Microestimation of Sulfw l n Plant Materials, Soils, and Irrigation Waters C. 31. JOHYSOY AYD HIDE0 NISIIlT.4 University of California, Berkeley 4 , Calif. Sulfur-deficient plant material may contain as little as 0.5 p.p.m. of sulfate sulfur. The soluble sulfate of soils giving rise to sulfur-deficient plants maj- be as low as 0.1 p.p.m. In studies of the sulfur nutrition of plants under conditions where growth is limited by their sulfur supply, often only small amounts of plant material are available for analysis. It was desirable to develop rapid and accurate methods for the determination of total sulfur and sulfate sulfur that w-ould be relatively specific for sulfate sulfvr in the presence of organic sulfur comp u n d s . Sulfate is digested at 115’ C. with a reducing mixture composed of hydriodic acid, formic
acid, and red phosphorus. The resulting hydrogen sulfide is determined spectrophotonietrically as methylene blue. From 1 to 300 micrograms of sulfate sulfur may be determined in 1 to 2 hours w-ith a precision of 2 to 5% without interference in the presence of cystine, cysteine, glutathione, methionine, and taurine. Total sulfur may also be determined after the sample is ashed. The method permits rapid determination of sulfate sulfur or total sulfur in samples containing as little as 1 microgram of sulfur. It should prove of value in studies of sulfur metabolism in plant and animal hiochenlistry and physiology.
R
ESEARCH programs involving sulfur metabolism in plants
and other biological systems have frequently been hampered by the lack of sensitive, rapid, and specific methods for the estimation of sulfur. Current research in these laboratories on sulfur deficiencies in field crops, R here it has often been necessary to work with small amounts of experimental material of low sulfur concentration, has prompted the development of a method that is sensitive, rapid, and reasonably specific for sulfate sulfur in the presence of a number of organic sulfur compounds. With minor adjustments in techniques, the method may be adapted to the determination of total sulfur as well as sulfate in biological materials in general, including soils and waters. Siegfriedt, Wiberley, and Moore (11) have briefly reviewed a YYI iety of methods for the determination of sulfur. This review in conjunction with those appearing in the annual review articles in AXALYTICAL CHEMISTRY make an extensive literature review unnecessary here. Because of the sensitivity of the methylene blue method for sulfide ( 4 ) and the rapidity with which the determination may be completed, it seemed desirable to develop a technique whereby sulfate might be easily and rapidly reduced to sulfide. St. Lorant (10) in 1929 described a method in which sulfate is reduced to SUIfide under relatively mild conditions compared to methods involving catalytic reduction on alumina ( S ) , or on a bed of quartz chips (6). These latter techniques have been developed primarily for vapbr phase reduction of sulfur compounds to hydrogen sulfide and would not be easily adaptable to the kinds of samples encountered in the present work. Zimmerman’s modification (IS, 1 4 ) of Burger’s method did not seem well suited for routine determinations of sulfate in waters, soil eutracts, and plant estracts and was not investigated. Luke (7’) used hypophosphorous and hydriodic acids in the reduction of sulfate to sulfide, but found that quantitative reduction was difficult to achieve. The authors’ experiences have indicated that the hypophosphorous-hydriodic reduction s o h tion is inferior to that used by St. Lorant ( I O ) and, therefore, the method described makes use of the reducing mixture proposed by St. Lorant, although it is more difficult to prepare and less convenient to dispense. St. Lorant’s techniques and reagents have been modified as necessary for use with plants and soils, so that from 0.5 to 300 micrograms of sulfur, either as sulfate or total sulfur, may be determined with a precision of approximately 2% at the 95% confidence interval. [While this manuscript was in preparation, a paper by Roth (9) appeared describing a similar
method, although using a different reducing agent for the determination of 10 to 40 micrograms in soil extracts and waters.] 4PP 4 RATUS
Digestion-Distillation Apparatus. The digestion-distillation apparatus as shoan in Figure 1 consists of a boiling flask, uaterjacketed condenser, connecting U-tube, gas-washing column, and detachable connecting tube (4 mm. in outside diameter) leading to a receiving vessel. 4 gas-washing bottle (not shown in Figure 1) is placed between the nitrogen tank and the inlet arm of the boiling flask. The incoming nitrogen is washed by bubbling through a solution of mercuric chloride in 2% potassium permanganate to remove possible traces of sulfur gases The sample flask is a 50-ml. r o u n d - b o t t o m e d boiling flask (Pyrex 90. 4326) fitted with a 7-mni. gas inlet tube as illusti ated in Figure 1. I n order to prevent excessive bumping. it is essential that the gas inlet tube terminate below the liquid level in the flask, approximately 3 to 5 mm from the bottom of the flask. Heat is supplied by a microburner p r o t e c t e d from drafts by a suitable shield. A4n asbestos pad must protect the b o i l i n g flask from direct flame.
INCHES
Figure 1. DigestionDistillation Apparatus 736
Special attention should be paid to the angle a t which the side arm emerges from t h e gas washing c o l u m n . T h i s a n g l e is necessary, in order that the side arm may remain filled with solution to prevent trapping of hydrogen SUIfide. The delivery tube leading to the receiving vessel is attached to the connecting arm of the gas washing column by a short piece of rubber tubing. The rubber
V O L U M E 24, NO. 4, A P R I L 1 9 5 2 41ould freecl of sulfur compounds by boiling with diluted alkali : i d thorough Xvashing. 13ec:iu;c zinc sulfide adheres to the portion of the delivery tube tlil)ping i n the zinc acetate solution, it must be disconnected and ullon-ed t o remain in the volumetric flask during the development of the methylene blue color. The tubes may be removed with \wghing :liter the development of the methylene blue color and Ijefore making to volume. If volumetric. flasks are selected so that the marks are approsi1n:itely i n t h e mnie position on the neck, and if the deliver>-tubes xi'c of the smie diameters and sufficiently lomg to extend above the gratlu:ition marks of the volumetric flasks, the tubes may be left, in the flasks throughout the whole procedure with negligible relntivc eri or i n final volumes of colored solutions. h
i
Spectrophotometer and Colorimeter. A Beckman llodel €3 spectrophotometer with 1-cm. cells was used throughout this Lvork. C'oinparative data over a limited range are also given for the Iilett-Sunimerson photoelectric colorimeter, using n 4-rm. ccll and S o . 66 filter. RE.AGESTS
Reducing Mixture. Place 15 grams of reagent grade red phosphorus, 100 ml. of hydriodic acid (specific gravity 1.7, niethoq-1 grade), and 7 5 nil. OOo/, formic acid (ACS reagent grade) in a 250-nil. boiling flask. Introduce a stream of nitrogen to the bottom of the flask, to remove evolved hydrogen sulfide and a1.o t,o prevent bumping. Heat the mixture slowly to 115" C. and hold at 115' to 117" C. for an additional 1 to 1.5 hours. 1,onger times of heat,ing result in an unnecessarily large redurtion in reagent volume; shorter times result in a reagent, of poor staI)ility and low reducing power. reduction in volume of approsiniately 30% occurs during preparation of t,he reagent. Reagent prc.pared i n this manner is stable for 2 to 3 weeks and can be used for longer times with reduced sensit'ivity and precision. C3ed reducing mixture may be saved and regenerated by adding to each 100 ml. of the used reagent 1 gram of red phosphorus, 10 nil. of h>-driodic acid, and 25 nil. of formic acid followed by digestion ior 1.5 to 2 hours a t 115' to 117" C. Regeneration of used reagent niay be repeated as many as four times, provided excessive amounts of perchloric acid have not been introduced by samples wet-ashed by perchloric acid techniques for total sulfur. Nitrogen Purification Solution. Add an excess of mercuric chloride, 5 t o 10 grams per 100 nil., to 2% potassium permanganate solution. Pyrogallol-Sodium Phosphate Wash Solution. Dissolve 10 grarlis of sodium dihydrogen phosphate and 10 grams of pyrogallol in 100 nil. of sulfur-free distilled water with the aid of a stream of nitrogen bubbling through the solution. Prepare fresh daily. Sulfur-Free Distilled Water. If necessary, prepare by distilling Xvater containing alkaline permanganate from an all-glass still. Zinc Acetate-Sodium Acetate Sulfide Absorbing Solution. Dispolve, in sulfur-free water, 50 grams of zinc acetate dihydrate and 12.5 grams of sodium acetate trihydrate. LIake to 1-liter volume and filter. Aminodimethylaniline Solution. Dissolve 2 grams of Eastman Kodak Co. S o . 1333 p-aniinodimethylaniline sulfate in 1500 nil. of distilled water. Add 400 nil. of concentrated reagent grade sulfuric acid. Cool and make to 2 liters. Ferric Ammonium Sulfate Solution. T o 25 grams of ferric ammonium sulfate add 5 ml. of concentrated reagent grade sulfuric acid and 195 ml. of distilled water. Standard Sulfate Solution. Prepare a stock solution containing 5.434grams of reagent grade potassium sulfate per liter, using sulfur-free distilled Jyater. One milliliter of this solution contains 1 mg. of sulfur. Dilute t'he st.ock solution with sulfur-free water, as necessary to give working solutions of the desired concentrations. Sulfur-Free Ground Joint Lubricant. Mix approximately 5 grams of Dow-Corning silicone stopcock lubricant with 10 ml. of an equal volume of hydriodic acid and hypophosphorus acid. Heat to boiling with frequent. stirring for about 45 minutes. A 100-ml. beaker fitted with a condenser made from a 50-ml. roundbottomed boiling flask filled with cold water is convenient in the preparation of the sulfur-free lubricant. At the end of the boiling period, pour off the acid mixture and wash the lubricant thoroughly with sulfur-free water. PROCEDURE
Lubricate all spherical joints with a minimal amount of the desulfured lubricant. Place 10 ml. of the pyrogallol-sodium phos-
737 phatc. rcagcmt i n the gas-washing column. Add 10 ml. of the zinc acetate-sodium acetate solution to 70 ml. of sulfur-free distilled water in a 100-ml. glass-stoppered volumetric flask, at'tach the small connecting tube to the side arm of the gas washing column, and put the receiving flask in place. Transfer an aliquot of the standard or sample, not exceeding 2 ml. in volume (if necessary to use larger volumes, evaporate to near dryness) and containing not more than 300 micrograms of sulfur, to t,he digestion flask. Resuspend the red phosphorus of the reducing mixture by shaking and add 4 ml. from a fast delivery pipet. Quickly attach the flask to the condenser and connect the tube leading from the nitrogen wash bottle to the side arm of the flask. Start the flow of cooling water in the condenser and adjust' the nitrogen flow through the system to a rate of 100 to 200 ml. per minute. Start heating and maintain at a low boil for I hour. Remove the receiving flask, leaving the connecting tube in the flask. TTsing a rapid delivery pipet, quickly add 10 ml. of the p-aminodimethylaniline solution to the receiving flask. Stopper, mix, add 2 ml. of the ferric ammonium sulfate solution, stopper and mix again, make to volume, and mix thoroughly. Read, after 10 minutes but within 24 hours, in a suitable colorimeter or spectrophotometer a t 670 mfi. PROCEDURE FOR SOIL EXTRACTS AND WATERS
The procedure for the determination of sulfur in irrigation waters and soil extracts is identical with that described for standards. The procedure will not distinguish, without preliminary chemical separations, sulfate, sulfite, sulfide, and possibly some very labile organic sulfur compounds. A number of organic sulfur compounds do not lilwate hydrogen si lfide in this procedure. PROCEDURE FOR SULFATE A S D LABILE SULFUR IN P L A S T MATERIAL
Sulfate sulfur can be determined directly in ground plant 1113terial or estracts thereof. Introduce directly into the digestion flask 25 rng. of finel>ground plant material. Add 2 nil. of sulfur free water and 4 nil. of the reducing misture and proceed as directed above. PROCEDURE FOR SA3IPLES COYTAINIZIG MCCH NITRATE
If more than 6 mg. of nitrate is present in the sample, the results will be low and erratic. Assuming a masinium sample size of 50 mg., an amount of nitrate in escess of 6 mg. will only rarely be esceeded. However, under certain conditions where deficiency of some essential element is limiting the normal metabolism of the plant, as, for esample, in severe molybdenum deficiency and possibly in severe sulfur deficiency, amounts of nitrat,e in the order of 6 mg. may be present even in 25 nig. of dry plant sample. Obviously, the simplest procedure is to reduce the sample size to the point u-here less than 6 mg. of nitrate is present. There niay be occasions; however, where it is desirable to utilize the increased sensitivity resulting from larger sample sizes. For this reason several methods of removing nitrate without affecting the recovery of sulfate sulfur Ivere studied. Sulfate sulfur concentrations were established for a sample of plant material that contained essentially no nitrate. Nitrate in escess of 6 mg. per sample was then added and t'he effects of the added nitrate and the treatments to remove nitrate were studied. Attempts to eliminate nitrate interference by reducing the nitrate with finely divided iron metal in acid solution were unsuccessful, as were procedures involving the use of Devarda's alloy. Removal of nitrate by taking t'he sample t'o dryness with hydrochloric acid was also unsatisfactory because of extensive hydrolysis of organic sulfur compounds. Luke ( 7 ) used formic and hydrochloric acids to remove nitrate from his inorganic solutions. This technique was also unsuccessful when applied to plant material because of extensive hydrolysis of organic sulfur compounds. The most successful method for the elimination of nitrate interference with minimum effect on the apparent sulfate content of the sample was the extraction of the sample with 10% barium chloride solution, followed by centrifugation and transfer of the
738
ANALYTICAL CHEMISTRY
solid residue of plant mateiial and barium sulfate to the digestion flask, and proceeding from there on as described previously. Transfer the sample to a 15-ml. conical centrifuge tube and moisten with sufficient 957$ ethyl alcohol to ensure that all the sample has been wetted. Add 5 drops of 10% barium chloride solution. Immerse the tube to a depth of 0.5 inch in a boiling water bath for 10 to 15 minutes. The alcohol and m-ater vapors condensing on the walls of the tube aid in wetting any sample particles adhering to the side of the tube. Add 5 ml. of 10% barium chloride solution and mix thoroughly. Wash doMn the sides of the tube with an additional 5-ml. portion of barium chloride solution and centrifuge. Discard the aqueous phase. Transfer the residue quantitatively to the digestion flask. Reduce the volume to 2 ml. or less and proceed in the usual manner. PROCEDURE FOR TOTAL SULFUR IN PLANT BIATERIALS
Total sulfur in plant materials niay be determined by the colorimetric method aftei oxidation of the sfimple by any one of a variety of oxidation methods, including the Association of Official A4gricultural Chemists' magnesium nitrate method ( 1 ) and Pari peroxide bomb method. Aliquots of ash solutions so obtained may be used directly for the digestion with the reducing mixture.
o
IO
20
30 -10 50 60 70 a0 MICROGRAMS SULFUR I N 1 0 0 ML.
90
Figure 2. Absorbancy of Methylene Blue Solutions as Function of Sulfur Taken through Procedure Beckman Model B spectrophotometer, 1-cm. cells, 670 rnw- 2.5' C .
The nitric acid-perchloric acid n e t digestion is desciihed in some detail because careful attention must he paid to prepai ation of an ash soIution that is sufficiently low in perchiorate and nitrate to prevent interference in the reduction of sulfate and synthesis of methylene blue. Wet Ashing of Plant Material. Transfer 100 mg., or less, depending on the amount of total sulfur expected, of the plant material to a 30-ml. micro-Kjeldahl flask, add 2 nil. of concentrated nitric acid, and digest on the steam bath for 30 minutes. Add 1 ml. of 60% perchloric acid and heat slowly over a microburner. Increase the heat and continue the digestion for at Ieast 1 hour after fumes of perchloric acid appear. Cool and add 3 ml. of 6 N hydrochloric acid to facilitate complete removal of nitrate. Heat the digest containing the hydrochloric acid again until copious fumes of perchloric acid again appear. Cool and make to 50 ml. in a volumetric flask. Take aliquots usually not exceeding 2 ml. for determination of sulfate. The size of the aliquot taken is determined by the amount of sulfur expected and also by the amount of perchlorate present.
hmounts of perchlorate equivalent to 0.1 to 0.2 ml. of 60% perchloric acid may be tolerated. Amounts in excess of this amount may lead to low recovery of sulfide. EXPERlMENTAL RESULTS
Standard Sulfate Solutions. Linear relationships between the amounts of sulfate carried through the entire procedure and the absorbancy of the resulting methylene blue solutions were obtained in the range of 1 to 50 micrograms of sulfur (expressed as S) (Figure 2). Measurements were made with distilled water as reference liquid in 1-em. cells at 670 mp in the Beckman Model B spect,rophotometer. The blue-sensitive phototube was used, with gain settings and slit widths such t,liat, according to the literature supplied with the instrument, effective band widths were j nip or less. Fog0 and Popoivsky (4)have published a spectral transmitt'ance curve for methylene blue prepared in a nlanner similar to that employed in the present investigation. An ident'ical curve has been obtained in these studies. The absorption maximum is rather sharp at 670 mp; consequently deviations from Beer's lam may be expected if instruments of low spectral purity are used. Amounts of sulfur greater than 50 micrograms yield methylene blue solutions that, deviate from linearity because of mutual interaction of the absorbing molecules. Dilution of the concentrated solutions with a solution of exactly the same acid concentration restores the linear relationship (Table I). It is convenient to dilute the concentrated solutions with distilled water containing the same concentrations of p-minodimethylaniline and ferric ammonium sulfate solutions, thus msuring identical conditions in solutions prepared directly from smaller initial amounts of sulfur and those prepared by dilution. Dilution of the concentrated methylene blue solution is satisfactory for amounts of sulfur up to approximately 300 niicrograms. Amounts in escess of 300 micrograms give low readinga; calculated values were 0.427. 0.470, and 0.491 compared with the observed readings of 0.411, 0.450, and 0.465 for 386, 424.6, and 443.9 micrograms of sulfur, respcctioely. The low readings are due to limiting amounts of p-:~n~inodimeth~laniline solution added when the color is first developed (p-aminodimethylaniline added in the dilution step does not react because escess sulfide has previously been oxidized by the added ferric iron). If a stoichiometric reaction between sulfidr and p-aminodimethylaniline is assumed-Le., 2 moles of p-aminodimethylaniline react with 1 mole of sulfide-it may he calculatrd that there should he sufficient p-aminodiniethylanilinr to react with 433 microgram of sulfate sulfur. However, it actually is observed that there is sufficient p-aminodimethylaniline to react completely with soniewhat less sulfide, p e r h a p heenuse the reaction is not complete unless excess reagent is present. The molar absorbanry indes for the methylene blue solution, calculated from average absorbancies at 670 mp over the range of 5 to 50 micrograms of sulfur, is high-in the order of 34,500. According to Liehhafsky and \T?nslow (6) and JPellon ( 8 ) molar absorbancy indexes of about, 35,000 are at the upper limit, of sensitivities in colorimetric methods. Measurements were also made in thc Klett-Summerson photoelectric colorimeter, employing &em. cells and SO.66 filter. Reference liquid mas distilled water. Linear relationship between amount of sulfate taken and scale reading was also obtained, although over x much shorter r a n g e f r o m 1 to 10 micrograms of sulfur in 100 ml. of colored solution (Figure 3). The deviation is not serious in solutions containing up to 16 niicrograms sulfur. However, it is recommended that more concentrat,ed solutions be diluted, and the acid concentration kept constant as described above, so that readings mill fall in the linear range. The calibration curves should be verified for each type of s p e c trophotometer or colorimeter used, as the spectral purity oh-
V O L U M E 24, NO. 4, A P R I L 1 9 5 2
739
method and by the colorimetric method are coinpared in Table 11. The results obtained by the two methods are in excellent agreement for three S i n 100 S i n 100 of the samples. The gravimetric results for water Absorbancy Absorbancy s $ . l : o 1\11. of ~ ~ l ~ colored ~ i ~ ~ Diluted, hbsorbanry, sample B are 12% lower than those obtained by Undiluted, COlorPd Solution .\, Solution -4 .I Diluted Solution Solution B. Solution the colorimetric method. No r w o n can be adi" Obsd. Calcd.. l : l O , y b Ohsd. Ca1cd.c yd Obsd. Ca1cd.E vanced for the lower gravimetric results. All water 48 Xi 0 516 0.534 4.825 0 . 0 3 6 0.053 4.823 0.053 0.053 .19963 ..SO 0.954 1.067 9 65 0.108 0 , 1 0 7 9.65 0.112 0.107 samples were boiled with hydrogen peroxide in 0 1.63 2.133 19 30 0 216 0 213 19.30 o 207 o 213 z89 5 T o o d a r k (3.2021 28.!J5 0 321 0 320 28.95 0.322 0.350 acid solution to oxidize any sulfide and sulfite to 686 0 Too d a r k (4.269: .38 60 0.411 0.427 38.60 0.421 0 427 sulfate prior to precipitation of barium sulfate, be424 (i Too d a r k ( 4 . 6 9 6 ) 42.46 0 . 4 5 0 0.470 42 40 0.470 0.470 443.9 T o o d a r k (4.910) 44.39 0.465 0 . 4 9 1 4 4 . 3 9 0.487 0.491 cause the colorimetric method would determine a Soliitiona A were prepared by reducing indicated a m o u n t of sulfate sulfur a n d iiiaking all three forms of sulfur. The water may have resulting methylene blue t o 100 ml. for measurement. 6 Prepared by diluting 10 ml. of methylene blue solutions of selutiohs 4 t o 100 ml. with contained some labile sulfur compounds which 10 mi. of p-aminodimethylaniline rolution. 2 nil. o i ferric ammonium sulfate solution, a n d distilled water. were not precipitated as barium sulfate but, which C Calculated from average abborbanc3- index for all iiieasurenientb on soliition B. appear as sulfide in the colorimetric procedure. d Pieriared bv reduction of indicated amount of aulfate sulfur. N a d r inetlivlene bhie so1:ition directl?. t o 100 nil. The chief advantages of the colorimetric method over the gravimetric are the rapidity with which Table 11. Sulfate in Irrigation Waters and Soil Extracts the colorimetric determination may be completed (P.p.m. sulfate sulfur) and the small sample sizes required for the deterGravimetric Method Colorimetric Method mination. The colorimetric determination was Confidence L i m i t s Confidence Limits Sample Mean 95% 99% Mean 95% 99% completed in less than 2 hours; the gravimetric Water sample Aa 122 0 1 2 0 . 0 118.5-121.5 117.7-122 3 method required an elapsed time of 21 hours. Less Watersample Bb 197:O 192.1lZOl.9 1 8 9 . 0 l i 0 6 . 0 221.2 216.5-225.7 214 5-227 7 than 1 ml. of water sample was used in each colori817-847 801-839 842 828-856 Soil extract C c 830 821-863 Soilextract Dd 93.2 80.1-106.3 68.2-128.2 94.4 92.7-96.1 92 4 96 4 metric determination, whereas 10 to 100 ml. of a Two gravimetric determinations, six colorinietric determinations. sample were required in the gravimetric deterb Three nravimetric determinations. ten colorimetric determinations. Three gravimetric determinations; sjx colorimetric determinations. mination. The use of sinall aliquots in the d Three gravimetric determinations, SIX colorimetric determination-. colorimetric procedure is of even greater advantage when sulfate is determind in saturation soil extracts, where difficulty is often esperienced in tainetl \vith different types oi' iiistrumc'nts mal. vary considerably. o1)t:iining sufficient, extract for a complete analysis bj- the classicd Th(wlf'ot,r the estent and di.grei> of' linrxar resuoiise must be gtxvimt~tric~ procedures. evaluatcd for each set of operating conditions. Table I.
Dilution of Concentrated Methylene Blue Solutions to Regain Conformity to Beer's Law
2,;;;;
~~
~
~
~~~~~~
C
~~
SL-LFATE I N I R R I G A T I O N U-ATERS a z i D SOIL E X T R A C T S
Thih apparent sulfate sulfur concentrations in irrigation ivaters and soil vstracts obtained hy the gravimetric* bai,iuni sulfate
/
d
/
? 0
z
2150W
a W
S I . L F 4 T E A N D LABILE SULFUR IN
DRY P L 4 Y T S I M P L E S
Tlic, prcJcixion obtained when sulfate and labile sulfur :we deterniintd hy direct digestion of dry plant material, containing less th:tri ci 1119. of nitrate per sample, is illustrated hy tl:tta in Table 111. The sample \vas a tomato blade (sample P.C.) initially Ion- in nitrate; therefore no estraction procedui~o\vas necessary. The data for the first item in thr table indicate that, for this tj-p,r of material and at this range of concentrations, a precision of better t'han 2% may be expected 95% of the tinie and that a precision of about' 3y0 may be obtained 99yoof the time (99% confidrncie limits of 26.0 to 26.8 with a mean of 26.4 micrograms of sulfur per sample). The first anti second itcxms of the table compare the apparent sulfate sulfur in the sanipl(~by the dirtArt digwtion method and by the barium chlot,itle estraction method a- described in the procedure for samples containing much niti,ntix. The apparent sulfate sulfur obtained whrn the extravtion procedure is used is itbout 10% lower than that obtained by the dirwt digestion procedure. The precision is also somewhat lon-er-compare standard errors of the means of 0.11 for the direct an4 0.41 for the estraction procedure. Tivo reasons
i
~ 1 0 0 -
Table 111. Sulfate and Labile Sulfur in Dry Plant Axaterial (Tomato blades, sample P.C., nitrate initially 1e.s t h a n 100 p . p . m . )
50
t/
O K
0
I
2
'
1
I
'
'
'
'
4 6 0 I O 12 14 16 1.3 2 0 MICROGRAMS SULFUR IN 100 ML.
Figure 3. Scale Reading of Rlethylene Blue Solutions as Function of Sulfur Taken through Procedure K l e t t - S u m m e r s o n p h o t o e l e c t r i c c o l o r i m e t e r , 4-om. cell, No. 66 filter, 25' C.
Treatinent S o nitrate added, direct digestion 1.0 nitrate added, BaC12 extraction 30 me. oi nitrate added, Ba 1 2 extraction 30 mg. of.nitrate added, n o extraction
Apparent Sulfate Fulfur in 2 5 - 3 l g . Sample Standarda %nfide-nceK!?rea Mean error oi mean 9.554 99% 26.4h
0.11
2fi.2-26.6
26.0-26.8
23.SC
0 41
22.8-24.8
22 1 - 2 6 . 5
23.6d
0 4 i
22,3-24,9
21.5-26.9
None
..
...
...
ANALYTICAL CHEMISTRY
740
are apparent for t,he lower results by the extraction procedure: First, labile soluble sulfur compounds would not be included in the e x h c t i o n procedure because they would be discarded with the supernatant after centrifuging. Secondly, rather small amounts of sulfate are involved and difficulty in obtaining complete transfer of barium sulfate and residual plant material may account in part for the lower value. hlso illustrated in Table I11 is the precision with 1vhic.h sulfate sulfur may be estimated in samples containing nitrate. The third and fourth items of the table compare the apparent sulfate values for this plant sample containing added nitrate. \\'hen nitrate was added, no sulfate could be detected in the sample using the direct digestion procedure. The barium chloride extraction procedure eliminat'ed the nitrate interference; values obtained by the barium chloride extraction procedure were 23.8 and 23.6 micrograms of sulfur in the absence and presence of added nitrate, respectively. Recovery of sulfate added to samples initially low in sulfate and then carried through the barium chloride extractioii procedure averages 90% (Table I V ) .
Table IV.
to 10 mg. of sulfur required for reliable giavimetric barium sulfate determinations. The met-ashing technique is m ~ l ladapted to use with the proposed method because of the ease mith which large numbers of n e t ashings may be carried out simultaneously, with only moderate attention a t the start of the digestion to prevent loss of sulfur Some additional care is required near the end of the digestion to ensure the complete removal of nitrate. Sitrate, from the nitric acid used in the digestion, is not removed completely even with prolonged boiling of the perchloric acid because of refluxing of nitric acid vapors in the neck of the flask. The addition of excess chloride, most easily from hydrochloric acid, effectively removes all ffitrate, presumably as the volatile nitrosyl chloride.
Table V. Total Sulfur in Tomato Plant Saniple (Comparison of ashing methods, colorimetric and gravimetric determination) s o . of Yo s __ Confidence Limits Nethod Detn-. l l e a n 957 99 R .40AC ashing, gravimetric BaSOi determination .40.4C ashing, methylene blue deterinination
8"
0 617
0.606-0.625
0 599-0.635
85
0 616
0.608-0.624
0.603-0.629
Recovery of Sulfate Added to Plant Material
(Barium chloride extraction method, 25-nig. samples of bean leaf tissue)
Mean Standard error of mean Confidence limits 95% 99%
S
S
S
S
Present, Y 2.4 2.7 1.1 2.3 1.6 1.9 2.0
.4dded,
Found,
Recorered,
%
Y
Y
y
19.3 19.3 19.3 19.3 19.3 19.3 19.3
18.5 18.3 19.4 19.3 21.5 19.9 19.5
16.5 16.3 17.4 17.3 19.5 17.9 17.5
Recovery 8.5.5 84.5 YO.0 89.6 101 . o 92.7 YO 6
0 47
0.24 1.4-2.6 1.0-3 0
.. ,
18.3-20.7 17.6-21.1
0.47 16.3-18.7 15.6-19.4
2.36 84,5-96.7 82.1-99.1
TOTAL SULFUR IN PLANT l I 4 T E R I A L
Total sulfur was determined on a sample of tomato blades (sample 195) employing three ashing methods, the AOAC magnesium nitrate method, the Parr peroxide bomb method, and the described \%-et-ashingtechnique. Gravimetric barium sulfate determinations as well as sulfur estimations by the proposed method were made on the ash solution from the magnesium nitrate ashings. Because of limited amounts of sample available from the other ashing methods, estimations by the proposed method only were done on the other ash solutions. The values for total sulfur in this sample are given in Table V. The results obtained by the proposed method for each of the three ashing methods are in good agreement and also agree well with the gravimetric values obtained on the samples ashed by the AOAC magnesium nitrate method. The confidence limits for all determinations overlap each other, indicating that one may expect substantially the same total sulfur values regardless of ashing method or final determination method. The AOAC magnesium nitrate method is somewhat tedious and time-consuming and occasionally loss of sample may occur in the preliminary ashing stages. Fairly large amounts of plant material may be ashed by this method, an important feature when gravimetric sulfates must be done on materials that are low in total sulfur but of minor importance if the final estimation of sulfur is made by the proposed colorimetric method. The peroxide bomb method is moderately fast, but can accommodate only limited amounts of sample, a factor that frequently limits its use when gravimetric sulfates are determined in low-sulfur samples. When the proposed method for estimation of sulfur is used, the sample size, for either method of ashing, may be scaled down to yield 1 to 300 micrograms of sulfur as compared to the 1
Eight separate ashings. duplicate BaSOa determinations on each ashing. 8 separate ashinge. F i r e separate ashings, six determinations of S as methylene blue. d Sixteen separate ashings, single determination of methylene blue on each ashing. 0
b Six replicate methylene blue determination on each of c
The wet-ashing method is limited by the amount of perchloric acid that can be tolerated in the final digestion of the sample or aliquot with the reducing mixture. Excess perchloric arid ill hot solution is an effective oxidizing agent and oxidizes part of the sulfide as it is formed and also oxidizes iodide to iodine which deposits in the condenser. The iodine deposit in the condenser also may oxidize the sulfide. The net result of excess perchloric acid is incomplete recovery of sulfide. APPARENT SULFATE SULFUR IN PLANT EXTRACTS
Extracts of the tomat'o blade sample 195 were made, using cold water, hot water, cold 5% t,richloroacetic acid, and hot 5% trichloroacetic acid. Four-gram samples were used in each case, and t,he final volume of the extract was 100 ml. The ext,racts were made by st,irring the sample with two successive 25-ml. portions of the extract'ants, filt.ering through KO.12 Whatman filter paper aft'er each extract'ion, and finally mshing t,horoughly on the filter. The hot extractions were made by holding the sample and extractant at a low boil for 5 minutes. hpparent sulfate sulfur n-as determined on appropriat,e aliquots of the extracts.
Table VI.
Apparent Sulfate Sulfur in Tomato Sample
(Comparison of extraction method and direct digestion) Sulfate Sulfur. % Confidence Limits Treatnient Uean 95% 99% Cold water extracta 0.381 0.368-0.394 0 363-0.399 0 377 0.363-0.391 0 387-0 397 Hot water extracta 0 . 3 8 2 0.371-0.393 0.367-0.397 Cold trichloroacetic acid extracta 0 . 3 8 4 0,373-0.395 0.368-0.400 Hot trichloroacetic acid extracta 0 . 3 8 9 0.386-0.392 0.384-0.394 Direct digestionb Q
b
Three separate extracts made: four sulfur estimations on each extract. Twelve separate direct digestions of 10-mg. samples.
The results, summarized in Table VI, indicate that the apparent sulfate sulfur values for this sample are independent of the method of extraction and also are essentially the same whether the determination is made on extracts or by the direct digestion
V O L U M E 24, NO. 4, A P R I L 1 9 5 2
74 1
method. I n order to avoid nitrate interference, 10-mg. samples mere used in the direct digestion method. These data indicate that, if sulfate sulfur only is to be determined on the sample, the direct digestion method is to be pieferred because of saving in time but that, if aqueous or trichloroacetic extracts of plant samples are t k n g made for other purposes, determination of sulfate sulfur in the extract may be the method of choice. The results obtained by the two methods will be essentially the same.
Table VII. Conipound Cystine Cysteine Glutathione Methionine Taurine Cystine Cysteine Glutathione Methionine Taiirine Cystine Cysteine Glutathione Methionine Taurine
Apparent Sulfate Sulfur in Organic Sulfur Compounds Type of Linkage
=C-S--S-C? =C--S--H EC-S-H =C--S--Cr =_C -~SOsH
Apparent
so+-sof
Treatinent
C o i n ~ o r i n d ,yo
Digested directly with reagent
0 001 0.001 0.057 0 001 0.001
. Pretreated acid
with formic
0.220 0 046 0.152
o:Ois PrPtreated ivith nitrate and formic acids
4 70
0 301 0 047 0 23.5 0.084
a freshly prepared sodium phosphate-pyrogallol a-ash solution \vi11 remain almost colorless for as many as six or eight runs. Preliminary tests such as the diphenylamine test for nitrate m t l other oxidizing agents should be made on samples of unkno~vn history. Yolatile compounds arising from the reaction of nitrate with the reducing mixture are incompletely trapped in the sodium phosphate-pyrogallol washing solution and prevent the synthesis of methylene blue, as can be demonstrated by the addition of known amounts of sulfide to the zinc acetate solution through Lvhich the nitrate reduction products have been passed. Addition of ;-aminodimethylaniline and ferric ion to this solution results in no methylene blue production. On the other hand some sulfide is produced when nitrate, sulfate, and the reducing mixture are boiled together, as can be demonstrated by darkening of lead nitrate-impregnated paper held in the issuing gas stream. Hydrogen Peroxide. Hydrogen peroxide interferes by ozidation of iodide to iodine in much. the same way as excess perchloric acid doe.. Therefore the use of hydrogen peroxide in the wetashing procedure is not recommended. Chromate. Moderate amounts of chromate ion appear not t o interfere. Chromate has been used in the barium chloride extraction procedure as a carrier for bariuin sulfate. The use of chromate, however, did not materially increase sulfate recovery and necessitated a considerable correction for sulfate impurities. Its use as a carrier has been clisc~mtinurd.
IVTERFERENCES
Organic Sulfur Compounds. Several organic sulfur compounds, illustrating four types of sulfur linkages, were digested \\-iththe reducing mixture to determine the extent of iriterfrrence that might be expected when apparent sulfate sulfur is tletermined in dry plant material by the direct digestion method. Cystine, cysteine, mrthionine, and taurine yielded no apparent sulfate sulfur when 10 mg. of the compounds Fvere smpended or dissolvetl in 2 nil. of clistilled water and digested with 4 ml. of the reduring mixture in the usual manner (Table CII). Glutathione romtained a small amount of reducible sulfur, probably sulfate impurity. If the rompountls were heated with 2 ml. of 90% formic acid on the steairi bath for 2 hours prior to digestion with t,he reducing niirtui.c, variable amounts of apparent sulfate sulfur were found. Similar ti~rntmentof these compounds in the presence of 30 mg. of nitrate resulted in even larger amounts of sulfate sulfur. The erratic labilizat,ion of organic sulfur thus prevented the use of formic ac4d treatment in removing nitrate interference in the direct digestion method. The use of formic acid in the removal of nitrate interference, from nitrate added to samples containing no nitrate initially, always resulted in apparent sulfate sulfur values that were erratic and 10 to 20% higher than that of the untreated lownitrate, 1o.rv-sulfate samples. The percentage error from this source would probably be small when the sulfat,e sulfur content of the sample is high, but may become apprecliahle in samples of low sulfate sulfur content. The nature and relative amounts of the very labile sulfur compounds in plant materials are not well known, but these results would suggest that substantial amounts of labile sulfur compounds, perhaps others than cystine, glutathione, etc., may be present in some samples. Paper chromatography techniques, especially using radioactive sulfur, as employed by Thomas, Hendrich, and Hill ( l a ) n-ill be of value in elucidation of this prohlmi. The formic acid incorporated in the reducing mixture does not labilize the sulfur of the compounds. No explanation for this anomaly may be offered a t the present time. Nitrate. More than 6 mg. of nitrate in the sample causes low apparent sulfur values. Evidence of nitrate interference is a rapid and pronounced darkening of the sodium phosphatepyrogallol wash solution. I n the absence of nitrate in the sample,
31ISCELL4NEOUS C O \ l \ l E S T S ON TECHNIQUE
Because of adsorption and solubility of hydrogen sulfide in the apparatus and wash solution, it is necessary, for work of highest accurary, to make a preliminary run n-ith a sample or standard containing a small amount of reducible sulfur. The sample need not hr taken through the developmrnt and measurement of color step. The digestion-distillation apparatus and wash solution aye equilibrated after the first run with respect to hydrogen sulfide, arid determinations may be carried on in the usual manner. The data of Tahle VI11 shon that results from the first run are about 5% lo\v-i.e,, about 1 microgram of sulfur was not recovered-but that revovery of sulfur in the next five runs was complete.
Table 1-111. Hecovery of Sulfur as a Function of History of Digestion Apparatus (Sbsoi bancy values are means of 6 replications for each r u n sequence, 19.3 y 9 w e d )
R u n Sequence First Second Third Fourth Fifth Sixth
.4bsorbancy 0,192 0.200 0.200 0.200 0.201 0 200
Confidence 96 % 0.184-0.200 0.195-0.203 0.195-0.206 0.198-0.202 0.197-0.205 0.19.5-0.205
Limits 99% 0.180-0.204 0.192-0.208 0.192-0.208 0 196-0.204 0.196-0.207 0.192-0.208
Methylene blue solutions are stable for a t least several days if stored in the dark. Exposure to sunlight, however, cauws rapid fading. The absorbancy of methylene blue solutionq, as pointed out by Fog0 and Popowsky ( J ) , is markedly influenced by acid concentration. Care must be exercised to maintain acld concentrations constant in all determinations. Dilution of concentrated methylene blue solutions should be made so that the final acid concentration is unchanged. The rate of heating of the digektion mixture is not extremely critical, provided free flame is not allowed to play on the surface of the flask above the level of the reducing mixture. Best results are obtained by the use of an asbestos mat on the top of the flame shield, as described previously.
ANALYTICAL CHEMISTRY
742 The rate of nitrogen flow through t h r apparatus should he sufficiently rapid to sweep the system completely free of hydrogen sulfide in a reasonable time. Sitrogen flow rat,es as high as 500 ml. per minute have been employed without loss of sulfide. Hexevcr, rates of 100 to 200 ml. per minute u e entirely arlequiate. Sloner rates require excessively long times to ensurr conipl(~tr transfer of hydrogen sulfide. LITERATURE CITED
(1) .kssoc. Offic. Agr. Chemists, ”Official and Tentative Methods of
bnalysis,” 6th ed., p. 127, 1946. ( 2 ) Brolvnlee, K. A , , “Industrial Experinlentation,” p. 33, Biooklyn, S . Y., Chemical Publishing c‘o.. 1949. (3) Field, E., and Oldach, C. S., IUD.Esc. CHEM.,A N ~ LF h . , 18, 668-9 (1946). (4) Fogo, J. F., and Popowsky, >I.,.\s.ir.
CHEM.,21, 732-4 (1949).
( 5 ) Ibid., pp. 734-7.
(6) Liebhafsky, H. 8 . , and Winslow, E. H., IND.Ex-tical step for its determination. The emission spectrograph possesses definite advantages over chemical methods for the analysis of minute quantities of inorganic substances: its specificity, speed and economy of analysis, and high sensitivity in detecting all the metallic elements \There only a small amount of sample is available. Hence, the emission spectrograph present,s an attractive means whereby minute quantities of petroleum ash matter, such as is obtained by the combustion of a practical quantity of Ion-ash content cracking stock, may he analyzed. The use of a common matrix for sample excitation in the arc is a well-known technique in emission spectroscopy (1, 3-5, 7-10, 14, 16, 1 7 ) . The common matrix usually is prepared by blending a powdered sample in known weight rat
