ra
Donald H. Wilkins, Program Chairman, General Electric Research Laboratory, Schenectudy, N . Y . Karl H. Robds,
Local Chairman, The McGean Chemical 60.) Cleveland, Ohio Plate Theory of Ion Exchange Chromatography. William Rieman 111, Rutgers State University, New Brunswick, N . J . New Ion Exchange Materials of Interest to the Analytical Chemist. Robert Kunin, Rohm & Haas Co., Philadelphia, Pa. Separations by Liquid Ion Exchange. e. F. Coleman, Oak Ridge National Laboratory, Oak Ridge, Fenn. Ion Retardation and Related Gel Chromatographic Separations Using Cross-Linked Polyelectrolytes and Simple Water Elution. M. J. Hatch, The DW Chemical Co., Midland, Mich. Gel-Liquid Extraction-Extraction and Separation of Some Metal Salts Using Tri-n-butyl Phosphate Gels. Hamish Small, The Dow Chemical Co., Midland, Mich. Fundamental Factors in Paper Electrophoresis. J. T. Edward, McGill University, Montreal, Que, Centrifugally Accelerated Chromatography and Electro-
Fourteenth Annual Summer Symposium was held a t Case Institute of Technology, Cleveland, Ohio, June 21 to 23, 1961. The 14 talks which were given without time restrictions stimulated active discussions of various problems in solution chromatography. William Rieman 111 opened the program with an interesting discussion on applications of plate theory. With a sufficiently small ratio of sample to resin, the chromatographic behavior of each constituent of the sample is independent of the presence of the other constituents. Therefore, the most efficient method of developing a procedure for the separation of a given (qualitatively known) mixture is to study the elution behavior of each constituent individually. When two constituents give overlapping graphs, the plate theory is very helpful in indicating what change should be made in the composition of the eluent. For example, oxalate and bromide ions gave overlapping graphs (the oxalate preceding) when eluted through Dowex HE
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ANALYTLCAL CHEMISTRY
chromatography. H. J. McDonald, Loyola University, Chicago, Ill. Zone Migration in Paper Chromatography. 0. H. Stewarf, Gonzaga University, Spokane, Wash. Study of Complex Ion Kinetics by Ion Exchange. D. W. Margerum, Purdue University, Lafayette, Ind. Kinetics of Ion Exchange with a Chelating Resin. William Rieman 111, Rutgers State University, New Brunswick, N . J . Gradient Elution of Aromatic Adsorbates from Composite Columns. C. W. Gould, General Electric Research Laboratory, Schenectady, N . Y. Ion Exchange Separations within the Transition Group Elements. J. 1. Hague, National Bureau of Standards, Washington, D. C. Ion Exchange Separation of Metal Ions Using Halide Complexes. J. S. Fritz, Iowa State University, Ames, Iowa Utilization of Zone Melting Techniques to the Resolution of Racemic Mixtures of Asymmetric Compounds and Their Diastereoisomers. Stanley Kirschner, Wayne State University, Detroit, Mich.
1 with 0.05M sodium nitrate. A change to 0.1M sodium nitrate gave a quantitative separation. Had the ion of greater charge followed the ion of lower charge, a decrease in the concentration of the eluent would have been called for. Similarly, the plate theory indicates when a change in the pH of the eluent and when the addition of a complexing agent is advisable. When these simple measures do not improve the separation, the column should be lengthened; and the plate theory enables the analyst to calculate the optimum length. Robert Kunin discuMed some of the developments in new ion exchange materials which are of interest to the analytical chemist. The extensive use of ion exchange resins has led to the development and commercial. availability of ion exchange materials specifically tailored to meet the needs of analysts. halytical grade resins, chromatographic grade resins, and ion exchange chromatographic papers are among the examples ob such materials. Specific examples of specialized resins
were given. The macroreticular resins will be of particular interest for work in nonaqueous solvents. These reshs have an unusually high surface area, reach equilibrium much faster, and function particularly well in nonpolar solvents. C. F. Coleman considered the liquidliqui 3 extraction systems that operate, a t lam formally, by interchange of ions a t the interface between an aqueous solution and an immiscible solvent, the extracting reagent showing negligible distribution to the aqueous phase. While extractions by liquid anion exchange with alkylamines or quaternary ammoniums and by liquid cation exchange with (especially) organophosphorus acids are being developed intensively for chemical processing, they appear as yet to be used considerably less than other types of extractions in analytical chemistry. It is suggested that this results from chance and habit rather than from any lack of potential usefulness, and that liquid ion exchange warrants increased attention from analytical chem-
ists. Some of the advantages that have proved important in chemical processing ould apply also to analytical use. When extractions reported from process and physicochemical applications are included along with ansblytical reports, a considerable range of extractions is already available to suggest and guide further uses. A representative compilation of such extractions was summarized by element and valence us. aqueous medium, and the controlling variables in some of the typical liquid ion exchange systems were reviewed. M. J. Hatch discussed ion retardation, a high capacity, inexpensive gel chromatographic method, employing modified ion exchange resins-snake-cage polyelectrolytes -to remove electrolytes from aqueous solutions of nonelectrolytes, and to fractionate electrolyte mixtures. Applicable snake-cage polyelectrolytes can be made, for example, by polymerizing anionic monomers such as acrylic acid inside conventional quaternary nitrogen anion exchange resins. The amphoteric snake-cage resins absorb electrolytes weakly and can be regenerated merely by water washing. This process was developed as an industrial processing tool and analytical applications have not been explored to any great extent. Examples of effective separations, using a polyacrylate quaternary resin: NaCl from glycerol or sucrose; small amounts of CaC12 from 220/, NaCl brine; small amounts of ZnCh from NHbCl; NaCl from NaOH of chlorine cell effluent concentrations up to 50% solids; NaC1 from NhSO,; fractionation of sea water into alkali metal chloride-sulfate, and alkaline earth chloride (only) fractions. The strength of the absorption of electrolytes, as seen in the above examples, generally parallels the ion exchange selectivities of the ion exchange resin components of the snakecage resin. Snake-cage resins containing carboxylate and quaternary groups protonate strongly in acidic media, and water regeneration is very inefficient, Resins containing sulfonate and quaternary groups were studied in separations of strong acids and their salts. Such separations conveniently may be designated as “acid retardation” separations. Effective separations were obtained on mixtures such as NH4N08 and “0, Fe(NO& and “Os, FeCh and HCI, NiSOl and &SO4. The resin used was made by polymerizing ar-vinylbenzyltrimethylammonium chloride inside conventional, 2y0 crosslinked, sulfonated, polystyrene cation exchange resin. In these separations, salt-acid mixtures (0.2 bed volume, 1 to 5 N ) were applied to the column, and followed by a water wash (0.6 bed volume) Surprisingly, studies of such
separations on simple quaternary anion exchange resins themselves, in the common ion form, showed that these anion exchange resins also physically absorbed the strong acids. Almost-as-good physical separations of salts and acids could be effected on the anion exchange resins as on the amphoteric sulfonatequaternary snake-cage resins. This behavior is consistent with the observation that “Donnan invasion” of quaternary anion exchange resins is unusually high, especially in the presence of high concentrations of neutral salts. Extremely high flow rates could be employed in “acid retardation’’ separations because of the high rate of diffusion of the acids in and out of the resin. The separations were not due to differential diffusion rates of the acids and salts, however, as shown by equally effective separations a t very slow flow rates. Hamish Small discussed gel-liquid extraction, the name given a new process wherein solvent swollen polymers are used in fixed bed operations to effect extraction and separation of aqueous borne solutes. In essence, what gel-liquid extraction involves is substitution of the organic liquid phase in a conventional liquid-liquid extraction process by an organic gel phasehence the name gel-liquid extraction. The gel is then manipulated in columns by familiar techniques and displays the high efficiency (low HETP) characteristic of column chromatography. The gels have been conveniently prepared by swelling styrene-divinylbenzene (SDVB) copolymers in the organic extractant of choice or in a mixture of the extractant and another solvent if the former proves t o have poor swelling power for these copolymers. Before swelling, the polymer beads are given a light sulfonation treatment which introduces a very thin shell of sulfonic,acid groups on the outside of the bead. This confers good water wettability on the final solvent swollen beads-a most important acquirement, since beads not treated in this way pack very poorly in columns, Copolymers (SDVB) swollen with mixtures of tri-n-butyl phosphate (a poor swelling agent) and perchloroethylene (a good swelIing agent) have been the most extensively studied in this work. They have been used to extract uranyl nitrate from nitrate systems and eluted with water to yield product streams many more times more Concentrated than the feed stream. The selectivity of TBP for one nitrate over another has been exploited to effect such separations as ferric nitrate from uranyl nitrate, uranyl nitrate from thorium nitrate, thorium nitrate from yttrium nitrate, and yttrium nitrate from ferric nitrate. Gels containing di(2-ethylhexy1)phos-
phorio acid have been employed t o extract and separate rare earths from aqueous solutions using acid eluents. This is an especially interesting system, since it holds promise of really exploiting the separating potential of these unique extractants. There are some problems of maas transfer in these “ion exchanging” gel systems which, however, require study and already have revealed intriguing wpects in their diffusional characteristics. Analytical chemists should find it profitable to extend the scope of gelliquid extraction to many other conventional liquid-liquid extraction systems. J. T. Edward discussed fundamental factors in paper electrophoresis. A satisfactory theory of paper electrophoresis should enable one to calculate the absolute velocity of a zone of ionic migrant in an electrical field. An attempt was made to provide such a theory by considering the possibilities of predicting the velocity of an ion in a uniform electrical field -Le., the ionia mobility-the velocity of the ion in a Bone surrounded by background electrolyte of different concentration and conductivity, as happens for electrophoresis; and the effect on the velocity of the ionic zone of the presence of the filter paper support. The mobilities of most inorganic ions may be calculated from conductivity data already in the literature. The mobilities of organic ions, when they are not excessively irregular in shape, may be calculated from their sizes and shapes using a modified Stokes equation. The mobilities of irregular organic ions may be calculated from their diffusion coefficients, when these are available. The potential gradient to which an ionic zone is subjected on moist paper may not be identical with the over-all measured potential gradient, depending on the relative concentrations and conductivities of the ionic zone and of the background electrolyte. An attempt was made to analyze the effect of these parameters on the zone shape and mobility, using t w o models representing limiting conditions of the zones. Zone shapes and mobilities are further affected by electro-osmosis, adsorption, etc., due to the presence of the paper. Adsorption effects are dependent on the solvent system used, and frequently can help t o increase the separation of ionic migrants. The velocity of all nonadsorbed migrants appears to be reduced to approximately the same extent by the obstructive effect of the fibrous network of the paper, and the extent of the reduction is related by a semi-empirical equation to the wetness of the paper. H. J. McDonald, Department of YO!,, 33, NO. 13, DECEMBER 1961
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1845
Biochemistry, Loyola University, Chicago,, reviewed the development Qf the techniques of centrifugally accelerated paper chromatography and planar electrophoresis. An early version of the apparatus was described in ANALYTICALCHEMISTRY31, 826 (1959). The apparatus for chromatography consists of rotating horizontally a circular sheet of filter paper while the developing solvent is delivered as a h e jet stream near the center of the disk, at right angles to its surface. 8 e p arations are comparable with those obtained by classical descending on ascending techniques, but development time is now reduced roughly in the ratio of 1 hour to 5 minutes or less. Fanning out of migrant zones as the chromatogram develops is prevented by cutting slits in the circular sheet to produce narrow spokes, which extend from near the center almost to the perimeter of the sheet. In centrifugally accelerated electrochromatography, or planar electrophoresis, a rectangular sheet of paper fitted with electrodes along its sides and connected to a direct current electric source, is rotated horizontally while deyeloping liquid is added as described above. The material undergoing electrochromatographic fractionation movea across the paper sheet under the stimulus of both electric and centrifugal forces. During the past year the apparatus has undergone extensive changes including a more functional size, improved electrode assembly, continuous sample application through a wick arrangement, and an improved collecting system. Dr. McDonald illustrated the great potential of planar electrophoresis by describing the fractionation of proteins and lipoproteins from human serum, which can be handled a t the rate of 1.5 ml. per hour. In one experiment, the fraction containing glutamic oxaloacetic transaminase was determined to have been increased 10-fold by B single pass of serum through the apparatus. George H. Stewart discussed zone migration in paper chromatography, the analysis of the partition coefficient, and the R, value in paper chromatograms by the method of Consden ff
=
2 (k-
1)
where AI/& is the ratio of the areas of cross section of the mobile and the stationary phase may be corrected for the nonconstant value of the concentration profile of the developing solvent over the length of the path. The R, shows the initial unsettled period during which it approaches a limiting value. Use of the diffusion analogy model for solvent flow allows calculation of this effect in close agreement with experimental observation. The model is 1846
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ANALYTICAL CHEMISTRY
extended to cases of radial flow including fioa control through the use of wick systems. The relationship between R values for rectangular and radial flow is demonstrated. The method of assigning a value t o A, requires close analysis and standardization before values of the partition coefficient may be interpreted in terms of solution theory. The ability to predict partition coefficients by static measurement to facilitate the choice of suitable solvent systems requires two pieces of information: cross-sectional area of the active stationary phase and the nature of the solvent in that phase The general assumption that the stationary phase is a thermodynamically homogeneous phase is a limited concept which leads to misinterpretation of apparent partition coefficients and an inability to predict R, values. D. W. RiIargerum presented a study of complex ion kinetics by ion exchange. Cation exchange resins can be used to establish a very low, but constant metal ion concentration in solution to follow the rate of reaction between a metal ion with ligands in solution. Concentrations of NiC2 ions between 10+ and 10-8 were used to study its reaction with EDTA a3 a ligand. It was noted that the concentration gradients across the resin-solution interface are small and that H + plays an important role in allowing Ni to diffuse inside the resin even though the resin was primarily in the Na+ form. Many complex formation rates are rapid and difficult to measure by other means. Resin-controlled coordination rates are applicable within the limitations of metal ion diffusion rates and the evidence of direct interaction between the ligand and the metal in the resin, Measurement of the rates of dissociation of metal complexes is also possible within the limitation imposed by direct resin catalysis. Both the formation and dissociation rates of complexes can play an important role in metal ion separations with ion exchange resins. William Rieman I11 continued the consideration of kinetics with a discussion on chelating resins. When ion exchange occurs between an ordinary ion exchange resin and an adequately stirred solution not less than about 0.05kf, the slow, rate-controlling step is the diffusion of the ions inside the resin. On the other hand, the slow step in exchanges involving Dowex A-1 and one or two chelating cations is the second-order chemical reaction. The active group in this resin is -CHzN(CHzCOO-)2. The exchange between the magnesium form of thi’s resin and hydrogen ion was studied under two sets of conditions: (A) with resin beads 0.022 om. in radius and 0.1130M hydrochloric acid and e
(B) with 0.016-om. beads and 0.1051N acid. In both experiments, the quantity of resin was equivalent to the acid. In a diffusion-controlled process, the smaller particles (B) would react more rapidly; but in a process controlled by B second-order chemical reaction, the more concentrated solution would react more rapidly (A). Actually, the reaction was faster in experiment A; and both experiments gave identical values for the velocity constant. C. W. Gould considered an instrumental approach to separations of organic components. The apparatus consisted of a gradient elution system and stainless steel composite columns. The columns consisted of sections of various volumes and decreasing diameters with mixing plates between sections to minimize uneven fronts in the chromatographic zones. An automatic fraction collector was used and after appropriate combination of fractions the concentration of each component -was dptermined spectrophotometrically. Data were given for the separation of an eight-component system consisting of benzene, styrene, anisole, benzaldehyde acetophenone, phenol, benzoic acid, and benzyl alcohol. John L. Hague discussed a wide variety of inorganic separations with principal emphasis on some complex metallurgical alloys. Flow sheots were given for alloys which contained mixtures of zirconium, titanium, molybdenum, tungsten, niobium, and tantalum. These elements were separated by using a strongly basic anion exchange resin and various mixed chioridefluoride solutions as eluents. In addition other elements such as antimony, bismuth, iron, phosphorus, and tin were considered to show where they would fall in the various elution mixtures The separation of zirconium and hafnium has been studied in sulfuric-hydrofluoric acid mixtures with good results for the determination of zirconium in hafnium. The separation is less precise wheI: zirconium is present in considerably larger amounts than hafnium. J. S. Fritz reviewed separations of metal ions using halide complexes. Examples were given of the use of fluoride solutions for separations on cation resins. These separations are based on the formation of soluble fluoride complexes which are not absorbed by the resin. One of many examples given was the separation of aluminum and titanium. Another technique discussed was the use of partially nonaqueous solutions as eluting agents. The elution mixtures consisted of water-hydrochloric acid and various amounts of isopropyl alcohol, ethanol, or acetone as nonaqueous solvents. The adsorption of metal ion halide complexes is generally increased when a nonaqueous solvent is introduced
into the system. Many interesting separations have been developed for metal ions using various compositions of hydrochloric acid in solvent mixtures of water and alcohol. Stanley Kirschner discussed the use of zone melting techniques for the resolution of two-component inorganic salt systems and racemic mixtures of o p tically active compounds and their
diastereoisomers. The technique, b brief, consists of freezing a solution, which contains two components, into the form of a bar and then causing B molten zone to traverse the frozen charge. The entire zone melting ~p paratus was immersed in a deep freeze chamber. The sample solution to be zone melted was contained in B Pyrex tube fitted with standard-taper glass
stoppers. The melted zone was made to traverse the tube by moving an electric heater along its length. Preliminary separations were made with simple binary mixtures in order to study characteristics of the apparatus and the technique. Partial resolution of D&[ C ~ ( e n ) ~Cis, ] racemic propylenediamine, and tris(acety1acetonate)-eobalt(II1) were given.
uantitative GasChain Fatty Acids as KURT O M E and E.
H. AHRENS,
Jr.
The Rockefeller Institofe, New York 27, N. Y .
,A simple procedure is described for quantitative formation, recovery, and gas-liquid chromatographic analysis of 2-chloroethanol esters of short-chain fatty acids. Esters of propionic and higher monocarboxylic acids are accurately quantified in ionization chamber detectors without recourse to empirical correction factors.
of methods have been developed recently for isolation and quantification of individual fatty acids in complex mixtures. The availability of these micro scale procedures has focused attention on the need for precise methods of defining double-bond structure on comparably small quantities of material. Previous studies on large samples have demonstrated that the method of choice is identification and measurement of split products after oxidative degradation. I t is now feasible on a micro scale to identify the split products by gas-liquid chromatography (GLC); indeed, GLC makes possible all preparative and analytical steps on a few milligrams of homologous acids of given chain length (10). Unfortunately, a number of technical difficulties have hindered the attainment of all the quantitative goals. NUMBER
Complete degradation is difficult to obtain without forming varying amounts of secondary reaction products. Complete recovery of split products, free of other reactants, is hindered by the volatility of the u s s 1 ' * lucts. Quan*; mdar weight unreli$?A? wa: detection evicea.'I J
I
LL-Ls
Previous methods have overcome some but not all of these problems. James and Martin (4) monitored their GLC separations by titrating the short-
chain acids produced from unsaturated acids by permanganate degradation, but relatively large amounts of starting materials were required, secondary products were prominent, and recoveries were incomplete. Losses of volatile split products prior to chromatographic analysis were reduced by Ralls' (9) method of producing ethyl esters from small molecular weight soaps by flash exchange a t the head of the chromatographic column. At present, the most sensitive GLC detection device in common use is Lovelock's ionization detector (6). Unfortunately, it is characteristic of this detector that methyl esters of low molecular weight are overestimated and fatty acids of low molecular weight are underestimated, requiring use of correction factors such as those presented by Bottcher (1) for analysis of mixtures of short-chain aliphatic acids. Craig, Tulloch, and Murty (g) state that these several interrelated problems may be resolved by formation of the phenacyl esters of the short-chain fatty acids. The resultant increase in their molecular weight has led to better recoveries and more reliable GLC quantification. The present report proposes a new and possibly more satisfactory solution to the double problem of quantitative formation and recovery of low molecular weight fatty acid esters, followed by reliable GLC microestimation with an ionization chamber. Aliphatic monocarboxylic esters formed with 2chloroethanol (CHz@lCHzOOCR) are sufficiently nonvolatile to permit their complete recovery from reagents and solvents. In addition, they are ionized in a linear manner-Le., area measurements of GLC curves of esters of propionic and higher homologs accurately reflect per cent compositions of mixtures (on a weight basis). The
technique can be carried out oil 1 mg. or more of acids. We have evaluated a number of esterification techniques for short-chain fatty acids, including methylation with methanol-HC1, a i t h diazcmethane, and with boron trifluoride in methanol; also butylation with diazobutane. None has proved as advantageous and simple as the procedure described. EXPERIMENTAL
Reagents. 2-Chloroethanol (Matheson, Coleman and Bell), purified by vacuum distillation a t about 15-mm. Hg pressure in a 70" to 80' bath. HC1 (5 to 7%, w./v.) in 2-chioroethanol, prepared with commercial HCl gas. Boron trifluoride (Matheson, Inc.) (9 to 11% w./v.) in 2-chloroethanol, according to Metcalfe and Schmitz ('7). 1 4 Petroleum ether (30' to 60") or pentane, glass-distilled. Procedure. From 10 to 25 mg. of fatty acid or soap is weighed into a 1- or 2-ml. glass ampoule, and 1 ml. of 2-chloroethanol-HCl, (CE-HCI) or 0.5 ml. of 10% 2-chloroethanol-boron trifluoride (CE-BFa) is added. [Smaller samples (down to 1 mg.) are esterified in sealed capillary tubes with about 0.1 ml. of either esterification reagent.] The ampoule is sealed, then heated in a boiling water bath (1 to 2 hours with CE-HC1, 10 minutes with CE-BFa). After cooling, the contents are transferred quantitatively to a test tube or separatory funnel with 5 volumes of water. The esters are extracted three times with volume of petroleum ether (for highest recovery of the esters of formic and acetic acids, four to five extractions with pentane are preferable.) The pooled extracts are washed once or twice with l/d volume of water, then dried over sodium sulfate crystals. For GLC analysis, the solvent is evaporated a t - 50 a t not more than 5-mm. VOL 33, NO. 13, DECEMBER 1961
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