April 15, 1930 I AY D I 'S T RI =I L d S D E S G I S E E RI S G C H E J f I S T R Y 191

Determination of Total Nitrogen of Plant Extracts in Presence of Nitrates'

George W. Pucher, Charles S. Leavenworth, and Hubert Bradford Vickery

BIOCHEMICAL

XTRACTS of g r e e n tobacco leaves grown E under cliff er en t c o iid i-

tiom hare been found to coli- tain widely variable propor- tions of the total nitrogen as nitrate (6). More than 30 per cent of thr. water-soluble iiitroeen is sonietinies nreseiit

0

111 this form. although .iiialler proportion- are perhaps more ii-ual. T h e preqeiice of nitrate nitrogen iiecesitate. the use of opecial method. for the determiriation of the total

LABOK.ATOK~, COSSECTICLTT .~GKICL' l ,TI .KAL EYPEKIMEST STATIOS. NEW' H.Il'ES. CONS

The observation of Ranker that the salicylic acid-zinc method does not give quantitative results when applied to aqueous extracts of plant tissue that contain nitrates has been confirmed. A n error due to the loss of from 40 to 95 per cent of the nitrate nitrogen may occur. The total nitrogen of such extracts may, however. be quantitatively determined if a preliminary reduction of the nitrates with reduced iron powder and dilute sulfuric acid is carried out, followed by Kjeldahl diges- tion with sulfuric acid in the customary way. This method was originally suggested by Olsen for use in determining the nitrogen content of soils and its application to plant extracts is herein described.

niany re-pet. tlic conipwi- tioii of a plant cxxtract. I t i-. clear that the analy-is by tlie d icy l ic a c i d - z i n c method of +iniple- of plaiit extract. that contain water cannot lie concliicted without the daii- ger of lo-i of riinch of the nitrate nitrogrn.

Development of New Rlethod

Alttentioii u a. therefore cli-

nitrocen in such extracts. axid the writers' attempts to employ the salicylic acid-zinc method ( I ) have substantiated the rewlts of Ranker (.?). who found that thiq niethod cannot he successfully used to determine nitrogen in aqueous solutions although it is per- fectly satisfactory when applied to properly dried material. The writers have therefore deJ-eloped a method for the de- terniination of nitrogen in the presence of nitrates that can lie applied directly to aqueous solutions. d description of this method and data illuitrating the errors that may arise when the salicylic acid-zinc niethotl is iiiiproperly used arr hrrriii premit et1 .

Analyses by Salicylic Acid-Zinc hlethod

Table I gives the rewlts of analyses by the -alicylic acitl- ziiic riiethotl of 10- arid 20-cc. portions of a series of tohacco- leaf extracts. Altliough dupli well with each other. it is clear occ~ i r during the digeqtion of 1iiore. a serious error even in t p l e ~ bcconiw evident ~vlien th t h k nietliod is coniparetl with the nitrogen content of the cxtract calculated froni the clifference in the amounts of nitrogen in the leaf material before and after extraction. T1ie.e results are in accord with Raiiker'.s view that water. prwent during tlie treatment of the saiiiplr with the acid reagent. gives rise to side reactions which involve lo;.: of nitrogen. The insidious nature of thi.: difficulty iq apparent ~vl ien it is recalled that such losses may escape detection cntirely if nitrogen balances are not calculated. Tests upon mtracts which contain a high proportion o f nitrate nitrogrn ciiipliasize thii. error, but it is of equal iniportaiice ill tlie trnalysis ~f extract> of Ion nitrate content.

In Table I1 are preRentetl data secured of mixtures of the product:: of acid h\-drol> edehtin XTith known amounts of potassium nitrate in ratios cif nitrate to total nitrogen Yarying from 8 to 50 per cent. The losses of total nitrogen ahon-n in coluiiin 5 varied froin 7 t o 22 per cent. On the assumption that the entire error is due to loss of nitrate nitrogen. the figures in column 6 shorv that this varied from 43 to 96 per cent. These data are especially significant since the solution analyzed simulates in

1 Received December 19, 1929. The expenses of this investigation were shared by the Connecticut Agricultural Experiment Station and the Carnegie Institution of Ivashington, D. C.

rected to the tiewlopnient of a procedure that iiiight be conveniently applied to aqueous solutions containing nitrates. llethoclb iiiT-olviiig the re- duction of the nitrate in the solution 1)y mean? of Dvarda's alloy were not concitleretl hecause of the clo,se attention required to the details of the prnceclure. Raiikcr's niethod of drying the $ample.: a t exact neutrality in the Kjeldahl flasks before digestion with salicylic aritl-thiosulfate iiiixture calls for a considerable expenditure of tiiiir. The present writer> therefore turiietl to the iihe of reduced iron powder in dilute acid as a nieaiii: for the reduction of the nitrate to aniiiionia hefore the application of the Kjrltlahl tligestioii proceqs.

T a b l e I-Analyses of Tobacco Ext rac ts b y Salicylic hc id-Zinc M e t h o d

I N FOVND IS 8 LITERS of s IN 8 E X T R i C T F K O X A S ~ L Y S I S Dr 'E To

OF i - ~ , OF: s \\lPI.E LITERS O F

10-cc. I ' 0 - c c . 10-cc. E X T K . A C T ~ I 20-cc.

E 110

u 177 , 127 8 143 1 2 i . 6 19 .; 1'8.4 I 4 2 0

The figures lor the total nitrogen in these extracts were calculated from the difference in the nitroien in the dry leaf before and af te r extrac- tion with hot water. Their accuracy was confirmed hy analyses h y the method dercrihed in this paper

T a b l e 11-Analyses of A q u e o u s S o l u t i o n s C o n t a i n i n g A m i n o Acids a n d P o t a s s i u m S i t r a t e b y t h e Salicylic Acid-Zinc

M e t h o d

~ O L I . M E TOTAL x SITRATE t i TOTAL N 1.0s~ O F Loss O F AKALYZED PRESEXT T ~ ~ ~ ~ % ~ FOCSD TOTAL N SITRATE S''

cc. Grams Per c e n t Grams P e r cenl Peu ct'ni 15.0 19 13 $1 75 1 4 . 9 1 22 06 42 63

"7 0 26 .98 14 49 23.29 1: 68 94.37 26 0 2 5 . 0 3 7 81 23.15 I . a 7 9 5 . 9 6

30 0 32 97 :x 03 2 5 2 8 2 3 . 3 1 7 7 . 6 7

~~ ~~ ~~

Calculated on the acsumption tha t the entire loss falls upon the nitrate nitrogen.

d iiiethod for the drterniination of tlie total nitrogen of >oil., which involved reduction of the nitrates with iron powder, has been described by Olsen ( d ) > but no data on the application of this procedure t'o the analyis of plant

192 A-YA4LYTZC'AL E D I T I O S T-ol. 2 , s o . 2

NITRATE h- PRESENT

extracts were given by him. The reaction of dilute sulfuric acid on reduced iron powder has been widely used for the quantitative reduction of nitrate to ammonia in analytical procedures. Jones (2 ) has reported quantitative recoveries of nitrate by this method, as have T'ickery and Pucher (6). McCandless and Burton (3) have reported incomplete recoveries by this method. The analyses in Table I11 were therefore conducted in order to furnish an additional clleck upon Olsen's statement that the amount of reduced iron employed influences the conipleteneqs of the reduction. In columns 2 and 3 are given results of the analyses of different amounts of potassium nitrate using 2 granis of reduced iron. The low recoveries indicate that this is insufficient. TVhen 3 grams of reduced iron were used, however, the aT.erage recovery was 99.46 per cent, which agrees n i t h the results of Olsen, who used 3 grams and obtained a recovery of 99.5 per cent. It is highly probable that different lots of reduced iron pon-der differ in quality, and before use each lot should therefore be tested in this respect as v-ell as for its content of nitrogen. The fact that the results are almost inT ariably slightly low suggests that a sniall but ordinarily negligible loss of nitrate nitrogen occurs.

2 GRAMS REDUCED IROX 3 GRAMS REDCCED IROU

Nitrate N Recovered Nitrate N found found

Table 111-Determinations of Nitrogen i n Potassium Si trate Solution Using Reduced Iron Powder .Method

Per cent M e . Per i z n l 9 . 9 0 9 .90

19.80 19.80 19.80 2 9 . 7 1 2 9 . 7 1 29,il 39.60 39.60 39 .60

9.iS 9.3;

16.03 1 6 . 7 4

2 6 . 8 4 2 7 . 1 2

3i : 06 35.50

. . .

. . I

98.79 94.64 91 .05 9 4 . 6 2

90: 36 91.31

93: i 7 89.64

. . .

9.80 93 99 9 .80 98.99

19 .74 99 i 0 19 60 98 OR 19.60 95 99 29 .54 09 43 29 .54 99 4,3

39 90 100 , 39.48 99 i t 1 39.48 99 7u

29 .54 90 2"

evaporation of the solution before the digestion is sometimes troublesome, since bumping may take place in the early stages and, as the acid concentration increases, a heavy froth may form. After this stage is passed, however, diges- tion runs smoothly and more rapidly than is usually the case with salicylic acid digestions. The addition of the sodium sulfate and of the mercury is essential to a smooth and rapid digestion. After the dilution of the digest any cake of salt a t the bottom of the flask must be dissolved before adding the alkali. The initiation of boiling during the distillation is sometiines accompanied by violent frothing, although this seldom becomes uncontrollable if due care is exercised. The contents of the flask should be black during the distillation; if they are red or brown an insufficient amount of alkali has been added. Blank determinations on the reagents, cspe- cially the reduced iron powder, must be conducted.

rlccuracy of Method

Table IV gives the results obtained on solutions containing inistures of the products of hydrolysis of the protein edestin ant1 potassium nitrate. The total nitrogen content of the hydrolysate was establi,+ed by ordinary Kjeldahl cleterinina- tions and four mixtures v i t h different ratios of nitrate nitro- gen to total nitrogen were prepared. The recovery of nitro- gen from these mixtures was extremely good an(l indicates that the method gives satisfactory results.

Table IV--Analyses of Mixtures of a Solut ion of Amino Acids with Potassium Nitrate by t h e Reduced Iron-Powder

Method

TOTAL S KITRATE K NlTR.4TE N TOT.AL N RECOVERY OF PRESEST PRESEXT TOTAL N FOUND TOTAL N

M g . .If 2. Per cent J i g . Pev cent

~ v e r a p e I 92.99 I 99 46

Average 100.78

It is clear, therefore, that, when properly used, reduced iron may be depended upon to effect practically complete reduction of the nitrates present in a plant extract and h c e these, in general, form only a small proportion of the total nitrogen, the small error may in most cases be safely dis- regarded.

Reduced Iron-Powder Method

The sample is delivered from a pipet into a Kjeldalil flaA (700 cc.) and diluted to 3&40 cc. with distilled water; 10 cc. of 1:1 sulfuric acid and 3 grams (10.3) of reduced iron powder are added. X funnel is placed in the neck of the flask, which is shaken at room temperature for 10 minutes and then s l o ~ ~ l y heated and the contents boiled for 5 minutes. The flask is cooled and 30 cc. of concentrated sulfuric acid. a drop of mercury (0.3 gram), a few angular quartz pebbles, and 5 grams of anhydrous sodium sulfate are added. The flaqli iq heated slorrly to evaporate the water and the contenti are digested in the usual way until the acid layer is clear and the precipitate assumes a yello\T color. Heating is then con- tinued for 1 to 2 hours more. .& few crystals of potassium permanganate are dropped into the hot acid, which is then cooled and diluted with 300 cc. of water; 3 to 3 grams of sodium thiosulfate and a small piece of paraffin are added. An excess of sodium hydroxide and a little zinc are then introduced and the ammonia is distilled into standard acid in

t h I t is important that the reduction of the nitrate be carried out with careful attention to the mescribed volumes of water

e usual way.

32.97 0 . 9 0 30.03 3 2 . 9 4 9 9 . 9 1 32.94 99 .91 32.90 9 9 . 7 9

Average 99.87 26.98 3 .91 14.49 27.12 100.5

26 .84 9 9 , 4 7 2 7 . 1 2 100.5 26.99 100.0

Average 100.01 25.05 1.9s 7.81 2 4 , 9 9 99,7.i

24.99 9 9 . 7 5 24.85 99 .45

Average

Grand average

99 .65 100.08

a 0.5 gram sucrose added before reduction.

Table V-Total Nitrogen i n Samples of Dry Tobacco by Reduced Iron- Powder Method a n d by Salicylic Acid-Zinc

Method

IROP REDUCTIOS SALICYLIC ACIPZIXC SAMPLE METHOD ~ I E T F I O O

As a final check upon the accuracy of the method a series of dry samples of tobacco was analyzed by the new method nnrl hv the st,andard salicvlic acid-zinc method. The samples and acid; otherwise loss of nitrogen may occur. The -i ---- -

April 15, 1930 I S D r S T R I , I L An'D ESGIA-EERISG CHEMISTRY 193

contained approximately 20 per cent of the total nitrogen in the form of nitrate. The data (Table v) show that the two methods lead to practically identical results; they further demonst,rate that the can be safely employed for the determination of the total nitrogen in solutions of the complex mixture of substances found in leaf tissue.

Literature Cited (1) ASSOCU. Official Agr. Chem., Methods, 1925, p. 9. (2) Jones, IND. ENG. CHEM., 19, 269 (1927). (3) McCandless and Burton, J . Assocn. O f i c i a l A u . Chem., 10, 216 ( 1 0 2 : : . (4) Olsen, Comfit. rend. W a y . lab. Carlsbevg , 17 [3]. 1 (1927). (5) Ranker, J , Assocn, 05ciai A g r , Chem, , ( 6 ) Vickery and Pucher, IKD. ESG. CHEM., Anal. Ed., 1, 121 (1929).

230 (192i)

Titrometric Determination of Magnesium' J. Stanton Pierce and 11. B. Geiger

GEORGETOWN COLLEGE, GEORGETOWX, KY

M AGSESIUlI has lieen determined titronietrically in the presence of calcium by the use of the hydrogen electrode (2 , d ) , Ti-ith trinitrobenzeiie as indicator

(T), and iii 66 to 75 per cent alcohol solution Tvith thymol- phthalein a3 indicator (.9). In the absence of calcium, mag- iiesiinn has !xen tleterniiiied 11s precipitation of the hydroside with excess alkali! separation of the precipitate and solution, and titration of the e w e s alkali in a n aliquot of the solu- tion i.3, S ) . Hou-euer, in tlie presence of a high concentra- tion of calciuiii salts t,his method fails unless some meaiis is used to indicate the completeness of precipitation of map- nesiinii, for on the addition of more alkali calcium also is precipitated.

Since calcium is associated so frequently with iiiagnesiuni in nature and in manufactured products, and since the de- termination of niagnesiuiii in such mixtures often is verp important, there is need for a practical, rapid, convenient, and fairly accurate laboratory method for the determina- tion of magnesium in the presence of calcium. Direct titra- tion of a neutral solution with alkali, using trinitrobenzene as indicator ( i ) , gives results accurate enough for most con- trol work, but requires a matching of colors. The method worked out in this laboratory for the determination of mag- nesium in cement' (6) gives fair results, but the filtrat'ion and washing of the gelatinous precipitate of magnesium hydroxide is tedious if this precipitat,e is large. Also, if the time re- quired for this operation is long, there is danger of adsorp- tion of carbon dioxide from the air and precipitation of calcium carbonate with the niagnesiuiii hydroxide.

Since the time factor determines the value of analytical procedures in industrial laboratories, the nTork on niagnesiuni was continued so that tlie tedious filtration could be avoided and the method made more universal.

In this n-ork magnesium is precipitated as the hydroside. Jt-itli standard carbonate-free alkali. When tlie end point for trinitrobenzene (a dark brick red) is obtained, the cal- cium remains in solution. The solution containing the pre- cipitate is diluted t'o a definite volume, an aliquot filtered. and the escess alkali titrated.

The accuracy of this method depends, not on the sharpness of the break of the neutralization curl-e in the titration of magnesium with alkali, but on the completeness of the pre- cipitation of magnesium, with calcium remaining in solu- tion, and the end point when the excess alkali is titrated with acid. This end point is around a pH of 4, and is quite definite. Iiolthoff (.$) states that calcium hydroxide does not precipitate in 0.01 -1- sodium hydroxide and in this work its concentration does not become half so great.

Experimental Procedure

The following procedure for limestone, with slight modifica- tion, may be used for many substances containing magnesium.

I Received March 21, 1930.

Dissolye sample of 0.500 gram in shout 50 cc. of 0.25 S hydrochloric acid, boil out carbon dioxide, add 5 or 6 drups of 0.04 per cent alcohol solution of broniothymol blue, and add alkali until a blue color, iiidicatiiig neutrality, is ob- tained. Filter and wash tlie precipitate, cat'ching the ~vash- iugs in the same beaker as the original filtrate. To the filtrate add 5 drops of 0.1 per cent alcohol solution of di- methylamiiioazoheiizene and 0.25 S hydrochloric acid to the appearance of a faint pink. Add 10 cc. of a saturated alco- holic solution of triiiit,robenzene and titrate wit'h 0.25 K carbonate-free sodium hydroxide (I?!) to the appearance of a deep red color. Transfer the solution t o a 100-cc. volumct- ric flask and continue titration, if necessary, t o hold the deep red color. Dilute to 100 cc.. mix, and filter through a dry filter paper into a dry beaker, keeping the funnel covered ivith a watch glass as much as possible. Pipet 50 cc. of the filtrate and add an indieator which a t a p H of about 4.0 has a color change easily recognizable in a reddish solution. Tit'rate with 0.25 S hydrochloric acid ( A , ) to the color change of tlie indicator.

X sufficiently close approximation of the per cent mag- nesium oside is as follows:

B1 - 2A1 = per cent MgO Solutions of samples containing 110 cation whose hydroxide

is insoluble a t neutrality are treated as is the neutral filtrate mentioned above.

LIMESTOWS TSCIIXICAL S A L T S OF hIAGNBSIEY C I I L O R I D B

3IgO 3IgO hlg 31g

s ,n 5% I < >

Sample present found Sample present found c- c i

1 1 . 0 1 . 0 4 7 4 7 4 (contains CaCI:I

3 1 6 . 8 l 6 . , 6 2 3 . 8 2 3 . 5 2 L O 4 . 1 5 12 2 1 2 . 4

With the same samples the inagiiesium was determined as described in tlie article on the determination of mag- nesium in cement (6). satisfactory data were obtained, but the method is much longer than the one given above.

Instead of filtering an aliquot of the solution above, the sample may be centrifuged after precipitation of the inag- iiesiuiii hydroxide and a sample for analysis pipetted. If a large number of samples are t o be ruii ill which the iiiag- iiesiuiii content is not very high, it may be advantageous to place each solution, after dilution to 100 cc., in a tall cylinder to allow the precipitate to settle, and to pipet a sample of the supernatant liquid for titration.

Discussion of Method

Bromophenol blue was used as indicator for obtaining the data given above. This is a satisfactory indicator if a n aliquot of the alkaline filtrate is titrated right away. Trini- trobenzene in alkaline alcoholic solution readily changes to some compound which retains a distinct red color after