1334
ANALYTICAL CHEMISTRY
benzaldehyde, and acetone, it has been found that these compounds are distilled with the formaldehyde and cause a serious inhibition of the development of the color with chromotropic acid. Rork is now in progress to eliminate this type of interference. ACKNOWLEDGMENT
The authors wish to acknowledge the assistance given by W. A. Vail and C. E. Langenhop in obtaining some of the results presented in this paper. Also, they wish to thank the Decyl Pharmacal Company. Charles Pfizer and Company, and C. J. Cavallito of the Sterling-U’inthrop Research Institute for some of the chemicals used i n this investigation. Finally, they gratefully acknowledge the financial assistance received from the grant made by E. I. du Pont de Semours & Company to sponsor chemical research at Princeton Universitv.
2nd Annual Summer Sqmmeium
LITERATURE CITED
(1)
Bricker, C. E., and Johnson, H. R., IND.ENG.CHEM.,ANAL.ED., 17,400 (1945).
Ellis, B. 9., and Jones, R. A., A n a l y s t , 61, 812 (1936). (3) Elving, P. J., Warshowsky, B., Shoemaker, E., and Marnotlit. J., ASAL. CHEY.,20, 25 (1948). (4) Hoepe, G., and Treadwell, W. D., H e h . Chim. Acta, 25, 353 (2)
(1942). (5) Johnson, M. J., IND. ENQ.CHEM.,~ A L ED., . 16, 626 (1944). (6) Kaufmann, H. P., and Baltes, J., Fette u. Seifen, 43, Nos. 6-7 93 (1936). J . Polvlmer Sci., 2, 206 (1947). (7) Kolthoff, I. M., and Lee, T. S., ENG.CHEM.,ANAL.ED.,18, (8) Reinke, R. C., and Luce, E. N., IND. 244 (1946). (9) Saffer,A., and Johnson, B. L., Ind. Eng. Chem., 40, 538 (1948) ENQ.CHEY.,ANAL.ED.. (10) Warshowsky, B., and Elving, P. J., IND. 18,253 (1946).
RECEIVED July 27, 1949,
- Organic Rennente
Preparation and Colorimetric Properties of Aluminon W. H. SMITH, E. E. SAGER,
AND
I. J. SIEWERS, National Bureau of Standards, Washington, D. C .
Commercial preparations of the dye called Aluminon vary in quality; some are worthless for analytical purposes. Methylenedisalicylic acid as prepared here, which with salicylic acid is used to make Aluminon, was found to contain a low amount of monomer probably because phenolic resins are formed during its preparation. I t is not expedient to remove all the resins but rather to keep the amount of polymer low. The molecular w4ght of methylenedisalicylic acid is 288. By using a product with an average value of 310 or less, a satisfactory reagent can be made and directions are given for its preparation. I t is more soluble in water than the acid form of commercial Aluminon and therefore an
T
HE reagent Aluminon is described as the ammonium salt of aurintricarboxylic arid. Preparations of it, offered for the rolorimetric determination of small amounts of aluminum, vary in quality, frequently lack sensitivity, and have caused considerable trouble in laboratories of the steel industry and elsewhere. In the experimental work reported here, a method is described for the preparation of a reagent with properties that may be reproduced well i n successive lots. Spectrophotometric measurements have been made of dyes prepared by this method and of two commercial preparations. PREPARATION OF A REAGENT
Two procedures ( 2 , 5 ) originallv proposed for the preparation of Aluminon yicald unsatisfactory products, and modifications (6, ‘7, 1%)of them appear to produce little improvement. In the method of preparation ( 5 ) devised by K. Sandmever, sodium nitrite is added to concentrated sulfuric acid and to this mixture salicylic acid is added. Formaldehyde is then slowly introduced. In the procedure of Caro ( I ) , sodium nitrite is added to concentrated sulfuric acid and an intimate mivture of equivalent amounts of methvlenedisalicylic acid and salicvlic acid is added. The second method seems preferable, but the dye formed lacks sensitivity. Holaday ( 7 ) improved this procedure
ammonium salt is not required. Spectrophotometric measurements of different lots indicate that a reagent can be satisfactorily reproduced. Studies were made in the ultraviolet and visible portions of the spectrum of aqueous solutions of commercial Aluminons and of others made by the method described. Changes in spectral characteristics with changes in hydrogen ion concentrations were also studied. Errors which may be introduced by the presence of the colorless, carbinol “faded” form of the dye are indicated. Changes in absorbancy which accompany metal complex formation are given and typical calibration curves a t selected wave lengths that may be employed with colorimeters are shown.
by adding a second portion of nitrite and sulfuric acid and in thimanner improved the dye. The amounts of monomeric aurintricarboxylic acid must br low in products made by Sandmeyer’s and Caro’s procedures for the following reasons. In the published discussions of the preparations of methvlenedisalicylic acid it appears to be assumed that only the monomer is present. However, when a reaction mixture containing salicylic acid, formaldehyde, and a mineral acid ic heated for several hours, resinous products result, doubtless of the phenol-formaldehyde type. Clemmensen and Heitman (2) who used a 50% aqueous solution of sulfuric acid as a condensing agent, obtained a product which after washing with hot water melted at 238” C. In the course of this work, methylenedisalicylic acid was prepared by their procedure, but only a small amount oi pure monomer could be isolated from it. Kahl ( 8 ) , who used hydrochloric acid as a condensing agent, purified the crude product of the reaction by dissolving it in a 50% hot aqueous solution of acetic acid, filtering, and precipitating the Toluble material by pouring the solution into an excess of distilled water In this wav, he obtained an acid that melted at 242’ C. A rapid method for the preparation of methylenedisalicylic acid is described below. One product of this procedure was found by titration to have an average molecular weight of 300. Its melting
V O L U M E 21, NO. 11, N O V E M B E R 1949 point was 247" C. The molecular weight of the monomer is 288. Methylenedisalicylic acid with an average mLlecular weight of 310 or less was found to be suitable for the preparation of Aluminon with satisfactory properties. I t is not necessnry to use a pure monomer because of uncertainties in the subsequent reaction when Aluminon is prepared. When monomeric methylenedisalicylic acid and salicylic acid are converted to Aluminon, two products can be formed. One is surintricarhosylic acid and the second is formaurindicarboxylic acid which is produced by reaction with methylenedisalicylic acid alone. Iiahl (8), who first prepared formaurindicarboxylic acid, observed that it forms colored lakes with aluminum, barium, and calcium. From several unsatisfactory commercial preparations of Aluniinon, it was possible to separate colored impurities in the following manner: An aqueous solution of the ammonium salt was acidified, and the precipitate separated by filtration. The precipitate was dissolved in acetone, precipitated with benzene, and filtered. After three or four such treatments, a black powder separated from the acetone, probably formcd by slow coagulation. When the powder was dissolved in a colorless solution of Aluminon huffered with ammonium acetate, a red color was produced. Such colored impurities may be resinous products formed when highly polymerized methylenedisalicylic acid is used to make lluminon. They arenot present if the acid has an average molecular veight of 300 or less. The following procedure for the preparation of a reagent is based on this fact. A product of this method is more soluble in water than the acid form of commercial Ilnminon. It is therefore not necessary to prepare the ammonium salt. Formaurindicarbosylic acid also was made according to this method. Alizarinsulfonic acid is another reagent for the colorimetric estimation of aluminum and its use suggests that B sulfonated .lIuniinon might be advantageous because it would not require the addition of a stabilizing protective colloid A mixture of equivalent amounts of methylenedisalicylic wid and 5-sulfosalicylic acid was treated by the following procaedure for the preparation of Aluminon. The product contained 0.99% sulfur instead of the theoretical 6.37%, if only a monosulfonated aurintricarboxylic acid had been formed. Presumably some condensation had occurred. The remainder was assumed to be formaurindicarboxylic acid. Aluminon was also directly sulfonated with fuming sulfuric acid. The percentage of sulfur found in different lots varied, but an aqueous solution of each preparation produced a color with at least 1 microgram of aluminum. During sulfonation and subsequent isolation of the sulfonated products, great care \vas required t o prevent contamination by traces of aluminum and of other elements which form colored complexes. No further work n-as done on this subject because the degree of sulfonation in different products varied too much to permit the preparation of a rwpent of uniform quality. PREPARATION O F METHYLENEDISALICYLIC ACID
Reagents used for the preparation of methylene disalicylic acid (3,3'-dicarboxy-4,4'-dihydroxydiphenylmethane)are: 80 grams of salicylic acid, 7.0 grams of trioxane, 100 ml. of glacial acetic acid, and 1 ml. of concentrated sulfuric acid dissolved in 5 ml. of glacial acetic acid, Add the salicylic acid and trioxane to the glacial acetic acid and heat the mixture to 95" C. Remove the source of heat and add to the mixture the solution of sulfuric acid in glacial acetic acid. The temperature will rise 5 or more. Probablv traces of acetvl sulfate aie formed which act as a catalyst for thcreaction. If the solution of sulfuric acid in acetic acid is heated before it is added, a violent reaction may result. After 5 minutes, pour the mixture into 4 liters of distilled water and allow to remain, or stir, until a clear supernatant liquid is O
1335 formed, and filter. Add the filtered material to 400 ml. of a mixture of equal volumes of glacial acetic acid and distilled water and again filter. Pour t,he filtrate into distilled water, separate the precipitate, and wash it with successive portions of hot distilled water until all acetic acid has been removed. Dry the precipitate in air and afterward in a vacuum with a desiccant, at room temperature. The yield is 20 grams or more. The moleculnr weights found by titration of several lots prepared by the procedure above varied between 298 and 304. With one of them a value of 301 was found; and with this lot, by ebulliometry, the Physical Chemistry Section of this bureau obtained a value of 300. PREPARITIOY OF ALUMINON
In the following procedure, a mixture of mcthylencdisalicylic acid and salicylic acid is treated twice as recommended by Holaday ( 7 ) . Reagent .4is made up of 20 grams of methylenedisalicylic acid prepared by the above procedure, and 10 grams of salicylic acid. The acids are finely powdered and intimately mixed. Reagent B. T a o portions are made: 60 ml. of concentrated sulfuric acid and 10 grams of sodium nitrite Cool the sulfuric acid to the temperature of crushed ice. Powder the nitrite in an agate mortar and mix quickly small portions with the acid to avoid the formation of red fumes. Surround with ice one portion of Reagent B contained in a 400ml. beaker. Stir small quantities of A into it with a thermometer a t a rate that permits the temperature to be held not higher than 5" C. After all has been added, allow the mixture to remain in ice for about 1 hour. Then add and mix the second portion of B which has been chilled to 5" C. or below. After another hour, remove the ice and allow the mixture to remain a t room tempcrature for about 12 hours. Pour the mixture, in small amounts while stirring, into 4 liters of distilled water previously cooled to about 2 " C. and filter a t once with suction. Add the filtered material to 2 liters of distilled water, stir well, and again filter. Wash the filtercd material once, and allow it to dry first in the air and then in a vacuum over a desiccant at room temprrature The yield is 22 grams or more COLORED COMPLEX OF ALUMINUM FORMED BY ALUMINON
Most dyes do not exist as "pure" organic compounds. Extensive studies have recently been made on the rate of fading of many sulfonephthalein indicators in strongly alkaline solutions (11). The indicators were of the highest purity obtainable, but the spectrophotometric data indicated that in the purified crystalline indicator some of the dye existed as a colorless carbinol form. The same may be true in the case of Aluminon. Changes in color of aqueous solutions may indicate instability. Not only may some colorless carbinol form of the dye be present but in this case some dicarboxylic acid may be mixed with tricarboxylic acid. The amount of colorless carbinol present in the dye may depend upon the methods and conditions under which the dye is synthesized. For instance, a freshly prepared solution of a dye containing some carbinol may deepen in color for a few days if the pH of the solution is favorable. For compounds of this type some knowledge of the various equilibrium relationships or the dissociation constants of the carboxyl groups and of the hydroxyl groups is essential for optimum utilization. The dissociation constants of a simple molecule such as salicylic acid are easy to evaluate.
O
O O OH
H
Expressed as pK, the negative of the logarithm of the dissociation constant, pK1 for the carboxyl group is about 4 and pKn for the hydroxyl group is about 10. In the case of aurintricarboxylic acid, however, the dissociation of the groups may not occur simultaneously and may overlap. Thus, the constants can only be approximated.
ANALYTICAL CHEMISTRY
1336
period of time even when buffered at different pH values. Some changes were noted in the ultre violet as well as in the visible portion of the spectrum. Hence, spectrophotometric measurements were made at frequent wavelength settings over a spectral range of A1 3H' 3 200 to 400 mp. The solution.. were prepared and kept at a constanl temperature of 25" C. Hydrogen. ion concentrations were controlled by use of acetic acid-sodium hydrox. 0 ide buffers or of various concentraH tions of hydrochloric acid or of Colored Metal Colored Dye sodium hydroxide. pH measure Al(OH): Complex II at or above menta were made of all solutions PH 3 by use of a commercial glamSome Colorless electrode assembly. Carbinol The curves in Figure 1 illustrate the differences in samples of Aluminon obtained from two different sources and in three different lotp One would not expect the formation of chelated compounds to of dye (free acid) prepared at this bureau. require much time. However, suppression of such complex formation may occur if the pH is substantially lower than that of the carboxyl dissociation. The possibility also exists that some of the MU 500 400 . 373 276 ZiO dye itself may be in its colorless carbinol (faded) form, and this I .o I would apparently retard the complete reaction. At pH 3 and higher, insoluble aluminum hydroxide is formed. At much higher pH, the dye itself develops a more intense color, but the 8rate of fading is also high. Therefore, maximum color development probably cannot be achieved and a compromise must be made. Using the reagent Aluminon as free acid buffered at pH 4.2, the complex formation with aluminum is practically complete >- .60 In 20 minutes at room temperature (25' C.). Under these conz ditions it is not necessary to use heat or stabilizers such as gelatin Q ffi or gum arabic. 0 .4-cc The introduction of p sulfonate group ortho to the central carm bon atom in the phthalein indicators greatly increased the solubilm d Ityand stability ( 3 ) . Thva we have the familiar series of stable indicators, the sulfonephthaleins. In an analogous case perhaps an orthosulfonic acid group in the aurintricarboxylic acid would make the acid stronger and more soluble. Assuming that a sulfonate group would also form chelated compounds, the reaction 15 20 25 30 35 40 could take place at a lower pH and a metal complex probably WAVENUMBER X 10-3 ( GM-I) would be more stable. However, the synthesis of such a comFigure 1. Comparison of Absorbancies of Samples of pound as an Aluminon reagent is a problem in itself. Aluminon from Different Sources In metal complex formation, the following reactions may occur :
'J
+
II
SPECTROPHOTOMETRIC MEASUREMENTS
Several workers have reported on the instability of the reagent Aluminon under various conditions. All used the ammonium salt of the reagent. Winter, Thrun, and Bird (IS), using a Duboscq colorimeter, reported maximum color formation in 10 minutes a t pH 4.5 to 5 after heating to 80' C. They noted that the dye itself is decolorfeed a t pH 7 and also that the lake begins to fade at pH 7.3. Craft and Makepeace (4) using a Coleman spectrophotometer also made an extensive study of the visiblr portion of the spectrum. Usin ammonium acetate buffers, they studied the effect of pH upon &e development of color which accompanies the formation of a metal complex. Roller (IO) noted that the vellow color of the dye in alkallne solution and its red color in acid solution practically neutralize each other at pH 6.3 and suggested the use of this pH in which the blank of buffer and dye is practically colorless. The importance of a precisely drtermined calibration curve is also emphasized by Olsen, Gee, and McLendon (9). Preliminary measurements with a Beckman spectrophotometer at this bureau confirmed these results and showed stock solutlons of the ammonium salt of aurintricarboxylic acid to be unstable. The absorbancies of the aluminum complexes changed over a
All the dyes were in 5 X molar aqueous solutions and were measured relative to water in %em. absorption cells. Transmittancy readings were made on the Beckman instrument at 2 to 5 mp intervals. In Figure 1 absorbancy values (absorbancy = -loglo transmittancy) are plotted as a function of wave number (l/X) with the corresponding wave lengths shown at the top of the graph. Curves 1, 2, and 3 in Figure 1 represent the dyes synthesized at the bureau, and it is readily seen that they are reproducible. Curves 4 and 5 represent the two commercial dyes and show differences throughout most of the spectral range. Curve 5 shows a shift in the wave length of maximum absorption in the visible, which also denotes a change in hue of this dye. Because metal complex formation increases the absorbancy values of the bands in the visible, it is easily seen that in the determination of the same amount of aluminum the two commercial dyes would give different results. The curves in Figure 2 show the absorbancies of formaurindicarboxylic acid (curve 1) and aurintricarboxylic acid (curve 2 ) . Changes in color of the reproducible free acid dyes with change in hydrogen ion concentration were then studied. Transmittancy measurements of 5 X M dye in buffers were made relative to the respective buffers in 2-cm. absorption cells at 25' C. The two sets of curves in Figure 3 represent the various
V O L U M E 21, NO. 11, N O V E M B E R 1 9 4 9 MU
I .o
500
400
1331 absorptim cells. The spectrophotoiiietric measurements were made as soon as possible after addition of the alkali, and at stated intervals thereafter. Curves 1 through 7 were made at the end of 0.5, 1, 2, 3, 4, 5, and 24 hours, respectively. The initial deep pink color in the visible gradually decreases until a slightly colored equilibrium mixture is obtained at the end of 24 hours. After this there is little change in the visible, but significant changes in the molecule are indicated by the ultraviolet data, particularly at about 380 and 275 mp. Curves 8 through 12 represent the faded dye after 2 , 5 , 7 , 9 , and 15 days, respectively. The use of stronger alkali would accelerate the rate of initial fading and perhaps show greater rhanges in the ultraviolet.
25
286
333 1
I
100 c
z
w
80
e
5
60
> V
$
40
L 5:
Comparison of Absorbancies of Formaurindicarboxylic Acid (1) and Aurintricarboxylic Acid (2)
Vigure 2.
9
eo
C
a
c
0 WAVELENGTH
m g e s in the overlapping dissociation of the cart)oxyl groups ill *iirintricarboxylic acid. Thcre is first. a slight increase in color with decrease in hydrogen ion concentration (increase in pH), and then a decrease in color. The first group of curves, I through 5, inclusive, shows the dye at pH values 3.4, 3.9, 4.2, 4.4, and 4.6, respectively. The 3econd group of curves, 5 through 12, represents the dye a t pH values 4.6, 4.8, 5.0, 5.2, 5.4, 5.5, and 5.8, respectively. At pH .5.8 the solution is almost colorless, as shown by the higher transmittancy values a t about 525 mp. The changes in the main band in the ultraviolet are slight. Above p H 7, the dye changes to a deep pink color. The pink color a t about pH 12.0, however, is fugitive. The fading reaction may be somedmt similar to that reported for the stable sulfonephthalein indicators in great excess b f alkali (II), or of phenolphthalein (3). In the case of Aluminon, only t,he practical aspects of the problem were considered, and the results of the fading reactlion are dhown in Figure 4. Transmittancy measuremeiits uf IO-' M dye with enough solium hydroxide added t,o givp a pH of 11.9 were made in l-cm.
f
0 100
i
80 4 L
i-
20-
1
01
/I
I
zoo
Figure 3.
i v I I I 300 400 WAVELENGTH IN
I
I
I
500
I
600
I
I 700
MILLIMICRONS
Effect of pH on .Spectral Characteristics of Aluminon
Figure I..
c
IN
MILLIMICRONS
4lkaline Fading of Aluminon at pli 11.9
c
WAVELENGTH
IN
MILLIMICRONS
Figure 5. Transmittancy of Aluminum Complex of Aluminon Formed at pll 4.2 Relative tn nve Alone at pH 4.2
The data demonstrate the seriousness of errors which can be introduced if the dye is subjected to treatment with alkali, or, if in making up stock solutions, alkali is added to dissolve the dyb, then allowed to stand before adjusting the p H downward with acid. In the case of the sulfonephthalein indicators, the reaction was for the most part reversible, and color could be regenerated by bringing the solutions again to the favorable lower pH. Aluminon, however, seems to be permanently changed after prolonged reaction with sufficient alkali. The technique formerly used in the determination of aluminum, which consisted of roughly adjusting the pH by means of ammonium hydroxide and hydrochloric acid, may be responsible for some variation in the results. Sot only should the stock solution of Aluminon reagent be promptly adjusted to a pH of about 4.2, but the test solution should also be brought to this same pH before formation of the q.omple~for the most accurate results. In studying the changes in spectral characteristics which aocompany aluminum complex formation with Aluminon, several aeries of observations were made throughout the ultraviolet and the visible, using different buffers and various amounts of dye and of aluminum. The curves shown in Figure 5 are representative of such a series made at pH 4.2. The transmittancy measurements of the aluminum complex were made relative to the initial amount of the dye itself, in buffer. It is recognized that some of
1338
ANALYTICAL CHEMISTRY
the dye is taken up by the complex and that the amount must vary with the amount of aluminum. Rater has been used in the solvent cell, or buffer, but the most precise calibration curves were obtained by using the buffered dye in the solvent cell. By following such a procedure the transmittancy values are as shown in the figure ' I he ultraviolet data are of little use h u t were obtained in order to get an over-all picture of the complex formation. The curves represent the addition of 0.5, 1, 2, 3, 4, and 5 ml. of 2 X 10-4 M aluminum chloride in 100 ml. of buffered dye. I
1
I
ALUMINUM CHLORIDE
Figure 7.
IN M I L L I L I T E R S
( I ML
= 0 01 M G A L )
Calibration Curves
the wave lengths of maximum absorption, straight lines show COIIformity to Beer's law, while some curvature may be expected over the steeper portions of the bands. For use with a colorimeter using u. 525 mp filter, for example, one would expect to obtain straight-line calibration curves if the photocell response of the particular colorimeter used is linear. It is not necessary to use a spectrophotometer for accurate results in routine analysis, but it is desirable to make a precise calibration curve for each lot of dye regardless of the instrument used. LITERATURE CITED
IO
450 W A V E LENGTH
IN
500 550 MILLIMICRONS
Figure 6. Absorbancy Values of Aluminum Complex of Aluminon Ilelative to Dye Alone
On the absorbancy basis, the same data are given in Figure 6 for the visible range The numbers on the curves are the same as those of Figure 5 and represent the same amounts of aluminum. T t is clearly seen that the metal complex makes an appreciable increase in absorbancp throughout the bands in the visible. Using the same data for calibration curves, the absorbancy values a t stated wave lengths are plotted as a function of the amounts of aluminum in 100 mi. of solution as shown in Figure 7. Any of the selected or other wave Jengths may be used. However, near
2nd Annual Summer Sgmpoeium
(1) Caro, K., Ber., 25,941 (1892). (2) Clemmensen, E., and Heitman, A. H., J . Am. Chem. Soc., 33 733 (1911). (3) Cohen. B.. Pub. Health Re&.. 41. 3051 (1926). (4) Craft, C. H., and Makepeace, G. R., IND. ENG.CHEM.,ANAL. ED.,17,206 (1945). (5) Geigy and Co., German Patent 49,970 (1889). (6) Heisig, G. B., and Lauer, W.M., Org. Syntheses, 9, 8 (1929). ( 7 ) Holada\, D. A.. J . Am. Chem. SOC., 62, 989 (1940). (8) Kahl, L., Ber., 31, 143 (1898). (9) Olsen, A. L., Gee, E. A., and McLendon, V., IND.ENG.CHEM. ASAL.ED.. 16, 169 (1944). (10) Roller, P. S., J . Am. Chem. SOC.,55, 2437 (1933). (11) Sager, E. E., Maryott, A. A , and Schooley, M. It., Ibid., 70, 732 (1948). (12) Scherrer, J. A , and Smith, W.H., J . Research S a t l . Bur. Standurds, 21, 113 (1938); Research Paper 1118. (13) Winter, 0. B., Thrun, W. E., and Bird, 0. D., J . Am. Chem SOC.,51, 2721 (1929). RECEIVED July 30, 1949.
Organic Reagent8
Spectrophotometric Study of Spot Tests E. H. WINSLOW
A
AND
H. A. LIEBHAFSKY, General Electric C o m p a n y , Schenectady 5 , N . Y .
BOUT ten years ago, an attempt \\as made i n this laboiatorg to answer certain fundamental questions about spot tests by measuring the spots on a General Electric recording spectrophotometer ( 4 ) . In particular, the authors hoped to discover whether the results of reflectance and transmittance measurements ever obey Beer's law, and whether one type of measurem e n t is preferable to the other. Two tests, radically different In their chemistry, were chosen for investigation-namely, the ,detection of cupric ion with a-benzoin ouime, and the identificattiop.Qf silver by the deposition of that element through the action of a developer containing silver ion on exposed silver bromide derived from the silver in the sample ( 2 , 3 ) . The results of this
work are intrinsically valuable, and they have a bearing 0x1 recent efforts to make filter-paper chromatography quantitative (2, 5). If a series of spots is to be evaluated on the spectrophotometei it is desirable to have the beam incident upon a constant fractiou of the sample. If this fraction is constant, it need not be known If it varies, its values will have to be known if the consequent deviations from Beer's law are to be calculated. The simplest way of satisfying the requirement of constancy is to distribute the spot uniformly over a constant area, which overlaps the incident beam. The copper spots were confined by using Yagoda test papers with an inner circle 1.2 cm. in diameter. The silver
