Paper Chromatography of Volatile Fatty Acids Himansu Mukerjee, University College of Science and Technology, Calcutta, India
of the fermenD tationinvestigations products of certain algae URING
(Chlorella vulgaris and Chlorella pyrenoidosa), it was necessary to separate and identify very small amounts of volatile fatty acids. It is convenient to use methanolammonia and methanol-acetone-ammonia as solvents for the paper chromatography of formate and acetate, when the methods of Kennedy and Barker (6), Hiscox and Berridge ( 5 ) , and Brown and Hall (8) are followed. Other methods were less suitable because they required large amounts of material (3) or because the volatile free acids cannot be applied as such t o the paper (7'). Conversion into hydroxamate (4) or ethyl esters (1) before chromatography is inconvenient. Formate has been satisfactorily separated from acetate by using methanol-ammonia solvent. Bromophenol blue, as used by Kennedy and Barker (6),is a very useful indicator. EXPERIMENTAL
Khatman No. 1 filter paper was used
throughout. To eliminate the formation of ghost spots, it was washed first with 1% oxalic acid, as recommended by Kennedy and Barker (6), then thoroughly with distilled water, and finally dried a t room temperature before use. For unidimensional descending chromatography a t 25-27' C., the following solvents were used: A, 100 ml. of absolute methanol with 1 ml. of 30% ammonium hydroxide; B, 70 ml. of absolute methanol with 30 ml. of acetone and 1 ml. of 30% concentrated ammonium hydroxide solution; and D, butanol saturated with 1.5N ammonium hydroxide. To the paper was applied 0.01 mi. of an aqueous solution containing 2 to 5pmoles of each acid as its ammonium salt. Solvents C and D used by Kennedy and Reid (8), respectively, take 6 to 8 hours for development. A and B require only 4 hours. After development of chromatograms, the papers were dried a t room temperature to reduce decomposition of the ammonium salts to the minimum. This takes from one to several hours. The spots were then located 11sspraying the
28" C. ButanolAmmonia New analysis R
Table I. I?,Values for Ammonium Salts of Fatty Acids at
Ammonium Salts Formic Acetic Propionic Butyric
MethanolAmmonia 0 56 0 65 0 70 0 73
MethanolAcetoneAmmonia 0 0 0 0
45 52 59 65
EthanolAmmonia New analysk K 0 0 0 0
28 30 44 51
0 0 0 0
31 33 44 54
0.09 0.10 0.18 0.24
0.10 0 11 0.19 0.29
papers with a solution of bromopheno blue (6). The indicator solution was prepared by dissolving 40 mg. of bromophenol blue in 100 ml. of 10% aqueous alcohol mixed with 200 mg. of citric acid. Intense blue spots appear against a pale yellow background.
R; values in different solvents are shown in Table I ; some values by Kennedy (K) and by Reid (R) are included to show to what extent they are reproducible under the conditions used in the present work. The values given are the average of four experiments Methanol-ammonia satisfactorily separates formate and acetate, but methanol-acetone-ammonia separates t8hesalts of all four volatile acids. LITERATURE CITED
(1) Boldingh, J., Discussions Faraday SOC. 7,162 (1949). (2) Brown, F., Hall, L. P., Nature 166, 66 (1950). (3) Elsden, S. R., Biochem. J. 40, 252 (1946). (4) Fink, K., Fink, R. M., Proc. SOC. Exptl.Biol. filed. 70, 654 (1949). (5) Hiscox, E. R., Berridge, N. J., Nature 166,522 (1950). (6) Kennedy, E. P., Barker, H. .4,, ANAL.CHEM.23, 1033 (1951). ( 7 ) Lugg, J. W.H., Overell, B. T., dustraliun J. Sci. Research A l , 98 (1948). (8) Reid: W.W., Nature 166, 569 (1950).
WORE done during a fellowship tenure at the Research Institutes, university of Chicago. FelE Fund, Chicago, Ill.
Simple Apparatus for Backwashing Chromatographic Columns with Inert Solvents Ralph L. Oannley and Bernard L. Weigand, Morley Chemical Laboratory, Western Reserve University, Cleveland 6, Ohio
with water has long B been recommended in the preparation of ion exchange columns to remove ACKWABHING
fines and provide maximum homogeneity (1-3), but has not been applied with organic solvents to familiar adsorbents such as alumina and silica. To backwash with an organic solvent. a pump must give sufficient liquid velocity to fluidize the adsorbent. Thesolvents, because of their cost, should be recycled after removal of adsorbent fines, and contamination should be avoided both to permit re-use and to prevent fouling the column. The apparatus diagrammed incorporates these necessary features and is easy t o construct and simple to operate. The diagram is not to scale, for the only critical size is that 1284
ANALYTICAL CHEMISTRY
of reservoir C, which must hold enough solvent to flood the entire apparatus while retaining a liquid level immersing the tip of M . Glass wool is used for filtering because it offers little hindrance to liquid flow and may he discarded after use. Apparatus. The solvent is drawn from reservoir C by pump A (Eastern Industries, Model E-1). This pump has performed very satisfactorily, although it requires priming and a needle valve to regulate the flow of liquid. Sufficient heat is generated t o vaporize more volatile liquids and cause vapor lock; therefore the solvents are passed through condenser B for cooling. Operation of the pump produces finely divided black particles which are removed by a glass
wool plug in an enlargement of the tubing a t G. After backwashing column D ,the solvent returns to reservoir C. R'hen it enters the base of C it passes through a main glass wool filter held in place by glass beads. To ensure complete removal of adsorbent fines, the exit tube, X,from C also contains a glass wool plug. Tygon tubing was used extensively in earlier models of this apparatus but extraction of plasticizer from the tubing contaminated the solvent. Ball joints a t G, N , and J reduced rigidity of the apparatus to permit almost complete use of glass tubing. As the pump causes vibration, however, four Tygon joints. F , were retained to give freedom of movement and simplify alignment. Only inch of Tygon is exposed to solvent and plasticizer extracted from
this has been so small as to escape detection. Use of stopcock lubricant should be avoided as much as possible, for it is a source of contamination. It may be omitted in the section used exclusively for backwashing, for loss of solvent is of no significance during this operation. Only the rim of the T joint, R,needs a trace of lubricant. Lubrication of the stopcocks is avoided by uce of Teflon plugs. Procedure. Pump A and attached apparatus from joint J to joint G are permanently assembled and clamped. In assembling the column, some glass wool is placed between two perforated porcelain plates, \\liich are then inserted in the bell of K . These disks I conimrrcially available porcelain glazed filter disks, 22-min. diameter) pass through an outer 24/40 T joint but seat themselves against the shoulder of the attached tubing. The inner joint a t the base of the column holds the plates firmly in place, as the internal diameter of the inner joint is too small to permit the disks to enter. A piece of coarse filter paper may also be inserted betneen the disks. Thc plates must be fixed in position, for displacement forces are exerted in opposite directions during backna3liing and elution. A sinteredglass plate (Corning column, A T 99930) could be used a t this point, but perforated plates are easier to clean and reduce the volume below the joint to a minimum. After the column has been approximately half-filled with adsorbent, it is aligned carefully, so that it may be connected to the fixed portion of the apparatus. Glass wool is now inserted in the open connecting tube a t (7. The column is
L
7
d:
I -
;L
(d, G . E., Schubert, F., Adamson, 0. IT.,7 . ;lm. Chem. SOC.69, 2818 (1947). ( 2 ) Spedding, F. H., Voigt, 0. F., Gladron, E Il.*Sleight, N. R., Ibid., 69, 2 i T i (1947). (3) Spedding, F. H., Voigt, 0. F., Gladrow, E. >I,, Sleight, E. R., Powell,
J. E., Wright, J. M Butler, J. O., Figard, P., Zhzd , 69, $786 (1947).
End Point location in Controlled-Potential Coulometric Analysis Louis Meites, Department of Chemistry, Polytechnic Institute of Brooklyn, Brooklyn 1 ,
THE; electrolysis current which is inte-
grated during a controlled-potential coulometric analysis ordinarily decays exponentially with time (2) a t such a rate that-depending on cell design. solution volume, stirring rate, and diffusion coefficient of the substance being determined-between 20 and 50 minutes are needed for the current integral to approach within 0.1% of its final value. Though Lingane (1) has correctly pointed out that the attention of the operator is not required during this period, it m u l d often be advantageous to be able to locate the end point more rapidly than this, even if a small amount of operator time were required. The technique suggested below is both simpler and more rapid than that previously described (6). It shares the assumption described by Equation 1 with the technique earlier described by 3Iach’evin and Baker
(S), but yields results much more accurate than were obtained by these authors. This is due in part to the different manner in which the mathematical relationships are applied, and also to the fact that errors arising from momentary fluctuations in the electrolysis current are minimized by the present treatment. When the electrolysis current obeys Lingane’s equation (a) i = ioe+ (1)
where io is the initial current, i is the current after t seconds of electrolysis, and k is a combination of several constant parameters, it is easily shown that Qt =
io - (1 -
L
~
4
6
)
(2)
and (S) Qm =
io -
k
(3)
N. Y. From these equations it is readily shown that (4)
Here Q m is the total quantity of electricity that will have been consumed at infinite time, and Q1,Q2,and Q3 are the quantities that have been consumed after tl, tz, and tS seconds, respectively. If ( t 2 - tl) = (t, - t J , Equation 4 becomes
The calculations involved in the use of Equation 5 are simplified by arbitrarily taking Q1 = 0; the value of Q m then found is the quantity of electricity that must be added to the quantity actually accumulated a t time t l . The data shown in Table I were obVOL. 31, NO. 7, JULY 1959
* 1285
