ANALYTICAL CHEMISTRY L
tI
2.
v,
boron per molecule of dianthrimide is about 8, whereas in B the ratio is 16, and in C is 32. Using data found in a discussion of the quinalizarin method published by Weinberg, Proctor, and Milner ( 7 ) the comparable ratio for this reagent a t the concentration studied was calculated to be approximately 125. Curve D shows the relationship between the maximum color developed and reagent concentration and it can be seen that the Lambert? Beer law holds in this case.
/
Finally, the instability of the colored boron complex should br noted. If an excess of boric acid is added to a solution of the reagent and heated, a very deep blue color results. If the solution is then poured into water, the reagent precipitates out ae fine red crystals, leaving a clear colorless dilute acid phase The reagent may then be filtered, washed a few times with water, dried, and redissolved in concentrated sulfuric acid and found to have the same absorption spectrum (300 to loo0 mw) ap the original reagent.
1.2
z W
0 -I
0.8
Q.
0 I-
n 0.4
0
ACKNOWLEDGMENT
0
I 60
1
0 0
I
I
20 40 80 100 BORON MICROGRAMS/S M L . 50 100 150 200 250 REAGENT MICROGRAMS/5 ML.
-
-
Figure 3. Relationship between Color Developed and Concentrations of Reagent and Boron wlll give a blue color with boric acid after several hours a t room temperature. To obtain the same intensity of color, however, 5 to 10 times as much boron is required as in the regular prncedure. In Figure 3 a family of three curves is shown.
Curve A was obtained by running a series of reactions using II reagent concentration of 200 micrograms per 5 ml. of acid and with boron (as boric acid) concentrations as indicated. After heating a t 80” C. for 5 hours the optical density was determined using a 620 mp filter and Evelyn colorimeter tubes. Curves B and C were obtained similarly, except that the reagent concentration waa 100 micrograms per 5 ml. in B and 50 micrograms per 5 ml. in C. In each case the maximum color was attained a t a boron concentration of approximately 40 micrograms per 5 ml. Calculation q h n w that at this point in the rase of A , the ratio of atoms of
2nd Annual Summer Sgmpoeinm
The authors gratefully acknowledge the help of E. I. du Pun1 de Nemours & Company which furnished six or eight dyestuffs and a special preparation of 1-amino-4-hydroxyanthraquinone; of the General Dyestuffs Corporation for several samples; of W. Bergmann, Yale, IT.F. Bruce, Cornell, L. F. Fieser, Harvard. and J. &I.Andreas, Pasadena, Calif., for many reagents otherwise difficult to obtain; and particularly the help of V. hl. King, Calco Chemical Division, American Cyanamid Company, who wpplied a large number of dyestuffs and dyestuff intermediates LITERATURE CITED
(1) Austin, C. M., and McHargue, J. S.,J. Assoc. Ofic.Agr. Chemists,31,254-5 (1948). (2) Berger, K.C., and Truog, E., IND.ENG.CREM.,ANAL.ED., 11. 540-5 (1939); Soil Sei., 57, 25-31 (1944). (3) Eckert, A., and Steiner, K., Monatsh., 35,1129-51 (1914).
(4) Naftel, J. A.,IND.EXG.CHEM., A N ~ LED., . 11,407-9 (1939). (5) Radley, J.A., Analyst, 69, 45 (1944). (6) Rowe, F.M., ed., “Colour Index,” Bradford, Yorkshire, England. Society of Dyers and Colourists, 1924. (7) Weinberg, S., Proctor, K. L., and Milner, O., IND. ENG.CHEM.. ANAL.ED., 17,419-22 (1945). (8) Winsor, H. W., ANAL.CHEM..20. 176-81 (1948) RECETVEn A l l K U S t
5 . 1949.
- Organ&Reagenta
Color Reaction of Beryllium with Alkannin and Naphthazarin Spectrophotometric Studies 4.
L. UNDERWOOL,
AND
M
W . F. NEUMAN, School of M d i c i n e ond D e n t i s t r y , University of Rochester, Rochester, N . Y
OST of the methods that have been proposed for the detarmination of beryllium have proved unsuitable for dealing with microgram quantities. The quinalizarin method of Fischer ( 8 ) can readily detect 0.5 microgram of beryllium, but the reagent is too unstable in the alkaline medium employed by Fischer to permit convenient quantitative work; furthermore, the color change from violet to blue is not a desirable one. The quinizarin2-sulfonic acid method proposed by Fairhall et al. (8) has been found extremely sensitive to salt concentration, and in general yields reliable results only when conditions are rigorously conrrolled (3). ilurintricarhnxylic acid (aluminon) has been tried in
?
chis laboratory as a colorimetric reagent for beryllium, but thr color reaction lacks sensitivity and is undesirable for several other reasons (9). The various fluorometric methods ( 7 , 8, I O , 11, 1 4 ) have been found inaccurate in the authors’ laboratory In the quinizarin method, for example, the optimal pH is about 11.5, and very small variations from this value lead to erratic results; it is difficult to buffer in this region (IS). Spectrographic methods (1, 2 ) are reported to be very good from the standpoint of sensitivity and specificity, but are somewhat lacking in precision. In view of the increasing interest in beryllium, and certain deficienciesin existing methods, it appears desirable to pub-
V O L U M E 21, NO. 1 1 , N O V E M B E R 1 9 4 9
1349
A rapid, sensitive, and accurate spectrophotometric method is described for the determination of microgram quantities of beryllium, using either alkannin or naphthazarin as the reagent. From 1 to 20 micrograms of beryllium in 20 ml. can be determined with a probable error of less than 3940 (standard deviation, 4.5%); as little as 0.1 microgram can be determined with somewhat less accuracy. Optimal conditions for the method have been found by studying the various factors that influence the color of the reagent
_ _ _...
with beryllium, such as pH, dye concentration, time of standing, and various extraneous ions. This method is subject to coneiderable interference from other elements, so that isolation of beryllium will be necessary in the analysis of biological samples, minerals, alloys, etc., but the method has proved extremely useful where the analysis of fairly pure beryllium solutions is desired; the accuracy, sensitivity, and ease of carrying out the procedure compare favorably with other methods.
.___-
winpound, naphthazarin (12). A solution of 0.4 tug per ml. in redistilled 1,4-dioxane was employed. Standard Beryllium Solutions. These mere prepared by dissolving about 1 gram of the metal in dilute hydrochloric acid, removing a slight insoluble residue, and diluting to 1 liter with distilled water. The residue contained beryllium and silicon, as shown by the spectrograph, but the solution was essentially pure with respect to beryllium. Samples of this solution were evaporated to dryness after addition of a little sulfuric acid, and ignited to beryllium oxide for accurate analysis. This fairly acid stock solution could be kept indefinitely; dilutions in the microgram range were freshly prepared frequently to eliminate the possibility of changes due to adsorption, base exchange with the glass container, etc. Borate-Mannitol Buffer. Various buffers were tried. Phosphate buffers interfered with the color reaction, as did bisulfite. A borate-mannitol buffer was found satisfactory. A solution of 0.5 hl boric acid was made up in 10% mannitol and adjusted to pH 5.0 with sodium hydroxide. A trace of thymol was added to inhibit microorganisms, and the solution was stored in the refrigerator when not in use. Under these conditions, it can be kept indefinitely. This buffer changes Figure 1. Absorption Spectra of Blank and Beryllium Solutions pH on dilution, so that solutions prepared for spectrophotometric analysis as described below had a final 4.65 Y of Be, 5 m l . of buffer, 8.2 m l . of g u m arabic solution, 1 mg. of alkannin Total TOlUIIle 20 d. pH 6.5. Read us. d i s t i l l 4 water pH of 6.5. Solid line, blank Gum Arabic. The beryllium-dye color lake would flashed line. beryllium sollitinn not remain in clear solution, but tended to flocculate on standing. This effect was easily countered bv the addition of gum arabic as a stabilizer. A 0.5% solut~onwas prepared for-this purpose lish a new spectrophotometric method which has proved rapid, sensitive, and accurate. EXPERIMEIVTAL Alkannin and naphthazarin were used in a qualitative test for Absorption Spectra. Absorption spectra were studied to deberyllium by Dubsky et al. (4, 6); these workers carried out the termine a suitable nave length for further studies. I t can be rest in the presence of ethylenediamine, which, they believed, reseen from Figure 1 that a t pH 6.5 there is a large difference be&ed with alkannin or naphthazarin to form a diimine which then tween blank and beryllium samples at 600 mp and that the formed a chelate complex with beryllium. In any case, alkannin or blank is fairly low a t this wive length. h similar picture is seen aaphthazarin gives a blue color with ethylenediamine which goes with naphthazarin. Other curves were run a t several pH values, mer to a purple in the presence of beryllium. This approach 2nd 600 nip was optimal in all cases, so that working a t this has the disadvantage that the reagents are unstable in the alkawave length in a further study of the effect of p H on the color line medium, and the colors fade rapidly. On the other hand. reaction was justified-that is, although the actual spectra are the authors have found that ethylenediamine is not essential. shifted by changing pH, the net values obtained by reading berylm d that a suitable color difference can be developed a t a lower pH lium samples against blanks will be maximal a t 600 mp. where the dye is stable. the red rolor going toward thp hlllP in thP Effect of pH. Figure 2 shows that with alkannin, the maximal Drpsrnrp of hrrvllium isolor is developed above a pH of about 5 , beyond which pH W P A K A T U S A.11) KEAGEhI'3 changes are not significant over a range of a t least 3 p H units A similar curve is obtained with naphthazarin. The reagents are .\I1 absorption spectra and spectrophotometric readings M ert unstable above a pH of about 8; the colors fade rapidly, and roumade with a Beckmari Model DU quartz photoelect'ric spectrophotometer. pH measurements xere made with a Beckmari tine quantitative wgrk would be difficult. These compounds laboratory model pH meter. are pH indicators; they change from red through a series of Alkannin. It was found possible to set up a quantitative purples to blue i:i the region of p H 8, and because of their instamethod for beryllium using an alcoholic extract of the roots of bility the alkaline region has not been investigated as regards a .ilkannu tinctoriu, but variations among various extracts made it desirable to isolate the pure compound alkannin (12). In the suitable wave length a t which to work. A pH of about 6.5 haE studies that follow, solutions of the pure material, 0.4 mg. per been found to be entirely satisfactory; the borate-mannitol ml. in redistilled 1,4-dioxaneJwere employed. buffer is efficient in this region, the colors are stable, and small Naphthazarin. Certain difficulties in obtaining pure alkannin variations in pH do not introrlnrr errors A aystrm of 1 p r t of irom va,rious bat.ches of roots led t,o synthesis of the analogous
Lspo,
.
. ...-
I
ANALYTICAL CHEMISTRY
1350
-
dioxane and 3 parts of water is involved, where the question of pH becomes somewhat complicated; directly measured, uncorrected pH values have been correlated with optical density, a procedure satisfactory for the purposes. 0 Effect of Dye Concentration. I t can be seen from > Figure 3 that alkannin alone does not conform to the Beer-Lambert law; the blank curve tends to flatten, so that it is possible to work a t a level of 2 mg. of dye without incurring too high a blank. This qusntity of dye has proved satisfactory with both alkannin and naphthazarin, in that it is a sufficient excess to give fairly linear standard curves over the range of beryllium concentrations in question. Effect of Time. Figure 4 shows that, after a small initial increase, the readings on a naphthazarin-beryllium 0.25 0 . 5 0 0.75 1.00 1.25 1.50 1.75 2.00 solution remained essentially constant for some time. M G ALKANNIN S o significant changes occur over the time normally reFigure 3. Effect of Dye Concentration quired to prepare and read a series of samples. Fading 4.65 y of Be, 5 m l . of buffer, 0.5 m l . of g u m arabic solution, varying a m o u n t s of of the colors is observed in solutions which have stood alkannin. Total volume 20 m l . pH 6.5. Read US. distilled water for longer periods-e.g., overnight. Alkannin behaves Upper. Be solutions Middle. Net values similarly. Lower. Blanks Effect of Heating. Heating up to 80’ C. was found to have very little effect on color development where 2 mg. - of alkannin were used. During some preliminary studies where 1mg. of alkannin was employed, a slower color development than that described above was observed, and it was found that heating the jolutions hastened the development of maximal intensity. Boiling the solutions resulted in almost immediate fading of the colors. Recommended Procedure for Analysis of Pure Beryllium Solutions. On the basis of the data described above, a method has been set up which has served very well in the analysis of pure beryllium solutions such as were obtained in certain physicochemical studies of solubilities of beryllium compounds, etc. 0.1
0’41
$t
;
To the beryllium sample, containing from 1 to 30 micrograms of beryllium in 9.5 ml. or less, 5 ml. of the buffer, 0.5 ml. of the gum arabic solution, and 5 ml. of alkannin or naphthazarin solution are added, and the volume is adjusted to 20 ml. with distilled water. This sample is read against a similarly prepared hlank on the spectrophotometer a t 600 mp.
0
‘ Y 10 20 30 40 50 60
0
TIME
IN MINUTES
Figure 4.
Effect of Time
4.65 y of Be, 5 m l . of buffer, 0.5 m l . of g u m arabic solution 2 m g . of naphthazarin. Total volume 20 m l . pH 6.3. Read tis. blank a t timed intervals after mixing
arabic are employed. Figure 7 shows a standard curve for the region below 1 microgram. Results. Three groups of “unknowns” were made up by a disinterested worker and analyzed by the above procedure (Tables I, 11, and 111). By comparing these tables, it can be seen that the absolute error increases with increasing beryllium content, but that the increase is not proportional, so that per cent error decreases with larger amounts of beryllium. Interferences. Several ions that might be encountered later were tested for interference with the determination of beryllium
c
Figure 2.
Effect of pH
4.65 y of Be. 5 ml. of buffer, 0.5 ml. of g u m arabic solution, 2 m 3 . of alkannin, adjusted to various pH values with hydrochloric acid or sodium hydroxide. Total volume 20 ml. Read us. blanks adjusted to s a m e pH values
Figure 5 shows a typical standard curve. Beyond about 10 micrograms the solutions become too dense for the most accurate spectrophotometric readings; the upper limit of the method is set by the instrument. Figure 6 shows a standard curve over that part of the range which may be considered as the most precise from the standpoint of the instrument. It is possible to go down to as little as 0.1 microgram of beryllium by reducing the final volume to 10 ml.; half-volumes of the buffer, dye, and gum
Figure 5.
Standard Curve with Alkannin
V O L U M E 21, NO. 1 1 , N O V E M B E R 1 9 4 9
1351 ~~
Tahle I.
Determination of Beryllium in Synthetic Unknowns (Range 0 to 1 Be Added. Y 0.93 0.28 0.39 0.19 0.61 0.79 0.49
A v . absolute error, y A v 70error
0.033
7.6
y
Table 11.
i n 10 ml.) Be Found, 7 0.93 0.33 0.44
Determination of Beryllium in Synthetic Unknowns (Range 1 t o 10 Be Added. Y
in 20 ml.) Be Found.
y
Y
0.19
0.65 0.82 0.55 Probable error, % Standard deviation, %
6.6 9.9
Table I V summarizes the effects of these ions in -rveral concentrations. It was found that alkannin and naphtham r i n had essentially the same susceptibility to interference by t h r w extraneous elements. l)v this method.
Av. absolute error, y A v . % error
Probable error, % Standarddeviation, 70
0.145 3.4
2.8 4.2
V.,
Table 111.
Determination of Beryllium in Synthetir Unknowns
(Range 10 t o 20 y in 20 ml.) Be Found, 1 Be Added, y 20.9 12.8 11 2 14.4 19.5 15.6 16.7 18.1 0.275 Probable error, o/o 1.4 h v . absolute error y 1.8 Standard deviation, 9’~ 2 . 1 Av. l o error
0.5
Tahle IV. Per Cent Error Introduced by Addition of Various Ions to 4.65 Rlicrograms of Beryllium [on Added. Ca++ .\1g++
Zn++
cu++
Fe+++ Al++ FPO>---
a
Y
1
10
0
0 +5
7-7 +8 +27 -8 0
100 0
+ 10
1000 0
+,27
+$45i50
+35 t77 - 56 -30
- 95 - 70
a a
Visible precipitate made colorimetric measuretnent impossible.
Figure 6. Standard Curve w-ith Alkannin
O0.0 * O I
o.of 0
0.1
Figure 7.
0.2
0.3 0.4 0.5 0.6 MICROGRAMS BERYLLIUM
Standard Curve in Range below 1 y with Naphthazarin
The effect of total salt concentration wab studied, using sodium chloride. This salt does not alter optical densities so long as the colored materials remain in solution, but everts its effect by causing flocculation of the latter. Thia effect depends on the amount of gum arabic added to stabilize the system. With no gum arabic present, the buffer alone is sufficient to cause precipitation. With the amount of gum arabic used in these studies, salt concentrations as high as 0.2 M (calculated for the final solution taken for spectrophotometry and exclusive of the buffer) can be tolerated. The amount of gum arabic used can be varied to meet the conditions encountered. The use of dioxane as solvent for the dyes might be mentioned. Not only are the free dyes more soluble in dioxane than in most common organic solvents miscible with water, but there is less tendency for the
ANALYTICAL CHEMISTRY
13%
beryllium-dye lake to come out of solution when dioxane i,i present.
tions must be observed to avoid contamination. These precautions have been well pointed out by Cholak and Hubbard (g).
DISCUSSION
In the analysis of fairly pure beryllium samples, alkannin and uaphthazarin yield results that compare favorably with other methods which have been proposed. Advantages of the method include the fact that color development is spontaneous and rapid, extremely rigorous pH control is not necessary, and the colored jolutions are stable for a sufficient time to permit convenient quanritative work. The sensitivity is as great as that of other meth')ds except the spectrographic, and the accuracy seems adequate. The two reagents serve equally well, conditions are essentially the 4ame for employing either one, and it becomes a matter of convenience as to which is to be used; naphthazarin is somewhat more easily obtained than alkannin, so that it may be the reagent of 'Bhoice. Like most of the beryllium methods previously employed, dkannin and naphthazarin are subject to considerable interfera c e from extraneous elements, and it appears that a t least a parrial isolation of beryllium will be necessary before application of rhe method to the analysis of such things as biological samples, minerals, alloys, etc. Tn analvzing such small amounts of material, sperial precau-
LITERATURE CITED
Cholak and Hubbard, . ~ N A I . .CHEM., 20, 73 (1948). Ibid., p. 970. Cucci, Neuman, and Mulryan, University of Rochester A t o m r Energy Project, Rept. UR-26 (1948). Dubsky and Krametz, Mikrochemie, 20, 57 (1936). Dubsky, Langer, and Wagner, Ibid., 22, 108 (1937). Fischer, 2.anal. C h m . , 73,54 (1928). Fletcher, White, and Sheftel, IND.ENG.CHEM.,ANAL. ED., 18 179 (1946).
Hyslop, Palmes, Alford, Monaco, and Fairhall, Natl. Ins1 Health,Bull. 181 (1943). Kosel and Neurnan, Cniversity of Rochester Atomic Enera! Project,Rept. M-1965 (1947). Sandell, IND. ENG.CHEW,ANAL.ED., 12, 674 (1940). Ibid., p. 762. Toribara and Underwood, A x . 4 ~ CHEM., . 21, 1362 (1949). Underwood, Neuman, and Carlson, University of Rocheatw Atomic Energy Project, Rept. M-1951 (1947). White and Lowe, ISD. ESG. CHEM.,ANAL.ED.,13, 809 (1941) RECEIVED August 1, 1949. Based on work performed under contract witL the United States Atomic Energy Commission at the University of Rochester htomic Energy Project, Rochester, N.Y
- Organic Reagenta
B n d Annual Summer Sump~sium
Preparation of Alkannin and Naphthazarin For Use us
Reagents f o r Beryllium
r. Y. T O R I B ~ R AAND A.
L. UNDERWOOD
?t*html oj Medicine and Dentistry, UniEersity of Rochester, Rochester, N. Y .
ikannin and naphthazarin were shown by Underwood and Neuman to be equivalent reagents for the microdetermination of beryllium. Alkannin (a substituted naphthazarin), which forms only a small fraction of the colored matter extractable from alkanet root (Alkanna tinctoria), was shown to be the only constituent active with beryllium. The bulk of the colored material appears to be a polymer of alkannin, and evidence indicates a dimer. The isolation procedure for alkannin was thoroughly stud-
I
N SEEKING a more satisfactory method for the microdeter-
mination of beryllium than any previously reported, Underwood and Neuman (8) developed first a method using alkannin and then an equivalent procedure using naphthazarin. Because alkannin is a substituted naphthazarin, the two compounds would be expected to behave similarly. 011
1
1 Alkannin
u
/I
ittr 0 I1 Naphthazarin
Formaaek (6) first referred to the color reaction of berylliuni with an alcoholic extract of the root Alkanna tinctoria. Dubsky and Krametz ( 4 ) used both alkannin and naphthazarin in the microdetection of beryllium in a solution containing ethylenediamine. The alcoholic extract of Alkanna tinctoria (roots were
ied. The difficulties in obtaining large amounts of alkannin made it desirable to find a reagent that could be more readily procured. Naphthazarin was synthesized according to the method of Zahn and Ochwat, but the purification procedure was modified. The synthetic method was found to be much simpler than extraction from the root. Absorption spectra of the compounds alone in carbon tetrachloride and at several pH's in aqueous medium, with and without beryllium, were determined.
obtained from the S. B. Penick Company) was tried, and a methoc for the microdetermination of beryllium was developed using such an extract. The variation from batch to batch of root led tc. the isolation of the active ingredient, which was alkannin -4lkannin itself forms only a few per cent of the highly coloreci material extractable from the root, and it was shown that the nonalkannin material gave no color reaction with beryllium. Brockmann (2) made a very complete study of alkannin ana its related compounds, and gave directions for its extraction and isolation. He extracted the root with petroleum ether and ther extracted the red material from the solvent with sodium hydroxide solution. After the deep blue alkali solution was washed severa! times with benzene and petroleum ether, the addition of acetic wid caused the precipitation of a red material. This was r e crystallized repeatedly from benzene to give pure alkannin which melted a t 148°C. Underwood and Neuman (9) attempted to follow Brockmann'c directions, but found that alkannin was too soluble in benzene to permit recrystallization. Evaporation of the solvent left a tarry, resinous mass. A different procedure was employed for the purificstion After precipitation with acetic acid, the solid w s
