indicated by the greater polarity of Ethomeen T-25 compared t o Ethofat 60/25. ACKNOWLEDGMENT
The author gratefully acknowledges the encouragement and valuable suggestions given by R. J. VanderTT’al of Armour and Co. LITERATURE CITED
(1) Ambrose, D., Keulemans, 8.I. ill., Purnell, J. H., ANAL.CHERI.30, 1582 (1958).
J. T., “Proceedings 2nd International Congress of Surface Activitv.” KID. 426-38, Butterworths, London,“i95?: (3) Greenwald, H. L., Brown, G. L., Fineman, M. S.,ASAL. CHEW 28,
(2) Davies,
atography,” pp. 161-80, Reinhold, Neiv York, 1959. (10) Knight, H. S., ASAL.CHEM.30, 9-14
(4)Greenwald, H. L., Kice, E. B., Kenly, pvl , Kelly, J., Ibid., 33, 465-8 (1961). i~, 5 i Griffin. tV. C.. J . SOC. Cosnietic Chemists 1,311-26(1919).
(1958). (11) Little, R. C., Ph.D. Thesis, Rensselaer Polvtechnic Inst.. Mic. 60-2689 i1960). (12) ?;;akagawa, T., Xakagawa, I., J . Chem. Soc. Japan, Ind. Chem. Sect 59, 1154-6 (1956). (13) Rohrechneider, L., 2. anal. Chem. 170,256 (1959). (14) Tenney, H. ill, A s . 4 ~ .CI-IEII. 30, 2-8 (1958). (15) \roodford, F. P., van Gent, C. >I., J . Lzpid Research 1, 188-90 (1960).
(8) James, -2.T., Xartin, A. J., Baochem. J . 50,6i9(1952). (9) Keulemans, A. I. h l . , “Gas Clirom-
RECEIVED for review October 3 , 1961. Accepted February 2, 1962.
1693-7 (1956).
(6) Zbid., 5 , 219-56 (1954). (7) Harva, O., Kivalo, P., Keltakallio, .4.,Suonaen Kemzstzlehtz, B 32, 52-54 (1959).
Volatility Effects in the Paper Chromatography of the Lower Fatty Acids ANDREW A. MOLLOY‘ and GEORGE N. KOWKABANY Department o f Chemistry, The Catholic University o f America, Washington,
b The effect o f temperature
and humidity acting individually and together in promoting the loss o f C14t a g g e d sodium salts of the lower fatty acids spotted on chromatographic p a p e r has been studied. The losses have been ascribed to volatilization of free acid formed b y humidityinduced hydrolysis o f the salts of the acids on the paper.
T
HE TOLATILITP of the lower fatty acids precludes their chromatography as such on paper. For this reason, among others, workers have converted them to nonvolatile derivatives or salts prior to application to the paper. A few of the more common derivatives and salts of the fatty acids prepared and used in paper chromatography are the hydrouamate (4) and the hydrazide (10) derivatives, and the sodium ( I ) , ammonium ( 5 ) > diethylamine ( 7 ) , and morpholine (9) salts of the acids. A strong atmosphere of ammonia, ethylamine’, or morpholine in the development chamber is an additional aid to cutting don n on losses of the acids from the paper during the development of the chromatogram. Also, after chromatography the spots may be fixed with a spray of potassium monohydrogen phosphate ( 6 ) . However, a n aqueous spray reagent may distort the spots. A study of the volatility effects accompanying the use of C14-tagged sodium salts of the lower fatty acids in paper chromatography of these com-
Present address, Poughkeepsie, N. Y .
Marist
College,
D. C.
pounds was prompted by the observation of substantial losses in count following a chromatographic run. Subsequent observations showed that a paper spotted with a solution of the tagged salt of one of the acids revealed a large decrease in radioactivity on being left out on a desk for a fen- hours. Variations in humidity and temperature were suspected to be responsible for these losses. It appeared extremely unlikely that the sodium salts of the fatty acids were themselves volatilizing off the paper. A detailed study of the efiect of humidity and of temperature on the amount of radioactive material lost and on the rate a t which these losses occur has been made. EXPERIMENTAL
Reagents and Apparatus. The CI4labeled sodium salts of butanoic, pentanoic, hexanoic, and octanoic acids used in this n-ork \\(:re obtained from Suclear-Chicago Corp. and had specific activities of 4.7: 2.65, 2.87, and 2.08 me. per mJd, respectively. The counting assembly used was of t’he type usually employed in work of this kind. Procedure. To study the effects of humidity, two identical set’s of desiccators containing saturated solutions of various salts chosen to give specific humidity conditions n’ere prepared (a), placed in a temperature controlled cabinet set a t 30” =!= 1’ C., and allowed to come to equilibrium. Other desiccators of the same size x-ere used to study the effect of temperature alone and in conjunction with high humidity. A 2-sq. cm. piece of Whatman No. 1 chromatographic paper was spotted in
the center n-ith a 1-~1.sample containing from 3 to 10 pg. of a CI4-labeled sodium salt of a lower fatty acid and placed in an oven for 15 minutes a t 105’ C. to dry. It \vas then removed. counted, and inserted in one of the desiccators for a period of 4, 8, 12, 16, or 24 hours. After the desired time had elapsed, it was removed and immediately recounted. The results for each salt studied were graphically recorded. Represcntative traces are presented in Figures 1 and 2. ,4 time lapse of 3 days was allowed following each humidity exposure to permit desiccators to re-establish equilibrium conditions. Care was taken to open the desiccator lids just sufficiently to insert and remove the papers. The humidity conditions in the desiccators were undoubtedly disturbed by sliding the covers open. Hon-ever, though the humidity in each of the desiccators may not have been exactly as specified, i t was a t least of the same magnitude. RESULTS A N D DISCUSSION
Studying the rate of loss of material as a function of humidity variation as recorded in Figures 1 and 2 reveals that large increases in rate of loss of material accompany each 10% increase in humidity. Also, the per cent of niaterial lost a t each humidity increase decreases with increase in acid chain length. The greatest and most rapid losses of material occurred a t humidities above 5O7& while below 30yC,the observed losses of material in the case of the four compounds studied were less than loyo. Planchets carrying water and others containing saturated solutions of calVOL. 34, NO. 4, APRIL 1962
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for example, was cut from about 90% to less than 10% by this addition. Papers which revealed a 90% loss of material after exposure to high humidity for 24 hours were sprayed with a color reagent (bromocresol purple in 95% ethanol-37yo formalin). The cation (Xa) spot appeared with an intensity comparable to that given by squares sprayed immediately after application of material. The color depends on p H and not on presence of the acid. All the above observations indicate that a hydrolysis process has been operative. It appears that the paper picked up moisture from the air and that in the presence of the latter, hydrolysis of the sodium salts to free acid occurred. The free acid then volatilized off the paper. The observed behavior of the compounds toward temperature and humidity variations as well as the effectiveness of the basic atmosphere in cutting losses supports the above conclusions. With what has been said, i t is not surprising to read that some losses of material were encountered by Fairbairn and Harpur (3) when one of the steps involved in the preparation of the fatty acids for chromatographic work entailed evaporating a sodium salt solution to dryness overnight in a hot air oven a t lloo c. Workers in paper chromatography
t (hrs.) Figure 1 . Sodium octanoate-1 -C14: rate of loss as a function of approximate relative humidity
cium hydroxide were placed in the high humidity (84%) desiccators for 24 hours a t 30' C. with the paper squares spotted with samples of the acid salts. After evaporation of the solutions to dryness, subsequent counts revealed significant increases in activity. It was noted that the planchets containing calcium hydroxide showed u p to a fourfold greater pickup of radioactivity than did the planchets containing the water. Papers spotted with the sodium salts of the acids and then placed in a vacuum desiccator over Drierite and pumped on for 14 hours at room temperature showed negligible losses in radioactivity. I n the study of the rate of loss of material as a function of temperature, i t was noted that when papers spotted x i t h either sodium or morpholine salts of the acids were placed in a desiccator over silica gel and placed in a n oven for 24 hours a t 105' C., the losses were less than 10% in all cases. Papers similarly spotted with sodium salts of the four acids and placed in a desiccator with a high humidity (about goyo) at 100' C. showed losses of better than 90% in 2 hours in all cases. Addition of ammonia to the atmosphere in the high humidity chambers emphatically inhibited the loss of material. The loss of sodium butanoate, 492
ANALYTICAL CHEMISTRY
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t (hrs3 Figure 2. Sodium butanoate-1 -C% rate of loss as a function of approximate relative humidity
who encounter certain losses of material might a t least consider the possibility of volatility effects even though a t first sight the compounds with which they are working exhibit no appreciable volatility. A note in a Xuclear-Chicago Bulletin ( 8 ) injects a similar word of caution stating that certain compounds considered nonvolatile will evaporate a t a significant rate when present as spots on paper. Methyl palmitate is cited as an example of a substance which exhibits such unusual volatility. While the compounds themselves may be relatively nonvolatile, when spread on the surface of paper and subjected to moisture and temperature variations, they may very well undergo an altera-
tion in chemical nature. This change may be similar to that resulting from the hydrolysis process discussed above and may yield products of a more volatile nature or products whose presence might explain other unexpected or undesirable effects.
(6) Kennedy, E. P., Barker, H. A.,
ANAL.CHEY.23, 1033 (1951). ( 7 ) Long, A. G., Quale, J. R., Stedman, R.J., J . Chem. SOC.1951, 2197. (8) Nuclear-Chicago Corp., Technical Bull. No. 4. 1959.
LITERATURE CITED
(1) Brown, F., Hall, L. P., Kature 166, 66 (1950). (2) Cam, D. S., Harris, B. L., Ind. Eng. Chem. 41, 2014 (1949).
(3) Fairbairn, D., Harpur, R. P., h’ature
166, 789 (1950). (4) Fink, K., Fink, R. M., Proc. SOC. Ezptl. Bzol. Med. 70, 654 (1949). (5) Hiscox, E. R., Berridge, N. J., Nature 166, 522 (1950).
RECEIVED for review September 13, 1961. Accepted February 15, 1962. Material for this paper was taken from the dissertation presented by Brother Andrew Molloy, F.M.S., in partial fulfillment of the requirements for the degree of Doctor of Philosophy, The Catholic University of America. Work was supported in part bv a research erant from the Division of Research Gracts, National Institutes of Health, U. S. Public Health Service.
Integral Chronoamperometry with a Simple Manual Apparatus ALVIN L. BElLBY and ALAN L. BUDD‘ Department of Chemistry, Pomona College, Claremonf, Calif.
b A simple manual apparatus has been developed for making integral chronoamperometric measurements at stationary solid microelectrodes. The results obtained are in good agreement with those obtained with complicated electronic apparatus used for integral chronoamperometry. The degree of precision is comparable to that obtained with a manual polarograph using a dropping mercury electrode.
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of the methods for making electrochemical measurements a t stationary solid microelectrodes in unstirred solutions with the potential of the R-orking electrode held constant is that of measuring the charge transferred during a short period of electrolysis. The charge is obtained by integrating the current during a period of the electrolysis. If the time of electrolysis is kept less than 10 seconds to irvoid convection, and a large amount of indifferent electrolyte is present to avoid electrical migration, the mass transfer process a t the electrode may be attributed solely to diffusion. This method should be designated as “chronoarnperometry” because it is a potentiostatic method with the experimental measurements concerned with current-time variations ( 5 ) . However, KE
Present address, Instruments, Inc., 2500 Fullerton Road, Fullerton, Calif.
since the actual experimental measurements involve the integration of currenttime functions to obtain the charge, the qualification “integral” may be added to chronoamperometry (4). Although this method has been used before, it is believed that this paper is the first instance in which the term “integral chronoamperometry” has been used. Following the recommendations of Delahay, Charlot, and Laitinen (6), it is suggested that the term integral chronoamperometry be used for designating this electroanalytical method in all subsequent work. The technique of integral chronoamperometry was first used by Booman, Morgan, and Crittenden ( 2 ) to measure diffusion charges a t cylindrical electrodes and by Morgan, Harrar, and Crittenden (9) to determine half-wave potentials. The necessary theoretical mathematical relationships for these studies with integral chronoamperometry were derived by these authors. The experimental results obtained were in good agreement with the theory. The best results TTere obtained when the charge was measured shortly after the beginning of the electrolysis, eliminating the integration of the large initial current. Beilby and Crittenden ( I ) used integral chronoamperometry in studies of nonadditive diffusion charges. With slight variations the technique was usedby Smith (IO)for studying the surface oxidation of platinum electrodes. Laitinen and Enke ( 7 ) also used a similar technique for their studies on
the formation and dissolution of oxide films on platinum. I n all these previous studies using integral chronoamperometry, electronic current integrators and electronic timing devices were employed for making the charge measurements. No simple mechanical type of instrument similar to the manual polarograph has been available for integral chronoamperometric studies a t stationary solid microelectrodes. One obvious method would be to use a ballistic galvanometer, IT hose purpose is to measure charge, coupled with a mechanical timing circuit in place of an electronically controlled timer. A more convenient arrangement would be t o adapt the galvanometer circuit of a conventional manual polarograph so that the galvanometer could be used in the sense of a ballistic galvanometer to determine the charge transferred a t an electrode for a short period of time. An existing manual polarograph could then be used for both conventional polarography using dropping mercury electrodes and for integral chronoamperometry using solid electrodes. EXPERIMENTAL
Apparatus. The modified circuit of a manual polarograph is shown in Figure 1. A Sargent Model I11 manual Polarograph n-as used. HOKever, any conventional manual polarographic setup should be satisfactory for the method. SFvitches SIand S 2 are microswitches operated by the mechanical timing mechanism. VOL. 34, NO. 4, APRIL 1962
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