirring is coiitiniietl.
(Disodium dihydrogen ethylenediamine tetraacetate standardized against Iceland sparu) Standard Sample NBS co. la 88 1099 1100 Seleniteb 54.28 30.49 32.57c CaO reported, % 41.32 30 49 b 21.83 0.85 21.48 RIgO reported, % 2 . 1 9 32.53 54.33 30.34 CnO found, "0 41.31 30 48 30 48 32.46 54 17 41.37 30 3 i 32.51 54.31 30 53 30 41 41.2.5 54.30 32.54 30.46 41 26 30 56 32.64 30 44 54.23 41.21 30 4 3 32.50 30 5 6 54.18 30 38 41.36 54.26 32.47 4 1 21 30 44 30 44 54 20 32.54 41.20 30 50 30 51 32.5'' 41.18 32.60 32.54 32 46 Av. 41.26 30.4ci 54.25 30.45 32 5 2 Range 0.19 0 19 0.16 0.22 0 18 AT. d e r . 0.056 0 044 0 054 0 061 0.039 0 070 0 059 0.065 0.075 0.054 Std. der-. a Transparent crystals; magnesium content determined spectrographically t o be 40 p.p.iii, b Transparent crystals of gypsum (selenite variety from Freedoni, Okla.) : niagnesiiim content determined spectrographically t o be 20 p . p . i n C Theoretical CaO content for Cas04 2HzO.
PREPAR.ATION O F I N D I C A T O R
llis 100 grams (0.3 mole) of fluorescein, 300 ml. of ethyl alcohol, 150 nil. of distilled xater, and 90 ml. of 307, sodium hydroxide. Add with stirring 87 grams (0.M mole) of iminodiacetic acid, dissolved in 105 ml. of 30% sodium hydroxide plus 120 ml. of distilled n.ater. Cool the mixture to 10" C. in an ice bath. Add dropwise 74 ml. (0.75 mole) of 3 i % formaldehyde, stirring vigorously. After ail of the i'ormddehyde has heen added, heat the mixture to 60' to 70" C. for 0 to 7 hours, stirring continuously. rlllow the solution to cool, then dilute to 3 liters. Add 1 to 1 h?-tirochloric acid, precipitating the indicator as the free acid. Filter and A-ash with distilled water. Redissolve the material in 3 liters of \\-ater containing 120 grams of sodium acetate. Precipitate it again with hydrochloric acid, filter, and wash. Transfer the material into 2 liters of ethyl alcohol, stir for 1 hour, and filter. Repeat the ethyl alcohol xvdiing: then dry the materid in a vaciium. The product is bright yellow. When heated, it begins to clccompose slowly a t ahout 185' C. It is apparently a mixture, with a compound predominating which contains t x o iminodiacetic acid residues, because analyses performed on materials ol)t:iinetl from various purification processes gave variable results (Tai)le I). T h e titration with sodium hydroside gave a titration curve having a single sharp break with the end point around a p H of 7.5. .Ittempts to determine the molecular weight. were unsurcessful. Though admittedly not a p\ire product, the material so prep : i d functions well as an indicator. It is also available from the G , I2reclericl; Smith Chemical Co., Columbus, Ohio.
Limestone. Keigh a sample of about 0.3 gram into a 400-ml. beaker. Add 20 ml. of 1 to 1 hydrochloric acid and evaporate to dryness. Redissolve the sample in 5 ml. of 1 t o 10 hydrochloric acid and then dilute t o 100 t o 200 ml. with distilled water. To this add 1 to 2 drops of the indicator solution or a scoop of the solid indicator mixture and about 5 ml. of 1OM sodium hydroside containing 5 grams of sodium cyanide per 100 ml. Titrate with 0.LV disodium dihydrogen ethylenediamine tetraacetate under the same conditions as for calcium in water.
Table 111. Titration of Calcium in Presence of Large Amounts of Magnesium and Sodium Salts Calcium, Taken 0.1188 0.1188 0.1188 0.1188 0.1188 0,0833 0.1176 0 0595 0 0.59:
Gram Found 0,1188 0 1191 0.1178 0.1190 0.1173 0.0827 0.1162 0,0585 0,0589
Magnesium, Grains
Sodium Chloride, Grams 1.0 3.6 3.0
2.b 3 0
Calciuin Recovered,
% 100 100 99 100 98 99 98 98 98
00 29 18 19 74 32 91 38 99
DETERIIIN.ATIOS OF CALCIUM
Preparation of Indicator. SOLCTIOS. Dissolve 2 grams of the indicator in 25 nil. of lL\- sodium hydroxide and dilute to 100 ml. with distilled water. Use 1 to 2 drops of this solution. SOLID. Grind thoroughly 1 gram of the indicator with 100 grams of potassium chloride.
RESULTS
Two limestone samples from the Bureau of Standards, two samples of limestone from the Standard Sample Co., h m e s , Iowa, and a sample of selenite (transparent variety of gypsum 1,
884
ANALYTICAL CHEMISTRY
obtained from the G. Frederick Smith Chemical Co., were analyzed for calcium. T h e limestones were analyzed b y the procedure given above. T h e selenite was dissolved in a n excess of standard disodium dihydrogen ethylenediamine tetraacetate at a pH of 12, the stirring being continued for 4 t o 6 hours, Excess standard calcium chloride was then added and t h e solution titrated with disodium dihydrogen ethylenediamine tetraacetate. The results reported were obtained using both the indicator solution and the solid indicator with and without charroal. T h e results on the samples from the Bureau of Standards were correctedfor the strontium present (0.0570 in sample l a and O.OlyG in sample 88). T h e results are given in Table 11. Titrations of k n o m amounts of calcium in the presence of magnesium and sodium chloride were carried out. T h e results of
these titrations are given in Table 111. S o interference n i t h the end point could bp detected. LITER4TURE CITED
(1) Biedermann, W.,Schwarzenbach, G., Chimia. (Swift.) 2, 56 (1948). (2) Diehl, H., Goetz. C. A , Hach, C. C., J . 4711.Water Works Assoc. 4 2 , 4 0 (1950). (3) Schwarzenbach, G., Ackerinann. H., Helz. Chim.-4cta 30, IT98 (1947). (4) Schwarzenbach, G., Anderegg, G., Flaschka, H., Salliman It., Ibid., 37, 113 (1954). (5) Schwarzenbach, G., Biedermann, W., Bangerter, F., Ibid., 29,811 (1946). (6) Schu-arzenbsch, G., Gysling, H., Ibid., 32, 1314 (1949). RECEIVED f o r review d u g u s t 15. 1955.
Accepted Fehruary 20, 1956.
Fluorometric Micromethod for Determination of Tryptophan GERALD D. MILLER
and
JOHN A. JOHNSON
Department o f flour and Feed M i l l i n g Industries, Kansas State College, Manhattan, Kan.
BYRON S. MILLER Federal Hard W i n t e r W h e a t Quality Laboratory,
U. S. Department
The measurement of fluorescence intensity of substances formed by the reaction of glucose and tryptophan is suggested as a method for the quantitative determination of microquantities of tryptophan. Tryptophan is separated from the other amino acids on a resin column of Dowex-50 (sodium form), and is made to react with glucose under optimum standard conditions. These conditions include the heating of 20 y or less of tryptophan with 0.8 gram of glucose at pH 1.38 and a temperature of 118' C. for 4 hours. There is a linear relationship between tryptophan concentration and fluorescence intensity which is read at pH 1.80. The standard error is *3qG for 12 y of tryptophan.
o f Agriculture, Manhattan, Kan.
fluorescent material (2, 4,5 ) . .Ilthough moJt of the amino acids react with glucose t o form fluorescent compounds, the fluorescence intensity of compounds resulting from the reaction of glucose and tryptophan is much greater than t h a t for the other amino acids (6). This reaction, therefore, appeared t o have several advantages over other methods. The object of the present study was to define the conditions affecting the development of fluorescent compounds resulting from the ieaction of tryptophan with glucose and t o apply the reaction to the quantitative estimation of tryptophan. The application of this method t o the determination of tryptophan in biological materials \vi11 be published later. APPARATUS AND MATERI.iLS
T
HE determination of ti yptophan, particularly in materials
containing carbohydrates, requires special analytical techniques as well as hydrolysis in an alkaline rather than in an acid medium. Both chemical and biological methods may be used for the analysis of tryptophan in pure protein. The many available chemical methods for the determination of tryptophan involve reaction with oxidizing agents, condensation with aldehydes, or diazotization reactions. These methods have the common limitation t h a t they are not sensitive enough to determine microquantities of tryptophan which occur in some biological materials. T h e ninhydrin reaction ( I O ) which has been used for assay of many amino acids is not sufficiently sensitive t o give satisfactory results for assay of tryptophan in the low concentrations present in flour. Portner and Hog1 ( 1 2 ) reviewed the chemical methods used in the determination of tryptophan and concluded t h a t the most advantageous method is t h a t of Spies and Chambers (14-17). This method involves condensation of tryptophan and p-dimethylaminobenzaldehyde followed by oxidation with nitrous acid. While the Spies and Chambers method is satisfactory for tryptophan assay of proteins, i t is not adaptable t o the assay of flour and similar materials. T h e limitations of available methods for the analysis of tryptophan in concentrations that occur in foods suggested t h a t a new approach t o the problem might be profitable. Tryptophan is known t o react with reducing carbohydrates ( 7 ) t o produce
A Coleman electronic photofluorometer, Model 12C, \vas ustd for t h e fluorometric analyses. I t was equipped with a S o . 5 8 i 4 Corning filter which transmits the 365-nip mercury line, and another filter consisting of tv-o Corning filters (Sos. 3398 and 4308) which absorbs below 425 mp. A reference standard consisting of a solution of 0.1 y per milliliter of sodium fluorescein in mater was used t o adjust t h e instrument to a given sensitivity. All values m r e calculated on the basis of a fised value of 40 for sensitivity. Dilutions of thp reaction niistures used to estahlish the standard curve were made with the buffer in order to adjust them t o the reading range of t h e photofluoronieter and to adjust the p H to permit reading at maximum fluorescence. Commei,cially available chromatographic columns (10 X 300 mm.) wew fitted into condenser jackets and used to remove tryptophan from other amino acids according to the procedure of Moore ailti Stein (11). One-ounce prescription bottles v i t h TeAon inserts in the caps were used as containers for t h e solutions containiiin tryptophan and glucose, which were heated in the autoclave to develop fluorescent compounds. A Beckman Model GS pH meter was used to measure the pH of all solutions. A saturated solution of potassium acid tartratc' (6) was used as a standard buffer (pH 3.57). A Coleman standaid buffer (pH 2.0) also was used. Tryptophan. Analytically pure b t r y p t o p h a n was obtaincd from Sutritional Biochemicals Corp., Cleveland, Ohio. .I sample of DL-tryptophan obtained from the Dow Chemical Co., Midland, hlich., ~ a recrystallized s twice from water and ethyl :I]coho1 followed by a rinse with anhydrous ethyl ether. Tlir purified DL-tryptophan was used as a standard. Sources of Other Amino Acids. All of the amino acids othcr than tryptophan normally found in food products were obtained from Xutritional Biochemicals Corp. and v-ere used without additional purification.
