April 15, 1930
I AYD I 'ST RI =IL d SD E S G I S E E RI S G C H E Jf I ST 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 LABOK.ATOK~, COSSECTICLTT . ~ G K I C L ' l , T I . K A L EYPEKIMEST STATIOS. NEW' H.Il'ES. CONS
The observation of Ranker that the salicylic acid-zinc niany re-pet. tlic conipwiXTRACTS of g r e e n method does not give quantitative results when applied tioii of a plant cxxtract. I t tobacco leaves grown to aqueous extracts of plant tissue that contain nitrates i-. clear that the analy-is by under cliff er en t c o iid ihas been confirmed. A n error due to the loss of from tlie d i c y l i c a c i d - z i n c method tiom hare been found to coli40 to 95 per cent of the nitrate nitrogen may occur. of +iniple- of plaiit extract. tain widely variable proporThe total nitrogen of such extracts may, however. be that contain water cannot lie tions of the total nitrogen as quantitatively determined if a preliminary reduction concliicted without the daiinitrate (6). More than 30 of the nitrates with reduced iron powder and dilute ger of lo-i of riinch of the per cent of thr. water-soluble sulfuric acid is carried out, followed by Kjeldahl digesnitrate nitrogrn. iiitroeen is sonietinies nreseiit tion with sulfuric acid in the customary way. This 111 this form. although .iiialler Development of New method was originally suggested by Olsen for use in proportion- are perhaps more Rlethod determining the nitrogen content of soils and its ii-ual. T h e preqeiice of application to plant extracts is herein described. Alttentioiiu a. therefore clinitrate nitrogen iiecesitate. rected to the tiewlopnient of a the use of opecial method. for the determiriation of the total nitrocen in such extracts. procedure that iiiight be conveniently applied to aqueous solutions containing nitrates. llethoclb iiiT-olviiig the reaxid the writers' attempts to employ the salicylic acid-zinc method ( I ) have substantiated the rewlts of Ranker (.?). duction of the nitrate in the solution 1)y mean? of Dvarda's who found that thiq niethod cannot he successfully used to alloy were not concitleretl hecause of the clo,se attention determine nitrogen in aqueous solutions although it is per- required to the details of the prnceclure. Raiikcr's niethod fectly satisfactory when applied t o properly dried material. of drying the $ample.: at exact neutrality in the Kjeldahl The writers have therefore deJ-eloped a method for the de- flasks before digestion with salicylic aritl-thiosulfate iiiixture terniination of nitrogen in the presence of nitrates that can calls for a considerable expenditure of tiiiir. The present lie applied directly to aqueous solutions. d description of writer> therefore turiietl to the iihe of reduced iron powder this method and data illuitrating the errors that may arise in dilute acid as a nieaiii: for the reduction of the nitrate to when the salicylic acid-zinc niethotl is iiiiproperly used arr aniiiionia hefore the application of the Kjrltlahl tligestioii proceqs. hrrriii premit et1.
E
0
Analyses by Salicylic Acid-Zinc hlethod
Table I gives the rewlts of analyses by the -alicylic acitlziiic riiethotl of 10- arid 20-cc. portions of a series of tohaccoleaf extracts. Altliough dupli well with each other. it is clear o c c ~ i rduring the digeqtion of 1iiore. a serious error even in t p l e ~bcconiw evident ~vlient h 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 ~vlienit 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 I o n 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. T h e expenses of this investigation were shared by t h e Connecticut Agricultural Experiment Station and the Carnegie Institution of Ivashington, D. C .
T a b l e I-Analyses
of T o b a c c o E x t r a c t s b y Salicylic h c i d - Z i n c M e t h o d
s IN 8
s \\lPI.E
I
N F O V NISD8 LITERSof E X T R i C T FKOX A S ~ L Y S I S OF:
LITERSO F EXTK.ACT~
I
E
110
u
177
,
-
20-cc.
~
10-cc.
127 8
143 1
1'8.4
I42 0
i
I
D r ' E To OF
, '0-cc.
2i.6
10-cc.
;.
19
The figures lor the total nitrogen in these extracts were calculated from the difference in the nitroien in the dry leaf before and a f t e r extraction with hot water. Their accuracy was confirmed h y analyses h y t h e 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 Method ~OLI.ME TOTAL x SITRATE t i TOTALN A K A L Y Z E D PRESEXT T ~ ~ FOCSD ~ ~
cc.
Grams
Per
15.0
19 13 32 97
:x
30 0 "7 0 26 0
Grams 14.91
cent
$1 75
25 28
03
14 49 7 81
26.98
25.03 ~~
23.29 23.15 ~~
1.0s~ O F TOTAL % N ~
P e r cenl 22 06 23.31 1: 68 I .a7
Loss
OF
SITRATE S''
Peu
ct'ni
42 63
77.67 94.37 95.96
~~
Calculated on the acsumption t h a t the entire loss falls upon t h e 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
A-YA4LYTZC'ALE D I T I O S
192
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 b y 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. I n 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. Table 111-Determinations of Nitrogen i n P o t a s s i u m S i t r a t e S o l u t i o n Using Reduced Iron Powder .Method N I T R A Th EPRESENT
9.90 9.90 19.80 19.80 19.80 29.71 29.71 29,il 39.60 39.60 39.60 ~verape
2 G R A MR S E D U C EIROX D
3 GRAMS REDCCED IROU
Nitrate N found
Nitrate N found
9.iS 9.3; 16.03 16.74
...
Per cent 98.79 94.64 91.05 94.62
26.84 27.12
90:36
: 35.50
93: i 7 89.64
3i 06
I
Recovered
. . I
Me. 9.80 9.80
19.74
91.31
...
92.99
I
19 60 19.60 29.54 29.54 29.54 39 90 39.48 39.48
Per i z n l
93 99 98.99 99 i 0
98 OR 95 99
09 99 90 100 99 99 99
T-ol. 2, s o . 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, digestion 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, cspecially 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 cleterininations and four mixtures v i t h different ratios of nitrate nitrogen to total nitrogen were prepared. The recovery of nitrogen from these mixtures was extremely good an(l indicates that the method gives satisfactory results. Table IV--Analyses of Mixtures of a S o l u t i o n of A m i n o Acids w i t h P o t a s s i u m Nitrate by t h e Reduced Iron-Powder Method TOTAL S PRESEST
K I T R A TK E PRESEXT
NlTR.4TE N
.If 2.
Per cent
Jig.
0.90
30.03
32.94 32.94 32.90
3.91
14.49
27.12
TOTALN
TOT.AL N R E C O V E ROFY FOUND TOTALN
43 4,3
2" ,
Mg.
Pev cent
it1
7u 46
100.78
Average
It is clear, therefore, that, when properly used, reduced
32.97
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 disregarded.
26.98
Reduced Iron-Powder Method
25.05
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 ~ ~ heated ly 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 continued 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 e usual way. t h I t is important that the reduction of the nitrate be carried out with careful attention to the mescribed volumes of water and acid; otherwise loss of nitrogen may occur. The
Average
26.84 27.12 26.99 Average
1.9s
7.81
Average Grand average
24,99 24.99 24.85
99.91 99.91 99.79 99.87 100.5 99,47 100.5 100.0 100.01
99,7.i 99.75 99.45 99.65 100.08
a 0.5 gram sucrose added before reduction.
T a b l e V-Total Nitrogen i n S a m p l e s of Dry Tobacco by Reduced IronPowder M e t h o d a n d b y Salicylic Acid-Zinc Method SAMPLE
IROPREDUCTIOS METHOD
SALICYLIC ACIPZIXC ~IETFIOO
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 t---h e st,andard salicvlic acid-zinc method. The samples -i
I S D r S T R I , I L An'D ESGIA-EERISG CHEMISTRY
April 15, 1930
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.
193
Literature Cited (1)
(2) (3) (4) (5) (6)
Official Agr. Chem., Methods, 1925, p. 9. Jones, IND.ENG.CHEM.,19, 269 (1927). McCandless and Burton, J . Assocn. O f i c i a l A u . Chem., 10, 216 ( 1 0 2 : : . Olsen, Comfit. rend. W a y . lab. Carlsbevg, 17 [3]. 1 (1927). 230 (192i) Ranker, J , Assocn, 05ciai A g r , C h e m , , Vickery and Pucher, IKD.ESG. CHEM.,Anal. Ed., 1, 121 (1929). ASSOCU.
Titrometric Determination of Magnesium' J. Stanton Pierce and 11. B. Geiger GEORGETOWN C O L L E G E , 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 thymolphthalein a3 indicator (.9). In the absence of calcium, magiiesiinn has !xen tleterniiiied 11sprecipitation 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 solution i.3, S ) . Hou-euer, in tlie presence of a high concentration of calciuiii salts t,his method fails unless some meaiis is used to indicate the completeness of precipitation of mapnesiinii, 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 determination of niagnesiuiii in such mixtures often is verp important, there is need for a practical, rapid, convenient, and fairly accurate laboratory method for the determination of magnesium in the presence of calcium. Direct titration of a neutral solution with alkali, using trinitrobenzene as indicator ( i ) ,gives results accurate enough for most control work, but requires a matching of colors. The method worked out in this laboratory for the determination of magnesium 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 required for this operation is long, there is danger of adsorption 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 calcium remains in solution. The solution containing the precipitate 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 precipitation of magnesium, with calcium remaining in solution, 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 modification, 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 obtained. Filter and wash tlie precipitate, cat'ching the ~vashiugs in the same beaker as the original filtrate. To the filtrate add 5 drops of 0.1 per cent alcohol solution of dimethylamiiioazoheiizene and 0.25 S hydrochloric acid to the appearance of a faint pink. Add 10 cc. of a saturated alcoholic 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. volumctric 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 magnesium oside is as follows: B1 - 2 A 1
=
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 3IgO Sample present 1
2 3
3IgO found
s
ci
1.0
1.0 4.1
L O
16.8
,n
l6.,
TSCIIXICAL S A L T S OF hlg Sample present
4 5
6
hIAGNBSIEY C I I L O R I D B
31g found
5%
c-
7 4
7 4 (contains CaCI:I
12 2 23.8
I
12.4 23.5
With the same samples the inagiiesium was determined as described in tlie article on the determination of magnesium 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 inagiiesiuiii hydroxide and a sample for analysis pipetted. If a large number of samples are t o be ruii ill which the iiiagiiesiuiii 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. Trinitrobenzene in alkaline alcoholic solution readily changes to some compound which retains a distinct red color after
