Spectrophotometric Method for Determination of Gibberellic Acid

Spectrophotometric Method for Determination of Gibberellic Acid A L L A N A . H O L B R O O K , W . J. W . EDGE, a n d FRED BAILEY

Downloaded by PURDUE UNIVERSITY on March 22, 2013 | http://pubs.acs.org Publication Date: January 1, 1961 | doi: 10.1021/ba-1961-0028.ch018

Pharmaceuticals Division, Imperial Chemical Industries, Ltd. Alderley Park, Macclesfield, Cheshire, England

A rapid quantitative method f o r the determina t i o n o f g i b b e r e l l i c a c i d is b a s e d o n t h e conversion of g i b b e r e l l i c a c i d t o g i b b e r e l l e n i c a c i d , f o l l o w e d b y measurement o f t h e a b s o r p t i o n o f t h e l a t t e r compound a t 2 5 4 mμ. The m e t h o d has b e e n s h o w n t o b e specific f o r g i b b e r e l l i c a c i d a n d toler ant of the presence o f o t h e r k n o w n i m p u r i t i e s a n d decomposition products. It h a s b e e n successfully applied to a wide range of fermentation samples.

permentation broth samples may contain, in addition to gibberellic acid, varying amounts of closely related but inactive compounds such as gibberellenic and isogibberellic acids, together with other acidic products of the fermentation. The resolution of these from the active constituent can prove extremely difficult and methods hitherto used for the determination of gibberellic acid in broths have not been completely satisfactory: Physical techniques employing infrared spectropho tometry (4), polarography (2), or fluorimetry (3) all lack specificity, while biological methods (I) are time-consuming and require greenhouse facilities, not generally available to the analytical laboratory, for their successful application. The present work was undertaken in an attempt to resolve these difficulties. Experimental Solutions of gibberellic acid in dilute mineral acid decompose slowly at room temperature, giving first gibberellenic acid and, on prolonged standing, allogibberic and gibberic acids. Gibberellenic acid absorbs strongly in the ultraviolet and has an extinction coefficient (E*^) of 613 at 254 τημ, whereas the other three acids are only very weakly absorbent at this wave length. It was thought that acceleration of this reaction might form the basis of a selective method of gibberellic acid determination, and the effects of acid and elevated temperature were studied using various strengths of hydrochloric, phos phoric, perchloric, acetic, formic, trichloroacetic, and methanesulfonic acids over a temperature range of 2 0 ° to 5 0 ° C . Hydrochloric acid proved to be by far the most effective, and decomposition 159 In GIBBERELLINS; Advances in Chemistry; American Chemical Society: Washington, DC, 1961.


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curves covering the conditions employed are illustrated in Figure 1. At 2 0 ° C . a reagent consisting of 3 volumes of concentrated hydrochloric acid and 7 volumes of water showed considerable promise, giving a sharp rise in gibberellenic acid concentration over the first 30 minutes, followed by a relatively flat portion where the rates of formation and decay of gibberellenic acid are almost equal. Addition of alcohol to the medium retards the rate of decomposition of gibberellenic acid, as shown by the increase in maximum absorption from a constant weight of gibberellic acid (Figure 2). A 10% (v./v.) alcoholic concentration in the solution gave the most satisfacto/y curve and all future work was carried out with this amount of alcohol present. A calibration graph was prepared from known aliquots of pure gibberellic acid and a linear relationship established over a concentration range of 0 to 4 mg. per 100 ml. with a reaction time of 75 minutes and a temperature of 2 0 ° C . The 160 In GIBBERELLINS; Advances in Chemistry; American Chemical Society: Washington, DC, 1961.


H O L B R O O K , EDGE, AND

BAILBY

Spectrophotometry

20 TIME

Τ

30

40

Determination

50

IN MINUTES

Figure 2. Effect of alcohol concentration on rate of de composition of gibberellic acid by hydrochloric acid • 2% v./v.

χ 5% v./v.

• 10% v./v.

• 20% v./v.

results of four replicate calibration graphs, given in Table I, illustrate the high degree of reproducibility. Table I. Gibberellic Acid, Mg. 1.0 2.0 3.0 4.0

R e p r o d u c i b i l i t y o f Points o n C a l i b r a t i o n G r a p h 1 0.219 0.431 0.644 0.863

Absorbance at 254 M 3 2 0.214 0.216 0.430 0.435 0.648 0.658 0.860 0.868

u

4 0.216 0.428 0.640 0.858

Influence of Impurities and Decomposition Products. G I B B E R E L L E N I C ACID. Gibberellenic acid decomposes slowly in the presence of strong concentrations of mineral acids and therefore, if this acid is initially present in the sample, the net absorbance due to gibberellic acid decomposition will be low by that fraction of the initial gibberellenic acid that has decomposed during the 75-minute reaction period of the method described below. The amount by which the net absorbance due to gibberellic acid decomposition is low can be calculated from the initial gibberellenic acid content and a simple correction applied. In practice it is rarely 161 In GIBBERELLINS; Advances in Chemistry; American Chemical Society: Washington, DC, 1961.

ADVANCES IN CHEMISTRY SERIES


2

-300

10 20 30 40 50 60 70 TIME IN MINUTES AFTER ADDITION OF HYDROCHLORIC ACID Figure 3.

Rate of decomposition of gibberellenic acid by hydrochloric acid

necessary to correct for gibberellenic acid, as it is present in significant amounts only in stored solutions or artificially decomposed samples. The rate of decomposition of gibberellenic acid has been measured under the conditions employed for gibberellic acid determination. The graph relating fall in absorbance with time is reproduced in Figure 3. ISOGIBBERELLIC A C I D (gibb-4-ene 1 3 lactone). This compound exhibits a small absorption at 254 πΐμ, but as it is unchanged by the conditions employed for the decomposition of gibberellic acid, no complication is introduced by its pres ence. ALLOGIBBERIC A N D GIBBERIC ACIDS. Both these compounds exhibit weak absorption in the ultraviolet region with maxima at 265 πΐμ. No change in absorp tion takes place on treatment with hydrochloric acid and hence no interference is caused by either compound. SUMIKI'S A C I D (5-hydroxymethylfuran-2-carboxylic acid). This acid is often present to a greater or lesser extent in fermenter broth and is occasionally found in solid isolates in significant amount. It absorbs strongly in the ultraviolet, exhibit ing a maximum at 260 τημ in acid solution (Ε ^ 950). The absorption is unchanged under the conditions employed for the gibberellic acid determination and hence this compound causes no direct interference. The close similarity of the spectrum of Sumiki's acid to that of gibberellenic acid, however, precludes the estimation of the latter compound by direct ultraviolet measurement for correction of the gibberellic acid content. Where these three acids are present in admixture, it is necessary first to estimate the Sumiki s acid and to subtract the contribution of this acid from the absorption curve of the sample before the gibberellenic acid content can be calculated. Sumiki's acid can be estimated by taking advantage of the difference in absorption spectra under acid and neutral-alkaline conditions displayed by this compound (Figure 4). The absorbance of the sample solution is measured under both neutral and acid conditions and the Sumiki's acid content calculated from the change in absorbance at 268 m/*. 1

χ

Application to Broth Filtrates Samples of gravity-filtered broth are invariably turbid and, without treatment, totally unsuitable for spectrophotometric work. The form of treatment depends 162 In GIBBERELLINS; Advances in Chemistry; American Chemical Society: Washington, DC, 1961.


HOLBROOK,

EDGE, AND BAILEY

230

240

Spectrophotometry

250

260

270

Determination

280

290

300

WAVE LENGTH nyi Figure 4.

Spectra of Sumikts acid under acid and alkaline conditions

largely on the composition of the culture medium used. Treatment with zinc acetate-potassium ferrocyanide has been found satisfactory for clarifying a wide range of media; on no occasion has it failed to give an optically clear solution. In a few instances, where complex media had been used, the absorbance of the filtrate was very high and prior separation of the gibberellic acid by extraction into ethyl acetate, followed by re-extraction into phosphate buffer, was preferred. Stronger hydrochloric acid was used to overcome the effect of the phosphate buffer and the reaction time was extended from 75 to 80 minutes. A series of broth samples taken at intervals during the course of fermentations using a variety of media has been examined after zinc acetate-potassium ferro cyanide clarification and hydrochloric acid treatment. Full spectra of blank and sample were measured in each case between 230 and 300 ταμ. On the assumption that the increase in absorbance at 254 ταμ was due solely to gibberellenic acid, the whole spectral curve was, with the aid of a reference curve for pure gibberellenic acid, corrected for the presence of this compound. The degree of coincidence obtained when the corrected curve was compared with that of the blank indicated that no other ultraviolet-absorbing substance was produced in significant amount. A typical set of spectra is illustrated in Figure 5 . Method On the basis of the foregoing experimental work, the following method was developed for the determination of gibberellic acid in solid samples and broth filtrates. Reagents. 1. Dilute Hydrochloric Acid, 30%. Dilute 300 ml. of concentrated hydrochloric acid (specific gravity 1.18) to 1 liter in a volumetric flask with water, cool to 2 0 ° C , and adjust the volume to 1 liter with water if necessary. 163 In GIBBERELLINS; Advances in Chemistry; American Chemical Society: Washington, DC, 1961.

A D V A N C E S I N C H E M I S T R Y SERIES

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Spectrophotometric Method for Determination of Gibberellic Acid

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