34
ANALYTICAL CHEMISTRY
allopregnane-11fl,21-diol-3,20-dione-21-acetate,and allopregacetate; Kenneth Savard for pregnane-llp,l7,a-21-trio1-3,20nane-17a,21-dio1-3,11,20-trione-21-acetate;Alika Hayano for dione-21-acetate; and Lederle Laboratories Division, American allopregnane-1 1p,21-diol-3,20-dione; Enrico Forchielli for allopregCyanamid Co. for ergostan-3-one. nane-17a,21-diol-3,2O-dione and pregnane-17a,21-dio1-3,20LITERATURE CITED dione; Harold Levy for allopregnane-3,20-dione and allopregnan17a-ol-3,20-dione; Ciba Pharmaceutical Co., for androstan-3(1) Dobriner, K., Katzenellenbogen, E. R., and Jones, R. N., “Infrared Absorption Spectra of Steroids, An Atlas,’’ Interscience, one, androstane-3,17-dioiie, androstan-17P-ol-3-one, and alloNew York, 1953. pregnan-21-01-3,20-dione-21-acetate;R. I. Dorfman for andro(2) Furchgott, R. F., Rosenkranta, H., and Shorr, E., J . B i d . Chem. stane-3,11,17-trione, pregnan-1 la-ol-3,20-dione, and pregnan163., 375 .(1946). , 20a-ol-3-one-20-acetate; Merck & Co. for etiocholan-17fl-ol-3(3) Jones, R. N., Herling, F., and Katzenellenbogen. E., J . Am. Chem. SOC.77, 651 (1955). one and pregnane-17aJ21-diol-3,11,20-trione; Parke-Davis Co. (4) Rosenkrantx, H.. and Skogstrom, P., Ibid., 77, 2237 (1955). for pregnane-3,aO-dione; Glidden Co. for etiocholane-3,17-dione; 25, 1025 (1953). (5) Rosenkranta, H., and Zablow, L., ANAL.CHEW. Frank Ungar for allopregnane-17a,21-diol-3,20-dione-21-acetate, RECEIVED for review June 29, 1965. Accepted October 17, 1955. Supported et iocholan-l7p-ol-3-one- 17-acetat e, and pregnane-1 7a,2 1-diol-3, by a grant from Medical Research and Development Board, Office of t h e 20-dione-21-acetate; Kicholas Saba for allopregnan-20a-ol-3-oneSurgeon General, Department of .4rmy under Contract No. DA-48-007-M D20-acetate; John Davis for pregnane-l~a,21-diol-3,11,20-trione-21-310.
Flame Photometric Determination of Sodium, Potassium, Calcium, Magnesium, and Manganese in Glass and Raw Materials NORMAN ROY Research laboratory, Thatcher Glass Manufacturing Co., Elmira,
Use of the hydrogen flame attachment and photomultiplier tube with the Beckman Model B spectrophotometer in the analysis of soda-lime and borosilicate glasses, as well as various naturally occurring materials, such as sand, slag, and feldspar, is described. The range of concentration of the various components is wide and the accuracy obtained in the determinations varies, but in most cases is within 0.3 to 0.5% of the amount of the component present in the sample. A single disintegration of the material with no preliminary separations is employed in determining all of the components listed.
S
OME flame photometric techniques involve preliminary
separations from interfering elements, whereas other techniques call for comparison of the unknown solution with a standard ivhich incorporates some or all of the interferences present in the unknown solution. Some authors add known or excess amounts of the interfering cations t o both the unknown and the standard solutions, with or without first completely removing the interferent from the unknown solution by precipitation or by an exchange resin. Although the flame photometric determination of the alkali, alkaline-earth, and certain other elements has considerably shortened analysis time, the preliminary separation and/or addition techniques detract from the speed and simplicity which might ultimately be attained. For this reason and because it was felt that the best accuracy could be obtained by making the standards as similar as possible to the sample solution as proposed by Gilbert m d others ( 6 ) , no preliminary treatment other than solution of the sample was employed in the method adopted and here described. hlost of the flame photometric methods described in the literature employ a water or acid solution, or acid extraction of the sample for the determination of the alkali and alkaline-earth metals. Sintering is also used to effect eolution of the sample for flame analysis. Where the sample is water- or acid-soluble, this technique is perfectly feasible. iicid extraction has the disadvantage of leaving part of the sample undissolved, and sintering of the sample is relatively time-consuming. The method of solu-
N. Y.
tion described herein takes less than an hour for most glasses and raw materials. The powerful oxidizing action of boiling perchloric acid combined with the volatilizing action on silica and boron of hydrofluoric acid provides a sample solution technique which ie sufficient for all glasses known to the author and for most glass raw materials. The development of the photomultiplier tube for use in connection with the spectrophotometer has unquestionably extended the usefulness of flame analysis. N o w it is possible t o use the broad selection of sensitivities of the photodetector on the spectrophotometer in analyzing a single solution containing a number of components of tvidely varying emissivity. Although the Beckman 3Iodel B spectrophotometer with No. 9125 flame attachment was used in developing this method, the Reckman Model DL with flame attachment can be used, with no essential change in the method. The Model B is a direct-reading instrument and might be slightly faster in reading than the DC; the D U is undoubtably more sensitive and selective.. GENERAL CONSIDERATIONS
In all quantitative analyses, the qualitative make-up aiid approximate quantities of the components present in a giveii substance must be knowi, if the analysis is to have reasonable cert,aint.y of success. This information must also be knoll-n in making up a standard for a glass or other material which is to be analyzed by the method described. The needed information may be obtained from analyses of similar material, such as glass from the same tank, or examinat,ion of the flame spectrum of a totally unknown material will give a quick qualitative analysis for sodium, potassium, calcium, magnesium, and manganese, as \yell as lithium, strontium, copper, etc., and then a rough quantitative analysis may be run off immediately for the elements detected. In the latter case, it is most convenient to make an approximately 0.1N hydrochloric acid solution of the material t o be examined and compare it with solutions of each of the chlorides of sodium, potassium, calcium, magnesium, and manganese following in outline the procedure for glass analysis. I n the case of a
35
V O L U M E 2 8 , NO. 1, J A N U A R Y 1 9 5 6
interference effect of any given component is but a small fraction Table I. Effect of Aluminum Salts on Intensity Readings of Synthetic Standard -
AlzOa, Mg./L. None 5P Intensity Readings
Standard Composition, lIg,/L.
5%
CaO XgO Naz0 Kz0
300 173 700 10
0 4 0 0
64 60 60 62
h. Ea L
CaO MgO NatO
400 150 700 10
0 0 0 0
66 61 60 63
.S.
5 0 0 0
GO 60 60 62
Net Effect of Additive,
%
0 0 0 0
- 7 0 0 0 0 0 0 0
0 0 0 0
- 9 1 0 0 0 0 0 0
100 mg /l.
I(Z0
0 0 0 0
GO 61 60 63
of the magnitude of the concentration of the component, and therefore a small variation in the concentration of the interferent results in a negligible change in the interference effect. The use of a blank in any given determination is an attempt t o eliminate the background effect. Theoretically the background effect a t any given emission peak should be very nearly the same for the sample being measured as for the synthetic standard. Thus the same amount may be subtracted from both the unknown and the standard readings in order to compensate for the energy reaching the photocell which is not relevant to the element in question.
200 mg./l.
EXPERIMENTAL WORK
Table 11. Effect of Iron Salts on Calcium and Magnesium Readings of Synthetic Standard btanddrd E,
*
F t
3"
L
Net Effect of Additix e
Composition >Ig / L
FenOa. hlg./L None 10 Intensity Readings
AlzOs
02 5 A2 5
62 5 62 5
0 0 0 0
62 0 62 0
62 0 62 0
0 0 0 0
MgO NaaO
100 400 150 700 10
CaO RlgO Nag0 K20
50 0 300 0 173 4 700 0 10 0
CaO
KzO .%Or
0 0 0 0 0
%
yiass, a convenient concentration of sample is 0.5 gram per 100 1111. Using this concentration as the feasible upper limit, the solutions of a single salt, used as a standard in the preliminary quantitative analysis need be no stronger. Starting Tl-ith the highest possible percentage of a given compoiient, which can be in the unknown, the standard salt solution is compared, then diluted down volumetrically until it reads Tvithin a factor of 2 of the unknown, and an approximate percentage of the desired component is calculated. I n this manner, all of the elements which show up in the flame spectrum of the particular unknown in question are determined. Even though they do not show up in the flame spectrum, the presence of any other elements in the sample under examinatiou should be known, PO that their inclusion in the standard may be decided upon on the basis of their effect 011the emissivity of the sample. Thus in the case of the totally unknown material, ari additional scheme of qualitative analysis probably has to be run through, t o obtain the information necessary for making up a standard. Such a standard ideally should be similar in composition t o the unknown solution with respect to the concentration of both the flame-emittent elements and those element9 affecting the emissivity of the unknown. Certain considerations simplify the making up of the standard. By definition, this excludes from consideration components that are present in too low a concentrat,ion t o show up in the flame spectrum, are not themselves excited by the flame, have no enhancing or depressing effect, and do not contribute noticeably to the flame background. This is illustrated later in the make-up of a glass standard. Because in many cases t'he relationship between the element concentration and the emission of that element is linear over a fairly wide range of concentration, especially i n the more dilute solutions, a further simplification is in the leeway that may be allowed between the sample and standard concentrations of emissive elements. This has been shoa-n hy numerous investigators, including Gilbert,, Hawes, and Beckman ( 6 ) )Close, Smith, and Watson ( 4 ) ,Close and Watson ( 5 ) ,and Beckman Instruments ( 2 ) . Still a third simplification lies in the fact that the
Aluminum does not shon up sufficient'ly in the flame spectrum at the concentration of 0.5 gram per 100 ml. of sample to be measured on the Beckman Model B, even in the case of feldspar and nepheline syenite, which contain some 20% alumina. HOWever, hlosher, Bird, and Boyle ( 7 ) have shown that aluminum in solution has a pronounced depressive effect on the calcium emission mhen calcium is present alone in solution and a somewhat lesser depressive effect when magnesium and iron are also in the solution. T h e depressive effect of aluminum on calcium has also been observed by Brabson and Wilhide ( 3 ) . Accordingly, the effect of aluniinum additions to the aynthehic standard was studied. Table I shows that addition of aluminum depresses the calcium emission, with no effect on the other components. This depression is proportional to the amount of aluminum present, but is also affected b y the other components. Figure 1 shows the effect of perchloric acid additions to the synthetic standard. The data for these curves were obtained t)y adding small increments of the 72% acid to the El, Ea, L synthetic standard. Kone of the components other than calciuni and magnesium changed in intensity and the dilution effect of the additions was negligible. I n the case of calcium, a limiting or saturation value is reached a t 0.01N perchloric acid; magnesium reaches a saturation value at about 0.05N. Continuat'ion of additions of perchloric acid results in no measurable further increase in emission intensity of either calcium or magneeium. The increase in calcium intensity is 12%, and the increase in magnesium intensity is 10% on the basis of the original emissions in the part,icular solution examined. 70
>
MAGNESIUM
I-
Z
60"
W +
-
z
z Z
0
50
-
40
-
CALCIUM
383 mp
422
m+
p 4 - 0
v)
Lo -
2 W
E,, E 3 , L
I
I
SYNTHETIC
I
STANDARD
I
I
I
blosher, Bird, and Boyle ( 7 ) have observed that iron depresses the calcium emission to some extent. Close, Smith, and Watson ( 4 ) have also shown that sulfate and manganese affect the calcium and magnesium emission in hydrogen flame spectrophotometry. Accordingly, the effect on the synthetic standard of iron and sulfate in the concentrations usually present in glass was studied with a view t o their possible exclusion from the standard,
36 Table I1 shows the rpsults of additions of iron to two synthetic standards; the equivalent of 0.2% iron oxide was added. The net' effect of these additions on the emission intensity is zero. The result' of additions of sulfate to the synthetic standards is shown in Table 111; the equivalent of 0.5% sulfur trioxide vias added. There is a depression of approximately 3% in both the calcium and magnesium readings with no perchloric acid present,, but no effect on the emission intensity with perchloric acid present. It seems that a sufficient excess of perchlorate anion is present a t a concentration of O.0125.br t,o exhibit a predominating effect over t>hatof the sulfate and iron ions, as has been suggested by Baker and Johnson ( 1 ) . T o determine whether the efiect of perchlorate on the calcium and magnesium emission intensity in the standard reaches a maximum value, after n-hich it levels off, it was decided t o test the intensity of the unknon-n by adding a large excess of perchloric acid to various typical g1 decompositions. I n Table I V under Unknown, the column headed 0.0625S perchloric acid represents the typical glass decomposition and the column headed 0.125.V perchloric acid represents an addition of a 100% excess concentration of perchloric acid. Evidently the glass solutions obtained by the procedure for glass analysis have enough perchloric acid in them to exhibit a limiting effect. The standards also show no increase in emission intensity of calcium and magnesium on addition of a 100% excess of perchloric acid over that shown in Figure 1. If the procedure for glass is to be extended to other materials, the amount of perchlorate in the residue of the perchloric-hydrofluoric decomposition, as illustrated in Table V, must be studied. The procedure used in determining the perchlorate here was Scott's ammonium chloride sublimation (8). Boron was found to depress t,he calcium readings in the synthetic standards. 111 an experiment, 25 mg. per liter of boric oxide as boric arid, equivalent to 0.5% boric oxide in the sample, \vas added to the El, Ea,I, synthetic standard; the calcium emission was found t o be depressed some loyo. The boron need not be considered when the sample is brought into solution hy perchloric-hydrofluoric decomposition, however. Phosphorus was found to lower the calcium eniission decidedly in one synthetic standard in which it was included as phosphoric wid, n-ith perchloric acid ai-iselit. dddition of perchloric acid to about 0.05S raised the calcium emission t o an extent even greater than 12%. This phenomenon was not studied any further, because there was apparently no perchlorate in the residue of this phosphate glass perchloric-hydrofluoric decomposition. Instead of perchloric acid, phosphoric acid was added to the standard for this glass, in an amount equivalent to the phosphorus in the glass; the calcium determinations by the flame then checked closely the standard titrimetric results. Potassium interferes with the manganese emission somewhat, because of the weak potassiuni emission line at 404 mp. When the potassium concentration becomes greater than 10 times the manganese concentration, the interference begins to become serious. The manganese oxide blank for the Sa synthet'ic standard, which standard contains the equivalent of 0.2% manganese oxide and 1.2% potassium oxide, definitely shows a peak at' 404 nip which is due to t,he potassium in the solution. However, the manganese peak in this particular standard is over and above the potassium at 404 mp; the manganese peak a t 403 mp] when present strongly, ('blots out" the potassium a t 304 mp with the Beckman Model B. An instrument with slightly greater resolving power than the Model B could separat,e these two peaks. Manganese interferes with the magnesium emission a t 383 mp by raising the background and, if present in widely divergent amounts in t,he st,andard and the unknoim, the determinations on magnesium are erroneous. The amount of emission intensity of each component of the standard, Tyhich is due to background radiation, is illustrated graphically for two different st,andards in Figure 2, 9 and B .
ANALYTICAL CHEMISTRY Table 111. Effect of Sulfate Salts on Calcium and Magnesium Readings of Synthetic Standard Component Measured
Standard
SOa, AIg /L. Sone 25 0 Intensity Readings
Ket Effect of Additive, 70
0.0125.Y HClOI
E:, E3. L
CaO 3IgO CaO
Si. S?
11go
60 0 58.0
60 0 38 0
0.0 0.0
.5S 0 .56, 0
38.0 56.0
0.0 0.0
No HClOa
Ei. Ei. L
CaO 1190 CaO MgO
91, S?
63.5
62.0 6 1 .5
-3.1 -3.2
64.0 63.5
62.5 61 d
-3 2
64.0
-2.4
Table IV. Effect of Perchloric Acid Additions to Sy-nthetic Standard and Unknown Solutions Solution Being Read Standard E1
Es,L
51,
sz
Intensity Readings Component Measured CaO JlgO CaO MgO
0.06253
0.125.V
HClOi 65 0 66 0 65,O
HClOi
Ket Effect,
%
65 0 66 0
0.0 0 0
66.0
65.0 66.0
0.0 0.0
CaO MgO CaO MgO CaO MgO CaO MgO CaO \lgO
60 0 62 0
60 0 62 0
0 0 0 0
60 62 61 62
60 62 61 62
0 0 0 0
1 nhnoun E1
E? 39
L Sa
0
0 0 0 0
0 0 0 0
fil 0 63 0
61 0 63 0
0 0 0 0
fil 0 63 0
61 0 113 0
0 0
0
0 0
0 0
Figure 2, -4,shows a high background effect for manganese present in very low concentration in the standard, an intermediate background effect for magnesium present in intermediate concentration, and an even lower background for potassium present in much lower concentration than magnesium. Sodium and calcium shoiv a negligible background. In Figure 2, comparing -4t o B illustrates the change in background with the change in concentration of a given element,. Here the magnesium emission beconies a tenth of that illustrated in Figure 2, d , and is impractical to measure with this instrument, the background effect being 95% of t,he emission a t full scale and the slope of background us. standard emission approaching 45 '. Douhling the manganese concentration results in a nice lowering of the hackground for that element and sodium, potassium, and calcium Iiackgrounds here are negligible. PREPARATION OF GLASS STANDARDS
.\liquets of the standards containing a single chloride salt were pipetted in order to make the composite solutions designated as synthetic standards. The known composition of the glass or glasses t o be analyzed is used in calculating the amount of each component in 1 liter of the synthetic standard a t the level of concentration of 0.5 gram of glass per 100 nil. of solution. The calculations are simply set up as in Table VI. This standard is used for a range of concentrations as follows: .Il*Or CaO MgO Na20 KsO \lnO
l . G to 2.1% 6 . 8 to 8 . 6 2 6 to 3..i 13.6 to 14.9 0.16 to 0.32 O.0Oto 0 . 1 0
The glasses for which the standard illustrated is used also contain silica, ferric oxide, barium oxide, sulfate, fluorine, arsenic pentoxide, and boric oxide. -4luminum ip included, but iron,
37
V O L U M E 28, N O . 1, J A N U A R Y 1 9 5 6 Table V.
Perchlorate Present in Hydrofluoric-Perchloric Residues Weight, G 0 12.50 0 do00 0.5000
Material Glass Slag
Table VI.
Perchlorate, G. 0.07 0 25 1 0
Chloride Negative 3-egatix.e
SegatiLe
Typical Glass Standard Compositionn
(El, Ea, L synthetic standard, prepared 6-7-54) % in Theoretical 11g. per Lite1 Glass in of Synthetic Component Standard (C) Standard 2 00 100.00 8 00 3.00 14.00 0 20 0 030 a
400 00 180.00
iOO.00 10.00 2.80
Plus 4.0 ml. of 7'2% perchloric acid per lite!.
barium, and sulfate are not included for the reasons developed in the experimental section of this paper. Silica, arsenic pentoxide, boric oxide, and fluorine are volatilized in the course of the perchloric-hydrofluoric disintegration \\-hen the remaining elements are also changed to their perchlorate salts. The theoretical equivalent of perchloric acid in the residue of 0.5000 gram of these particular glasses is approximatelv 0.28 gram agreeing with experimental determinations of perchlorate on the residue. This is an amount greater than that needed for a maximum enhancement of the calcium and magnesium emission. In order t o achieve the same effect in the standard, 4.0 ml. of 72y0 perchloric acid must be added per liter. In the case of a phosphate glass, a perchlorate assay of the residue from the disintegration of the particular glass in question indicates the steps to be taken in making the synthetic standard. The blanks are made u p exactly the Sam? its the synthetic standards, except for omission of the component for n.hich the background effect is 1)eing measured.
gen and 10 pounds per square inrh of oxygen are used. .Is a general rule, the sensitivity control on the inst.rument and the phototube voltage are set sufficiently high t o get the narroTyest possible slit opening, but not below 0.01 mm. Sodium is read a t 589 mp, potassium a t 767, calcium a t 422.7, manganese a t 403, and magnesium a t either 383 or 371 m p . After the desired emission peak is located exactly, the wave-length setting is not disturbed until all of the determinations on that particular element are accomplished. h l l solut,ions t o be analyzed may be lined up in 5-ml. beakers covered with small watch glasses t o minimize concentration of solute by evaporation. The wave length having been set, the slit is adjusted so that the galvanomet'er response is around 60 t o 80% transmittance; the slit is then not changed while a given sample is read. Greater accuracy of the readings is obtained the higher up the per cent transmittance scale the readings can be taken; holTever,. the needle fluctuation becomes greater the higher up the scale one goes. With the particular instrument used in this work, 60 t o SO'% transmittance was a likely compromise between accuracy of the reading and the needle fluctuation. For the most precise work, the synthetic standard solution is read coincidentally. A4fter a stable reading of the standard in the flame is obtained, the standard is immediately removed, the aspirator is given a 1 to 2-second rinse ivith distilled water, and the unknown is placed in the flame. .4ft,er a stable reading of the unknown is obtained, the capillary is rinsed and the standard measured again t o check the first reading. If the two standard readings do not agree, the unknonm reading is repeated, followed by another reading of the standard, and so on, until the standard readings before and after the unknown reading agree nithin =k 0.5% transmittance. The capillary is always rinsed for 1 to 2 seconds Ti-ith distilled water between readings, and the dark current is zeroed while doing this. A blank is read in conjunction with each determination, or thp value for any given determination is read from a prepared curve, .such as Figure 2.
100
/ PROCEDURE FOR GLASS AIYALYSIS
Perchloric-Hydrofluoric Decomposition. Keigh out a 0.5000gram sample of powdered glass (200-mesh or finer) on a damped balance and transfer t o a 60-ml. platinum evaporating dish. Add i rnl. of perchloric acid and 5 ml. of hydrofluoric acid. Grasping the dish Tyith platinum-tipped tongs, swirl the contents t o mix and set on the edge of a n asbestos-covered hot plate. When the effervescing dies down, move the dish more t o the center of the hot plate and take down t o first dense white fumes of perchloric acid. Remove from the hot plate and cool the platinum in a small water bath, and when cool examine for undecomposed silicates. If the material is not, completely dissolved at this point, add 1 to 2 ml. more of hydrofluoric acid and repeat the evaporation to perchloric fumes. -4s soon as the material is completely decomposed, take down to dryness. Cool the dish in water again, remove from the water bath, and add 1 to 2 ml. of 6.V hydrochloric acid and hot water t o dissolve. Transfer to a 100-ml. volumetric flask, dilute t o volume, and mix. The solution should be completely clear a t this point. .4n alternative procedure is to decompose a 0.1250-gram sample and make up to a final volume of 25 ml.; in either case, t'he final concentration of sample is t>he same. The smaller sample uses less time and materials in t,he decomposition with still enough solution for the determination of sodium, potassium, calcium, magnesium, and manganese. A 1 to 40 dilution of this solution is made for taking the sodium readings, vhich are read against a 1 to 40 dilution of the synthetic standard. The dilution is best made by transferring 5 ml. of the more concentrated solution to a 200-m]. volumetric flask and diluting t o volume. Flame Photometric Measurements. The gas pressures employed are 4 pounds per square inch of hydrogen and 10 pounds per square inch of oxygen for Podium, potassium, calcium, and manganese; for very !Teak concentrations of the preceding elements arid for magnesium, 5 pounds per squarc inch of hydro-
40 STANDARD
60 80 100 EMISSION INTENSITY
Figure 2. Typical background readings of synthetic standards A.
B.
El, E3, L synthetic standard composition (see Table VI) Sa aynthetic standard composition, milligrams per liter A1203 130 0 Sa20 700.0 CaO 500 0 K?O 60 0 MgO 15.0 1InO L O
I n calculating the results, use is made of the fact that, in the case of the usual glass and in a sufficiently dilute solution, there is a straight-line relationship between the emission intensity and the element concentration. I t was found in numerous euperi-
38
ANALYTICAL CHEMISTRY
ments in the laboratory that, in the case of sodium, potassium, calcium, magnesium, and manganese, the straight line is fitted very closely by the following equation over a relative deviation from the standard concentration of at least f 25%.
where c' S
B
C
V
V = -U - B x c S - B = reading of sample = reading of standard = reading of blank = per cent of component being measured which is present in the synthetic material in the standard = per cent of component being measured which is present in the unknown sample
tate from the glass analysis, whereas no peaks a t all in this region showed up in the reagent precipitate. T o test this explanation further, Bureau of Standards glass No. 80 was analyzed for sodium oxide, potassium oxide, calcium oxide, and magnesium oxide by the flame procedure. The results (Table VIII) show close agreement in all cases between the flame analysis results and the bureau's certified values.
Table VIII,
Comparison of Flame Analysis of Standard Glass with Wet Analysis Procedure
Component CaO
hZg0
NazO
KZO
MnO
Wet 8 19 3 07 14 27 0 24 0 02
Analysis,%
Flame 7 98 3 32 14 25 0 25 0 02
No. of Detns. 4 4 6
Investigation of the calcium oxalate precipitate in the wetanalysis method showed coprecipitation of some magnesium oxalate and thereby explained the discrepancy in the results by the two methods, as the filtrate from the calcium oxalate precipitation had been used for the magnesium precipitation. The calcium oxalate precipitate was tested by dissolving it in dilute hydrochloric acid and examining for peaks of magnesiuni on the flame spectrophotometer. Reagent grade calcium chloride was dissolved and precipitated as the oxalate by the wetanalysis procedure used for the glass, and this precipitate was examined in the same manner on the flame spectrophotometer. Definite peaks at 383 and 371 mp were observed for the precipi-
4.66
MgO NrtrO I120
Table IX.
Component CaO MgO NazO
KtO
MnO b
Sample 9 4.57 3.26
7c
crto
DISCUSSION OF RESULTS
Table VII.
Value.
Component
The background effect is compensated for by the blank. All other interferences are incorporated in the standard. These effects include line spectral interference, stray light, band spectral interference, continuum interference (including anionic effect), and temperature and viscosity effects.
A single scale reading on the Model B cannot be read closer than 0.5% transmittance when the flame is used, but the average of three or more readings gives a result accurate to about 0.2 division. When the readings are taken at 80% transmittance a photometric accuracy of 2.5 parts in a thousand is given. The accuracy is less when the blank is greater than zero, becomes progressively less with increasing background, decreases with lower scale readings, and increases with higher scale readings of the sample. Almost always precision has been found to be within photometric accuracy. A standard glass xhich has been analyzed in this laboratory a number of times by a wet-analysis procedure, was used to compare the wet-analysis method t o the proposed procedure. The results in Table VII shoa. good agreement with sodium oxide, potassium oxide, and manganese oxide determinations, but low calcium oxide and high magnesium oxide results. In order to check the consistency of the discrepancy between the two methods, the calcium oxide content of eight different glasses was determined on many occasions by the flame and by the wet-analysis method. The flame results were consistently below the wet analysis by 0.2 t o 0.3% in seven out of the eight glasses; in the eighth case, the flame results were higher than the wet analysis, and i n this case the glass was also very low in magnesium oxide.
Flame Analysis of National Bureau of Standards Glass Sample 80 Certified Flame Analysis, yo
3.23 16.65 0.04
16.62
0 039
Sample
B
4.60 3.26 16.62 0.038
Comparison of Standard Glass Flame Analysis with Results of Other Laboratories Flame Analysis.
%
7 98 3 32 14 25 0 25 0 02
Lshor2tory A,
Laboratory
%
B,b %
8 03 3 20
8 08
14 30 0 22
S 40 14 12 0 19
Laboratory C,C
%
7 3 14 0
64
20 30 3i
Hartford-Empire Co. Testing Laboratory Sharp-Schurtz Co. Testing Laboratories. Solvay Process Division Laboratories.
In general, analytical results often vary from one procedure t o another, but the above account is a good illustration of how selective flame photometric analysis can be, and how the flame photometer can be used as a tool in a rapid check of gravimetric precipitates for purity. Further comparison of results with independent testing laboratories is shown in Table IX, where it is apparent that the results of laboratories .4,B, and C all fall within the range obtained by this method. EXTENT OF APPLICATION
Sarid, dag, and feldspar have been analyzed successfully by applying the procedure outlined. 4 further technique which may be used with many materials, especially with minerals, is to dissolve a standard sample of the material to be analyzed, such as National Bureau of Standards dolomite or magnesite, compare the flame emission of the st,andard with the unknoivn solution, and use the certified analysis values for calculation of the unknown sample. The background effect may be measured very closely in these cases by taking readings just before and jupt, after the emission peak, where the intensity levels off and there are no immediately adjacent emission peaks. In the case of broad oxide bands, however. the t'echnique of estimating the background is not so simple. Sodium may be determined in materials such as salt cake and soda ash by comparing flame intensities of their solutions at the proper dilution with similar solutions of the analyzed laboratory reagents-in this case sodium sulfate and sodium carbonate, respectively. Other materials which have been analyzed successfully by this method include nepheline syenite, razorite, and cryolite. I n analyzing materials, such as aluminum silicates, that oannol be readily dissolved by the perchloric-hydrofluoric disintegration met.hod used in the glass analysis procedure, the following modification is often found to work well. Enough hydrofluoric acid alone is added to the sample to volatilize all the silica and boron. The sample is then cautiously
V O L U M E 2 8 , N O . 1, J A N U A R Y 1 9 5 6 taken down to dryness on a medium hot plate and cooled. T o the platinum dish are added, by running down the sides, about 5 ml. of perchloric acid and 2 ml of hydrofluoric acid. The dish is swirled t o mix, and again taken down t o dryness and cooled. The residue should now yield to solution in dilute hydrochloric acid. ACKNOWLEDGMENT
The advice and encouragement of R. L. Wilson during the progress of this work are gratefully acknowledged, as well as the courtesy of R. S. Arrandale in permitting its publication. LITERATURE CITED (1) Baker, G.
L., and Johnson, L. H., ANAL. CHEM.26, 465 (1954).
39 Beckman Instruments, Inc., South Pasadena, Calif., Bull. 278, May 1952. Brabson, J. A,, and Wilhide. W. D., ANAL. CHEM.26, 1060 (1954).
Close, P., Smith, W. E., and Watson, M. T., Jr., Ibid., 25, 1022 (1953). Close, P., and Watson, M. T., Jr., J . Am. Ceram. Sac. 37, 235 (1954). Gilbert, P. T., Jr., Hawes, R. C., and Beckman, A. 0.. ANAL. CHEM.22, 772 (1950). Mosher, R. E., Bird, E. J., and Boyle, A. J., Ibid., 5, 715 (1950). Scott, W. W., “Standard Methods of Chemical Analysis,” 5th ed., p. 275, Van Nostrand, New York, 1945. RECEIVED for review A4pril30, 1955. Accepted Ootober 17, 1955. Division of Analytical Chemistry, 127th Meeting, ACS, Cincinnati, Ohio, MarchApril 1955.
Colorimetric Determination of Cycloserine, a New Antibiotic L A W R E N C E R. J O N E S Commercial Solvents Corp., Terre Haute, Ind.
A specific method of analysis was needed for cycloserine, a new antibiotic, as an aid in chemical evaluation. Cycloserine reacts with sodium nitrotopentacyanoferroate in slightly acidic medium to give an intense blue-colored complex suitable for quantitative measurement at 625 mp. The color deviates slightly from Beer’s law but is reproducible in the range of 5 to 200 y of cycloserine. The test quantitatively determines the antibiotic in amounts as little as 2 to 3 y and has an accuracy within +2% and a precision within *l%. The method is specific for the cycloserine molecule and has been adapted to all phases of its production and use. The results are in good agreement with the bioassay.
C
YCLOSERINE is the generic name of an antibiotic isolated from a culture of Streptomyces orchidaceus by Harned, Hidy, and LaBaw (3). Preliminary clinical evaluation has demonstrated its effectiveness in pulmonary tuberculosis ( 2 ) and certain genitourinary infections (9). Cycloserine is a cyclic hydroxamic acid derivative of serine, determined to be D-4-amino-3-isoxazolidinone ( 1 , 4, 5 , 7 , 8), and has the formula:
”,
SH*
I
H-C-C=O
A-H
Hz-h
=
H-
A
-C-OH
H,-J
0 ‘’
$
0 ‘’
A specific method of analysis was needed for cycloserine in the presence of amino acids, other antibiotics, degradation products, and compounds of biological origin. A micromethod most suited the several requirements. Therefore, colorimetric methods were examined, because these are the most universally applicable. Cycloserine reacts with sodium nitritopentacyanoferroate in slightly acidic medium to give an intense blue-colored complex suitable for quantitative measurement a t 625 mp. The color develops rapidly and is stable for several hours. It deviates slightly from Beer’s law but is reproducible in the range of 5 to 200 y of cycloserine. The method is specific and sensitive for the cycloserine molecule and has been adapted to all phases of its production and use. The results are in good agreement with the bioassay.
CENTRIFUGE, any laboratory type for 15-ml. tube VACUUM OVEN,60” C. WEIGHINGBOTTLE,glass-stoppered DEsICC~TOR,with phosphorus pentoxide desiccant PIPETS, 1.0, 2.0, 3.0, 4.0, and 10.0 ml. Normax brand or equivalent BURET,50 ml., Normax brand or equivalent REAGENTS
CYCLOSERINE STANDARD. Repeated biological assay, elemental analyses, optical rotation, titration, and other tests indicate that the purity approaches 100%. SODIUMHYDROXIDE, 4.000 and 0.100N solutions ACETIC ACID,3.000 and 1.000N solutions SULFURIC ACID,0.666N solution Na2W04, 10 j = 0.5% aqueous solution SODIUMTUNGSTATE, TUNGSTIC ACID REAGENT. Mix equal volumes of the sodium tungstate and sulfuric acid solutions. This reagent must be made fresh daily. SODIUMNITROPRUSSIDE, N a 2 F e ( C N ) a 0 .2H20. Prepare a fresh 4 f 0.5% aqueous solution every 2 weeks and store in a glass-stoppered brown bottle. CYCLOSERINE COLORREAGENT, Nac [Fe( CN)bNOz]. Mix equal volumes of the sodium nitroprusside and 4 V sodium hydroxide solutions just prior t o use. The reagent is ready for immediate use and must be discarded after one set of determinations because of instability. ACTWATEDCHARCOAL, Darco G-60 or equivalent, PREPARATION OF CALIBRATION CURVE
Add 0.10 to 0.12 grams of the crystalline cycloserine to a tared glass-stoppered weighing bottle and place in’ the 60’ C. oven at 10 to 15 mm. of mercury pressure for 2 hours. Cool in the desiccator. Prepare a solution of cycloserine standard in 0.100N sodium hydroxide solution t o contain 1.0 mg. per ml. (This solution is stable for several weeks if kept refrigerated.) Transfer by means of a buret, 0.0-, 2.5-, 5.0-, 7.5-, 10.0-, 12.5-, 15.0- 17.5-, and 20.0ml. portions of the standard solution to separate 100-ml. volumetric flasks. Dilute each to volume with 0.100N sodium hydroxide. Transfer 1.0 ml. of each dilution into a test tube. Add 3.0 ml. of 1.OOOX acetic acid and 1.0 ml. of color reagent and m x . Allow the solution to stand a t room temperature for 10 minutes. Transfer to a 1-em. Corex cell and read the absorbance a t 625 mp, using the solution containing 0.0 ml. of standard as the blank. Plot concentration against absorbance on linear graph paper. The curve deviates slightly from a straight line. The above standards equal 0, 25, 50, 75, 100, 125) 150, 175 and 200 y of cycloserine, respectively. DETERMINATION
APPARATUS
SPECTROPHOTOMETER, Beckman Model DU, with 1-cm. Corex cells
The determination is the same as the calibration, except the sample is prepared so that a 1.0-ml. aliquot of 0.100N sodium hydroxide solution does not contain more than 1507 of cyclo-
