Analysis of Plant Tissue - Analytical Chemistry (ACS Publications)

THE EFFECTS OF INDOLE-3-ACETIC ACID AND COMMON ORGANIC ACIDS ON THE RESPIRATION OF SLICES FROM TOMATO STEM AND CROWN GALL ...
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Analysis of Plant Tissue Application of a Semi-Micro-Kjeldahl Method W. W. UMBREIT AND V. S. BOND, University of Wisconsin, Madison, Wis.

A semi-micromethod, suitable for the determination of nitrogen in plant tissues, is described. The method is rapid, inexpensive, suitable for small samples, and facilitates determination of large numbers of samples. Its precision and accuracy on the basis of a statistical analysis of the data are given.

without evidence that the method so modified is applicable to the new conditions. Critical examination of macro- and microprocedures for determination of nitrogen have frequently been made, but similar work on semi-micromethods is not available. Moreover, it is fallacious to assume that the existing semi-micromethods developed for the analysis of pure compounds or for animal tissue are suitable for the analysis of plant tissue without some modification. Before acceptance it is essential that evidence be presented concerning the applicabiIity, accuracy, and precision of the modified method when used for routine analysis under given conditions-e. g., in plant metabolism experiments.

R

ESEARCH on the nitrogen metabolism of plant tissues demands a method for accurate determination of total nitrogen on relatively small samples. The limitations imposed on plant culture work-. g., the necessity for constant environmental and nutrient conditions-do not admit the use of large samples in metabolic studies in which frequent sampling of specific tissues is necessary. Further, as the number of plants in such an experiment is increased the variable factors introduced by varied environmental conditions-e. g., l i g h t p l a y an increasingly important part in determining the extent of growth, and hence results of the experiment. I n addition to being applicable to small amounts of material, it is essential that the method be rapid, simple, inexpensive, and adapted to routine determinations on a large number of samples. This need is most readily met by use of semi-microprocedures. Equal speed and ease of manipulation are possible with microtechnics, but these often require expensive apparatus and specialized skill not always available. I n this paper is detailed a semi-micro-Kjeldahl method suitable for plant metabolism studies; this method is essentially a combination of standard procedures used in the macroanalysis, but which have been modified to meet the requirements already outlined. It should be emphasized that semi-micromethods involve more than mere reduction in size of macroprocedures. Methods are too often transferred from macro- to micro- or semi-microtechnic and from one type of tissue to another TIHCAND RITE or AER~TION

Efrccr

OF

Estimation of Ammonia Of the numerous methods described for the estimation of ammonia, only a few were found to be suitable. The steam distillation recommended by Parnas and Wagner (6) and distillations of other types, while excellent for microwork, require considerable attention and are not adapted to routine determinations on a large number of samples. In addition they are not particularly suitable to the estimation of ammonia nitrogen in undigested plant saps. An atteinpt t o simplify the procedure by simple distillation from the semi-micro-Kjeldahl flask through a microdistilling head into standard acid, as in the macromethod, entailed a loss in precision, as is shown by the relatively large standard deviations of the mean (Table I). Aeration into standard acid was found to be satisfactory from the point of view of ease and speed of manipulation, precision, and accuracy. The apparatus used is essentially that of Van Slyke and Cullen (11). A bank of 24 to 36 units is easily operated by a simple water aspirator and the rate of airflow measured with a standardized flowmeter. As will be seen from Table I, the precision and accuracy of this method are entirely satisfactory. TABLE I. NITROGEN DETERMINATION Method Sim le distiaation

EXCESS ALKALI

ABration

0

I‘

4I

Material (NHa)zSO( Digested soybean sap (NHSzSOr

Macro-Kjeldahl

No. of Nitrogen Found Samples (Semi-Micro)

1.94 t 0 . 0 1 n 0.97 t 0 . 0 1 2.27 * 0.02 0.91 * 0 . 0 2

26 25 19 9

1.96 t 0 . 0 5 a 0.91 t 0 . 0 3 2.36 + 0.03 0 . 9 9 AO.06

0 . 3 9 * O 01 0.97 0.01 1 94 * 0 . 0 1 2 27 k 0 . 0 2 0.91 * 0.02 0.45 * 0.02

3 3 3 30 4 10

0.37 0.95 1.94 2.26 0.92 0.46

Digested soybean sap The deviation given is the standard deviation of the mean.

*O.OO

kO.01 *0.01 t0.01

==o.oo 0.01

=t

The rate and time of aeration, the concentration and volume

of the sample solution, the temperature, and the pH of the sample,

CFfecT of VOLUME of

C f FECT OF S A L T @NCENTPAllON

O N nccoveuy

S O L U T I O N ON QECOWCaY

0 5~

j

,

a

w

a

r

all influence recovery. These factors have been discussed by many workers, particularly Folin (4) and Sessions and Shive to Figure 1 it will be seen that a rate of 1.5 (F) Byer reference liters minute at room temperature gives complete recovery in sligEtly over 3 hours, 4 hours being entirely adequate. Since, longer aeration does not alter the final results, samples may be left aerating as long as is convenient-+. g., overnight-providing the aeration time is at least 4 hours. At aeration rates above r minute, addition or omission of inorganic salts does Ac%igence the results. The pH of the sample should be a t least 8.0. In aerating from plant juices, excess alkali is to be avoided. For quantities of nitrogen greater than 5 mg. the boric acid absorption of Meeker and Wagner (6) may be used; phenol is somewhat better than boric acid for this absorption. However, in the range from 0.5 to 5.0 mg. of nitrogen the precision of such

n

TOTAL VOLUME Of SOLUTION

M a t 1 2 3 4 5 & + u.. r o h A PpRCENT NaCI

FIGURE1. EFPECT OF VARIOUS FACTORS ON RECOVERY OF NITROGEN BY AERATION 276

JULY 15,1936

ANALYTICAL EDITION

absorptions is not as great as is the 0.018 N acid-alkali titration. For smaller quantities of nitrogen the absorption bulbs of Folin (4) are necessary and nesslerization is convenient.

Digestion of Sample The conversion of nitrogenous compounds into ammonia by digestion with concentrated sulfuric acid in the presence of a catalyst does not always proceed quantitatively and in these cases certain modifications have been introduced. The general method of analysis will be described in detail, then certain modifications indicated for different types of samples.

277

is washed clean and its contents are collected in the flask. Evaporation, digestion, and nitrogen estimation proceed as in the general method. Precision and accuracy are discussed below. BASICNITROGEN FRACTION (PHOSPHOTUNGSTIC ACIDPRECIPITATES). There is perhaps no group of reduced nitrogenous compounds less susceptible to Kjeldahl nitrogen determination than those found in the fractions precipitated by phosphotungstic acid. Modifications suitable for quantitative determination of total nitrogen in these are discussed by Umbreit and Wilson (10) in connection with the semi-micromethod for basic nitrogen.

Discussion

The precision of the method on various types of samples determined by a comparison of duplicates is fundamental t o METHOD FOR TOTAL NITROGEN.The sample, containing from the interpretation of any results obtained through its use. 0.5 to 5.0 mg. of nitrogen, is weighed or pipetted into a 100-cc. semi-micro-Kjeldahl flask and 5 cc. of catalyst-digestion mixture The method has now been used for over a year in routine are added. This mixture consists of 2 grams of copper sulfate, analysis on several thousand samples of plant materials, 2 grams of selenium oxide, 100 grams of sodium sulfate, 500 cc. of soils, and bacteriological media, as well as in research on water, and 500 cc. of concentrated sulfuric acid. Modifications to nitrogen metabolism. The estimations of precision given in suit specific materials are discussed below. Digestion is carried out over a low flame until almost water-white. For most maTable I1 are obtained from the regular routine work of the terials this entire digestion is completed in 30 minutes. The laboratories now using the method and as such represent the sample is allowed to cool, diluted with about 20 cc. of water, and precision under varied conditions of analyst, laboratory, and washed into an aeration tube. The total volume of sample and sample type. The correlation coefficient, T , between the washing should not exceed 50 cc. The neutralization of the digestion mixture is carried out in nitrogen content of the samples and the difference between two stages: (1) sufficient 10N alkali is added to change the duplicates showed these to be independent. The values of crystal violet 1drop of 0.2 per cent solution) from yellow through T given in Table I1 are not significant as judged by the t test green to blue [this preliminary addition of alkali is insufficient to (8, 4). The weighted averages of the differences between cause the evolution of ammonia but accounts for the greater amount of the heat of neutralization). (2) The sample is allowed duplicates are given in Table 11; in taking two duplicates from to cool before making completely alkaline to avoid condensation of the same sample one can normally expect them t o agree moisture in the trap between the sample and receiving tube. within 0.03 or 0.04 mg. of nitrogen, the percentage deviation This trap may be omitted when the aeration apparatus is used varying with the nitrogen content (for the 2 mg. of nitrogen solely for Kjeldahl digest but is necessary in aeration of ammonia from plant juices. The sample tube, then, is cooled, placed range this deviation would be 1.5to 2.0 per cent of the nitrogen in the aeration rack, made entirely alkaline to phenolphthalein, in the sample). and aerated (1500 cc. per minute) into 25 cc. of 0.018 N sdfuric acid for at least 4 hours. The acid is back-titrated with 0.018 N TABLE11. PRECISION OF METHOD sodium hydroxide using bromocresol green. DRYSAMPLES WITHOUT NITRATE. From 50 to 200 mg. of the Weighted .4verage sample, ground to 200 mesh, are weighed into the flask, the Number of Difference in catalyst-digestion mixture is added, and the samples are analyzed Type of Sample N Range Duplicates r Duplicates as described above. Mo . DRYSAMPLES CONTAINING NITRATE.The semi-micro salicylic0.50-4.00 207 +- 0.073 0.031 Dry * Nos sulfuric acid analog of the macromethod is entirely applicable, Wet NO1 0.33-5.28 108 0.05 0.041 Some slight modifications are necessary, since water must not be added until after nitration and reduction. The sample is weighed as usual, 2.5 cc. of the salicylic-sulfuric acid mixture The accuracy of a method is something apart from its preused in the official macromethod (1) are added, mixed well, and cision, and before the data may be relied upon it must be the whole is allowed to stand for 30 minutes. A small crystal shown that it is actually, determining the component it purof sodium thiosulfate is added and the mixture heated carefully ports to measure. The method here described gives virtually for 5 minutes. After cooling, 5 cc. of modified digestion mixture (exactly the same as the normal digestion mixture except that the same results as does the macro-Kjeldahl method. In those the 500 cc. of concentrated sulfuric acid are replaced by 500 cc. cases where the macro-Kjeldahl disagrees with the Dumas of water) are added, evaporated until white fumes come off, method (9) the results obtained by the use of the semidigested, and aerated as usual. On dry samples containing nimicromodification described agree with the macro-Kjeldahl trate the same precision has been found with this modification as with dry samples containin no nitrate. data. The method is therefore subject to the limitations of WET SAMPLES WITHOUT &*RATE. The general method is its macroanalog, though it is not as readily influenced by entirely adequate. The data given in Table I are typical of the slight changes in procedure as is the macromethod. results obtained. When plant sap samples are compared with The semi-micromethod is adaptable t o all the modifications similar samples containing nitrate, both should be run by the nitrate modification, even though one is free from nitrate nitrogen. of the macro-Kjeldahl method. From the data in Table 111, WET SAMPLES CONTAINING NITRATE. It has been shown by it will be seen that in the case of soybean materials, combiRanker (7) that the salicylic acid method is not applicable to nations of mercury and hydrogen peroxide with selenium give wet samples and reduction by other means is necessary. This no better results than selenium alone. Likewise, mercury reduction may be carried out in acid or alkaline solution, the latter being preferable on plant saps in that it eliminates and hydrogen peroxide alone, permanganate, persulfate, the possible source of error due to the loss of nitrogen from dichromate, phosphoric acid, and copper selenate gave no the reaction of nitrous acid on free amino groups present. The better results than selenium dioxide with nitrogenous mamethod used is that of Davisson and Parsons (2), modified for terials from soybeans. semi-microwork. The sample is made 0.125 N to 0.1 N with sodium. hydroxide, 150 m of Devarda's alloy are roughly measured in, and tak flask is attached to the TABLE 111. ADAPTABILITY OF METHOD Davisson and Parsons tower, which contains 5 cc. of (Based on quadruplicate samples of each treatment) the normal catalyst-digestion mixture. The flasks are Selenium Selenium Selenium Alkaline Hydrolysis allowed to stand in the cold for about 10 minutes, (as SeOl) + Mercury + Hg + HzOz Se + H g Selenium then warmed slitzhtlv until the active e v o l u t i o n O f hydro@;enceauses"(10 to l5 minutes)* Foaming soybeansap 5 89 * 0.02 5.84 t 0 . 0 2 5.91 r 0 . 0 2 5.88 * 0.01 5.90 i 0.02 may be prevented by adding a drop of capryl alcohol Soybertn aap 1 :79 * 0.02 1.79 * 0.03 I . 80 * 0 . 0 2 1.80 A 0.03 1.83 * 0.02 Basic fraction to the sample at the start. The heat is removed 2.70 * 0 . 0 2 2.72 * 0 . o2 2.68 * 0 . o2 2.83 * 0.04 2 . 8 7 * 0.05 and the catalyst allowed to suck back into the flask. 2 . 5 1 * 0 . 0 2 2.52 h O . 0 2 2.50 * 0 . 0 2 2.53 * 0.02 2.65 * 0.02 P,($Y~~) With repeated heatings and washings the entire tower Yeast 1.71 h 0.01 1.81 10.03 ...... 1.73 J . O . 0 1 1 . 7 2 0 . 0 0 J.

VOL. 8, NO. 4

INDUSTRIAL AND ENGINEERING CHEMISTRY

218

Finally, use of the modifications for nitrate nitrogen in addition to total nitrogen gives quantitative estimation of both forms, as will be noted in Table IV. The authors have found no evidence that the organic matter present is able to reduce the nitrate during the process of digestion, and reduction with nascent hydrogen must be resorted to. TABLEIV. NITRATE NITROQEN DETERMINATION -Nitrogen FoundNo. of Nitrogen Present ReSamples Organica Nitrate Total Nitrate covery Mg. Mg. MQ. Me. % Pure NaNOs 6 0.45 0.45 *0.02 0.45 *0.02 100 2125 0.45 2.69 *0.06 0.44 *0.06 97 Sap NaNOs NaNos 2.25 0.20 2.45 * 0.03 0.20 * 0 03 100 a Determined on sap before addition of nitrate. Material

‘5

+

Literature Cited (1) Assoc. Official Agr. Chem., Official and Tentative Methods, 3rd ed., 1930. (2) Dsvisson and Parsons, J. IND.ENG.CHIM., 11, 306 $:919). (3) Fisher, “Statistical Methods for Research Workers, London, Oliver and Boyd, 1930. (4) Folin, J. Bid. Chem., 97, 141 (1932). (5) Meeker and Wagner, IND. ENG. CHEM.,Anal. Ed., 5, 396 (1933). (6) Parnas and Wagner, Biochem. Z., 125, 253 (1921). (7)Ranker, E.R . , Ann. Missouri Botan. Gardens, 13, 391 (1926). (S) Sessions and Shive, Plant Physiol., 3, 501 (1928). (9) Smgth and Wilson, Biochem. Z.,282, 1 (1935). (10) Umbreit and Wilson, to be published. (11) Van Slyke and Cullen, J. Biol. Chem., 19, 218 (1914). R E C E I V ~April D 23, 1936. Herman Frasch Foundation in Agricultural Chemistry, Paper No. 117: Contribution from the Departments of Agricultural Bacteriology and Agricultural Chemistry, University of Wisconsin.

Determination of Hydroxyl Groups in Organic Compounds M. FREED AND A. M. WYNNE, Department of Biochemistry, University of Toronto, Toronto, Canada

D

URING an investigation of the enzymic synthesis of

glycerides it was desirable to have information as to the degree of esterification accomplished at various stages of the reaction. For this purpose a method for the determination of the hydroxyl content of the product, based upon that of Verley and Bolsing {2), was devised. The method of these workers, as originally described, required more material than could be conveniently obtained in the synthetic experiments and, in addition, was not sufficiently rapid. The use of the reagent of Verley and Bolsing-namely, acetic anhydride in pyridine solution-has been applied by other workers to the analysis of small amounts of material [of. Peterson and West ( I ) , and West, Hoagland, and Curtis (S)],but it seemed possible that some of the precautionary measures, such as the use of condensers and ground-glass stoppers, adopted by these workers to prevent possible losses of reactants might

not be necessary in the analysis of many nonvolatile compounds, especially in view of the fact that the original authors found such measures unnecessary, Moreover, the modified methods were found to be still somewhat time-consuming. Because of the observation that no loss of titratable acid occurs even on fairly vigorous boiling of the acetic acidpyridine reagent in open vessels without condensers, it has been possible to simplify and shorten the analytical procedure very considerably without loss of accuracy. Before using the method for the purpose for which it was designed it was tested on a number of representative compounds, with the results recorded in Table I.

Experimental Methods REAQENT.This was a solution of acetic anhydride (either 12 or 20 per cent) in dry pyridine, which was prepared by redis-

TABLEI. HYDROXYL CONTENT OF ORQANICCOMPOUNDB

Compound 1-Naphthol Hydroxyiscbutyric acid Salicylic acid Catechol

Phloroglucinol Arabinose

Xyloee

-OH -OH EqmvaEquivalents lents per Mole, &?e Aver- Theoreti- Error ’ Founb age cal Per CeAt 20 Per Cent Reagent 1.000 1,000 0.996 0.998 0.995 0.997 0.098 0.909 0.998 2.05 1.96 1.93 1.92 2.12 2.99 3.03 3.88 3.98 4.14 3.94 3.90 4.00 4.00 4.18 3.82 4.01 4.13 3.89 3.94 3.94

-OH

Source of Compound

Equivalents per Mole Compound Founh Glucose

0.998

1,000

-0.2

Eastman

0.997

1,000

-0.3

Kahlbaum

Mannitol

0.999

1,000

-0.1

Merck

Sorbitol

2.00

2.00

0.0

Eastman

3.01

3.00

f0.33

Eastman

Dulcitol

Trichloro - tertbutyl alcohol 0 . 0 0 Monobutvrin Triolein

3.97

4.00

-0.75

Pfanstiehl Cholesterol Oestrone Pregnandiol

3.99

4.00

-0.25

Pfanstiehl

5.00 4.99 5.00 5.10 5.90 5.83 5.83 5 89 A RA -.I5.84 5.85

2.06 1.97 0.35 0.44 0.41 1.018 1.004

-OH Equivalents Der hlole, AverTheoreti- Error, age ad Per Cent 20 Per Cent Reagent (Cont’d)

Source of Compound British Drug House8

5.02

5.00

+0.40

5.85

6.00

-2.5

British Drug Houses

5.86

6.00

-2.4

Pfanstiehl

5.84

6.00

-2.4

Pfanstiehl

. ..

..

1.00 12 Per Cent Reagent 2.02 2.00 1.0

+

0.40

0.00 1.00 1.00

*.

+1.8 4-0.4

Eastman Schuchardt Kahlbaum British Drug Houses Isolated from pregnant mares’ urine Iaolated from pregnant humun urine

1.67a 1.44 2.00 -28.0 1.30a 1.96b 1.97 2.00 -1.5 1.99b Determinations made by the standard method. b The reaction mixture, after the preliminary heatinq, was allowed to stand for 13 hours at room temperature before the final titration. @