DESALTING AND RELATED TECHNIQUES

CHAPTER

3

DESALTING AND RELATED TECHNIQUES /.

Smith

DESALTING is almost always an essential preliminary t o t h e achieve­ ment of satisfactory chromatograms of organic compounds and the adverse effect of salts has already been mentioned (see p . 36). Those techniques which result in t h e removal of inorganic ions or salts while leaving the organic ions and molecules either in the original solution or in a condition where they can be quantitatively recovered free from inorganic compounds are referred to as "desalting techniques," and these fall into three main classes, viz.: electrol3rtic desalting, ion ex­ change separations and organic solvent extraction.

ELECTROLYTIC DESALTING Add

me»nbrAhe movihg H9 surface

Hg + CATIONS

Ιηβί-ΑίΙκ"*" CATIONS

F I G . 3 . 1 . T h e principles of electrolytic desalting. Metallic cations discharge a t a n d are carried a w a y b y t h e flowing m e r c u r y c a t h o d e w h i c h is t h e n w a s h e d free of t h e cations a n d returned t o t h e cell. A n i o n s , inorganic a n d s o m e organic, p a s s t h r o u g h t h e ion-perme­ able m e m b r a n e , discharge a n d are w a s h e d a w a y b y t h e flowing acid anode w h i c h is r u n t o w a s t e . Organic cations a n d neutral molecules remain in t h e cooled sample c h a m b e r .

The principle of electrolytic desalting is t h a t a direct current is passed through the solution to be desalted and electrolysis occurs (see Fig. 3.1). The metal cations discharge a t a flowing mercury cathode in which they dissolve, and are carried away; the inorganic anions pass to the anode, which is separated from t h e main solution by an ionpermeable membrane, and are carried away by a continuous stream of dilute acid flowing past the anode. Thus t h e inorganic ions are removed from solution in a cell which is a modified, microversion of 41

42

CHROMATOGRAPHIC T E C H N I Q U E S

the Kastner-Kellner cell. The organic cations also discharge a t t h e mercury cathode but, being insoluble in it, they remain in solution as neutra] molecules or zwitterions, or they m a y precipitate. Organic

F I G . 3.2a. The electrolytic desalter, (α) Complete apparatus includ­ ing power p a c k ; (6) enlargement of sample c h a m b e r illustrating anode and c a t h o d e flow, cooling j a c k e t a n d t h e level mercury surface o n which t h e s a m p l e solution (not s h o w n ) rests. (1) W a t e r -f cations out. (2) Trap. (3) W a t e r in. (4) Levelling t u b e . (5) Cathode. (6) Mercury. (7) H2SO4 + anions o u t . (8) 2 % H2SO4 in. (9) A n o d e . (10) Cooling jacket. (11) Sample chamber. (12) Membrane.

anions derived from strong acids (e.g. sugar phosphates, sulphates, etc.) pass through the membrane and are lost, whereas weak acids and their anions are retained in the solution. The Apparatus. The original apparatus of Consden et al.^'^^ has been greatly improved by Astrup et al}^^ who introduced t h e flowing anode.

DESALTING AND R E L A T E D T E C H N I Q U E S

43

by Dent^^^ and by Stevens et alM^ who introduced the cooHng jacket and modified previous designs in order to be able to deal with small volumes (1-5 ml.) of solution. The following discussion refers in detail to the Stevens apparatus, as this is compact, mechanically stable and is known to perform well* (Fig. 3.2a). Recently a miniature desalter operating on the same principle has become available! (Fig. 3.26). The desalter (see Fig. 3.2a) requires some 50 ml. of mercury which is added through the reservoir or levelling tube, the coiled flexible P.V.C. tubing acting as a cushion to break the fall. The mercury is caused to circulate by t a p water (functioning as a lift pump) which is passed in

FIG.

3,2.6.

through the first vertical tube from the left and raises it u p t h e second vertical tube when it falls down into the third. This mercury is then forced u p the inner tube of the cell where it overflows down the outer tube and is drawn back to the flrst for recirculation. I n this way a continuous, flowing mercury cathode is circulated through the appar­ atus such t h a t the mercury surface, which should appear practically motionless, is just above the level of the inner tube of the cell. This level is adjusted b y determining the volume of mercury necessary and then altering the water pressure so t h a t the mercury is raised as a fairly fine stream; after water has been flowing for some minutes the final adjustment is made with the levelling tube. The circulating water passes through two traps before it empties away and, consequently, no mercury is lost down the sink; a t the end of an experiment the water is * From Aimer Products Ltd., 5 6 - 8 Rochester Place, London, N . W . I , t S h a n d o n Scientific Co., 6 5 P o u n d L a n e , L o n d o n , N . W . I O , a n d Sewickley Pa.,

1 5 1 4 3 , U.S.A.

44

CHROMATOGRAPHIC T E C H N I Q U E S

turned off and the apparatus left. The lower horizontal tubes, through which the mercury circulates, lead to the back of the apparatus where they are connected by means of P.V.C. tubing to a three-way t a p so t h a t the mercury can be completely drained from the apparatus and, when necessary, the apparatus can be rapidly and simply dismantled; as it is rarely necessary to do this, all joints should be well greased. The desalting chamber can be removed and cleaned without disturbing t h e main apparatus as the two are connected b y a ground glass joint (a spare chamber is shown in the lower middle of Fig. 3.2a). Where the water pressure varies it is convenient to place a screw clip, almost completely screwed shut, on the mercury outlet of the cell (behind the apparatus) to ensure t h a t a high back pressure, which confers opera­ tional stability on the mercury surface, is built up. Mercury which slowly appears in the t r a p may be periodically returned to the reservoir, and water which occasionally appears on top of the reservoir can be rejected. J u s t before use, an ion permeable membrane (a piece of dialysis tubing or, better, a shaped "Viskap" size 00—supplied with the appar­ atus) is placed over the open end of the anode and bound firmly with many turns of an elastic band. Dilute sulphuric acid is then passed down the inner tube of the anode (from an aspirator—not shown in the picture) and flows u p the outer tube to waste; 1-2 per cent, v/v, con­ centrated acid is added to the flowing anodic liquid to render it conduct­ ing and a flow rate of about 100 ml./minute is suitable. From 1-5 ml. of the solution to be desalted, p H > 7, is then pipetted on to the mercury surface from a Pasteur pipette, the anode is swivelled round in its clip, lowered down into the surface of the sample and the current is applied. Practically no change in volume occurs during short periods of desalting (up to 15 min.) although over longer periods some dilution due to osmotic flow may occur. Much heat is generated during this process and therefore it is essential to keep the sample cool so a simple cooling jacket surrounds the electrode chamber, this being sufficient to prevent the temperature of the solution ever rising above 30°C. When desalting is complete the viskap is removed from the anode before allowing it to dry out or the tube is immersed in water in the cup provided. One viskap can be used for a number of desaltings, provided t h a t it is well washed between uses, b u t should not be kept overnight. Desalting, or electrolysis, occurs when the two electrodes are con­ nected to a D.C. supply and, from the point of time of desalting and cost, the most suitable supply is 200-250 volts D.C. either direct from the main supply or after rectification. The desalter should be connected through an ammeter (scale 0-2 amp.), a 2 a m p . fuse and a second switch to the mains—it is useful to place this switch close to the apparatus. The commercially available apparatus incorporates these parts in a single unit which also includes a bulb indicating when the mains connection is live; a unit including a rectifier is also available for connection to the A.C. supply. Current passes from the flowing acid anode through the sample to the mercury and, at this stage, the anode solution forms the main electrical resistance. Thus, during desalting a high steady current is indicated on

DESALTING AND RELATED TECHNIQUES

45

the ammeter even though salts are bemg lost continuously. However, near t h e end of t h e process t h e sample resistance becomes increasingly greater t h a n t h a t of t h e anode acid a n d so t h e current falls until t h e desalting is complete when a low steady reading is obtained; t h e func­ tion of t h e ammeter is, therefore, t o indicate when desalting is over. Immediately t h e current is switched off t h e anode must be removed otherwise t h e acid will diffuse rapidly into t h e sample a n d vitiate t h e process. Originally, 1 per cent acid was used t o wash t h e anode b u t this was later increased t o 2 per cent in order t o speed u p desalting as it was found t h a t no extra heat was generated in t h e solution due t o t h e very ef&cient cooling system. The current m a y rise as high as 0-8-1 a m p . a t t h e beginning of desalting a n d will remain a t this value until just before desalting is complete. Over t h e course of a few minutes it t h e n falls t o a new low level, which is characteristic of t h e particular sample, and quite wide variations in this value are found; t h u s a normal b u t dilute urine m a y finally register 0-1 a m p . whereas a n abnormal a n d concentrated urine or a concentrated plant extract m a y n o t go below 0-25 a m p . However it has always been found t h a t both chloride a n d sulphate ions are absent when t h e low steady current is attained. Frothing of t h e sample often occurs with concentrated extracts of high conductance b u t this can readily be broken with a few drops of acetone. The mercury is best cleaned with nitric acid (1 vol. concentrated acid a n d 6 vol. water) a n d the apparatus can be filled with acid without dismantling, although this is seldom necessary. To sum up, a desalting should be carried out in the following sequence: 1. Start t h e mercury circulating. 2. Turn on water jacket. 3. Fill aspirator with anodic acid. 4. F i t new viskap t o anode chamber, if necessary. 5. Level 'the mercury in t h e cell. 6. A d d sample t o be desalted. 7. Start acid flowing through anode a n d ensure free flow t o waste. 8. Place lower end of anode just below surface of sample a n d t u r n on current. 9. Continue desalting—^replenishing acid in aspirator if neces­ sary—until ammeter indicates a low, steady current. 10. Turn off current, remove anode immediately t o its cup a n d stop acid flow. 11. Withdraw sample with a Pasteur pipette t o small bottle or centrifuge tube. 12. Wash out desalting chamber with water and, after removing this, t u r n off both water taps.

The Fate of Various Ions (a) Inorganic Anions. All inorganic anions diffuse through t h e Viskap membrane, discharge a t t h e anode a n d are washed away b y t h e flowing acid. (b) Organic Anions. I t is too seldom realized t h a t , provided t h e

46

CHROMATOGRAPHIC T E C H N I Q U E S

organic anion is small enough t o pass through t h e membrane pores, losses of t h e anions of strong acids occur; t h u s both indoxyl sulphate and glucose phosphate, when present as salts, are rapidly lost a n d it can be shown t h a t this is a physical loss through t h e membrane a n d not a chemical conversion as t h e compounds can be found quantitatively in the efläuent anodic acid. No appreciable loss occurs when weak organic acids or their salts are similarly treated, a n d this m a y be because t h e solution rapidly approaches neutrality when these acids will be union­ ized or, as has been suggested, t h e membrane assumes a small charge which is suf&cient t o repel t h e weak acids or their ions. (c) Inorganic Cations. All cations travel t o t h e flowing mercury cathode and discharge. Inorganic cations dissolve in t h e mercury as soon as they are discharged forming amalgams which then circulate with t h e mercury until they meet t h e water when they dissolve o u t t o form hydroxides a n d are carried away as such. I n this way, t h e water serves t h e dual purpose of circulating t h e mercury round t h e apparatus and of removmg t h e unwanted metals before returning it t o t h e cell. The one exception t o this rule is t h a t of t h e ammonium ion as t h e am­ monium amalgam formed is not destroyed b y water and so is con­ tinuously circulated: this amalgam tends t o stick t o glass, t o coagulate and t o conduct t h e current erratically so t h a t desalting cannot be completed, a n d as a general rule, therefore, solutions containing much ammonia cannot be desalted electrolytically (once contaminated with ammonium amalgam, mercury can only be purified b y vacuum distilla­ tion). (d) Organic Cations. After discharge t h e great majority of organic cations form amines or zwitterions which remain passively in solution during further desalting (chemical conversions consequent on electro­ lytic desalting are discussed under a separate heading). Many naturally occurring compounds are soluble in t h e original extract although insoluble in water, and so m a y precipitate if t h e desalt­ ing is complete. F o r example, tyrosine occurs in certain abnormal urines a n d it has been found occasionally t h a t , after desalting, a pre­ cipitate which can be shown t o be tyrosine slowly forms; cystine m a y fail t o redissolve after storage of t h e sample in t h e deep freeze. Pre­ cipitations of this type are more obvious when a solution containing only one organic compoimd is so treated a n d all t h e halo-tyrosines respond in this way; insoluble guanidines, such as glycocyamine, react similarly. Such precipitates should be redissolved with t h e minimum of HCl. Many proteins are denatured during this process a n d , although it is not possible t o generahze on the basis of present knowledge, it appears t h a t this m a y be a simple method of deproteinizing prior to chromatography. (e) Neutral Organic Molecules. Although a study of various types of neutral molecules has not been made, i t appears t h a t no osmotic diffusion occurs. Certainly t h e sugars yield chromatograms quaHtatively identical before a n d after electrolytic desalting although, of course, t h e desalted solution yields much more compact spots. How­ ever, m a n y pigments are precipitated rapidly—^thus desalted urine a n d plant extracts often appear much hghter a n d clearer t h a n t h e original.

DESALTING AND R E L A T E D T E C H N I Q U E S

47

(/) Proteins. Proteins are usually precipitated during desalting and small amounts can be accommodated without preliminary ultra­ filtration. However, large amounts of protein m a y precipitate on the

h m ^ n

Sulphate

QlüCOSE é-PHOSWATE (Casnir)

8uA t Ehrlich

itdhi^

9 I Ζ S BINS* OES ALTIN«

t

t

t

t

o

a

Φ

t

MIN«. NSALTIN«

F I G . 3.3. Illustration of t h e loss of strong organic anions during desalting. A l i q u o t s were r e m o v e d periodically from t h e desalter a n d either c h r o m a t o g r a p h e d (left-hand picture), or e x a m i n e d w i t h a v a r i e t y of reagents t o determine residual material. I t c a n be seen t h a t a t o t a l physical loss occurs a n d t h a t chemical reaction is n o t responsible for t h e decrease in colour i n t e n s i t y after t h e reagent is applied. T h e original c o m p o u n d s c a n b e s h o w n t o be present in t h e effluent acid.

membrane and cause it t o clog, particularly if t h e molecule carries a net negative charge, and so the solution should be first ultra-filtered as described on p . 63. Urine. For routine chromatography of urine, 1-2 ml. of desalted solution is usually adequate and desalting takes between 5-15 minutes. Quite often the urine will foam b u t t h e addition of 1-2 drops of acetone is sufficient t o break this without any adverse effect on t h e compounds of interest. When desalting is complete t h e solution will be hghter in

48

CHROÍ^IATOGRAPHIC T E C H N I Q U E S

colour and free of protein, b u t m a y need centrifuging. Urine suspected of containing ethanolamine phosphate cannot be desalted b y this method; taurine and the indole-strong acid conjugates are also lost but these have no diagnostic significance at present. All t h e other aminoacids, indoles and sugars so far known t o occur in m a n are unaffected. The fate of phenohc acids, barbiturates, steroids, etc. undergoing this process is not known, b u t as they are present in such small amounts they are usually concentrated, prior t o chromatography, by solvent extraction procedures which effectively eliminate inorganic salts. For those compounds which, a t present, dominate t h e field of clinical interest both losses and conversions are t h e exception rather t h a n the rule, and in our opinion, t h e electrolytic method is t h e most useful for desalting provided t h a t t h e above remarks are borne in mind and t h a t results are interpreted with care a n d understanding. Plant Extracts. Wide use of this method has been made with valuable results. Desalting times of u p t o four hours m a y be necessary t o remove all t h e salts and t h e final solutions are usually much freer of pigment.

Chemical Conversions Occurring During Electrolytic Desalting Although one or two particular chemical reactions have been reported to occur during desalting, it was assumed until recently t h a t these reactions were exceptional a n d t h a t once an organic ion h a d discharged, the neutral molecule or zwitterion remained passively in solution. This is now known to be a naive assumption a n d careful watch should be kept for t h e possible occurrence of desalting artifacts when interpreting chromatograms of solutions desalted in this way. Many of t h e con­ versions which occur are rapid, complete, and quite unaffected by t h e amount of cooling, b u t this does not imply t h a t t h e desalting method is of little value. The absence of a particular compound from the chroma­ togram of a desalted solution, for example, will indicate t h e absence of the parent compound in t h e original undesalted liquid. The first example of a chemical conversion occurring during electro­ lytic desalting was t h a t of arginine t o ornithine. <2, 5) This has been studied b y a number of workers, b u t results reported are difficult to correlate as the apparatus used b y t h e various workers were of quite different dimensions—a factor of great importance in t h e study of rates of conversion. I n t h e apparatus described here, there is no significant loss of arginine in t h e time necessary t o desalt most urines (up t o 15 minutes) provided t h a t t h e mercury and anode acid are circulating correctly and t h a t water is flowing rapidly through t h e cooling jacket. During electrolysis, large amounts of nascent hydrogen are liberated and, therefore, a very strongly reducing atmosphere exists in t h e solu­ tion being desalted. Thus compounds which are easily attacked by nascent hydrogen generated b y t h e more usual procedures will be similarly attacked here, and a number of general examples are quoted. Identical results are obtained in aqueous-organic solutions although a t a somewhat slower rate.^^'^ All compounds containing an activated double bond are rapidly a n d

D E S A L T I N G AND R E L A T E D T E C H N I Q U E S

49

cleanly reduced to t h e di-hydro-derivative, e.g. urocanic acid is con­ verted to di-hydro urocanic acid or imidazolyl propionic acid,^^^ cinnamic acid and its derivatives form the corresponding propionic acids and the pyridine mono-carboxyhc acids each form a number of products as yet unidentified. Where only one double bond is present in the molecule, i.e. where only one reduction product is possible, t h e absence of the reduction product on the chromatogram of the desalted solution will mean t h a t the unreduced compound is absent in the ori­ ginal. The presence of a reduced compound, such as imidazolyl propionic acid, m a y imply t h a t the compound originally present was the unreduced one, i.e. imidazolyl acryhc or urocanic acid, and therefore this will have to be confirmed b y the use of another desalting technique. I n a search for pjrridine acids, this method of desalting is contraindicated. Hippuric acids yeild glycine for reasons unknown. Certain halogenated compounds are reductively de-halogenated in the desalter and, although a t present only iodo- and bromo-compounds are known to respond in this way, care must be t a k e n even when dealing with other halogen derivatives. Compounds so far investigated include the halo-tjrrosines (iodo-, bromo-, chloro-, and fluoro-tyrosines) and t h e iodo-thyronines when it was found t h a t iodine and bromine atoms were split off the parent molecule in a rapid stepwise manner b u t no evidence of chlorine and fluorine cleavage was obtained (see Fig. 3.4).

Electrolytic Desalting as a Preparative Technique When Consden et alM^ first described their electrolytic desalter they remarked, prophetically, t h a t " t h e field of use probably greatly exceeds the use here described," i.e. the removal of sulphate ions. The value of the apparatus for the removal of all inorganic ions was soon appreciated, b u t the fact t h a t " u n w a n t e d " reactions could also occur has only recently been realized. Smith, Stevens and Jepson,^"^^ were t h e first to investigate the general principles governing losses and conversions in the desalter, and from their work has emerged the possibility of using the apparatus for chemical synthesis. ^^"^^ I n chromatography the need often arises for t h e preparation of small amounts of compounds for use as chromatograpMc markers a n d the desalter appears t o be very suitable for such small scale preparations, although there is no reason why it should not be used for the preparation of gram quantities of material. The apparatus, as described, can be used for work on this scale even if this means preparing t h e compound in three or four lots, although t h e cooling jacket and sample chamber could be removed and replaced b y a larger chamber without cooling. Present knowledge suggests two main types of preparation both based on reduction as, it has previously been remarked, a strongly reducing atmosphere exists during the passage of the current. Reduction can be effected in aqueous, ethanol, glacial acetic acid and even ethyl acetate solution provided t h a t a few drops of concentrated HCl are added in order to render the solution conducting; reduction could probably be effected in a n y other organic solvent which will dissolve small amounts of concentrated acid. I n order to carry out an electroljrtic reduction it is, of course, not

50

CHROMATOGRAPHIC T E C H N I Q U E S

necessary to use t h e desalter as a flowing mercury cathode is no longer required. A simple test-tube or boihng-tube containing about 10 ml. of mercury has yielded identical results with those obtained in t h e desalter in two test cases. Although it is unnecessary t o cool t h e tube with a water jacket (as in t h e desalter) it is as well t o use a flowing acid anode in order t h a t secondary reactions due t o t h e nascent chlorine do not complicate t h e result. Simple Hydrogenation. I n a previous section t h e reduction of com­ pounds with an activated double bond has been mentioned. This method has t h e advantage of performing such reductions cleanly and without t h e need of introducing materials which are difficult t o remove before t h e product can be chromatographed, e.g. salts of t i n or zinc. Thus, 10 mg. of urocanic acid in 1 ml. water, containing sufficient HCl to dissolve it, is completely converted in about two minutes t o its dihydro derivative a n d t h e solution obtained can be chromatographed directly. There appears t o be no reason why 5 ml. of saturated solution should not be similarly reduced a n d t h e reduction product isolated in a pure state either by neutrahzing residual acid, or by continuing desalting until all acid is removed, when it will precipitate. Reductive Dehalogenation. Once it has been established t h a t a com­ pound can be prepared in t h e desalter it is n o t necessary t o isolate it from a simple mixture before using it as a chromatographic marker and, therefore, t h e method can be used t o prepare mono-iodo- a n d monobromo-tyrosine a n d perhaps even the iodo-thyronines for use in this way. This example is chosen because these partially halogenated compounds are difficult and laborious t o prepare, although admirable methods have recently been described for t h e synthesis of some of these compounds. Ten mg. of di-iodo- or dibromo-tyrosine in 1 ml. of water, containing a small amount of acid t o dissolve it, is rapidly dehalogenated although the process can be stopped before t h e reaction has gone t o completion (see Fig. 3.4) and, in t h e course of a few minutes, sufficient for a marker can be prepared. If it is desired, t h e simple mixture m a y be chromato­ graphically separated a n d t h e mono-compound eluted in a pure form as described in Chap. 38.

Reduction of Hydrazones to the Corresponding Aminoacids. The 2 : 4

dinitrophenylhydrazones of ketoacids are rapidly split a t t h e nitrogennitrogen bond t o form t h e corresponding aminoacid—this is discussed more fuUy in Chap. 14. There can be little doubt t h a t m a n y compounds containing this type of bond will react similarly.

Reduction of a-Ketoacids to Hydroxyacids. Indolyl-pyruvic and

indolyl-glyoxylic acids have been reduced t o t h e corresponding a-hydroxyacids in amounts sufficient for chromatography—^the reaction did not go t o completion. Purines, Pyrimidines and Nucleosides. All purines, pyrimidmes and nucleosides are rapidly reduced with loss of ultra-violet absorption capacity. Sulphur Compounds. Bisulphides a n d mercury complexes are reduced t o thiols. This is a very satisfactory method of ehminating mercury with t h e liberation of t h e original thiol. Reductive Methylation. On "desalting" a solution of glycine in

DESALTING AND RELATED TECHNIQUES

51

formaldehyde sarcosme is obtained, b u t it is n o t yet known whether di-methyl glycine is also formed. Preparation of Free Bases. The necessity for preparing aminoacids and related compounds free of acid arises as a preliminary t o t h e

• 1»

iilHiiiiÉilB^^ F I G . 3.4. R e d u c t i v e d e h a l o g e n a t i o n . P u r e di-iodotyrosine (DIT) or di-bromotyrosine (DBrT) w a s d i s s o l v e d i n w a t e r a t a concen­ tration o f 10 m g . / m l . a n d a f e w drops o f c o n c e n t r a t e d H C l were a d d e d t o cause s o l u t i o n a n d render t h e w a t e r c o n d u c t i n g . A l i q u o t s were w i t h d r a w n periodically, c h r o m a t o g r a p h e d directly and located with ninhydrin. The chromatograms show that a s m o o t h stepwise d e h a l o g e n a t i o n h a s occurred a n d t h i s w a s con­ firmed b y CO-chromatography w i t h a u t h e n t i c t y r o s i n e ; n o other c o m p o u n d s are d e t e c t a b l e after t h e application o f a n u m b e r of different reagents.

determination of structure as well as for other reasons. Until recently t h e methods for achieving this were tedious a n d much loss occurred although it can now be achieved simply b y pouring t h e solution down a column of anion exchange resin in t h e free base form. However, an

52

CHROMATOGRAPHIC T E C H N I Q U E S

equally satisfactory result is obtained by completely "desalting" a solution of the compound provided t h a t the free base is not volatile. ELECTRODIALYSIS Until recently, desalting by electrodialysis has found limited applica­ tion mainly because of the permeability of the membranes then avail­ able, the difficulty of preparing them satisfactorily and reproducibly and the high resistance with consequent heating effects in the sample. Recently, however, the commercial availability of ion-exchange membranes has refocused attention on the possibility of desalting solutions electrodialytically. Considering the desalting of neutral compounds, this method possesses many advantages over both the non-ionic membrane electrodialytic method, and the simple ion-exchange method for the following reasons. The ion-exchange membrane functions as an ion filter, i.e. a cation exchange resin membrane allows the passage through it of cations only under the influence of the electric current, anions are repelled and the membrane is impermeable to neutral compounds; the membrane can be used many times and requires no chemical regeneration; the time taken for desalting is very much smaller t h a n with the previous methods and compared with the non-ion membrane method the heat generated is usually much smaller, particularly on the large scale; the sample does not come into contact with strongly acid or basic resin; the apparatus is simple and cheap to construct in the laboratory. I t cannot be to strongly stressed, however, t h a t this apparatus is quite useless for desalting ionic compounds such as aminoacids; manu­ facturers claims to the contrary are incorrect. An apparatus built in the author's laboratory, and designed primarily for the desalting of about 1-5 ml. of urine, is described. I t is a microversion of t h a t described by Wood. The dimensions of this apparatus can be scaled up to deal with volumes of hundreds of miUilitres and, in this case, both the voltage and amperage can be increased because of the greater cooling possible. The dialyser consists of a central pear-shaped compartment (to permit complete removal of the contents) and two electrode chambers, 2-5 cm. diameter, closed at the outer sides with further perspex sheets cemented to these chambers. A coiled platinum electrode is fitted into each outer chamber and connects with a small metal screw which leads out to the electrical contacts. Between the central chamber and the two outer compartments are placed ion-exchange membranes which need be only slightly larger t h a n the chamber diameter. The assembled appar­ atus is held together b y wing nuts running on threaded rods passing through four holes drilled near the corners of the blocks. Samples are applied to and removed from the central chamber by means of a hole drüled down into the perspex, and water flows through the electrode compartments via similar holes drilled through the top and bottom of the blocks. Perspex, glass, or metal tubes are cemented into these latter holes and are connected either to a single water supply or to a

D E S A L T I N G AND R E L A T E D T E C H N I Q U E S

53

single effluent tube by means of glass T-pieces. Details of construction can be seen in Fig. 3.5. On such a small scale the electrical heating effects are large and so it is essential to have efficient cooling as otherwise the sample tends to boil. More important, however, is the fact t h a t the membranes lose their selectivity above 60°C. Cooling is attained by running cold t a p water

F I G . 3 . 5 . A p p a r a t u s for electrodialysis (not available commerci­ ally). T h e a p p a r a t u s is built from p e r s p e x s h e e t 11 m m . t h i c k a n d e a c h block is 8 c m . square. T h e m e m b r a n e s a n d t h e rods a n d n u t s are n o t s h o w n . A , s a m p l e c h a m b e r ; B , electrode c h a m b e r ; C, coiled p l a t i n u m electrode w i t h electrical c o n t a c t t h r o u g h p e r s p e x s h e e t t o outside t e r m i n a l s ; D , w a t e r o u t l e t ; Ε a n d F , p e r s p e x t u b i n g c e m e n t e d into a p p a r a t u s for w a t e r inlet a n d outlet.

through t h e electrode compartments when t h e temperature of the sample never rises above 35°C and this also serves to remove strong acid and alkali which would otherwise prevent complete desalting of the sample in the centre compartment. As a further aid to cooling a resistance is placed in parallel with t h e appara,tus t h u s stabilizing the voltage when the current through the cell drops during the desalting. Because of the original difficulties encountered during the operation of such a small unit a circuit diagram (Fig. 3.6) is shown and, of a number of methods and resistances tried, the most satisfactory is t h a t using a simple 500 W. electric fire element. The element is arranged such t h a t

54

CHROMATOGRAPHIC T E C H N I Q U E S

four of the lengths of wire are in series and the fifth in parallel with cell and ammeter. Spiral platinum electrodes are used and these lead through the back of the electrode compartments, into which they are sealed with perspex cement, and wind round a screw which is threaded into t h e back of the compartment and which serves as the electrical contact. Fast flowing t a p water is led into the electrode compartments and fiows out to waste. The cell is arranged in t h e order: cathode, cation membrane, centre compartment containing sample, anion membrane, and anode

F I G . 3.6. Circuit diagram suitable for small scale electrodialysis w h e n u s i n g t h e m a i n s p o w e r s u p p l y . C = cell (Fig. 3 . 5 ) ; Μ = a m m e t e r reading t o 500 m . a m p s ; R = variable resistance; F = fuse.

compartment. To check t h a t the current is being passed through the cell in the right direction, distilled water is placed in the centre when the original small current falls rapidly to zero; if current is being passed in the wrong direction the small initial current rapidly increases and a deposit of sodium sulphate m a y appear in the centre. At the beginnmg of the experiment the current should not be greater t h a n 200 m.amp. and the potential drop across the cell should be not more t h a n 45 volts; figures greater t h a n this cause the temperature to rise above 35 °C with consequent loss in membrane selectivity. I t is usually possible to distinguish between the anion and cation membranes by inspection as the former is lighter in colour. However, the type of membrane can be readily determined b y immersing each in acid for a short period when the cation resin originally in the sodium form is converted to the acid form and the anion resin is unaffected. The resins are then washed until the ^ H of the supernatant is neutral and a few

DESALTING AND RELATED TECHNIQUES

55

grams of sodium chloride is added t o each. The cation resin is recon­ verted t o t h e sodium form with t h e hberation of hydrogen ion a n d t h e supernatant quickly becomes acid whereas t h e anion resin supernatant remains neutral. The anion resin yields a positive response if alkah is used instead of acid when t h e final supernatant will become alkaline. I t is, a t present, worthwhile t o check each new batch of resin in this way. A piece of membrane only just bigger t h a n t h e outer compartments is sufficient a n d it is quite unnecessary t o use a piece as large as t h e outer dimensions of t h e apparatus. When in continuous use t h e membranes occasionally adsorb coloured material from t h e sample a n d after some time t h e small piece of membrane can be discarded. However, larger pieces can often be cleaned b y passing t h e current in t h e reverse direc­ tion for a period, washing out t h e centre compartment a n d then re­ converting t h e membranes t o their original form b y placing a sodium sulphate solution in t h e centre a n d passing t h e current in t h e usual direction. When not in use t h e membrane should be stored under water. Desalting Solutions of Neutral Compounds. I t would appear t h a t this method is t h e most satisfactory for desalting solutions of neutral molecules such as sugars as t h e sample rapidly attains a neutral ^ H a n d remains in this condition until desalting is complete. Thus Anderson and Wylam^^^^ have shown t h a t t h e method is completely satisfactory for a variety of mono- a n d di-saccharides a n d even sucrose is recovered completely after 45 minutes desalting a t a potential difference of 306 volts b u t in a much bigger cell t h a n t h a t used here. Similar results were obtained after desalting a n 80 per cent ethanolic extract of grass.

Desalting of Aminoacids and other Organic Ions. The electrical

mobilities of small inorganic ions are greater t h a n those of larger organic ions a n d zwitterions a n d therefore it is t o be expected t h a t t h e former will move out of t h e sample compartment first. However, in model experiments on mixtures containing strong a n d weak cations and anions, it has been found t h a t all ionic materials are lost although at different rates. I t must also be realized t h a t t h e simple process of placing a n ion-exchange resin membrane into a n ionic solution wiU permit t h e exchange of ions onto t h e resin with consequent loss of material from solution; this has indeed been shown t o be so with both basic a n d acidic aminoacids. This apparatus is, therefore, quite useless for desalting solutions of organic ions even if only qualitative information is required. However, none of t h e chemical conversions occurring in t h e electrolytic desalter can occur in this apparatus and, therefore, it would be quite possible t o check whether such conversions have occurred b y repeating t h e desalting in this apparatus a n d then examining for t h e original, i.e. t h e unreduced, material. The original paper should be consulted for further information on this subject. ^^^^ Desalting Urine. Approximately 1-5 ml. of urine requires between 5-10 minutes for the sample t o become chloride-free. I t is n o t necessary to continue until t h e current has dropped as low as 20 m.amp. as normal urines appear t o be chloride-free a t about 70 m.amp. a n d perhaps, after this reading, t h e extra material lost m a y b e mainly organic. The method can be used with confidence for desalting urines prior t o identifying the sugars a n d other neutral molecules present.

56

CHROMATOGRAPHIC TECHNIQUES ION EXCHANGE DESALTING*

A modern cation exchange resin^^^^ m a y be considered as a very high molecular weight organic acid of which the anion is completely in­ soluble and whose formula may be written, R c H . The R denotes the whole of the anion, the subscript c shows t h a t only cations are exchanged and the Η indicates t h a t the only cation on the resin, at this stage, is the hydrogen ion. Complete insolubility of the resin is, of course, necessary to the success of this technique and is achieved by the use of polymerized long chain organic compounds, the chains being linked together (cross-linked) t o increase insolubility and t o decrease t h e swelling which occurs on wetting. Thus the cation exchange resin known as ZeoKarb 2 2 5 , I R C 120 or Dowex 5 0 is synthesized b y poly­ merizing vinyl-benzene in the presence of divinyl-benzene and the polymer is then sulphonated, t h e formula being represented as shown in Fig. 3.7. Modern resins are usually synthesized in t h e form of micro-spheres or beads, the beads consisting of an open lattice-work with t h e majority of the exchangeable hydrogen ions present within the lattice. Further­ more, the resins contain only one type of acid group (sulphonic, carboxylic, etc.) and are referred t o as monofunctional. When a dilute solution of cations, containing one or more cationic species, is passed down a column of such a resin in the hydrogen cycle they displace t h e hydrogen ions and are retained in stoichimetric ratio, t h u s : RcH++

X+ - ^ R c X + + H+ (influent) (effluent)

In this way, if the solution is sufficiently dilute and passes down t h e column slowly enough for exchange t o occur t h e n all t h e cations, inorganic and organic, are removed quantitatively from the solution; the most satisfactory practical results being attained when an excess of resin, over the amount required t o exchange all the cations in t h e particular solution, is present. The resin has a finite exchange capacity, depending on the number of ionic centres present, and which is expressed either as milli-equivalents per d r y gram, or as milli-equivalents per ml. of wet resin. At this stage t h e resin will contain all the cations originally present plus the excess of hydrogen ions; all t h e original anions, neutral molecules, etc., and the displaced hydrogen ions will have passed through the resin or are removed b y a final water wash. However, t h e tenacity of the resin varies for different ions, and, in general, organic ions are held less strongly t h a n t h e inorganic ones. Thus, it is possible t o remove the organic cations while leaving the inorganic ones still on t h e resin and this m a y be done either by elution or displacement. Ions are displaced from t h e resin b y percolating down t h e resin column a solu­ tion of some ion which is more strongly held, e.g. acidic and neutral aminoacids m a y be displaced by a solution of ammonium ions, t h u s : R c N H 3 . C H 2 . C O O H + N H ^ O H - H ^ RcNH4 + N H a - C H g . C O O H + HgO (glycine) * Much detailed information is available from t h e manufacturers w h o u s u a l l y s u p p l y free b o o k l e t s o n e a c h resin o n a p p l i c a t i o n — s e e reference 12.

DESALTING AND RELATED TECHNIQUES

57

Ions are eluted from the resin b y passing down the column a n excess of ions which, although they are held less strongly b y the resin, neverthe­ less, remove the ions on the basis of the mass action effect; thus aminoacids m a y be eluted by a solution of hydrogen ions.

STYRENE ' DIVINYL. BENZENE CATION

8 % DIVINYL-BENZENE

LARGER SCALE

2 0 % DIVINYL-BENZENE

F I G . 3.7. S c h e m a t i c representation of cross-linked s u l p h o n a t e d styrene exchangers. N o t e t h e m u c h m o r e rigid cross-linking for a high di vinyl-benzene ( D V B ) c o n t e n t . (Reprinted from Glueckauf, E . , Endeavour, 1951, 40.)

Of the two processes the displacement method is the faster as once an ion is displaced it remains in the solution of the displacing ion and simply washes off the column, whereas in the elution process t h e eluted ion is continually exchanging on to the resin lower down t h e column and again being eluted. I t m a y also be an advantage to use an aqueous alcohol solution of the displacing agent as the organic ions m a y still

58

CHROMATOGRAPHIC T E C H N I Q U E S

be quite soluble b u t will show decreased basic ionization whereas t h e inorganic ions are much less soluble a n d probably tend t o be retained on t h e resin even more tenaciously. When all of t h e organic ions have been removed, t h e inorganic ones may be eluted with a n excess of strong acid when t h e column of resin will then be in its original form (hydrogen form or cycle). This process is referred t o as regeneration a n d t h e resin m a y thus be used many times for t h e same purpose. Similarly, anion exchange resins of formula RaOH m a y be prepared a n d used for t h e exchange or desalting of organic anions although it is more usual t o prepare a n d keep these in the chloride cycle, RaCl, as this is more stable. Neutral molecules m a y be completely desalted b y passing first down a resin of one type a n d then down a second resin of opposite type. Thus, a solution passed down a cation exchange resin will yield an efluent containing all t h e neutral molecules plus an amount of free acid corresponding t o t h e total salts originally present. On passage of this effluent down a n anion exchanger, t h e effluent will consist only of t h e neutral molecules present. The reverse process of passage down an anion exchanger yields an initial alkaline effluent which is then passed down a cation exchanger t o yield a final neutral effluent. However, in the case of compounds labile in t h e presence of free acid or alkali (e.g. sugars) neither of these methods is satisfactory a n d recourse is made t o the "mixed b e d " or "mono-bed" method. Here a mixed resin, contain­ ing both anion and cation exchangers in equivalent ionic amounts, is used and on passage of the original solution down a bed of this material both cations a n d anions are simultaneously removed a n d t h e effluent contains only t h e neutral molecules. The removal of ions from solution is much more complete in t h e case of a single bed resin when t h e solu­ tion is allowed to flow down t h e resin t h a n when t h e solution a n d resin are simply mixed a n d shaken. However, in t h e case of a mixed bed resin for removal of cations a n d anions the process of mixüig and shaking (batch process) is usually quite adequate. Mixed bed resins can be regenerated b u t as t h e two t3φes of resin have first t o be separated from each other it is n o t usually worth t h e time a n d trouble on t h e small scale required for urine desalting, a n d so t h e resin m a y be rejected after use.

Preparation of Resins for Desalting Cation Exchange Resins. I t is most satisfactory t o purify a n d clean the resin on t h e large scale (1 lb.). The resin is suspended in distilled water and, after stirring, t h e smallest particles, which do not settle rapidly (fines), are poured off. The resin is then suspended in a volume of 4N HCl so t h a t a t least an equal volume of acid remains above t h e resin surface and t h e temperature is raised t o about 100°C. After occasional stirring over about 1 hour, t h e supernatant, which is usually yellow in colour, is decanted and t h e process is repeated several times until t h e supernatant is colourless. The resin is t h e n well washed and the process repeated with 2N or 10 per cent N a O H , when t h e pure resin is converted completely t o t h e Na+ form. I t m a y then be washed and stored in this form or legenerated t o t h e Η form, washed, a n d stored. If it is intended t o use aqueous alcohol on t h e resin it should also be well

DESALTING AND RELATED TECHNIQUES

59

washed with alcohol before storing. Weak acid resins (COOH or OH) are not usually stable t o heat and should be purified a t room tempera­ ture. Columns are prepared by suspending t h e resin in water or dilute acid a n d pouring into a glass tube, 15 X 0-9 cm. being a satisfactory size for desalting of urines, b u t columns much bigger t h a n this should be poured in sections. The column should have a t a p below a n d it is essential never t o let t h e resin dry or t o allow t h e head of t h e solution to sink below t h e surface of t h e resin as, under these circumstances, channels form a n d t h e solution t o be desalted m a y t h e n flow down t h e channel without exchanging ions a t all. After pouring t h e resin column

F I G . 3 . 8 . Simple c o l u m n for i o n e x c h a n g e desalting. T h e o u t l e t t i p is a b o v e t h e level of t h e u p p e r g l a s s w o o l p l u g s o t h a t t h e colimcin c a n n o t r u n d r y .

it should be recycled with dilute HCl, washed a n d converted t o t h e N a form, once or twice, before finally converting t o t h e Η form a n d wasliing again for use. Anion Exchange Resins. Strong anion exchange resins m a y be similarly treated with carbonate-free 2N N a O H and HCl, although it is advisable n o t t o use temperatures above 40°C. Mixed Bed Resins. These should be well washed with a large excess of water, again carbonate-free.

Desaltmg Aminoacid Solutions. A column, 15 cm. χ 0*5 cm., of t h e

resin ZeoKarb 225 is prepared as previously described. 1 ml. of urine, cerebrospinal fluid, etc. is slowly passed through t h e column a n d residual solution is washed through with 15 ml. of water; anions, proteins, a n d neutral molecules wash through. Aminoacids, a n d other organic molecules, are t h e n displaced with 2N ammonia; inorganic cations are retained on t h e column. The first 3 ml. of eluate is discarded and t h e next 12 ml., which contains t h e aminoacids, is collected and evaporated down t o 1 ml. for later chromatography. Taurine is lost through t h e column a n d arginine is retained on t h e resin.

60

CHROMATOGRAPHIC T E C H N I Q U E S

More concentrated ammonia can be used t o displace arginine and other strongly held compounds. The author has not used this procedure, which was reported for use with cerebrospinal fluid only,^^^^ a n d it should certainly be checked for quantity of solution against amount of resin, before being p u t into general use.

Desaltmg Sugar Solutions. See Chap. 13.

Desaltmg with an Ion Retardation Resin, AG 11A8. This resin is formed by polymerizing an acrylic monomer, i.e. a weakly acidic cation exchanger, inside Dowex 1 which is an anion exchanger. Thus a resin is obtained which is capable of exchanging both anions and cations and where, in the absence of other ions, t h e two groups tend to neutralize each other. However, when a solution containing both types of ions is brought into contact with t h e mixed resin, these ions can exchange onto the active sites. The ions so held exhibit weak absorption and so can be eluted from t h e resin with water leaving t h e latter in its original form. I n practice this allows desalting t o occur as inorganic ions are held back (retarded) on the resin whereas aminoacids, peptides, sugars, etc., pass through.^^^'^o) Method. Prepare a 10 X 1 cm. column by slurrying the resin into a suitable tube. Pass 9 ml. urine through the column a t a rate of 2 ml./min. and continue to wash with water. Discard the first 5 ml., collect the next 5 ml. which contains the desalted urine specimen b u t continue washing the resin with another 100ml. water to regenerate i t ; t h e washing stage can be speeded u p to 10 ml./min. The resin tends to adsorb urine pigment but a pretreatment with 20 ml. urine followed by regeneration leaves a pigment-saturated resin which can be used for desalting without interference. A D S O R P T I O N DIALYSIS The process of simple dialysis for t h e separation of large molecules from small ones is well known. The main disadvantage of this tech­ nique, however, is t h e fact t h a t very large volumes of dialysate are obtained during t h e process, if quantitative extraction of t h e small molecules is required, and t h a t this volume must be correspond­ ingly concentrated before a chromatographic analysis is possible. W^en t h e technique is coupled with t h e use of an ion-exchange resin the equilibrium between small molecules inside and outside t h e dialysis tubing is so displaced, due t o t h e exchange of these ions on t o t h e resin, t h a t diffusion continues t o completion and all t h e ions are bound t o t h e r e s i n . T h u s , t h e diffusate volume is reduced from perhaps many litres t o t h e "volume'' of the resin and both anion and cation fractions, as well as t h e residual non-diffusible material, are all available for examination. A 100 ml. wide-necked stoppered bottle a n d a length of 18/32 dialysis tubing equal t o t h a t of the bottle is convenient for this extrac­ tion, although t h e scale can be increased as required. 5-10 gm. of t h e solid t o be extracted is stirred into water a n d placed in t h e dialysis tubing which is then tied with cotton; solutions can be used directly. About 5 g. of resin (say ZeoKarb 225 in t h e hydrogen form) are placed

DESALTING AND RELATED TECHNIQUES

61

in t h e bottle, t h e dialysis tubing is stood in position a n d water is added up t o t h e neck. The bottle is stoppered, sealed with Sellotape, fixed t o a vertical turntable a n d rotated for 24 hrs. The tubing is then with­ drawn a n d t h e resin poured directly into a wide glass column with a sintered glass disc or cotton plug a n d a t a p a t t h e lower end. The effluent a n d dialysis tubing are returned t o the bottle with a further 5 g. of resin and t h e process is repeated when a quantitative extrac­ tion of cations is obtained. The resin columns are then washed with water a n d t h e organic cations are displaced with 5N ammonia; about 90 ml., a t a flow r a t e of 1-2 ml. per minute, is suf&cient for a 5 g. column. The eluate is concentrated under vacuum and made u p t o a suitable volume for chromatography either in water or in water con­ taining a little dilute HCl. If desired, t h e process can be repeated with an anion-exchange resin which takes u p t h e anions a n d these can then be displaced with an acid such as formic acid. The resins pick u p small molecules, such as aminoacids, b u t t h e fate of diffusible ions of intermediate molecular size has n o t yet been ascertained. The technique has been applied successfully t o extract aminoacids (and related nitrogenous cations) from a sohd commercial food, and from various flours and starches, on t o ZeoKarb 225. SOLVENT E X T R A C T I O N T E C H N I Q U E S The principles of solvent extraction are too well known t o be dis­ cussed here, a n d t h e discussion will therefore be confined t o t h e use of particular methods for different tjrpes of compound. 1. Aminoacids and Related Compounds. Aminoacids m a y be ex­ tracted from solid mixtures with a number of different solvents. The solution is first taken t o dryness a n d t h e n extracted in t h e usual way. (a) Acetone or methyl ethyl ketone containing 5 per cent of 6N HCl quantitatively extracts aminoacids a n d related compounds from solid containing inorganic salts. After three extractions ( 3 x 5 ml. of solvent per 1 ml. of original urine), a small volume of water is added, t h e ketone is blown off in t h e cold a n d t h e residue can be used directly for chroma­ tography. The method is not satisfactory in t h e presence of indoles as some condensation m a y occur. (6) Ethanol containing 10 per cent of Ν HCl m a y be substituted for the above ketone solutions with similar results. (c) Butanol saturated with I N HCl or 0-lN HCl (85/15 volumes) m a y be used as above. The method is particularly useful for aromatic compounds although many, if not all, aminoacids are extracted b y this procedure and, providing t h e solution is kept well cooled, it is useful for indoles. This method m a y equally well be carried out on t h e original solution, as t h e butanol is immiscible with water, although it is necessary to adjust t h e solution t o be I N or 0-lN with respect t o HCl before extraction with t h e acid butanol. The above methods have all been used with success t o desalt plant extracts and hydrolysates containing much salt. The butanol method has been found most satisfactory for extracting iodoaminoacids from alkaline hydrolysates. Although these methods have not been used by

62

CHROMATOGRAPHIC T E C H N I Q U E S

t h e author t o desalt urme, they have been used b y other workers a n d should be as valuable for urme as for plants. 2. Sugars. Sugars are very soluble in pyridine containing small amounts of water. Four volumes of pyridine are added t o t h e original solution (urine m a y be concentrated first) when inorganic salts slowly precipitate a n d are centrifuged off. The supernatant m a y be used directly for chromatography if the resultant sugar concentration is high enough. Alternatively, solids m a y be extracted b y warming with 80-100 per cent pyridine, or by refluxing with pyridine alone, as sugars are soluble in the boiling liquid. Epimerization m a y occur (Chap. 13).

3. Barbiturates, Phenolic Acids, Ketoacids, etc. The quantities of

these materials present in urine are usually very small and concentration is an essential prerequisite t o chromatography. Therefore, these materials are extracted into organic solvents for concentration and this effectively desalts t h e m a t t h e same time (see t h e relevant chapters).

Choice of Desalting Technique The choice of a desalting technique will depend on t h e apparatus available; on whether t h e desalting is being used as a preliminary to chromatography (when only small volumes need t o be treated), or t o isolation (when large quantities of sohd or solution are t a k e n ) ; a n d on whether a n y chemical conversion or loss of some of t h e substances of interest occurs. Desalting Prior to Chromatography. There can be little doubt t h a t electrol3Í}ic desalting is t h e most suitable a n d convenient technique for routine work. The time of desalting varies from 5-15 minutes (for 1-2 ml. of urine) u p t o a number of hours (concentrated plant extracts) but, in spite of this, there is practically no dilution of t h e original vol­ ume of solution a n d recovery is in t h e order of 100 per cent. Provided t h a t a minimum of 2 ml. is placed in t h e desalter it is rarely necessary to do more t h a n replenish t h e aspirator acid occasionally a n d t h e ammeter indicates when desalting is complete. B o t h ion exchange and solvent extraction methods require much more of t h e operator's time and they usually result in dilution of t h e original; t h e latter method may be t h e least ef&cient in certain cases (e.g. ketone-HCl or ethanolHCl extractions are not as satisfactory as t h e other methods for aminoacids) a n d suffers from t h e further disadvantage t h a t pigments are also extracted a n d m a y interfere with t h e chromatography. All sugars a n d t h e great majority of aminoacids are chemically unaffected b y electroljrtic desalting and, in m a n y instances, t h e actual conversion of one compound into another is of little consequence for the purposes of identification, provided t h a t t h e existence and nature of the conversion is known (see pp. 48-52). All strong acid conjugates are physically lost a n d so t h e method cannot be used for desalting these compounds, although it might be useful t o determine whether an un­ known compound is indeed a conjugate of this type. Desalting Prior to Isolation. This question is dealt with elsewhere (see Chapter 38). I n t h e study of a particular family of compounds, say t h e naturally occurring sugars in plant and animal juices, it m a y be of value t o

DESALTING AND RELATED TECHNIQUES

63

eliminate not only inorganic salts b u t also as m a n y other types of compounds as possible and, therefore, the ion exchange methods, or the solvent extraction procedures, m a y yield a more suitable product t h a n the electrolytic one even though this necessitates greater expenditure of time. However, in certain clinical conditions, say galactossemia, it is of value to study the aminoacid p a t t e r n as well as the sugar present and, in these cases, the electroljrtic method AIR I N L t r is the only one which allows of the removal of inorganic salts without t h e simultaneous separation of aminoacids TO D E S I C C A T O R « from sugars. There can be little doubt t h a t , as our knowledge of metabohc variations due t o disease increases, it VISKING T U B I N G will become necessary t o study not only the variations within a particular family, such as the aminoacids or indoles or sugars, b u t also t h e varia­ tions and interrelations between these ÜLTRAFILTRATE families and, at present, t h e electro­ lytic method is t h e one which results in the least loss of potentially interesting F I G . 3.9 A p p a r a t u s for ultrafiltra­ compounds. tion. T h e larger t u b e containing t h e V i s k i n g t u b i n g and ultrafiltrate is s t o o d in a v a c u u m flask containing ice. T h e lower figure s h o w s t h e m e t h o d of inserting t h e dialysis t u b i n g right through t h e rubber b u n g a n d t h e m e t h o d of holding it in position b y m e a n s of t h e glass tubing.

ULTRAFILTRATION

Biological fluids of high protein content need to be deproteinized before they can be analysed b y chromatographic technique. Depro­ teinizing should be carried out before the biological fluid is desalted. Ultrafiltration is the best method for removal of the protein as under the conditions set out below it does not alter t h e relative or absolute composition of t h e fluid as far as t h e constituents of low molecular weight are concerned. A further advan­ tage is t h a t the ultraflltrate is for all practical purposes sterile and therefore has much improved keeping properties (see Vol. 2, Chap. 1). Method. Ultrafiltration is carried out through Visking seamless cellulose tubing as used for dialysis ;* a 10 cm. length of J in. diameter tubing has a capacity of 2-5 ml. The tubing is first softened b y thorough wetting in distilled water. One of the open ends of the tubing is then inserted into the hole in the rubber bung and pushed right through until it projects at the top of the bung. A glass tube of appropriate size is inserted through the tubing and into the bung, thereby holding the membrane in position. The opposite end of t h e tubing is then knotted and pulled tight. Care should be taken to ensure t h a t creases in the membrane have not produced weak points. This assembly is mounted inside an 8 X 1 in. test tube with a. side arm, t h e glass tubing projecting through the rubber bung t h a t closes the test tube * Obtainable from H u d e s Merchandising Corporation, L t d . , 52 Place, L o n d o n . W . l .

Gloucester

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CHROMATOGRAPHIC T E C H N I Q U E S

(see Fig. 3.9). The side arm of the test tube connects with a desiccator. Before filling the Visking tubing the desiccator moistened with water on the inside is exhausted at the water-pump and with its t a p closed is connected with pressure tubing to the side-arm of the large test tube. The Visking membrane is now filled with the solution through a fine teat pipette, with each addition the vacuum is momentarily turned on as this will extend the membrane to full capacity and at the same time test it for leaks. When the membrane has been filled to capacity, the glass tube projecting from it is connected, as shown, to a second test tube containing a small amount of water. The t a p on the desiccator is then turned to the open position. If the binding at the junction of the membrane with the glass tube is defective it will be at once apparent, as a continuous stream of air bubbles will be seen passing through the water. The test tube containing the membrane is stood in a vacuum flask containing ice (not shown in figure). Filtration proceeds slowly under these conditions. As it takes place a t 0°C the filtrate does not become concentrated, evaporation being reduced to a minimum by keeping some water inside the desiccator. Fluid t h a t has filtered through the tubing is replaced by air which has passed through water. A moisture saturated atmosphere in this part of the system prevents changes in concentration by evaporation of the original solution. The advantages of this arrangement over others is (a) volume changes in the original fluid and filtrate are reduced to a minimum; (b) once set up it needs no attention; (c) a vacuum of 50 cm. mercury, which can be produced by any good water-pump, is sufficient to operate the process and there is no danger of bursting the membrane. The filtration rate for various biological fluids depends on t h e exact composition of the fluid and particularly on the nature of the protein present, e.g. the higher the mucoprotein content the slower the filtration rate. Working with a 10 cm. length of ¿ in. diameter tubing 1-2 ml. of filtrate are obtained at 50 cm. Hg. vacuum in a period of 4 hours from various biological fluids of differing protein content (plasma, urine, cerebrospinal fluid, gastric juice, saliva, and a suspension of faeces). Volume changes in the ultraflltrate are often of no consequence and, in these cases, the method can be simplified and considerably quickened. Thus the air saturator (right-hand smaller tube in the figure) and the vacuum flask can be eliminated and the apparatus connected directly to the water p u m p . A few ml. of urine will filter in as many minutes while serum will require about an hour for 3-4 ml. to filter. Three or four such tubes can be set up in series, using two-armed boiling tubes, with little loss in the time of filtration. This procedure is even more valuable on the large scale. A five foot length of membrane can be used with a tube of 4 cm. diameter and a 1 or 2 litre flask below, and is set up exactly as described for the smaller tube. One such tube filters over a litre in 8 hours with continuous water p u m p suction when the top end of the glass t u b e is connected by means of a sjrphon to a urine reservoir. For overnight runs a pinch cock is used to close the outlet and filtration continues,

DESALTING AND RELATED TECHNIQUES

65

albeit a t a slower rate. A number of large tubes can also be set u p in series. Thus very large volumes can be efficiently ultrafiltered in a very cheap apparatus without t h e tedium of t h e more expensive ultra­ filtration apparatus. The simplest procedure for fiUing t h e membrane tube is as follows. An all polythene wash bottle is filled with t h e solution to be ultrafiltered, the nozzle is inserted through t h e t o p glass tube and down to the top of the membrane when, on squeezing in t h e usual way, the solution runs into t h e sack without t h e formation of air bubbles. When the sack is quite fuU the nozzle is slowly withdrawn, thus filling t h e glass tube as well. T h e tube is connected b y means of the syphon to t h e reservoir, t h e pressure sloΛvly applied a n d filtration commences. Deproteinization can also be carried out with picric acid as described in Vol. 2, Chapters 1 and 5, p . 176. Concentration of Protein Solutions. This is discussed in detail in Vol. 2, Chap. 1. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Consden, R., Gordon, A . H . , a n d Martin, A . J . P . Biochem. J., 1947, 4 1 , 5 9 0 . A s t r u p , Τ., Stage, Α . , a n d Olsen, Ε . Acta Chem. Scand., 1 9 5 1 , 5, 1 3 4 3 . D e n t , C. E . R e c e n t A d v . in Clin. P a t h . 2 n d E d n . , 1951, p . 2 5 2 . S t e v e n s , B . J . , S m i t h , I., a n d J e p s o n , J . B . Chem. and Ind., 1954, 2 4 3 . Stein, W . H . , a n d Moore, S. J. biol. Chem., 1 9 5 1 , 190, 103. Zenisek, Α . , a n d Krai, J . A . Biochem. Biophys. Acta, 1953, 12, 4 7 9 . S m i t h , I., S t e v e n s , B . J . , a n d J e p s o n , J . B . Biochem. J., 1956, 62, 2 P . P i t t - R i v e r s , R . Chem. and Ind., 1956, 2 1 . R o c h e , J . , a n d Michel, R . Bull. Sac. Chim. Biol., 1955, 37, 8 1 9 . W o o d , T. Biochem. J., 1956, 62, 6 1 1 . Anderson, A . M., a n d W y l a m , C. B . Chem. and Ind., 1956, 1 9 1 . P e r m u t i t I o n E x c h a n g e — a series of booklets issued b y T h e P e r m u t i t C o m p a n y , London, W . 4 . W h i t e h e a d , T . P . a n d W h i t t a k e r , S. R . E . J. Clin. Path., 1955, 8, 8 1 . Woolf, L. I . Nature, 1953, 171, 8 4 1 . Gross, D . (Persona] commxmication.) H u n t e r , I. R . , H o u s t o n , D . G., a n d K e s t e r , E . B . Anal. Chem., 1955, 27, 965. Allen, M. J . , Organic E l e c t r o d e Processes. C h a p m a n & H a l l , 1958. I o n E x c h a n g e — A L a b o r a t o r y Manual. S a l m o n , J . E . , a n d H a l e , D . K . B u t t e r w o r t h , L o n d o n , 1959. Rollins, C , J e n s e n , L., a n d Schwartz, A . N . , Anal. Chem., 1962, 34, 7 1 1 . W e i s s , J . B . , 1967 (personal c o m m u n i c a t i o n ) . M c E v o y - B o w e , E . Nature, 1 9 6 1 , 192, 1072.