V O L U M E 28, N0:6,
J U N E 1956
1035
Table \'. Determination of Silicon and Copper in Titanium Metal by Neutron Activation Analysis Assay fur Si
KO.of Determinations
3 3
CU
Concentration, P.P.M. Activation Spectrographic" 12.3,12.3, 1 2 . 1 3%.5,33.4,33.1
10 30
Average of assays from several laboratories.
a
Table VI.
Determination of Vanadium in Titanium Oxide by Yeutron Activation -4nalysis Sample 1
irradiation can usually be easily separated from other induced radioactivities in the sample and its radioactivity can be measured without difficulty. Also, the radioisotope can be distinguished from other radioactivities by its own specific half life and the radiations it emits in its decay. Thus, activation analysis is unusually free from the contamination difficulties experienced in conventional methods of analysis, unless the contaminant has been added to the sample before the irradiation. T h e sensitivity of activation analysis for most elements is potentially greater than for most other analytical methods.
Vanadium Concentration, P.P.11. 35.4 10.9
Table VII. Determination of Nickel and RIanganese in Titanium Oxide by Neutron 4ctiwation Analysis Assay for
Silicon and Copper. Another sample of titanium metal was analyzed for these elements. The results of these assays are given in Table V. The comparative data were obtained by spectrographic analyses. Vanadium. Several samples of titanium oxide were analyzed for vanadium (Table VI). T h e value given is the average value of several determinations on each sample. T h e mean deviation of these results was no greater than =t5.0%. Nickel and Manganese. Several other samples of titanium oxide were analyzed for nickel and manganese (Table V I I ) . Comparative data were not available. Qualitative Analysis. I n the study of several samples of titanium metal and titanium oxide, mnny of the elements shown in either Table I or Table I1 were identified as being present. I n addition to thePe, other elements such as sodium and thorium wei'e found to he present DISCUSSIOY
The iesults of the analyses reported above indicate that the neutron activation analysis method can easily be applied t o the determination of submicrogram and microgram amounts of many diffei ent elements n-hen these elements appear as impurities in titanium metal and its alloys or compounds. A4ctivationanalysis is favored by its specificity and sensitivity. T h e artificial radioisotopc(s) induced into the element being determined during the
S I
hfn
Sample 1 23 29 2 9,2 8,3 3
Sample 2 18 13 1 4 1 5 1 4
ACKSOWLEDGRIENT
Acknowledgment is gratefully given to TV. T. Mullins, J. H. Oliver, L. M. Frakes, and N . B. Tuck for their assistance in these analyses. LITERATURE CITED
(1) Boyd, G. E., AKAL.CHEM.21, 335 (1949).
(2) Brooksbank, W. -A,, Jr., Leddicotte, G . W., lIahlman, H. il., J . Phys. Chem. 57, 815 (1953). (3) Brooksbank, W. A., Jr., Reynolds, S.A,, Leddicotte, G . W., C . S. Atomic Energy Commission, S E C Rept., ORNL-1880 (1955) (unpublished). (4) Hevesy, G., Levi, H., KgZ. Danske V i d e n s k a b . Selskab, Mat.-fUs. M e d d . 14, KO.5 (1936). ( 5 ) Hollander, J. M., Perlman, I., Seaborg, G . T.. Revs. M o d . P h y s . 25, 469-651 (1953). (6) Isotopics, Announcements of the Isotopes Division, CSAEC, Oak Ridge, Tenn., 2, No. 2, 4 (1952). (7) Leddicotte. G. W., Reynolds, S. A , , .Vucleonics 8, KO.3, 62 (1951). (8) Reynolds, S. A,, Record Chem. Progr. 16, 99 (1955).
R E C E I V Efor D review Kovember 1 4 , 1955. .iccepted March 2 2 , 1956. D i r i sion of Analytical Chemistry, Symposium on the Analysis of Titanium and I t s Alloys, 128th Meeting. d C S , Minneapolis, I f i n n . , September 1955.
Microdetermination of Calcium and Magnesium in Tissue Ashes ROBERT L. GRISWOLD' and NELLO PACE Department of Physiology, University o f California, Berkeley, Calif.
A method is described for the separation of calcium and magnesium from each other and from phosphate, and for their titration. The method meets the special problenis arising in the analysis of small tissue samples, in which the calcium or magnesium content is less than 1 micromole.
M
ICROGRAII quantities of calcium can be determined in seruni and other relatively simple systems nithout much difficulty, but determination in tissue ashes presents a number of problems. The presence of a large excess of magnesium makes a t least t n o precipitations of calcium oxalate necessary, if the oxalate method is used. This method also has other disadvantages when very small quantities are involved. T h e direct titraPresent address. Department of Biochemlstrj , IValter Reed Army Instit u t e of Research, Walter Reed Army Medical C e n t e r , Washington 12, D. C 1
tion of calcium by the Schwarzenbach method, using murexide (ammonium purpurate) as the indicator, is also obviated by a large excess of magnesium, as well as by the phosphate invariabl) present Thus, in the course of an investigation of the metal ions of liver cell fractions, the need became apparent for a method of cleanly eeparating small amounts of calcium and magnesium from each other and from phosphate. These requirements were fully met by the use of a cation exchange column. Although ion exchange chromatography is widely used as an adjunct t o analysis, its application to this problem has not been reported. T h e Schwarzenbach method ( I ) , using Eriochrome Black T as indicator, was settled upon as a convenient means of finally determining both calcium and magnesium. OUTLINE OF ,METHOD
Samples of rat liver homogenate containing 0.5 to 1 gram of liver were digested as follows. A quantity of concentrated
1036
A N A L Y T I C A L CHEMISTRY
Table I.
Sequence of Elution of Ion Exchange Columns
Size of Column, Cm. 0 . 5 x 5.0
0.5
a
b
x
10
Elution Timea, Rlin. 0-43 46-166 165-226 225-285 0-45 45-150 150-285
Normality of HC1 0.9 1.5 1.5
6.0 1.0 3.0 3.0
Content of Effluent Phosphate Magnesium Iron b Calcium Phosphate Magnesium Calcium
Flow rate, 4 ml. per hour. Incompletely resolved.
nitric acid equal to about half the volume of the sample was added, and the mixture v a s heated a t 70" C. until the sample was dissolved. Thereafter, the temperature as gradually raised t o 100' C., effecting progressive digestion and evaporating the sample to dryness. If the residue was charred, another 5 ml. of nitric acid was added and evaporated down again. When the residue was brown or l-ellow, 1 ml. of perchloric acid ( i o to i2Y0) lyas added and the temperature raised to 120' C. The sample was then evaporated until a white crystalline residue remained. At this point, it was found important to leave the residue moist with acid. If dried completely, it became insoluble, but left slightly wet, it remained readily soluble in a small amount of water. The ashed residue of the liver homogenate sample was dissolved in water and diluted to 50 ml. A 10-ml. aliquot of this n a s applied to a column of Dowex 50 in the hydrogen form. All the cations as well as phosphate became bound to the resin. Phosphate magnesium, and calcium were then eluted one a t a time with hydrochloric acid. The latter two samples were collected, evaporated to dryness, redissolved in 11ater, and titrated with the chelating agent disodium ethylenediamine tetraacetate, using as indicator a solution of Eriochrome Black T in a buffer of p H 10.5. REAGENTS
Water. All water used in column operation was freshly redistilled in an all-glass still, from a solution of sodium hydroxide and potassium permanganate in house-supplied distilled water. Hydrochloric Acid. Concentrated (1221') hydrochloric acid was diluted with an equal volume of redistilled water, and this constant-boiling mixture was freshly distilled in an all-glass still. The distillate (6S)was diluted as needed with redistilled water. Buffer. I n distilled water, 6.8 grams of ammonium chloride, 57 ml. of concentrated ammonium hydroxide, 0.20 gram of hydroxylamine hydrochloride, and 6.6 grams of potassium cyanide were dissolved and diluted to 1 liter. This provides a buffer solution in the pH range 10.0 to 10.5, which is a favorable p H range for the titration. Indicator. Powdered Eriochrome Black T was dissolved in a few milliliters of buffer solution (about 0.001%7,)to make a blue color Thich experience dictated ii-ould be easily read during the titration. Titrating Reagent. I n 100 ml. of Lvater, 4.5 grams of the chelating agent ethylenediaminetetraacetic acid (disodium salt,) were dissolved. This solution was standardized by titrating against a standard magnesium solution. Standard Magnesium Solution. d sample of approximately 1 gram of reagent grade magnesium sulfate heptahydrate was placed in a weighing bottle in an oven a t 260" C. overnight; this treatment drove off the water of hydration. The resulting magnesium sulfate Tvas cooled in a vacuum desiccator and rapidly weighed. It was then dissolved in nater and diluted to 1000 ml., resulting in a solution of known concentration containing approximately 0.1 mg. of magnesium per ml. ION EXCHANGE COLUMN
The cation exchange resin Dox-eu 50 (200 to 400 mesh) was n-ashed several times 4 ith freshly distilled 6 S hydrochloric acid, until the washings were free of iron, and next with freshly redistilled water until the washings were neutral in reaction. The resin was then in the hydrogen form. TTO sizes of column were used with equal success. The first was 0.5 cm. in diameter and 5 cm. high; magnesium and calcium could not be separated conveniently by chromatographing with one strength of acid, but could be eluted separately by using first a dilute, then a more concentrated acid solution. The second type of column was of the same diameter, but 10 cni. in height;
here the two ions could be conipletelv separated by chromatographing with one intermediate strength of acid. The flow through the columns was by gravity, and the rate of flow was adjusted to approximately 4 ml. per hour by varying the level of the fluid reservoir. Each column was tested individually, as different degrees of packing of the resin resulted in different rates of flow under the same pressure. Tests were performed to determine the proper lengths of time for elution with the various strengths of acid and for collecting the samples. Table I shows the sequence finally settled upon for the t n o column sizes. Iron, zinc, and copper, which are knonn to interfere Fith the titration, cannot be entirely separated from magnesium and calcium by this process as described. Their presence is therefore unavoidable in the magnesium and calcium samples. The 1 . 5 5 hydrochloric acid was, however, allon-ed to run for an extra hour after the magnesium was eluted from the 5-cm, column in order to minimize the amount of iron in the calcium effluent. Interference by the remaining iron and by copper was eliminated by the addition of cyanide to the buffer ( d ) , interference by the small amount of zinc present was found to be negligible.
Table 11. Values for Magnesium and Calcium after Separation of Known Mixture on Each of Four Ion Exchange Columns Column 3-0,
Ion Mg++
1
2 3 4
C8++
1 2
3 4
Alicromole Found 0 139 0 138 0 138 0 140 0 139 0 134 0 134 0 136
% Deviation from Expected Value -2.3 -2.7 -2.7 -1 1 3 9 -0 3 -0 3 1.7
TITR4TION OF RIAGNESIUXI AUD CALCICXl
The strongly acid samples collected from the ion exchange column cannot be titrated directly, both because of their large volume (4 to 8 ml.) and because the titration must take place in a basic solution. Therefore, the samples, which were collected in conical-tipped, 12-ml. centrifuge tubes, Tvere evaporated to dryness in an oven a t 100" to 105' C. If they Tvere heated to higher temperatures, the Eriochrome Black T did not give a satisfactory end point. The reason for this is not known. -1few drops of n-ater Tvere then added and the tip of the tube was heated over a hot plate; the distillate was allowed to condense on the sides of the tube, dissolving the invisible film of salts left after evaporation. These droplets were then centrifuged to the bottom of the tube. The titrations were perfornied from a capillary buret, the tip of which reached to the bottom of the centrifuge tube. Stirring Tvas accomplished by bubbling air or gas through the sample. The capillary tube of the buret had a bore of about 0.66 mm., and delivered about 85 pl. a t full scale, or, in other !vords, enough to titrate about 1 micromole of magnesium or calcium. Because somewhat different procedures vere f o l l o ~ e din the determination of magnesium and calcium, they are described separately. Magnesium. -1sample of tissue ash which contained enough calcium for accurate determination usually contained too much magnesium for convenience in titration. Therefore, the redissolved sample of magnesium Tvas redried. (-111 the sample was now found in the tip of the tube.) From a silicone-coated capillary pipet of the Cunningham-Kirk type, 0.300 ml. of water was added. From this solution 0.100 ml. (or in some cases 0.025 ml.) was taken and transferred to another test tube. -1bout 0.5 ml. of indicator-buffer solution was added, and the mixture \vas titrated to the blue end point. Calcium. Because the red calcium-dye compound is unstable
1037
V O L U M E 2 8 , NO. 6, J U N E 1 9 5 6 Table 111. Results of Analyses of Five Rat Liver Homogenates Calciuni 0 0 0 0 0
(Xicroinole per mg. nitrogen) Magnesium 0 300 0 330 0 378 0.296 0 323
046‘3 0349
0387 0414 OFYR
and gives a poor end point, a small amount of standard magnesium solution (0.025 ml.) was added t o each calcium sample. The titration was then carried out as for magnesium, and from this titer (expressed in moles) n a s subtracted the moles of magnesium added. This procedure is successful because the chelating agent reacts first Jl-ith calcium, and only then n i t h magnesium. Thus the end-point change is due to the chelation of magnesium, n-hich provides a good color change. RESULTS
Trials of the above separation and titration procedure on solutions of known concentration indicated recoveries of about 100%
for calcium and 97% for magnesium, with deviations of less than 5%. X typical set of data demonstrating this is shown in Table 11, which gives the micromoles found after ~eparation,on each of four columns, of identical known mixtures of calcium and magnesium. T h e results of analyses done on some samples of normal rat liver homogenates are given in Table 111. Before homogenization, t,hese livers were perfused free of blood with 0.25.1f sucrose solution. l l o r e detailed results on rat liver honiogenates and cell fractions will be presented elsevihere. LITERATURE CITED
(1) Biedermann, W., Schwaraenbach, G , Chirnia (Sicztz ) 2 , 56 (1948). ( 2 ) Diehl, H., Goets, C. .1., Hach, C. C . , J . A m . Tl’afer Works Assoc. 42, 40 (1950). RECEIVED for review December 16, 1955. Accepted 3Iarcli 12, 19%. Supported b y funds under Contract Konr-222((31) between the Office of Kava1 Research and the University of California. R . L. Grisnold was r,9 . Public Health Service postdoctoral research fellow 1952-33.
Detection of Carbohydrates on Paper Chromatograms M. L. WOLFROM and J. B. MILLER Department of Chemistry, The O h i o State University, Columbus 70, O h i o
Carbohydrates and related substances are well indicated on developed paper chromatograms by successively applying aqueous sodium metaperiodate, aqueous potassium permanganate, water, and an acid solution of benzidine.
A
S A means of detecting material on paper chromatograms, the joint use of sodium metaperiodate and potassium permanganate is particularly attradive because of its a i d e applicability. Thus, such treatment should detect not only compounds that are easily oxidized by permanganate but also compounds whose periodate cleavage products are so oxidized. The permangmate-periodate system as an oxidizing medium for olefins has been investigated in detail by Lemieux and coworkers ( d ) , who have also reported its use in the detection of carbohydrates on paper chromatograms (S). A similar procedure, which has been in use in this laboratory for some time, is somewhat faster and less sensitive to an arid atmosphere, and tem of reagents Jl-hich results in a dark blue spot. The chromiitograms are permanent for some months and the sensitivity somexvhat exceeds that of the Lemieux and Bauer method. EXPEXIAIENTAL
The production of blue spots results from the oxidation of benzidine to “henzidine blue” by manganese dioxide. The benzidine reagent has the i‘olloil-ing composition ( 1 ) : 1 gram of benzidine, 8 grams of trichloroacetic acid, 20 ml. of anhydrous acetic acid, 12 nil. of xvater, and 160 nil. of absolute ethanol. T h e air-dried chromatogram is first sprayed v-ith a 17caqueous solution of sodium netaperiodate. ;ifter 3 to 4 minutes, the chroniatogram is sprayed with a freshly prepared 1%; aqueous solution of potassium permanganate. I n 5 minutes, sites of the largest amounts of material viill show as green, yellow, or bro\l-n spote. The paper is then washed free of permanganate color with distilled water. This treatment will frequentl?. reveal faint broxvn spot? previouely covered bT- the excess of permanganate solution. hfter air-drying, the chromatogram is sprayed ivith the benzidine reagent, Xl-hich instantly converts the bron-n spots to dark blue ones and frequently reveals spots not previously
seen. The background is initially light blue, changing to nhite on drying and then to broan after about 1 aeek. This method will detect 0.5 y of mannitol, 2.4 y of glucose, and 7 . 8 y of sucrose, as 5 X 6 mm. oval spot., xl-hen these substances are placed directly on K h a t m a n S o . 1 paper. Distilled water on R h a t m a n KO. 1 paper may give a faint blue ring (not a spot). DISCUSSION
The sensitivity, wide applicability, and speed of this method make it attractive. The benzidine reagent (I) may be used alone as a supplementary test. As with any aqueous spray reagent, too heavy application will cause the spots to dipperse, thus decreasing the sensitivity. Failure to obtain a white background, indicating the presence of manganese dioxide, n-ill lessen the contrast and hence the sensitivity. T h e source of such manganese dioxide may be: permanganate oxidation of the periodate cleavage products of the paper ( 2 , 5); colloidal manganese dioxide in the permanganate reagent; failure to n-azh the paper thoroughly; or failure to cover the paper completely Tvitli the periodate spray, thus leaving those uncovered portions open to rapid permanganate oxidation. Exposing the paper, particularly when wet, to a reducing atmosphere such as one containing hydrogen sulfide, d l destroy the manganese dioxide spot.. ACKNOWLEDGMENT
One of the authors (J. B. 11.)is pleased to :tcknon-ledge the support of t h e Procter & Ganihle Co. fellomhip held during 1955-1966. LITERhTL-RE CITED
(1) Bacon, J. S. D., Edelnian, J., Biocherri. J . (Lordon) 48, 114
(1951). Cifonelli, J. d.,Smith, F., ;\XAL. CHEST. 26, 1132 (1964). Lemieux, R . U., Bauer, H. F., I b i d . . 26, 920 (1954). (4) Lemieux, R. U., Rudloff, E. \-on, Can. J . Chem. 33, 1701 (1955). (5) IIetzenberg, R . L., IIitchell. 13. K., J . .4m. C h e m . SOC.76, 4187 (1954). (2) (3)
RECEIVED for revieiv December 1 3 , 1955, Accepted \ l a r c h 8, 1956.
