Quantitative Determination of Serum Proteins by Paper Electrophoresis Rapid Dyeing Method KATHERINE M. FORMUSA, RUTH R. BENERITO, W. S. SINGLETON, and J. L. WHITE Southern Utilization Research and Development Division, U. S. Department of Agriculture, New Orleans, l a .
b A quantitative dyeing technique was required to measure the changes in serum protein fractions accurately and rapidly after administration of intravenous fat emulsions. Under rigidly standardized conditions, the dye uptake is proportional to the concentration of protein nitrogen, regardless of the type of serum protein. Direct scanning of the dyed and oiled strips by transmission densitometry results in a linear variation of integrated area with the concentration of protein nitrogen over a larger range of protein values than usually considered valid.
T
effects of the addition of fat emulsions prepared for intravenous alimentation to normal human serum and rat serum in vitro, and to dog serum in vitro and in vivo were investigated by paper electrophoresis. The mobilities and relative percentages of the protein fractions and lipides in normal serum and in serum-emulsion mixtures were determined. Several published methods for location of the separated proteins were used. I n all cases, the mobilities of the various fractions were reproducible, but quantitatively reproducible results were not obtained with any published dyeing technique. Therefore, a quantitative dyeing technique was designed to measure accurately and rapidly the changes in serum protein fractions in the presence of fat emulsions. Much has been written on the use of staining techniques and direct scanning methods for the quantitative determination of proteins after paper electrophoresis. The literature presents many conflicting statements on the following points: Proportionality of dye uptake to protein concentration Variation of dye uptake with class of the protein Validity of Beer’s law in the case of absorbances obtained by direct scanning as compared with those obtained by elution of the dyed proteins from the paper fitrips HE
Crook and coworkers (5, 6) state that detailed experimental justification is needed before it can be said that the 18 16
ANALYTICAL CHEMISTRY
absorbances from dyed paper strips follow Beer’s law. They concluded that bromophenol blue gave unreliable densitometer readings even when oiled strips were scanned, and that the albumin fraction was less chromogenic. Jencks, Jetton, and Durrum (12) state that the quantity of bromophenol blue bound by the denatured proteins on filter paper is dependent on the time and temperature of denaturation of the proteins and the durations of staining and rinsing off the excess dye; these variations differ with the various types of proteins. These workers concluded that bromophenol blue does not follow Beer’s l a v when the papers are directly scanned, that the deviations were greatest for the densely stained albumin fraction of normal serum, and that another error in the direct scanning of the dyed paper strips was due to the 3 to 5% of trailing of the albumin fraction. Similarly, Franglen and Martin (8) found that, with their technique, there was no linear relationship between protein concentration and dye uptake, but that the dye uptake depended upon the area of paper to JThich the protein Tyas adsorbed. Recently, Rees and Laurence (16) scanned oiled strips dyed for proteins with Amidoschwartz 10 B or -4zocarmine B. They found that Beer’s law held for absorbances up to 1.4 when dmidoschwartz 10 B mas used and up to 1.1 when ilzocarmine B was used to dye the proteins. Other investigators (3, 10, 11, 14, 17) concluded that Beer’s law was justified as long as transmission densitometry was used. Von Frijtag Drabbe (18) concluded that the average uptake of Amidoschwartz 10 B by albumin was 1.28 times greater than that for gammaglobulin when their dyeing technique was used, and that the length of time that the proteins are exposed to the dye solution influences the uptake of the dye to an extent that differs for various proteins. Kunkel and Tiselius ( I S ) used the elution method and bromophenol blue as the dye for albumin and globulins, which they concluded did not take up the dye to the same extent per milligram of protein. They concluded that
the binding of the bromophenol blue by proteins was enhanced in acid solution and that a critical part of the staining technique was the use of a 0.5y0 acetic acid solution for rinsing. The techniques used to standardize a rapid and quantitative method for the dyeing of serum proteins are described in detail here. Certain modifications in the paper electrophoretic method were found essential to the reproducible fractionation of serum proteins. EQUIPMENT
The Junior Ionograph model (Precision Scientific Co.) was used to prepare the paper electrophoreograms. The dyed electrophoreograms were scanned in a balance beam type of photoelectric recording photometer called the Analytrol Model R (Spinco Co.). This instrument has automatic scanning, integrating, and recording mechanisms which measure the dye uptake as an integrated area in square centimeters (X10-I). Accurately measured volumes of all samples were applied to the paper strips by means of the Spinco stripper. REAGENTS
Buffer. T o prepare a buffer of pH 8.6 and a n ionic strength of 0.05, 20.618 grams of sodium diethyl barbiturate and 3.684 grams of diethyl barbituric acid were dissolved in distilled water and diluted t o 2 liters. Bromophenol Blue Dyeing Solution. One gram of bromophenol blue
(tetrabromophenolsulfonphthalein, p H range 3.0 t o 4.6) was dissolved in 1 liter of 95% ethyl alcohol which had been saturated rrith mercuric chloride and contained 5 ml. of glacial acetic acid. Rinsing Solution for Strips Dyed with Bromophenol Blue. Eighty
grams of anhydrous sodium acetate were dissolved in water containing 400 ml. of glacial acetic acid and diluted t o a final volume of 4 liters. Solution to Minimize Scattering of Light. Two hundred milliliters of xylene (Baker’s analyzed reagent grade) were added to a solution containing 1 liter of paraffin oil (white, domestic, viscosity 125/135) and 500 ml. of CYbromonaphthalene (Fisher reagent
chemical). Seventeen grams of Span 80 (Atlas Chemical Co. commercial emulsifier) were added to the final solution. Protein Samples. Purified human albumin and gamma-globulin were obtained from the Cutter Laboratories. These fractions were better than 98% pure. Pooled normal human sera samples, an albumin fraction, and a fraction consisting of a mixture of alpha-, beta-, and gamma-globulins separated by salt fractionation were used. PROCEDURE
Paper electrophoresis of serum proteins, human serum albumin and human serum globulin rvas carried out by a modified procedure similar to that described by ,Durrum ( 7 ) and Cremer (4). I n every experiment, four horizontal strips of Khatman's 3 A I 1 1 filter paper, 50 em. long with 5-em. drops on either side to dip into two buffer vessels, were clamped into place on a rack and pulled taut. Exactly 500 ml. of buffer solution were put into each of the two buffer vessels containing the paper strips and into each of two electrode vessels. The electrode vessels were connected to the other two buffer vessels by two U-tubes containing the same buffer solutions. A very light pencil mark in the center of each strip indicated the point of application of the sample, but was not dark enough to be detected when the paper n a s scanned in the photometer. The rack was placed in the electrophoresis cabinet and leveled with the aid of a spirit level. The buffer solution m s allolved to wet the strips by capillary action. Usually, about 3 hours were allowed for equilibration. After the strips had been allorTed to equilibrate, they were again pulled taut, and the samples (10 to 40 pl.) were applied to the centers of the strips. The sample to be subjected to electrophoresis was applied in a film across the wires of the Spinco stripper by means of a graduated micropipet, preferably, not graduated to the tip. The sample was then transferred in a strip across the midpoint of the paper. Electrophoresis was allowed to continue for 16 to 18 hours a t a constant potential of 350 volts and a t a constant current, which varied from 3 to 4 ma. in different runs. The experiments were carried out a t 25" C. A helium atmosphere was maintained in the cabinet, in order to dissipate the heat generated on the strips during electrophoresis. This kept evaporation and resulting chromatographic effects as well as changes in electrical resistance a t a minimum. The atmosphere mas kept highly saturated with water vapor by immersing the buffer vessels in a water bath. Upon completion of a run, the papers were dried in an oven a t 110" to 120° C. for 20 minutes to denature the proteins. The dried strips were placed in the bromophenol blue dye solution and heated for 10 minutes a t 70" C. in a covered vessel over a steam bath. After removal from the dye bath, they were
rinsed three times in the buffered rinsing solution. Ten minutes were allowed for each rinse. At the end of the final rinse, the strips, which were now free of all background color, were blotted on a piece of filter paper for 5 minutes and then returned to the oven for 5 minutes. The slightly damp strips were exposed to ammonia fumes until the dyed areas became a uniform, bright blue color. -4n additional 10 minutes of oven drying were required to completely dry the dyed papers. The strips were rendered transparent by immersing them in the paraffin oil1-bromonaphthalene-xylene-Span 80 solution. After immersion in the oil, they were blotted gently, the xylene n-as allon-ed to evaporate for 20 minutes, and they TTere scanned in the photometer. No filters were used. For quantitative comparisons, the protein content of the samples was determined by a micro-Kjeldahl method and the Kingsley biuret method as modified by Gornall, Bardawill and David (9). Biuret determinations were carried out using a Beckman \Iode1 B soectroohotometer a t 540 mu. DISCUSSION OF PROCEDURE
.4n electrophoresis apparatus allowing the use of horizontally suspended strips was selected, in order to eliminate the gravitational gradient, The cabinet was large enough to allow immersion of the buffer vessels in a water bath and a large space above and below the freely suspended strips. This free space was enveloped with an atmosphere of helium which bubbled through the cabinet. I n preliminary experiments, it was found that the atmosphere of helium vas essential for good resolution and reproducibility of results. When nitrogen gas or air instead of helium was bubbled through the cabinet, the electrophoreograms showed evidence of streaming and the protein fractions Ivere diffused. Evidently, helium, which has a much higher thermal conductivity than nitrogen or air, was essential for maintenance of a constant temperature and constant electrical resistance, and elimination of diffusion effects due to temperature gradients. I n an ideal case of electrophoretic migration in free electrophoresis, the electrophoretic mobility of an isolated particle in an electric field is measured. I n practice, this hypothetical case cannot be emulated, as too many retarding forces oppose the electrical forces on a charged particle. There are the retarding effects due to the inherent properties of the dispersing medium, such as viscosity, dielectric constant, and ionic strength. Other retarding effects are due to the forces of attraction between the colloidal particles and the paper and between the particles themselves. I n paper electrophoresis, it is best to minimize these retarding forces as much as possible. One way of
minimizing the greatest retarding force is to use a solution of low ionic strength. Some electrolyte is essential for a good conducting medium, but too high an ionic strength increases the forces of attraction between colloidal particles and ions, and causes an increase in current and a resulting increase in heat liberated during electrophoresis. I n this investigation, it was concluded that the optimal ionic strength of buffer was 0.05 on the volume basis. Preliminary experiments also proved that agar bridges containing potassium chloride should be eliminated. During an electrophoresis experiment over an interval of 16 to 18 hours, the current gradually increased a t constant voltage when potassium chloride salt bridges were used to connect the buffer vessels. Khen there was this increase in current during an experiment, there ITas a loss in reproducibility and in sharpness of resolution. This error was eliminated by the substitution of bridges containing onlv the buffer solution for the aear and potassium chloride salt bridges.- After the salt bridges were eliminated, every electrophoresis experiment mas run a t constant amperage as well as a t constant voltage and resulted in excellent resolution and reproducibility. The photometer selected for this investigation u-as of the double-beam type and, therefore, problems due to the fluctuation in light source or in line voltage were eliminated. A cam (called B-3 by the manufacturer) was used, which gare a plot linear t o concentration of the protein sample. For the proper functioning of this apparatus, the light falling on the undyed strip should be transmitted. There should be no scattering of light. K i t h a dyed strip, the only decrease in the intensity of light falling on the measuring photoelectric cell should be due to the absorption of light by the dye absorbed in the protein. Therefore, it was found necessary in preliminary experiments t o fill the interstices of the cellulose paper strips with a solution of oils before scanning the dyed strips. The xylene acted only as a volatile solvent in aiding the penetration of the oils into the paper. The Span SO was selected as one of the oils because of its affinity for the paper. The solution of oils selected n-as such that its index of refraction was very close to that of the cellulose ( n = 1.55). Therefore, the amount of scattered light was minimized. If the dyed papers were not oiled before they were scanned, the linear variation of area with concentration of protein applied to the strip did not hold. Preliminary dyeing experiments were performed with Amidoschwartz 10 B, as well as Kith various concentrations of bromophenol blue in the presence of bivalent zinc or mercuric ions. Once a VOL. 29, NO. 12, DECEMBER 1957
1817
Toble I.
Relationships of Protein Concentrations to Absorboncies by Biuret Reaction and to Scanned Areos of Electrophoreograms Absorbancy of Prot,ein Inteerated Protein A&., Solution, Nitrogen Nitrogen, Biuret Appliedto Sq. Cm. G./100 MI x 10-1 Kind of Protein Solution Reaction Strip, y 130.0 Pure human albumin 140.7 1.4070 0.249 ~
Pure human gamma-globulin
1.1256 0.8442 0.5628 0.4221 0.5760 0.4220 0,2880 0.1440
0.193 0.144 0.086 0.065 0.111 0.077 0.056 ...
...
... ... Pooled normal human serum
Albumin fractionated from pooled human serum Globulins fractionated from pooled human serum Pure human gamma-globulin in presence of pure human albumin
Pure human albumin im presence of pure human gamma-globulin
1.1290 1,1290 0.9032 0.6774 0.4516 0.3387 0.1129
0.205 0.205 0.160 0,110 0.064 0.046
...
0.133
...
...
...
0.072
0.325
0.055
0.280
0.048
...
good dyeing technique with the bromophenol blue dye had been established, the use of Amidoschwarts 10 B was abandoned. The bromophenol blue dyeing method mas chosen chiefly hecausc of its relative simplicity. Dyeing techniques involving Amidoschwartz 10 B have many undesirable features. The rinsing involves use oi large quantities of methanol for the elimination of the background color. However, even after exhaustive washing, the remaining background color is undcsirably prominent. Strips dyed with bromophenol blue are washed easily in an aqueous solution of acetic acid and sodium acetate with the complete elimination of background color. In this investigation it was found that the bromophenol hluc dye vas bound more strongly in the prcscnce of mercuric ions than in the prcsence of zinc ions, the use of which was suggcstcd in a rcccntly published dyeing procedure (g). This is easily understood in the light of data published by several investigators ( 1 , 1 5 )who were concerned with quantitative studies of the avidity of amino acids for himetallic ions. It is generally agreed that the mercuric ions are more tightly bound than the zinc ions. With a standardized method, reproducible results were obtained with
,1818
ANALYTICAL CHEMISTRY
...
112.6 84.4 56.3 42.2 57.6 42.2 28.8 14.4 115.2 172.8 230.4 225.8 112.9 90.3 67.7 45.2 33.9 11.3 78.0 156.0 42.0 84.0 32.5 65.0 97.5 130.0 162.5 28.0 56.0 84.0 112.0 140.0
102.0 83.0 50.0 35.0 52.0 43 30 12.0 112.0 148.0 206.0 204.0 104.0
... ... ... ... ...
72.0 144.0 40.0 78.0 33.0 67.0 96.0 120.0 157.0 26.0 53.0 68.0 101.0 131.0
lo-, 20-, 3 0 , and 40-pI. samples of pooled serum. All samples mere applied in 10-pl. aliquots in ordcr to keep the area occupied by the original serum band a t a minimum. After reproducibility was established, 20-pl. samples of serum mere used. EXPERIMENTAL RESULTS
Electrophoreograms illustrating the resolution of scrum protein fractions (Figure 1) were obtaincd with 20 pl. of
pooled normal human sera. However, fractionations on lo-, 30-, and 40.~1. samples were equally successful. Typical data obtained in this investigation are recorded in Table I. All data recorded for the pure human serum albumin and gamma-globulin were obtained in triplicate experiments. Two different samples of pooled normal human sera were used. Ten., 20-, 30-, and 40-pl. quantities of each sample of pooled scra wcre uscd to standardize the dyeing method. Several electrophoreograms on each of these amounts of sera were made in order to compare the relative percentages of each fraction. The relative percentages and mobilities of the various protein fractions as well as the albumin to globulin ratios of both samples of pooled sera are given in Table 11. The recorded values were the averages from ten reproducible electrophoreograms of each pooled sera sample. The precision among the electrophoreograms for the alpha-1 globulin fraction of a given serum was as good as that for the albumin fraction, even though these two fractions differ widely in amounts. To ascertain the reproducibility of the dyeing technique, the following protein solutions were used: (1) a solution of purified human serum albumin, containing approximatcly 10 grams of albumin per 100 ml. of 0.9% saline solution; (2) a solution of purified human serum gamma-globulin, containing approximately 4 grams of globulin per 100 ml.; (3) pooled normal human sera samples; (4) albumin fractionated from pooled normal human sera and (5) a mixture of alpha-, beta., and gamma-globulins fractionated from pooled normal human sera. The albumin and globulin fractions of the pooled human sera were separated by salt fractionation, using 22.6% sodium sulfate. Under these conditions the globulins were precipitated, and the albumins remained in solution. By use of the Gornall (9) method, the albumin and globulin contents of the pooled
Figure 1. Reproducibility of electrophoreograms from two runs
Table II.
Pooled Sera Sample
Relative Percentages A41pha- hlpha1 2 Beta 3 2 5 1 4.5 2 7 9 0 11 0
-41-
bumin 63 6 62 8
A B
I
I
Paper Electrophoretic Results on Pooled Normal Human Sera Sq. Cni. hIobilitie s
I
I
I
I
I
I
,
Gamma 23 6 14 5
Alpha-
Al-
L U G
Ratio
humin 2 8 2 9
1 75 1 68
1
2 1 2 2
x 105 (Sec. Volt) Alpha2 Beta Gamma 0 8 -0 6 1 3 0 9 -0 4 1 5
200
- 180 /"
- 160 z
+
m 140
,s-
w
5
0
XI75
- 120;
W
- 100 R
W
0 I
-80
-
60
5 9
a W
I
- 40
o' 6
UP
04
06
od
in
LP
14
LS
in
o
0
PROTEIN NITROGEN-G/IOO ML
Figure 2. Analyses performed b y biuret reaction and by direct scanning after electrophoresis
A
Albumins Gamma-globulin A Pooled serum proteins 0
B 0 dlbumins
Gamma-globulin A Pooled serum proteins
human sera were determined and compared with the results obtained by electrophoretic analyses. The total nitrogen and nonprotein nitrogen contents of the protein solutions were also determined by the microKjeldahl method. The moisture contents of the albumin and gammaglobulin samples n ere determined, and the necessary corrections were made. Tarious dilutions of these sample solutions were used to standardize the determination of protein nitrogen by the biuret reaction (9). The protein nitrogen content of each solution in terms of grams per 100 ml. and the absorbance of each solution obtained by the biuret reaction are recorded in the second and third columns of Table I. Various dilutions of the standard albumin and gamma-globulin solutions n ere mixed in 1 to 1 ratio and subjected to electrophoresis. Figure 2 shons that there is a linear variation of the integrated areas recorded by the automatic integrator and the concentration of the protein nitrogen when 10 pl. of the various protein samples are subjected to electrophoresis, dyed, oiled, and scanned according to a standard technique. This linear relationship holds for pure human serum albumin, pure gamma-globulin, pooled serum proteins, globulins fractionated
'0
25
50
75
100
125
150
175
200
225
EX
MICROGRAMS OF NITROGEN APPLIED TO STRIPS
Figure 3. Variation of scanned areas with concentration of protein nitrogen
e Pure albumin fraction @ Gamma-globulin in alhmmin-garnma-globulin mixture 0 ;ilbuniin in albumin-gamma-globulin misture Pure gamma-globulin fraction A Pooled serum proteins
from pooled sera, albumin fractionated from pooled sera, and the mivtures of pure gamma-globulin and pure albumin solutions. The absorbances of the same protein solutions as determined according to the biuret reaction are also plotted against the Concentration of protein nitrogen in Figure 2. This relationship is also linear. Further determinations of nitrogen content of the 1-arious sera and protein solutions were made by the biuret reaction, as this method !vas quicker than the Kjeldah1 method. Because of the limited solubility of some of the protein fractions, it was necessary to apply different volunies of the sample solutions to the paper strips in order to cover a wide concentration range. Figure 3 shows that the linear variation of the integrated areas with the concentration of protein nitrogen holds for the concentration range of both albumin and globulins found in normal sera when as much as 20 p l . of serum is applied to the strips. This linearity holds over a larger range of protein d u e s than usually considered valid. To subject a large concentration of a protein to electrophoresis, the papers were striped several times with 10-pl. portions of protein solutions.
Ten-microliter aliquots were applied rather than a single larger volume in order to minimize the area of the sample on the strip. The total amount of sample in terms of micrograms of protein nitrogen applied to the strip in each electrophoresis experiment is given in the fourth column of Table I. The average value n a s 1.1 i 0.1 y of protein nitrogen per one integration unit (0.1 sq. cm.), regardless of the kind of protein sample. The method of least squares m s applied, to obtain thc cquation of the best straight line through the experimental points plotted in Figure 3. The equation Y = 0.922x
(1)
represents the data where Y is the integrated area in square centimeters X 10-1 and X is the concentration of the protein subjected to electrophoresis eypressed in micrograms of protein nitrogen. LITERATURE CITED
Albert, -I.,BiochenL. J . 50, 690 (1952). (2) Block, ,R. J., lhrrum, E. L., Zweig, G., Manual of Paper Chromatography and Paper Electrophore-
(1)
VOL. 29, NO. 12, DECEMBER 1957
1819
sis,” p. 4018, Academic Press, Xew S’ork, 1955. (3) Cooper, G. R., hladel, E. E ., J .
Lab. Clin. Med. 44, 636 (19541. Bioc:hem. (4) Cremer, H. D., Tiselius, 8., 2. 320, 273 (1950). (5) Crook, E. M.,Harris, H., Hassan, F., Warren, F. L., Biochem. J ‘. 56, 434 (1954).
Crook, E. ll.,Harris, H K arren, F. L., Ibid, 51, xxvi (19k2). Durrum, E. L., J . Am. Chem. SOC. 72,2943 (1950).
Franglen, G. T , Martin, N. E., Biochem. J . 57, 626 (1954).
Gornall. A. G.. Bardawill. C. J.. Grassmann,’k., Hannig, K., Klin. Wochschr. 32, 838 (1954).
(11) Griffiths, L. L., J . Clin. Pathol. 5, 296 (1952). (12) Jencks, W. P., Jetton, M. P., Durrum, E. L., Biochem. J . 60, 205 (1955). (13) Kunkel,’ H. G., Tiselius, A,, J . Gen. Phwiol. 35, 89 (1951). (14) Latner, A. L:, Molyneux, L., Rose, J. D., J . Lab. Clin. Med. 43, 15i (1954). (15) Mellor, D. P., Maley, L., A-ature 161, 436 (1948). (16) Rees, V. H., Laurence, D. J. R., Clinical Chem. 1, 329 (1955). (17) Sommerfelt, S. C., Scand. J. Clin. and Lab. Invest. 5, 299 (1953). (18) Von Frijtag Drabbe, C. A. J., Reinhold, J. G., ANAL.CHEX 27, 1090 (1955).
RECEIVED for review October 25, 1956. Accepted June 17, 1957. Presented in part at Fortieth Annual Meeting, Federation of American Societies for Experimental Biology, Atlantic City, N. J., March 1956. Investigation supported in part by funds from the Office of the Surgeon General, and carried out in one of the laboratories of the Southern Utilization Research Branch, Agricultural Research Service, U. s. Department of Agriculture. Trade names have been used only to identify equipment or materials actually used in the work, and such use does not imply endorsement or recommendation by the U. S. Department of Agriculture over other firms or similar products not mentioned.
Classification of Organic Compounds Based on Behavior of a Solvochromic and Thermochromic Indicator System SAUL SOLOWAY and PERRY ROSEN Deparfmenf o f Chemistry, City College, College o f the Cify of New York, New York, N.
b
Organic compounds are classified according to their effectiveness in promoting or inhibiting chelation between ferric chloride and n-propyl gallate. If this classification is used in conjunction with others based on solubility, elementary analysis, and acid-base indicators, 19 out of 28 types of compounds are identifiable as unique. Some examples are alcohols, phenols, carboxylic acids, amides, nitro compounds, and nitriles. Some aliphatic compounds may be distinguished from their aromatic counterparts on the basis of their stronger inhibitory properties. Explanations are offered for the apparently anomalous behavior of some functions which behave as promoters or inhibitors of chelation, depending on their concentration,
F
qualitative analytical purposes organic compounds are grouped according to their elementary composition, solubility in specified solvents (IO), and acid-base strength ( 2 ) . Such preliminary classifications are not only helpful in suggesting possible functional groups in a n unknown compound, but may also give indications of molecular weight, polyfunctionaIity, and bond type. Recent work on the solvochromic and thermochromic behavior of an indicator system ( l a ) suggested another preliminary classification to aid the analyst OR
1820
ANALYTICAL CHEMISTRY
further in inferring the presence of certain functional groups. The indicator system consists of ferric chloride, npropyl gallate, and o-chloroaniline. The solutions may be blue or yellow throughout the entire temperature range of the liquid state, or they may exhibit a reversible thermochromic transition from blue to yellow. The solvent and temperature control the extent of chelate formation between the indicator components. As the stoichiometry of the reaction is not precisely known, the chemistry is summarized by the following word equation. Ferric chloride (yellow)
Y.
dants, organic as well as inorganic, caused irreversible destruction of the indicator. This was recognized by the formation of a permanent red-brown color. EXPERIMENTAL
Indicator Solutions. Indicator A was made by dissolving 2 grams of anhydrous ferric chloride and 4 grams of n-propyl gallate in 100 ml. of warm glacial acetic acid. Then 1.5 ml. of acetyl chloride were added, and the solution was quickly filtered through a large piece of fluted paper, t o mini-
+ propyl gallate + chloroaniline e chelat,e- + chloroanilinium ion (colorless) (colorless) (blue) (colorless)
The designation of the products as ions is a matter of conjecture. It is thus written to emphasize the function of the amine as a proton acceptor. The effectiveness of a given compound toward promoting or inhibiting chelate formation in the indicator system was studied in two solvent media as a function of temperature: one in which the compound itself was substantially the total solvent, and the other in which it was a further diluent to a bromobenzene solution of the indicator. Although most substances were either promoters or inhibitors of chelation in both media, most alcohols and amides were promoters as bulk solvent but inhibitors in dilute hromobenzene solution. Some oxi-
+
mize contact with moisture in the air. The final solution was a light brown. It darkens rapidly on exposure t o a moist atmosphere, because water is a strong promoter of chelate formation. This indicator has been used t o detect water in alcohols and t o determine their relative basicities ( I S ) . Indicator B was prepared b y adding 2 ml. of indicator A, 1 ml. of benzene sulfonyl chloride, and 1 ml. of ochloroaniline t o 90 ml. of bromobenzene. The final solution or possibly colloidal dispersion was blue. As some solid matter separates with time, the mixture is well shaken before use. The change of color with temperature was somewhat sharper after t h e solution had been aged several days. T h e relation of this phenomenon t o sulfon-
