Table 11.
Titrations Using Various Masking Agents
Masking Agent 2,4-Pentanedione, pH 7
+
2,4-Pentanedione citrate Sulfosalicylate, pH 4.5
Mixture, Ion Masked -4173 A1 +3 1--3 -11 -3 Zn +2 -11 - 3 Zn '2 KO2 -2 Zn *2 -11-3, Th+4 UOn -2 La+3 Ce - 3 U02'2 Dy '3 TO2 +2 Detd. La'3 Sm+3
\* -3 y + 3
Tartrate, pH 7
Yb + 3 Yb + a
-11 + 3 COAL? AI 1 3 C02-
Zn+ 2
110+e
Zn'2 Zn'2
Sb'j Sb-3
Zn'2
thorium, zirconium, and iron that can he tolerated is more limited. Sulfosalicylate. This complexing agent will not effectively mask ions such as thorium(IV), zirconium(IV), or iron(II1). A t p H 4 . 5 , i t masks sluniinum and uraniuni(V1) effectively but permits t h e titration of rare earths, yttrium, and thorium. Several bivalent metal ions can also be titrated. Yttrium and the higher rare earths have large end point breaks, but the lower rare earth end points cannot be located so precisely (Figure 6). 2,CPentanedione. T h e most iniportant use for 2,4-pentanedione is in the titration of Iare earths in t h e
K T 6
Theory 7 97 20 00 05 05 05 2 17 2 94 3 98 3 33 4 5 5 5 5
3 33 3 06
3 5 5 5 5
06
07 07 07
07
MI. Found 7 94 4 20 5 00 5 5 5 2 2
06 07
3 3 3 5 5
33
08 16
96 4 00 3 32
06 06 10 09 5 06 5 07
Diff. -0.03
*o.oo 10.00
$0.01 $0.02 $0.03
-0.01 $0.02 +o, 02 -0.01 f0.00 10.00 *o. 00 $0 03 $0 02 -0 01 1 0 00
presence of aluininum (Figure 7 ) . T h e lower rare earths give about as sharp an end point as t h e higher members of t h e series. Thorium, iron, and zirconium are incompletely masked and interfere. Rare earths can also be titrated in the presence of uraniuni(V1). The end point break is short but easily detected. Zinc can be titrated in the presence of rare earths or uranium. Copper is too strongly complexed by 2,4-pentanedione to be titrated, but it is too Jveakly complcxed to be effectively masked. All titrations with 2,4pentanedione are carried out in the p H range from 6.5 to 7 . 5 . Other Agents. Although tartrate is
related t o citiate in structure and chelating properties, the tartiate complex of a n y given metal is usually less stable than the citrate complex. Thorium, zirconium, ironfIII), and other metals that form very strong EDTA complexes are masked by citrate but not sufficiently by tartrate to aroid interference. However, metals such as uranyl (11) , antimony (111), niobium (V) , niolybdenum(T'I), and tungsten(V1) are masked just as effectively by tartrate as by citrate. At p H 4.5, aluminum is effectively coniplexed by fluoride, permitting the titration of zinc and copper. Zinc can be titrated successfully in the presence of both thorium and aluminum a t pH 7 . Citrate is added to mask the thorium, and 2.4-pentanedione is added to complex the aluniinun]. Data for titrations using sulfosalicylate, 2.4-pentanedione, and tartrate are given in Tahle 11. LITERATURE CITED
(1) Fritz, J. S , Ford, J. J., A A A L .CHEV. 2 5 , 1640 (1953).
(2) Reilley, C. N.,Porterfield, K. IV.. Zbzd., 28, 413 (1956). ( 3 ) Reilley, C. S . , Schmid, R. it-.,Zbzd., 30, 947 (1958). (4) Schmid. R. K.. Reillev. C. Y.. J . Am. Che,?;. SOC.78, 5513 (1956). (5) Siggia, S., Eichlin, D. W.,Rheinhart, R. C., ANAL.CHEJI.27,1745 (1955). RECEIVED for revieTv Xovember 29, 1957. -1ccepted .Ipril 7 , 1958. Work performed in the Ames Laboratory of the U. 8. .Itomic Energy Commission. \
,
Qualitative and Quantitative Determination of Aliphatic Carbonyl Compounds as 2,4-Dinitrophenylhydrazones KENNETH J. MONTY McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Md. )By the combined use of partition chromatography and spectrophotometry, the joint qualitative and quantitative microanalyses of mixtures of saturated aliphatic carbonyl compounds may, b e effected. The 2,4dinitrophenylhydrazones formed from the mixture are fractionated on the basis of the molecular weights of the parent carbonyl compounds, using partition between nitromethane and petroleum ether on kieselguhr columns. Spectrophotometric examination of the chromatographic fractions in the visible range permits the differential determination of the aldehydes and ketones represented by each fraction.
1350
ANALYTICAL CHEMISTRY
The method is applicable to the study of fractional micromoles of carbonyl compounds up to about 14 carbon atoms. HE 2,4 - dinitrophenylhydrazones ha1.e been used to separate, identify, and determine carbonyl compounds. The spectral properties of these derivatives have been examined under a variety of conditions and from the ultraviolet through the infrared regions (1, 5, 6, 11, 13). The different spectral properties of the dinitrophenplhydrazones in alkaline solution n-ere made the basis for a differential determination of
T
saturated aliphatic and olefinic aldehydes (4). For the separation of carbonyl co~iipounds, paper chromatographic methods involving the 2,4-dinitrophenylhydrazones have met Tvith varying degrees of succcss ( 7 , 9, IO), but they are more applicable to the derivatives of relatively low molecular IT-eight compounds. The nietliod of Ellis, Gaddis, and Currie (2) for the chromatographic separation of 2,4-dinitrophenylhydrazoneson filter paper, like the present one, is applicable to the derivatives of aliphatic carbonyl coinpounds of u p to 14 carbon atoms. The ease with which materials may be compared by chromatography on filter
paper may make their method a valuable adjunct to tlie described procedure in establishing with greater certainty t,lie configuration of carbonyl coiiipounds. Various column chrornatograptiic nietliods have also been described (3, f ,?'), but the most successful has been t,liat, of Iiramer and van Duin (9). Tliese investigat'ors employed a partition betn-een petroleum et'her antl nitroIiietliane on silica gel columns to fractioiiate homologous series of 2,4-diiiitroplieiiI\-lliydrazones of n-aliphatic :ildeliytles antl methyl ketones of chain lcngths u p t o 18 carbon atoms. The present method uws partition clironiatogrnpliy and spectrophotoiiictric measurements for t!ie qualitative deterniination of s:iturated alipliatic riioiiocarlionyl compounds as their 2]4t.litiitroplifn!-lliy(~razones. The clironiatographic system is a nioclification of that of Iiraiiier arid van Duin (S), ~vlieretlie use of a comniercially avail:iblc> diatoniaceous earth as supporting nicdiuni circumvents the problem of obtniiiing reproducibility betivecn different i)reljaratioiis of silica gel. The spwtral
pounds \\-it11 chain lengths up to about 14 carbon atoms. The spect8ropliotonietric niensurenient' ferentia1 deterininat inicroinoledes of aldehydes and ketones :IStheir dinitropheii!-lliyc~razoii~~. 2,4-DINITROPHENYLHYDRAZONES
Preparation. Dinitrophenylhydrazones nere prepared by t h e method of S h i n e r a n d Fusoii ( I d ) , using 2,4dinitrophenvlhydrazine twice recrystallized from methanol. Technical grade carbonyl conipounds n-ere used ivlitxre more purified compounds were not available. T h e 2,4-dinit1ophenylhydrazones were recrystallized a t least tuice froni ethanol or froni a suitable niixture of ethanol and water. Each sample 11as examined for chromatographic homogeneity before being subjected to spectrophotometric study. Tlie use of melting points as criteria of purity i n s abandoned, because chronistographically inhomogeneous samples often failed to yield melting point depressions. Before weighing, the derivatives were dried for 24 hours under vacuum and in the presence of calcium chloride. ASSEMBLYASD OPERATIOSOF COLUMS. Hyflo Super-Cel (Johns-lIanville, S e n . Tork. S.Y.)is used as supporting nicdium. K i t h stirring, 6 nil. of nitromethane are added t o 6 grams of Super Cel. A slurry is made by suspending this mixture in the mobile phase, petroleum ether (boilingpoint, 30 to 60" C.) saturated with nitromethane. This nil1 pack a column 9 X 250 mm. A
Table I. Molar Extinction Coefficients for Some 2,4-Dinitrophenylhydrazones
Extinction Coefficient x 10-4" 425 mp 530 mp 0 863 2 06 0 813 2 08 0 898 2 16 0 843 2 14 0 868 2 08 0 873 2 17 0 900 2 17
VOLUME ELUTED MILLILITERS
Figure 1. Chromatographic behavior of 2,4-dinitrophenylhydrazones
C -0
0-0
A -A
n-Aliphatic aldehydes Methyl ketones Symmetrical ketones
tamping rod is indispensable in achieving uniforni packing of the column. Before introducing the sample, the column is prenashed nith 15 to 20 ml. of the niohile phase. The colunin iq operated a t 25' C. a t a flow rste of 0.8 to 1.2 nil. per minute. T o make full use of the resolving capacity of the system, sniall volumes (1 nil.) should be collected. Chromatographic Behavior. Figure 1 demonstrates t h e ability of the chioniatogiaphic system t o resolve derivatives of t h e homologs of three series of aliphatic carbonyl conipounds: the n-aliphatic aldehydes. methyl-n-alkyl ketones, a n d synimetrical dialkyl ketones. Tlie indicated elution volumes reflect the position of the center of t h e fraction. T h e elution volumes were measured from the time of introduction of t h e sample and are not corrected for t h e volunie of mobile phase on the column. T h e variation in nine experiments v a s i. 1 inl. for the derivative of tetradecyl aldehyde and i.5 nil. for that of butyraldehyde. Variations for other niembers of tlie three homologous series studied were proportional to the volunies n i t h nhich they were eluted and were comparable to those cited. The system permits the resolution of differences of one carbon atom for chain lengths up to 10 carbon atoms, and differences of two carbon atoms for chain lengths betiyeen 10 and 14 carbon atoms. There is no effective resolution for chain lengths longer than 14 carbon atoms. Spectrophotometric Measurements. T h e chioniatogiaphic fractions a l e evapoiated t o dryness, as nitroniethane nil1 interfere n i t h t h e spectiophot o m et ri c determinations. The residue is dissolved in benzene, and t h e desired alkaline conditions are oli-
Parent Compound Acetaldehyde n-Propionaldehyde n-Butyraldehyde n-Heptaldeh? de n-Octyl aldehyde ?a-Decylaldehyde n-Dodecj 1 aldehyde 0 850 n-Teti adecj 1 nldehyde 2 08 0 871 .Ildehyde av. 2 13 0 850 1 79 .icetone 1 79 Methyl etlij 1 ketone 0 ,S i 0 Netlir 1 n-but\ 1 ketone 1 86 IIethkl x-heskl ketone 1.91 0,910 llethyl n-nonj-1ketone 1.90 0.873 Diethyl ketone 1.81 0.865 Di-rz-propyl ketone 1.80 0.923 Di-ri-butyl ketone 1.85 0.913 Methyl isopropyl ketone 1.88 0.863 1.8T 0.888 Ketone av. a ;Ihscrbsnce per inole per liter per 1cm. light pat,h, as determined at 25' C. taiiied by adding 0.5 nil. of 4ccpotassium hydroxide in absolute ethanol. The absorbance of each sample a t 425 aiid 530 niF is followed as a function of time after the addition of the potas>ium 11) tlro-de. The zero-time absorliances a t t.:ic.li nave length are obtained by grapliic extrapolation of the plot absorlxtnee us. time. Measurements n ere niatle n ith a Beckman IIodel DU spectrophotometer, a t a controlled temperature of 25' C. Tlie aero-time molar extinction coefficients for the 2,4-dinitrophe1iylhydrazone' of a number of aliphatic carbonyl conipounds n ere cleterrnined (Table Ij. As observed by Jones, Holmes, aiid Seligiiian (6). the rate of fading of the alkaline color is different for the derivatives of aldehydes and ketones. Apparent first-order reaction coilstants 2 303 (calculated as F; = log 81, A nere
+
">
estimated to be 4 to 6 X lop3per second for the aldehyde derivatives and 2 to i x 10+ per second for the ketone derivatives. Exceptions were found in tlie short-chain members of the t n o series. Tlie derivatives of formaldehyde, acetaldehyde, and acetone exhibited fading rates n hich were atypically rapid. DIFFERENTIAL DETERMINATION OF ALDEHYDE AND KETONE
A differential determination of the aniount of aldehyde and ketone represented in each fraction may be made. because the extinction coefficients are different for the derivatives of aldehyde. and ketones. From tlie average values for tlie molar extinction coefficientq a t the t n o wave lengths (Table I). empirical expressions for the amount of aldehyde and ketone represented by a given sample may lie VOL. 30, NO. 8, AUGUST 1958
1351
Table II.
Analysis
of Mixtures of Dinitrophenylhydrazones Mole Fraction of Total Carbonyl Derivatives Theoretical 0.7 @mole* 1.4 pmoleb
Parent Compounda Tetradecyl aldehyde Decyl aldehyde Methyl hexyl ketone Heptaldehyde Diethvl ketone a
*
0 0 0 0 0
128 236 214 213 208
0 0 0 0 0
117 243 197 194 249
0 0 0 0 0
120 265 223 162 231
Listed in order of elution from column. Total amount of dinitrophenylhydrazones in sample analyzed.
and encouragement in this work, and t o Cesia Swartz, Marlene Brinley, and Julia Phifer for their technical assistance. LITERATURE CITED
(1) Braude, F. A., Jones, E. R. H., J . Chem. SOC. 1945 ,498. 12) Ellis. R.. Gaddis. A. 11.. Currie. G.’T.. k x - 4 ~CHEW . 30. 475 11958’): ( 3 ) Gordan; B. E., F o p a t , F’., Bu;nham, H. D., Jones, L. C., Zbid., 23, 1754 (1951).
derived from the zero-time absorbances a t the two x a v e lengths. Assuming the identity
A4 = concn.ald X
Esld.
+
concn.ket. X
Eket.
(1)
where E is the average extinction coefficient, equations for the two vave lengths niay be constructed. The solution of these as simultaneous equations leads to the empirical expressions for the number of micromolecules of aldehyde and ketone derivatives per 3-ml. volume, the volume used in examining the chromatographic fractions: Concn.aid = Concn.ket. =
A425
- 2.10 X As30 ( 2 )
2.45 X
,4630
- A425
1.02
From the volume a t which each chromatographic fraction is eluted from the column, the chain length of each is predicted. APPLICATION OF METHOD
A mixture of dinitrophenylhydrazones rvas assembled from standard solutions. Aliquots containing a total of 0.70 and 1.40 pmoles were subjected to chromatographic and spectrophotometric determinations. The chromatographic separation obtained with the smaller sample is depicted in Figure 2. The results (Table 11) compare the theoretical and observed mole fractions of each constituent as determined with the two different samples. Deviations from the theoretical values result from errors incurred in assembling the mixture and in eluting from the column and assaying spectrophotometrically the 90 chromatographic fractions. The methods described are being successfully applied t o a study of the formation of carbonyl compounds in animal fats as a result of exposure t o high doses of ionizing radiation (16). They have also proved useful in characterizing carbonyl compounds from bacterial systems ( l e ) . I n these cases, the 2,4-dinitrophenylhydrazones were formed quantitatively from appropriate extracts of the specinieii to be analyzed, evaporated to dryness under vacuum, and redissolved in the mobile phase of the chromatographic system.
1352
ANALYTICAL CHEMISTRY
VOLUME ELUTED - MILLILITERS
(3)
Figure 2. Chromatographic fractionation of 2,4-dinitrophenylhydrazones
of a mixture
Peaks (from left to right) represent derivatives tetradecyl aldehyde, decyl aldehyde, methyl hexyl ketone, heptaldehyde, and .diethyl ketone
Tolatile acids are used for the formation of the dinitrophenylhydrazones t o prevent their interference with the chromatographic system. Excess dinitrophenylhydrazine does not interfere, as its mobility is considerably lon-er than that of any of the derivatives studied. The method described is applicable to the study of saturated aliphatic carbonyl compounds only, and provides a means for the differential estimation of aldehydes and ketones. The analytical methods do not provide data concerning the configuration of the hydrocarbon chain, but do estimate the total number of carbon atoms with reasonable accuracy. Although the derivatives of olefinic carbonyl compounds have not been examined, the methods should be extended to these with similar success. As the dinitrophenylhydrazones of aromatic carbonyl compounds have a negligible mobility in the present chromatographic system, their study would require a different approach. ACKNOWLEDGMENT
The author wishes t o express his gratitude to Paul B. Pearson and Robert van Reen for their continued interest
Henick, -4. S., Benca, 11.F., Mitchell, J. H., Jr., J . Am. Oil Chemists’ SOC.31, 88 (1954); 33, 35 (1956). Johnson, G. D., J . Am. Chem. SOC. 75,2720 (1953).
Jones, L. A,, Holmes, J. C., Seligman, R. B., .&SAL. CHEM.28, 191 (1956).
Klein, F., Jong, K. de, Rec.trao. chim. 75, 1285 (1956).
Kramer, P. J. G., Duin, H. van, Zbid., 73, 63 (1954).
Lynn, W. S., Jr., Steele, L. A., Staple, E., A x . 4 ~ . CHEM. 28, 132 (1956).
Meigh, D. F., Suture 170, 579 (1952).
Mendelorritz, A , , Riley, J. P., Analyst 78, i o 4 (1953).
Rogers, P., Ph.D. dissertation, Johns Hookins Universitv. Baltimore,
Md., 1957.
ROSS,J. H.,
ANAL.
CHEX. 25, 1288
(1953).
Shriner, R. L., Fuson, R. C., Curtin, D. Y. , “Systematic Identification of Organic Compounds,” Wiley, S e w York, 1956.
Strain. H. H.. J . Am. Chem. SOC. 5 7 , 7 5 8 (1035). (16) Tappel, A. L., Monty, K. J., unpub-
lished data. RECEIVED for review February 28, 1957. Accepted March 25, 1958. Presented in part at the conference on the Radiation Preservation of Foods, Gatlinburg, Tenn., January 1957. Work supported in part by a contract from the Office of the Surgeon General, Departmmt of the Army.
