Sensitive N e w Methods for Autocatalytic Spectrophotometric Determination of Nitrite through Free-Radical Chromogens EUGENE SAWICKI, T. W. STANLEY, JOHN PFAFF, and HENRY JOHNSON Division of Air Pollufion, Robert A. r a f t Sanitary Engineering Center, Cincinnati26, Ohio
b Many of the methods presented here are more sensitive than any described in the literature. A molar absorptivity of 1,270,000 can be obtained in the 1 -methyl-2-quinolone azine procedure, while in most other procedures molar absorptivities average over 200,000. The following reagents are compared: 1 -methyl-2 quinolone azine, 3-methyl-2-benzothiazolinone azine, glyoxal bis(N,Ndiphenylhydrazone), 3-methyl-2-benzothiazolinone picrylhydrazone, phenothiazine, N,N,N',N'-tetramethy1-4,4'diaminostilbene, N,N' diphenylp phenylenediamirle, N,N,N',N'tetramethylbenzidine, N,N,N',N'-tetramethyl p phenylenediamine, and N,N,N' trimethyl p phenylenediamine. The syntheses of some of the reagents are given. Evidence i s presented that free iradicals are obtained in all the procedures. The advantages and disadvantages of the methods are discussed. Recommendations are given for application of some of the procedures ta the analysis of solutions containing dyes, large amounts of sulfite, or minute amounts of nitrite.
-
-
-
A
-
-
-
-
large number of methods are available for the determination of nitrite ion and its precursors (14). I n more recent work 52 spectrophotometric methods for thc determination of nitrite ion have been c mitically compared (16). I n this paper some new methods, which are more sensitive than any available in the litemture, are introduced and their advantages and disadvantages are discussed. VERY
EXPERlMEIqTAL
Reagents and A.pparatus. Triphenylamine, 2 - chlorophenothiazine, and N,AJ,N',N'-tetr:imethylbenzidine mere obtained from Aldrich Chemical Co., Milwaukee 10, Wis. The latter two compounds and phenoxazine (K and K Laboratories, Jamaica 33, K. Y.) were crystalized from ethylcyclohexane t o a constant melting point. Phenothiazine (Laboratory Services, Inc., Cincinnati '3, Ohio) was crystallized from heptane-ethylcyclohexane to a constant melting point of 183-84' C.
X ; N , N ' - Trimethyl - p - phenylenediamine dihydrochloride and the N,N,N', "-tetramethyl analog were obtained from Distillation Products, Rochester 3, N. Y., and K and K Laboratories, respectively. Both compounds were crystallized once out of methanol. N,N'-Diphenyl-p-phenylenediamine was obtained from the United States Rubber Co., Kaugatuck, Conn. Colorless crystals were obtained after two crystallizations from ethylcyclohexane. Phenoselenazine (Z),m.p. 193-94' C., and 4,4'-bis(dimethy1amino)stilbene (16, 17), m.p. 253-54' C., cor., were prepared by literature procedures. Preparation of 1-Methyl-2-quinolone Azine. Add 2.0 ml. (0.04 mole)
of 98% hydrazine hydrate to 100 ml. of a warm aqueous solution of 7.62 grams (0.02 mole) of 2-iodo-1-methylquinolinium methosulfate. [This salt was prepared by warming equivalent amounts of 2-iodoquinoline (Distillation Products) and methyl sulfate a t about 80' C. until the liquid solidified (-30 minutes).] Stir for approximately 1 hour. Filter the red-brown precipitate and crystallize from ethylcyclohexanebenzene (2 to 1). The yield is 11/4grams (40%) of glistening red plates, m.p. 257-58" C. cor. Lit. map. 257"-58" C. (3). Preparation of 3-Methyl-2-benzoAdd 2.75 ml. thiazolinone Azine.
(0.054 mole) of 98y0 hydrazine hydrate slowly t o a hot stirred solution of 36.7 grams (0.1 mole) of 3-methyl2-methylthiobenzothiazolium p-toluenesulfonate (IS) in 60 ml. of water. Boil the mixture for approximately 5 minutes. Add 28yGammonium hydroxide until the mixture becomes alkaline and then allow the precipitated material to cool. Filter, wash with water, and then crystallize from dimethylformamide. For additional product, add mater to the boiling dimethylformamide solution till definite turbidity is obtained. Total yield is 8.6 grams (53'3,) of pale yellow plates, m.p. 260' C. cor. Lit. m.p. 230' C. (11). Calculated for C16HI4N~S2:C, 58.9; H, 4.30; N, 17.2; S, 19.6. Found: C, 58.9; H,4.37; N, 17.1; S, 19.6. Preparation of 3-Methyl-2-benzoAdd thiazolinone Picrylhydrazone. a solution of 8.4 grams (0.034 mole) of picryl chloride in 9570 ethanol to 50 ml. of a hot aqueous solution of 6.45 grams (0.03 mole) of 3-niethyl-2benzothiazolinone hydrazone hydro-
chloride. Stir the mixture 30 minutes. Crystallize the precipitate from xylene. Eight and eight-tenths grams (75% yield) of glistening black needles, m.p. 216-18' cor., is obtained. Calculated for C M H ~ ~ N ~ OC, OS 43.1 : ; H, 2.56; N, 21.5; S, 8.21. Found: C, 43.3; H, 2.74; Tu', 21.3; S,8.30. Preparation of Glyoxal Bis-(1,ldiphenylhydrazone). Add 2 ml. (0.01 mole) of 30% aqueous glyoxal t o a hot stirred solution of 4.44 grams (0.02 mole) of 1,l-diphenylhydrazine hydro-. chloride in 25 ml. of 95% ethanol. During addition keep temperature below 65" C. Extract the collected precipitate with hot 2-methoxyethanol, add charcoal, and filter hot. Add excess water to the filtrate. Refilter and wash residue with aqueous 2-methoxyethanol (1 to 1). Crystallization from heptane-ethylcyclohexane (1 to 1) gives 2 grams (51% yield) of colorless needles, map.202-03" C. cor. Lit. m.p. 207' C. (19). Detection of Nitrite.
T o 9 drops of reagent solution on a spot plate add 1 drop of aqueous test solution. In the presence of nitrite the following reagents give a blue color: N,N'diphenyl-p-phenylenediamine (0.1% in acetic acid containing 20% perchloric acid), 3-methyl-2-benzothiazolinone azine (0.01% in acetic acid containing 10% propionic acid), and N,N,N',N'tetramethyl-p-phenylenediamine dihydrochloride (0.1% in acetic acid). With 3-met hyl-2benmt hiazolinone 1-met hyl2-quinolone azine a purple color is obtained after a Sminute standing period. The detection limit for nitrite with the first-named compound was 0.02 pg.; with the other reagents it was 0.009 fig. Reaction of a drop of reagent solution with a drop of test solution on filter paper increased detection limits by about a factor of 10. The blanks were all colorless. 1-Methyl-2-quinolone Azine Procedures. PROCEDURE A. Dilute 1 nil. of aqueous test solution t o 10 ml. with a n acetic acid solution containing 0.1% azine. Read a t exactly 1hour a t Amax 520 mill (Figure 1). Alternatively, 1hour after the reagent solution had been added, add 1 ml. of 0.1% sulfamic acid (w./v.) in acetic acid containing 25y0 mater (v./v.). The color intensity is stable for a t least 1 hour. Read absorbance a t ,A, 520 mp within this time. PROCEDURE B. Add 5 ml. of an aqueous 0.1N sodium hydroxide test solution to 5 ml. of O.lyoreagent in acetic acid, VOL. 35,
NO. 13, DECEMBER 1963
2183
Me2$-
(CH=CH)qONMe,
7 =O
X = O , S or Se
i
?'I
X =(CH=CH),Y=S
X=Y=(CH=CH)
P
R
:N - N C H - C H = N - N -
I
I
2 2
I 1
I
4
6
8
2a
36
48
Figure 2.
Postulated chromogen structures
HOURS
Figure 1.
of 70% aqueous perchloric acid (v./v.) in glacial acetic acid. Read absorbance 65 minutes later a t the mave-
1 -Methyl-2-quinolone azine Procedure A
Change in absorbance at A,, 5 2 0 m p with time after mixing of reagent and test solution 0.23 fig. NO;, 48-haur study - - - - 0.69 M g . NO;, 8-hour study , , 0.345 pg. NO;, 6-hour study
length maximuni.
.. ..
In 60 minutes read absorbance at A, 520 mp. The spectrophotometric constants obtained in the determination of nitrite with different reagents are compared in Table I.
Table 1.
N NR;~
ri=Me,Me + R'=Me,Me R=Me,Me + R ' = M e , H R=+,H + R ' = + ,H
d I
,
N,N,N',N' - Tetramethyl - 4,4'diaminostilbene. Dilute 1 ml. of aqueous test solution to 10 ml. with a reagent solution containing 0.05% of the stilbene derivative and 20%
Phenothiazine. Dilute 1 ml. of aqueous test solution t o 10 ml. with 0.1% phenothiazine in distilled glacial acetic acid. Read absorbance exactlv 1 hour later at, , ,A 518 mp. Phenoxazine, 2-chlorophenothiazine, and phenoselenazine can be substituted for phenothiazine.
-
KJIT1 Diphenyl - p - phenylenediamine. PROCEDURE A. Dilute 1 ml. of aqueous test solution t o 10 ml.
Comparison of Spectrophotometric Methods for Determination of Nitrite mP
Range 0.1 to2.0), x 10-4
520 520
33-127 38-76
4.6 2.0
10 2
33-127 190-380
0.14 0.12
62 62
514 517
38-72 18-47
6.4 5.1
10 10
38-72 18-47
0.12 0.96
67 62
720 705
9.2-46 -15-51
11 6.3
10 2
9.2-46 77-255
0.50 -0.3
62
614 614 612 735 540 1152
10-20 15-38 8.1-16 13-52 10-37 7.1-36
5.4 7.6 7.0 6.5 5.5 6.7
10 10 1.11 10 10 10
10-20 15-38 73-144 13-52 10-37 7.1-36
0.45 0.30 0.30 0.35 0.44 0.65
32 32 22 62 7 7
518
8.4-39
9.5
10
8.4-39
0.55
62
406
7.7-32
4.5
10
7.7-32
0.60
32
473 1018 472
4.9-20 4.9-20 7.2-10
3.7 11 3.0
10 10 2
4.9-20 4.9-20 36-50
0.94 0.94 0.64
2 2 2
0.98-1 . 7 7-12.7
4.7 6.0
3
Amax,
Reagent 1-Methyl-2-quinolone azine A
B
N,N,NtJN'-Tetramethy1-4,4'diaminostilbene Phenoselenazine N ,N'-Diphenyl-p-phenylenediamine A B N ,N ,N N'-Tetramethyl-p-phenylenediamine A B
:,
3-Methyl-2-benzothiazolinone azine Glyoxal bis(N,N-diphenylhydrazone)
(A
=
Rel. std. dev.
Dil. factor"
Sensitivityb
Proc. Determ. time, limit; fig, min.
63
Phenothiazine
A
3-Methyl-2-benzothiazolinone picrylhydrazone N , N ,N',N'-Tetramethylbenzidine A A
B N,N,N'-Trimethyl-p-phenylene-
Pmine B
0
10 0.98-1.7 2.7 578 1.11 0.77-1 .4 4.7 578 Essentially final volume/test solution volume. e x 10-3 Sens. = dilution factor ' Total micrograms of nitrite ion in test solution giving absorbance of 0.1 in 1-cni. cell.
2184
ANALYTICAL CHEMISTRY
2
with a reagent solution containing 0.05% of the diamine and 20% of 70Y0 aqueous perchloric acid (v./v.) in glacial acetic acid. Read absorbance 60 minutes later at, , ,A 386 or i20 mp. PROCEDURE B. To :I ml. of aqueous 0.lN sodium hydroxide test jolution add 5 ml. of a n acetic acid c,olution containing 0 057, of the dian ine ($5 / v.) and 207, of iO% aqueous perchloric acid (v./v.). Read absorbmce 60 minutes later a,t, ,A 705 mp. N,N,iV',N' - Tetramethyl - p phenylenediamine. P R o c m u m A. Dilute 1 ml. of aqueous test solution t o 10 ml. with a solution containing 0.17, of the diamine dihydrochloride in glacial acetic acid. liead absorbance 30 minutes later at, , A, 614 nip. The color intensity is stab111 for a t least 60 minutes. PROCEDURE B. Use the qame procedure with a reagent solution containing 0 17, of A',N,N'.A '-tetramethyl-pphenylenediamine dihrdrochloride in glacial acetic acid containing 25% water (v./v.). PROCEDURE C. To 9 ml. of an aqueous 0.1.t7 sodium hydroxide solution add 1 ml. of a n acc tic acid qolution containing 0.2% reagent and 10% concentrated hydrochloril? acid ( v . , ' ~ . ) . Read absorbance 20 minutes later a t , , A, 612 mp. 3 Methyl - 2 - benzothiazolinone Azine. Dilute 1 ml. of aqueous test solution t o 10 ml. with a glacial acetic acid solution contain ng 0.1% azine (w./v.) and 107, propionic acid (v./v.). Read absorbance eyactly 1 hour later at, , ,A 735 mp. Glyoxal Bis-Y,.Y-diphenylhydrazone. Dilute 1 ml. of aqueous test solution t o 10 nil. ivith a glacial acetic acid solution containing 0.017, reagent and 10% of aqueous 70% perchloric acid. Allow solution t o stand 5 minute?; read absorbance at 540 mp . 3 Methyl - 2 - benzothiazolinone Picrylhydrazone. Dilute 1 nil. of aqueous test solution t o 10 ml. with a glacial acetic acid solution containing 0.017, reagent (n ./v.) and 207, of 70% aqueous perchloric acid (v./v.). AUlowthe solution to stand 30 minutes, then read the absorbaice at, , ,A 405 inp . N,N,N',S'- Tetramethylbenzidine. PROCEDURE A. Dilute 1 nil. of the aqueous test solution to 10 ml. with a glacial acetic acid solution containing 0.1% of the benzidine derirative. Read absorbance a t 475 or 1018 mp immediately. PROCEDURE B. Lldd 5 ml. of an acetic acid solution containing lY0 of the benzidine derivatixe to ml. of an aqueous 0.1S sodium hydroxide test solution. Read immediately at 4 i 2 or 1018 mp. N,iV,N' - Trimethyl - p - phenylenediamine. PROCEDURE A. Dilute 1 ml. of aqueous test solution to 10 ml. with 0.27, X,S ;Y'-trimethyl-pphenylenediamine in acidc acid. Read 578 abxrbance immediately a t , , ,A mp. PROCEDURE B. To 9 ml. of aqueous test solution add 1 ml. of 0.27, N , N , N ' trimethyl-p-phenylenediamine dihydro-
~~
~~
~~~
~~~~~
Table II. Spectral Evidence for Free-Radical Structure of Chromogens Obtained in Some of the Procedures A,, ml.c (relative intensity or A ) Semiquinone Lit. Present work Phenoxazine 522-532 (strong)" 530 ( 0 . 6 4 ) * Phenothiazine 432-440 (strong)a 440 ( 1 , 3 3 ) 454-460 (very weak) 465 ( 0 . 9 4 ) 472-478 (weak) 483 ( 1 . 1 1 ) 492-498 (strong) 502 ( 1 . 4 8 ) 518 (2.00) 510-516 (very strong) Phenoselenazine 428-438 (strong)" 432 ( 1 . 3 ) 476-486 (very weak) 487s ( 1 .2)c 505-525 (very strong) 517 ( 2 . 4 5 ) N,N,.V '-Trimethyl-p-phenylenediamine
500s (-0.
500s ( 0 . 8 4 ) ~
:V, W,N',N'-Tetramethyl-p-
536 ( 1 . 2 7 ) 580 ( 1 . 2 9 ) __ 525s (-0.8)' 565 ( 1 . 2 5 ) 614 ( 1 . 2 5 )
537 (1.46) 577 ( 1 . 5 2 ) , g s (0.S6)p 565 ( 1 . 2 0 ) 614 (1.20) 425s 438 459 473 800s
phenylenediamine
-
N,M,N',AV'-Tetramethylbenzidine
450,h 472' 790h 9ooj l0lOi 390 (2 5 ) k 710 ( 1 . 3 7 )
A',N'-Diphenyl-p-phenylenediamine a
-
c
ssj 1018 386 (0.48)' 720 10.27)
(7). Also weaker band at 406 mp and inflection at 500 mH. s =
shoulder.
(18). j
In EPA a t 70" K. ( 5 ) . In acetic acid 10-4M (8).
' 10-'M
Not-.
chloride in glacial acetic acid. Read absorbance immediately at, , ,A 578 mp.
-
MECHANISMS
A
I
. :
j'
35ty \ ~
i
i
i;,
,.'
:i'
\ \
.
' \
\\
I
ii
'.. %' 1.
7
*.
\\
', 52C
,
'3
'\
The structures of the different types of chromogens are shown in Figure 2. The abqorption spectra of the phenoxazine, phenothiazine, and phenoselenazine semiquinone radicals have been reported (9). These free radicals were obtained by oxidation of the parent compound with bromine in 8 0 5 acetic acid. The absorption spectra rrere measured with a hand spectroscope. The absorption spectra obtained in the present work in the determination of nitrite with phenoxazine, phenothiazine, and phenoselenazine were closely similar to those reported bj- the Alichaelis group (Table 11). Other oxidizing agent. gave similar spectral bands but with very much weaker intensities. Even in the attempted preparation of 3H-3-phenothiazone from the reaction between phenothiazine and ferric chloride a t room temperature, the absorption spectrum of the solution was that of the free radical. K h e n this solution was boiled, the absorption spectrum of 3H-phenothiazin-3-one was obtained.
'., L4=-J.\
j.,.'
,
i
%/
~
I
600
400
hmp
Figure 3.
1 -Methyl-2-quinolone azine
-
Procedure A 0.69 fig. NO? - - - - _1 ml. of aqueous solution of 4 6 0 f i g . N O i diluted to 10 ml. with acetic acid solution of lo-' reagent, Run immediately 5 X 1 0 - 6 M reagent in ethylcyclohexane or 2-methoxyethanol
. .. . . .
VOL. 35, NO. 13, DECEMBER 1 9 6 3
0
2185
I
I
334
I
1
I
I
I '018
-4
~
Me
Me M e z N w N M e 2
I\
Lw Amp
Figure
4.
Figure 5.
3-Methyl-2-benzothiazolinone azine
-
Procedure for nitrite determination 0.92 pg. NO;, A=, 3aa mp, A = 2.45 To 1 ml. of aqueous 1% sodium nitrite a d d 8 ml. of 2 X 10-5M reagent in acetic acid followed b y 0.5 ml. of 70% aqueous perchloric acid. Read immediately , , 4 X 1 O-6M reagent in 2-methoxyethanol or acetic acid
-___
N,N,N',N'-Tetramethylbenzidine
Procedure A -4.6 pg. NO; ---- Dilute 1 ml. of aqueous solution containing 4 6 0 pg. NO; to 10 ml. with 1 O-'M acetic acid solution of reagent . . . , . . 5 X lO-%Ireagent in 2-methoxyethanol [in acetic acid, A,, 31 3 mp, c =
12,000)
.. . .
Relatively weaker bands were found a t approximately 600, 660, and 750 mp in the absorption spectra obtained in the determination of nitrite with phenothiazine. Illumination of a solution of phenothiazine in EPA a t 90' K. gives a spectrum containing the 532-mp band of the semiquinone and faint bands a t 606 and 654 mp (6). The bands around 400 to 518 mp are apparently derived from the neutral free radical. The origin of the weak longwavelength bands is uncertain. I n the references given in Table I1 evidence has been presented for the free-radical structure of the partially oxidized products (usually obtained by means of bromine or photo-oxidation) of phenoxazine, phenoselenazine, N,N,N' trimethyl - p - phenylN ,N ,N', Nl-tetramethylenediamine, pphenylenediamine, N, N, N', "-tetramethylbenzidine, and N,N'-diphenylp-phenylenediamine. The reported absorption spectra closely match the spectra obtained when the same reagents are used for the determination of nitrite (Table 11). It is postulated that the remainder of the reagents also form free radicals in the procedures for the determination of nitrite. I n all cases small amounts of various types of inorganic oxidizing agents gave exactly the same spectrum as obtained with nitrite. Further evidence for the structure of the chromogens can be deduced from the reaction of five of the reagents with excess nitrite (or bromine) to give an entirely different type of spectrum, which is believed to be derived from dicationic quinonic dyes. For example, under these conditions 1methyl-2-quinolone azine, N,N,N',N'-
-
2186
ANALYTICAL CHEMISTRY
tetramethyl-p-phenylenediamine, 3methyl-2-benzothiazolinone azine, N ,N ,N',N'-tetramethylbenzidine, and N, N'-diphenyl-p-phenylenediamine give bands a t 402 (Figure 3), 340, 510, and 825 (Figure 4)) 464 (Figure 5), and 510 mp, respectively. Titration of these solutions with aqueous sodium bisulfite brings back the spectrum of the free radical. The absorption spectra of the free radicals, many of which are reported for the first time, are very distinctive and reproducible. With N, N, N', N1-tetramethyl-4,4'-diaminostilbene two types of free-radical spectra are obtained. I n acetic-perchloric acid solution a red color, Amax 514 mp, is obtained, for which the structure
~ e i a - c H = c H O - & H M e *
is postulated. This dicationic structure is consistent with the spectra of the reagent in a neutral and acidic solvent (Figure 6). The latter shows a violet shift due to salt formation. I n aceticpropionic acid the reaction between nitrite and reagent is characterized by the presence of an initial red color for about 20 seconds, then a blue color (main .A, 600 mp) for about 20 seconds, followed by decolorization, and then the slow development of a dark green color, Amsx 447 and 715 mp. The latter bands are probably derived from the free radical, M~*N--CH=CH+ -
---NM~~
for when enough perchloric acid is added to form a 2001, solution, a red 514 mp, is formed with an color, A, absorption spectrum closely similar to that of the dicationic free radical. The phenoxazine and phenoselenazine free-radical spectra obtained in the determination of nitrite resemble that of the phenothiazine free radical (Table 11)* The distinctive absorption spectrum of the free radical obtained in the determination of nitrite with glyoxyal bis(1,l-diphenylhydrazone) is shown in Figure 7. Although the reagent does form a salt in acetic acid-perchloric acid solution (see Figure 7), the free radical is postulated as mainly the simple monocation. The absorption spectra indicate the complexity of this problem, in that the relative intensities of the two bands in the nearinfrared region approach unity with a decreasing concentration of nitrite. The absorption spectrum of 3-methyl2-benzothiazolinone picrylhydrazone 430 mp) or acetic acid in xylene (A, (A, 425 mp) closely resembles that of picramide in xylene (A,, 415 mp). In acetic acid containing 2001, perchloric 255, 295 mp) the absorption acid (A, spectrum is drastically changed, indicating salt formation. consequently the spectrum obtained in the nitrite determination (A, 406 mp) is probably that of the free-radical cation. In the determination of nitrite with any of the reagents the formation of the free radical is autocatalytic, as shown by the extremely high molar absorptivities obtained in the procedures. With the type of reagent containing an NH group the mechanism is postulated as taking the course shown in Figure 8. With the other reagents the mechanism
uI I200
imp
Figure L m p
Figure bene
7.
. . . . ..
-0 . 9 2 pg. NOT
Recommended pracedlJre Dilute 1 ml. of aqueous solution of 46 pg. of NO; to 10 I T ~ .with acetic acid solution containing 0.5% ireagent and 20% propionic acid
----
. . .. ..
-
Recommended procedure 2.3 pg. NO; 2.8 X 10-5M reagent in acetic acid containing 10% of 70% aqueous perchloric acid (v./v.) 1 O-5M reagent in 2-methoxyethanol
-.-.-
6. N,N,N’,N’-TetramethyI-4,4,’-diaminostil-
-.-.-
Glyoxal bis(N,N-diphenylhydrazone)
(V/V.l
1.88 X 1 O-6kl reagent in acetic acid containing 20% of 70% aqueous perchloric acid (v./v.) 5 X 1 O-sM reagent in dioxane
is similar except for the absence of the N-nitroso derivative. EFFECT OF VARIABLES
1-Methyl-2-quinolone Azine. Unless otherwise stated, the following discussion applies t o Procedure 4 . T h e optimum intensity was obtained with 0.02 t o 1% reagent. With 0.1% reagent a better blank and slightly lower intensities werc obtained as compared t o the 1% leagent. Since the absorbance increascd over a fairly long period of time ( F i g r e I), 1 hour was chosen as a convenient time t o make a reading. Figure 1 shows that the sensitivity could be doubled by choosing a longer standing period. If there is a necessity to st zbilize the color intensity, sulfamic acid can be added to the solution 1 t o 2 holm after mixing (Figure 9). As in almost all the procedures described in this paper, the slope of the calibration curve decres sed with lower concentrations of nitrite. This effect was more drastic in Procedure A. I n Procedure B there was a straightline relation from 0.1 to l . 3 pg. of nitrite. I n Figure 3 the absorption spectra of this newly described stable free radical, the starting reagent, and the more completely oxidized quinonic structure are compared. Kitrates, aldehydes, ketones, alcohols, and hydrocarbons gave negative results. Inorganic oxidizing agents, such
as periodate, iodate, permanganate, and chromate, gave positive results in Procedures d and B. ;In attempt to collect ozone in 0.1N sodium hydroxide solution and then analyze by Procedure B gave negative results. The presence of very large amounts of sulfite interfered with the determination of nitrite. The interference of sulfite was augmented with an increase of water in the final analyzed mixture. I n Procedure B, the presence of 50 parts of sulfite to 1 of nitrite decolorized the mixture. The spectrophotometric constants for all procedures are reported in Table I. N,N,N’,N’ Tetramethyl - 4,4’diaminostilbene. Optimum intensities were obtained with 0.025 t o 0.07% reagent and 207, perchloric acid in the reagent solution. This solution was stable for a t least 20 hours. When distilled acetic acid was used to make up the reagent solution, turbidity was found occasionally in the final red solution. Addition of about 1% water to the reagent solution preventcd the formation of this turbidity. Once reagent and test solutions were mixed, approximately 60 to 70 minutes were necessary for the development of nisximuni intensity. After the solution had stood for 24 hours, all peaks between 400 and 800 mp had disappeared. The concentration-absorbance curve resembled that obtained in the other procedures. The
-
absorption spectra of the reagent and the two types of derived free radicals are shown in Figure 6. I n Figure 6 an alternative procedure is given for the determination of nitrite. This method was not explored further. Exposures of about 10 to 60 minutes to instrumental light at the wavelength maximum accelerated the increase in intensity. Kitrates, aldehydes, ketones, alcohols, and hydrocarbons gave negative results. Inorganic oxidizing agents gave positive results. I n the presence of sulfite a t 400 to 1 (sulfite-to-nitrite ratio), nitrite was determined with no loss in intensity (Figure 10). Phenothiazine. These procedures were standardized with phenothiazine. The percentage of reagent was not critical in the range of 0.05 to 0.8%. A maximum color intensity was reached a t 60 t o approximately 105 minutes. The slope of the calibration curve decreased with lower concentrations of nitrite for both the phenothiazine and phenoselenazine methods. With phenoxazine as the reagent the maximum intensity was reached in 10 minutes; 2.3 pg. of nitrite ion gave a molar absorptivity of 128,000 a t A, 530 mp. With 2-chlorophenothiazine as the reagent the maximum intensity was reached a t 30 to 40 minutes; 2.3 pg. of nitrite ion gave a molar absorptivity of 168,000 a t A,, 525 mp. Negative results were obtained with sodium nitrate, formaldehyde, potassium iodide, sodium chlorate, and magnesium perchlorate. Ozone gave a t most a very weak reaction. I n proportions of a t least 2000 to 1, VOL. 35, NO. 13, DECEMBER 1 9 6 3
2187
HONO
c Me
tie
/
\
I \
~
i NO
2
NO2
Figure 8. Mechanism for formation of phenothiazine-type free radicals I
sulfite had no effect on intensity (Figure 10). “Ar,N’ C ’ Diphenyl - p - phenylenediamine. PROCEDURE A . Optimum intensities were obtained from 0.01 to 0.1% reagent, with 0.05% reagent giving slightly higher intensities. The color intensity gradually increased with time and did not reach a maximum in 4 hours. I n both procedures the slope of the calibration curve increased with higher concentrations of nitrite (Figure 11). I n a procedure similar t o Procedure A, except t h a t excess nitrite and a much smaller concentration of reagent are used, a band was obtained a t 510 mu which is
TIME ,HOURS
-
IO0
20c
300
’
3
2
Figure
9. 1 -Methyl-2-quinolone
azine Procedure A
Change in absorbance with time after mixing in determination of 0.46 fig. NO? at Amsx 5 2 0 mp 1 ml. of 0.1% sulfamic acid in 25% aqueous ocetic acid ( 2 5 ml. of acetic acid diluted to 1 0 0 ml. with water) added a t : Minutes offer mixing
-
A.
0
D. 60
B.
15 30
E.
C.
probably derived from the quinonic compound. Nitrates, perchlorates, phenols, alcohols, and hydrocarbon gave negative
120
results. Most inorganic oxidizing agents gave positive results. At 800 to 1, sulfite did not interfere with the determination (Figure 10). PROCEDURE B. At least 20% of 70% aqueous perchloric acid was required t o keep the reagent in solution during the analysis. The highest attainable concentration of reagent was 0.0570. With lower concentrations of reagent,
400
pg SO;- per 1u.g NO;
Figure 10.
Effect of sulfite on determination of nitrite
0.92 pg. NO;. N,N,N’,N’-Tetramethyl-4,4’-diaminostilbene procedure, A,, 5 1 4 mp Phenothiazine procedure A, Ax, 51 8 m p 1.84 pg. NO,. 1 .E4 pg. NO;. 3-Methyl-2-benzothiazolinone azine procedure, Amax 7 3 5 mp 0.72 pg. NO;. N,N’-Diphenyl-p-phenylenediamine procedure B, Amax 7 0 5 mp; Procedure A, A, 7 2 0 mp 0.50 pg. NO;. 9.25 pg. NO;. Sulfanilic acid plus N ( l -naphthyl)ethylenediamine procedure ( 1 2 ) Am,x 5 4 9 mp 1.38 pg. NO;. 3-Methyl-2-methylbenzothiazolinone picryl405 mp hydrazone A, 2.3 pg. NO;. Glyoxal bis-(N,N-diphenylhydrazone), A,*, 5 4 0 mp
21 88
ANALYTICAL CHEMISTRY
Figure 1 1 . nitrite
Change in absorbance with concentration of
. .. . . . N,N’-Diphenyl-p-phenylenediamine Procedure A at 7 2 0 m p . .-. .- Procedure B at 705 mp -3-Methyl-2-benzothiozolinone picrylhydrazone procedure at kmax 406 m p
0 - - - N,N,N’,N’-Tetrameihylbenzidine
A --- N,N,N’,N’-Tetramethylbenzidine
.-.- Procedure B ot Amzx 4 7 2 m p
Procedure A at A,, Procedure A a i A,
4 7 3 mp 1018 m p
lo\\c’r intensities mere obtained. The re.igeiit solution x a s stitble for at least 8 hour.; The blank was colorless to faint blue. As in Procedure A, the color intensity increasc.d with time, so a 1-hour standing pericd mas chosen for the development of tolor. The concentration-absorbance curve is shown in Figure 11. Sitrate gaT-e negatiTTe results; other inorganic oxidizing agents gal e positive re-ult- Sulfite had very little effect (Figure 10); a t 2500 t o 1 absorbance decredoed from 0.52 to 0.40. Y,.Y,AY’sAYt Tetramethyl - pphenylenediamine. PROCEDURES A ASD B. Increasing the concentration oi reagent from 0.005 t o 0.270 increased the color intensity. Since 0.2% reagent soluticn gave a lightbluish blank, 0.1%; reagent was chosen. T h e reagent solution turned liqlit blue after 6 hours. Xs the miourit of diatilled w a er in the reagent solution was increased (from 0 to 25%), the intensity increasec . With 50% or greater amounts of distilled water in the reagent, the blank became blue, pw1i:ips because of the prespnce of minutc amounts of ozone in the water. Once the nitrite and reagent solutions were mixed, the color intensity increased rnpidlr for about hour and then much inore gradually for the next hour. The slope of the concenti ation-absorbance curves for the two provedures decreased I !ith lower concentrations of nitrite. 111a procedure similar to A except that excess nitrite and a xruch smaller concentration of reagent are used, a band derived from the quinonic compound n a - obtained at 340 rlip. The reagent in acetic acid gave no band aboTe 300
-
mp
Sitrate gave negati i e results; many other types of inorganic oxidizing agents gare positive results. At 1000 to 1, sulfite had no efYect 011 the intensity in Procedure 8. PROCEDURE C. Optimum intensities were obtained with 0.270 reagent and 20 minutes’ reaction time. Interferences nere the same as for l’rocedures A and E.evcept that sulfite lad a much more ic effect: At 60 to I, the absorbnnce decreased from 0.’*5to 0.08. 3 Methyl - 2 - benzothiazolinone Azine. Optimum intensities were obhilied with 0.005 to 0,0270 reagent. Teii per cent propionic acid was necessary t o dissolve the reagent. T h e older of addition of reagents was important, since changes in order caused some differences ir intensity. A t tc.niperatures higher than 40’ F. the intensities decreased The intensity n a b also affected by exposure of the olution over a pericd of minutes to iiidrumental light a t the wavelength maximum. Seven hours after mixing of the reagent and nitrite test solutions, tlir intensity was stil on the increase.
-
The absorbance-concentration curve is similar to that obtained with the other reagents. Addition of 1 ml. of 0.1% sulfamic acid in 25% aqueous acetic acid a t 15, 30, 45, 60, and 120 minutes after mixing reagent and test solutions stabilized the intensities for a t least 30 minutes. Common nitrates, iodides, perchlorates, chlorates, aldehydes, ketone,, alcohols, hydrocarbons, and selenium dioxide gave negative results. Other inorganic oxidizing agents, especially iodate and periodate, gave positive results. The absorption spectra of the reagent, the free radical obtained in the analytical procedure, and the fully oxidized quinone are shomm in Figure 4. The amount of interference of sulfite in the determination of nitrite is s h o m in Figure 10; at 500 t o 1, absorbance droppedfrom 1.16 to 0.78. Glyoxal Bis-(;\i‘,N-diphenylhydrazone). Maximum intensities were obtained with 0.01% of the hydrazone and 10 t o 207, perchloric acid in the reagent. The reagent was stable for 1 hour. Maximum intensities were obtained 2 to 6 minutes after mixing the reagent and test solutions. The concentration-absorbance curves obtained with this compound a t wavelengths 540 and 1152 have slopes somewhat similar t o those of the other reagents. The absorption spectra of the reagent, its salt, and the free radical are shown in Figure 7 . Sodium nitrate, potassium iodide, cupric chloride, iodine, magnesium perchlorate, potassium and ammonium persulfate, selenium dioxide, sodium chlorate, acetone, alcohol, and phenol gave negative results. Oxidizing agents, such as ferric chloride, potaqsium permanganate, potassium chromate, potassium ferricyanide, potassium iodate, potassium periodate, and -It bromine gave positive results. values up t o 100 to 1, sulfite did not interfere with the determination of nitrite (Figure 10). I t 500 to 1, the absorbance decreased from 1.82 to 1.35. 3 - Methyl - 2 - benzothiazolinone Picrylhydrazone. Optimum intensities were obtained with 0.01 t o o.o5yOof t h e hydrazone and 20% of 70% aqueous perchloric acid in the reagent solution. A much paler yellow blank was obtained with 0.01% reagent. T h e reagent solution was stable for at least 24 hours. The color intensity increased rapidly for the first 30 minutes and then much more slowly for the next 90 minutes. The slope of the calibration curve increases with higher concentrations of nitrite (Figure 11). The reagent has bands at 255 and 295 mp in acetic acid-perchloric acid, so it does not interfere with the determination of nitrite a t 406 mp. Kegative results were obtained with
codiuin nitrdte, potassium periodate, potassium iodate, cupric chloride, iodine, bromine, potassium persulfate, and m a g n e w m perchlorate. Positive results were obtained with potassium permanganate, pota\sium chromate, potas+ m i ferricyanide, and ferric chloride. T p to 200 to 1, the interference from Gulfite wa. negligible (Figure 10). At 900 to 1. the absorbance dropped to 0 8 i from an initial value of 1.48 (no sulfite). h’,S,A~’.S’ - Tetramethylbenzidine. PROCEDURE -4. Optimum intensities nere obtained with 0.01 t o 0.1% reagent. K i t h the higher percentage the color intensity reached a mavimuni in 1 t o 3 minutes and then faded much more gradually than with the lower percentage. The reagent is stable for about 30 minutes. T h e elope of the calibration curve decreased with a lower concentration of nitrite (Figure 11). Sitrite can also be determined in highly colored solutions. For example, it was readily and accurately deter1018 mp in a dark bluish mined a t A,, green colution containing the dye LY,A\7,2\7’,A\7’ - tetrampthyl - 4,4‘ - diaminoazobenzene. Segatire results mere obtainfd with d i u m nitrate, sodium chlorate, magne4um perchlorate, cupric chloride, potassium persulfate (-3000 pg.), and ferric chloride (-3500 pg.). Positive results were obtained with potassium chromate, potassium iodate, potassium permanganate, potassium ferricyanide, and bromine. -it 50 to 1, sulfite did not interfere in the determination of nitrite; with much larger amounts of sulfite there wag some interference. PROCEDCRE B. Optimum intensities were obtained with 1% reagent. The color intensity was stable for about 5 minutes and then gradually faded. Exposure to instrumental light at the wavelength maximum over a period of minutes accelerated the fading. The slope of the calibration curve decreased ivith a lower concentration of nitrite (Figure 11). The addition of sulfite interfered more drastically in this procedure by decreasing the intensity and increasing the rate of fading. N,N,.V’ - Trimethyl p phenylA. Optienediamine. PROCEDURE m u m results were obtained with 0.05 t o 1% reagent. T h e color intensity was stable for about 7 minutes and then gradually faded. The concentration-absorbance curve was linear from 4.7 to 50 pg. of nitrite; in Procedure R it was linear from 6 to 50 pg. of nitrite.
- -
COMPARISON OF METHODS
Thus far, the highest molar absorptivity obtained in the determination of nitrite was with the 1-methyl-2-quinolone azine Procedure A; with 0.72 pg. VOL. 35, NO. 13, DECEMBER 1963
2189
of nitrite a molar absorptivity of 1,270,000 was obtained. If the reaction were allowed to continue for about 7 hours, the molar absorptivity would be approximately doubled. Other methods with molar absorptivities of over 50,000 are the N,N,iV’,N‘-tetramethyl4,4’-diaminothiobenzophenone ( I @ , 1methyl-2-quinolone azine B, N,N,N’,N’tetramethyl - 4,4’ - diaminostilbene, N,N‘-diphenyl-p-phenylenediamineB, and 3-methyl-2-benzothiazolinone azine procedures. The autocatalytic methods have, on the whole, much higher molar absorptivities than the various nitrosation methods previously described (15). The molar absorptivity obtained in a method can be considered as the potential sensitivity of a method where the test solution volume and the final volume are identical. In most spectral methods of analysis these two volumes are not equal, however. Therefore the sensitivity of an analytical method can be defined as the molar absorptivity multiplied by the fraction of test solution volume in the final analyzed volume. To compare various analytical methods for sensitivity the following arbitrary formula can be used: Sensitivity =
e
x
10-3
dilution factor
where the dilution factor is final volume/ test solution volume. For a method in which Beer’s lam is complied with, the sensitivity is the same over the entire range of determinable concentrations of analyzed substance. For most of the methods in this paper the slope of the calibration curve, as well as the sensitivity, decreases with lower concentrations of nitrite. The signal obtained on the chart-Le., the absorbance per equimolar amounts of material in a 1-em. cell-is relatively greater at higher concentrations of material. Of all the methods in the literature the 1-methyl-2-quinolone azine Procedure B is the most sensitive. Its sensitivity ranges from 190 a t an absorbance of 0.10 to 380 at a n absorbance of 2.0. The remainder of the procedures described in this paper range in sensitivity from 0.98 to 255. In the previous paper (16) the most sensitive azo dye method reported for the determination of nitrite was the p-phenylazoaniline plus I-naphthylamine procedure, with a sensitivity of 31; the most sensitive diazonium cation method wm the chloro-p-phenylenediamine procedure, with a sensitivity of 31.6. The new methods with the highest sensitivities reported in that paper are the N,N,iV‘,N‘ - tetramethyl - 4,4‘ - diaminothiobenzophenone, sensitivity 56, and the azulene, sensitivity 37.5. The determination limit is essentially the number of micrograms in the analyzed test solution giving an absorbance of 0.10 in a 1-cm. cell. I n the 2190
ANALYTICAL CHEMISTRY
previous paper the lowest determination limit (0.10 pg.) was reported for the azulene procedure. I n the work described in this paper the phenothiazine Procedure B gave a determination limit of 0.07 pg. If all volumes in this method were decreased to 0.4 of the original, the determination limit would drop to 0.028 p g . In a similar fashion determination limits could be decreased in any of the other procedures described in this paper. Except for the A-,A-,X‘trimethyl - p - phenylenediamine procedures the determination limits for procedures reported in this paper ranged from 0.07 t o 0.94 p g . ; for those in the pre1ious paper, limits ranged from 0.1 to 66 pg. I3eer’s law was complied with in a very large number of the methods reported in the previous paper; it was not in any of the free-radical methods reported h u e . K’oteworthy is the autocatalytic method for nitrite using the A-,AY$Al”jAY’ - tetramethyl - 4,4’ - diaminothiobenzophenone procedure, because of its straight-line relationship between concentration and absorbance over a wide range of absorbance-from about 0.02 to 12 (15). In the analytical procedures increasing the amount of water tended to make the relationship between concentration and absorbance more linear. On the average better precision mas obtained with the nitrosation and diazotization type methods described previously than with the autocatalytic methods. All the autocatalytic methods involve simple procedures. About one third of the methods require a procedure time of a little over 1 hour; another third require 22 or 32 minutes, and the remainder require 2 or 7 minutes. These latter procedures take such a short time because the color starts fading within 5 or 6 minutes. I n the others the color intensity increases over a more extended period before it starts fading. With some of the procedures the addition of sulfamic acid at an appropriate time stabilizes the color intensity. I n respect to color stability, many of the azo dye methods reported in the previous paper (15) had color stabilities greater than 1 hour. -4 few were stable for more than 16 hours. I n most of the autocatalytic methods the presence of large amounts of sulfite caused little, if any, interference; in azo dye methods sulfite and other reducing agents would be expected to interfere. The presence of a larger percentage of water in the autocatalytic methods increased the interfering effect of sulfite. Many types of inorganic oxidizing agents gave the same type of spectra as obtained with nitrite. This kind of phenomenon is absent in the azo dye methods.
RECOMMENDATIONS
The Ai,”-diphenyl-p-phenylenediamine Procedure B is recommended for the determination of nitrogen dioxide in the presence of large amount,s of suIfur dioxide. This type of method would be of value in t’he analysis of source and atmospheric samples containing relatively large concentrations of sulfur dioxide. A few other methodse.g., iY,r\i‘-diphenyl-p-phenylenediamine Procedure 1 and phenothiazine Procedure .I-are even less affected by sulfite, but are somewhat less sensitive to nitrit’e. These latter procedures are also less sensitive than the most sensitive azo dye methods. The 1-methyl-2-quinolone azine Procedure B is recommended for the determination of 0.1 to 1.3 pg, of nitrite (or nitrite precursor) in the absence of inorganic oxidizing agents and high concentrations of sulfite. The intensity is not affected in the presence of a rat’io of sulfite to nitrite of about 2 ; wit>ha ratio of 10 to 1 the ir~t~ensity is decreased by one bhird. By use of smaller volumes of test and reagent solutions, 0.04 to 0.52 fig. of nitrite couId be determined. The S,S,S’,S’-tetramethylbenzidine procedures are recomrnendcd ivhere a quick, simple procedure is il(4red for t’he determination of nitritc in the absence of inorganic Oxidizing agcnts and very large amount,^ of sulfite. Some less sensitive methods with fairly short procedural times have been described (15);these methods are relatively insensitive to inorganic oxidizing agents and have much more stable colors. The S,~~-,i~-’!,~’-tetramethyl-4,4’diaminothiobenzophenone procedure (15) is recommended where it is desirable to have a very large increase in ab ance with a small increase in 11 concentration. Where it is desirable to determine nitrite in highly colored solut’ions, the glyoxal his(-VI',"-diphenylhydrazone) procedure a t A,, 1152 mp and the N,X,S’,S’-tetramethylbenzidine pro1018 mM are recomcedures a t , , ,A mended. By a slight modification of the procedures, electron paramagnetic resonance spectrometry could be used in the determination of nitrite, nitrite precursors, and some of the inorganic oxidizing agents. Some of the methods could be modified for use in the determination of oxidants, For example, the l-methyl2-quinolone azine reagent solution reacted strongly with ozone. The reaction of the 3-methyl-2-benzothiazolinone azine solution with ozone was fairly strong also. Further work on the reaction of the various reagents with oxidizing agents and with transient free radicals should prove of value.
The relatively stabje free radicals introduced in this paper should prove of value in other fields of research. LITERATURE CITED
( 1 ) Albrecht, A. C., Sinipson, W.T., J. Am. Chem. SOC.77, 4454 (1955). (2) Cornelius, W., J . Prulzt. Chem. 88, 395 (1913). (3) Fuchs, K., GrauauE:, E., Ber. 61B, 57 (1928). (4) Hunig, S., Richter, P., Ann. 612, 272 (1958). (5) Lewis, G. N., Bigelcisen, J., J . Am. Chem. SOC.64,2808 (1942).
(6) Ibid., 65,2419 (1943). (7) Lewis, G* Ne, LiPk'n, D., Ibid.9 64, 2801 (1942).
(8) Linschitz, H., Rennert, J., Korn, T. M., Ibid., 76, 5839 (1954). (9) Michaelis, L., Granick, S., Shubert, M. P., Ibid., 63, 351 (1941). (10) Michaelis, L., Schubert, M., Granick, S., Ibid., 61, 1981 (1939). (11) Riemschneider, I. R., Monafsh. 89, 683 (1958). (12) Saltzman, B. E., ANAL. CHEM.26, 1949 (1954). (13) Sawicki, E., Hauser, T. R.1 Stanley, T.W., Elbert, W., Ibid., 33,93 (1961). (14) Sawicki, E., Pfaff, J., Stanley, T.
W., Rev. Univ. Ind. Santander 5, 337 (1963). (15), Sawicki, E., Stanley, T. W., Pfaff, J., D Amico, A,, Talunta 10, 641 (1963). (16) Stewart, F. H. C., J. Chem. SOC. 1957, 1026. (17) Stewart, F. H. C., J . Org. Chem. 26, 3604 (1961). (18) Wieinger, R., Angew. Chem. 68, 528 (1956). (19) Wohl, A,, Xeubert, C., Ber. 33, 3107 (1900). RECEIVEDfor review April 8, 1963. Accepted July 22, 1963. Eleventh Detroit Anachem Conference, October 21 to 23, 1963.
Direct Qulantitative Isolation of Monocarbonyl Compounds from Fats and Oils D. P. SCHWARTZ, H. S. HAILER,' and MARK KEENEY2 Dairy Products laborat'ory, Eastern Utilization Research and Development Division, Agriculfural Research Service, U. S. Department of Agriculture, Washingfon 25, D. C.
b A quantitative procedure is described for the direct isolation of carbonyl compounds from fats and oils. Carbonyl compounds in the fat are converted to their 2,4-dinitrophenylhydrazones, subsequently freed of fat, and fractionated by adsorption on activated magnesia and partially deactivated alumina The fat-free monocarbonyl fraciion is then separated into classos on magnesia and the members of each class are obtained by column partition chromatography and identified by supplementary techniques. The procedure is ideally suited for m a l l samples of fat, but can be applied to kilogram quantities. Advantages and limitations of the method are discussed.
T
paper describes a method for the direct quantitative isolation of monocarbonyl compounds from fats and oils. The method was developed in conjunction with a s t i d y on off-flavor development in stored whole milk powder. Carbonyl compounds are usually isolated from autoxidized fats and oils by some form of distillation, although other methods such as extraction of water-soluble derivatives have been reported (tl, 11, 12). Over 95% of the carbonyl compounds occurring in many fats and oils are nonvolatile when the umal methods of distillation are used (9, 10). The nature HIS
Present address, Bureau of Program Planning and Appraisa, Food and Drug Administration, Depar;ment of Health, Edu_cation, and Welfarc!, Washington 25, u. c;. * Present address, Department of Dairy Science, University of Maryland, College Park, Md.
and role of these nonvolatile carbonyl compounds had not been elucidated a t the onset of this work, although it has been suggested that part of them, a t least, might be involved in fat oxidation ( 2 ) . I n the proposed method, distillation and extraction techniques are circumvented, and, theoretically, all carbonyl compounds capable of forming a 2,4-dinitrophenylhydrazone under the conditions outlined are isolated lipidefree, regardless of the amount of starting material. EXPERIMENTAL
Materials. Seasorb 43 (activated magnesia, Fisher Scientific Co., Silver Spring, Md.) is used as received. Hexane (Phillips' high purity grade) and benzene (ACS grade) are rendered carbonyl-free by the method of Schwartz and Parks (17). Celite 545 is dried a t 150' C. for 24 hours. Nitromethane (Fisher's highest purity) and chloroform (ACS grade) are used.
Scope of Method. The method for isolation of the monocarbonyl fraction from fats and oils may be divided into six steps: (1) reaction of the carbonyl compounds in the fat with 2 , 4 dinitrophenylhydrazine; (2) adsorption of the resulting derivatives onto activated magnesia while eliminating the bulk of the fat or oil, followed by (3) desorption of the derivatives; fractionation of the derivatives on weak alumina; (4)adsorption of the monocarbonyl derivatives on an anion exchange resin, if necessary; (5)separation of the monocarbonyl derivatives into classes on magnesia; and (6) separation of the members of each class by liquidliquid partition chromatography.
Step 1. Reaction with 2,CDinitrophenylhydrazine. A column of Celite impregnated with dinitrophenylhydrazine, phosphoric acid, and water (reaction column) is prepared as described by Schwartz and Parks (17). The column is flushed with 50 ml. of benzene, followed by hexane until a colorless effluent is obtained. The fat or oil is dissolved in hexane and passed over the column. When the last of the solution has just entered the column, the sides of the tube are washed down with hexane and the washings allowed to enter the column. Fifteen milliliters of hexane are added and permitted to drain into the column by gravity. The column is then flushed with hexane, using NQ pressure until the effluent emerges colorless or has the same absorptivity (at or near 340 mp) as the effluent from a blank column, which should always be run simultaneously. The hexane-fat effluent now contains all of the original lipide, the 2,4dinitrophenylhydrazine derivatives of the monocarbonyls, semialdehyde, and ketoglycerides (7, 8), and other classes of carbonyl compounds whose derivatives are soluble in the fat-hexane solution, a small amount of dinitrophenylhydrazine, and traces of the decomposition products of dinitrophenylhydrazine. Remaining on the column are those carbonyls whose derivatives are insoluble in the fat-hexane solution. QUANTITATIVE ASPECTS. The quantitative aspects of reaction of carbonyls in fat with dinitrophenylhydrazine on the column were thoroughly studied from two standpoints. First, carbonyl compounds in the form of pure semicarbazone or other suitable derivative were added singly to monocarbonyl-free, butter oil. Schwartz (15) had shown that micro amounts of the semicarbaVOL. 35, NO. 13, DECEMBER 1963
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