By HORACE D. GRAHAM and LINNIE B. THOMAS? Gibberellic acid reacts with acidic 2 .... all additions. 2,F-Dinitrophenylhydrnzine Reagent.-Five milli-. 44 ...
Rapid, Simple Colorimetric Method for the Determination of Micro Quantities of Gibberellic Acid By HORACE D. GRAHAM and LINNIE B. THOMAS? Gibberellic acid reacts with acidic 2,4-dinitrophenyIhydrazineto yield a product, presumably the 2,4-dinitrophenylhydrazoneof gibberic acid which, when treated with alcoholic potassium hydroxide, gives rise to a stable wine-red color the intensity of which is proportional to the amount of gibberellic acid present when measured at 430 or 540 mp. The temperature of heating the gibberellic acid with acidic 2,4-dinitrophenylhydrazineis very critical. At 70' very little or no color development occurs. At 80" much longer periods are required for maximum color development on alkali treatment as compared to heating at looo, where only a fiveminute period of heating is necessary for maximum color development. The effect of several variables on the reaction was thoroughly investigated and conditions for reproducibility established. Indoleacetic acid, 2,4-dichlorophenoxyacetic acid and kinetin failed to give the typical wine-red color when heated at 100' for five minutes under identical conditions.
ESPITE A PLETHORA of
literature on the effect of gibberellic acid on plants, the mechanism of action of this plant growth regulator has not yet been elucidated. Recently, studies on growth responses resulting from gibberrellic acid and 2,4-dichlorophenoxyaceticacid interaction have been reported (1) and the stability of gibberellic acid t o heat at various pH levels has been questioned (2). To facilitate studies on the mechanism of action of gibberellic acid, and for its rapid assay i n pure solutions or in the presence of other plant growth regulators, much simpler and more rapid and sensitive tests than those now available (3-7) are desirable. This communication describes an approach t o such a need. Gibberellic acid, when subjected t o acid hydrolysis a t 100' produces gibberic acid, a ketonic compound. The use of 2,4-dinitrophenylhydrazine t o isolate and characterize small quantities of aldehydes and ketones responsible for off-flavors in foods, food products, and other biological materials is well known (8-16). Spectrophotometric procedures based on the alkaline 2,4-dinitrophenylhydrazine method have been applied t o t h e quantitative determination of aliphatic and aromatic carbony1 compounds (17-21) and steroids @). Preliminary experiments showed that, with adequate control, the wine-red color produced Received May 6, 1960, from The George Washington Carver Foundation, Tuskegee Institute, Alabama. Accepted for publication June 24, 1900. t National Science Foundation undergraduate research partidpant. The authors wish t o thank Merck and C o . , Inc., Abbott Laboratories, Eli Lily and Co., and Velsicol Chemical Corp. for samples of gibberellic acid, gibberellin, gibberellin Ax, and Gibrel. A portion of this research was supported by a grant from the Research Corporation, N. Y .
44
on the addition of alcoholic potassium hydroxide t o the product resulting from heating gibberellic acid with acidic 2,4-dinitrophenylhydrazine at 100' for five minutes could be made the basis of a rapid, simple, reproducible, and highly sensitive test for gibberellic acid, gibberellin, gibberellin A1, and the potassium salt of gibberellic acid. Moreover, under the prescribed conditions, indoleacetic acid, 2,4-dichlorophenoxyacetic acid, kinetin, and other auxins do not give the characteristic wine-red color.
EXPERIMENTAL Materials.-Gibberellic acid, supplied by Eli Lily and Co. and Abbott Chemical Laboratories. Gibberellin, supplied by Velsicol Chemical Corp. and Chas. Pfizer and Co., Inc. Potassium gibberellate, 75y0 potassium salt, supplied by Nutritional Biochemicals Corp. Gibrel, supplied by Merck and Co. Gibberellin A I , supplied by Abbott Laboratories. Unless otherwise stated, stock 400 mcg./ ml. solutions of the above were prepared in carbonyl-free alcohol. When necessary, further dilutions were made with carbonyl-free alcohol. 2,4Dinitrophenylhydrazine (2,4-DNPH), supplied by Eastman Organic Chemicals. Carbonyl-Free Methyl Alcohol QY Carbonyl-Free Ethyl AZcohoL-Five hundred inilliliters of the desired alcohol, 5 Gm. of 2,4-dinitropIienylhydrazine, and 1.0 nil. of concentrated hydrochloric acid were refluxed for three hours. The alcohol was distilled, the fraction boiling at 64.7-65" collected and redistilled. Alcoholic Potassium H31droxide lO%.-Ten grams of potassium hydroxide (U. S. P. pellets, Fisher Scientific Co.) was dissolved in 20 ml. of doubledistilled water and the contents made up to 100 ml. with carbonyl-free alcohol. The 100-ml. volumetric flask used was kept in an ice bath during all additions. 2,F-Dinitrophenylhydrnzine Reagent.-Five milli-
Vol. 50, No. I , Janztary 1961 liters of concentrated hydrochloric acid w a s added to 100 mg. of 2,4-dinitrophenylhydrazine and 50 ml. of carbonyl-free alcohol. The mixture was gently heated until all the 2,4-dinitrophenylhydrazine was dissolved, cooled, and diluted to 100 ml. with carbonyl-free alcohol. A new batch of reagent was prepared every five days. Equipment.-Pyrex, 20-ml., ground-glass-stoppered test tubes, constant temperature water bath or suitable oil bath, volumetric pipets, and Coleman Universal spectrophotometer, model 14. Procedure.-A 1-ml. portion containing 40-400 mcg. of gibberellic acid or gibberellin was placed in each of seven Pyrex, ground-glass-stoppered test tubes. A control tube containing 1 ml. of carbonylfree alcohol instead of gibberellic acid was included. One milliliter of the 2,4-dinitrophenylhydrazinereagent was added to each tube and the mixture heated a t 100" for exactly five minutes. Zero time was taken as thirty seconds after the immersion of the last tube. At the end of this period, the tubes were rapidly removed and placed in an ice water bath for five minutes. A thick oily droplet condensed and settled to the bottom of the tube. Five milliliters of of 10% potassium hydroxide in 80% carbonyl-free methyl alcohol was rapidly added t o each tube; the contents were mixed well and allowed to stand for five minutes. A wine-red color developed indicating a positive test. Fifteen milliliters of doubledistilled water were added to each tube, the contents mixed well, diluted 1:2 with distilled water, and the intensity of the color measured at 430 and 540 mfi with a Coleman Universal spectrophotometer, model 14, using a reagent blank. The wavelength of maximum absorption was determined by plotting the absorbance as a function of the wave length as shown in Fig. 1. Here the total volume of the colored solution was 20 ml. and distilled water was used as the blank.
45 scribed by the following logarithmic least squares equations: x 4 ,
=
X b 4 0
=
2.0031 - log y 0.072124
o20
1.99062 - log 7 0 TX.540 0.04914
where X = concentration of gibberellin in mcg./ml. and 430 and 540 mp are the respective wavelengths a t which the intensity measurements were made. Similar equations can be derived for gibberellic acid, gibberellin AI, and potassium gibberellate. For the assay of unknown samples, the same procedure can be used after any necessary dilution. The amount of gibberellic acid present can then be determined by use of the standard curve or the logarithmic least squares equation. TABLE RELATIONSHIP BETWEENCONCENTRATION OF GIBBERELLIN AND COLOR INTENSITY Concentration of Gibberellin, mcg./ml.
1.0 2.0 4.0 6.0 8.0 10.0
Transmission, % (Average of Triplicate Readings) At 430 mp At 540 rnF
86.5 71 . O 52.0 37.0 26.5 18.9
88.5 78.0 61.5 50.0 40.5 31.0
INFLUENCE OF VARIABLES ON COLOR DEVELOPMENT Development of the wine-red color is dependent on close control of several factors. Since any change of several variables will either influence or totally eliminate color development, i t was found necessary to study critically such variables and select appropriate conditions for reproducibility. Temperature of Heating.--In order to study this variable, 400 rncg. of gibberellin in a volume of 1 ml., and 1 ml. of the 2,4-dinitrophenylhydrazine reagent were treated according to the general procedure a t 100, 90, 80, and 70'. The final concentration after dilution with double-distilled water was 10 rncg./ml. (total volume 40 ml.). The results recorded in Table I1 indicate that heating for as short a period as five minutes a t 100" TABLEII.-EFFECT OF TEMPERATURE OF HEATING ON COLOR DEVELOPMENT
WAVELENGTH (Mp)
Fig. 1.-Absorption spectrum of color produced by gibberellin in the alkaline 2,4-dinitrophenylhydrazine method. I, 0 mcg./ml.; 11, 2 mcg./ml.; 111, 4 mcg./ml.; IV, 6 mcg./rnl.; V, 8 mcg./ml.; VI 10 mcg./ml.
Over the concentration range employed, Beer's law was obeyed as indicated by the linear relationship resulting when the logarithm of the per cent transmission is plotted as a function of concentration (data in Table I). This relationship can be de-
Duration of Heating, mill.
I
Transmission at 430 mq, yo Temperature, OC. 90 80
70
1
No 53 .color _ . ~ . ~ ..
3
No color Nocolor No color Nocolor Nocolor Nocolor No color No color No color Very little color
5 7 10 15 20 30 40 50 60
_._
34 2
...
28.5 26.5 21.0 19.5 19.0 19.5 19.0
48.0 41.5 29.2 28.5 20.5 20.0 19.5 19.5 19.0 19.8 19.2
100
42.0 20.5 20.0 20.2 20.0 19.6 19.8 20.0
.. .. ..
46 -
Journal of Pharmaceutical Sciences TABLEIII.-INFISJENCEOF VARIABLES UPON COLOR DEVELOPED IN TESTFOR GIBBERELLIC ACID
Variable
Limits for Reptoducibility Min. Max.
Range Investigated
Concentration of 2,4-DNPH, mg./rnl. 0.1-2.0 Concentration of KOH in 80% alcohol, yo 1 .O-20 Final alcohol concentration, % 1.0-80 Diluent for color developed HzO, carbonyl-free McOH, alcoholic KOH Acidity of 2,4-DNPH reagent, N 0.1-2.0 Color stability 0.0-48hr.
produced maximum color development. This condition was selected as the most suitable since even after twenty minutes heating at this temperature very little difference in absorbance was noted. Failure of color development at 70" and the comparatively slower color development at 80 and 90" make these temperatures less suitable for rapid work. Concentration of Potassium Hydroxide.-From Table 111, it is evident that under the experimental conditions employed, the concentration of potassium hydroxide employed should be between 515%. Below 5%, color intensity was reduced due, apparently, to incomplete alkalinization of the 2,4dinitrophenylhydrazone. A t a concentration of 20%, color intensity increased slightly. In view of the constancy of color intensity when the potassium hydroxide concentration is 5-15%, a concentration of 10% was selected. Concentration of 2,4-Dinitrophenylhydrazine.With a maximum final concentration of 10 mcg./ml. of gibberellic acid, in a total volume of 40 ml., 0.41.0 mg. of 2.4-dinitrophenylhydrazine should be present in the 1 ml. of the reagent used; 1 mg. per ml. was selected for use. Table I11 summarizes the results. Influence of Alcohol Concentration.-When the potassium hydroxide concentration was kept cotistant at loyo, maximum color intensity was realized at alcohol concentration of 20-80Y,. At alcohol concentrations below 20y0 color intensity decreased, and above 80% precipitation of salt occurred. The results are summarized in Table 111. Influence of Acid Strength of the 2,4-Dinitrophenylhydrazine Reagent.-The acidity of the 2,4dinitrophenylhydrazine reagent proved critical for color development, as shown by Table 111. For good color development, the reagent should be 0.2-1.5 N with respect to hydrochloric acid. Below 0.2 N poor color development, or no color development a t all, occurred. Above 1.5 N color intensity diminished, as compared to that obtained with 0.21.0 N . For future experiments a reagent which was 0.5 N with respect to hydrochloric acid was chosen. the Stability of the Color Developed.-Under experimental conditions employed, the color developed was found to be stable for as long as fortyeight hours when allowed to stand a t room temperature (28 =!= 1") in ground-glass-stoppered test tubes. Therefore, measurement of color intensity can be made at any time within this period without incurring serious errors. Selection of a Suitable Diluent for the Color Developed.-For diluents the alcoholic potassiuni hydroxide, carbonyl-free methanol, or distilled
0.4 2.0
.. . . ..
0.2
...
Value Selected
2 . 0 1.0 20.0 10.0 80 12 . . . HzO Selected 1.5 0.5 10 min. after color development
...
water could be used. With alcoholic potassium hydroxide, further addition may lead t o nonreproducibility of results due to salt formation. Carbonyl-free alcohol may also cause difficulties in salt formation, hence water was selected as the most readily available and suitable diluent. Influence of Water in the Reaction Vessel.-When water was present in the medium, color development was poor or totally eliminated. For this reason, all reagents were diluted with absolute methanol or ethanol. For practical purposes, however, experimental gibberellic acid systems are usually prepared in buffers or systems of low alcohol concentration. By evaporating solutions containing gibberellic acid or gibberellin a t 105" for as long as twenty-four hours, then adding 1 mI. of carbonylFree alcohol to the dry system, the color developed according to the general procedure showed no important deviation from that given in the absolute systems (Table IV). Therefore, this procedure is recommended for direct assay of aqueous systems.
TABLE IV.-EFFECT OF DEHYDRATION AT 105°C. O N COLOR INTENSITY OF POTASSIUM GIBBERELLATE SOLUTIONS Transmission, % A t 540 ma
Time of Heating, hr.
At 430 m u
1 2 3 4 8 12.0 16.0 24.0
36.5 20.5 20.8 20.4 20.2 20.5 20.2 22.5
55.5 38.5 38.0 38.2 39.0 39.0 40.0 40.5
Influence of Various Salts Commonly Used as Buffers.-Gibberellic acid, when applied t o plant systems as sprays or droplets, is usually incorporated in buffers of various sorts. It was, therefore, advisable to assess the effect of these on the intensity of the color developed. One milliliter of each of the buffers listed in Table V was placed into glass-stoppered test tubes. One milliliter containing 400 mcg. of potassium gibberellate dissolved in water was added to each test tube and the contents of the tube evaporated to dryness in an electric oven at 105". After evaporation, 1 ml. of carbonyl-free methanol and 1 ml. of the 2,4-dinitrophenylhydrazine reagent were added t o each test tube and the color developed according to the general procedure. From Table V it is seen that the citrate and
V d . 50, No. 1, January 1961
47
Table V.-EFFECT OF VARIOUS SALTSON COLORINTENSITY IN THE DETERMINATION OF POTASSIUM GIBBERELLATE B Y THE ALKALINE2,4-DIXlTROPHENYLHYDRAZINE METHOD 7 -
-
Transmission at 430 mp. % Normality0.10 0.05
0.01
Salt Used
0.2
Ammonium citrate Ammonium sulfate Ammonium acetate Disodium hydrogen phosphate Sodium chloride Potassium acid phthalate Potassium chloride Sodium carbonate Sodium bicarbonate Boric acid HzO potassium gibberellate Carbonyl-free MeOH potassium gibberellate Carbonyl-free MeOH only HzO only
12.2 20.8 20.0 22 .o 20.4 10.0 22.0 22.0 21 .o 20.8 20.8
14.2 20.2 20.6 20.8 20.6 10.6 20.6 22.0 21.2 20.6 20.2
14.6 20.4 20.2 20.6 20.2 11.0 20.2 21.8 20.4 20.2 20.4
14.4 20.0 20.6 20.2 20.0 14.0 20.0 22.0 20.2 20.2 20.2
...
20.4 80.4 79.8
...
...
...
...
+
+
...
...
phthalate ions will interfere rather seriously in color development. Such interference is due t o colors produced by the ions themselves on heating and not to any interaction with the gibberellic acid. The absence of interference from several other salts, however, makes it possible to conduct direct assays in a variety of buffer solutions. Tween 20, commonly incorporated into gibberellic acid buffered media, when subjected to the same experimental conditions, gave an intense color which will inter/ere severely. Significance of a Small Volume.-Although 5-10 ml. of alcohol can be used with the same ultimate relative results, due mainly to the low-boiling point of methanol, the total volume should preferably be minimized to 0.5-2.0 ml. in order to make the blank reading as low as possible. This objective is also further attained by diluting up t o 40 ml. after addition of alkali. In addition, such extensive dilution with water facilitates solution of any salt formed in the alcoholic medium. I n cases where the assay of lower concentrations of gibberellic acid must be performed, the procedure can be conveniently scaled down to accommodate determinations. Relationship Between Concentration of Gibberellin and the Intensity of the Color Developed.After the conditions for optimal color development were established, aliquots necessary t o give final concentrations of gibberellin shown in Table I were placed in duplicate glass-stoppered test tubes and the color developed and measured according t o the general procedure. I t can be seen that when the logarithm of the per cent transmission is plotted as a function of the concentration, a straight line results indicating compliance with Beer's law over the concentration range used and that a quantitative relationship exists.
...
...
Nature of Color Produced
Off-color Wine-red Wine-red Wine-red Wine-red Off-color Wine-red Slight off-color Wine-red Wine-red Wine-red Wine-red
...
...
hydrochloric acid has also been recorded (23). It is. therefore, postulated that the 2,4-dinitrophenylhydrazone formed is that of gibberic acid. This is substantiated by the fact that a t 70°, the wine-red color was not obtained and at 80 and 90' much longer heating times were required as compared to heating at 100'. When gibberdlic acid was treated with nonacidic 2,4-dinitrophenylhydrazine and alcoholic potassium hydroxide added as in the general procedure, the wine-red color did not develop. Control tubes containing 2.4-DNPH only gave a yellowish-green color on the addition of potassium hydroxide. This constitutes evidence in favor of the proposed mechanism. The characteristics common to the chromogen from gibberellic acid, gibberellin, and the potassium salt of gibberellic acid lend further credence to the postulate that the 2,4-dinitrophenylhydrazoneof a common ketonic compound, gibberic acid, is formed. However, final and unequivocal proof of this postulate will depend upon more comprehensive characterization of the hydrazone. The exact mechanism for formation of the wine-red color of the 2,4-dinitrophenylhydrazones in strong alkali is not known. Friedemann and Haugen (11) think that it is due to substitution of the nitro group in the para position of the benzene ling. Chataway and Clemo (25) presented an alternate explanation which has been widely endorsed by other workers (19,26). According to this school of thought, the color change results from the extraction of a proton and the formation of a resonating quinoidal ion. The data presented indicate clearly that, under the specified conditions, the proposed reaction can be used for a rapid, simple, reproducible, and highly sensitive method for the quantitative determination of gibberellic acid. Since gibberellin, gibberellin A1, and the potassium salts of gibberellic acid gave DISCUSSION AND SUMMARY positive tests, the method may be used to estimate Mechanism of the Reaction.-Gibberellic acid all these forms. Although the reaction must be readily loses carbon dioxide in the presence of hot carried out in an anhydrous medium, the simple and hydrochloric acid, producing allogibberic acid, from expedient process of dehydrating aqueous solutions a t 105", without significant destruction of the growth which gibberic acid, a ketonic compound, can result through the Wagner-Meerwin rearrangement (23, regulator, permits ready assay in experimental buf24). Direct production of gibberic acid from fer systems. In addition, most of the common salts gibberellic acid under the influence of hot dilute used as buffers do not interfere severely when present
Journal of Pharmaceutical Sciences
48 TABLEVI.-SUMMARY
OF REACTIONOF VARIOUS conditions were found t o give optimal and rePLANT GROWTHREGULATORS IN ALKALINE2,4producible results: heating a n anhydrous, alDINITROPHENYLHYDRAZINE METHOD coholic solution of the growth regulator with Production of 1 ml. of alcoholic 2,4-dinitrophenylhydrazine Wine-Red Compound Tested X Maximum Color reagent for five minutes, adding 5 ml. of 10 per Gibberellic acid 430,540 cent alcoholic potassium hydroxide, dilution Gibberellin A1 430,540 with distilled water, and measuring the color 430,540 Gibrel (dissolved in alcohol) f 430,540 intensity at 430 or 540 mp against a reagent Gibrel (dissolved in H20) Potassium gibberellate blank. Direct assay of aqueous or buffer sys(dissolved in H30) 430.540 tems can be accomplished after dehydration Gibberellin 430; 540 Indoleacetic acid 390 a t 105' for two hours. Most common salts Indolebutyric acid 390 used as buffers do not interfere. Indolepropionic acid 390 Kinetin 390 Indoleacetic acid, 2,4-dichlorophenoxyacetic 2,4-Dichlorophenoxyacetic acid, and other plant auxins do not give the 390 acid characteristic wine-red color. Tween 20 and Tryptophan 390 other polyoxyethylene-type compounds will interfere. T h e Spans do not interfere. in the medium a t the tested concentrations of 0.01REFERENCES 0.2N. Citrate and phthalate should be avoided (1) Clor, M . A,, Currier, H . B., and Stocking, C. R . , as these give rise to severe off-colors. Tween 20, Gas., 120, SO(1958). a nonionic surfactant, widely used as an emulsifier Bofan. (2) Henderson J. H . M Nature, 185,628(1960). (3) Arison B . ' H . Spe;h. 0. C., and Trenner, N. R., in gibberellic acid experimental solutions interferes Ckem., $0,1083(1958). drastically. All polyoxyethylene surface-active Anal. (4) Baumgartner W. E. Lazer L. S. Dalziel A. M. agents should be avoided. If necessary, the Spans, Cardinal, E. V., and Varner: E . L.,' J . A&. Food kkem., 7: 422 (1959). sorbitan-fatty acid surfactants, are recommended. (5) Head, W. F.. THISJ O U R N A L , 48,631(19591. (6) Kavanagh, F., and Juzel, N. R . , J . Agy. Food Ckem., Negative results obtained with important plant 6,459(1958). auxins (Table VI) are of particular significance in (7) Washburn, W. H., Scheske, F. A., and Schenck, J. view of the fact that fundamental studies on gib- R. J., ibid., 7,420(1959). (8) Case, E. M., Biochem. J . , 26,753(1932). berellic acid-auxin interactions are now gaining (9) Day, E. A., Bassette, R., and Kenney, M., J . Dairy Sci.,43,463(1960). accelerated prominence. (10) Ellis, R . , Gaddis, A. M., and Currie, G. T., Anal. As seen from Fig. 1, color intensity measurements Chem., 30,475(19,58). (11) Friedemann T. E., and Haugen, 0 . E., J . B i d . may be made at 430 or 540 mp. The former peak Ckem.. 147.415f194kl. is the sharper of the two but is relatively closer t o (12j F r i k J.' S., ~Yumamura,S. S., and Bradford, E . E., the absorption maximum of the blank which is at Anal. Chem., 31, 260(1959). (13) Lu, G. D . , Biockem. J . , 33,249(1939). 390 mp. Despite its lack of incisiveness, the (14) Steinherg, M . A . , Livingston, G . E., and Fellers, 540 mfi peak permits measurement in a range C . R . , Food Teck., 10,475(1956). (15) Walker, J. R. L., and Harvey, R . J . , J . Dairy Research, where the blank contribution is almost negligible. 26.266f19.59) ~,~ ~ ~ , ~ . . (16) Witting, I.. A., and Schweigert, B. S., J . A m . Oil The proposed method is suitable for the assay Chemzsfs' Soc. 35,415(1958). of extracted samples of gibberellic acid or gibberel(17) Greenberg, L. A., and Lester, D., J . B i d . Ckem., 154, lins and will lend itself to fundamental studies on 177(1844). Kolecnikov, P. A., Biockem., (English translation), (18) plant auxin interaction studies. 22,580(1957).
++
+ + -+
CONCLUSIONS
(19) Lappin, G. R., and Clark, L. C., Anal. Chem., 23, 541 (1951). (20) Mendelowitz, A,, and Riley, J. P., Analyst, 78, 704
(1953) ~-- __, .
The alkaline 2,4-dinitrophenylhydrazine method which has been used for the estimation of carbonyl compounds and steroids, has been successfully adapted t o the quantitative estimation of gibberellic acid, gibberellin, gibberellin A,, and potassium gibberellate. The following,
(21) Monty K . J. Anal. Ckem. 30 1350(1958). ( 2 2 ) Gornali, A. 'G., and Ma'cDAnald, M. P., J . Biol. Chem., 201,279(1953). (23) Grove, J. F., and Brian, P. W., Endeavour, 16, 161 (1957). (24) Stowe, B. B., and Yamaki, T., A n n . Rev.Plant Pkysid.,8 , 181(1957). (25) Chataway, F. D . , and Clemo, G. R . , J . Chem. Soc., 123,3041(1923). (26) Roberts, J. D., and Green, D. C., J . Anz. Ckem. Soc., 68,214(1946).
