343
V O L U M E 2 5 , NO. 2, F E B R U A R Y 1 9 5 3 nitrogen per 50 ml. permits the successful determination of nitrites in samples containing from 3 to 10 mg. of nitrate nitrogen. COYCLUSIONS
An ultraviolet absorptiometric method is described for the determination of nitrites by measuring diazotized 4-aminobenzenesulfonic acid versus a reagent blank. The method does not approach the sensitivity of the Griess method for nitrites since the molar absorptivity of the Griess method is 41,000 and that of the described method is 15,500. The new method does have the possible advantage of applicability a t higher nitrite doncentrations, where the uncertain precipitation of the relatively insoluble azo d!-e may present a problem. Another advantage is the rapidity with which sample determinations can be made. The time involved is considerably shorter than that required for the Griess method in that there is no coupling reaction. As is obvious from the absorbance maximum used, the sample should be relatively free from organic matter. RECOMMENDED PROCEDURE
Sample. Weigh or measure by volume an amount of the sample containing from 0.003 to 0.05 mg. of nitrite nitrogen. Any
interfering ions which may be present should be made to conform to the permissible concentrations given in Table 11. Desired Constituent. Place the sample in a 50-ml. volumetric flask and adjust the p H to 1.4; for previously neutralized unbuffered samples, 1 ml. of the hydrochloric acid solution is sufficient. To the acidified sample add 1.0 ml. of 4-aminobenzenesulfonic acid reagent, dilute to volume, and mix the contents. After 3 minutes, and less than 15 minutes, measure the absorbance of the diazo compound a t 270 mp in 1-cm. quartz cells versus a reagent blank. LITERATURE CITED (1) American Public Health Association, Xew York, “Standard Methods for the Examination of X-ater and Sewage,’’ pp. 71, 121, 1946. (2) Griess, P., Be?., 12, 427 (1879). (3) . . Kolthoff, I. M.. and Sandell. E. B “Textbook of Quantitative Inorganic iinalysis,” p. 603, S e w Tork, Macmillan Co., 1943. ( 4 ) Rider, B. F., thesis, Purdue University, 1944. (5) Rider, B. F., with Mellon, M. G., ISD. ENG.CHEM., ANAL.ED.,
.
18,96 (1946). RECEIVEDfor review August 4, 1952. Accepted October 22, 1952. hbstracted from a thesis presented by J. AI. Pappenhagen t o the Graduate School of Purdue University in partial fulfillment of t h e requirements for t h e degree of doctor of philosophy.
Microgram and Submicrogram Determination of Phosphate -4pplications of Sealed Tube Digestion and Capillary Cell Spectrophotometry FREDERICK L. SCHAFFER’, JEAN FONG, AND PAUL L. KIRK Dit-ision of Biochemistry, University of Calqornia Medical School, Berkeley, Calif.
CONSECTIOS it-ith the analysis of needle biopsy samples IXfrom the liver, in the study of infectious hepatitis and other
influences, it became necessary to determine microgram and submicrogram quantities of phosphorus. The tissue samples available, with a dry weight of about 1 mg., contain adequate phosphorus for microgram spectrophotometric analysis. The fractions in which the phosphorus exists, when analyzed separately require that the lower limit of analysis be reduced. Various phosphorus-containing fractions of tissue are of the greatest biochemical and possibly clinical interest, including nucleic acids, phospholipids, coenzymes, and metabolic intermediates. The method of Berenblum and Chain ( 1 ) involving extraction of the phosphomolybdic acid with isobutyl alcohol has several fundamental advantages over methods in which the molybdenum blue is developed in the aqueous phase. The medium is essentially constant in composition and the molybdenum blue appears to be in molecular solution. The method is also valuable for analysis of phosphate in colored solutions (9) and in the presence of labile phosphate compounds ( 3 , 9, 11, 12). When this procedure is employed with capillary absorption cells in the spectrophotometer, large blanks are obtained which reduce greatly the analytical range of concentration, and introduce additional uncertainties in the results. These difficulties have been overcome in the method described here by employing noctyl alcohol as the extracting agent. S o t only does octyl alcohol extract phosphomolybdic acid readily, but it apparently does not extract any appreciable quantity of molybdic acid, which is not the case with butyl alcohol. Martin and Doty (Q), who employed a mixture of isobutyl alcohol and benzene, indicated that various solvents may be applicable for the extraction of phosphomolybdic acid. The technic of extraction when the amount of sample is very 1 Lt. USNR. Office of Kava1 Research Unit S o . 1, University of California, Berkeley, Calif.
small has been approached with a number of extractor designs ( 7 ) . With amounts of phosphorus not less than 1 microgram, the extractor of Kirk and Danielson (8) is very satisfactory. In the submicrogram range, all liquid-liquid extractors for lighterthan-water solvents suffer from the necessity of unit operation and constant attention, combined with manipulative difficulties in some instances. An extraction technique for as many as eight simultaneous samples is presented here. hnother problem of importance is the method of digestion of biological samples prior to the determination of phosphorus. Digestion in a sealed tube, which has proved very satisfactory for the Kjeldahl determination of nitrogen on the microgram scale (6), may also be used for phosphate. In this paper are described procedures for analysis of phosphorus in quantities ranging from a few micrograms down to 2 my, making use of sealed tube digestion with sulfuric acid alone, extraction of phosphomolybdic acid with octyl alcohol, and final spectrophotometric measurement which in the lower ranges requires the use of capillary absorption cells. APPARATUS
Extractors. Shown in Figure 1, extractors (hIicrochemica1 Specialties Co., Berkeley, Calif.) were constructed of borosilicate glass tubing with over-all height of 90 mm., length of upper chamber of 47 mm., and outside diameter of 14 mm. The capillary bore of the large extractor, A , was 1 mm., and of the small extractor, B, 0.3 mm. The bulb capacities were 500 and 62.5 pl., respectively (to levels falling within the capillaries). The extractors were designed to fit into 15-ml. cups of the International clinical centrifuge. Centrifugal Pipets. Special pipets (Microchemical specialties Co., Berkeley, Calif.) of short length and 200 and 25 pl. capacities were constructed as shown in Figure 1. Each was provided with a plastic collar which was seated in the top of the extractor. Shaker. The edges of a hard rubber stopper were cut to form a pentagon. The stopper was then mounted on the shaft of a laboratory motor in a horizontal position.
344
ANALYTICAL CHEMISTRY
Micropipets and Volumetric Flasks. Various sizes of standard capillary transfer type pipets and micro volumetric flasks (7) were employed. Spectrophotometers. A Beckman Model DU spectrophotometer, fitted with capillary absorption oells of 5-om. light path and 2-mm. bore (7), was used with the smallest quantities of phosphorus. A Beckman Model B spectrophotometer with I-cm. Carex cells was used with larger quantities. Horizontal Amineo absorption cells with 5-cm. light path and 10-mm. outside diameter ere used n i t h intermediate quantities. Adaptors to hold these eella in the Model B speetrophotometer were machined from aluminum rad. A hole was bored along the axis to fit the body of the cells and in the top to fit the filling neck. The upper half of the adaptor was cut away along thc greater length to the top hole. The spring clip of the spectrophotometer cell carriage was used to hold the cells and adaptors in place. The assembly is shown in Figure 2. A mask, with a squatre hole, cut from heavy black paper to limit the height of the light beam to about 7 mm. was aligned and fastened to the inside of t,he cell compartment. It was unnecessary to rcrnovn the mask u-hile using thc I-cm. Cores cells.
i
L5 cm
Ammonium molybdate, 5 % solutions, both aqueous and acid, were made by dissolving 2 grams of C.P. grade ammonium molybdate in 40-ml. quantities of water and 2 N sulfuric acid, respectively. These reagents were stored in polyethylene bottles and were stable for many months, though some hatches gave poor results. Alcohols. Esstman practical n-octyl alcohol; C.P. grade n-butyl alcohol; and 95% ethyl alcohol were used. These solvents were redistilled and stored over copper pellets. This
of concentraxed hydrochloric acid. It was stored in a brown
Hydrochloric acid, 1 N . C.P. grade hydrochloric acid Was diluted with 11 volumes of water to a concentration of about 1N. PROCEDURE
One Tenth to Five Micrograms. The sample was transferred to the upper ohamber of a large extractor, A , Figure 1, and sufficient water NLXadded to bring the volume to 150 PI. To this were added 50 PI. of molybdate reagent and the mixture was centrifuged into the lower bulb. With acidic samples (containing about 100 p eq. of acid) the aqueous reagent was used, and conversely the acidic reagent was used with neutral samples. Next, 200 pl. of ootyl alcohol were added by means of the special centrifugal pipet or with an ordinary transfer pipet. Following centrifugation of the oetyl alcohol, the phosphomolybdic aoid was extracted by holding the lower bulb of the extractor against the rotating shaker for a period of 15 t o 30 seconds. To \rash and displace the alcohol phase sufficient 1 N hydrochloric acid (approximetely 300 PI.) to bkng the interface near the upper end of the capillary was added and centrifuged dawn. With s capillary pipet, the octyl alcohol N&S transferred to a glass-stoppered 4-ml. volumetric flask to which 2 PI.of dilute stannous chloride had been added. The upper chamber of the extractor N ~ S rinsed severd times with butyl alcohol, the rinsings being transferred to the flask. The flask was shaken to develop the blue color, and about 200 PI.of ethyl alcohol were added to assure solution of any aqueous droplets present. The volume was then adjusted with butyl alcohol. The optical density was read a t 725 mp with butyl alcohol in the reference cell. Tho absorption cells of I-cm. light path were used with quantities of 1 to 5 micrograms of phosphorus, while the 5-cm. Aminco cells were used with 0.1- to 1.0-eamma auantities.
Apparatus for Cleaning Extractors. Xetal clips to hold the extractors were fastened t.o 8. wooden block, behind which was mounted a manifold connectod to an evacuated waste bottle. Glass capillaries to reach into the bulbs of the extractors were connected to the manifold rjith 10 to 15-cm. lengths of small flexible plastic tubing (Transflex). For easy replacement when broken, large numbers of capillaries were prepared a t one time from ordinary tubing. A short constriction to fit the plastic tubing was pulled in each where the larger tubing narromed dowr t o the cqiilary. REAGENTS
Standard phosphate solution containing 1 mg. of phosphorus per ml. was prepared by dissolving 2.194 grams of primary potassium phosphate in water and diluting to 500 ml. Appropriate dilutions were made for the ranges used.
aneleor biholdinait wainst-the shaker. ~.. .. .~ ,.~., . .
~~~
~
~~~~
~
~
T h e e x t r a c t a r b then
~~
a capillary absorption cell for measuremedt
of the color.
345
V O L U M E 25, NO. 2, F E B R U A R Y 1 9 5 3 The importance of proper positioning and alignment of the capillary absorption cells has been studied, and certain modifications have been made (IO). A mask with a 1.5-mm. hole was fastened t o the inside of the cell compartment so that the same portion of the light beam was always used. The best match for .a pair of cells, as determined by light transmission, was found by reversing their direction and exchanging them in the holders. With the cells filled with pure solvent, horizontal, vertical, and rotational (about the axis of the cell) adjustments were made to obtain maximum transmittance with each cell. I n use, the cells were always returned to the same positions, including the detail of pushing them t o the right until the shoulders of the cells were against the holders. To reproduce the rotational positions, marks made on the cells were aligned with pointers fastened to the carriage, or alternatively with the tops of the V notches of the cell holders. I n the latter case small mirrors mounted on the carriage were used to aid observation. After all adjustments were made, one cell was selected as a reference and the optical density of the other measured, thus giving a correction to be applied to the color measurements. This correction was checked daily or oftener. Two Thousandths to Eight Hundredths Microgram. The small extractor, B, Figure l j was used Ivith the following quantities: sample and rinse 20 p l . ; molyhdate reagent, 5 pl.; octyl alcohol, 25 MI.; hydrochloric acid, 45 p l . Theextraction procedure was carried out as described for the large extractors, followed by reduction with 1 p l . of the dilute stannous chloride reagent in the upper chamber aa described above. h 200-p1. pipet Tvas used for dilution t o volume. About 15 pl. of ethyl alcohol were first drawn into the pipet, followxl bv the octyl alcohol extract and several butyl alcohol rinses. After adjusting to the mark, the excess hutyl alcohol was aiped from the pipet tip and the contents were mixed by exlielling into a small test tube and "puniping" them into and out of the pipet. The same pipet n-as used for transfer to the capillary absorption cell for color measuremcnt. Cleaning of Extractors. The capillaries of the cleaning apparatus were inserted into the bulbs of the extractors and suction \vas applied. Successive washes of ethyl alcohol, sodium hydroxide (ea. 15yo), wat,er, nitric acid (ea. 3 N ) , water three times, and ethyl alcohol were added to the upper chamber from >\-ash bott,les and drawn off through the capillaries. The extractors were dried in a n oven or \Yere emptied completely by inverting on a clean piece of tissue and centrifuging. Digestion. For the determination of total phosphate in biological samples, digestion was performed with sulfuric acid in sealed tubes a t 450" t.o 470" C. for 30 minutes as described elsewhere ( 6 ) . Liquid samples were dried in the digestion tubes over sodium hydroxide pellets a t 90" to 100" C. after the addition of the sulfuric acid. The quantity of sulfuric acid employed depended upon the amount of organic material present and the extractor used. ( I t yay be calculated that about 5 t o 6 pl. of sulfuric acid are used in the oxidation of 1 nig. of proteinaceous materinl, while 8 to 9 pl. are required for l rng. of phospholipide. .4n ample excess should bc provided for complete digestion.) With samples for analysis in the large extractors, 10 p l . of sulfuric acid were generally used for each digestion, each digest Tvas diluted to 200 pl., and a 75-pl. aliquot was taken for analysis. \Then diluting the sulfuric acid digest t o a given volume in a micropipet, or volumetric flask, t h e precaution of thorough mixing and cooling n-:is observed before final adjustment to the mark. \Vith s:mples contailling millimic~ograni ainount,s of phosphorus in volumes not rsccetiiiig 20 to 25 pl,. digestion tubes of 4 mm. outside diameter were eniployed i n t e x d of the usual 7mm. tubes. I n this case, 1 p l , of :i digestion mixture of 1 part. of sulfuric acid and 2 parte of watei' (I)!. volume) was used. Thus thc proper quantit,y of :icitl for the analysi.~\\'asobtained without taking an aliquot of the digest. The digwts were transferred to the small extractors by centrifugation. The top of each small digestion tube v a s discarded and :tplirosimat~ely1/3 of a 2O-pl. drop of water W:LS alloi\-ed t,o run don-n one side of the tube from a pipet, diluting the acid digeat. The position of a mark on the tube was noted, and it was then inverted in the upper chamber of the extractor and centrifuged. The digestion tube \vas rinsed and emptied t x o more times in a similar manner, rotating the tube 120" from each previous position. After opening the digestion tubes, heating t'he digest to drive off acid gases ( A ) {vas omitted unless nitrogen was also to be determined. After centrifugation of the sample and rinsing into the lower bulb, the extractor was placed in a boiling water bat>hfor a fcw minut,es to hydrolyze pp-ophosphoric to orthophosphoric :tcicl. .After cooling, the usual procedwe \vas followed. Use of Desicote. Pipets used for aqueous solutions were treated with the water repellant DePicote ( 4 , 6 ) , to obtain complete or nearly complete delivery. Self-filling, self-adjusting pipets (1 and 2 ul.) were treated only on the outside of the tips
einre the action of these pipets depends upon wetting of the capillary walh. RESULTS
Unsuccessful attempts were made to extract phmphomolybdic acid by a countercurrent method l)>- centrifugation through a c;tpillary. However extraction b>- mechanical shaking, or by hand for a longer period of timr, : i d separation in the centrifuge irere very effective. Several 4-,5-, 6-, and 8-carbon alcohols were tested and all \vere effective for the extraction of phosphomolybdic a(:id. .Us0 effective 11-as a 1 to 1 mixture of isobutyl alcohol and benzene :IS used by llartin and Doty (!/). The IoTvest blanks, indicating ininimum extraction of uncombined molybdic acid, were obtained with n-octyl alcohol. The absorption maximum of molybtlcnum blue in octyl alcohol was found to be 725 m , ~as , in butyl dcohol. Some of the solvents tested, including octyl alcohol, gave emulsions that separated very slowly under the force of gravity but were easily broken in the centrifuge. Hydrochloric acid was substituted for sulfuric acid for washing the alcohol phase because of the lower density of hydrochloric acid solutions. T h e n 1 sulfuric acid was used, reducible molybdic acid remained a t the interface owing to mixing with the aqueous phase in the extractor bulb. I n displacing the a1coho], hydrochloric acid tended to form a layer on top of the ammonium molybdate-sulfuric acid mixture in the bulb. Csing dilute stannous chloride (0.270 in 1 N sulfuric acid) as in the original method of Berenblum and Chain, i t &'as found that reduction to molybdenum blue in butyl alcohol was complete t)y displacement through a capillary. However with ortyl alcohol, shaking t o mix the phases was necessary. Mixing of :t very small volume of a more concentrated reagent was technically more practical. Also, the technique of reducing to molybdenum blue in the volumetric flask (9) was easily adapted by adding a emall amount of ethyl alcohol. Use of the concentrated re:igent in concentrated hydrochloric acid gave erratic results because of the high acidity. The best results were obtained by dilution of the concentrated reagent with 4 volumes of water. The maximum amount determinable by the method as described was about' 5 to 7 y of phosphorus. For larger quantities it was necessary t o use more than 2 p1. of the stannous chloride reagent for maximum color. Beer's l a x apparently did not hold for quantities of phosphorus greater than 0 to 107 wheri dilut,ed to 4 ml. Hoxever, a t least 157 of phosphorus can be extracted in 200 pl. of octyl alcohol, and may be determined with proper quantity of reducing agent and dilution. By using an esact quantity of octyl alcohol, an aliquot could be taken for measurement after color development. The total content of a capillary pipet may be delivered by centrifugation with no significant error ( 6 ) . This principle \vas applied by using a short pipet fitting into the upper chamber of the extractor. It \vas noted that the volume of the alcohol phme during extraction incremxl because of diff erent mutual solubilities of the phascs. The results n-ere reprodurihl(~and a satisfactoi.>. standard curve was prepared. K i t h the smallest quantities of phosphate the small extractor gave better results than the large extractor because of Ion-er Iilanks and less variability. This extractor was designed to be used with the photomultiplier tube adaptation of capillary tube spectrophotometry of Craig, B ~ r t e l l and , Kirk ( g ) , b u t has not as yet been tested with that instrument. I n the preparation of samples for digestion, preliminary esperinients indicated that complete wetting of the samples with suli'uric acid was necessary. Failure t o wet the sample resulted in a xhite deposit (presumablj. salts fused onto the glass) after digestion. This deposit \vas not removed by ordinary rinsing or by heating n-ith dilute acid. Recoveries as low as 2?iyO accompanied this effect. Aft,er discovery of this effect, the sulfuric acid was added prior to removing water from the samples to ensure complete wetting with the acid. Small quantities of water
ANALYTICAL CHEMISTRY
346
Table I.
Analysis of an Extract of Dried Lamb’s LiverD
Form of Phosphate Inorganic Totalb
Total extract Diluted 1: 100
Sample Size, PI. 75 75 0 40 40 40 60 60 60 0 0
5 5 0
PAdded, y
Average No. of P found, Samples y
0
0.0020 0,0020
y
1
1.76 3.83 2.03 0.79 1.56 2.33 1,26 1.95 2.77 0.76 1.46
2 2 2
0 . 0 0 2 5 0.00014 0.0046 0.00007 0.0019 0,00014
0 2.00 2.00 0 2.00 4.00 0 2.00
4.00 2.00 4.00
Std. Dev.,
1
0.018 0.037 0.000 0.026 0.030
.....
..... .....
.....
P in Extract, ,y/All. 23.5 24.4
...
52.8 54.0 55.3 56.1 53.4 56.6
... ...
51 52
.
Approvimately 42 mg. of powder extracted with 3 ml. of 5% trichloroacetic acid. b Digest diluted t o 200 PI.; 75 pl. taken for analysis.
recovery of phosphate due toinhibition of the extraction at highsalt concentrations. Concentrations of sodium sulfate less than 0.15 M had no effectupon the extraction. The third alternative, digesting with the amount of acid required in the analytical procedure and total transfer of the digest to the extractor, was employed with the millimicrogram range samples cited in Table I. The extraction of phosphomolybdic acid is quantitative over a broad range of acid concentration. Berenblum and Chain ( 1 ) found complete extraction with sulfuric acid concentrations from 0.05 to 2.0 N . Approximately the same range was found to apply with the procedure described here. If silicate is likely to be present, lo~vacid concentrations should be avoided to obviate the extraction of silicomolybdic acid. DISCUSSION
a
remaining in the acid had no apparent effect upon the digestion. Unsatisfactory results were obtained when only 1 pl. of sulfuric acid was used for digestion in the regular tubes, and with samples in volumes of water greater than 20 to 25 pl. in the small digestion tubes. Apparently this was due to the failure of the small volume of acid to cover sufficiently the sample spread over a relatively large area. The top portion of several regular digestion tubes, used for digestion of microgram quantities of standard phosphate, were tested for the presence of phosphate. Amounts ranging from 0.2 to 2.5y0 of the total sample were found, indicating the necessity of rinsing the upper portion. Assuming similar percentages would be lost by discarding the tops of the small tubes, the loss in this case wTould be of doubtful significance. Extraordinary care must be exercised in handling the smallest samples to assure that the water, reagents, and glassware used are free of contamination. Molybdenum blue was slowly developed when acid molybdate solutions were in contact with cellulose materials, presumably due to hydrolysis of the cellulose to reducing sugars, Therefore, contamination by cellulose fibers was avoided. Calibration curves were drawn by the method of least squares for known phosphate solutions in the three ranges discussed. In all instances straight line relations were obtained between the optical density and concentration. Capillary cells 5 cm. in length and containing 0.2 ml. covered the range 0.002 to 0 . 0 8 ~ per 0.2 ml.; horizontal cells 5 cm. in length and containing about 3 ml. were used in the range 0.1 to 1.0y per 4 ml.; and Corex cells were employed in the range 1 to 57 per 4 ml. Because of the increase in volume of the alcohol phase, the slope was not so great when sliquots were drawn without dilution toafinal volume. Tests of the method were made on a 5% trichloroacetic acid extract of dried lambs liver, with and without added phosphate. The results are shown in Table I. Inorganic phosphate was determined on aliquots transferred directly to the extractors. Total phosphate was determined after digestion of aliquots of the extract. The practice of drying the samples over sodium hydroxides pellets a t 90” to 100’ C. served to remove the relatively large quantities of trichloroacetic acid as well as the water. The amount of sulfuric acid used for digestion (other than that required to cover the sample) should provide an ample excess over the amount necessary to destroy the organic material present; while the aqueous phase during extraction should be about 0.5 iV with respect to hydrogen ion. Keeping the requirements for digestion in mind, three alternatives for providing the proper acid concentration for analytical procedure were tested. The first, digestion with a large excess of sulfuric acid, dilution to volume, and selection of a suitable aliquot, was employed in the microgram range samples cited in Table I and also when nitrogen was to be determined on the same digest (6). The second alternative, neutralization of excess acid, resulted in low
The procedures presented provide relatively simple and rapid ultramicrodeterminations of phosphate, incorporating the advantages of the alcohol extraction of the Berenblum and Chain method. The apparatus is not complicated and multiple samples are easily handled. Modifications to fit other needs could easily be made. Extractors of similar design, but larger in size could be applied to milliliter size samples for the range of 1 to 100 y of phosphorus. Other photometric or colorimetric instruments could be used within their ranges of applicability. Absorption cells of 4-mm. bore ( 7 ) would be particularly useful in the range of 0.05 to 0.27, however the cells with 2-mm. bore Tvould also serve in this range if the alcoholic molybdenum blue were diluted to 1 ml. instead of 0.2 ml. as described. The method could easily be applied to the determination of inorganic phosphate in the presence of labile phosphorus compounds by reversing the order of addition of acid molybdate reagent and octyl alcohol and performing the extraction and centrifugation without delay. Although other methods of digestion of organic samples could be used in conjunction with the analytical procedure, the use of sealed tubes offers the advantage of being able to determine nitrogen and possibly other constituents in the same digest. There is no danger of contamination while the tubes are sealed and no danger of loss of phosphorus pentoxide. The samples may be stored in the sealed tubes until it is convenient to perform the digestion or analysis. Further advantages are that no difficulties are experienced in the breakdown of organic materials and no additional oxidizing agents need be added. ACKNOWLEDGMENT
This investigation was made under contractual support by the Veteran’s Administration contract No. V 1001M-1979; and with aid of the University of California Committee on Research and the Office of Xaval Research. LITERATURE CITED (1) Berenblum, I., and Chain, E., Biochem. J . , 32, 295 (1938). (2) Craig, R., Bartell, A. H., and Kirk, P. L., J. Sci. Instruments, in
press.
(3) Ennor, A. H., and Stocken, L. A . , S t ~ s t r a l i a nJ . Exptl. Bid. Med. Sci., 28, 647 (1950). (4) Gilbert, P. T., Jr., Science, 114, 637 (1951). (5) Grunbaum, B. TV., and Kirk, P. L., Mikrochemie z’er. Mikrochim. Acta, 39,268 (1952). ( 6 ) Grunbaum, B. W., Schaffer, F. L., and Kirk, P. L., ANAL. CHEM.,24,1487 (1952). (7) Kirk, P. L., “Quantitative Ultramicroanalysis,” New York, John Wiley & Sons, 1950. (8) Kirk, P. L., and Danielson, M., Anal. Chem., 20, 1122 (1948). (9) Martin, J. B., and Doty, D. M., Ibid., 21, 965 (1949). (10) Vaughan, B. E., and Kirk, P. L., unpublished experiments. (11) Weil-Malherbe, H., Biochem. J., 48, xxiv (1951). (12) Ibid., 49,286 (1951). RECEIVEDfor review July 10, 1952. A4ccepted October 7, 1952. T h e opinions expressed in this article are the private ones of t h e writers and are not t o be construed as official or reflecting the views of the N a v y Department or t h e naval service a t large.
