ANALYTICAL CHEMISTRY
1566 pairs of the adsorbent. Hence the high observed R j values for adsorptives developed with methanol and acetone, particularly, and with ethyl acetate and ether to a lesser extent, as noted in Table I. The dipole moment and dielectric constant of nitrobenzene are larger than that of any of these solvents, yet its developing power is considerably below that of the solvents containing carbon-oxygen linkages. The data from Table I11 are plotted in Figures 5 and 6. Petroleum ether-butyl ether mixtures show a very nearly linear relationship between R, values and per cent butyl ether. This solvent combination offers a wide range of developing power and for this remon should find general applicability. Ethyl alcohol mixed with petroleum ether or benzene could be used where a stronger developer is desired. Figures 1, 2, 3, and 4 are graphical presentations of the results of various runs from threecomponent solvent mixtures.
ACKNOWLEDGMENT
The authors wish to express their appreciation to the Office of Naval Research for financial assistance provided under Subcontract N7onr-356, T. 0. IV. LITERATURE CITED (1) (2) (3) (4) (5)
Elder, A. L., and Springer, R. d.,J . P h y s . Chem., 44, 943 (1940). Heymann, E., and Boye, E., 2. p h y s i k . Chem., 150A, 219 (1930). Hoyer, H., Kolloid-2.. 121, 121 (1951). Jacques, J., and Rlathieu. J. P.. Compt. rend., 221, 293 (1945). LeRosen, A. L.. Carltoii, J. K., and Moseley, P. B., ANAL.
CHEY.,25, 666 (1953). (6) Patrick, W.A,, and Jones. D. C . , . I . P h y 8 . Chem., 29, 1 (1925). (7) Roychoudhury, S.,Kolloid-Z., 57, 308 (1931). (8) Schroeder, W. A , , J . -4m. Chem. Soc.. 73, 1122 (1951). RECEIVED for review March 1, 19.54. .4ccepted July 22, 1954.
Immobile Phase in Chromatography on Silicic Acid-Gel ite Role of Water in the Mechanism of Development LOIS
M. KAY and KENNETH N. TRUEBLOOD
Department o f Chemistry, University o f California,
Lor Angeles 24, cdlif.
The unusual increase in chromatographic adsorptive strength of silicic acid with increased content of adsorbed water which occurs when certain highly polar developers are used has been investigated to elucidate the role of adsorbed water in chromatographic debelopment on polar adsorbents. Frontal rates of the active components of various developers have been measured and used in calculating approximate volumes and compositions of liquid layers immobilized on silicic acid under different conditions. Wide variations occur in the volumes of the immobile phases and in the ratio of the volumes of the immobile and %owing phases. These variations are attributed primarily to the exceptional hydrogen-bonding capacity o€ water. Observed development rates of adsorptives on untreated (hydrous) silicic acid with certain highl? polar ternary de,elopers are quantitatively explained in terms of a liquid-liquid partition of the adsorptive on the column. With either anhydrous adsorbent or less polar derelopers, adsorption is of primary importance, partition effects alone being insufficient to account for the obserred rates of movement.
A
COMMOK procedure for crontrol of the activity of polar inor-
ganic adsorbents is variation of the adsorbed (“free”) ( 1 4 ) water content-for example, by heating at 120’ to 200” C. or by prewashing with appropriate dehydrating solvents. Under most conditions, the adsorptive strengt,h derreases as the adsorbed \rater content is increased ( I , . 6,24, 32, 3.9, 48, 51, 52, 56); this behavior occurs with such diverse adsorbents as silicic acid or silica gel, alumina, calcium sulfate, calcium carbonate, magnesium carbonate, magnesium oxide, magnesium silicate, and bentonite, and for gaseous adsorptives as well as those dissolved in organic solvents. Recently Green and Kay ( 1 7 ) reported that dinitrophenylamino acids chromatographed from acet,ic acidacetone-ligroine developers were more strongly adsorbed on untreated silicic acid-Celite, which contained about 8 % free water, than on the corresponding anhydrous material. The present Rtudies have been made to ascertain the role of water on the adsorbent and so explain the unusual effect found by Green and Kay. Comparatively few systemat,ic experimental investigations of
the fundamental factors important in so-called adsorption chrcmatography have been made (5,29, 3O), although t8herehave been a number of qualitative discussions of the role of hydrogen bonds :tnd other forces in chromat,ographic adsorption (7, 8, 21, 43, 49). Ilecent,ly there has been a gron-ing recognition that the traditional lines of demarcation betireen adsorption, partition, and ion exchange chromatography are not sharp (8, 5O), and especially that, adsorption can play an important role in the mechanism of paper and starch “part,itioii” chromatogranis (3,6, 10, 20>26, 34), :ind of ion exchange chromatograms ( 2 , 37). The present, work provides evidence for overlap of partition and adsorption phenoincana in cshromatography on silicic arid-Celite under ronditions cwtoiiiarily considered those for adsorption chromatography. Theae studies vxre initiated with the observation that the relative xisorptive strengt,hs of untreated and prewashed silicic acid columns were primarily dependent upon the type of developer. Highly polar ternnrl- developers, such as the acetic acid-acetone-ligroine mixtures used by Green and Kay (1?), tended to give t,he unusual slo\r-er i~itcson hydrous adsorbent with :I variety of adsorpt,ives. Consrcluently, the behavior of the drvelopers themselvep on the c.olunin \vas investigated. Frontal rates of the polar components of hiria nd ternary developers e of various alcohols, which cont,:iined ligroin? and O I ~ Por organic acids, or acetone i r e r c measurcd on adsorbent, pretreated in different !rays. From t,hese rates, the compositions and relative volumes of t,he fixed- :md inohile-liquid phases in each chromatogram were calculated. 1’:rrtition roefficients were then measuwd for several solutes in ii number of these pairs of liquid phases, arid t8heobserved d lopment rates of the solutes were comparcd with those to be expec*t,pdon the basis of partition alone. Ohserved developmeiit ratrs of adsorptives on untreated (hydrous) silirica acid with cci,t:iin highly polar ternary developers :ire quantitat,ively explained in t,crmF of u liquid-liquid partition of t,he adsorpt,ive on the column. In contrast to the view expressed by Craig ( I O ) , the authors 1)rlieve i t to he distinctly advantageous t o consider that liquidliquid partition sometimes plays a rignificant role in chromatography. For example, partition is of demonstrated importance in chromatograms on hydrous silicic acid from highly polar solvents which can appreciably swell the irnmobile-liquid phase on the adsorbent while rem:iining rssentially immiscible with it.
V O L U M E 26, NO. 10, O C T O B E R 1 9 5 4
1567
were packed as described earlier (59). Volumes of chromatographic solvents are given in uriits of V,SO, where VISOis the volume of solvent needed to wet a 150-mm. column of adsorbent Limit of (33). I n most experiments with untreated, Detection, Test Mg./Mm of Literature heated, or methanol-treated a d s o r b e n t , t h e Reagent To Detect Positive Negativo Column Cited column was wetted with 0.5 VISOof ligroine and Orange or 28,% (SHa)zCe(NOa)s .4lcohols Yellow 0.0006 (IS) Vljo of developer before introduction of the in 2 N HC1 red-orange" 1% a q . NazFe(CN)s- Acetone sample; in most experiments with prewashed Pink or Light 0.70 0 10 0.68 0 . 6 2 0 . 6 3 >0.68 1.1 Q - Bi; thus, CY^ = CY^ - (Q 1 0 0.16 0.12 0.15 1.3 - Bl)/d. Then 012 is used in 2 0 0.24 0.23 0.26 1.0 4 0 0.34 0.35 0.38 0.39 1.0 turn to approximate B, (which 8 0 0.43 0.52 0.59 0.8 equals azcod) and consequently 4 0 0.34 0.35 0.38 0.39 1.0 the total volume immobilized, 4 4 0.33 0.50 0.54 0.7 0 . 4 1 0.50 0.53 0 8 4 8 0.32 0.62 0.64 0.55 0.5 0 . 6 1 0.66 0 . 6 9 >0.68 0.8 Q - BP. The calculation con4 20 0.38 0.74 0.82 0.5 0.76 0.82 0.9 verged in three approxima>0.70 1.2 0 4 0 . 4 7 0.40 0 . 4 2 4 4 0.33 0.50 0.54 0.7 0 . 4 1 0 . 5 0 0.53 0.8 tions. 8 4 0.35 0.66 0.66 0.46 0.5 0 . 3 5 0 . 5 5 0.60 0.60 0.6 Two assumptions were im2 8 0.24 0.54 0.60 0.4 0.60 0 . 6 1 0 . 6 4 1 0 plicit in the present calcula4 8 0 . 3 2 0.62 0.64 0.55 0.5 0 . 5 1 0.66 0.69 >0.68 0.8 tions-that any water initially Conipositiom in volume %. b Values of Ro could not be calculated for methanol-treated adsorbent because t h e distribution of this material in the adsorbent remained throughout t h e column was n o t determined. Reported frontal rates on this adsorbent are merely average values for distance moved. It was assumed t h a t P for a column of methanol-treated adsorbent is equal t o t h a t for a prethere, and that no ligroine washed column minus volunie occupied b y methanol; thus a * 0.041 ml./mm. was immobilized. The first a s s u m p t i o n is c e r t a i n l y justified; t h e s o l u b i l i t y of d for the higher alcohols. Coniparialcohol, snd then is rcv water in the developers used is so low that the normally used volume of even the most polar could dissolve less than 5 % of son of the individual values of ROrather than of the ratios shows even more pronounced increases with increasing molecular the water adsorbed on the untreated column. The assumption weight; however, this effect,occurs with both types of adsorbent that no ligroine is immobilized is not critical. I t is prohably a and is the familiar general reversal of Traube's series ( 7 ) . The fair approximation when the immobile phase contains water.; in any event, the calculated quantities of acetic acid, acetone, and data in Tables I1 and 111 also show that, for every compound btudied, the ratio decreases progressively as the concent'ration of water in the fixed phase will be substantially the same nhether or not a small quantity of ligroine is also present. the compound increases. The implications of thcsc observations are discussed below. The result's of the calculations are presented in Table V. The The frontal rates of the polar components of various wetonevolumes of the liquid phases immobilized on prewashed adsorbent ligroine, acetic acid-ligroine, and acetic acid--ncetone-ligroine vary comparatively little with change in developer; on the other developers on prewashed, heated, untreated, and methanolhand, the volumes of the immobile phases on hydrous untreat,ed treated adsorbent are presented in Table IV. These types adsorbent vary by more than a factor of 5 among the different of developers were studied in det'ail because the firpt behaves developers investigat'ed. The adsorbed water manifestly exerts a very pronounced effect, in immobilizing othcr polar liquids, normally and moves adsorptives more rapidly on unt'reated adsorbent than on prewarhed, while the latter ta-o generally move The thickness of t'he advorbed layers may be roughly estimated adsorptives more do\vIy on untreated adsorbent. Thus, these from t,he calculated volumes of the layers and the approximate systenia bridge the gap between the normal behaviol :tiid the surface area of the adsorbent. Reported specific surface arcas measured in different ways on many differently prepared samples unusual behavior observed by Green and Kay ( 1 7 ) . With the ternary developers 011 unprewashed adsorbent (Table of silicic acid and silica gel vary by less than a factor of 3 (4,11, IV), an increase in conceiitration of one polar component (acetic /to, 44). A representative value for a commerci:il sample of acid or acetone) often causes a decrease in the frontjid r:ite of the silicic ucid ( 4 4 ) is 300 to 400 m . 2 per gram. The thickness of other component. The ef-fect is especially marked when the the adsorbed liquid layer may thus be estimated to vary from about 4 A. (one or hvo molecules) for the free water nurm:illy acetic acid concentration of the developer is increased :It constant acetone concentration ; the frontal rate of acetone decreases by present, or for thc, liquid immobilized on prewashed adsorherlt, to about 25 A. for tlic most voluminous immobile phase found. as much as 25% when the acetic acid concentration is increased from 0 to 8%. This behavior is in marked cont'rast to bhat with ~-.~ . prewashcd or heated a.dsorbent, on which an inrrcwe in polnrity of the devcloper normally increases the rate of niovrmcnt of till Table V. Calculated Immobile and Mobile Liquid Phases substances prearnt,. .L\n intcrprctation of these okmrwtioiw is (For various acetic acid-acetone-limoine devdooers) prepent,ed below. Prewashed", hll. Untreated"8b. %I1 Immobile Liquid Phases. The composition and rolumc of the Developer, % Immobile hfobile, Immobile-__ p,:obilr, A.4 A AA A vl50 AA d VlX c liquid phasc which is iinmobilizcd on the adsorbent in any chro0 0 (7.00) G.G5 matographic systeni mny IIC calculated from observed frontal 2 0 0.43 6.57 0.49 G 20 ratw . The total volunic, &, of polar component, P,which en4 0 0.51 6.49 0.52 0 13 8 0 0.53 ters the column is equal to i'coIxhere v is the total volume of solu8.47 0.70 5 80 tion used and ce is the volume concentrat'ion of P. The volume 0 2 0.36 6.64 0.2ii ii30 0 4 0.41 R.SQ 0 32 (i.83 of I' immobilized is then equal to Q minus the volume; B, still 0 10 0.49 6.51 0.34 6.31 dissolved in the solution which occupies the interstitial space 2 8 0.12 0.42 6.46 0.42 0.43 5.80 4 8 0 . 2 0 0 . 3 2 6 . 4 8 0 . 6 0 0.63 5.42 behind the front. To estimate B , one must know t,hc volume 4 4 0.28 0.28 6.44 0.58 0.42 2.03 4 4 0.36 0.23 conraentr:ition of the solution in the region behind the front, vihich 6.41 1.10 0.56 4~. 9'1 1-olumes fur 9 X 150 mm. column, which contains 4.48 g. of 2 : 1 Qiliric arily be co ( 9 ) .and the effective value of 01 in this region. acid-Celiie, or approximately 2.G0 g . of dry silicic acid. Any immobilized ligroine is ignored. Hon.erc,r, CY itself depends upon the quantity of liquid adsorbed, b Immobile phase for ungrewashed column contains in each case 0.35 ml. because t,he free volume decreases as the quantity of adsorbed of water in addition. C VIM,free interstitial volume in colurnn, i i calculated bv subtracting volliquid increaseLC. Consequently, B can best be rstimated by ume of adsorbed phase from 7.00, which is approximate VM f o r prewashed successive approsinintions. Initially, it is assumed that B1 = column. .. crlcod,where B1 is the first approximation to B , 011 is CY for the .
Table IV.
Frontal Rates of Acetic Acid and Acetone
~
,
0
~~~
~
ANALYTICAL CHEMISTRY
1570 Table VI. Approximate Compositions of Bulk Immobile Phases (Before and after equilibration with ten successive portions of developer”) More Polar Immobile Phase, % Vol., Developer hll. AA A HzOb Lb 8.4.4-4.4-L Initially 10 55 28 17 0 Finally 24 57 26 ea. 6 ea. 11 4.4.4-8.1-L Initially 10 38 40 22 0 Finally 24 39 47 ca. 8 ca. 6 10.4-L In.tially 10 0 49 51 0 Finally 37 ca.61 ea. 2 7 0 a Each portion of developer was 60 ml. b Concentrations of water and ligroine in final solutions are only estiiuates; % H10 precise t o only ea. +15X (relative).
Alt,hough these estimates represent only orders of magnit,ude, they do provide insight into the nature of the adsorbed layers. A hydrogen-bonded layer, only one or two molecules thick, probably retains fen- properties of the bulk liquid. On the other hand, the outer portions of the voluminous phases on the untreated adsorbent are probably five to ten molecules distant from the silicic acid surface and more nearly resemble the normal liquids. These large immobile phases are formed by gradual transfer of acetic acid and acetone from a flowing ligroine solution to a surface already covered with a layer of water. Consequently, it can reasonahly be inferred that they are dist,inct,ly nonuniform in composition, with the concentrat,ion of water highest near the adsorbent surface and the concentration of organic substance highest in the outer portions of the layer. Experimental evidence consistent with t’hese deductions has been obtained. Bulk liquid phases which initially had the cbmposition of certain of the calculated immobile phases were shaken in separatory funnels with successive fresh portions of the corresponding developers and the resulting changes in composition were determined. Fresh portions of developer were used because in a column the conditions on the adsorbent necessarily approach those corresponding t,o equilibrium with unchanged developer. The important role of the adsorbent on pren-ashed columns was manifested by the comp1et.e miscibility of all of the mobile and fixed liquid phase pairs for prewaehed adsorbent. rllt,hough none of the phase pairs calculated t,o be present, on untreated adsorbent were coiiipletely miscible, some solvent was transferred b e h e e n them, as expected if the above-dpscribed imhomogeneity actually esists on the adsorbent, for t’lie bulkliquid esperiments are necessarily performed n i t h homogeneous samples. The cornpositmionof each final equilibrated phase was determined (Table 1‘1). The percentages of arctic acid and acetone in the bulk polar phases which are in approsimate equilibrium with 84.4-4-4-L and 4AA-81-L differ little from those of the calculated immobile phases; the concentration of water, however, is only about one third of that calculated, with ligroine presumably making up the difference. These results arc mnsistent with the hypothesis that the outermost part of the immobile phase, which is in contact with and presumably in equilibrium with the flowing developer, consists primarily of acetic. acid and acetone, x i t h some ligroine, and a little wat,er. The portion of the layer near the surface of the adsorbent presumably is much richer in wat,er and contains almost no ligroine. With 10A-1, as developer, the immot)ile phase is far smaller in volume, anti presumably even its out,er portion is comparatively rich in water and contains lit,tle ligroine. As the volume of immobilized liquid increa,ses, t,he partition phenomenon would be expected to become increasingly important in the retention of chromatographed substances. The data of Tables V and VI1 show that AA-A-L developers on unt,reated adsorbent give the largest immobile phases and consequently the smallest volume ratio of flowing phase to immobile phase. The fractional distribution of a solute between the mobile and fixed phases in ana- rhromatograrn depends both on the ratio of its con-
centration in the two phases [a partition coefficient in a partition chromatogram, or f(c)/c in Equation 1 for a n adsorption chromatogram] and on the relative extent of the two phases (the ratio of the volumes in a partition chromatogram, or M / a in Equation 1). Hence, an increase in the volume of the immobile liquid phase relative to that of the flowing phase will cause a decrease in development rate of the solute if this increase more than overbalances any opposing change in the distribution coefficient. The striking differences in the relative phase volumes for a given developer on the different adsorbents (Table VII) suggested that the unusual behavior observed by Green and Kay might be caused by just such an effect, with a partition mechanism of primary importance. Consequently, this possibility was investigated experimentally.
Table VII. Variation of Relative Volumes of Mobile and Immobile Phases with Different Developers and Adsorbent s a Developer, yo -
A4.0
Prewaslied Adsorbent ~Vol. Mob. Vol. lmni. R I , ~XI, ~
4
96 92
16.1
4
8
4
88
11.5 10.9
0.50 0.6Ij
4
0 4 8
96 92
12.i
0.35
4 4
a
4
~
0.40
0.50 0.55 0:50 0.66
0.50
11.5
88 12.5 O.G2 Calculated from data of Table V.
Untreated Adsorxcs Vol. Mob. Vol. Imm. RIAL RIA 9.4 0.47 4.2 O:i3 0 . 4 1 2.5 0.35 0.35
~
7.0 4.2
3.4
0.84
0 . 3 3 0:41 0 . 3 2 0.51
Development Rates and Partition Coefficients of Adsorptives. The term “adsorptive” is used here, without implication as to mechanism, to denote a substance being chromatographed at low concentration in an elution analysis ( 9 ) experiment. The ndsorptives chosen for the presmt studies, 2,4dinitrophenylalanine, 4-nitronnilineJ and isatin, were selected because of their color, diversity of structure, and appropriate adsorption affinity. Development. rates wit11 IOA-L, 4.1.\-8.I-L, and 8AA-4h-1, were measured on untreated :ind pi,rn-:Lslieti :tdsorbent (Table TTII). Since each adsorptive iiiovc~more sloivly than any developer component., it is alwa!.s in an environiiieiit i n which the free interstit,ial volume remains esseiit,ially constant. Consequently, in calculating development rates, allon.arice was made for thc known volume of the ininiobile phases coiwsponding to the different developers.
Table VIII.
Development Rates of Typical Adsorptive8 DSP-Alanine6
RO Developer L-ntr. Pren.. 10A-L 0 . I6 4AA-8A-I, 0.054 0122 8.4.4-4.4-L 0.051 0 . 1 8 D N P means S-2,4-dinitropiien)
4-Xitroaniline . _ _ -
RO
RO
Untr. Prew. 0 22 0.090 0.0lill 0.OSli
Isatin ~
0.18 0.12
Untr. 0.14
Prew 0.092 0.066 0.19 0.053 0 . 1 4
1
The importance of the partition mecthanism was evaluated with the aid of partition csoefficieut,s for the adsorptives of Table VI11 l)et,ween each of the three developers used and the corresponding polar liquid phase found to be in approsimate equilibrium with it on untreated adsorbent (T:tt)lr VI I. The relative phme volumes in these experiments approximated those calculated to be present in the chroniat,ographic clspcBriments. The adsorptive concentrations were never greater than 0.1%; analyses were made spectrophotometrically. Kach measured partition coefficient was used to calculate a hypotheticd development rate, R d e d . , which should approximate the observed rate if a partition niechanism is of primary iniport:ince. If the partition coefficient, Ibest be interpreted in t’erms of a combination of adsorption and partition mechanisms. As t,he quantity of adsorbed water increases, the partition mechanism becomes relatively more iniport,ant : indeed, silica gel wet with 70% it,s weight of water !vas the original immobile phase in partition chromatography (%), and sirnil:?rlywet silicic acid has b ~ e usedforpartition n chromatography of organic acids (22: S6, 4 2 ) , phenols ( 5 7 ) , and other pol:ir substances. Certain of t>he present data (Tables V and I S ) indicate that as the immobile phase swells appreciably, partition of adsorptives between liquid phases plays a major role. On the other hand, when the fixed liquid phase is very thin, a s is the layer of water on untreat,ed adsorbent’, partition as such does not play an important role. Thus, from the partition coefficient of 1-pentanol between ligroine and water, it may be calculated that, if partition in the usual sense were responsible for the inimobilization of I-pentanol on unt,reated adsorbent, it should be
developed a t about R = 0.97 to 0.99-that is, it should scarcely be retained by the column a t all. I n actuality, the front of 1pentanol in a 1% solution in ligroine moves a t R = 0.16. Although the water on untreated adsorbent acts unlike bulk water, it nonetheless plays a critical role, for only when it is present do the enormous increases in fixed-phase volumes occur with highly polar developers. This enhancement of “adsorption” of the more soluble alcohols and organic acids by the adsorbed water seems better attributed to the unique polydentate hydrogen-bonding ability of water than to partition in the usual sense. Water alone of the liquids here studied can participate in three or even four strong hydrogen bonds, and can donate hydrogens in two strong bonds. Furthermore, the increased bulk of the alkyl groups in the higher alcohols and organic acids may diminish the retention of these compounds by the adsorbent, relative to that of water, through both steric interaction and an orientation effect. The hypothesis that the effects of mater are attributable to its unique structure and hydrogen-bonding capacity is further substantiated by comparison of the frontal rates on adsorbent which is covered with a film of methanol, in place of the usual free water, with the corresponding rates on untreated and pi(,washed adsorbent (Table IV). If a partition mechanism alone were operative, the development rate of acetic acid or acetone on methanol-treated adsorbent would presumably be about the same as, or even less than, that on the usual untreated adsorbent (wet with an equimolar quantity of water). However, with each binary developer tested, the frontal rates of acetir acid arid acetone are greater on methanol-treated adsorbent than on untreated adsorbent. Methanol presumably interfereq A ith direct adsorption, as does water, b> occupying adsorptioii sit()., however, while water can sometimes more than compensate f 01’ this reduction in the number of rites by acting as an adsorbmt itself through hydrogen bonds, methanol is much less effective in such a role because of its significantly lesser potentialities for participation in hydrogen bonds. If the methanol molecule is bound to the surface by a hydrogen bond in which it donates the hydrogen atom, it can only accept further hydrogen bonds anti presumably could thus retain specifically acetic acid but riot a(+ tone. On the other hand, if the bond which holds the mPthanc.1 molecule involves a hydrogen atom from the adsorbent, the mrltlianol could then form bonds with either acetic acid or acetoriv The pertinent frontal rates in Table IV are in qualitative agreement with these views. James and Martin ( 2 2 ) have noted it similar effect in their observations of retention volumes of various aniines in gas-liquid partition chromatograms, LITERATURE CITED (1) Bastick, J., Bull. soc. chim., 1953,437. (2) Baunian, W. C., Anderson, R. E., and Wheaton, R. M., Ann. Rev.P h y s . Chern., 3 , 120 (1952). (3) Bentley, H. R., and Whitehead, J. K., Hiochem. J . , 4 6 , 341 (1950). (4) Boreskov, G. K., Borisova, M.S., Dshigit, 0. AI., Dsis’ko, V. A , ,
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P h y s . Chem. (U.S.S.R.), 22, 603 (1948). (5) Brockmann, H., Discussions Faraday Soc.. F o . 7 , (1949), 58. (6) Burma, D. P., - ~ N A L .CHEX.,25, 549 (1953).
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(8) Chem. ETLQ..Vews, 30, 4244 (1962). (9) Claesson, S., A r k i t Kemi Mineral. Geol., 23.1, F o . 1 (1946). (10) Craig. L. C., . ~ N A L . CHEY.,22, 1346 (1950). (11) Dieta, V., Ann. ‘V. Y.Acad. Sci., 49, 315 (1948). (12) Elsden, S.,Biochem. Soc. Sumposia, 3 , 74 (1950). (13) Feigl, F., “Spot Tests,” 3rd ed., Kew York. Elsevier Publishing Co., 1946. (14) Fells, H. A,, and Firth, J. B., J . P h y s . Chem., 31, 1230 (1927). (15) Fink, D. F., Lewis, R. TV., a n d Weiss. F. T., A N . ~ LCHEY., . 22, 850 (1950). (16) Freymann, AI., and Freymann, R . , Conipt. rend., 232, 401 (1951). . 24, 726 (1952). (17) Green, F. C., and Kay, L. M., h . 4 ~ CHEM., (18) Haak, F. A, and van Xes, K., private communication, 1950.
1572
A N A L Y T I C A L CHEMISTRY
(19) Hahn, F. L., and Klockmann, R., 2. p h y s i k . Chem., 146-4, 373 (1930). (20) Hanes, C. S., and Isherwood, E. .L.,Snfrire. 164, 1107 (1949). (21) Hoyer, H., Kolloid-Z., 116, 121 (1950). (22) James, A. T.. and Martin. .I.J. P.. Anulyd, 77, 915 (1952). (23) Kiselev, A. V.,Colloid J . (C.S.S.R.),2 , 17 (1936). (24) Kiselev, 9.V., T’orms, I. A.. Iiiseleva, V. V,, Kornoukova, V. N., and dhtokish, E. A , , J . Phya. Chenr. (C.S.S.R.),19, 8 3 ( 1945). (25) Kistler, 9. S..Fischer, E. A , , and Freeman, I. I t , , J . Ani. Chem. SOC.,65, 1909 (1943). CHEM., . 2 4 , 643 (26) Kowkabany, G. S . , and Carsidy, H. G.. A I x a ~ (1952). (27) LeRosen, A., J . Ani. Ckeni. Soc., 64, 1905 (1942). (28) I h i d . , 6 9 , 8 7 (1947). (29) LeHosen. A. L., Carlton, J. K.. and llosoley, P. B., .4~.41.. CHEY.,2 5 , 6 6 6 (1953). (30) LeRosen, A. L., Monaghaii I-’. H., Rivet, C. -I.,and Smith, E. D., Ibid., 23, 730 (1951). (31) LeRosen, A. L., llonaghan, P. H.. Rivet. C. .\., Smith, E. D., and Suter, H. -1.. I b i d . , 22, 809 (1950). (32) XIcGavack, J., Jr., and Patrick, W. A. J . Bm. Chent. Soc., 4 2 , 9 4 6 (1920). (33) llalmberg, E. W.,Trueblood, K. F.,and Waugh, T. D., .LNAL. CHEM..25,901 (1953). (34) Martin, A. J. P., A m . Reu. Riochem., 19, 517 (1950). (36) Martin, A. J. P.. and Sgnge, R. L. AI., Biochem. J . (London), 35, 1358 (1941). (36) Marvel, C. S.,and Rands, R. D., J r . , J . Am. Cheni. Soc.. 72, 2642 (1950).
(37) (38) (39) (40) (41) (42)
(43) (44) (45) (46) (47) (48) (49) (50) (51) (52) (53) (54) (55) (56) (57)
Xoore, S.,and Stein, W. H., J . Bid. Chem., 192, 663 (1951). Mukherjee, J . N., and Chatterjee, B., :Vatwe, 155, 85 (1945). Patrick, W. h.,and Long, J. S., J . Phys. Chem., 29, 336 (1925). Plank, C. J., J . Colloid Sci.,2 , 413 (1947). Plank, C. J., and Drake, L. C., Ibid.,2 , 399 (1947). Rainsay, L. L., and Patterson, W.I., J . Assoc. Ofic.Agr. Chemisis, 28, 644 (1945). Schroeder, W.A., J . Ani. Chem. Soc., 73, 1122 (1951). Shapiro, I.. and Kolthoff, I. 31.. Ibid., 72, 776 (1950). Shapiro, I., and Weiss, H. G.. .I. Phys. Chwn., 57, 219 (1953). Shull, C. G., Elkin, P. B.. and Hoess, L. C., J . Am. Chem. Soc., 70, 1410 (1948). Sporer, .I.,unpublished data. Stewart, A , Discussions F u m d a y SOC.,KO.7, (1949), 13.5. Strain, H. H.. J . Am. Chem. Soc., 70, 588 (1948). Tiselius, A , Discussions Furudu.y Soc., No. 7, (1949), 7. Trappe, W., Biochem. Z . , 306, 316 (1940). Trueblood, Ia i s of certain mixtures of chloral hydrate and dichloro- or chloroacetaldehyde. Mixtures of dichloro- and chlornacetaldehy-de cannot be analyzed by direct polarographic methods, for the two compounds give coincident wales in the usual buffer systems. The precision for determiuation of the inditidual chloroacetaldehyde alone or in the presence of others as specified is about 3Yo.
MA“
methods have been reported for the quantitative determination of chloral hydrate. Those most frequently used are based upon its reaction with sodium hydroxide to form chloroform and sodium formate, followed by dctermination of the sinount of: (A) alkali consumed (E-6, 8-i1, 15, 14, 16-18, 25, 24, 27, 30, 32, 5.5, J6> 38, 3 0 ) ; (B) sodium formate formed (16, 38); or (C) chloroform produced (30, 58). Method A has several drawbacks-e.g., esperiniental condit.ions such as time, temperature, and stirring must be carefully standardized because the chloroform produced usually reacts to some extent with the excess alkali; the volatility of the chloroform may affect the extent of such reaction; and ot.her substances which independently react with alkali must be absent. Method B is based upon the reducing power of formic acid; therefore, other
reducing agents cannot be present,. Method C is generally inferior to the other procedures, owing to the volat.ility of chloroform and to its possible reaction wit,h excess alkali. Chloral hydrat,e can be oxidized to trichloroacetic :wit1 with iodine (18, 28, 31, 38), bromine (SI), permanganate ( 3 2 ) , or persulfate ( 2 8 ) ; the excess st,andard oxidant is then determined by back-titrat,ion. Other reducing agents must be absent. An obvious procedure is based on the determination of t,otal chlorine as chloride ion. The conversion of ehloral hydrate to chloride ion may be accomplished by coiiiplete hydrolysis with alcoholic alkali (6, 20, S 7 , 3 9 ) or by reduction with zinc in sulfuric acid solution ( 2 2 ) . The chloride is then determined by conventional methods ( 2 2 , 26, 35). This procedure has the distinct disadvant,age that no other halogen compounds ran be present. Other methods for t,he determinat,ion of chloral hydrat,e use color reactions (1, 12, 15, g l ) , specific gravity (S4), and surface tension (8). Several of the methods described could also be used successfully for the determination of either dichloro- or chloroacetaldehyde if it vere the only chloroacetaldehyde present. Dichlor- and chloroacet,aldehyde can also be assayed by an aldehyde determination. These methods described cannot be uscd directly for the analysis oi mixtures of the chloroacetaldehydes. In view of the successful polarographic determination of acetaldehyde and other aldeha-des, an investigation of the feasibility of the polarographic technique n-as desirable for the analysis of mixtures of the chloroacetaldehydes as well as for the rapid determination of tthe individual aldehydes. EXPERLWENTAL
Stock aldehyde solutions (about 10 d ) were repared from U.S.P. chloral hydrate (Merck & Co., Ino., 9 9 . 5 8 pure), a re-
