New Techniques for Adding Organic Acids to Silicic Acid Columns VLADlMlR ZBINOVSKY and R. H. BURRIS Department o f Biochemistry, University o f Wisconsin, Madison, W i s .
In chromatographic separation of organic acids on columns of silicic acid, it has been customary to introduce the sample in an organic solvent to avoid a n y disruption of the equilibrium between phases. A method is described for successfully adding the organic acids or their sodium salts in the aqueous phase, a procedure which facilitates the quantitative introduction of the acids into the column and eliminates any difficulties from dehydration of the silicic acid. The acids or their salts also may be introduced as a uniform, narrow band by adding them in blotter disks. The techniques should be applicable to all water-soluble organic and inorganic compounds separable on columns of silicic acid.
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N CURRENT techniques for chromatographic separation of free organic acids on silicic acid columns, the sample is usually introduced in the nonaqueous phase. Bulen et al. ( 1 ) employed, as a modification, the mixing of an aqueous acidified solution of organic acids with silicic acid and the addition of this to the top of the column. It has been considered that the introduction of a ssmple in aqueous solution results in a water-logged silicic acid column, disrupts completely the equilibrium between the aqueous and nonaqueous phases, and thus prevents any separation of organic acids. This belief, which is probably based on previous unsuccessful attempts to introduce the sample in this manner, is incorrect, and in this investigation methods were developed for overcoming these difficulties and obtaining good separation.
vent even introduction of the water, so it is important that I top and the sides of the column should be absolutely free of t solvent. To introduce an aqueous sample, place a blotter disk (with a diameter 0.2 to 0.3 mm. greater than that of the column) on the top of the column while a 3-cm. depth of solvent is still present. (A light gray blotting paper without embossed pattern, 140pound stock, obtained from the Wrenn Paper Co , Middleton, Ohio, has proved satisfactory; other highly porous blotters will doubtless serve The disks should be cut accurately with clean edges, and this is best done with a frequently sharpened cork borer turned by a drill press.) Press the blotter disk on the top of the column with a glass tube whose outside diameter is nearly the same as the inside diameter of the column; inside the tube should be a cork, flush with the bottom, which has a 1-mm. central hole. All air bubbles are removed when the blotter disk is pressed into position. Close off the bottom of the column with a pinch clamp, and wait approximately 1 minute. Pour out or pipet off the excess solvent from the top of the column, and remove any residual solvent with a bacteriological inoculating needle carrying a plug of absorbent cotton. The top of the blotter disk should still be moist. The handle of the inoculating needle should be split and should have a narrow strip of qualitative filter paper twisted around the handle and then bent back on itself; wipe drops of solvent from the walls of the column with this paper. To add the azmple, introduce the pipet into the column, without touching the walls, to a distance about 2 mm. above the middle of the blotter disk. Open and close the pinch clamp quickly to draw any excess butyl alcohol-chloroform away from the upper surface of the blotter disk; a t the same time be careful not to admit any air into the blotter disk. (If the blotter disk is not drained sufficiently, drops of butyl alcohol-chloroform may appear on top of the disk.) Now immediately introduce all of the sample, covering the blotter disk evenly with aqueous phase. While the pinch clamp is still closed, the undersaturated column starts to absorb the sample. Rapidly open and close the pinch clamp to allow control!ed addition of the sample into the column and to avoid overrunning
REAGENTS
Technical grade n-butyl alcohol (W. H. Barber Co., Chicago) and chloroform (Merck) were used without redistillation. Mallinckrodt's analytical reagent 100-mesh silicic acid (batch ZVV-I) was used; the line particles were removed from the silicic acid by repeated suspension in distilled water and decantation. The silicic acid then was dried overnight at 110' C. EXPERIMENTAL PROCEDURE
Preparation of Column. To avoid a water-logged column an undersaturated column is prepared, although a water level approaching saturation is used to improve the resolution of the organic acids. A silicic acid holding a maximum of 0.8 ml. of water per gram can be used successfully when 0.6 ml. of water per gram is added initially and not over 0.15 ml. of aqueous sample is added subsequently (usually much smaller volumes of sample can be employed). Good separation is achieved when complete hydration of the silicic acid does not extend below the top half of the column. Four grams of silicic acid mixed with 2.4 ml. of 0.5.4' sulfuric acid (referred to as the aqueous phase) was made into a slurry with the nonaqueous solvent and was added to a 10-mm. (inside diameter) glass column provided with a sealed-in porous disk. The length of the silicic acid bed. was 11.5 to 11.6 cm. Two pounds' pressure wm used for packing the column and 1.5 pounds for developing the column. The temperature of operation ranged from 25' to 27" C., and each separation required approximately 1 hour. Two milliliter fractions of developing solvent were collected. The developing solvent contained 35 volumes on n-butyl alcohol to 65 volumes of chloroform. Four volumes of the solvent mixture was equilibrated with 1 volume of 0.5N sulfuric acid. The nonaqueous solvent used for introducing acids in comparative tests consisted of a mixture of equal volumes of chloroform and 2,2-dimethyl-l-propanol (terl-amyl alcohol) ( 2 ) . Introduction of Free Organic Acids in Aqueous Phase into Column. Commonly, the sample of organic acids has been introduced in a solvent miscible with the nonaqueous solvent used in slurrying and developing the silicic acid column (2),and no special difficulties have been encountered. In this procedure it is necessary to ~ A o wexactly the described technical details. Two immiscible solvents are used-Le., butyl alcohol-chloroform and water, and in no case should these immiscible solvents be present together on the top of the column. Small drops of butyl alcohol-chloroform remaining on the top of the column will pre-
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B
B
IC'
0.5 MI. of aqueous sample was added to a IO-mm. diameter 4-gram column of silicic acid
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V O L U M E 26, NO. 1, J A N U A R Y 1 9 5 4 with accompanying introduction of air. Wash the sample in with developing solvent according to the standard procedure described by Isherwood ( 2 ) , and develop the column as he describes. With a little practice, an even introduction of an aqueous sample can be achieved. The aqueous sample will saturate the upper portion of the column and produce a white horizontal band; the developing solvent will carry excess viater into the lower, unsaturated part of the column. The hydration of the column is rapid in the first stages of development, but progresses slovJy durin the rest of the run (Figure 1). If too much aqueous phase is aided, the excess will pass completely through the column, and the sulfuric acid in this aqueous fraction will be titrated together with the last organic acids eluted from the column. When the hydration of the column is controlled by proper introduction of the sample, a separation of acids as shown in Figure 2 is achieved.
209 diameter column). Follow this by washing with three 0.1-ml. portions of solvent. Even and quick removal of the indicator from the disk will show that the size of the disk was suitable and the preparation of the sample was correctly performed. Good separation is obtained because the sample is dispersed inside the disk very evenly; such even dispersal is difficult to achieve by introducing the sample as a powder or as crystals. When no convenient solvent can be found in which to introduce the sample into the column, this method allows direct addition of the sample in a concentrated and yet evenly dispersed form. An example of a separation of free organic acids added in a blotter disk is shown in Figure 3. Introduction of Sodium Salts of Volatile or Nonvolatile Acids in Filter Paper Disks. Suspend qualitative filter paper in a saturated solution of sodium bicarbonate with a Waring Blendor and compress the heavy suspension in a hydraulic press to a thickness of 2 mm. Dry the pad in the oven a t approximately 40" to 50" C.! and cut disks of a suitable diameter with a corkborer BS described. Introduce sodium salts of organic acids into these disks as described. Place 0.1 ml. of 911- sulfuric acid above the column and immediately add the disk; while 1 mm. of acid still remains above the column add another 0.1 ml. of 9 N sulfuric acid. Escaping carbon dioxide expands the disk sufficiently to allow quick and even access of sulfuric acid into the disk. No detectable carbonic acid passes through the column DISCUSSION OF RESULTS
When organic solvents have been used for sample introduction, it has been accepted that they should be added in minimum volume. When the sample is added in a polar nonaqueous solvent, an increased amount of this solvent increases the Rf values of organic acids and simultaneously broadens the base of the acid peaks; both these effects harm good resolution. FRACTION NUMBER
Figure 2. Separation of Organic Acids (25 hlg. of Total Acids) Introduced in Free Form in 0.5 M1. of Aqueous Phase on 10-Mm. Diameter &Gram Column of Silicic Acid Fractions 1 to 9, 1.0 ml.; 10 to 63, 2.0 ml.; 64-84,2.25 ml. Acid peaks. 1, propionic; 2, succinic; 3, oxalic; 4. glycolic; 5 , malic; 6, citric; 7, isocitric
Introduction of Sodium Salts of Organic Acids in Aqueous Phase. The treatment of the column before adding the sample is the same as described for the separation of free acids. Sodium salts of nonvolatile acids can be acidified before placing the solution on the column, and the method described in the preceding .&ion then can be followed. The sodium salts of volatile acids should be acidified directly on the top of the column in the following way: .ldd 1 drop of phenol red indicator to the solution of sodium salts. Proceed in the previously described manner, but before introducing the sample add 0 2 ml. of 9.V sulfuric acid (saturated with developing solvent) to the top of the column. Follow this immediately with the aqueous solution of sodium salts and then with 0.1 ml. of 9 N sulfuric acid. When the color change from red to yellow to orange-red occurs, it will indicate that the organic acids are liberated from their salts (pH below 1.0). Open the pinch clamp and allow the m i y ture to soak quickly into the column. When the aqueous phase is all in the column, then develop as described by Isherwood (2). Introduction of Free Nonvolatile Organic Acids in Blotter Disks. The sample is placed on the column without solvent. Cut a disk of even thickness having the same diameter as the inside of the column from a 1-mm. thick white porous blotter. Thread a 22gage Xichrome wire, sharpened a t one end and flattened like a pin head a t the other, through the center of the disk. .4dd 0.5 ml. of the solution of free organic acids (25 mg. of acids) containing 1 drop of phenol red indicator to the disk by alternatelr allowing a few drops to soak into each side of the disk, and drying it under an infrared lamp. Remove the wire, and place the dry disk on the column upon which a 1-mm. layer of solvent still remains. Push the disk down with the glass tubing and cork device described for addition of the blotter disk until it is 1 to 2 mm. above the solvent; then push it down evenly with a pointed inoculating needle. Release the pinch clamp a t the base of the column and wash the organic acids from the disk into the column by slow dropwise addition of the developing solvent (totnl volume shout 0.2 ml. for a 1-cm
0.6 Z
0 4
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0.2
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Figure 3. Separation of Organic Acids (25 Mg. of Total Acids) Introduced in Free Form on Disk of Blotting Paper T w o milliliter fractions collected from IOmm. diameter 4-gram column of silicic acid Acidpeaks. 1, succinic; 2, oxalic; 3, malic; 4. citric
Several quantitative experiments were performed to determine the effect of the amount of the aqueous sample on the separation of organic acids. The resolving power of columns to which aqueous samples were added was superior to that of siniihr columns receiving samples in tert-amyl alcohol-chloroforii~, and no increase in R, values was observed with increasing amounts of water. With increasing water the bases of the pe:rks were broadened, and the limit of aqueous phase which can be added was indicated by this broadening of the peaks of the slowest moving acids. The best results n ere obtnined when the column
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ANALYTICAL CHEMISTRY
was prepared with the maximum amount of water (consistent with the previously discussed criteria) and the aqueous sample was introduced in a minimum volume. With the silicic acid employed, 15 to 30 mg. of organic acids can be separated very effectively on a 10-mm. diameter column containing 4 grams of silicic acid. Emergence of the acids (as in Figure 2 ) is reproducible to a single tube if care is employed in adhering to standard operating conditions. The recovery from the column of added known acids is 90 to 100%. Advantages of Introducing Sample in Water Phase. The new technique for the introduction of samples in the water phase constitutes a simplification of the generally used methods. It eliminates the tedious and often nonquantitative transfer of the eample from the aqueous to the nonaqueous phase (b). As the sample is introduced in the water phase, acidification of the sodium salts directly on the column becomes possible, and the loss of volatile acids is decreased appreciably. The method permits the eeparation of compounds, such as glyoxylic acid which tend to form hydrates and are not readily extractable by organic solvents. Direct addition of the sample to the column in the aqueous phase should be useful in the routine and exploratory separation of organic acids from such aqueous biological materials as fermentation media, vegetable and fruit juices, plant saps, and deproteinized extracts from animal tissues. This chromatographic technique has been used for the separation of organic
acids, but it may be applied generally to water-soluble organic and inorganic compounds. The new procedure gives a t least as good separation and recovery of organic acids as is obtained when the acids are introduced in an organic solvent. Dehydration of the column, which is often troublesome with samples added in an organic solvent, is eliminated in this method. The only apparent disadvantage in the use of the method described is that skill must be developed in the addition of the samples. Advantages of Introducing Sample on a Paper Disk. Introduction of the sodium salts of organic acids on paper disks followed by acidification has essentially the same advantages as those cited for the introduction of sodium salts in the aqueous phase. Free organic acids introduced as solids in paper diskR are separated better than when they are introduced in the aqueous or organic phase. Compounds soluble only in a large amount of organic solvent can be concentrated in a paper disk, and this decrease in sample volume improves their separation. LITERATURE CITED (1) Bulen, W.
.4.,Varncr, J. E., and Burrell, R. C., Ax.4~.CHEJI.,24,
187 (1952). ( 2 ) Isherwood F. A , Biochem. J . (London),40,688 (1946). RECEIVED June 22, 1953. Accepted October 19, 1953. Published with t h e approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part b y the Research Committee of the Graduate School from funds supplied b y the Wisconsin Alumni Research Foundation.
Determination of Oxygen in Sodium 1. C. WHITE, W. 1. ROSS, and ROBERT ROWAN, J R . ~ Analytical Chemistry Division, O a k Ridge National Laboratory, O a k Ridge, Tenn.
.4 method for the determination of sodium monoxide in sodium depends upon the reaction between sodium and excess n-butyl bromide (I-bromobutane) in hexane solution. Sodium monoxide does not react with the reagent and can be determined, after the addition of water, by titration. Both oxygen and other impurities can be determined on the same sample, and the method requires only the simplest equipment and is easily adaptable to the analysis of large numbers of samples. The standard deviation is 0.003 to 0.005%. The method has been applied to sodium that has been sampled in glass and in metallic containers. It is believed that the method will be applicable in the range 0 to 0.02%, if large samples are used.
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HE oxygen content of sodium is of interest because of the
extreme reactivity of sodium monoxide a t elevated temperatures. The oxide reacts with all the common metals (including the platinum group metals), graphite, and ceramic materials, and because of this extreme reactivity, other impurities besides oxygen are often determined in sodium. Ideally, determinations of both oxygen and impurities should be made on the same sample, thus providing a sounder interpretation of data as well as economy of sample. This investigation was undertaken for the s p e cific purpose of determining oxygen and other impurities on the sample of sodium. A method which has been previously developed for this determination is that of Pepkowitz and Judd (4). Essentially, this method depends upon the extraction of sodium with mercury in an inert atmosphere and the separation of the residual sodium monoxide, which is insoluble in mercury. With this method, however, it is not practicable to determine oxygen and impurities on the same sample. A modification of the Pepkowitz and Judd method has been reported by Williams and Miller (6). A new method has been developed which is based upon the fact t h a t n-butyl bromide (1-bromobutane) reacts with sodium to 1
Present address, Standard Oil Development Co., Linden, N. J.
form the neutral salt, sodium bromide, but does not react with sodium monoxide. Following this reaction, dissolution of the residue in water yields titratable sodium hydroxide (from the monoxide) in a solution of sodium bromide. The development of this method has, accordingly, entailed a study of the reaction of n-butyl bromide with sodium and with sodium monoxide. REACTION OF n-BUTYL BROMIDE WITH SODIUM
In the Wurtz synthesis of hydrocarbons ( I ) , an excess of sodium is present. A series of tests was conducted to determine whether the reaction proceeds to completion when an excess of the alkyl halide is present. Small amounts of sodium were added to large excesses of propyl, butyl, and amyl chlorides, bromides, and iodides (chloro-, bromo- and iodo-propanes, butanes, and pentanes). 2Na RX + R S a SaX (1) R X -+ S a S R-R RNa (2) In all cases sodium was converted to the halide with explosive violence. The rate of reaction varied little with the halide used. Diluents in the form of hydrocarbons, which are inert to sodium, were added to the halides in order to decrease the reaction rate. Hydrocarbons with boiling points higher than that of
+ +
+ +
