The lack of a peak absorption at 10.3 microns and the singlet at 10.4 microns are indicative of vitamin D3. The peak at 10.4 microns is neutralized by the addition of 5 mg. per ml. of vitamin D3 to the reference cell. Here, too, the form and the approximate amount of vitamin D is elucidated by the technique of spectrophotometric neutralization. The application of these procedures to the determination of the form and the approximate amount of vitamin D present in some commercial preparations is summarized in Table I. In some cases, inspection of the infrared spectrum was sufficient to identify the form of vitamin D present; in other cases, spectrophotometric neutralization was necessary. Thus, the finding by infrared spectrophotometry that in poultry feed concentrates the vitamin D present in the D3 form is confirmed by the chick bioassays. Despite the relative insensitivity of spectrophotometric neutralization in the infrared as compared to that in the ultraviolet,
the results obtained by this procedure are comparable to those obtained by the chemical assay for total vitamin D by the method of U.S.P. XVI (13). ACKNOWLEDGMENT
The authors are indebted to Ronald Yates, Division of Cosmetics, Alma Hayden and Jonas Carol, Division of Pharmaceutical Chemistry, and Daniel Banes, Bureau of Biological and Physical Sciences, all in the Food and Drug Administration, for their interest and suggestions during the course of this investigation. LITERATURE CITED
(1) Assoc. Official Agr. Chemists, Washington 4, D. C., “Official Methods of Analysis,” 8th ed., 1955. (2) Bartlett, J. C., Chapman, D. G., J . Agr. Food Chem. 9, 50 (1961). (3) Fred, M., Putscher, R., ANAL.CHEM. 21, 900 (1949). (4) Haenni, E. O., J . Assoc. Oj%. Agr. Chemists 42, 215 (1959).
(5) Johnson, J. L., Grostic, M. F Jensen, A. O., ANAL.CHEM.29,468 (1957). ( 6 ) Jones, J. H., Clark, G. R., Harrow, L. S., J . Assoc. Ofic. Agr. Chemists 34, 136 (1951). ( 7 ) Jones, R. K., Chem. in Can. 2, 94 (1950). (8) Powell, H., J . A p p l . Chem. 1956, 488. (9) Report of the Spectroscopy Committee, J.A.O.C.S. 36,629 (1959). (10) Schmall, M., Senboroski, B., Colarusso, R., Woolisch, E. G., Schaefer, E. G. E., J . Am. Pharm. Assoc., Sci. Ed. 47, 839 (1958). (11) Theivogt, J. G., Campbell, D. J., AXAL. CHEM.31, 1375 (1959). (12) United States Pharmacopeia XV, 1955. (13) United States Pharmacopeia XVI, 1960. (14) Wilkie, J. B., Jones, S. W.,Kline, 0. L., J. Am. Pharm. ASSOC., Sci. Ed. 47, 185 (1958). (15) Wilkie, J. B., Jones, S. W.,Morris, W.W.,J. Assoc. O j i c . A g r . Chemzsts 42, 422 (1959). RECEIVEDfor review September 6, 1961. Accepted December 19, 1961. In part: 5th International Congress on Kutrition, September 1960, and 74th Annual Meeting of the Association of Official Agricultural Chemists, October 11, 1960.
Spectrographic Solution Procedures for the Determination of Some Less Familiar Elements in Iron and Nickel Base Alloys J. P. McKAVENEY and G. L. VASSILAROS Crucible Steel Co. of America Research laborafory, Pitfsburgh 7 3, Pa.
b Spectrochemical analysis by the solution-rotating disk technique i s proposed for the determination of less familiar elements in typical iron and nickel base alloys to relieve the wet chemist from the long and difficult task of developing a separation scheme for each new alloy composition. Chemical procedures required for the sample solution preparation are outlined for beryllium, niobium, magnesium, silver, yttrium, and zirconium. Chemical aspects of the solution are empirically examined both from the view of solvent effect on spectrographic response and chemical interaction between solvent and metallic ion.
T
HE TECHNIQUE of direct spectrographic analysis of solutions is fairly well known for most of the common alloying elements present in iron and nickel base alloys, but little information is available for the less familiar elements. This has not been because of lack of investigation of the physical
384
ANALYTICAL CHEMISTRY
aspects of the problem, as seen from the excellent work of Feldman (3) m-ith the porous-cup technique, Pagliassotti and Porsche (8, 9) with the rotating disk, and Zink ( 2 1 ) with the vacuum-cup technique. Also on the physical side, Margoshes ( 5 ) has worked on improved source techniques to increase sensitivity. However, on the chemical side, only the work of Baer and Hodge ( 2 ) has considered one aspect of the problem, namely, the effect of the solvent composition on spectral response. It is the authors’ conviction that the physical and chemical aspects of the problem should go hand in hand for successful application of the spectrographic solution technique. Too often in the American metals industry there is complete separation of chemical and spectrographic laboratories. The spectrographer who has been trained only in the solid specimen technique cannot solve analytical problems for less familiar elements by the solution technique because of lack of experience with chemical solutions. On the other hand, while use of a met chemist as a consult-
ant might be of advantage, this approach often fails because he does not understand the spectrographic technique. Success, therefore, will lie in the hands of the spectrographer with the chemical background. The spectrographic solution technique can be a porerful tool because of the advantages for minimizing segregation and metallurgical history as well as eliminating the need for preanalyzed chemical standards. Also, another point often orerlooked is the nearly complete elimination of interelement effects, e.g., carbon on niobium because of niobium carbide formation, or sulfur on manganese through formation of manganese sulfide in the solid solution of the metal. The proper chemical solvent will decompose completely the intermetallics, and total niobium or manganese can be determined easily. The specialty steel analyst is often challanged when analyzing research heats for less familiar elements because of the lack of solid specimen spectrographic standards or a chemical procedure free of interferences. I n this labora-
tory the rotating disk technique has been used with great success in overcoming the problem. By coupling chemical procedures and techniques of solution preparation with the rotating disk spectrographic technique, the problem of less familiar element analysis can be solved.
Table I. Volume of HNOs (Ml. of 1.42 SP. gr.) 0.0 0.6
APPARATUS AND REAGENTS
5.0 10.0
-4 Jarrell-Ash Co. (JACO) Custom Model LA 7101 Varisource was used a t a high voltage, air-interrupted spark setting. The spectrograph was a JACO Model JA 7101 3.4-meter Ebert with a reciprocal dispersion of 5.20 A. per mm. and a grating of 15,000 lines per inch blazed for the 2800-A. region second order. The lower electrode is a carbon disk ('/z inch diameter, inch wide) of high purity graphite and the graphite upper electrode is 1/4 inch in diameter and 21/2inches in length with a sparking surface having a 120' included angle tip. The carbon disk electrode is attached to a JACO Model 19390 rotating disk assembly which provides a constant rotation of the tantalum spindle of 10 r.p.m. Sample solutions are contained in a glazed porcelain combustion boat 55 mm. long, 9 mm. wide, and 8 mm. high. The spectra are recaorded on a Kodak SA1 plate (4 by 10 inches). Magnesium Standard Solution: 0,100 mg. of Mg per ml.; dissolve 100 mg. of pure magnesium metal in 50 ml. of 1:9 hydrochloric acid and dilute to 1 liter with water. Electrolytic Nickel. Sodium Tungstate Solution (NazW04. 2H20): 10 mg. of W per ml.; transfer 1.7945 grams of salt to a 250-ml. beaker and dissolve in 50 ml. of water, transfer to a 100-ml. volumetric flask and dilute to 100 ml. with distilled water. Beryllium Standard Solution: 0.100 mg. of Be per ml.; dissolve 0.100 gram of high purity beryllium metal in 50 ml. of 1: 9 hydrochloric acid and dilute to 1 liter. Exercise caution when Lvorking with beryllium metal. Zirconium Standard Solution: 0.100 mg. of Zr per ml.; dissolve 0.3533 gram of ZrOCl? 8H20 (c.P.) in 50 ml. of 1:9 hydrochloric acid and dilute to 1 liter with distilled water. The purity of the salt was checked by removing aliquots for gravimetric analysis with cupferron. Siobium Standard Solution: 0.100 mg. of Nb per ml.; dissolve 0,100 gram of high purity niobium metal in 15 ml. of 48y0 hydrofluoric acid and 5 ml. of concentrated nitric acid in a platinum dish, transfer to a I-liter volumetric flask, dilute to volume with distilled water, and store in a polyethylene bottle. SILVER STAKDARD SOLUTION:0.01 mg. of Ag per ml.; dissolve 0.1575 gram of AgiXOa (c.P.) in 5.0 ml. of 1:1 nitric acid and dilute to 1 liter. Pipet 10 ml. from this solution into a 100-ml. volumetric flask, and dilute to volume with distilled water. Yttrium Standard Solution: 1.000 mg. of Y per ml.; dissolve a 1.000-gram piece of high purity yttrium metal in 1:9
1.o
Table 11.
of HCI-HN03 on Spectral Response of Beryllium in Iron Volume of HC1 (MI. of %T Intensity 1.19 SP. gr.) Be 3131.07 Fe 3020.49 Ratio 15.0 57.0 72.0 1.35 15.0 55.0 70.0 1.35 15.0 54.0 69.0 1.34 15.0 63.0 76.0 1.34 15.0 58.2 71 .O I .30
Effect
Spectrographic Settings
Discharge voltage, volts (approximately) Ca acitance, pf. Incfuctance, ph. Secondary resistance, ohm Radio-frequency - current, amperes Discharges per half cycle Spectral region, A. Slit width. microns Slit height, mm. Spark preburn period, see. Spark exposure period, sec. Analytical gap, mm. Auxiliary gap, mm.
13,000 0.005 40 Residual 5 3 2800-3400 30
2.5 10 15 3 4
hydrochloric acid and dilute to 1 liter with distilled water. Potassium Dichromate Solution: 50 mg. of Cr per ml.; dissolve 14.1425 grams of K2Crz07 (c.P.) in 50 ml. of distilled water and dilute to 100 ml. EXPERIMENTAL PROCEDURE
Iron Base Alloy Acids. Hydrochloric acid is the principal solvent used for the dissolution of iron base alloys. Varying amounts of nitric acid ranging from a few drops t o nearly 1:1 by volume are used for oxidation purposes in conjunction with hydrochloric acid. Although the work of Baer and Hodge ( 2 ) indicated little effect on the spectral response for fairly wide concentrations of the individual acids, the effect of varying mixtures of the two acids was unknown. As beryllium was one of the first elements investigated by the rotating disk technique in our study, its spectral response in different hydrochloric-nitric acid mistures was examined. Table I indicates the results using 1gram of iron and 1mg. of beryllium (0.10% beryllium) in a total volume of 100 ml. The spectro-
Table Ill.
Volume of H3P04 (Ml. of
1.72 sp. gr.) 3.0
4.0 5.0 10.0
Effect of
ii3P04
Volume of HNOi (ml. of 1.42 SP. gr.) 15.0 15.0 15.0 15.0
graphic settings listed in Table I1 were used. Nickel Base Alloy Acids. I n the case of nickel base alloys, nitric acid is the most rapid solvent for acid dissolution. Phosphoric acid is often used with nitric acid t o retain alloying elements such as molybdenum and tungsten in solution as phospho complexes. Table I11 indicates the spectral results obtained using magnesium in the presence of varying ratios of nitric and phosphoric acids. The data are for 0.15 mg. of magnesium (0.037, Mg) in solution with 480 mg. of nickel and 20 mg. of tungsten a t a total volume of 50 ml. The spectrographic settings of Table I1 were used except with a radio-frequency current of 10 amperes and preburn of 5 seconds. Spectrographic Conditions. The excitation and exposure conditions used for the determinations in t h e experimental and analytical procedures are listed in Table 11. Exceptions for each element are given in the separate procedures for the elements in Table IV. ANALYTICAL PROCEDURE
Chemical Preparation of Standards. A weight of analytical element-free matrix material is transferred in accord with Table IV to six 250-ml. beakers. Often the matrix sample can be obtained from the melting department just prior to addition of the desired constituent. Also it is easy to prepare a synthetic matrix by direct weighing because of the recent availability of high purity metal powders. Aliquots 1Thich cover the desired range of the element are transferred to five of the beakers containing the matrix material. The sixth beaker serves as a blank. The beakers are placed on a hot
on Spectral Response of Magnesium in Nickel
Mg 2852.13 42.6 63.6 75.0
%T
Ni 2821.29
Intensity Ratio
41.2 -~
0.98
62.5 74.0 91 .o
0.99 0.98 0.96
90.0
VOL. 34,
~
NO. 3 , M A R C H 1962
c
385
Table IV.
Element Be
Range 0.05-0.25
Be
Same aa above
Nb
0.10-1
Nb
Same aa above
.oo
0.01-0.10
Summary of Chemical and Spectrographic Analytical Conditions
Matrix Fe:50 Al, Nil Co, Zr :50 Fe:95 Cu, Cr, Ni, Mo:5 Fe :68 Cr, Nil Mn:30 Fe :93 Cr, Mol W:5 Fe:95 Cu, Cr, Mo:4
Weight (Grams) 0.100 0.100
0.100
0.100
.oo
0.50-2.00
Fe :68 Cr :30
1
Mg
0.005-0.15
0.500
Zr
0.01-0.12
Ni :92 Cr, W:6 Ni :76 Si, Fe, Cr :22
Zr
Same rn above
Ni : 76 Co, Cr, W:22
1.00
386 *
ANALYTICAL CHEMISTRY
30 ml. 1 : l HC1 2 ml. " O B 0.5 ml. H F 5 ml. Hz02 Same as above
1.oo
100 100
100 100
0.100
Y
plate and the samples dissolved in the appropriate solvent mixture of Table IV. Hydrofluoric acid and hydrogen peroxide are usually added when solution is nearly complete. The beakers are removed from the hot plate, cooled, and the solutions transferred to appropriate volumetric flasks. Solutions are diluted to volume with distilled water, mixed, and set aside for analysis. Sample Chemical Preparation. Samples are dissolved and prepared in exactly the same way as the matrix material for the standards except the addition of standard metal solution is omitted. BERYLLIUM, This metal is usually present in iron base alloys in such a form that it easily dissolves in mixtures of hydrochloric and nitric acids. NIOBIUM. Niobium may be partially present in steel as a stable carbide which requires hydrofluoric acid for complete solution in acid media. Hydrogen peroxide also aids in solution of niobium carbide through complex formation and was used with hydrofluoric acid in this Rork because it does not attack glassware. SILVER. Silver-bearing steels had to be dissolved without the use of hydrochloric acid because of the danger of loss through precipitation as silver chloride. Nitric acid (1: 1) was a satisfactory solvent in the low alloy composition examined. More highly alloyed compositions would require the use of hydrofluoric acid, as it is the only halide acid which forms soluble silver salts. Dilute perchloric acid with hydrogen peroxide was examined as a solvent but results were erratic. YTTRIUM.The yttrium-bearing stainless composition examined was soluble in the hydrochloric-nitric acid mixture.
Solvent 30 ml. 1 : l HC1 2 ml. HNOI Same as above
Volumetric Dilution (bfl.) 100
30 ml. 1: 1 HC1 5 ml. "03 5-ml. Hz02 20 ml. 1: 1 HNOI 3 ml. HsPO, 30 ml. 1:5 HNOI 1.O ml. H F
100
Same as above
100
However, dissolution could not be accelerated with hydrofluoric acid because of the insolubility of yttrium fluoride in acid solution. MAGNESIUM.As most alloy compositions which could serve as matrices are melted in refractories containing magnesia, it is difficult to obtain a matrix material sufficiently free of magnesium. Therefore, a matrix was synthesized by direct weighing of electrolytic nickel with sodium tungstate and potassium dichromate added for the chromium and tungsten present in the sample alloy. Phosphoric acid was used to retain tungsten in solution as the phosphotungstate complex. However, as shown in Table 111,phosphoric acid should be dispensed from a buret because of the critical depressing effect on the entire spectrum. ZIRCONIUM.Tungsten is often present as an alloying element with zirconium in nickel base alloys. To prevent the hydrolysis of tungsten and the separation of zirconium from solution with it, possibly as zirconium tungstate, hydrofluoric acid is used to form the soluble fluoro complexes of tungsten and zirconium. Hydrofluoric acid will also ensure the dissolution of any zirconium carbide or nitride in the alloy. Phosphoric acid is not used to retain tungsten in solution in this instance because of the possible formation of insoluble zirconium phosphate. Spectrographic Analysis. A portion of the solution from the volumetric flask is transferred to the porcelain boat and the lower electrode disk on the shaft of the rotating assembly submerged in the solution. The analytical gap between the upper electrode and the rotating-disk is 3 mm. The spectra are produced and re-
50 100
Spectrographic Data Be 3131.07/Fe 3020.49 20-see. exposure Same aa Table I11 Be 3131.07/Fe 3020.49 Nb 3194.98/Fe 3083.74 5-sec. preburn, 10 amp. Nb 3130/Fe 3094.9 5-sec. preburn, 10 amp. Ag 3280.68/Fe 3280.26 10-amp., 5-sec. preburn 30-sec. exposure Y 3195.61/Fe 3183.11 7.5-amp., 5-sec. preburn 10-sec. exposure Mg 2852.13/X 2821.29 10 amp., 5-sec. preburn Zr 3393.19/Ni 3391 10-amp., 20-sec. preburn 40-sec. exposure Same aa above
corded according to the conditions in Table I1 with elemental changes being made in accord with Table IV. Duplicate exposures are made for each sample. Fresh electrodes and solutions are used for each separate exposure. The emulsion is processed and calibrated in accordance with recommended practices (1). The transmittance of the analytical lines and the internal standard lines are converted t o log intensity ratios using the emulsion calibration curve. Analytical curves for the standards are prepared by plotting the log intensity ratio us. the log concentration for each element, For samples, the intensity ratio is obtained and referred to the analytical curve to obtain the concentration. PRECISION A N D ACCURACY
Table V shows some of the results obtained using the rotating-disk technique. Chemical methods (underlined results) were used for cross-checking the spectrographic results of Table V for all elements except beryllium and magnesium. An attempt was made for 2 weeks to develop a chemical separation of beryllium from the large quantities of aluminum, iron, and zirconium present in the first sample but was abandoned when the spectrographic method was developed in one day with synthetic standards. Similarly for magnesium, the presence of tungsten in the sample prevented success with the chemical method because of suspected occlusion of part of the magnesium when the tungsten was separated by hydrolysis. Chemical values for niobium were ob-
tained using the hydroquinone spectrophotometric procedure (6). Silver was determined chemically (4) by precipitation and weighing as silver chloride. Yttrium was determined by precipitation as a fluoride and ignition to the oxyfluoride (7) following prior separation of chromium with perchloric acid as chromyl chloride and iron by electrolysis with a mercury cathode. Zirconium was determined by a method using the pyrocatechol procedure developed by Raber (IO). While most of the data are given as typical results obtained, a statistical analysis was made on 16 beryllium determinations a t the 0.17070 concentration level and 20 magnesium determination a t the o.o?5y0 level. The relative standard deviation was 1.98y0 for beryllium and 2.20y0 for magnesium. This kind of precision can be considered as good or better than most conventional met chemical procedures.
_____
Table V.
~
~~
Rotating-Disk Results for Iron and Nickel Base Alloys
Sample Composition,a yo Al,25 Co, 15 Xi, 1 Zr, 50 Fe, 0.120Be5 0.2 Cu, 0.5 Mo,0.5 Ni, 2 Cr, 95 Fe, 0.070 Be* 0.2 Cu, 0.5 Mo,0.5 Ni, 2 Cr, 95 Fe, 0.120 Be5 0.2 Cu,0.5Mo,0.5 Si, 2 Cr, 95 Fe,0.170Be5 0.2 Cu, 0.5310,0.5 Ni,2 Cr, 95 Fe, 0.230 Be5
Analytical Results, yo
0.120,0.118,0.115,0.116 Be 0.070,0.068,0.070, 0.071Be 0.112, 0.110,0.110,0.112Be 0.150,0.150,0.145,0.150Be 0.230,0.225,0.215,0.225 Be (~.B.S.123B)18Cr,11Ki,0.18TV,0.20Ta,0.006 0.72,0.75,0.73,0.73Sb Ti, 68 Fe, 0.75 Kb 0.2 Mo, 1.0W,3 Cr, 93 Fe, 0.15 S b 0.13,0.14,0.15 Nb 0.2 M o , 1.0 W,3 Cr, 93 Fe,=b 0.24,0.24,0.23Nb 0.2Cu, 0.5 Mo,2 Cr, 95 Fe, 0.054 A g 0.052,0.054,0.056,0.056 Ag (G.E. 446)30 Cr, 68 Fe, 0.62Y 0.58, 0.60 Y (G.E. 446) 30 Cr, 68 Fe, 0.75 Y 0.74,0.74,0 . 7 7 Y (G.E. 446) 30 Cr, 68 Fe, l m 1.54,1.58 Y 1 W, 5 Cr, 92 Ni, 0.075 IClgb0.076,0.072,0.072,0.075blg 1 W,5 Cr, 92 Xi, 0.140Mg5 0.131,0.131,0.132,0.135 Mg (N.B.S. 169) 20 Cr, 0.5 Fe, 1.5Si, 76 Ni, 0.042 Zr 0.040,0.041,0.039,0.039 Zr 4 W,5 Co, 13 Cr, 76 Ni, 0.064Zr 0.066,0.067,0.067 Zr
8
a
b
Com ositions are approximate, except where underlined. Syntgetic standards submitted as unknowns.
DISCUSSION
The data of Table I indicate that the of the beryllium and iron lines changed very slightly while the intensity ratio remained constant over a wide range of nitric acid concentrations. Hence the dispensing of the respective acids (hydrochloric and nitric) does not have to be carefully controlled as different acid mixtures will not affect the spectral response. Because of the high sensitivity of beryllium in solution a t the 0.10% level, the 3020.49-principal line of iron had to be used as an internal standard for the 3131-line of beryllium. Even so, an excellent analytical calibration curve was obtained. I n contrast to the negligible effect of varying ratios of nitric or hydrochloric acids, the data of Table I11 indicated a pronounced effect by phosphoric acid on spectral response. Hence, if phosphoric acid is necessary in a procedure, it should be dispensed from a buret because of its critical depressing effect on the entire spectrum. hlthough the yoT data of Table I11 varied widely, the intensity ratio remained constant. As noted it was not possible to develop chemical procedures for beryllium and magnesium in a reasonable period of time. The complexity of the alloy compositions precluded this. However, the excellent data from the synthetic standards on beryllium and magnesium indicate two major advantages of the spectrographic solution technique. First, the elimination of previously analyzed chemical standards from the usual spectrographic viewpoint and second, the no longer required need to develop urgently a chemical procedure from the \vet chemical viewpoint. The data on niobium indicated another advantage of the spectrographic solution technique over chemical pro-
7, T
cedures. The spectrophotometric procedures for chemically similar elements such as niobium, tantalum, titanium, and tungsten often have absorption bands in the visible range which overlap and hence these procedures fail unless a lengthy chemical separation procedure precedes the spectrophotometric finish. While it is possible for two elements to have spectrographic lines falling a t nearly the same wavelength and not capable of resolution, usually other principal or secondary lines can be chosen. Hence, unless it is a matter of spectral sensitivity, alloys containing mixtures of elements such as niobium, tantalum, titanium, and tungsten do not require a chemical separation and can be analyzed easily by the spectrographic solution technique. The data for silver and yttrium offer a slightly different aspect of the advantage of the spectrographic solution technique over conventional chemical procedures. There are no specific chemical procedures for either silver or yttrium. Elements such as lead and mercury can, if present, interfere with the silver determination. The rare earth elements and thorium likewise interfere with the chemical method for yttrium. Fortunately, in the alloys examined, the chemical interferences were not present but, even if they had been, the spectrographic solution method would still have been applicable. The chemical determination of zirconium offers a particular challenge when the zirconium content is low and tungsten is present. The zirconium will separate into two fractions, soluble and insoluble. The soluble fraction will remain in solution while the insoluble fraction will result from inclusion of part of the zirconium with hydrolyzed tungsten.
As a result, two separate chemical determinations have to be made for total zirconium. No such difficulty occurred in the spectrographic solution procedure examined as no separations are required and both elements are easily retained in solution with hydrofluoric acid. Perchloric acid, which was not examined by Baer and Hodge, was found qualitatively in our studies to exhibit erratic results on a solution spectrum. illthough the present authors did not use a wide concentration of hydrofluoric acid (because of attack by excess on glassware), there appeared to be little effect on the spectrum for the elements examined. Hydrogen peroxide, while not an acid, also did not appear to affect the spectral response. I n general, it appears that wide changes in concentration of volatile acids such as hydrochloric, hydrofluoric, and nitric do not affect spectral response in solution while the concentration of high boiling acids such as phosphoric, perchloric, and sulfuric (examined by Baer and Hodge) must be carefully controlled. The rotating-disk technique from the few examples cited above has proved to be valuable for the rapid determination of less familiar elements in highly complex iron and nickel base matrices. The recent availability of high purity metal powders and compounds of these elements has enabled the analytical chemist to synthesize his own standards by direct weighing and dissolving in a suitable acid solvent, thus eliminating the wet standardization required for solid specimen spectrographic techniques. The only critical requirement for the spectrographer is a wet-chemical background to enable him to choose the proper solvent for each alloy combination. VOL.
34, NO. 3, MARCH 1962
e
387
LITERATURE CITED
(1) Am. SOC. Testing Materials, Philadelphia, Pa., “Methods for Emission Spectrochemical Analysis,” Deaignation E-115-59T, p. 1, and E-116-59T, p. 12 (1960). (2) Baer, W. K., Hodge, E. S., A p p l . Spectroscopy 14, No. 6, 141 (1960). (3) Feldman, Cyrus, ANAL. CHEM. 21, 1041 (1949). (4) Lundell, G. E. F., Hoffmann, J. I., Bright, H. A., “Applied Inorganic An-
p,” second ed. revised pp. 205-7, iley, New York, 1953.
(5) Margoshes, M., Chem. Ens. N e w s 39, . No. 38; 94 (September 18, 1961). (6) McKaveney, J. P., ANAL.CHEM.33,
744 (1961). (7) McKaveney, J. P., Crucible Steel Co. of America, Research Project 110, Research Book No. 20, p. 99, August 5,
----.
1Q59
(8) Pagliassotti, J. P., ANAL.CHEM.28, 1774 (1956). (9) Pagliassotti, J. P., Porsche, F. W.,
Zbid., 23, 198 (1981). (10) Raber, W. J., Crucible Steel Co. of America, Final Report Test 6175, pp. __ 3-7 (September 1, i96l). (11) Zink, T. H., A p p l . Spectroscopy 13, No. 4, 94 (1959).
RECEIVEDfor review May 24, 1961. Accepted January 5, 1962. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., March 3, 1961.
Continuous Analysis by Measurement of the Rate of Enzyme Catalyzed Reactions Glucose Determination W. J. BLAEDEL and G. P. HICKS Chemistry Departmenf, University of Wisconsin, Madison, Wis.
b An instrument is described which permits the continuous measurement of the rates of many enzyme catalyzed reactions. The instrument is designed specifically for the routine assay of enzymes or substrates. Illustrative application is made to the determination of glucose by the glucose oxidaseperoxidase coupled system. Glucose in aqueous solutions up to 60 p.p.m. may b e determined with a standard deviation of 1 p.p.m. Samples may b e analyzed a t the rate of 15 per hour, with a readout time of 4 minutes per sample. Faster analyses are possible. Glucose is also determined in 0.2-ml. samples of blood plasma. Sample and reagent manipulation b y the technician is kept to a minimum through use of a flowing system. Calibration is performed with a standard sample to permit direct readout on unknown samples, with no calculation.
E
are extremely useful in clinical and analytical chemistry ( I ) . Since the analytical and clinical applications are increasing rapidly, the need for instrumentation is apparent. Some automated procedures have been developed, which minimize effort and manipulation on the part of the technician ( 2 , 3 ) . Other instruments have been developcd which automatically measure the rate of an enzyme reaction by measuring the time required for the systrm to change from one particular composition (measured by absorbance or electrode potential) t o another (4, 6). Since the change is small, and since measurements are made 388
very near initial velocity, the instrument readout is inversely proportional to the initial rate, and therefore also inversely proportional to the concentration of sought-for substance. In this paper, an instrument is presented which gives a continuous record of the rate of a chemical reaction, provided that it is accompanied by a change in absorbance. The principle of the method is shown in Figure 1. A sample stream containan ing the sought-for substance-Le., enzyme or substrate-flows at a constant rate t o meet and mix with a reagent stream also flowing at a constant rate. The reagent stream contains fixed concentrations of all of the other components necessary for the reaction,
NZYME-CATALYZED REACTIONS
a
ANALYTICAL CHEMISTRY
UPSTREAM DELAY UPSTREAM CELL INTERCELL DELAY DOWNSTREAM CELL WASTE Figure 1. Outline of continuous measurement of reaction rates
except the one in the sample stream. As the reaction occurs, the absorbance of the resultant stream changes continuously as it flows a\?ay from the mixing point. Since the flow rate is constant, the time interval betnecn the two cells is fixed, and the steady state absorbance difference between the photometer cells is proportional to the reaction rate. The absorbance difference is measured with a sensitive differential recording filter photometer. The delay betwecn the mixing point and the upstream cell is made long enough to overcome any nonlinear induction periods, if such exist. The intercell delay is made long enough to obtain an accurately measurable absorbance difference for the range of concentrations to be determined. Several benefits and advantages over existing methods are inherent in the scheme of Figure 1 and are summarized here : Through use of a sensitive differential photometer] the time interval over which the rate is measured can be made very small. Thirty seconds is typical for the glucose determination. The extent of reaction is kept small, and the measured rate is virtually an initial rate, with very small changes in reactant concentrations. Such circumstances give the best chance of achieving direct proportionality between the measured absorbance change and the concentration of the sought-for substance. bleasurement of the absorbance difference largely eliminates errors due to absorbing, but nonreactive, impurities in the sample and reagents. Separate blanks for each sample are not required.
