January 19691 PREPARATION OF HIGH PURITY SUBSTANCES 5 Preparation of High Purity Substances The following are summaries of some of the papers presented at a Joint Meeting of the Midlands Section and the Special Techniques Group with the Midlands Section of the Royal Institute of Chemistry held on September 25th 1968 and reported in the October 1968 issue of Proceedings (p. 195). The Separation of Milk Proteins by Column Chromatography BY L. IRONS AND M. JONES COLUMN chromatography is one of a number of separation methods dependent on migration for which the driving force is the flow of moving phase relative to the stationary phase and the resistive force is one of attraction to the stationary phase. The types of column chromato- graphy commonly used for protein separations based on the interaction between protein and stationary phase are adsorption ion exchange and molecular sieve chromatography (gel filtration).Adsorption chromatography has been used successfully for many enzyme preparations with calcium phosphate as adsorbent but difficulties are often encountered arising from irreversible adsorption of the protein and lack of reproducibility in the behaviour of the adsorbent. Ion-exchange chromatography is the most widely used technique for protein separations ; the supporting phases in common use being ion-exchange resins and ion-exchange celluloses. Limitations to the use of resins for protein separations are imposed by their cross-linked struc- ture low exchange capacity and hydrophobic nature but they have been used successfully for the separation of small molecules such as peptides and for some proteins especially those of high-isoelectric point and low molecular weight.The modified celluloses carboxymethyl cellulose and diethylaminoethyl cellulose have been widely applied to protein separations as their swollen hydrophilic matrices are readily permeable to large molecules. Elution from these adsorbents is achieved by changing the pH or increasing the salt concentration of the eluant to reduce the number of electrostatic bonds between the protein and the cellulose. Column chromatography on diethylaminoethyl cellulose can be used to purify the maj or casein and whey protein fractions of milk. Whole casein consists of a mixture of proteins which are separated from milk by acidification to pH 4-6 a t room temperature. Moving- boundary electrophoresis resolves whole casein into a- P- and y-caseins.The a-casein fraction can be further separated into calcium-insoluble as-casein and calcium-soluble K-casein. The full complexity of whole casein can only be demonstrated by the method of zoneelectro- phoresis in starch or polyacrylamide gels under conditions in which the casein aggregates are dispersed (7M urea). Whole casein is then resolved into about 20 components the high resolu- tion of the methods being obtained by a combination of charge and molecular-sieving effects. Crude forms of ccs- and p-caseins can be separated from whole casein by their different solubilities in aqueous urea solutions and then purified by chromatography on columns of diethylaminoethyl cellulose. Elution is carried out with 3 . 3 ~ urea and 0 .0 1 ~ imidazole buffers (pH7) and linear sodium chloride gradients ranging from about 0.1 to 0 . 3 ~ . The samples obtained in this way give mainly single bands on zone electrophoresis and are con- taminated with only trace amounts of other proteins. The major whey proteins P-lactoglobulin and a-lactalbumin can be obtained in a cry- stalline form by salt fractionation methods from the protein solution left after removal of casein from milk. However the proteins obtained are often impure even after several recrystallisations. They can be purified further by chromatography on diethylaminoethyl cellulose or by gel filtration on columns of Sephadex GlOO or G200. Separation of proteins by gel filtration depends only on molecular size and different molecules appear in the eluate in order of decreasing molecular weight.With whole whey the immunoglobulins are eluted first followed by bovine serum albumin P-lactoglobulin a-lactalbumin and finally the phos- phoglycoproteins. By using gel filtration as a final purifying stage samples of P-lactoglobulin and a-lactalbumin can be prepared from the impure crystalline preparations which give only single bands on polyacrylamide gel electrophoresis. (Unilever Reseavch Laboratovy The Fvythe Welwyn Hevts.) 6 PREPARATION OF HIGH PURITY SUBSTANCES [Proc. SOC. AnaZyt. Chem. Preparative-scale Gas Chromatography BY R. G. PLEVEY (Department of Chemistry The University Birmingham 15) GAS - liquid chromatography can be used to separate substantial amounts of material in two ways. In the first a chromatographic column of about the size conventionally used for analysis is used.Successive samples of the mixture are applied to the column and the chosen components collected by condensation from the gas emerging from the column. In the second method columns of much greater diameter (30 to 75mm) are used and to these are applied larger samples (1 to 50g) so that substantial amounts of chosen components can be obtained in a single pass by condensation from the emerging gas stream. Apparatus for the first method is available commercially. This paper is concerned with columns of the second type made in this department. The basic design of these instruments is similar to that of gas - liquid chromatographic apparatus available commercially. However larger columns either 30 or 75mm have generally been used. The columns are constructed from two 2-5-m lengths of glass or copper tubing joined together at one end copper tubes are preferable.The columns are packed with a stationary phase supported on Celite except for a portion below the inlet that is filled with metal shot. The columns are arranged vertically or horizontally in electrically heated ovens maintained at suitable temperatures. Samples fall by gravity from a glass vessel (designed so that there is little disturbance of the high back pressure of carrier gas at this point) on to the heated metal shot. The metal shot is usually heated to a temperature some degrees higher than the boiling-point of the least volatile component and this system allows a rapid transfer of heat to the injected sample. The rate of volatilisation is a factor that determines the efficiency of the column.At the other end of the chromatographic column a by-pass transfers about 1 per cent. of the effluent through a katharometer detector. Electronic amplification and recording on a moving-chart recorder of the signals resulting from thermal-conductivity changes in the effluent gas permit the controlled trapping by freezing into glass traps cooled in liquid air of components from the main gas stream. Optimum flow-rates of carrier gas for the smaller column (30mm) are about 15 to 20 litres per hour and for the larger column (75mm) are about 50 to 70 litres per hour. In the small columns from 0.5 to 3g of a single compound in a mixture can be dealt with and in the large columns about 10 to 25g. Fig. 1. The plateage (N) of a gas-chromatographic column Some idea of the efficiency of the columns can be obtained by comparing the performance of analytical columns with the 30 and 75-mm preparative tubes.The simplest way is to compare plateages of the columns working under optimum conditions. The plateage N is defined by the expression- January 19691 PREPARATION OF HIGH PURITY SUBSTANCES 7 Analytical columns usually accept samples of about 5p1. With the same sample loading per unit cross-sectional area of the column it is found that columns have plateage values of about 1000. However bearing in mind the different lengths of the columns (analytical 2m; preparative 5m) it appears that there has been a significant increase in the value of the height of the equivalent theoretical plateage given as the ratio of the column length to plateage.This decrease in efficiency even for relatively small samples has not been a serious problem as successful separations have been performed on 100 to 200-g samples in the 75-mm columns. The recovery from the columns varies between 75 and 95 per cent. depending to some extent on the retention times of compounds. In general separations take from 1 to 8 hours for completion. It should be assumed that materials may be contaminated with stationary phase or its decomposition products ; distillation in vacuo from phosphorus pentoxide is recommended. This bleed-off of stationary phase the so-called “ageing” of the tube is reflected in the gradual shortening of retention times leading to lower plateage values. Experience shows that this process does not seriously affect the performance of columns even after 2 or 3 years or more.The use of large diameter columns in preparative gas - liquid chromatography is a very effective method for separating considerable amounts of volatile materials (boiling-point less than 130 “C) and frequently adequate separations of less volatile mixtures are obtained. Laboratory Techniques of Metal Purification BY I. A. BUCKLOW (Metals Research Ltd. Melbouvn Royston Hevts.) SMALL-SCALE techniques for purification suitable for amounts of up to say 1 kg are described and reference is made solely to purification of metals and not to their compounds. Purification reactions are best conducted in the molten state when reaction rates and mobilities are high but the high temperatures normally required and the reactivity of molten metals often cause considerable difficulty in the choice of a suitable container; containers are therefore best avoided.Levitation would be the ideal state but it has considerable limita- tions and so compromise methods,in which the molten metal comes into contact only with its own kind are used. This is principally achieved in two ways viz. the floating zone and the water-cooled copper crucible. FLOATING-ZONE METHOD- First developed on a large scale for silicon the method involves the use of a vertical rod of metal supported at both ends by chucks that can rotate and move vertically. A small zone of molten material is established across the rod and held in place by surface tension; the zone traverses the rod and purification occurs partly by zone-refining (i.e. fractional crystallisation) and partly by preferential evaporation of impurities (in a vacuum) or by chemical reaction with an atmosphere.In practice the length of the zone is about the same as the rod diameter but as the molten metal is supported only by surface tension a practical limit on rod diameter is reached. The necessity for a sufficient surface tension restricts the method to metals with melting-points above about 1150" C. Rod dimensions are say up to 15mm diameter by 400 mm long and traverse speeds are from 2 to 20 mm per hour. The two principal methods of heating the zone are by electron- beam and radiofrequency induction heating. EZectyon-beam heating-This method requires an extremely good vacuum system (low leak-rate clean and well trapped) if back-streaming pump-oils etc. are not to introduce considerable contamination; beam heating is efficient and power supplies are not too expen- sive.The method is suitable for refractory metals where impurity evaporation is probably as important as zone refining. Radiofrequency induction heating-The generator is expensive but the heater can now be outside the reaction chamber therefore zoning can take place in neutral or reactive atmos- pheres at positive pressure. Evaporation losses of the parent metal are reduced and impuri- ties are removed by reaction with the gas (e.g. carbon is removed from iron as carbon mon- oxide by zoning in wet hydrogen). Induction heating can give some support to the 8 GEL FILTRATION AND GEL PERMEATION [Proc. SOC. Analyt. Chem. molten zone but heating is not very efficient and the method is most often used for metals melting at the lower end of the range.WATER-COOLED COPPER CRUCIBLE- Developed by Standard Telephone and Cables Research Laboratories Harlow Essex the crucible forms either a horizontal trough or a basket of water-cooled copper tubes; the metal charge is melted by an external radiofrequency induction coil. On an atomic scale there is only point contact between the crucible and molten metal within; the charge is frozen at these contact points which due to stirring in the melt are always changing hence the whole charge is effectively molten normally there is no contamination. Reactive atmos- pheres can be used and with the trough several zones can be passed at one time. SOLID-STATE ELECTROLYSIS- Solid-state purification is best avoided because it is slow inefficient and usually limited to impurities that can be converted into gases but solid-state electrolysis is a relatively new method and holds great promise; M.Whittaker at Metals Research is applying it very successfully to rare-earth metals (to be published in “Proceedings of the Second International Congress on Crystal Growth Birmingham U.K. 1968,” North Holland Publishing Co. Amsterdam). A rod is held vertically in a reaction chamber under pressure of argon (again good vacuum standards are required to prevent the ingress of oxygen) and a d.c. current of about 500A per cm2 is passed to heat the rod nearly to melting-point. After about 100 to 150 hours considerable proportions of the impurities the non-metallics especially have drifted to the anode end and are precipitated as inclusions. Crystalline perfection and purification levels are much higher than can be achieved by zoning and rod diameter is not limited by surface tension. SEMANTICS OF METAL PURIFICATION- The speaker thought that probably the only metal to have been truly purified to 99-9999per cent. (i.e. 6N) was tungsten; even the electronic metals which are quoted as 7N or even 9N are purified to this degree only in respect of electronically-active impurities. And yet some of the most reputable suppliers still claim 6N for a wide range of metals. The process that leads to these figures is one of “purification by omission,” in which the supplier (often unwittingly) omits to define the limits of analytical detection and often forgets altogether the existence of for example surface oxide on powders. A note of warning on the meaning of “purity” was given. Let the buyer beware!