Determination of Guaiacol in Presence of large Amounts of Catechol DAVID H. ROSENBLATT, MARY M. DEMEK, and JOSEPH EPSTEIN Chemical Corps Medical Laboratories, A r m y Chemical Center,
A method is described for the determination of guaiacol in the presence of catechol. The reagent used is 4aminoantipyrine, with which guaiacol gives a chloroform-extractable dye.
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T WAS of interest to this laboratory to estimate small quantities of guaiacol in the presence of relatively large amounts
of catechol. Of all known methods for determining phenols, the Emerson method (%, 4 ) alone was known to give virtually negative tests rvith catechol. Thus it was to be expected that under suitable conditions only the guaiacol of a guaiacol-catechol mixture xould give a positive test-namely, a chloroform-estractable dye, with 4aminoantipyrine.
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Pipet off each chloroform layer and measure its absorbance a t 460 mp. Construct a curve by plotting absorbance readings versus guaiacol concentration. Determination of Unknown. Adjust the unknown solution to the catechol concentration used in preparing the standard curve, The method for determining catechol concentration is referred to below. Proceed as above, and read the concentration of guaiacol from the standard curve. It is recommended that prior to the preparation of the standard curve the catechol concentration of a portion of the unknown solution be adjusted to 2.5 X 10-4AI, and the present procedure followed. If the absorbance is above 1.5, the unknown solution should be diluted and the procedure repeated. The standard curve should be made a t the diluted catechol concentration. EXPERIMENTAL
APPARATUS
The Beckman Model G p H meter with No. 1 l i O calomel electrode and No. 1190-42 glass electrode was used for pH measurement. Reactions were run in 400-ml. beakers with Tefloncovered magnetic stirring bars for mixing. A Klett-Summerson photoelectric colorimeter with a No. 44 filter and a Beckman Model DU spectrophotometer with quartz cells of 1-em. light path were used for photometric measurements. REAGENTS
Sodium carbonate, reagent grade, 2M. ilmmonium hydroxide, reagent grade, 6M. Catechol, recrystallized twice from toluene, melting point 105" C. Guaiacol, N.F. VIII. 4-Aminoantipyrine reagent, a O.i5% solution, melting point 108" C., in distilled water, made up freshly each day. Potassium ferricyanide reagent, a 2% solution of potassium ferricyanide, c.P.,in distilled water. Chloroform, C.P. Kater, distilled and then redistilled from alkaline potassium permanganate.
The present work is an adaptation of the procedure of Ettinger et al. ( 4 ) . Some simplification of that procedure was effected by the use of a single chloroform extraction carried out in a beaker, with vigorous stirring. This was done to avoid contamination by stopcock grease, which was found to impart color to the chloroform extract. As a base, sodium carbonate gave lower reagent blanks and greater consistency than did ammonia (Figure 1). The choice of pH 10.45 to 10.55was made because lower pH values gave higher ratios of blank to net reading, Tvhile higher values lowered the sensitivity of the test. As indicated by previous authors ( 4 ) , the chloroform solutions do not fade noticeably in 24 hours. 7,
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PROCEDURE
Preparation of Standard Curve. Nake up fresh solutions containing a constant concentration of catechol (not more than 2.5 X 10-4X) and concentrations of guaiacol varying from 0 to 0.6 p.p.m. To 250 ml. of each of these solutions in 400-ml. beakers add sufficient 2M sodium carbonate to adjust (with p H meter) to pH 10.45 to 10.55. Add exactly 2.0 ml. of 4-aminoantipyrine reagent to each solution and stir well To each solution add 5 ml. of potassium ferricyanide reagent, stir vigorously for a few seconds, and immediately add 12.0 ml. of chloroform. Stir well for 5 minutes and allon- the chloroform to settle 0.5 minute.
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* - Na2C03 ADJUSTMENT
NH40H ADJUSTMENT
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Figure 1. Variation of Reagent Blanks with pH
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CATECHOL
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400
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WAVE LENGTH
460
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Figure 2. Absorption Spectra of Chloroform Solutions Resulting from Reaction with 4-Aminoantipyrine
Spectral examination showed that the optimum wave length for spectrophotometric determinations is 460 mp (Figure 2). When the test was run on solutions containing various amounts of catechol and no guaiacol, spectrophotometric values reached a maximum a t about 3.5 X 10-4M catechol (Figure 3). With a constant quantity of guaiacol and varving amounts of catechol, the spectrophotometric readings passed through a maximum at 2.5 X 10-4M concentration of catechol. Net values for guaiacol -i.e., absorbance of catechol-guaiacol solutions minus absorbance of corresponding solutions without guaiacol-were constant up to about 2.5 X l O - 4 X concentration of catechol, showing a decrease thereafter. The decrease can be explained by the competitive consumption of 4-aminoantipyrine by the large amount of catechol. 1655
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ANALYTICAL CHEMISTRY
The curve of net absorbance versus guaiacol concentration a t 10-4M catechol concentration in Figure 4 shows adherence to the Beer-Lambert law up to a t least 0.6 p.p.m. of guaiacol. Experiments were not extended above this figure because of the decreasing accuracy of color measurement a t low transmittances. Sensitivity (net values) over the range 0.09 to 0.67 p.p.m. of a guaiacol was 0.252 absorbance units per 0.1 with the spectrophotometer and, over the range of 0.02 and 0.45 was 129 colorimeter units per 0.1 p.p.m. with the colorimeter, both in the presence of 1 x 10-4M catechol. Sensitivity was virtually the same in the absence of the latter. A comparison of results obtained a t 0.122 p.p.m. of guaiacol and 1 X 10-4M catechol from 17 samples showed a mean net absorbance of 0.32, and a standard deviation of 0.02 for the Beckman spect,rophotometer; results from 37 samples read on the Klett-Summerson colorimeter gave a mean net of 162, and a standard deviation of 5 colorimeter units. Uniform results were obtained only if the solutions containing catechols were less than 3 hours old.
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MOLARITY
Figure 3.
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OF CATECHCL X l O '
Effect of Variation of Catechol Concentration on Color Developed at 460 -Mp
Where concentrations of guaiacol higher than 0.6 p.p.m. are to be determined without prior dilution, it is possible to use more chloroform, thereby bringing the absorbance within the instrumental range. It is conceivable that cases may exist where the concentration of catechol is not previously known. As it is important that not too much catechol be present, its concentration should be determined by some reliable method; the Arnow test ( 1 ) proved satisfactory for the purpose in this laboratory. [A reviewer has pointed out that the Emerson method ( 2 ) ,without chloroform extraction, could be used to estimate catechol plus guaiacol. This is probably correct.] Alternatively, an attempt was made to apply the Gibbs method (3,5 ) to the determination of catechol (or of catechol plus guaiacol). The lavender color produced by the reaction of catechol with 2,6-dibromoquinonechloroimide was not extractable with 1-butanol, although guaiacol reacted to give an extractable blue dye. The aqueous catechol dye solution was so sensitive to light that i t was impossible to determine its absorption spectrum; the absorption maximum of the aqueous guaiacol derivative occurred a t about 580 mp. However, a solution containing the Gibbs reagent, 0.1 p.p.m. of guaiacol, and 20 p.p.m. of catechol exhibited an unexplained new maximum near 420 mp. rllthough catechol did not give an extractable dye, it was impossible to use this property for the estimation of guaiacol in the presence of catechol. Thus 5 p.p.m. of guaiacol gave four times as much extractable blue color as the same concentration in the presence of 10 p,p.m. of catechol. DISCUSSION
The basis for the difference between the reactions of guaiacol and catechol with 4-aminoantipyrine under the influence of
PARTS PER MILLION GUAIACOL
Figure 4. Color Developed with Varying Concentrations of Guaiacol In presence of 10-4 M catechol
alkaline ferricyanide lies primarily in the nature of the dye formed. The dye derived from catechol is extremely acidic, existing as an anion a t a high pH, while the guaiacol derivative, which has no ionizable hydrogen, is neutral and preferentially soluble in chloroform under comparable conditions (see Figure 5). The method presented should therefore be applicable, with but slight modification, to mixtures other than those of catechol and guaiacol. The behavior of certain other polyphenols should be analogous to that of catechol; thus resorcinol should form a chloroform-insoluble dye like the catechol derivative shown in Figure 5. Many monophenolic substances-such as those already investigated by previous authors ( 2 , 4)-may prove determinable in the presence of catechol and other polyphenols. I n these cases it will be necessary to use the wave lengths of maximum absorption of the particular dyes formed, since these are known to vary (4).
GUAIACOL DERWATIVE
CATECHOL DERIVATIVE
Figure 5. Dyes Formed in .41kaline Solution with 4-Aminoantipyrine
I t is believed that the present method will be of value in the estimation of small amounts of monophenolic impurities in certain polyphenols, as well as in the study of the kinetics of reactions between these polyphenols and 0-alkylating or acylating reagents. ACKNOWLEDGMENT
The authors wish to express their appreciation of Privates First Class Peter Hlinka, Roy Campbell, and Francis Costello for their technical assistance. LITERATURE CITED (1) Arnow, L. E . , J . Bid. Chem., 118, 531 (1937). (2) Emerson, E.,J . Org. Chem., 8, 417 (1943). (3) Ettinger, M . B . , and Ruchhoft, C. C., A x . 4 ~ .C H E b f . , 20, 1191 (1948). (4) Ettinger, M . B . , Ruchhoft, C. C., and Lishka, R. J . , Ibid.,23, 1783 (1951). (5) Gibbs, H . D . , J. B i d . Chem., 72, 649 (1927). RECEIVED for review December 21, 1953.
Accepted J u n e 16, 1954.
