Column chromatography of lipids

COLUMN

CHROMATOGRAPHY

OF

LIPIDS

ROBERT A. STEIN and VIDA SLAWSON

I. INTRODUCTION THE purpose of this writing is to review methods found to be successful in separating lipid components by column chromatography. Separations of steroids or carotenoids will not be specifically considered. Since column chromatography was last reviewed in Vol. I of this series, techniques such as frontal analysis (and others) have fallen into disuse as methods of separating lipids. Through persistent use, adsorption and partition chromatography have become very powerful techniques in the separation of all types of lipid components. The practising chromatographer may categorize the method he is using as adsorption, partition or reversed phase chromatography, but the evidence indicates that these categories are only arbitrary and that there is a continuous gradation of processes between adsorption chromatography and separation by way of liquid-liquid partitioning. In many instances chromatographic techniques were developed empirically with little attention being paid to the variables later found to be important. Such methods, though not to be discounted as valuable tools to an individual worker, are difficult to describe successfully in the literature because of the effects of the important but uncontrolled variables. Into such a category can be placed the variability of pore and particle size of solid supports, the chemical nature of the surface, and the degree and type of solvation of the support or adsorbate. A general consideration of molecular happenings during the process of chromatography should assist in evaluating new systems or new applications for existing ones. The very nature of chromatography depends importantly on the ability of a solvent to solvate the solute molecule. The facility with which solvation occurs and the competition with different solvent species for adsorbent sites, that will participate in various bonding interactions, can be considered as the molecular basis for chromatography. These interactions include (a) van der Waal's forces, (b) hydrogen bonds, (c) ion exchange, and (d) covalent bond formation. 183)In most cases there is only a qualitative estimate of the direction and magnitude of these effects, but their significance can not be overlooked.

A. Hypothetical Model By considering a molecular model of the chromatographic process, the distinction between types of chromatography can be made to disappear. The chromatography of a compound (A), on a stationary phase (S), will be the result of a 375

Progress in the Chemistry of Fats and Other Lipids number of sequential and reversible steps as A approaches S. As in chemicaI kinetics, the overall rate of the process will be determined by the slowest step o f the sequence. Because A is initially completely surrounded by solvent, as it approaches S either a portion of the solvent shell may be removed, permitting direct adsorption of A on S, or the solvent shell may remain intact, and A, together with its shell, be adsorbed as a unit on S. Kl

Solvent-A-solvent + S

Solvent-A-solvent -- S K3

Solvent-A + S

Solvent-A -- S

Chromatographic separation depends on the fact that the equilibrium constants (K) for these processes are, in general, not the same for different types of compounds. It is not evident as to which step is the slow one. This will be a function of the immobile phase, solvent, temperature and compound being chromatographed. In lipid chromatography there does not appear to be sufficient data to evaluate these variables quantitatively. However, these are the variables with which practising chromatographers contend. To some extent sufficient evidence is available to allow some quantitative estimates of the results of the overall sequence. In this regard, attention may be directed to a theory that permitted prediction of the retention times of many solute compounds, tgse Evaluation of the various energy terms used to define the chromatographic process gave approximations of the quantitative contributions of functional group affinity, activity of the adsorbent, and nature of the eluent. B. Limitations

The two limitations that seem most pertinent to lipid chromatography are sample size and ability to separate compounds from one another. The adsorption affinities of the complete range of lipid classes vary so greatly that arbitrarily limiting the sample load so that it conforms to a linear distribution isotherm may not be necessary. In most instances of lipid chromatography, the isotherms of individual compounds have not been determined. Thus, the selection of the proper column load is necessarily based on conditions that have proved effective in the past. In general, the nearer the adsorption affinities of a pair of compounds are to each other, the greater the necessity for operating a column under optimum conditions. On silicic acid, for example, a great overload of a mixture of glycerides and sterol esters, which have similar adsorption affinities, might not be separated, but these two could be easily separated from phospholipids on such an overloaded column. Pertinent data concerning column loads are discussed below. A second problem that confronts the lipid chromatographer is deciding what type of structural features are distinctive enough to allow the separation of a mixture into pure compounds. It appears self-evident that in order to contribute 376

Column Chromatography of Lipids to a separation, a structural feature of a molecule must be adsorbed to some degree as the molecule is migrating on the column. This is an obligatory condition but not necessarily a sufficient one. Most severe difficulties in natural lipid separations arise from structural features of the fatty acyl portion of such substances. Positional isomers of unsaturated or substituted fatty acids are not usually separated on columns. When the structural feature is situated near either end of the molecule, there are adsorptivity differences of sufficient magnitude to allow separation, but all columns seem relatively insensitive to positional changes in the middle of the molecule. II. CHARACTERISTICSOF VARIOUS COLUMNS A. Silicic acid columns For lipid chromatography, the most widely used stationary phase is some form of amorphous silicon dioxide. The terms silicic acid, silica gel and silica are frequently used interchangeably to describe a general class of partially hydrated silicon dioxide. Various preparations of these hydrated silicas differ in surface area, pore diameter, particle size, bulk density, p H and trace metal content. Columns of hydrated silicas have been standardized with dyes to give a series of activities similar to that established for alumina, s7 SiO2 dried at 300 ° for 3 hr had activity I. Ten grades in all were defined, with the water content increasing to approximately 50 per cent. Accepted views of the nature of the association of water with silica have undergone evolutionary changes. It has long been appreciated that water molecules are either chemically bonded or physically adsorbed to the SiO~. Iler in 1955 reviewed thought concerning bonded water up to that time. 100b At 115 °, physically adsorbed water was believed to be removed, while water lost between l l5 ° and 600 ° represented dehydration of adjacent silanol groups without general reduction in surface area. Above 600 ° sintering was observed with further loss of water and decrease of surface area. The siloxane groups, S i - - O - - S i , were not believed to be rehydrated at 40-50 per cent relative humidity, which was consistent with data obtained at 33 per cent relative humidity, e5 This stability of siloxane bonds to hydrolysis by water vapor is in apparent contrast to the situation in liquid water. The equilibrium solubility of amorphous silica in water amounts to 0.01--0.012 per cent, z indicating constant hydrolysis and formation of siloxane bonds in the presence of liquid water. In connection with the loss of water by heating, it is interesting to note that the heat associated with rehydrating silica in liquid water is a function of the temperature used in outgassing the adsorbent. Following treatment at temperatures in the range of 200-300 ° or higher, there is a decrease in the heat of immersion compared to that obtained with silica degassed at a lower temperature. This was attributed to irreversible loss of hydroxyl groups from the surface at such temperatures with consequent reduction in hydrogen bonding capacity. 2et More recent experiments 11° have modified the concept of bound water and 377

Progress in the Chemistry of Fats and Other Lipids suggest that it is as difficult to remove adsorbed water as to dehydrate silanol groups. The surface of the "activated" adsorbent may thus be a mixture of silanol groups and adsorbed water. It has also been reported that after heating in a vacuum from 100-350 °, free water molecules were still adsorbed on the surface. 13 The optimum hydration is not the same for all silicic acid and must be determined empirically for each batch. 110 The large pore gels have a greater range of deactivation than small pore gels. Optimum pore size and surface area needed for each type of separation are probably different. 1°6 If the molecular area of the compound being adsorbed is low, a fine pore adsorbent offers a better degree of resolution, but if the molecular area is large, better resolution is achieved by a large pore gel. 1°9 It is, therefore, unlikely that an adsorbent of unsatisfactory pore size will be much improved by the usual activation procedures. The surface of adsorptive silicic acid is convoluted and contains S i - - O - - S i and S i - - O H groups as well as adsorbed water. The relative amounts and arrangements of these surface components are functions of the manner of preparation and of subsequent activation processes. The activation process involves adjusting the amount of water associated with the surface. As mentioned above, this water may be either chemically bonded or adsorbed. Four methods are used to obtain the desired state: (1) heat, (2) addition of liquid water, (3) application of controlled humidities and (4) solvent washing. For a given silicic acid preparation, it is likely that these different modes of activation will give surfaces with different properties. Liquid water is more likely to hydrolyze siloxane bonds than is water vapor, thus altering the bonded to adsorbed water ratio. The relative activation energies of removing chemically bound and adsorbed water will probably be different at various temperatures, thereby affecting the ratio of the type of water present in the silicic acid obtained by the heat activation method. The solvent activation method requires additional consideration. I f stringent reproducibility is desired, it is important to control the water content of the activating solvents. Variability in the water content of solvents leads to different residual moisture contents of the activated silicic acid and thus to different activities. 2° Washing silicic acids from several sources containing 0.8 to 5.1 per cent water (Karl Fisher determination) with a methanol-acetone-etherpetroleum ether series led to identical residual water contents (1.6 per cent). However, this final value depended on the water content of the solvents. By adjusting the water content of methanol and ether to 0.14 and 0.02 per cent, respectively, and omitting acetone from the series, a final water content of the silicic acids of 1.6 per cent was obtained. Although omitting the acetone from the washing sequence did not alter the final moisture content appreciably, the chromatographic characteristics of the adsorbent were different. Apparently methanol is displaced by acetone, but not by ether from the column, and its continued presence on the adsorbent's surface imparts different properties. Solvents that are capable of participating in hydrogen bonding can remain on 378

Column Chromatography of Lipids the adsorbent after the washing process, thus changing the surface character. This phenomenon is not restricted to the activation process. During a chromatographic run, the solvents used can change the adsorbent during the chromatography itself. This has been shown clearly by the preferential adsorption of one compound of a solvent pair. 21~ The ether-ligroin and ether-benzene systems give a solvent front emerging from the column that is about 10 per cent poorer in ether than the original mixture. A portion of the ether that remained on the column must be bound by hydrogen bonds. If subsequent compounds passing over this column surface do not displace the ether, the chromatographic surface will be different from what it was before the ether was present. In connection with changes of column characteristics induced by actions of solvents, the appearance of silicic acid in fractions eluted by methanol should be mentioned. This occurrence has been noted by many investigators. It does not appear to involve mechanical transfer of particles, but rather the dissolving and reprecipitation of silicic acid. Recalling the reversible relationship between structural and adsorbed water that was mentioned above, it is entirely consistent with this process to expect methanol to form esters on the silicic acid surface. The formation of enough of these will permit an incomplete ester of a small polysilicic acid molecule to dissolve in the methanol and be eluted. Incomplete esters of polysilicic acid are known to polymerize on standing into insoluble polymers, l°°a This repolymerization completes the process whereby methanol can continually "bleed" silicic acid from a column. For some lipid separations the method of column activation is seemingly unimportant. For others it can be the determining factor in whether separations are possible. The approach to these separations is still largely empirical and is destined to continue in this manner until a greater knowledge of the adsorbent's surface is obtained.

I. Modified silicic acid columns Substitution of a portion of the hydroxyl groups on the silicic acid surface can change the surface's adsorption properties. Finely ground silicic acid in an ethereal solution of diazomethane was etherified to the extent that there were 0.6 meq/g of methoxy groups associated with the silicic acid. 183 The adsorption of methanol on the etherified gel was only one-fourth of that on the untreated gel. Other organo derivatives of silanol groups have been prepared by reacting acetic anhydride, benzoyl chloride and alcohols with silica gel. 47 Of much greater significance for lipid investigations are the columns modified by the addition of silver nitrate to the silicic acid adsorbent. 219, 220 Silver ions are available to participate in complex formation with the ~r bonds of unsaturated fatty acids. Since geometric isomers as well as numbers of multiple bonds are complexed preferentially, separations of these classes of compounds are possible. No evidence is available that pertains to the distribution of the silver on the silicic acid. It is possible that the silver ions are either adsorbed on the surface, incorporated into the lattice of the silicic acid, 157 or form salts with the silanol 379

Progress in the Chemistry of Fats and Other Lipids groups. In connection with the latter possibility, it may be noted that silicic acid acts as a weak acid ion exchanger for many cations of inorganic salts. For the equilibrium "\ \ - ~ /~/ SiOM + H a, j SiOH -t- M e -~____ the law of mass action appears valid2 2. Structure-specific silicic acid cohlmns Silica gel prepared from sodium silicate solution in the presence of d-camphorsulfonic acid had a distinct preference for adsorbing the d isomer from a dl mixture, d/-Mandelic acid was also partially resolved in this manner. The enrichment amounted to 30 and l0 per cent for the camphorsulfonic and mandelic acid, respectively.43 Similarly, specifically prepared silica gel gave a partial resolution of 2-butanol. 1°8 The resolution of racemic compounds does not pretend this use for the lipids, but there are intriguing possibilities that such a technique could be used for preparing adsorbents for specific separations of other types. The precipitation of silica gel in the presence of pyridine, quinoline and derivatives gave gels with the effective diameter of the pores proportional to the size of the molecule present during the precipitation. The gels had a specific affinity for the molecules that were present during their preparation, e2z Although the importance of pore diameter in the adsorption process on silica has been questioned, 196measurements of the heats of adsorption of hydrocarbons of various sizes on silicates in gas chromatography have shown a dependence of pore size and heat of adsorption. 1°6 B. Alumina columns The activity of alumina columns has been established for a series of dyes, resulting in the Brockmann Index. 24 When the adsorbent is deactivated by water, the number and strength of the adsorption sites is decreased. 195 Although the Brockmann Index covers the water added to the alumina from 0--15 per cent, it has been shown that water above the 5 per cent level is eluted by benzene and n-hydrocarbon solvents. 195 During the chromatography of lipids using this type of solvent system, it may be expected that the adsorbent will be modified in character as the water is removed during the chromatographic process. Such a change in the adsorbent during the chromatography can be expected to cause difficulty in obtaining reproducible columns. This will be the case particularly when the water content is not known or specified, as in many instances of the reported use of alumina for lipid separations. Dry alumina (340 ° for 15 hr) has a slight affinity for an increasing number of methylene groups in normal hydrocarbons. 5z A mixture of normal odd-chain hydrocarbons from C2z to C33 was eluted with heptane followed by chloroform. The heptane eluate contained a higher concentration of C23 to C27 and the chloroform contained more Cz7 to C33. A similar chromatographic run on silicic 380

Column Chromatography of Lipids acid (dried at 425 ° for 6 hr) did not show this partial separation. Superficially it might appear that the London dispersion forces between CH2 groups and A12Oa were greater than between CHz groups and SiO2, but this difference may be equally well attributed to difference in pore and particle size of the respective adsorbents, which could control the extent of dispersion forces in effect at any one time. This general increase of adsorption with increasing number of CH2 groups in a homologous series has become known as Traube's rule and applies to nonpolar adsorbents. The reverse order has been demonstrated in homologous series on polar columns. The failure to note a progression on SiO2 may be indicative of the difficulty in removing all of the surface OH groups from silicic acid. 145e It has been noted that 1-C14-octadecane was not eluted completely from an alumina column by heptane, v8 Despite prior purification of the octadecane by sulfuric acid treatment followed by alumina and silicic acid column purification, about 1 per cent of the C 14 material was eluted by solvents subsequent to heptane. Although a molecular sieve effect was advanced for this phenomenon, 7s another likely explanation may be the self-induced radiation damage of C 14labelled compounds. Such a phenomenon caused an appreciable autoxidation rate of C14-1abelled cholesterol compared to the unlabelled analogue, z09 An alumina column was also used to chromatograph 1-C14-stearic acid. TM Unfortunately, it was not known if a portion of the stearic acid suffered the same fate as octadecane. The acetic acid necessary to elute the stearic acid also eluted some aluminum ion indicating a breakdown of the alumina surface, which would preclude rational chromatographic interpretations. Alumina columns have been used to separate phosphatidyl choline and phosphatidyl ethanolamine from one another; the phosphatidyl ethanolamine was retained on the column longer than the choline derivative. In neither fraction was there a separation of the accompanying lyso compounds, indicating the very minor r61e the presence or absence of a hydroxy group or fatty acid moiety plays in these separations. 120, 170 Since alumina can give alkaline reactions, there is some concern that hydrolysis of ester bonds occurs during chromatography. Rechromatographing phospholipid samples under conditions that did not distinguish between the diacyl and the lyso compounds did not result in a significant change in the fatty acid to phosphorus ratio. 17° This stability of phospholipids toward hydrolysis was also observed earlier. 83 On the other hand, the hydrolysis of L-a-dipalmitoyl phosphatidyl choline to the lyso compound on alumina was estimated at about 1 per cent per hour at 22°. 168 Destruction of phosphatidyl ethanolamine and serine has also been reported. 1~6, 1~7,174 Chemical reactions have also been observed during the chromatography of glycerides,16 and a pentabromide precursor of 1-C14linoleic acid, in which some hydrolysis of the primary bromide occurredP 7 The apparent difference in the stability of the same type of chemical bonds to different alumina columns must be reconciled on the basis of different amounts of chemisorption occurring on various preparations of alumina. The transition 381

Progress in the Chemistry of Fats and Other Lipids between reversible adsorption and chemisorption, which involves covalent bond breaking and making, is a function of the intimate character of the surface of the adsorbent. There is insufficient knowledge of the surface structure to know if the ease of transition between adsorption and chemisorption is inherent in the alumina surface or related to specific features that are only present in some preparations. The diversity of results obtained by using alumina columns suggests that the latter reason may be the correct one. C. Other adsorbents Magnesium silicate, perhaps because of its more recent availability as a chromatographic adsorbent, has not been used for lipid separations as frequently as silicic acid and alumina. Florisil is the registered trademark of the Floridin Company for a coprecipitate of silica and magnesia. Because of its high capacity for phospholipids, it has been used in conjunction with mixed ion exchange resins for removal of lipids interfering with the anthrone reagent from cerebrosides. 167 It has also been used in the isolation of cerebrosides. 1°7, 165,166 Advantages in the separation of neutral lipid classes were rapid flow rates and good recoveries (95-105 per cent). 3° Separation of these classes followed the usual silicic acid pattern of hydrocarbon, cholesterol ester, triglyceride, cholesterol, diglyceride, and monoglyceride. Fatty acids were bound considerably more firmly than to silicic acid and consequently did not interfere with triglyceride fractionation as may occur on silica. High heat treatment provided an adsorbent active enough for complete separation of paraffin from squalene. Extraction of Florisil with concentrated HC1 increased its similarity to silicic acid, possibly by the extraction of magnesium and sodium sulfate) 1 The possibility of variability of the adsorbent has been considered a potential limit to the usefulness of Florisil in routine analysis. 19v In addition, the acidity of the surface may lead to the chemisorption of basic solutes. 19v The migration of ketones and esters on Florisil was not strongly influenced by carbon chain length.aS~. 193 Florisil proved superior to silicic acid and alumina in permitting reproducible detection of esters by hydroxamic acid formation. 153 Strong adsorption of phospholipid on Florisil necessitated addition of water to the eluting solvent of chloroform-methanol for removal of lecithin, inositol phosphatide, and sphingomyelin. 93b Separation of a number of complex lipids from mycobacteria on magnesium trisilicate (Magnesol, a product of the Westvaco Chemical Company) has been reported291 Magnesol-separated fractions of lipids obtained from 72 strains of mycobacteria were purified further on silicic acid or alumina. A variety of other adsorbents have been explored for lipid separations. A sugar column (saccharose) offered impractically low capacity for separations of phospholipids from fatty acids and glycerides, and MgO used for the same purpose irreversibly adsorbed some phospholipid and fatty acids. 15 Magnesium sulfate was found adequate for separations of 2,4-dinitrobenzenesulfenyl chloride derivatives of mono-, di- and triene fatty acids whereas silicic acid,

382

Column Chromatography of Lipids magnesium phosphate, calcium sulfate, alumina, magnesium oxide, and sucrose were unsuccessful. 189 Probably the most bizarre of column adsorbents is Teflon. Its well known nonwettable and unreactive nature notwithstanding, a column of 60-100 mesh Teflon effected a separation of vitamins A and D, cholesterol and coprosterol which were eluted in that order by a gradient of aqueous methanol. 5 A novel support for the separation of normal saturated acids from branched chain acids has been applied to the purification of the acids of the tubercule bacillus. 33 The different capacity of urea to form inclusion compounds with normal, branched, and unsaturated molecules was the rationale for use of a urea column to separate tuberculostearic acid from stearic acid. Columns of 100 mesh urea, on elution with 1 per cent methanol in isooctane, retained stearic acid completely and allowed most of the branched chain acid to be removed. The effectiveness of a column method based on the successful liquid-solid distribution of fatty acids with urea was not shown. TM Cellulose columns have been used for the separation of gangliosides from each other and from other lipids. 203, 205 Selection of proper column load and solvent changes was necessary, 173 but reproducibility was poor because of channelling, which contraindicated routine use. 174 Gangliosides chromatographed differently on different kinds of cellulose. 23° Polyglycerophosphatides and phosphatidyl choline were eluted before phosphatidyl serine, ls6 Considering the number of hydrogen bonds possible between cellulose and sugar derivatives chromatographed on cellulose columns, it is not an unexpected observation that inositolcontaining lipids were lost on such columns, 6~ and that cotton wool had a particular affinity for phosphoinositides, sl Contrary to this, however, it has been reported that the loss of sugar-containing lipids, and indeed all lipids, was very small on cellulose columns. 2° D. Partition chromatography

Partition chromatography is the term usually applied to chromatography with a fixed liquid phase. It has been pointed out 145e that the use of the designation "liquid-liquid chromatography" is more precise. Since any form of chromatography involves partition between mobile and immobile phases, the use of the term partition to refer to the use of liquid-liquid systems may be regarded as a conventional rather than an intrinsically specific term. In general, the partition columns separate compounds on the basis of their relative solubilities. With homologous series such as the fatty acids, the solubilities are directly related to the chain length and separations are easily made on this basis. Because of the limited solubility of fatty acids and lipids in polar solvents, partition chromatography with a polar immobile phase is somewhat restricted to water-soluble material. 145g One of the more efficient systems involves use of ethylene glycol monomethyl ether-water, 9:1, as the stationary phase. 2a5 383

Progress in the Chemistry of Fats and Other Lipids Monocarboxylic acids from C2 to C14 and dicarboxylic acids from C2 to C14 are separable on a jacketed column in a pressurized system. Addition of bromcresol green to the stationary phase makes the acid bands visible. Many other partition columns using silica gel to hold a stationary phase of citrate buffer,8, 3~,88,192 aqueous NaOH in methanol, 214 aqueous glycine,3s and methanolic ammonia 149, 150 have been used in separating short chain monoand dicarboxylic acids. In partition columns, the ratio of immobile liquid phase to solid support may not exceed a certain value, which is probably governed by interfacial tension. 95, 139 The general range varies from 0.5 to 1.0 part of stationary phase per part of supporting material, 27a although a specially prepared silica gel had a superior capacity.49,150 The stability of a partition system may be disturbed by an excessive sample load 112 or by temperature fluctuation. 95 1. Reversed phase columns

Reversed phase refers to a specific type of partition column in which the stationary phase is non-polar. Because these have been used extensively for fatty acid separation, there is ample justification for considering them by themselves even though the material composing the stationary phase may be of various types. For the separation of common saturated and unsaturated fatty acids, probably the favored stationary phase is mineral oil on a solid support of siliconized silicate. 95 Using mobile phases of aqueous organic solvents decreasing in water content as the chromatography proceeds, n-fatty acids are eluted in order of increasing chain length. Using aqueous acetone as eluent, caprylic and shorter acids are eluted together by 45 per cent acetone (v/v) with capric being separated from the shorter homologues. Increasing the acetone to 90 per cent in small increments makes it possible to separate the even numbered n-fatty acids up to

C24.164,188 One severe limitation to the use of the reversed phase columns is the inability to resolve some mixtures of fatty acids. With mineral oil as stationary phase, for example, oleic acid, threo-9,10-dibromostearic acid and palmitic acid are eluted together, 95 and linoleic acid and myristic acid are superimposed. 42 The temptation to correlate the order of elution with the number of C - - H bonds of the fatty acid chain leads to the empirically useful observation that the introduction of a double bond is equivalent to the loss of two methylene groups. Such a correlation, of course, oversimplifies the effect of the many attractive forces that ultimately determine the partition coefficient. The effect of a change in geometry of oleic and elaidic acids is made manifest by a slight retardation of the trans isomer with respect to the cis 95 although no difference is found between a- and fl-eleostearic acids. 42 The effect of conjugation of multiple double bonds is also slight. Despite the great difference in polarizability of the conjugated triene system compared to the methylene-interrupted triene, eleostearic and 384

Column Chromatography of Lipids linolenic acids are eluted nearly together. 42 Following further the correlation of C - - H bonds with elution, it would be predicted that an acetylene bond would be equivalent to two double bonds, and such is the case for stearolic acid and its isomer, linoleic, are eluted with myristic acid. 42 To some extent the problem of determining the composition of heterogeneous peaks has been solved by chemically changing the double bonds and rechromatographing the sample. Oxidation with permanganate 42, 179 and catalytic hydrogenation143, 164 have been used for this purpose. The use of alternative stationary phases for resolving the overlapping compounds has not received much attention. However, it has been noted that by using benzene-swollen rubber as a stationary phase and eluting with methanolacetone (13:1), oleic acid was eluted between palmitic and stearic acids instead

Table 1. The partitioning of fatty acids as a function of phase proportion and presence of support (Gunstone and Sykes) TM Acid Aqu. Me2CO/Paraffin Kieselguhr K (67~ aq. Me2CO) Girl

10:1

2-1/2:1 2-1/2:1 C18

"10:1

2-1/2:1 2-1/2:1

Present Present Absent

0.16 0.19 1.03

Present Present Absent

0.07 0.09 0.44

o f with palmitic acid as on mineral oil columns, and linoleic acid travels nearly as fast as palmitic instead of with myristic. 14 Considering the polyisoprene nature o f natural rubber, it appears that the tendency of the unsaturated acids to be ¢luted more like the saturated parent on the rubber than on mineral oil suggests an enhanced association between the olefins of the sample and stationary phase or between the methyl branches of the rubber and the double bonds of the acids. The solvent to use for the best separations may be determined by measuring the partition coefficients for the several components to be separated. This has been applied to the common fatty acids as well as a number of oxygenated ones.70 In determining K, the distribution coefficient, conditions should be used as close as possible to those actually encountered in operating the column. Table 1 shows that K is nearly independent of relative solvent concentration but is affected by the presence of solid support, indicating the functioning of :adsorption phenomena in reverse phase systems of this type. 70 With paraffin columns, an acid is eluted satisfactorily by a given acetone concentration if the partition coefficient is 0.16 or larger and will be separated from compounds trailing it whose coefficients are not greater than 0.08. Even though a solvent concentration is found that gives the acceptable K values for effective separations, other factors impose practical limits. The 385

Progress in the Chemistry of Fats and Other Lipids mobile phase must not be so high in organic solvent concentration that it removes stationary phase or so low that the rate of elution is impractically slow. The maximum acetone concentrations found useful for paraffin, castor oil and acetylated castor oil columns are 90, 75-80 and 80-85 per cent, respectively. 7° A silicone stationary phase has been used with an aqueous acetonitrile eluent to separate methyl esters of fatty acids from 12-20 carbons. 6z Mixed stationary phases are not too suitable because of their differential removal by the mobile phase. Dinonyl phthalate and several polyesters were found too soluble to be useful. TM Factice (sulfur-polymerized vegetable oil) was superior to many other hydrophobic polymers for the liquid-gel partition chromatography of non-polar lipids. 89 The mechanism of separation was attributed to simple liquid-gel partition. In the absence of phospholipid and free fatty acid, the neutral lipid classes of natural lipids were almost completely separable by elution with aqueous acetone. As in other reversed phase systems, members of individual classes could be separated by differences in fatty acid composition. However, the chromatographic equivalence of two methylene groups with one double bond resulted in traditional superimpositions. This system could be applied to the subfractionation of all classes except cholesterol esters, and notice was taken of the separation of 1,2- from 1,3-diglycerides. Columns of 152 × 1 cm tolerated as much as 100 mg without overloading, and could beused indefinitely providing the same solvent system was used. Because of the instability of reversed phase columns composed of keselguhrparaffin in the presence of high concentrations of acetone, 200 mesh polythene powder was used instead of the mixture of liquid and solid. 67 Acids from C6-C2o were separated with better than 90 per cent recovery by increasing concentrations of acetone in water.

2. Ion exchange columns Ion exchange resins have been used for columns that do not depend on the polymer's ion-exchange capacity. Separation of compounds that are related to the lipids such as short chain fatty acids (C1-C10), lvs, 185 and the separation o f glycerol from glycols and simple alcohols 177 and monoethanolamine from other amines has been achieved. 176 Two elution techniques are used with these columns. One method, termed "salting-in chromatography", TM is to elute the samples with decreasing concentrations of a salt solution. At high salt concentrations the water available (water not involved with solvating the salt ions) to partition the solute between the resin and aqueous phase is less than at low salt concentrations. The ehromatographed solutes are thus eluted in order of decreasing extent of water solvation (e.g. glycerol before propylene glycol). The short chain acids (C1-C4) are not well separated by this technique probably because of their participation in an ion exchange process as well as salting-in phenomenon, a78 The alternative to aqueous salt solutions as elution solvents is aqueous organic 386

Column Chromatography of Lipids solvents. The term "solubilization chromatography" has been applied to this technique, TM which appears the same in character as the reversed phase method of Howard and Martin. Instead of a non-polar stationary phase as in the reversephase system, the ion exchange resin serves in this capacity. The even numbered fatty acids (C4-C10) are eluted in order of increasing chain length by aqueous acetic acid solutions increasing from 2 to 10 molar, ls5 Capric acid was the upper limit of chain length because lauric acid did not move from the top of the column. Although the reported conditions gave complete separations of the even numbered acids, separations did not appear to be sufficiently great to enable acids differing by one carbon to be separated without some overlap with adjacent homologues. For a general discussion and for references to other compounds whose separations are amenable to this type of column chromatography, attention may be directed to further work in this area. 23 Ion exchange resins have been used for removal of free fatty acids from mixtures with neutral lipids. Amberlite IRA-400 was used for the removal of free fatty acids from lower glycerides 15s with no detectable isomerization of 2-monoglycerides on passage through the column. 17, is0 Elution of free fatty acids from IRA-400 columns with HCI in ethanol-ether caused partial esterification, ts° and complete recovery was not possible, z° Attention has been drawn to the possibility of hydrolysis or isomerization of phosphate esters on acidic ion exchange resins. ~2 The anionic form of IRA-400 had no catalytic activity for esterification of free fatty acids shaken with methanol although it did catalyze the transesterification of triglycerides. TM Separation of glyceryl-phosphoryl inositol, glycerylphosphoryl serine, and glycerophosphate on Dowex-2 acetate eluted with a sodium borate-ammonium formate system was reported, s5 The behavior of these phosphates was different on Nacite SAR and Dowex-1 even though these resins contain the same quaternary ammonium group as Dowex-2, thereby indicating the importance of the structure of the resin. When choosing basic ion exchange resins, cognizance should be taken of the fact that strongly basic resins react readily with chlorinated hydrocarbons that might ordinarily be used as solvents. ~66 Derivatives of the hydroxy groups of cellulose that have ion exchange capacity have been found superior to ion exchange resins for the chromatography of large molecules. This has been credited to a greater accessibility of active sites. 145'~, 173 Diethylaminoethyl (DEAE) cellulose has advantages over other ion exchange celluloses. 173 The acetic acid salt of DEAE cellulose was particularly useful in the preliminary separation of complex brain lipids into the following groups: (a) lecithin, sphingomyelin, ceramide, cerebroside, cholesterol and lsyolecithin; (b) phosphatidyl ethanolamine; (c) water-soluble non-lipids; (d) phosphatidyl serine and ganglioside; (e) cerebroside sulfate, inositol phosphatide and cardiolipin. However, some doubt has been expressed about an adequate separation of gangliosides on DEAE or silicic acid, particularly in connection with their partial degradation. 210, 23o Preliminary separation of anemone lipids on DEAE before purification on silicic acid permitted isolation of a new sphingolipid, 387

Progress in the Chemistry of Fats and other Lipids

ceramide aminoethylphosphonate. 174 Similar elution of a Dowex-2 column witb acetic acid-ammonium acetate buffer allowed separation of glycerophosphoryl inositol, diglycerophosphate, glyceryl glycoside sulfonate, and glycerophosphate. 123 By using Dowex-1 formate columns and elution with a gradient of ammonium formate, separations of p32 and S3S-labelled glycerophosphoryl glycerol, sulfodeoxyhexosyl glycerol, and a cyclic glycerophosphate were obtained. 122 A combination of basic Duolite A-7, acidic Dowex-50 × 4 and Florisil was used to remove ionic lipids such as strandin, ganglioside, and cerebronsulfonic acid before anthrone determination of the remaining cerebroside. 167

3. Gel filtration A three-dimensional polymeric network, in general termed xerogel,54 can be used as a molecular sieve. The relatively large molecules do not diffuse or diffuse poorly into the gel network and are eluted before the smaller molecules that wander in and out of the labyrinth channels of the gel. Polystyrene swollen with benzene has been used for the separation of phospholipids from triglycerides, sterols and sterol esters. 2°8 Although the molecular weight differences between these species are relatively small, the separation is effective because the phospholipids have a micellular structure in non-polar solvents whereas the other compounds do not. The large micelles are retarded less in the passage through the column than the independent molecules. Further discussions of the theory and experimental implications are available. 117 In a method similar in some respects to gel filtration, polystyrene was used for "inverse phase" partition separation ofcerebroside from cholesterol and esters. 167 The upper phase of a mixture containing toluene-ethanol-water, 1:4:5, was mixed with polystyrene and served as the stationary phase of the column. The toluene probably swelled the polystyrene as the benzene did in the example above. The lower phase of the toluene-ethanol-water mixture was used as the eluting agent for cerebroside. The addition of chloroform-ethanol-water, 10:8:1, caused elution of cholesterol and esters. III. EXPERIMENTALCONSIDERATIONS Because of the multiplicity of variables involved in the chromatographic process, a discussion of some of the more important practical considerations may facilitate choice of appropriate conditions for successful separations.

A. Use of Adsorption Data An adsorption isotherm shows the dependence of the total amount of solute present in a system to the ratio of the amount adsorbed on some insoluble phase compared to that in solution. The shape of the curve obtained from such data has important implications with respect to the loading of columns. Ideally the amount of sample is selected so that there is a linear relationship between the adsorbed/dissolved ratio and weight of sample up to that used for the column 388

Column Chromatography of Lipids load. There appears to be very little data on lipids that permit selection of column loads on this basis. However, linear adsorption isotherms have been demonstrated for other types of compounds, a , 213 For adsorbents with markedly heterogeneous surfaces, as alumina or silica, the linear capacities for pure compounds are relatively low, of the order of 10 -4 g/g.198a The linear capacity of these extremely heterogeneous surfaces may be improved as much as 100-fold by partial deactivation with water, 198a but the total capacity is decreased. ~94 The relationship between the adsorbed/dissolved ratio and a changing solvent concentration has more immediate practical importance for lipid separations than the adsorption isotherms. The amount of one compound adsorbed as a function of solvent concentration is not the same for different compounds. A curve of per cent adsorption on silicic acid against solvent concentration has been used to show the value of such measurementsP 0 In Fig. 1 the curves for cholesterol

I

Adsorption

Values

o Tr'ipolmitin z~ Cholesterol o Dipalmitir) .

0\0

r~

o 80

60 @ o

40 20 I

10

i

I

i

~

i

t

I

20 50 40 50 60 70 80 Per' cent ethyl ether,/pet, etheP

i

90

i00

FIG. 1. The percentage of lipid adsorbed at 20 ° by 4 g of silicic acid from 25 ml of a 50 mg/100 ml solution (Hirsch and Ahrensa°).

and dipalmitin cross. The concentration of solvents at this intersection should elute both of these compounds together from the column. A lower ether concentration should elute cholesterol before dipalmitin and a higher concentration after the glyceride. Similar consideration of paraffin, squalene and cholesteryl palmitate, Fig. 2, shows that the maximum separation of squalene and cholesteryl palmitate occurs between 0.5 and 1 per cent ether, and that no separation would occur at 2 per cent ether. A chromatographic run with 1 per cent ether in petroleum ether showed an excellent separation. When applying empirical curves obtained with single pure compounds, it 389

Progress in the Chemistry of Fats and other Lipids 70

Val tae.s

5O

Adsorption Paraffin " 5quale~ne Cl C h o l e s t e r y l ~tate

o

.o 50 ,J 0-

o

40

x3 d

30 @ 0 0

20

10 1

2

3

Pep c e n t e t h y l e t h e i , / p e t , e t h e p FIG. 2. The percentage of lipid adsorbed at 20 ° by 4 g of silicic acid from 25 ml of a 100 mg/100 ml solution (Hirsch and Ahrensg°). must be remembered that the intermolecular associative effects that are possible during the chromatography of a complex mixture can alter the expected separations.

B. Solvent gradients The elution of compounds that differ markedly from each other in adsorption or partition coefficient will generally require a change in solvent. The change to a new solvent can be achieved in either a discontinuous or continuous manner. Each method has its apparent advantages. From the standpoint of automatic operation, both methods are practical. The advantages cited 59, 90, 231,232 for gradient elution over the discontinuous method are sharper peaks, less tailing, and avoidance of spurious peaks. A similar recitation of advantages is presented along with an additional consideration of why multiple zoning does not occur with continuous gradients. 3, 145b Multiple zoning, the appearance of the same component in different solvent eluents, can cause inhomogeneity in succeeding fractions. The greatest disadvantage of the continuous gradient method is the possible loss of ultimate resolving power for difficult separations. 145b The importance of choosing a gradient suitable for a specific separation was also stressed, zs2 The devices for varying solvent concentration have many designs. The mechanically implemented methods are exemplified by solenoids to switch solvent reservoirs 2°6 and time-controlled motor-driven stopcocks. 124 Several simple pieces of apparatus have been described that are capable of 390

Column Chromatography of Lipids producing linear or non-linear continuous gradients of aqueous systems, 4 butyl or amyl alcohol in chloroform 4s or methyl alcohol in chloroform. 228 When the term "gradient elution" method was proposed, 3 it was mentioned that gradient elution could not improve the separation of compounds over that of standard techniques if the compounds had linear isotherms. The adaptation of a system used for blending organic solvents 46 was simple and trouble-free. The slope of the convex gradients was adjustable by alteration of the mixing chamber. Production of linear gradients was possible with the Parr combination of adjacent cylinders. 156 However, hydrostatic equilibrium restricted linearity to solvents of equal density. Utilization of vessels of different shapes allowed production of linear and concave, as well as convex, gradients. 12 It was observed that with a reservoir cross-section area smaller than the mixing chamber area, convex gradients could be obtained with the Parr apparatus, but density differences still caused complication. A more complex arrangement with considerably greater flexibility was subsequently described. 159 A series of nine mixing chambers was capable of producing a wide range of concave gradients, both compound and simple. Density deviations caused by hydrostatic levelling affected this system, as well as an all-glass simplification. 116 The effect of either a continuous or discontinuous gradient procedure on the resolution of some lipids is not completely understood. It has been reported that gradient elution of serum phospholipids gave a less sharp separation than stepwise elution ;90 that gradient elution caused considerable overlapping of the phospholipid classes that were completely separable by stepwise elution ;81 and that separation of egg phosphatidyl choline from lysophosphatidyi choline on silicic acid was adversely affected by gradient elution with chloroformmethanol. 22a On the other hand, the superiority of gradient elution for certain fractionations has been as clearly demonstrated. A gradient of chloroformmethanol with increasing amounts of water with an alumina column gave a better separation of the sugar-containing cerebrosides and sulfatides from ethanolamine-containing phospholipids than stepwise elution. 12s Cerebroside and sulfatide were then completely separated on silicic acid, with an increasing gradient of methanol in chloroform. Similar gradients allowed separation of esters, phosphatidyl ethanolamine + ethanolamine plasmalogen, and sulfatide + lysophosphatidyl ethanolamine. 127, 129 The ameliorating effect of gradient elution on the separation of sphingomyelin from phosphatidyl choline was depicted in an application of a concave gradient to complex lipid chromatography. 231 Separations of carboxylic acids obtained with one of the very earliest devices for automatically increasing solvent polarity were indicative of the method's potential.aS, 13s The enhanced resolution of the short chain acids was attributed to the balancing of the deterioration of the peak by diffusion with an increase in solvent polarity. Major technical problems in the application of gradient elution to lipid chromatography are the use of solvents of unlike density and the necessity for 391

Progress in the Chemistry of Fats and other Lipids all-glass systems to eliminate introduction of contaminants. A simple method for production of continuous concentration gradients from pairs of eluents of unequal density has been described. 233 Linear, concave, and convex gradients can be produced by alteration of the dimensions of the reservoir and mixing chamber. An "empirically modified" equation describes relationships in the system, and a nomograph is provided for choice of proper dimensions of reservoir and mixing chamber. Actual gradients produced with common pieces of laboratory glassware agreed well with calculated values. A homograph for determining the composition of solvent leaving the mixing chamber in a constant volume system is available. 223 lts utility for predicting optimal solvent mixtures for stepwise elution schemes was demonstrated. Variation of solvent concentrations is a very flexible aspect of elution chromatography. Generalizations about the resolving power of several procedures seems pointless, however, unless the comparisons are made between methods giving optimum results. C. Flow rates

The flow rate that allows equilibrium to be established in the smallest effective theoretical plate will give the most efficient column. 217 At a very slow flow rate, diffusion along the direction of flow of the mobile phase will limit the plate height. 14° As the flow rate is increased, the rate of diffusion between the phases of a partition column or to the adsorbing surface becomes the controlling factor. A flow rate faster than that required for establishment of equilibrium in the smallest possible plate height will adversely affect column efficiency. The effect of flow rate is shown in Fig. 3. 1-Nitronaphthalene eluted from silicic acid by methylene chloride-isooctane shows that the theoretical plate height is a function of the eluent velocity398b

I0-

o_ 6

•

w z

4

I00 ELUENT

VELOCITY

200 (rnm/rnin)

500

FIG. 3. The effect of flow rate on the theoretical plate height. 1-Nitronaphthalene eluted by 15 per cent methylene chloride-isooctane from silicic acid (SnydedgS). 392

Column Chromatography of Lipids

A summary of flow rates for several types of partition columns14~f indicates that the optimum will be in the range of 2-4 ml/hr cm2 of cross-sectional area. The effect of flow rate on elution from partition columns has been described.27b Using a reference compound, columns run at different flow rates gave a family of elution curves. For practical purposes, the optimum rate was obtained when the peak approached complete symmetry (Fig. 4). The optimum rate at which

70

60

,50

~.0

Effluent volume,

50

20

IO

mL

FIG. 4. Finding the optimum flow rate for a partition column. Curves 1, 2 and 3 represent elution curves for a dye using successively slower flow rate (Bush~Vb).

adsorption columns may be used is probably faster than is the rate for partition columns. On a silicic acid-Celite column, equilibrium was established in less than 10 sec.213 A rapid indication of column efficiency, and thus of the effect of flow rate, may be obtained by using dyes. Inspection will show channelling, band diffusion and front irregularities. However, Zechmeister has pointed out that the bands of color around the outside of a column are not necessarily an indication of the shape of the zone inside the column.2z6 Because of the dissimilarity in structure, polarity, solubility and bonding tendencies, it has been pointed out20o that no systematic relationship between separability of various molecular structures has been demonstrated. Extrapolations from conditions found ideal for reference compounds to lipid components should be drawn with reservations. The use of chromatographic systems under optimum conditions has not been a forte of lipid chromatographers. Flow rate is just one of several variables that is virtually unexplored with respect to lipid separations. Fortunately, many of the lipid classes differ enough in character to enable good separations to be made under non-ideal conditions.

D. Sample size It is difficult to generalize about appropriate sample size without rigorous definition of the chromatographic system and separation desired. One of the 393

Progress in the Chemistry of Fats and other Lipids major disadvantages of adsorption chromatography is non-linearity of elution with sample size. The shape of the adsorption isotherm indicates whether a large sample will travel faster or slower than a small one. If a large sample travels faster than a small one, the chromatographic peak will have a steep front and a long trailing edge. If the large sample travels slower than the small one, the leading edge will be long and diffuse and the trailing edge will be sharp. 14~a The adsorption isotherm is a function of the chemical nature and topography of the adsorbent and their relation to the shape and chemical nature of the adsorbed molecule and solvent. The elution of a compound can be perturbed by a change in any of these variables. In many instances, the practical aims of lipid chromatography have dictated an empirical approach to selection of sample size rather than a more rigorous one. This can be seen from the range of sample sizes used by various investigators. The best possible load is a compromise. Adjacent peaks must not overlap due to excessive overloads on the column, and the irreversible adsorption, sometimes found with excessively small samples, must be at a minimum. 7, 40, z26 Overlap is not the only effect that can occur when a column is overloaded. By applying an alkali metal salt of phosphatidyl serine to a silicic acid column, the ion exchange ability of the silicic acid gave the serine carboxyl group as the free acid, and the compound was eluted as such. 173 The multiple peaks from silicic acid columns for the salts of phosphatidyl serine 1~5, 160 were attributed to exceeding the ion exchange capacity of the silicic acid. It has been reported that basic nitrogen compounds were not completely eluted from silicic acid, and that this effect was exaggerated at low load. 196 Low recoveries were also demonstrated for relatively non-basic substances3 97 With increasing sample size, losses became relatively less. Capacity of an adsorbent is appreciably greater for a mixture of compounds than for a single one. 90, 23~ Reduction of linear capacity for pure compounds was attributed in part to the fact that only a small portion of a chromatographic column was occupied by a given elution band at any instant of the chromatogram.194 Possible intermolecular interactions that can change the chromatographic behavior are not taken into account when adsorption isotherms are measured with pure compounds. The value of these isotherms on "pure" lipid compounds would be questionable because with the more complex lipids, chromatographic homogeneity is frequently used as a criterion of purity. Some approaches to a more systematic choice of sample size have been made. The sensitivity of chromatographic systems to sample size has been demonstrated in the separation of polar and non-polar lipids on alumina. 190 Different proportions of neutral lipids, free fatty acids and phospholipids were chromatographed on Brockmann Grade I alumina. The chief factor influencing separation of uncontaminated triglyceride was the amount of phospholipid on the column although increased amount of free fatty acid also had a deleterious effect. By maintaining a constant amount of material placed on the column, and varying 394

Column Chromatography of Lipids the amount of other components, it was found that increasing the proportion of phospholipid led to contamination of the neutral fraction with phospholipid as well as an incomplete recovery of the neutral material. These Grade I columns failed as the lipid phosphorus load approached 0.4 mg/g alumina. The column loads reported in Table 2 are values that have been used successfully by many investigators. They may be taken as points of departure for definitive work on the exact effect of column load. E. Autoxidation

The susceptibility of polyunsaturated fatty acids toward autoxidation requires that preventive measures be taken during their handling. Chromatography of lipids containing these readily autoxidized compounds is no exception. When deemed necessary, chromatographic separations can be performed at a low temperature on a gas purged column with deoxygenated solvents under a blanket of inert gas. 20, 131, 134, 171,173 Phosphatidyl ethanolamine eluted from a silicic acid column in a system that had not been deoxygenated underwent some autoxidation (as indicated by an increased UV adsorption) compared with material eluted from a deoxygenated system. 118 The autoxidation of unsaturated fatty acid on chromatographic columns was noted very early by Trappe, 211 and the possible autoxidation of 1-C14-octadecane on alumina has been mentioned above. Since autoxidation of unsaturated fatty acids produces polar groups (hydroperoxide, alcohol and carbonyl), these products formed from the simple lipids will have different elution characteristics and will be readily separated from the unoxidized material. The polarity of these new polar groups may not be sufficient to allow separation from the more polar lipid parents, such as the phospholipids. Peroxides have been removed from alumina after being adsorbed. 44 The implication of this is that peroxides in solvents or lipid samples that are removed on a column will be available to initiate radical reactions in subsequent compounds passing them. The addition of antioxidants to limit autoxidative change in susceptible lipid material has been recommended. 19, 21, 27e The addition of 0.005 per cent of 4-methyl-2,6-tert-butylphenol (BHT) was useful in inhibiting autoxidation during the chromatographic process. TM Since BHT is eluted from silicic acid, like squalene and wax esters, with 1 per cent ethyl ether in light petroleum, it will be found in the eluate of any compound moving with this, or possibly with more polar eluents. F. Sample introduction to column

The introduction of a sample to the top of a column should occur in the narrowest possible band. This presents no problem when samples are completely soluble in a small amount of eluting solvent. When the solubility is low, the sample is frequently spread over a large surface and the adsorbed sample placed 395

Progress in the Chemistry of Fats and other Lipids

Table 2. Sample size for chromatographic columns

Adsorption Columns Si02 Si02 Si02 Si02 Si02 SiO2 Si02 SiO2 Si02 Si02 SiO~ SiO~ Si02 Si02 SiO2 Si02 Si02 Si02 SiO2 SiO2 SiOz SiO2 SiO2 SiOz SiOz SiOz SiOz SiOz SiOz SiO2 SiO2 SiO2

LipM Compound(s)

Load (Sample wt/column wt)

10-15mg/g Neutral lipids Neutral lipids 18-20mg/g 17-22mg/g Neutral lipids Naturally occurring lipids Less than 50mg/g Neutral lipid from 100mg/g phospholipid Neutral lipid from 30 mg/g phospholipid 16.4-17.7 mg/g NonsaponifiabIes and neutral lipid for class Nonsaponifiables for class 16.8 mg/g Neutral lipid for class 34.9 mg/g 16.7 mg/g (no more than Serum lipids 2.8 mg of any component) 15 mg/g Serum lipids 4.5 mg/g Plasma lipids Plasma !ipids 4.8 mg/g Methyl esters, Cls, C18:1, 98 mg/g El 8:2, C18:3 Cholesterol esters, C16, 0.6-2 mg/g ClS:I, ClS:2, C20:4 Sterol esters from 14 mg/g glycerides + sterols Pkospholipids 0.3-0.5 mg P/g i 0.35 mg phospholipid P/g I Phospholipids Phospholipids 0.8-1.0 mg P/g Phospholipids Less than 1 nag phospholipid P/g Phospholipids 7.5-8.75 mg/g Phospholipids 6.25-25 mg phospholipid/g Phospholipid from 160 mg/g triglyceride 1.4 mg phospholipid P/g Serum phospholipids 4 mg/g (Twice this amount Sphingolipids gives overlap) Homologous cerebrosides 200 mg/20 × 400 mm col Gangliosides 10 mg/g Lipid from 1 mg Ganglion lipids ganglion/200 mg Purification of mono0.5-0.6 mg P/g phosphoinositide Lipids from blood, liver 5.6 19mg/g intestine, marrow, adipose tissue Lipids of cytochrome 18 mg/g oxidase Nonsaponifiable lipids 33 mg/g Mercuric acetate adducts 1-6 mg/g of unsat, methyl esters 396

Reference 84 7 93b 232 93b 15 114 142 90 40 130 53 115 111 20 84 147 81 79b 93b 187 187 147 226 92 204 116 82 94 137 29 65

Column Chromatography of Lipids

Table 2--continued " Adsorption Columns SiO2~elite SiO2-CaSO4 SiO2-AgNO3 SiO2-AgNO3 SiO2-AgNOa Si02-AgNOa

SiO2-AgNO3 A1203 AlcOa A1203 A1203 A1203 A1203 Florisil Florisil Hydroxyapatite

Lipid Compound(s) Phospholipid separation i Lipid of human serum and lipoprotein Methyl esters, by unsaturation, configuration Methyl esters by unsaturation Sterol esters by unsaturation Triglycerides, by unsaturation, configuration! Cls methyl ester, by unsaturation, configuration Serum triglycerides, by general unsaturation ~o-Hydroxy esters from reduced ozonides Glyceryl ethers Separation of phosphatidyl serine from other lipids Phospholipid (egg) Phospholipids Brain lipids Neutral lipids Neutral lipids a~-lipoprotein from fl-lipoprotein

Load (Samplent/column wt)

Reference

10-15 mg/g 7-15 mg/g

146 41

9-15 mg/g

220

8.6 mg/g

39

40 mg/10 × 120 mm col

71

12-15 mg/g

219

19.5/zM/g

169

1 mg/g

56

20-25 mg/g 30 t~M phosphatidyl serine/g

76, 77 126

50 mg/g 1-2 mg P/g 10 mg/g 6.7 mg/g 10 mg/g 0.25-0.5 ml serum/g

83 79a 45 30 31 18

C~2-~2 acids

1 mg/ml liq phase

95

C~6 24 even acids

3.7-7.4 mg/g liq phase

188

Cls-22 even acids

1.4 mg/g liq phase

199

Partition Columns Paraffin-hydrophobic Celite Paraflin-hydrophobic Celite Mineral oil-hydrophobic Celite Polyethylene-Celite Silicone (DC 200)-Celite Neutral oil~rubber Rubber (from lab. tubing) Reversed phase 20 % MeOH in benzeneSiO2

Chlorophenacyl esters of 0.5-1 mg each compound/I C10-1s, 18:1, 18:2, 18:3 20 g ', Clz-20 methyl esters 4-28 mg/g liq phase 2.9 mg each acid/ml liq C8-24 acids phase Triglycerides 0.291 g/1.2 × 95 cm col. Methyl esters 2.5 g/6 × 46 cm col 2 mg/g Ca-is even acids Fatty acids/hydroper2.5-5.0 mg/ml liq phase oxides/secondary autox prod Fatty esters/hydroperoxides/secondary autox prod

104 62 215 212 164 57

(continued overleaf) 397 2A

Progress in the Chemistry of Fats and other Lipids Table 2--continued

Adsorption Columns

Lipid Compound(s)

Load (Sample ~t/column wt)

Ethylene glycol, monomethyl ether-SiO2

C2-14 monocarboxylic 1.8 mg/ml liq phase acids C2-22 dicarboxylic acids Citrate buffer-SiO2 Decandioic acid from 2- 5.9 mg/ml liq phase decendicic Citrate buffer-SiO2 Ca lo dicarboxylic acids 0.4-0.8 mg/g liq phase Glycine buffer-SiO2 C1-1o monocarboxylic 2.25 mg/ml liq phase acids Cl1-16 dicarboxylic acids Ion exchange (IRA 400) Free fatty acid from 1.0 g/2 x 10 cm col neutral fat Ion exchange (IRA 400) Fatty acids from 30 mg/g glycerides Ion exchange resin Glyceryl phosphoryl 12 mg P/I × 15 cm col (Dowex-1) inositol Ion exchange resin Separation of ganglioside Lipid from 1 kg of brain and other acid lipids on 25 × 250 mm col from total lipid Neutral lipids from Ion exchange resins 1.7 mg/1 × 5 cm col phospholipids and (Duolite-A-7 + Dowex50-x4-Florisil) acidic lipids Fatty esters 300 mg/1.3 x 200 cm col Ion exchange resinAgNO3 moncenes: cis from trans; dienes: trans, trans from trans, cis qcis, trans; cis, cis bound on col Lipid class separations Ion exchange-DEAE 18 mg/g cellulose

Reference

235 224 88 38 16 180 85 202 167 50

174

on the c o l u m n as it is. This is achieved by e v a p o r a t i n g a solution o f the sample in any solvent in the presence o f Celite or the c o l u m n p a c k i n g material. P r e s u m a b l y a thin layer o f the sample coated on the Celite is r a p i d l y dissolved during the c h r o m a t o g r a p h y . A similar system o f e v a p o r a t i n g a solution in a glass thimble a n d placing the thimble on the c o l u m n has been r e c o m m e n d e d . 116 It a p p e a r s obvious that a very small sample a p p l i e d in this m a n n e r w o u l d form an a d v a n t a g e o u s l y smaller b a n d t h a n a large sample. A n alternative m e t h o d is to a d d the sample in a solvent m o r e p o l a r than the first eluting solvent. One p r o b l e m o f this m e t h o d is the tendency to form a wide b a n d due to the partial m i g r a t i o n d o w n the c o l u m n which is caused by the dissolving solvent. G. Column containers

N e a r l y all c o l u m n c h r o m a t o g r a p h y o f the lipids is p e r f o r m e d in glass tubes. It is c o m m o n practice to use the containers repeatedly without m u c h regard to 398

Column Chromatography of Lipids them except for size, but it is interesting to note that the surface of Pyrex tubes used for silicic acid columns was changed with use to the extent that a zone being chromatographed travelled more slowly at the periphery of the column than in the center. This cone-shaped zone did not form when new Pyrex or metal columns were used. 213 An interesting departure from tubular shaped columns was used in preparing large amounts of oJ-fluorooleic acid 75 in a rectangular-shaped container. When the isotherms are not linear, attention to certain mechanical details will improve separation. Necessity for such attention is governed by the difficulty of the proposed separation. In some instances it has been shown that deviations from uniform cylindrical columns are advantageous. Decreasing the diameter of lower portions of a segmented column with mixing spaces between each division had a front sharpening effect.35 Use of a segmented column, which had units joined by capillaries, had a similar effect in elution, frontal and displacement procedures. 72 (See Vol. I, p. 113.) Division of adsorbent by "nodes" of glass wool was beneficial. TM Advantages of such physical alterations were combined in a system designed for preparative isolations. 2°7 Glass sections varying in size between 4 × 20 cm and 8 × 40 cm were connected by neoprene or silicone rubber O-rings. Division of the adsorbent into sections in this manner minimized the increased resistance to solvent flow due to the weight of large quantities of adsorbent. A flow rate of 2-4 liters per hour through 3-4 kg of 100-200 mesh adsorbent has been achieved. Meticulous levelling at the column top is also beneficial. This can be done by adding a layer of Celite, z13 a metal screen disk, 2~3 or a filter paper disk 74 to the column top to prevent disturbance of the column packing. Use of filter paper is sometimes inadvisable for chromatography of phospholipids since cellulose may have a preferential adsorption of certain phospholipid fractions, sl A sample applicator, consisting of a glass stopcock with a liquid reservoir and a bent tip was designed to prevent disturbance of the column top in sample introduction. 28

H. Thermostating The adsorption process is thought to proceed with essentially no activation energy. TM Thus the rate of adsorption will be temperature independent. However, the processes immediately preceding adsorption, such as diffusion and solvation, are temperature dependent, and the desorption process does involve an activation energy. Thus the overall adsorption chromatographic process is temperature dependent. Partition columns being diffusion and solvation controlled are also temperature sensitive. Reproducible chromatographic columns of all types require temperature regulation. Many adsorption columns operated for only a relatively short time during a day are run at ambient conditions with the assumption that the temperature is constant. Time-consuming adsorption and reverse phase columns are routinely thermostated. In some instances, an air jacket around the column is deemed sufficient94 while in other instances, a circulating water jacket is necessaryP o 399

Progress in the Chemistry of Fats and other Lipids IV. SEPARATIONS

A. The effect of intermolecular association on chromatographic behavior Carboxylic acid groups usually exist as dinaers in solvents that are not good hydrogen bond participants. There appears to be no evidence bearing directly on the question of the state of the acids at the time of adsorption from solutions or transfer between phases of partition columns. A mixture of two acids, A and B, can form three possible dimers. If the lifetime of these dimers is longer than the chromatographic process, one might expect to find all three species being eluted from columns. If the lifetime were shorter than the chromatographic process, mixing of the acid moieties would occur, and the eluent would contain evidence of this as demonstrated by the appearance of a single peak. An example of a part of this phenomenon is well illustrated by the chromatography of a

10% SOLVENT

5% SOLVENT Z

o 2~

i!

I--

I~=

I

n~ ~J

CL "10

AND I FORMIC FUMARIC

FORMICAND FUMARIC

r~

- FORMIC

A

~

Z

iI ,FOM'R'O,

0 O w ...1 0 >

FUMARIG

, A, 30

IO

2O

!

40 0 I0 20 FRACTIONNUMBER

30 40

FIG. 5. Chromatography of formic and fumaric acids on a silica gel partition column with 5 and 10 per cent l-butanol in chloroform (Bulen28).

mixture of formic and fumaric acids, Fig. 5. Using a 5 per cent 1-butanol in chloroform solution on a silica gel partition column, the formic and fumaric acids ran together in a single peak despite the fact that when run by themselves, they had different elution characteristics. 26 This indicates that in this solvent, the acids are associated with one another and that exchange of neighbors occurs rapidly. By increasing the amount of butanol to 10 per cent, the carboxyl groups of molecules are probably completely solvated by the alcohol, therefore do not dimerize, and the two acids are completely resolved from one another on the column. Each one travels as a solvated monomer. Influence of similar nature has been noted in the retardation of monocarboxylic acid elution by the presence of dicarboxylic acids on silica gel partition columns, as The change in chromatographic behavior of a substance in the presence of 400

Column Chromatography of Lipids other components appears to be a general phenomenon. The association of the carboxyl group of phosphatidyl serine was used to rationalize the elution of phosphatidyl serine before phosphatidyl ethanolamine on a silicic acid column. 17a Phospholipid has appeared prematurely in etherco-ntaining eluents from a silicic acid column, 125 and a phosphorus-containing lipid has been eluted by light petroleum from a similar column. 172 A phosphatidyl inositol fraction was found to contain free glucose even after a Folch wash. 8° Further examples of unexpected elution of compounds have been noted. 64, 190 All of these examples can be attributed to strong associations between lipid components. The solvent used either in the isolation or in the chromatography or the adsorption process has not been sufficiently polar to solvate the individual molecules so that they behave independently. The importance of the general nature of these associations is further shown by the solubilizing effect that an unsaturated phosphatidyl choline displays in making sodium chloride, serine, glucose and saccharose extractable into chloroform and ether. 6 This ability of lipids to form complexes can also lead to increased solubilization of silicic acid. Using Si81-silicic acid, it was shown that lecithin, cholesterol and liver fat increased the solubility of silicic acid in ethanol by as much as 30 times more than controls. 91 The association of lipids with non-lipid material can be effectively broken by passage through columns used as filter aids rather than as chromatographic adsorbents. Non-lipid nitrogenous substances were removed from egg yolk phospholipids by passage through cellulose or ion exchange columns more effectively than by water washing or dialysis, n9 B. Fatty acid derivatives

One of the major motivations for chromatography of a derivative rather than the original compound is to effect a better separation. In circumstances where the original compounds are to be recovered, simple quantitative procedures are desirable for the derivative formation and reversal. Addition of mercuric acetate to olefins is effective in increasing the relatively small chromatographic differences in esters with different degrees of unsaturation. Under certain conditions, the reaction rates of trans isomers, conjugated dienes, and substituted molecules with mercuric acetate are appreciably slower than reaction rates with simple cis forms. 34, 87 Addition to conjugated double bonds gave unstable products containing less than the theoretical amount of mercury. 162 Mercuric acetate also reacts with acetylene bonds, but the acetylene bond is not reformed when the adduct is decomposed. TM Saturated methyl esters from brain cerebrosides were separated from the unsaturated-mercuric acetate adducts by chromatography on Florisil. 1°7 From Florisil, saturated esters are eluted with Skellysolve B, or Skellysolve B/ethyl ether, 9:1, while the adducts are eluted with ethanol/chloroform/conc. HC1 (10:8:1). The hydrochloric acid decomposed the adduct in the receiving flask. 401

Progress in the Chemistry of Fats and other Lipids Separations have been made on the basis of the number of mercuric acetate groups adding to fatty esters containing different numbers of double bonds. 1°~ This effectively represents a separation according to the multiplicity of unsaturation. From a silicic acid column, the adducts derived from monoene fatty esters are eluted by 50 per cent ether in pentane, those from dienes by 0.2 per cent acetic acid in ethanol, those from trienes by 1 per cent acetic acid in ethanol, and those from tetraenes and higher polyenes by 5 per cent acetic acid in ethanolY Preparative separations have been made on aluminum oxide adjusted to 9.5 -~ 0.2 per cent H20 and pH 6.5 ± 0.3.113 There was little geometric isomerization in the regenerated material. The appearance of 2-4 per cent free fatty acids in the unsaturated fractions was attributed to the acidity of the eluting solvent (1:10 HCl/methanol) and to the acidity of the adsorbent. Some loss of unsaturated hydroxy acids accompanying the formation, silicic acid chromatography, and rectification of their mercuric acetate adducts has been reported. 224 The importance of proper activity and pH of alumina for separation of the mercuric acetate adducts has been emphasized. 22s Low recoveries may be the result of irreversible adsorption or partial ester hydrolysis on alumina. Although complete separation of methyl palmitate and the mercuric acetate adducts of methyl esters of oleic, linoleic, and linolenic acid was not achieved on a single column of alumina (Woelm, Grade II, neutral), substantial fractionation was obtained. Recoveries were 90 per cent or better. The solubility of fatty esters containing three or more acetoxy-mercuric groups per molecule in aqueous solvents were utilized for the preparation of 99.3 per cent pure methyl linolenate by continuous extraction of an ether solution with 10 per cent methanol in water. 229 Thus one might anticipate that liquid-liquid partition chromatography could be a successful method for fractionation of these adducts. Introduction of a hydroxy grouping into a molecule increases its polarity substantially and such substituted molecules are easily separated from unsubstituted ones. Normal and hydroxy fatty esters have been separated on Florisil. 107 The columns were somewhat sensitive to overloading; the maximum recommended sample was 10-15 mg per gram of adsorbent. Silicic acid chromatography of the dihydroxy esters was utilized for the separation of saturated esters from monoenoic esters in overlapping pairs collected from a reversed phase column. 10 Alumina chromatography of the reduced ozonides of ethyl petroselenate afforded separation of the major products, lauryl alcohol and ethyl hydroxycaproate, from each other as well as from methyl esters and diethyl adipate) 6 The adsorption chromatography results, except for diethyl adipate, were reasonably equivalent to the results obtained by gas chromatography) ~ Isolation of mono- and dihydroxy compounds from complex mixtures by chromatography of the nitrate derivatives has been reported. 132, 2z7 Acetyl nitrate reacts with alcohols to yield nitrate esters, and these behave differently from the parent alcohols during chromatography. The original alcohols may be regenerated by lithium aluminum hydride reduction or catalytic hydrogenation of the nitrate ester. Acetyl nitrate also reacts with olefins to give allylic nitro 402

Column Chromatographyof Lipids compounds by a substitution reaction and acetoxynitro and nitro-nitrate by addition reactions. The allylic nitro compounds derived from methyl oleate, namely methyl 8-nitro-9-trans-octadecenoate, 9-nitro- l O-trans-, l O-nitro-8-transand 11-nitro-9-trans- isomers were all eluted from a silicic acid column with 5 per cent ether in petroleum ether. 133 Although these derivatives do not give back the original olefins by a simple reaction, their separation by chromatography offers a further variable to be used in the separation of olefinic fatty acids. The separation of the p-phenylazophenacyl esters of fatty acids by silicic acid chromatography is somewhat more satisfactory for separation of the shorter rather than the longer homologues. A one-carbon difference in chain length suffices for separation of the valerate from butyrate, and a two-carbon difference for the intermediate length esters, but in the sixteen or more carbon region, a difference of four carbons is necessary for complete resolutionP9, 10a The elution pattern is interesting in that the longest fatty esters are eluted first with a benzene/ Skelly B solvent mixture. The chain of the fatty acid derivatives is not polar enough to compete with the solvent for adsorption sites, but should be well solvated in this solvent system. Thus the longer the chain, the greater the tendency is to solvate the complete molecule. The greater the degree of solvation, the greater will be the elution rate. The wide range of melting points for some new branched, unsaturated, or oxygenated derivatives furnishes a useful adjunct to other methods of identification, particularly for certain of the positional isomers. 21s The p-phenylphenacyl derivatives behave similarly on silicic acid. x05 With careful attention to detail, the chlorophenacyl esters of fatty acids ranging from C-10 to C-18 can be separated on a polyethylene column. TM Rectification of the methyl esters of saturated dibromo- and tetrabromostearic acids was obtained by elution from alumina with diethyl ether-pentane mixturesP 6 These separations depend on the adsorption of the carbon-bromide dipole, and the molecules with the fewest bromines are eluted first. Another method of separation of polybromo derivatives of stearic acid has been developed. TM A one-gram mixture of stearic and dibromo-, tetrabromo-, and hexabromo-stearic acids has been resolved on a 12 × 0.8 cm column. The column packing materials which worked equally well were either cellulose powder, Celite, or an equal mixture of Celite and magnesium silicate. The brominated acids were eluted in order of decreasing solubility. Petroleum ether eluted the dibromo compound before the saturated acid. The tetrabromo stearic acid was eluted by ether, and the hexabromide by warm ethylene chloride. These separations appeared to be due to differential solubility and not to adsorption effects. The preparations were free from the more soluble and low melting isomers that probably would have been eluted differently.

C. Lipid separations Many examples of separation of the lipid compounds are given in Table 3. The table has been organized according to type of compounds separated. With 403

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