ANALYTICAL CHEMISTRY
1526
dioxide by the copper oxide even a t elevated teniperatures. This was noticeable until the wire gauzc. plug 0, Figure 1, a t the bottom of the quartz tube was placed in the hot section of the furnace, so that absorption by relatively cool copper oxide \vas eliminated. Thc large amount of oxygen used to 1 0.34 9 9 . 1 7 1.13 !i8 11 4.94 9 4 . 2 4 11.72 87.17 21.95 7 7 . 7 8 flush out the quartz combustion tube tended to 2 0 . 3 5 99.1.5 1 36 97.89 4 . 9 5 94.18 11.66 86.59 22.11 7 7 . 7 2 $', 0 . 4 0 99.10 1 . 3 0 07.!l.i 4 . 9 6 94.27 11.53 87.26 2 2 . 1 3 77.70 reduce the volume of absorbed carbon dioxide to i 0 . 3 8 99 12 I 33 ox nt 4.93 94.29 11 5 9 8 7 . 2 4 .. an absolute minimum. * 1)rr. 0 02 0 . 0 3 0 . 0 4 0 07 0 01 0 . 0 4 0.01; 0 10 0.08 0.03 Increasing the time of flush-out for a fixed gas ;\veragp deviation = .tO.05 flow did not change the final analytical results to _ ___ -_ . anv extent, $0 that error due to 'carbon dioxidr adsorption \vas not appreciable. 3. Any dissociation of copper oxide a t this temperature would obviously not interfere with this method. volume per cent was o1)t:iincd hy substituting in the following 4. Further accuracy would be obt,ained if molar volume corformulns: rections for deviations from ideal behavior were made on thr initial sample volume. If these corrections were made on the samples analyzed, they would be less than 0.1% for the carbon dioxide composition and would not appreciably change the h a 1 results. Table
1. Precision Obtained with Gravimetric Gas Analysis Apparatus
~
% s,= 100 - (% ( ' 0 2
+ % PO)
Sitrogmn is obtained by diflerrnce. DISCUSSION
T h r usual precautions taken for any accurate gravimetrir itnnlysis, such as combustion carbon-hydrogen analysis, pertain to this method. Besides these precautions, blank analysis had to be made periodically. When tank oxygen was passed through the : h o r p t i o n bulbs for any period of time, an increase in w i g h t was oljtained, proportional to the volume of oxygen used. The blanks \yere obtained a t a controlled flow rate for a specific time and were xpplied to each analysis. The blnriks under the esperinient:il vonditions discussed were approximately 1 mg. or less for e:ich bull) and were found to tie suq)risingly c~onaistrnt. Several possible dific.ultiea pertaining to this method of oxidation of carbon monoside have hrrn listed l)y vwious investigatow.
1. The caonibustion of ca~,l)onnlonosidc b y caoppcr oxidr is retests showed no inported to IF plow: hoivevc3r, i~u~)c~~~iment:tl fluence of various flow ratrs of itlrlitio:il gas samples passed over the hrated coppt'r osidc. KOdiffct~encesin results Twre obsei~vetl for various oxygen flush-out rates w h m suitoble blank corrections \vrw made. 2. -4 large error may he introduced k)y the absorption of rarbon
inalyses has been made using this apparatus. The gas samples contained 75.0 to 99.5% carbon monoxide with the balance carbon dioxide and small percentages of nitrogen. The deviation from the average composition of any series of analyses made on the same gas mixture was from 0.02 to 0.10%. The average deviation of all the :inalyses made was *0.05%. Several typical series of :inalyses :ire shown in Table I, w r h except series E comprising four separate determinations. The gravimetric gas an:dysis procedure would appea~.to I)e generally applicable with suit:hl)le modifications to the precise nnalysis of g:iseous mi\tui (8s othei. than those studied iii thi< invrst iga t ion. LITEH4TURE CITED
(1) .Joi.dan. L., and Ecknian E., Bur. Standards, Scz'. Paper 514, 445-82 (1924-26). (2) King. J. G.. and Edgcombe. L. J.. Dept. Sci. Ind. Research, Fuel Research, Tech. P a p e r 33 (1931). ( 3 ) Lunge, G., and +%mhler,H. R., "Techilical Gas rinalysis." p. 159, New York, D. Van Sostrand (70..1934. (41 O t t , E., J . Gas6dl~tcIit..62, S9-90 (1919). ( 5 ) Rodhe, O., U. S. Patent 1,385,150 (.July 19. 1921). (6) \Vii~kelmann,H., T V r r v n ~ ?i i . Kiiltctt'ch., 23, 229-30 (1921). HECF.IVKD FRbrriary li, 1949. Prmented a t t h e Third Annual Sympusiilin on Analytival Chemistry, l ' i t t ~ l > i l r d iScrtion, . & v E R I C h N CHEMICAL SOCIETY. PittatiiirRli, Pa.,February 12 a n d 13. 104X. From a thesis submitted in partial fiilfilllnent of t h e reqiiireinents for the degree of doctor of si'ienre a t Carnrnir Iii>titiite of Terhnology.
Determination of Nitrate in Plant Material C. M. JO€IYSON A Y D 4LBEHT VI,HICfI, I.niveraity of California, Berkeley I, Calif.
1 rapid, routine modification of the phenoldisulfonic acid method for the determination of nitrates in dried plant material is described. The success of the method, when applied to plant materials containing considerable amounts of chlorides, color, and easily oxidizable organic matter, depends on the rapid remo, a1 of chlorides w-ithout subsequent interference by silver ions and on mild alkaline peroxide digestion to destroy the organic matter.
A
S h PART of itn extensive study o f the fertilizer requii~>ment of crops (f&f8), nitrates, in preference t o other forms of
nitrogen (16, page 180), are determined in plant materials. S i trate is estimated by a modified phenoldisulfonic acid method ( 2 , 15,page 190) because of its sensitivity for small samples and the speed with which large numbers of analyses may be done. Frequently, for diagnostic and comparative purposes, the ahsolute accuracy of the analysis is regarded as of secondary importance to speed and precision, t,ecanse the field saniplirig errors are
usually much greater than the normal analytical et'rors. It is desirable, however, to increase the analytical accuracy and pi'ecision as much as rompatible with maximum speed, in ordei, that refinements in field experimental technique may he utilized to the fullest estent. This paper describes t,he techniques that have been found necessnr>- to the successful estimation of nitrate in plant materials. Phenoldisulfonic acid has been used for many years as a reagent for the dptrrminatioti of nitrate (6, 9-ff),hut Chamot and Po-
V O L U M E 2 2 , N O . 12, D E C E M B E R 1 9 5 0 \vot,ket,s(5-8) were the first to study atically the composions, and the preraution of the reagent, the nature of th tions necessary to the accurate determination of nitrates in ~ a t r r 1)). thi.; method. Roller and IlcKaig (13) have studied the fartors involved in the use of the method in the determinntion of nitr:Ltr i n soil extracts rontaining considerable color and organic material. The clrtermination of nitrate nitrogen in plant tissurs h y this nietliod hns twrn less common, probably because of the greater tliHiculty in wot,king with cstrarts containing large amounts oi' or~gariir matter. Ashton ( 1 ) and Rurstrom (4)havr tlesc*rilwl their p r o c d u r t ~ susing the phenoldisulforiic~arid nlethocl for the tlrterniination of nitrate in plant material. ..\rluenus extracts of many dried plant materials with whirli tlir authors have worked contain considrrahle amounts of ot.ganic matter :tnd i'requently amounts of chloride ranging from 1 to 15% on t i dt,y weight hasir. The presence of the organic, mnttei,, n.lic.thc~t~ pigmented or not, may lead to errors of t\vo kinds: (1 j ('olors :iriuing from plant pigments md from tht, c*hauirig of i~:i;ily oxidizable organir matter by the concentrated sulfuric arid of tlie phenoldisulfonic acid reagent interfere with thr accurate niwwrrment of the color intrnsity of the yrllow salt of t h r nitrophctioldisulfonic acid; and (2) nitrate is usually lost, presuma1~l~i)y reduction, when t h e ~~lietiol~lis~tlfonic ac,id reagent is p1:trcd o i i the (tried extract contsining osidizrihle organic matter. C'hlorides may cause losscs of nitrate when tlie dry residue is tiyated with the phenoldisulfonir ticid i,c>ngnitthrougli thr formation of volatile nitrosyl c,hloritlr. 3C1-
+ NO,,- + 1I-T+ =
c'l.!
A
SO('1
+ 21I4
I'oi,tunately, the shove rrartion is not stoichiometric, under rirrunistances of the procrdure, so that the presence of less than 1 nig. of (ahloride in thc residur dors not cause ot)servahle error. (If t h e renction were qunntitative, 1 my. of chloride would cause the loss of 0.58 mg. of niti,:rtr.) K i t h larger amounts of chloridr< t h r loss of nitrate m:iy Iw h r g ~:ind rscwdingly vnriahle. esprc,i:illy if the phenoltii~iilfonic:icid is not added rapidly in s w h :I ninnnrr as to ?over all tlic rc~siduc~ quic,kly :ind iwmpletely. In orcl(,r to rt'niovc' colorcd rii:itter, t h c x proposed nirthod ures :in :ilk:iline osidation \ritlr hytlrogcn prroside, t use of rilagents surh a s r:irIion tllark, alumina tntr. c t r . . whirh havc, lwcn suggest& for thr remora1 of colored i1i:tttt.r from water arid soil solution snniplrs. T h r nirthod ('onqtitutes a niutlific*ationof tlio pi~orrrlurrsof .\qhtori ( 1 i.Burstrom ( f , , :rnd Roller and l!vT- the .9.0. EQUIPMENT
In addition to tlw usuul lzihoi,:itory rquipment, 25-1111. autoniatic pipets for disprnsing t,he silvrr sulfate, a mechanical 4iaking machinr, nnd a photoc.lrctrir colorimetcr with a filter trlinsniitting 410 to 430 nip are dwir:il~lc, The rrsults hrre i.eportet1 ~vrreoht:iincd with a 1ilett-Sunimc.r~nncolorimeter rquippcd with :I S o . 12 filtrr and A 20-mm. crll. REAGEYTS
Extracting Solution. Dissolve 3.6 grams of silver sulfate per litrr of water. Solution of the salt is greatly facilitated by heating. Phosphate Solution. Dissolve 138 grams of sodium dihydrogen
1527
phosphate monohydrate in 500 nil. of distilled water. Adjust I O pH 6.5 with concentrated sodium hydroxide and make to 1000 ml. Hydrogen Peroxide. This reagent should contain less than 0 p.p.ni. of nitrate nitrogen, be low in rtcidhy, and assay a t least 28YG H?O,. Hydrogen peroxide of the quality supplied t)y the Buffalo I.~lectrocheniical Company and by Merck & Company, Ttic. (microchemical grade) has been found satisfactory. Filter Aid. Calcium sulfate has been found t o he the most suitable filter aid. If necwsary, nitrate should be removed from the calcium sulfate hy washing tn-ice with distilled watet,, drying :it 60" to 70" C., and grinding to a fine powder. Calcium Carbonate Suspension. Suspend 1 gram of ralriuni carbonate powder in 200 ml. of distilled water. Phenoldisulfonic Acid, prepared according to the directions of ' ('hamot ~t nl. (8). Dissolve 25 grams of pure white phc~noli n 150 ml. of concrntitated sulfuric acid and add 75 nil. of fuming sulfuric acid (13YGSO1). Mix well and heat at 95' to 100' C', fot. 2 howi: or morr. Store in a tightly stoppered brown bottle. Sequestering Solution. Suspend 20 grams of Scqqucistrrnc A.4 [(ethylenedinitrilo)tetraacetic acid, obtained from the hlrose C'hemical Coinpany] in 50 ml. of distilled water. rZdd 1 1 ammonium hydroxide until the Sequestrene discolvr~. 1Iake to 100 nil. with distilled wat,rr. Dilute 5 nil. of this stock solution to 1 liter with distilled water for usc in diluting the nitrated phenoldisulfonic acid prior to adding the miinonium hydroxide. Otliri, sequestering agrnt,!: of the ethylenediamine tetraacetatr type may be usrd equnlly well. The polvphosphate (('nlgon, et(..) used, but lesr; suroessfully. type of srquestering agent has a l ~ berri o Ammonium Hydroxide (1 1). Dilutr concentratcd animonium hydroxide with an equal volume of distilled watei.. Nitrate Nitrogen Standard. Dissolve 7 . 2 2 grams of reagent grade potassium nitrate in tlistillrd watcr and makr to 1 liter. Dilutr' 5 ml. of thr stock standard t o 1 liter with distilled water. Thr n-or1;1nc standaril c*otrtainsR niicrocram~of n i t r o ~ e nn r r ml.
+
+
PROCEDURE
Extraction of Nitrate from Plant Samples. The procedui~routlined helow is usrd n . l r t ~ t i t h r samples at'r known to roiltain :imounts of c~hloridc in rscess of 1 % on the d r y weight I If tho saniplrs :LI'P knoivn to he lo\vcv in chloi,idcs, the proc~edulr may lie simplifird by nqing tlistilled Ivater as the estractirig liquid and omitting thr UT of rilvc.1. sulfate, thr phocphate, and thia wquwtc~ring:igrnt, Thc~p t ' o ( ~ r , c l i i wi* otlici,n.ipc t h r w m c . Weigh into 1)ottle- 01' f h i k s 0.100 grain of the dried m:ttr~rial prrviously ground t o p:~ssthc 40-mesh scrrrti of the iritrrmrtiiatr Kiley mill. If c*c~n11~ifugirig rquipnirnt ir avitilittilr. \vc>igh the samples dirtvtly into ivnti~ifugf~ tuhw, eliminating the nred for filtration aftrr thr tx~tr:ic.tiniiwith the dv(Li, sulfatc solution. JYitli :I small mrnwring -'Ixion, :idti 0.8 to I .0 g fate: the e s w t :mount i i - ~ ~:I-l ii f i l t r v aid Phoulti 1x1 uniform f o i . a l l sxniplw .%(Id 25 nil. wlution, swirl, :ind :idti 1 nil. of tlic phosphate mlution. Stoppc~i~ and shake in n nirchunir:il shakei. for 5 to 10 minutes. Either filtc,r through fnltlrtl So. 12 \Vli:itnian fi1tr.r papri' 01'(c~ritrifugein t50-nil. tuhrx at 1000 x gmvity for 10 niinut(Js. Removal of Organic Matter. Timwfw an aliquot of t h e filtrate or crnti~ifugatc,containing 5 t o 500 micrograms of niti~atrnitrogcn. t o a 100-nil. t)eaki,r or waporating diiih. Add 2 nil. of the calcium carhonate suspension and rrduce the, volume to 10 ml. hy cvnpor:ttion if necessary. Add 1.00 nil. o f the 30YGhydrogen ~ 1 , vrswl 17-ith n watch glass and digest on the pcroxido. ( ' 0 ~ ~ the strain hath for 2 houi,s. lirniove the x x t r h glass, take to dryness, and cantinuti heating tiit, tlrv rrsidur for :in additional 30 minutes t,o remove the last trncw o f p t ~ r o s i d r ~ , Nitration of Phenoldisulfonic Acid. T o t hr cooled i,c.siclur, atid 2.5 nil. of the phenoldisulfonic arid reagent fwni a rapid delivery pipet, buret, or tuhc. Thc exact amount of thc reagent added is of less iniportanrr than the need for rapidly flooding the dry residue. LIix thc. reagcnt and the, r with a glass rod to hasten solution of thv re,~iduc~.0cc:a y n small amount of thrless add 70 nil. of rwidue does not ooniplctrly dissolve ; rring solution within 5 to 10 minutes aftrr t,he citl \vas :idtl(d. Prolongrtl contnct of thr, conarid 01' the i.r:tgt>nt with th(a dry resitluc may lead to charring :ind lo\v i~c~sults..4dd itti excew of thr 1 1 ammonium hrdtnsidr : 13 ml. are usuallv sufficitJnt. Make sure that an excessof ammonium hydroxide has hrrn added (test paper or odor of ammonia). If tui1hii.t~devrlops, add dropwise sufficient of the conccmtr:ttrd srquestering solution to dissolvfb the precipitate. Colorimetric Estimation of Nitrate. ;\fter the colored solution has cooled. makr it up to a volumr that will give a rrliahlr reading
+
1528
ANALYTICAL CHEMISTRY
with tho colorinictcr and calibration curve in use. Read the intensity of the yelloa- color in the colorinieter, using a blue filter transmitting in the region of 400 to 420 mg. The transmittances of the colored solutions are not strictly proportional to the concentration of nitrat,e taken; hence, for very accurate work, the unknown solutions should be compared with standard solutions of approximately the same concentration. Standard nitrate solutions are carried through the procedure in the same \vay-i.e., 1 nil. of the phosphate solution and the calcium sulfate are added to 25 ml. of the nitrate standard in the extraction vessel and carried through t,he shaking and filtering steps. Blanks on the reagents should a k o tie run a t the same time and the appropriate correction applied to the samplr and standard readings. DISCUSSION
Extraction of Nitrate from Sample. The scheme as desciibed above measures the nitrate in the equilibrium extract of the plant material. Kitrate is easily and rapidly extracted from the fincaly ground material. Five minutes' contavt betiwen the sample and extracting liquid is ample, although when large numbers of samples are prepared the contact time will probably be longer. Longer times of contact do not change the apparent nitrate values of the sample. Extremelj- short contact times of approximately 1 minute gave results that n.ere 5 to 670 lower than those obtained \\-ith the regular extraction times. Removal of Chloride and Excess Silver Ions. L\fter removal of chloride b!. adding silver sulfate, the complete removal of silver ions is extremely important, because in the digestion step, in the presenre of excess ralciuni carbonate, the following reaction occurs ( l e , page 32): AgzO HIO? = 2Ag 0 9 HIO
+
+ +
The deconiposition of the hydrogen peroxide is veq- rapid and little or no oxidation of the organic matter in the sample takes place. I n addition, the metallic silver protiably reduces the nitrate to a mixture of nitrogen, nitrous oxide, nitric oxide, and nitrogen dioxide when the residue is treated with conrentrated acid ( 1 2 , page 197), so that low results arc obtained when free silver ion is present in the extract. Digestion with Peroxide. The removal of coloring matter and easily oxidizable organic substances prior to treating the residue with the phenoldisulfonic acid reagent is cwential. If the acid concentration and temperature are such that charring occurs, nitrate is reduced. In the authors' experience the color produced by the charring is insufficient to compensate for the loss of color vaused by the reduction of nitrate, and IOK results are found. Large ewesses of hydrogen peroxide, aside from being R asteful of an expensive reagent, may cause low results probably because of loss of nitrate by entrainment, inasmuch as the large excaess of peroxide decomposes n-ith considerable effervescence. One milliliter of peroxide has been found adequate for all samples encountered in the authors' work, although the use of as much as 2 ml. is acceptable. The equivalent of 40 nig. of dry samples of sugar beet petioles, grape petioles, wheat and barley blades, alfalfa stems, lettuce leaves, and soil extracts has offered no difficulty from charring when this amount of peroxide was used. Occasionally a pink color develops tvhen the nitrated phenoldisulfonic acid is made alkaline mith ammonia. This pink color has been attributed by Roller and McIiaig (13) to the oxidation of the phenoldisulfonic acid reagent by peroxides remaining in the dried extract. The authors' experiences seem to verify this; the pink color never appears when the residues are dried on the steam bath for more than 0.5 hour in the presence of excess ralcium carbonate. Roller and NcKaig ( I S ) and Burstroni ( 4 ) mention the occasional spontaneous combustion of the residue, with the consequent loss of nitrate, as the digestion and drying period nears completion. They recommend dilution of the nearly dry residue with water (or carbonated water) once or twice before taking the residue to complete dryness. The authors have been able to prevent deflagration by the preliminary 2-hour digestion of the
covered sample, followed by the evaporation and final dehydration procedure. This procedure is somewhat more satisfactory than that recommended by Roller and McKaig and Burstrom in t,liat it requires less of the analyst's personal attention. It is necessary t o neutralize the extracts prior to evaporation, as has been pointed out by many workers. Sufficient base is required to neutralize any acid that maj- be present in the peroxide as a stabilizer and, in addition, to neutralize acids developed as a result of the peroxide digestion. For example, the pH of a plant extract before the digestion was 5.3 and dropped to 4.3 after the digestion when no calcium carbonate had been added. The use of calcium carbonate effectively neutralizes acids originating from reagents, t'he original plant extract, and acids resulting from digestion of organic matter. I t also appears to aid the decomposition of the peroxides in the final drying step. The more alkaline hydroxides are not so satisfactory, because the prroside deroniposes too vigorously. Table 1. Effcct of Xitrogenous C o m p o u n d s on Apparent K i t r a t e Nitrogen C o n t e n t of Plant >\laterial (Indicated aiiioiint of nitrate free coinpnund added to 100 ing. of d r y plant nraterial. Nitrate determined b y proposed method) Conll~ollrld
Son? (;elatin
(SH4)rBOr (;lutamine Gliitaniic a r i d
Irnoiint .If g .
Al>i,arent Sirrate S 1'. p , m ."
...
94.5
4
. .
10
07.5
18
3.2
3 . 32t1
10 100 2 10
045 1la6
36 00
...
23.4
0.00e 4.63J
950 1109
2i 0
17.3
2
993 1038
37 20
7
!I81 IO66 (15.5
IO
1031
41 li 1% 16
10
hsliaragine Alanine
2 10
rrea
"
2 10 Alean of f o i i r replications.
at'
1nrrea.e
tC
0
1.5 3.1
9.8 3.8 12.8
1.1
11.2
961
27
1.7
1147
-14
11.4
1 . o o p
8 2 . oo/ 2.60d 9.121 1.73" 13.88: 1 52; 8 42 1.19' 9.161
d-5: -I
srandard deviation = : wliere I = value of single de(71 - 1) termination. I = mean value of n replications. a n d n = number of replications. 1 of Student's test (3). d Mean is significantly different a t 5% lex-el: rerlriirpd value of t i s 2.306. e S o significant difference in inean valiie as result of adding coinpound in amount indicated. / > l e a n is significantly different a t 17' level: required valrie of t is 3.365. u =
Addition of Phenoldisulfonic Reagent to Dried Residue. I n order to minimize the possihilit?. of charring the residue by the concentrated sulfuric. acid of the phenoldisulfonic~ acid reagent, the dried residue should be allowed to cool to room temperature before it is added. The time of contart hetLveen the reagent and the residue should not be longer than 5 to 10 minutes. In the authors' experience, also verified by that of Chaniot rt ai. ( 8 )and Roller and AlcKaig ( I S ) , contart time of aliout 2 minutes is enough to dissolve the residue and nitrate the phenoldisulfonic acid. The amount of phenoldisulfonic acid reagent added is not critical, except that an exress of the reagent mu3t he provided. When 0.1 mg. of nitrate nitrogen was present, 1.2 to 6.0 ml. of t,he reagent gave the same final color intensity. However, for convenience in later neutralization with ammonium hydroxidr, it is best to add approximately the same amount of the :icid t o each sample. The use of ammonium hydroxide as the neutralizing h e is mandatory, because sodium or potassium hydrosides lead to the formation of a precipitate even when sequestering agent, has been added to the solution. Were it not for the formation of these precipitates, sodium or potassium hydroxides would be preferable because of the freedom from the objectionable ammonia fumes and also because, as pointed out by Chamot et al. ( 7 ) :a
1529
V O L U M E 22, NO. 1 2 , D E C E M B E R 1 9 5 0
ride contents, by the proposed rriclthod of e\traction which removes chlorides and by simple aqueous extraction n-hich does not. All other de(Comparison of aqueous a n d Iiroliosed extrartion methods. P.p.m. of nitrate nitrogen on d r y tails of the procedure \\ere the same. Khen weight basis) Errnr the amount of rhlorides in the sample is amall, Difference Due to sample Chloride, Aqueous Extraction Cl,lorides, materials containing large amounts of nitrate _ _ _ ,Proposed ~ Extraction bet\yeen KO. % Mean" o h C.l'.C .\leann nb C.V.C Means 70 (samples 3697 and 3698) are less subject to erroi C.S. 0 1,089 37 3 . 4 0 1,046 37 3 . 5 4 43; than those containing little nitrate (samplc 715-5 1.7 190 32 16.84 319 18 5 . 6 4 129 40 3697 5,O 12,302 153 1 24 14,110 192 1 . 3 6 1808? 13 715-5). However, when only small amounts of 2266e 16 ! . 3 2 14,402 226 1 . 5 7 8 , s 12,136 160 3698 nitrate are present the differences in the results '2 3 13 n.22 332 23 6 . 9 3 249 33-11 11.1 83; 80 4 , .j 1,758 71 4.23 16 0 . 9 1 13.5 1,678 33-6 obtained by the two methods of extraction are 33-8 14.6 1,026 31 3 . 3 1 1,218 37 3 . 0 4 192 e 16 large a t both high and low chloride levels (samples M e a n f i v e replications. 715-5 and 33-11). Unless the phenoldisulfonic b where z is valrie of single determination, is mean vahlf. of 71 replicationn, a n d arid reagent is added very rapidly to the residue ti is number of replications. from the aqueous estract containing chloride, the c Coefficient of variation = b-x 100 ( 2 4 ) . results obtained for the aqueous evtract are much mean d &leans a r e not significantly different as determined b y Student's t teat. more variable than here indicated. Flooding of 3Ieans are significantly different a t 1% level ( 3 ) . the dried residue is, however, only partially effec__ tive in the prevention of loss of nitrate when chloride is present, and the removal of rhloride by greater color intensity is obtained with the latter bases. The use thr proposed extraction method is recommended for maximum accuracy and precision. of a large excess of ammonium hydroxide does not increase the The data of Table 111 further illustrate the order of precision final intensity of the color, but sufficient should be added to that may be obtained for a number of samples of widely varying assure the development of color (tested either by residual odor nit r a t e contents , after thorough mixing or 1)). the use of an indicator paper). Interferences. Inasmuch as Roller and McKaig ( I S ) pointed Of primary interest are the data in the column of confidenw out that the use of ammonium hydroxide for neutralizing the limits. For example, for sample 715-5, if we say that the trur aridity of the extracts prior to evaporation with hydrogen perpopulation mean lies betu;een 297 and 3-11 p,p.m. of nitrate nitrooside resulted in the oxidation of some ammonium ion to nitrate, gen, the probability of our being correct is 0.95. The values for the confidence limits show that the nitrate contents of samples and Ashton ( I ) showed that amide and amino nitrogen LV 719-5 and 33-11 are not really different, because there is a osidized to nitric nitrogen to some extent upon alkaline digestion probability of 0.95 that the true population mean lies between of plant samples with peroxide, the following tests for interfering almost the same limits-i.e., 297 to 341 p.p.m. for sample 715-5 nitrogen containing substances \yere made. and 303 t,o 361 p.p.m. for sample 33-11. On the other hand, we may be reasonably confident that the nitrate contents of samples A number of nitrogenous compounds, initially free of nitrate, C.S., 33-8, and 33-6 are really different from each other and the were added to plant, material and carried through the proposed others listed in th2 table, because the colifidence limits a t the procedure. The data of Table I indicate that small amounts of 0.95 prohabilitv level do not overlap. I t is also apparent that, it is each of the materials with the esception of glutamic acid and not possible td state unequivocally that sample5 3697 and 3698 redly differ in nitrate content, hecause the higher confidence limit gelatin do not lead to significant errors. Gelatin, of course, of sample 3697 is greater than the I o w r limit of sample 3698. would not be found in plant materials, but was used as an 9sHoxever, it is very certain that the values for these samples are ample of a soluble proteinaceous material. The smaller amounts entirely different from thosr for all the cther samples listed in the table. of the other organic nitrogen compounds are in excess of those to be found in normal plants. Plants grown in culture solutions LITERATURE CITED ceontaining large amounts of ammonium ion may contain more glutamine, glutamic acid, asparagine, and soluble amino acids .Ishton, F. L.. J . Sot. Cheni. 171d., 54,389-90T (1935). than was added in the test solutions. Serious errors would tw Assoc. Offic. Agr. Chemists, "Official and Tentative Methods of encountered if this method were used for the determination of Analysis," 6th ed., p. 631, 1945. Brownlee, K. A., "Industrial Experimentation," Brooklyn, nitrates in such material. S . Y., Chemical Publishing Co.. 1949. Burstrom, H., Swnsk. A-em. Tid., 54,139-45 (1942). Chamot, E. &I., and Pratt, D. 6 . . J . A m . C h e m . Soc., 31,922-8 Table 111. Precision of Nitrate Determinations as (1909). Function of Kitrate Content of Sample I b i d . , 32,630-7 (1910). Sample Confidenw so. Nitrate S Uh C.V.C Lirnitsd Chamot, E. M.,Pratt, D. 8.. and Redfield, H. W.,Ihid.. 33, P.p.m." I'.p. in. 366-81 (1911). 18 5.6; 297-341 310 715-5 Ibid., pp. 381-4. 23 6 . 9 3 303-361 332 33-11 Davis, C. X., I n d . Eng. Chem., 9, 290-5 (1917). 3; 3.54 1.001-1 ,OR 1 1.046 C.S. 3i 3.04 1.173-1,263 1,218 33-8 Fraps, G. S.,and Sterges, -4.J . . Terus d g r . E x p t . S t n . Bull. 439 33-6 1,758 16 0.91 1,738-1,778 (1931). 14,110 192 1.36 13,871-14,349 3697 210 1 . ,56 11.120-11.684 14,402 3698 Harper, H. J., I n d . Eng. C h e m . , 16, 180-3 (1924). Latimer, W.M., and Hildebraiid. d. H . , "Reference Book of Inorganic Chemistry," Xew York, Macmillan Co., 1940. Roller. E . A I . , and AIcKaig, T..Jr., S o i i S c i . , 47, 397-407 (1939j. Snedecor, G. \I-.,"Statistical lIethods," Ames, Iowa, Collegiate Press, 1946. S o error would result from the amount of ammonium found in Ulrich, A . , in "Diagnostic Techniques for Soils and Crops." Tashington, Am. Potash Inst., 1948. plant material, but the data confirm Roller and hlcKaig's obseIUlrich, A , , Proc. Am. SOC.Hort. Sci.. 4 1 , 213-18 (1942). vation that ammonium hydroxide must not be used in neutralizUlrich, .i.,Proc. A m . SOC.Sugar Bert Technol., 4, 88-95 (1946). ing the acidity of the extracts. ISlrich, A , , Soil Sci., 55,101-1'7 (1943). Precision. The data of Table I1 compare nitrate values ob-
Table 11.
Effect of Chloride on Apparent Nitrate Nitrogen Content of Dried Plant Material
'
tained for a number of d r y plant materials having differrnt (hlo-
R E C E I V E.\pril D 18, 19AO.
