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A rapid method for the determination of organic nitrogen and phosphorus based on a single perchloric acid digestion
3GcJ
ANALYTICA
A RAP111 METHOD AND I’HOSPHOHUS
FOR ‘l-HE I>ETEl
CHIhlICA
ACTA
NI’TIZOGEN DIGESTION
During iin investigation c)n milk lipicls in tliis laboratory, it was found necessary to clcvclop a mcthocl for the rapicl &termination of nitrogen using very small samples of lipid fractions. It is obvious that the lack of such a mctlmd is the main mason why most of the pul~lisliccl column chromatograT~l~y, or other fractionation expcrimcnts on lipids have been followed almost cxclusivcly by phosphorus estimations. In the early stages of tllcsc invcstigntions a method was tlcvisccl~ permitting the clctermination of amounts of nitrogen of the orclcr of 203opg in (lipid) samples of a. very low nitrogen content (0.003 to o.oInJ,). Since this mctliocl was time-consuming (IC-N-C<, >C= N-C<, --C= N). 5 -C\ It is cspccially useful when rapidity of operation is required. ‘l-his is III(JSt important in rcscnrcli on natural products, so that fractionation schemes may 1)~ followccl. Furthermore, our findings concerning the possibility of using pcrchloric acicl for nitrogen clctcrmination should IX of general interest, consiclcring the great number of publications on this subject.
A potassium sulfate-l)otassiunl hyclroxiclc mixture for the rcrnovrd of perchlorate was preparccl by mixing 4 volumes of 5 N potassium hyclroxiclc with one volume of G N sulfuric ncicl. Ncssler rcagcnt was prcparccl as clcscribccl by LANG?. Ammonium molybclatc (x.25:!,) and I-amino -5naphthol-4-sulfonic acid (0.0640/;,) reagents were prepared as clescrilxcl by SPEI~RY:~. Pyrex tubes 25 x 200 mm and cold fingers of the appropriate size (Fig. I) wcrc used for the digestion of the samples. A~rrcC.Clrirn. Actn,
34 (x966)
3Go-3Gd
I’EIZCHLOIUC
ACII)
DIGES1’ION
IS
ORGh&IC
ANALI’SIS
361
Digestion Place the sample (containing 5-15 pug of organic nitrogen, and phosphorus in a quantity of the same order of magnitude) in a Pyrex tube and evaporate the solvent if the sample is in solution. Add 0.75 ml of G7-70(x, pcrchloric acid and clrivc the cold finger into the tube (Fig. I). Start the digestion with very mild heating (at 60-70~ for 5 min), continue it at a moderate temperature until the mixture is colorless (or slightly colored), and then boil it vigorously for 5 min after it h,ascompletely clarificcl. In most cases, the whole digestion procedure takes no more than 12-15 min, because the amount of perchloric acid used is much mom than the very small amount of organic matter being digcstecl.
After cooling, rcmovc the tube while rinsing the cold finger with distilled water, and adjust the contents of the tube to a volume of 10-12.5 ml. This is cffcctccl tither with the aicl of a mark on the tube at IO, II, or 12.5 ml, or by rinsing the cold finger with x0 ml of water measurccl accurately with a pipette. Nitrogen dcternriwation Transfer 4 ml of the digcstctl sample into another tube the potassium sulfate-potassium hydroxide reagent. Transfer natant solution into a photometer tube, add 2 ml of water and ancl measure the optical density at 420 rnp, or at 500 m,u for
and add slowly I ml of 3 ml of the clear superI ml of Nessler reagent, denser solutions”.
Phosphorits determination Mix 5 ml of the digested sample with I ml of ammonium molybdatc reagent, add 0.1 ml (or 2 drops) of the aminonaphtholsulfonic acid solution and heat the mixture in a boiling water bath for 7 min. After cooling, measure the optical density at 830 rnpd. Stanclard curves are prepared by the same methods using sarnples prepared as d49rul. Chins.
Ada,
34 (rgG6)
3G0-306
D. S. GALANOS,
362
V.
>I. KAPOULAS
follows: IO ml each of aqueous solutions containing o, 4, 8, 12, . . . pg of nitrogen and phosphorus (as (NH&S04 and NanHl’Od) arc pipetted into a series of Pyrex tubes containing 0.75 ml of the perchloric acid used for the digestion of the samples. Morlificalion for the milligram rawge The procedure described may be modified so that it can bc used for any range of nitrogen (and phosphorus) content in the samples under investigation. In the milligram rang-c the method for nitrogen clctermination can bc simplified as follows. After digesting the sample as described above, dilute it in a volumetric flask, and add Nessler reagent (3 ml) directly to a s-ml aliquot of the diluted sample (without previous precipitation of the pcrchloratc). The limits for such modifications arc : 0.1-0.2 r.o--2.5 Quantity of nitrogen in the sample (mg) o.z-0.5 0.5-r .o Maximum volume (ml) of 67-70’): pcrchloric acid (digestion) I 10 2 4 Minimum volume (ml) of volumetric flask (dilution) 100 25 50 250 Phosphorus may be determined on a s-ml aliquot of the diluted sample as described above, after adjusting its PH to 0.7-1.3 by adding 67-70(x, lX!rchloric ad. Standard curves must bc prepared by the same proccdurcs. I~ESUL’I’S
AND
IlISCUSSION
A large number of natural products and pure organic compounds was analyzed by both the present method ancl the conventional one using sulfuric acid digestion. The results thus obtained (Table I) suggest that the analytical data obtained by the perchloric acid method are equivalent to those obtained by the Kjcldahl sulfuric acid method for all kinds of organic compounds, This is also true for such compounds as hydroxylaminc, nitrobenzcnc, triphcnyltctrazolium chloride, azobenzcne, ancl all compounds where nitrogen is bound by covalent bond(s) to atoms of the same or grcatcr elcctronegativity (i.e. N, 0, , . .). Such nitrogen was completely lost by both cligestion procedures (Table I). Particularly concerning heterocyclic nitrogen strong evidence was obtained that perchloric acid digestion has aclvantagcs over the sulfuric acid method. Thus, perchloric acid digestion of nicotinic acid (milligram range) gave reproducible results, being in excellent agreement with the theoretical nitrogen content of the sample, whereas the results obtainccl by sulfuric aciddigestion were IO-I~~)$ lower, unless strong heating of the colorless digest was prolonged; by heating the sulfuric acid digest for 1-2 11after it had become colorless, good results were also obtained. The results were similar for caffeine and coffee estracts (comparison of the results obtained by the two digestion proceclurcs). The methocl 11,a.s been used in this laboratory for nearly 4 years, during which time at least IOOO samples have been analyzed in duplicate or triplicate. It has proved highly satisfactory in both the microgram and the milligram range. Analyses of most of the compounds shown in Table I were repeated using Go-72% perchloric acid. The results were found to be equally satisfactory. Although the present method does not - at first sight - add much as far as Awl.
Chim.
Ada,
3.1 (rgGG)
3Go-366
PERCEILORIC
ACID
DIGESTIOS
IS
ORGASIC
363
AMALYSIS
accuracy is concerned, one of its advantages is that under the conditions described the solutions always remain quite clear after nesslerization. Most micromethods based on direct nesslerization after sulfuric acid digestion have a. problem at this point, because
COhlPARISON SULFURIC
OF ACID
RESULTS
ORTAI.Nl~I>
BY
THE
I’ERCIILORIC
ACID
PROCIZI~URI~
AND
TIIE
KJELDAtlL
MISTHOD
(The nitrogen contents found by the pcrchloric acid mcthotl arc csl~~~~ccl as a pcrccntngc of the nitrogen contents clctcrminctl by the same ncsslcrizntion proccdurc after tligcstion with sulfuric ;rcitl) _____.__ _--..-_. ..-.___-.. ..- ._.... .-_.--.. .-. . ..-.. ..- .- ..---.-.-... ..--Xilvogerr recowv) Sumpfe (% ) _______ ___. .._..______._ --_ .._. . -. .._....-. .-._--- -- . . --..---------.-----_------..-.--Total lipids of egg yolk I’olar lipids of milk Milk lipid fractionsa: I~olyglyccropl~ospl~;~titlc plus ccrcl)rosit!c I’hosphntidylcthanol;lt~~inc l’hosphntitlylscrinc I nositol phosphnticlcs l,ccithins l.ccithins plus sphingomyclins Splringoniyclins plus mucolipicls blucolil~icls Whole milk 1Jrinc I3lootl wru ni ‘Tea extract hlcat cxtract~’ Coffee cxtractl’ Cot-n flour*1
100.2 97.9 99.7 99.4 100. I Q9.7 98.7 Q9.0 90-S g8.h 99.0 90.8 100.0
9K.l 102.<)
Glycinc.llCI Asparaginc. I-ICI Argininc. I-ICl Caffcinc Nicotinic iICit1 H-Quinolinol Potnssiuni
(39.5 9s. 7
cyanitlc
09.7 99.9 gH.9 M.3 lOl..l 100.4 lOO.<)
Azobcnzcnc
In all cast’s the total I-lvtlroxyli~niinc.)_lC1 amount of nitrogen was lost l’;iphcnyltctrazolium chloride I Nitrobcnzcnc ------_ ..._.__.____.__ ____._. _.- .._._._._.. -_- .--. . --._-.--n lsolatccl by silicic wit1 chromntogmphy as rcccntly tlcscribcclfi. each fraction are mcntionctl in the ‘I‘illllC. 18Conirncrckil
The
main lipid
component(s)
of
products.
of the well-known sensitivity of Ncsslcr reagent, and its tendency to form turbid solutions. For the same reason, nitrogen determinations on samples of very low nitrogen content. - which are problematic or even impossible by direct ncsslcrization after sulfuric acid digestion - are possible by the present method. I’erchloric acid has been used previously for digestion of organic matter in Kjcldahl nitrogen determination, either alone or mixed with sulfuric acida. It has been also used - but with questionable success - added clropwise during the last stage of the conventional sulfuric acid digestion, as an aid in completing the destruction of organic matter’-Q. However, many workers in the past have discouraged the Aptal.
Clriwz. Ada,
34 (xgG6)
3Go-3GG
D. S. GALANOS,
364
V. hf. KAI’OULAS
general use of pcrchloric acid for nitrogen determination (for a review on this subject see RRA~STIWIY~*~), because it is known to be an extremely powerful oxidizing agent and ought to be used with caution, particularly from the standpoint of losses of nitrogcn cithcr tlirougll formation and subsccluent decomposition of ammonium pcrclllorate or oxidation of ammonium salts to nitrogen. A detailed stutly of the action of pcrchloric acid on ammonia in this laboratory (‘l’ablc II) showed no indications that either pcrchloric acid itself, or its thermal clccomposition products, llad any oxidativc effect on ammonia. However, the data given in Table II suggest that ammonia can cscapc from the digestion mixture even when a rcflux condcnscr is used; but this takes place only in pcrchloric acid-sulfuric
11151’LlJ.S l’BRCIILORIC
01’
hhfhIONIlJhl ACID
ANI>
SULFATIS
\VITII
SUI,FlJRIC
ACID,
I’ISRCIILOI~IC OR
I
N
ACID SODIUM
ALONIZ,
AND
WIT11
A
hll.KTURE
OF
IIYI’OCIILOllITl’:
(Apparatus: Fig. 2; ;~mmonium sulfntc (dry), 50 mg; 70% pcrchloric acid, 5 ml; cont. sulfuric acid, ml (SW below) ; rcflux on sand bath, r 11; cooliny; quenching of the digest with water; sh0rt l>oilinI: of both digest and clistillatc, to rcmovc chlorinc~. Digest (,\) : Precipitation of pcrchloratc dilution to I 1; ncsslcrization. I)istillotc (Is): by additkm Of 20 Ill1 of 6 N pcJtaSSillnl hydroxide; Dilution to x00 ml; ncsslcrizntion) ______ _._.._ .._.-____ __._._.._._- _..._....._ ..__._ - -._- _-__ - .--- -._.._._._. ..----------. .Exp~riwcrrl COIIC. I
5 0
0
Slow
0
Slow
2
7.5*3 87.4 99.3 go.5 7za
2.t.o
80.0 gS.8
1g.r
I 0
2
I I
5
l
I?. ‘3
5 5
I
;:
2
0
‘2
’ ‘I
Slow
C#.()
0.5
I oo..t
0.1
13.6 I .0 0.3 27.0
2.0 0.0
100.1
pit1
8fj.2
‘5 16
2
liil
x
linpid
93.7
5.8
‘7
8
SIOW
97.2
1.7
IH
8
Slow
gs.2
liapitl
63.4
10
‘20
10
I .t..t
I .()
30.0
IO
lii~pitl
gz.1
8.3
II
IO
Slow
22
I0
Slow
9.~4
3.7
_-----._
2 .o
98.2 _ ___.
-_---.______-
____ --
.
.__
15sccss of chlorine \vM always’fauntl in both flask t\ (“tligcst”) nnrl bcakcr I3 (“distillntc”). 11Consirlcrablc or complctc loss of’ammonia from the tligcst was obscrvctl in all cases when the rcflux conclcnscr was omittccl~ cvcn in the ciwc of pcrcliloric acid nlonc. u Compared to a stanckirtl solution prcpnrctl by dissolving 50 1~16of ammonium sulfntc in ca. zoo ml of water and then adding the rcngcnts given above, but omitting hating. ‘1 Compnrcd to a standard solution prcparcd by dissolving 10 1116of ammonium sulfntc in I0 ml of I .N sulfuric acid and clilutctl to 100 ml with wrrtcr. 0 Sodium liypochloritc (x N) to tlic amount of 10 ml added. 1’
I’ERCHLORIC ACILYIUIGESTIOX IN ORCASIC ASALYSIS
3%
acid mixtures where the dehydration of HC104 - 2HzO by sulfuric acid leads to a quick decomposition of ammonium perchlorate and subsequently to a loss of small or large amounts of ammonia, which is mechanically aided to escape by the fumes of perchloric acid and the excess of chlorine produced. The mechanical removal of some of the constituents left in acid mixtures by acid fumes has already been discussed by BETHGE~~. It is Supported by the finding that considerable transfer of ammonia from the “digest” to the “distillate” (Table II) takes place only when the thermal decomposition of perchlorate proceeds rapidly, and even then, it takes place in a nonreproducible manner. The present data and conclusions conccrrling the action of perch!oTic acid and chlorine to ammonia are obviously only in rough agrccmcnt with the findings of MOORE ;\sl) DIEHL~~‘. However, any slight differences appearing a+e eliminated by considering the nfore-mentioned non-reproducibility of ammonia losses from unrefluxed pcrchluric acid solutions, or from pcrchloric acid-sulfuric acid mixtures even under rcflux. The same reason might also explain the discrepancies in analytical results reported by various workers. For instance, MOORE ANI) DIEIIL~~ obtained good results with acetanilidc, urea and ammonium sulfate, for which pcrchloric acid digestion had previously been reportcd*a as giving unsatisfactory results. Consccluently, the only clucstion was to ascertain whcthcr destruction of organic matter by pcrchloric acid proceeds as far as nitrogen is concernccl in the s;Lme manner as when sulfuric acid is usccl. The agreement of the analytical dntn dbtainccl by the 2 digestion procedures (‘Table I) - even for caffeine, nicotinic acid, cyanide salts and choline which contain representative types of tertiary and ciuatcrnary nitrogen - strongly supports it. This investigation was financccl Hellenic I&search l;oundation.
by research grant
no. 350 from the
I
SUblhIAR\ Xcsults of nitrogen determinations obtained by pcrchloric acid digestion of organic matter under reflux were found to be crluivalcnt to those obtained by the conventional sulfuric acid I
Les Gsultats obtcnus pour le dosage de l’azotc par digestion h rcflux de la substance organique dans l’acicle perchloriquc, correspondent d ceux de la mdthocle de et le phospliore Kjeldahl. On peut ainsi doscr rapidement l’azote par “nesslerisation” par 13 mCthode conventionnelle au bleu de molybdbne. La m&hode propos6e a don& des rdsultats satisfaisants pour une grande vari&tC de produits naturels et de compos& organiques purs, comprenant des substances avcc azote tertiaire. Anccl.
Ckinr.
Aclu,
34 (xgG6)
3Go-3GG
D. S.
366
CALAXOS,
V.
h!.
ICAPOULAS
ZUSAhIhIENFASSUNC
Be%mmungcn dcs Stickstoffs durch l~chancllung organischcr Matcrinlien mit untcr Rtickfluss licfcrtcn die gleichcn Ergebnissc wie mit der konvcntioncllen Schwcfclsiiuremethodc. Einc schncllc Bcstimmung von Stickstoff uncl Scbwefel war so nach eincr einfachcn l+rchlors~urebchandlung moglich. Stickstoff wurde mit dem Ncsslcrschcn Reagent und Phosphor nach dcr Molyl~d~inblaumcthocle bcstimmt. Die Methodc ist s?uCricdcnstellcnd fur tine grosse Anzahl nattirlichcr Produkte und reincr organischcr Verbindungen cinschliesslich Stoffcn, die tertiiircn Stickstoff cnthalten. rcrChlorS:iUrc
/I~rrrl. Chim
Acta,
3,) (rgf80)
3Go-3GG
ANALYTICA
A RAP111 METHOD AND I’HOSPHOHUS
FOR ‘l-HE I>ETEl
CHIhlICA
ACTA
NI’TIZOGEN DIGESTION
During iin investigation c)n milk lipicls in tliis laboratory, it was found necessary to clcvclop a mcthocl for the rapicl &termination of nitrogen using very small samples of lipid fractions. It is obvious that the lack of such a mctlmd is the main mason why most of the pul~lisliccl column chromatograT~l~y, or other fractionation expcrimcnts on lipids have been followed almost cxclusivcly by phosphorus estimations. In the early stages of tllcsc invcstigntions a method was tlcvisccl~ permitting the clctermination of amounts of nitrogen of the orclcr of 203opg in (lipid) samples of a. very low nitrogen content (0.003 to o.oInJ,). Since this mctliocl was time-consuming (I
A potassium sulfate-l)otassiunl hyclroxiclc mixture for the rcrnovrd of perchlorate was preparccl by mixing 4 volumes of 5 N potassium hyclroxiclc with one volume of G N sulfuric ncicl. Ncssler rcagcnt was prcparccl as clcscribccl by LANG?. Ammonium molybclatc (x.25:!,) and I-amino -5naphthol-4-sulfonic acid (0.0640/;,) reagents were prepared as clescrilxcl by SPEI~RY:~. Pyrex tubes 25 x 200 mm and cold fingers of the appropriate size (Fig. I) wcrc used for the digestion of the samples. A~rrcC.Clrirn. Actn,
34 (x966)
3Go-3Gd
I’EIZCHLOIUC
ACII)
DIGES1’ION
IS
ORGh&IC
ANALI’SIS
361
Digestion Place the sample (containing 5-15 pug of organic nitrogen, and phosphorus in a quantity of the same order of magnitude) in a Pyrex tube and evaporate the solvent if the sample is in solution. Add 0.75 ml of G7-70(x, pcrchloric acid and clrivc the cold finger into the tube (Fig. I). Start the digestion with very mild heating (at 60-70~ for 5 min), continue it at a moderate temperature until the mixture is colorless (or slightly colored), and then boil it vigorously for 5 min after it h,ascompletely clarificcl. In most cases, the whole digestion procedure takes no more than 12-15 min, because the amount of perchloric acid used is much mom than the very small amount of organic matter being digcstecl.
After cooling, rcmovc the tube while rinsing the cold finger with distilled water, and adjust the contents of the tube to a volume of 10-12.5 ml. This is cffcctccl tither with the aicl of a mark on the tube at IO, II, or 12.5 ml, or by rinsing the cold finger with x0 ml of water measurccl accurately with a pipette. Nitrogen dcternriwation Transfer 4 ml of the digcstctl sample into another tube the potassium sulfate-potassium hydroxide reagent. Transfer natant solution into a photometer tube, add 2 ml of water and ancl measure the optical density at 420 rnp, or at 500 m,u for
and add slowly I ml of 3 ml of the clear superI ml of Nessler reagent, denser solutions”.
Phosphorits determination Mix 5 ml of the digested sample with I ml of ammonium molybdatc reagent, add 0.1 ml (or 2 drops) of the aminonaphtholsulfonic acid solution and heat the mixture in a boiling water bath for 7 min. After cooling, measure the optical density at 830 rnpd. Stanclard curves are prepared by the same methods using sarnples prepared as d49rul. Chins.
Ada,
34 (rgG6)
3G0-306
D. S. GALANOS,
362
V.
>I. KAPOULAS
follows: IO ml each of aqueous solutions containing o, 4, 8, 12, . . . pg of nitrogen and phosphorus (as (NH&S04 and NanHl’Od) arc pipetted into a series of Pyrex tubes containing 0.75 ml of the perchloric acid used for the digestion of the samples. Morlificalion for the milligram rawge The procedure described may be modified so that it can bc used for any range of nitrogen (and phosphorus) content in the samples under investigation. In the milligram rang-c the method for nitrogen clctermination can bc simplified as follows. After digesting the sample as described above, dilute it in a volumetric flask, and add Nessler reagent (3 ml) directly to a s-ml aliquot of the diluted sample (without previous precipitation of the pcrchloratc). The limits for such modifications arc : 0.1-0.2 r.o--2.5 Quantity of nitrogen in the sample (mg) o.z-0.5 0.5-r .o Maximum volume (ml) of 67-70’): pcrchloric acid (digestion) I 10 2 4 Minimum volume (ml) of volumetric flask (dilution) 100 25 50 250 Phosphorus may be determined on a s-ml aliquot of the diluted sample as described above, after adjusting its PH to 0.7-1.3 by adding 67-70(x, lX!rchloric ad. Standard curves must bc prepared by the same proccdurcs. I~ESUL’I’S
AND
IlISCUSSION
A large number of natural products and pure organic compounds was analyzed by both the present method ancl the conventional one using sulfuric acid digestion. The results thus obtained (Table I) suggest that the analytical data obtained by the perchloric acid method are equivalent to those obtained by the Kjcldahl sulfuric acid method for all kinds of organic compounds, This is also true for such compounds as hydroxylaminc, nitrobenzcnc, triphcnyltctrazolium chloride, azobenzcne, ancl all compounds where nitrogen is bound by covalent bond(s) to atoms of the same or grcatcr elcctronegativity (i.e. N, 0, , . .). Such nitrogen was completely lost by both cligestion procedures (Table I). Particularly concerning heterocyclic nitrogen strong evidence was obtained that perchloric acid digestion has aclvantagcs over the sulfuric acid method. Thus, perchloric acid digestion of nicotinic acid (milligram range) gave reproducible results, being in excellent agreement with the theoretical nitrogen content of the sample, whereas the results obtainccl by sulfuric aciddigestion were IO-I~~)$ lower, unless strong heating of the colorless digest was prolonged; by heating the sulfuric acid digest for 1-2 11after it had become colorless, good results were also obtained. The results were similar for caffeine and coffee estracts (comparison of the results obtained by the two digestion proceclurcs). The methocl 11,a.s been used in this laboratory for nearly 4 years, during which time at least IOOO samples have been analyzed in duplicate or triplicate. It has proved highly satisfactory in both the microgram and the milligram range. Analyses of most of the compounds shown in Table I were repeated using Go-72% perchloric acid. The results were found to be equally satisfactory. Although the present method does not - at first sight - add much as far as Awl.
Chim.
Ada,
3.1 (rgGG)
3Go-366
PERCEILORIC
ACID
DIGESTIOS
IS
ORGASIC
363
AMALYSIS
accuracy is concerned, one of its advantages is that under the conditions described the solutions always remain quite clear after nesslerization. Most micromethods based on direct nesslerization after sulfuric acid digestion have a. problem at this point, because
COhlPARISON SULFURIC
OF ACID
RESULTS
ORTAI.Nl~I>
BY
THE
I’ERCIILORIC
ACID
PROCIZI~URI~
AND
TIIE
KJELDAtlL
MISTHOD
(The nitrogen contents found by the pcrchloric acid mcthotl arc csl~~~~ccl as a pcrccntngc of the nitrogen contents clctcrminctl by the same ncsslcrizntion proccdurc after tligcstion with sulfuric ;rcitl) _____.__ _--..-_. ..-.___-.. ..- ._.... .-_.--.. .-. . ..-.. ..- .- ..---.-.-... ..--Xilvogerr recowv) Sumpfe (% ) _______ ___. .._..______._ --_ .._. . -. .._....-. .-._--- -- . . --..---------.-----_------..-.--Total lipids of egg yolk I’olar lipids of milk Milk lipid fractionsa: I~olyglyccropl~ospl~;~titlc plus ccrcl)rosit!c I’hosphntidylcthanol;lt~~inc l’hosphntitlylscrinc I nositol phosphnticlcs l,ccithins l.ccithins plus sphingomyclins Splringoniyclins plus mucolipicls blucolil~icls Whole milk 1Jrinc I3lootl wru ni ‘Tea extract hlcat cxtract~’ Coffee cxtractl’ Cot-n flour*1
100.2 97.9 99.7 99.4 100. I Q9.7 98.7 Q9.0 90-S g8.h 99.0 90.8 100.0
9K.l 102.<)
Glycinc.llCI Asparaginc. I-ICI Argininc. I-ICl Caffcinc Nicotinic iICit1 H-Quinolinol Potnssiuni
(39.5 9s. 7
cyanitlc
09.7 99.9 gH.9 M.3 lOl..l 100.4 lOO.<)
Azobcnzcnc
In all cast’s the total I-lvtlroxyli~niinc.)_lC1 amount of nitrogen was lost l’;iphcnyltctrazolium chloride I Nitrobcnzcnc ------_ ..._.__.____.__ ____._. _.- .._._._._.. -_- .--. . --._-.--n lsolatccl by silicic wit1 chromntogmphy as rcccntly tlcscribcclfi. each fraction are mcntionctl in the ‘I‘illllC. 18Conirncrckil
The
main lipid
component(s)
of
products.
of the well-known sensitivity of Ncsslcr reagent, and its tendency to form turbid solutions. For the same reason, nitrogen determinations on samples of very low nitrogen content. - which are problematic or even impossible by direct ncsslcrization after sulfuric acid digestion - are possible by the present method. I’erchloric acid has been used previously for digestion of organic matter in Kjcldahl nitrogen determination, either alone or mixed with sulfuric acida. It has been also used - but with questionable success - added clropwise during the last stage of the conventional sulfuric acid digestion, as an aid in completing the destruction of organic matter’-Q. However, many workers in the past have discouraged the Aptal.
Clriwz. Ada,
34 (xgG6)
3Go-3GG
D. S. GALANOS,
364
V. hf. KAI’OULAS
general use of pcrchloric acid for nitrogen determination (for a review on this subject see RRA~STIWIY~*~), because it is known to be an extremely powerful oxidizing agent and ought to be used with caution, particularly from the standpoint of losses of nitrogcn cithcr tlirougll formation and subsccluent decomposition of ammonium pcrclllorate or oxidation of ammonium salts to nitrogen. A detailed stutly of the action of pcrchloric acid on ammonia in this laboratory (‘l’ablc II) showed no indications that either pcrchloric acid itself, or its thermal clccomposition products, llad any oxidativc effect on ammonia. However, the data given in Table II suggest that ammonia can cscapc from the digestion mixture even when a rcflux condcnscr is used; but this takes place only in pcrchloric acid-sulfuric
11151’LlJ.S l’BRCIILORIC
01’
hhfhIONIlJhl ACID
ANI>
SULFATIS
\VITII
SUI,FlJRIC
ACID,
I’ISRCIILOI~IC OR
I
N
ACID SODIUM
ALONIZ,
AND
WIT11
A
hll.KTURE
OF
IIYI’OCIILOllITl’:
(Apparatus: Fig. 2; ;~mmonium sulfntc (dry), 50 mg; 70% pcrchloric acid, 5 ml; cont. sulfuric acid, ml (SW below) ; rcflux on sand bath, r 11; cooliny; quenching of the digest with water; sh0rt l>oilinI: of both digest and clistillatc, to rcmovc chlorinc~. Digest (,\) : Precipitation of pcrchloratc dilution to I 1; ncsslcrization. I)istillotc (Is): by additkm Of 20 Ill1 of 6 N pcJtaSSillnl hydroxide; Dilution to x00 ml; ncsslcrizntion) ______ _._.._ .._.-____ __._._.._._- _..._....._ ..__._ - -._- _-__ - .--- -._.._._._. ..----------. .Exp~riwcrrl COIIC. I
5 0
0
Slow
0
Slow
2
7.5*3 87.4 99.3 go.5 7za
2.t.o
80.0 gS.8
1g.r
I 0
2
I I
5
l
I?. ‘3
5 5
I
;:
2
0
‘2
’ ‘I
Slow
C#.()
0.5
I oo..t
0.1
13.6 I .0 0.3 27.0
2.0 0.0
100.1
pit1
8fj.2
‘5 16
2
liil
x
linpid
93.7
5.8
‘7
8
SIOW
97.2
1.7
IH
8
Slow
gs.2
liapitl
63.4
10
‘20
10
I .t..t
I .()
30.0
IO
lii~pitl
gz.1
8.3
II
IO
Slow
22
I0
Slow
9.~4
3.7
_-----._
2 .o
98.2 _ ___.
-_---.______-
____ --
.
.__
15sccss of chlorine \vM always’fauntl in both flask t\ (“tligcst”) nnrl bcakcr I3 (“distillntc”). 11Consirlcrablc or complctc loss of’ammonia from the tligcst was obscrvctl in all cases when the rcflux conclcnscr was omittccl~ cvcn in the ciwc of pcrcliloric acid nlonc. u Compared to a stanckirtl solution prcpnrctl by dissolving 50 1~16of ammonium sulfntc in ca. zoo ml of water and then adding the rcngcnts given above, but omitting hating. ‘1 Compnrcd to a standard solution prcparcd by dissolving 10 1116of ammonium sulfntc in I0 ml of I .N sulfuric acid and clilutctl to 100 ml with wrrtcr. 0 Sodium liypochloritc (x N) to tlic amount of 10 ml added. 1’
I’ERCHLORIC ACILYIUIGESTIOX IN ORCASIC ASALYSIS
3%
acid mixtures where the dehydration of HC104 - 2HzO by sulfuric acid leads to a quick decomposition of ammonium perchlorate and subsequently to a loss of small or large amounts of ammonia, which is mechanically aided to escape by the fumes of perchloric acid and the excess of chlorine produced. The mechanical removal of some of the constituents left in acid mixtures by acid fumes has already been discussed by BETHGE~~. It is Supported by the finding that considerable transfer of ammonia from the “digest” to the “distillate” (Table II) takes place only when the thermal decomposition of perchlorate proceeds rapidly, and even then, it takes place in a nonreproducible manner. The present data and conclusions conccrrling the action of perch!oTic acid and chlorine to ammonia are obviously only in rough agrccmcnt with the findings of MOORE ;\sl) DIEHL~~‘. However, any slight differences appearing a+e eliminated by considering the nfore-mentioned non-reproducibility of ammonia losses from unrefluxed pcrchluric acid solutions, or from pcrchloric acid-sulfuric acid mixtures even under rcflux. The same reason might also explain the discrepancies in analytical results reported by various workers. For instance, MOORE ANI) DIEIIL~~ obtained good results with acetanilidc, urea and ammonium sulfate, for which pcrchloric acid digestion had previously been reportcd*a as giving unsatisfactory results. Consccluently, the only clucstion was to ascertain whcthcr destruction of organic matter by pcrchloric acid proceeds as far as nitrogen is concernccl in the s;Lme manner as when sulfuric acid is usccl. The agreement of the analytical dntn dbtainccl by the 2 digestion procedures (‘Table I) - even for caffeine, nicotinic acid, cyanide salts and choline which contain representative types of tertiary and ciuatcrnary nitrogen - strongly supports it. This investigation was financccl Hellenic I&search l;oundation.
by research grant
no. 350 from the
I
SUblhIAR\ Xcsults of nitrogen determinations obtained by pcrchloric acid digestion of organic matter under reflux were found to be crluivalcnt to those obtained by the conventional sulfuric acid I
Les Gsultats obtcnus pour le dosage de l’azotc par digestion h rcflux de la substance organique dans l’acicle perchloriquc, correspondent d ceux de la mdthocle de et le phospliore Kjeldahl. On peut ainsi doscr rapidement l’azote par “nesslerisation” par 13 mCthode conventionnelle au bleu de molybdbne. La m&hode propos6e a don& des rdsultats satisfaisants pour une grande vari&tC de produits naturels et de compos& organiques purs, comprenant des substances avcc azote tertiaire. Anccl.
Ckinr.
Aclu,
34 (xgG6)
3Go-3GG
D. S.
366
CALAXOS,
V.
h!.
ICAPOULAS
ZUSAhIhIENFASSUNC
Be%mmungcn dcs Stickstoffs durch l~chancllung organischcr Matcrinlien mit untcr Rtickfluss licfcrtcn die gleichcn Ergebnissc wie mit der konvcntioncllen Schwcfclsiiuremethodc. Einc schncllc Bcstimmung von Stickstoff uncl Scbwefel war so nach eincr einfachcn l+rchlors~urebchandlung moglich. Stickstoff wurde mit dem Ncsslcrschcn Reagent und Phosphor nach dcr Molyl~d~inblaumcthocle bcstimmt. Die Methodc ist s?uCricdcnstellcnd fur tine grosse Anzahl nattirlichcr Produkte und reincr organischcr Verbindungen cinschliesslich Stoffcn, die tertiiircn Stickstoff cnthalten. rcrChlorS:iUrc
/I~rrrl. Chim
Acta,
3,) (rgf80)
3Go-3GG