Recent advances in capillary gas chromatography applied to lipid analysis

Prof. Lipid Re$. Vol. 26, pp. 257-280, 1987 Printed in Great Britain.

0163-7827/87/$0.00+0.50 ~ 1987 Pergamon Journals Ltd

RECENT ADVANCES IN CAPILLARY GAS CHROMATOGRAPHY APPLIED TO LIPID ANALYSIS H . ~ Nestl~ Research Center, Nestec Ltd., Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland CONTENTS I. II. III. IV. V. VI. VII.

VIII. IX. X. XI.

INTRODUCTION FUS~ SILICA AS CAmLLAgY CoLt~t~ MATERIAL C A g g ~ GAS I~CnON T~cn~ot~s Dgr~c'rogs GAS CH~OMATOORAPHY/MAss SPECTROt~-rRY A~ALYsts OF V~OUS I.apm CLASSES A. Fatty acids B. Fatty acid methyl esters C. Other fatty acid esters ANALYSISOF TRIGL¥CI~tlDES ANALYSISOF MONO- AND DIGLYCERIDESTOGETH~ wrrH TR1GLYCERIDES ANALYsls oF PROfrANOmS NEw T~h'DS Pd~n~NCm

257 260 261 262 263 263 264 264 265 268 269 273 273 277 277

1. I N T R O D U C T I O N

Gas liquid chromatography (GLC), ever since its very beginning, has always been a preferred analytical tool for the qualitative as well as quantitative evaluation of complex fat mixtures. This was valid in the days when only packed columns were available and this is even more true today when capillary columns have become an easily available and applicable analytical instrument. When Golay, in 1957, introduced his first capillary columns made from steel or nickel,~s it was at the time a very much overlooked progress in gas chromatography.42"~'~s'7 Only later, with the introduction of glass capillary columns, was the major breakthrough in capillary gas chromatography (CGC) achieved,n'31 In the early days, glass capillaries were basically used for gas solid chromatography (GSC) and surface roughening was the issue. Everything that could possibly attack glass was tried and obviously hydrogen fluoride (HF) was used quite a lot for that purpose. H5'~45Gaseous HC14'5'~35 as well as aqueous NaOH or NH31°9 solutions were also applied and gave varying results. Impressively enough, very fine separations of o- and p-hydrogen were achieved on such columns, as were separations of compounds with different degrees of deuteration such as CHCI3 and CDC13) °9 Already, at the very outset of using glass capillaries, attempts were made to coat them to obtain stationary phases for gas liquid chromatography (GLC). 1959 saw Desty and his co-workers coat apolar phases on untreated glass. 27 The activity of such columns compared to today's standard was quite high and coating efficiencies low, which led to the idea of a pretreatment of the glass surface. Grob in 1965 first tried a carbonization 5! whereas Tesarik and Novotny in 1968 applied HF-etching and used the roughened surface as a support for the stationary phase.m45The idea from the beginning was to increase the wettability of the glass surface in such a way that the polymers which were used as stationary phases spread well and homogeneously. This was and is relatively easy for apolar substances with small contact angles, whereas more polar substances with large contact angles tend to droplet formation, or in the best cases, only form islands of different sizes. 9°,j42 Activity of such columns is still high and, without deactivation of the surface, they are hardly useable. Alexander and Rutten in 1973 and 1974 developed a method to grow NaC1 crystals on the glass surface to obtain the necessary roughening. 4'5 These columns are very sensitive J.v.La. ~/*--A

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(b) FIG. 1. Pyrex glass surface (a) ammonia etched (magnification 12,000 x ) and (b) untreated. [See Refs 109, 155.]

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to water traces in the chromatographic samples. Similar approaches were chosen by Schieke and co-workers in 1975 by growing "whiskers" on the inner glass surface TM and by Grob in 1976, who introduced the BaCO3 pretreatment of the glass surface as a means of roughening to permit coating with medium polarity stationary phases. 54.6°m All these attempts were made to increase the wettability of the glass surface but did not necessarily influence its activity. Due to the presence of silanol groups in the glass matrix, either free or bound with metal ions, the glass surface itself participates in the chromatographic process in such a way that it irreversibly or reversibly retains the various compounds to be chromatographed. Thus, relatively early, capillary column development efforts were undertaken to deactivate the glass surface. Very often Carbowax (polyethyleneglycol) was used as deactivating agent 23 but basically two weak points led to its rejection: Carbowax in its highest molecular weight form as 20 M still has a relatively low degradation temperature of around 220-230°C and, when apolar phases such as methyl polysiloxanes are coated, there is a breakthrough effect of Carbowax through the polymer matrix and thus polarity can be modulated uncontrollably. On the other hand, the glass surface contains varying but significant amounts of different metals such as Na, K, Mg, Ca, Pb, and boron. In hard glasses such as Duran or Pyrex, a noticeable amount of boron replaces the silicon and is an integral part of the glass structure. The alkali metals in particular are bound to the siloxane and will also give interactive reactivity with a stationary phase such as catalytic attack and consequently breakdown of the polymer. Uncontrolled amounts of these metals present in the glass surface thus led always to column stability problems and methods were sought to eliminate these metals from the glass surface. Grob et al. in 1977 proposed an acidic leaching procedure which aimed at the exchange of metal ions against protons. 6° This procedure is carried out in a closed system with aqueous HCI at elevated temperatures (up to 180°C) and over a period of time of ca. 15 hr. This provides a cleaned (leached) glass surface with a sufficient number of free silanol groups which can be further reacted. Before this further reaction can take place, the glass surface has to be dried at a sufficiently high temperature to eliminate even monomolecular water layers, but not exceedingly high in order to avoid intramolecular elimination of water, leading to so-called strained silica groups. These groups are highly reactive and active and would finally participate in the chromatographic process. Optimum drying temperature was found to be 250°C in vacuum. Glass surfaces which have been pretreated by this method are ready for high temperature silylation 4m'6~'m39"~62and for further coating with apolar or only slightly polar stationary phases in the range between SE-30/OV-1 up to OV-17. However, it is impossible to coat medium to high polarity polymers from Carbowax to OV-275 on such surfaces6~ which again necessitates a roughening pretreatment to increase the wettability. An interesting and elegant alternative has been proposed by Traitler and Prevot in 1981 m which combines ammonia etching as roughening and high temperature silylation as deactivation procedure, and thus consequently enables the coating of medium polar to highly polar stationary phases. The latter can only be coated by this procedure, when high temperature silylation agents of mixed functionality such as phenyl-methyl or vinyl-methyl disilanes are applied. 59 These silylation agents lead to an increased wettability p e r se and the combined effect together with the surface roughening produces sufficient coatability for stationary phases such as Silar 10C or OV-275. Silylation at ambient temperature was proposed by Traitler in 1983 and applies to silane monomers which are normally used as coupling agents in fibre-glass reinforced polyester technology. ~'~One factor common to all stationary phases which have been coated by one of the methods initially described is their non-inertness towards organic solvents. Phases which have undergone a long-term chromatographic procedure are partly stripped off and, moreover, are polluted with material that could not be chromatographed, which did not elute and thus underwent degradation and remained in the column. These degradation products participate in the chromatographic process as part of the stationary matrix and influence elution shape and pattern. Generally this influence is highly negative

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and leads to prolonged retention time, to lower resolution and to irreversible and reversible adsorption of chromatographed substances. 66'68'7° Such columns, in general, must be rejected and in most cases cannot be revived at all. Some 10 yr ago the first attempts were made to create so-called bound or immobilized phases to obtain better inertness and, moreover, washability of stationary phases. First attempts included the ex-situ preparation of polymers which, once fully polymerized, were no longer easily soluble in organic solvents. 1°3"~°4For practical purposes later on, higher polymerization degrees which consisted in cross-linking polymer chains were only carried out in-situ, i.e. in the capillary column itself. 13'14'~5-59"62"127-129'158 For a long time, this cross-linking was only successfully applied to methylpolysiloxane phases and included radical reaction being initiated by various radical starters.~8 The latter can be peroxides ~5'S5-Ss'62's9'~28 or azo compounds H9 and one tried to enhance cross-linking by U.V. light, although a considerable amount of this energy is lost by passing through the glass wall in the case of glass capillaries. Phases of the methylsiloxane structure can be cross-linked by these methods, whereas further polar phases such as polyethylene glycol have to undergo other treatments to cross-link and immobilize them. In 1983, Traitler et al. introduced immobilization of polyethyleneglycol by in-situ grafting of silane-monomers onto the polymer chain;~48 cross-linking subsequently takes place between these grafted units and the whole procedure is done in a one-pot in-situ reaction. This method allows the production of Carbowax columns or mixed stationary phases (e.g. Carbowax and OV-275) at varying ratios which show high temperature resistance (up to 280°C), solvent inertness and no surface activity. II. FUSED SILICA AS CAPILLARY COLUMN MATERIAL Ever since 1960, glass has taken preference over stainless steel and nickel as column material in chromatography and after 1979 it was largely replaced by fused silica. Dandenau et al. 25'26 introduced this material as a chromatographic tool which, until that time, was used exclusively in fiber optics. Purity of the inner surface and flexibility of the material, due to the outer coating with polyimide, are the two basic advantages of fused silica. Overall, heat stability as compared to glass is not as good due to the polyimide outer coating, which can be a problem during high temperature silylation° and during long term utilization at elevated temperatures of around 350°C. The fact that fused silica is a much cleaner material than glass makes pretreatments such as acidic leaching unnecessary. Initial concentration of metals of various sorts are negligibly low and stationary phases of almost every type can be coated with less difficulty than with glass columns. As mentioned above, high temperature silylation at around 400°C is impossible, because it leads to a destruction of the outer coating and thus cannot be applied for fused silica capillary columns. Whether or not this silylation is already efficient at temperatures around 350°C is still a topic of discussion,69 but from personal experience fused silica columns coated with apolar stationary phases such as SE-30 seem to have a higher residual activity when compared to similar glass capillary columns. The conclusion to this may be that in the case of apolar stationary phases, which have to be used at very high maximum oven temperatures of up to 350°C, glass capillary columns are superior to fused silica columns. Very recently fused silica columns, with an additional outer reinforcement of steel wires--the so-called Western guitar-string columns--were introduced by Sandra and they seem to overcome to a great extent the high temperature limitations of normal fused silica columns. 127 Thus, it seems that the fused silica capillary column has left its childhood, and is now maturing. To many chromatographers, capillary columns are still somewhat enigmatic, dubious, unreliable, more difficult to handle than packed columns and very often give too much information. It seems as if many chromatographers still regard capillary columns the same way as one synthetic organic chemist did, who used to apply a 6 foot !/4 inch stainless steel packed column to verify the purity and the yield of a synthetic substance. He found

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one big mass of over 90% and one small one of less than 10%. After long discussions with many pros and cons on capillary gas chromatography, he was finally convinced to evaluate his substance mixture on a capillary column. To his great surprise and even greater deception, he found some 10 peaks with still one major peak, but this time far from being 90%. His first reaction was to blame capillary GC for this result and to go back to the packed column. This story has a happy ending, for he could be convinced to continue his purity check on capillary columns and finally he got an even better synthetic result. To overcome the antipathy which still exists with many chromatograpbers who work on packed columns to switch over to capillary columns the so-called "wide-bore" fused silica columns were created) '~43 These columns have an inner diameter of 0.50 mm to 0.53 mm and are coated with films of around 1 g m thickness. Capillary column producers never fail to tell repeatedly how the performance of wide-bore columns is almost equal to that of capillary columns of diameters between 0.2 and 0.3 mm and film thicknesses between 0.1 and 0.3/~m. From theoretical considerations the resolution power of a chromatographic column in GLC is inversely dependent on film thickness and on column diameter, and it is obvious that the best way to increase a column's separation efficiency is by decreasing its internal diameter as well as its film thickness. This is valid for the two types of capillary columns, the support coated (SCOT) '6 and the wall coated (WALCOT) ~ open tubular columns. SCOT columns are hardly used these days and most of the following as well as the foregoing considerations are based on WALCOT columns. The tendency to smaller column diameters is obviously limited to a certain minimum value at which gas flow as well as pressure regulation becomes a problem. Today this lower limit is considered to be around 50 #m i.d. for GLC. For liquid chromatography (LC) in open tubular capillary columns, 50 gm is the upper level which affords a suitable phase ratio between stationary and liquid mobile phases. Column diameters of 10-20 #m would probably be optimal but again are difficult to handle to provide a pulse-free constant flow. m~3For GLC, diameters between 200 and 320/~m have proven to be the most practical as far as gas flow control and manipulability with the columns themselves are concerned. Also the loadability of these columns with sample amounts necessary for gas chromatography/mass spectrometry (GC/MS) is sufficient. Film thickness, which is another critical parameter in most cases is optimal between 0.1 #m up to 0.3 gm and depends very much on the chromatographic behavior of the compounds to be analyzed, i.e. volatility and polarity. Overall column length was an issue in the very early days of capillary columns but has proven to be less critical in terms of resolution power. Column lengths of 100 m and more are applied today 1"4 only for very special applications of difficult isomer separations of fatty acids. For example, even on a relatively short (10 m) capillary column, separation of cis-trans fatty acid isomers can be achieved. Today column lengths between 10 and 30 m have proven to be most practical and are most probably also those most widely used throughout lipid laboratories. IlI. C A R R I E R GAS

The choice of the carrier gas has been a problem and is still today very often determined by practical security rather than by chromatographic considerations. Taking into consideration the large quantities of carrier gas used in packed columns, between 20 and 50 ml/min, it was quite understandable that nitrogen was used as mobile phase. Viscosity and dissolving properties of nitrogen rather disqualify it as an ideal carrier gas in GLC and very early in GLC history, alternatives such as helium or hydrogen were sought. Helium is expensive and, with the same flow as hydrogen, gives higher k-values for a given substance but has the advantage over hydrogen of being inert and not flammable. This latter property discourages many chromatographers from using hydrogen as mobile phase although it gives the best result in the shortest time at equal flow rates compared to helium and nitrogen. Hydrogen detectors are available for nearly every gas chromatographic instrument, and thus the security issue becomes less important.'14 Hydrogen is the carrier gas of choice for all chromatographic analyses on capillary columns.

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FIG. 2. Polarity test mixture according to Grob on an acidic leached, high temperature silylated SE30 glass capillary column of 12 m length and 0.30 mm inner diameter and a film thickness of ca. 0,12 mere. TZ (Kaiser) between El0 and E2 = 29. Chromatographic conditions: 50°C, 2'iso, 3.5°C/min.-150°C. H2:CH4.-elution after 28s. 10--decane, al--nonanal, ol--l-octanol, 1l--undecane, P--2,6-dimethylphenol, A--2,6-dimethylanihn, S---2-ethylhexanoic acid, Ei0----Cl0-acid methylester, am.---dicyclohexylamine,El2---Cl2-acid methylester.

IV. INJECTION TECHNIQUES Basically, we distinguish between two general types of injections in capillary gas chromatography: evaporative injection, and injection and deposition of the liquid sample. Evaporative injection works with heated injectors and with the possibility of splitting the sample at a preset ratio. 3~'32'65All types of hot injectors which include a septum, possibly a septum purge, a controllable split, 3t'77 a p r o g r a m m i n g device to increase temperature of an initially cold injector after injection, ~6'133 splitless devices 53'63"Ta't37as well as the falling needle method are evaporative injection techniques. Liquid injection has several requirements: a liquid sample plug has to enter the column via a cold injector and evaporation of substances other than the solvent only begins after the start of a temperature program. 64'~36 Physical transport takes place immediately and a sort of " c a r b u r a t i o n " takes place with the solvent. To guarantee the introduction of a liquid sample plug, the point of injection must be cold. 64,~36This can be achieved by either a secondary cooling device 35'36 or the simple removal of the front part of the column from the otherwise hot oven. On-column injection (OCI) has the advantage of giving quantitative linearity over a wide range of molecular weight as discrimination of low volatile

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compounds is much less pronounced compared to hot injection techniques: 2,~,lu Some very low volatile, high molecular substance-classes are very difficult if not impossible to chromatograph by the hot injector technique, and, thus necessitate the utilization of an on-column injector. Typical compound classes which are ideally chromatographed via OCI are triglycerides, derivatized or underivatized sterols, long chain alcohols, hydrocarbons, esters and prostanoids. Also, on the other hand, compounds with a very high volatility can undergo a strong discrimination when injected in hot split injector mode. Again, for these compounds, it is strongly recommended to inject via an OCI. Typical lipids in this compound class are short chain fatty acids. In general, it can be said that the OCI technique is widely applicable and can basically be used for all types of lipid analyses. Transfer of low volatile impurities into the column, which are not eluted is obviously a problem linked to on-column injection. This problem can be overcome by cutting off a small piece (50 era) from the column inlet after some time of utilization or, in the case of immobilized phases, to wash the column, e.g. with methanol, in the direction detector to injector. Probably the most elegant and also the most widely accepted method is the application of a pre-column. This pre-column is only deactivated and has no stationary phase. At the same time, this pre-column functions as a deposition site for low volatiles, not chromatographing substances, as well as providing a so-called "retention gap". 67'74 This retention gap has the function of refocusing the injected sample in order to avoid so-called peak splitting which can result from cold on-column injections. 95 This peak splitting is due to a physical separation of the injection sample at the column inlet into two zones of different density. These two zones migrate in parallel throughout the whole chromatographic process and finally give rise to a split peak. This phenomenon is principally due to the fact that, at the very beginning of the capillary column, there is a stationary phase which can interact with the sample deposition mechanism. Apart from using the above mentioned pre-column, it would also be sufficient to wash 50-100 cm of the column inlet phase-free. With the recent tendency towards immobilized phases, this latter possibility has become practically impossible. V. DETECTORS

In gas chromatography, flame ionization detectors (FID) are the most widely used detector types. In capillary gas chromatography, they are very often combined with an inert auxiliary gas such as nitrogen, in order to optimize the flow rate of the arriving carrier gas. Detector geometry is also clearly different for a FID used for capillaries or for a FID for packed columns. It is absolutely essential to place the column exit as close as possible to the flame; this requires a jet configuration which allows the column to enter the jet and thus reduce the dead volume to a strict minimum. The auxiliary gas provides a fast flow from the column exit into the flame and, at the same time, prevents remixing as well as reversible or irreversible adsorption. Other detector types which can be used in combination with capillary columns include electron capture detectors, ~3° hot wire detectors or other specific detectors such as sulfur, phosphor or nitrogen detectors, or ideally, a mass spectrometer. VI. GAS C H R O M A T O G R A P H Y / M A S S SPECTROMETRY

GC/MS has become a widely accepted analytical technique in the last l0 yr and has dramatically increased the reliability of the analytical results, in particular from a qualitative point of view. The most stimulating factor for this development in the GC/MS technique was definitely the qualitative evolution of capillary columns during the same period. Hardly has any analytical method undergone such a rapid development and has found such a widespread acceptance as CGC and GC/MS. The first attempts which were undertaken to combine gas chromatography with mass spectrometry date back to a time when only packed columns were widely available for these purposes and capillary columns were reserved for some enlightened specialists) s.~'92

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Gas flows eluting from a packed column obviously were a problem for the vacuum pumps and this could only be overcome by the application of molecular separators, e.g. the jet separator type) 2°'12~'t23Very often these interfaces separated in such a way that they became blocked, inhibiting arrival in the ion source of the mass spectrometer of any substance eluting from the chromatographic system. Frequent cleaning operations of the separators were most important in this method. Only with the introduction of capillary columns was it possible to abandon jet separators or similar instruments. ~°~ Capillary columns, having typical flow rates of 3 to 5 ml/min, allowed the direct connection to the ion source of the mass spectrometer without any complicated coupling device. Different coupling systems are applied today which include open or closed systems, with or without prior splitting of the column effluent, and, if necessary, via a restriction tube. GC/MS has developed to a stage where the mass spectrometer is only considered as a very specific and reliable detector and recently such mass spectrometer detectors have been developed and commercialized as independent gas chromatography detectors with the obvious consequence of being far more expensive than all other existing detectors. The fact that these detectors are all of the quadruple type implies some restrictions as far as the mass range is concerned, which in the presently available instruments is at a maximum of 600-800 amu. This is definitely not the molecular weight limit of chromatographable substances and thus this has to be looked upon as a considerable limitation in some cases such as triglyceride or prostanoid analysis. Typical cases where GC/MS is of absolute importance are complex lipid mixtures extracted, e.g. from biological tissues where impurities of unknown nature can easily co-elute with fatty acids--derivatized in whatever form---or can even falsely be interpreted as a relevant compound and consequently lead to misinterpretations. Specific detectors such as a mass detector provide an elegant means to overcome this problem and to render the results obtained by this analytical method more reliable. Preferably, mass spectrometry is performed in the single-ion-monitoring mode which increases the sensitivity--and more important, the specificity of the analysis. For this purpose, the most prominent fragment ion or several prominent fragment ions of a substance class are chosen and mass fragmentograms are specifically traced on these ions. Typical characteristic fragment ions in the case of fatty acid methyl esters are the McLafferty fragment at 74 amu as well as the ion at 87. t°'1°7:22'141 Other important examples are fragment ions at 524 amu for prostaglandin PGE2 and 526 amu for PGE~ in negative ion chemical ionization modes (NICI). ~6° This will be discussed in more detail in a later paragraph. VII. A N A L Y S I S O F V A R I O U S L I P I D C L A S S E S

A. Fatty Acids In the past some attempts have been made to analyze underivatized fatty acids t~8 and some stationary phases have been developed in particular for this application. These phases include the free fatty acid phase (FFAP) which is equivalent to the SP 2100 phase. As in most cases, the fatty acids to be analyzed originate from triglycerides or phospholipids or other esterified forms, it appears that a transesterification step is much easier to achieve than hydrolysis of the esters. °'2~'st This has led to decreased interest in hydrolysis except for very specific applications such as measurement of very short chain volatile fatty acids from different biological origins:s'~°~'~39 Under normal conditions, C-2 and C-3 fatty acids cannot be chromatographed as methyl esters and with usual injection solvents such as hexane or heptane. In this case, it is preferable to analyze these acids in an underivatized form. Various stationary phases are suitable, and phases like OV-351 ~ or Carbowax 20M (immobilized)34have been used with success. The preferred injection technique for this type of analysis is the cold on-column injection and it seems to be advantageous to inject in autosampler mode from closed sample vials to minimize losses of the acids due to their high volatility.34Typical injection

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Fla. 3. Chromatogram of standard VFA (WSFA-2, SupelcoInc.) with isocaproicacid as internal standard. Peak identification: first large peak probably formic acid, 1 = acetic; 2 = propionic; 3 -- iso-butyric;4 = n-butyric; 5 = isc~valeric;6 = n-valeric;7 = iso-caproicacid. Apparatus: Carlo Erba Mega 5160 model, equipped with automatic cold on-columninjector type AS 550. Column: 22m × 0.32 mm, Carbowax 20M immobilized; film: 0.25mcm; cartier: H2 at 0.60bar; Chromatographic conditions: 65°C, 2 rain. iso, 8°C/min. to 145°C hold.

solvents in the case of short chain fatty acids are acetone or acetonitrile, both mixed with significant amounts of water and hydrochloric acid. 34 It is quite obvious that only highly inactive and, moreover, extremely stable stationary phases o f the immobilized type will withstand regular injections of such sample mixtures over a reasonable period of time. '4s

B. Fatty Acid Methyl Esters The analysis of fatty acid methyl esters or FAMES, as they are commonly known, is certainly one o f the most widely investigated and used techniques in gas chromatography and many attempts have been made to "organize" this wide field of analytical interest. sJT'u'137 Looking into recent developments on this subject, it seems to be obvious that there is one predominant problem, i.e. the separation o f various positional and geometric isomers. 76,83. I .~i, 164 Thus, different stationary phases have been investigated, which would have the potential of separating all these different isomers. 7'|6':"~'°'~'97"l°|'|'s'm Prime interest here lies in the group o f 18:1 isomers and many isomeric forms may be present in a lipid sample. The more common double bond locations are at A6, A9 and AII and these occur in both cis and trans forms. Many attempts have been made in the last few years to obtain a satisfactory gas chromatographic separation o f these isomers, but it still seems to be impossible to separate

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them all in a mono-dimensional analysis without the combination with other chromatographic methods. It seems very promising to apply thin layer (planar) chromatography prior to the gas chromatographic analysis,s6 Argentation chromatography has proven its validity in preseparating cis and trans classes. 9 RP-18 plate material may be used in a similar manner but has not yet been extensively evaluated. Consequently, double bond isomers can be separated gas chromatographically on stationary phases of medium to high polarity such as Carbowax 20M, '4s Silar-5C, 3 Silar-10C, 7'm OV-275, SP-2340 ~°t or, as recently developed, on SP-2560)" The latter shows potent separation efficiency also for direct isomer analysis, but separations are still not base-line and quantification of the single fractions remains difficult. Column length still has to be long (up to 100 m) leading to prolonged retention times (around 28 min for the group of C18). Typical examples are shown in the following Figs. It should be emphasized that for most purposes, i.e. control of technological treatment such as hydrogenation, a group separation of total trans fatty acids or of total isomers other than oleic acid is sufficient for reaction feedback. Taking the nutritional issue of trans fatty acids into consideration, it seems appropriate to get detailed information on the composition of the total C18 fatty acids present in an

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FIG. 5. Chromatogram of Supelco positional c/s and trans standard (Cat. No 4-5170) [Ref. 144]. Column: 100m x 0.25ram SP-2560, film 0.20mcm, Split injection: split ratio IOO:I, injector: 2OO°C, Chromatographic conditions: 175°C iso, Detection: FID at 2OO°C.,Carrier: H2 at 20 cm/sec.

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FIG. 6. FAME c/s and t r a n s : isomer separation on a short, medium polarity column. Column: Carbowax 20 M, prepared by the cold silanization method, glass capillary column 10 m x 0.30 mm, film 0.12 mcm split injection 20:1, carrier Hz at 0.15 bar. Program: 90°C, 2 rain iso, 3°C/min to 190°C, hold.

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edible fat or oil. For this purpose, a combination of analytical methods still seems appropriate to achieve this goal. In addition to the planar chromatography combination with GC/MS of certain particular fatty acid derivatives, multiple methods could be an interesting and promising approach. 149This aspect will be treated in more detail later. The separation of branched fatty acids of the iso and the ante-iso types 1'79 is an easier task to perform using capillary columns coated with the stationary phases mentioned above. More diffÉculty may be faced with the analysis of very long chain fatty acids such as C22, C24, C26 or even longer. 2'~n'~4°In this case, either cross-linked (immobilized) medium polar stationary phases must be used to reach maximum allowable oven temperatures of around 280°C or more, or less polar or apolar columns must be chosen to allow oven temperatures of up to 350°C and more. Recent development of externally aluminium coated fused silica columns with appropriate stationary phases may even allow oven temperatures of 450°C, but instrumental problems seem to be unavoidable in the long run. ~°2Even so, the very long chain fatty acids may be difficult to analyze and to quantify because long retention times and high oven temperatures may cause irreversible adsorbtion, degradation and breakdown of products which affects quantitative results and long-term column performance. It is especially the very long chain fatty acids such as 20: 5, 22: 6 and even higher carbon numbers which are of greatest interest. This is true for lipid evaluation--in terms of its fatty acid composition--in all biological samples of tissues or plasmas. Prime interest here lies in minute amounts of these fatty acids (e.g. between 0.1 and 2%) and their possible changes due to pathological situations or dietary manipulations. It is absolutely essential that column performance is in accordance with these demands and that there is no preferential adsorption or decomposition of the highly unsaturated fatty acids, for in such a case, interpretation of the biochemical status based on the lipid analysis would be very difficult, if not impossible. C. Other Fatty Acid Esters The recent trend to fast chromatography has led more and more to chromatographic conditions which render the correct and quantifiable elution of very short chain fatty acids

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FIG. 7. F A M E - - s e p a r a t i o n on a long medium polarity column. Glass capillary column Superox 4, 54 m x 0.30 mm, film 0.20 mcm, cold on-column injection. Carrier gas: H 2 at 0.8 bar. Program: 80°C, 1 min iso, 12°/rain to 140°C, 1 min iso, 4°C/min to 250°C, hold.

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as methyl esters an almost impossible task. In particular, butyric acid, which is of prime interest in the analysis of samples from dairy origin, under fast chromatography conditions, almost co-elutes with the solvent or is only resolved partly or in an unsatisfactory manner. This leads to problems in quantification due to to extremely high and even varying response factors for butyric acid. Even caproic acid methyl ester under these conditions still has response factors as high as 3. ~ Taking into account that very short chain fatty acids in many cases of dairy analysis are important analytical parameters, it is obvious that emphasis must be put on reproducible and quantifiable methods for these fatty acids. Optimum resolution of the short chain fatty acids from solvent peaks can be achieved by increasing their molecular weight. 6'~7'8~ Butyl- and pentyl-esters are ideal derivatives and can be prepared by several methods, m~ One of the simpler methods is probably the transbutylation or transpentylation of triglycerides in a manner analogous to that used for methyl esters, ms4 This method includes the preparation of sodium iso-butylate or sodium iso-pentylate and subsequent reaction with the corresponding triglycerides such as milk, cream, butter, buttermilk, skimmed milk and human milk. It was shown that, in the case of pentyl-esters, response factors for butyric acid were close to 1 and resolution from solvent peaks (including unrcacted iso-pentanol) was base-line on immobilized Carbowax columns. TM Attaching a pentyl group instead of a methyl group on a fatty acid not only increases the molecular weight, consequently prolonging retention times, but decreases the polarity of the molecule of any chain length, counterbalancing the prolonged retention times to a certain extent. For short chain fatty acids, the additional molecular weight amounts to 48% with butyric acid and pentyl ester--compared to 16% for butyric acid and its methyl ester. Decreasing polarity leads to shorter retention times on polyethylene glycol stationary phases. For butyric acid, the molecular weight increase is more effective than is polarity decrease, thus leading to markedly longer retention times. For a long chain fatty acid (C16 or C18), polarity decrease seems to be more important than molecular weight gain as the shift towards longer retention times of pentyl-esters, compared to the corresponding methyl esters, is not equivalent to 4 carbons but only to slightly over 2. Consequently, overall retention times are not prolonged dramatically, thus still enabling fast CGC for the routine automatic analysis of fatty acids as esters. ~

VIII. A N A L Y S I S OF T R I G L Y C E R I D E S

Many efforts have been made in the last 7 yr to optimiz¢ and to standardize triglyccride (TG) analysis on capillary columns) 7'39 One of the main obstacles to any gas chromatographic TG analysis is an appropriate injection technique. Is9 Flash evaporizing injectors of the split/splitless type or also the falling needle type require extremely high injection temperatures of near or above 400°C. I1° This induces an extremely high thermal shock, particularly dangerous for polyunsaturated TGs. Monseigny et al. showed interesting results of capillary column application in TG analysis, n° They used a heated injector with injection temperatures as high as 400°C Non-polar columns of the dimethylpolysiloxane type were used in all cases. Although they could show an acceptable linearity of response factors of the different carbon number TGs, resolution of different degrees of unsaturation was not achieved. Grob, K., Jr. et al. showed, in 1980, the application of a cold on-column injector to TG analysis and demonstrated, under the conditions which he had applied, a partial separation of TGs of different degrees of unsaturation. 75Maximum oven temperature was above 300°C and TGs of carbon number 52 and 54, respectively, eluted in this high temperature range between 300 and 330°C. Traitler and Prevot published a base-line separation of TGs differing in carbon number as well as in degree of unsaturation. Is°'ls2 Final oven temperatures in this case were 295°C and 50, 52 and 54 carbon TGs, respectively, ¢luted during the isothermal hold period. Also, in this case, cold on-column injection was chosen and was proved to be by far the best

270

H. Traitler

34

3(

38

50

48 52 40 46

\

32 42

44

54

I

0

I

I0

I

;tO

L

30

I

40

M in

FiG. 8. Separation of triglycerides: Ghec-butter fat. Column: 13mx0.30mm SE-30. Acidic leached, high-temperaturesilylated, static coated film thickness 0.11 mere. Apparatus: Carlo Erba 4160, cold on-column injector equipped with a high oven-temperature secondary cooling device which allows injection on-columnat oven temperatm~'sabove 300°C without pulling the injection point of the column out of the oven (see R©fs 150, 152). Chromatographic conditions: H2 at 0.75 bar, 240°C, l min iso, 3°C/min to 340°C, hold. Legend: 4---butyric acid, 12--laurie acid, 14---myristic acid, 18--stearic acid, 30-58---carbon numbers of triglycerides.

technique for a damage-free sample transfer into the analytical column in the case of low volatile substances such as TGs. Separation of compounds with different degrees o f unsaturation was only achieved based on the total number of double bonds in one triglyceride molecule, but there was no distinction between triglyceride isomers with unsaturated acids in different positions. Under those conditions separation of triglycerides with different isomers of unsaturated acids was not possible. Separation of positional isomers of TGs was shown by Traitler and Rossier (1982) on specially prepared capillary columns containing inorganic salts such as AgNO3, CuCI2, CoCI~ or other similar salts in the dimethyipolysiloxane matrix. ~55 Retention times, which are already long under the above described conditions (40-50 min), for T G analyses, become even longer in the case o f the application of mixed organic/inorganic phases and are around 80-90 min. This and the fact that, in the case of complex mixtures, an overlapping o f different species makes clear-cut separations very difficult, disqualifies this method for routine analyses of T G isomers. However, it can still be very helpful in the evaluation o f preparations such as cocoa butter or palm midfraction in which the positional isomer is of prime interest, because for melting quality reasons, symmetrical isomers of these triglyceride mixtures must be present and must also be controlled analytically. In all cases described so far, dimethylpoly-siloxane--as such or mixed with inorganic salts--has been used for the gas chromatographic analysis of triglycerides.

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271

(b) PPO

(o) PPO

80

POP

85

Min

I

50

55

I

60

Min

FIG. 9. Triglyceride separation: positional isomers. A" separation of positional isomers on a mixed phase column SE-30/AgNO3. 9 m x 0.30ram. B: on a OV-I column, 15m x 0.30ram.

Recently other approaches with stationary phases were chosen. Instead of using apolar stationary phases of the SE-30 or OV- 1 type, phases of "quarter-polarity" were used such as OV-17 or OV-17-1ike phases. OV-17 has a very high temperature stability and can be used at oven temperatures as high as 360°C. Due to its increased polarity, this phase resolves substances varying only very slightly in polarity. 3s This polymer, which is a mixed methyl-phenypolysiloxane has another interesting property. At temperatures of up to 250 ° or 300°C, it shows an elution sequence behavior which is in the normal order for this polymer, i.e. unsaturation before saturation. This sequence is completely changed at high temperatures, i.e. saturation before unsaturation, which possibly indicates a polarity change of the polymer matrix, but even more likely, a restructuring of the polymer which then brings about the changed elution sequence. Consequently, this means that during a temperature program over the full temperature range from, e.g. 80 to 350, there is a point at which this sequence switch will take place, leading to remixing or reapproaching of the separated compounds still in the column, thus decreasing or destroying the resolution. What has been observed, and is valid for stationary phases of the OV-17 type, could consequently also be valid for other stationary phases. Whether there is a critical sequence-inversion temperature for a given polymer matrix during which elution patterns are completely or partly affected has not been studied. If this critical temperature is above the maximum allowable oven temperature of a stationary phase, then obviously there is no danger or risk of degradation of resolution. If this is not the case, this temperature must be determined and evaluated for such stationary phases. Consequently, a stationary phase such as OV-17 or similar phases should only be used at temperature ranges entirely above or below the inversion temperature, not using both regions in one run. For TG analysis the use of the high temperature range between 300 and 360°C is necessary. Sample transfer onto the column at such an elevated temperature becomes a problem and requires a special injection technique. On-column injection technique is the method of choice, but conventional devices to cool injector ports will necessarily fail to cool the injection spot of the column so as to guarantee the sample transfer into the column

272

H. Traitler

POP

PO0

PPP )LP D34

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PLS

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i

D3S

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~IOP

ooo

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soo|

i MLPPPS MLO

SLO

I ~AO0 T46

T4s

T~o

TS2

T~4

T~s 16 man |

340 =

355"(:

FIG. 10. Separation of triglyccrides: palm oil (Ref. 38). Column: 25 m x 0.25 mm RSL-300 (comparable to OV-17) immobilized phase. Chromatographic conditions: H: at ca. 1 bar inlet pressure, cold-on column injection with a movable injector. 340°C, I min iso, l°C/min to 355°C, hold. Legend: MPmyristic acid, P--palmitic acid, P--palmitoleic acid, S---stearic acid, A - arachidic acid, O--oleic acid, L--linoleic acid.

PLL

PLP PLO

OLL

LLL

POP . SLO MLO

~'00 II

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1

330 °

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|V T~;~

P.LL

l~

.....SLL

,oo T54

T~o

IBmin r ...........................

344"C

J- ......

FIG. 11. Separation of triglycerides: cottonseed oil (Ref. 38). Cottonseed Oil. For conditions and legend, see Fig. 10 except: 330°C, I min iso, l°C/min to 344°C, hold.

Advances in capillary gas chromatography

273

in the liquid state. Therefore, it is appropriate, if not necessary, to use a movable on-column injector to be able to completely pull out of the oven the part of the column where the sample deposition takes place, or to keep the site of injection sufficiently cool (ca. 60°C) even with oven temperatures of around 300°C. 2°,72'73'9t This enables solvent migration from cold to hot zones and total elution of the solvent before the injector is pushed down to introduce the sampling zone of the column into the chromatographic oven. Due to its increased polarity under the described conditions, this stationary phase gives excellent results as far as resolutions of differently unsaturated triglycerides are concerned. Moreover, total retention times of even high carbon number triglycerides 54"57are reduced dramatically and a TG analysis can be performed in ca. 15 min without losing the advantages of high resolution. 3s Figures 10 and 11 show typical examples of triglyceride analyses performed under the above conditions with an OV-17 type column. Note the perfect resolution between the various triglyceride species of identical carbon numbers as well as the relatively short retention times. IX. ANALYSIS OF MONO- AND DIGLYCERIDES TOGETHER WITH TRIGLYCERIDES Many problems of the quality of fats and oils are linked to the presence of free fatty acids (FFA) and mono- and diglycerides. In general, it is the latter two which cause problems in technological treatments of fats and oils such as fractionation or emulsification. Unlike FFA, they are not removed from an oil by simple refining techniques and their abundance in refined oils, especially the less volatile diglycerides, reflects to a certain extent the degree of hydrolysis in the crude oils or fats. The correct analysis of mono- and diglycerides is an important issue and can be performed in a very elegant way by gas chromatography on capillary columns. 24'112 It could be shown (Traitler/Prevot) that underivatized mono- and digiycerides together with triglycerides can be separated on high temperature stable non-polar stationary phases such as OV-1 or SE-30.~30'~52However, monoglycerides show reversible or even irreversible adsorption and require the application of elevated correction factors, and a decreased resolution of the monoglycerides. Consequently, it appears appropriate to apply derivatized mono- or diglycerides to gas chromatographic analysis, decreasing adsorption and resolution problems. 99'"7'z63 Silylation is the derivatization method of choice. 9s,~24,m6mTwo major requirements must be met in this type of silylation: (1) long-term chemical stability of the derivatives and (2) low contamination, preferably volatile by-products, to eliminate interference with the products to be chromatographed and quantified. An ideal derivatization reagent which fulfills the described requirements and which is easy to apply is BSTFA (N,O-bis trimethylsilyltrifluoroacetamide). ~°° Response factors are evaluated for the various mono-, di- and triglycerides and good linearity was observed between mono- and diglycerides, including triglycerides of carbon number up to around 42. Only higher trigiycerides then showed identical deviation to those already observed for triglycerides alone. 3° A typical chromatogram of silylated mono- and diglycerides together with triglycerides is shown in Fig. 12.

x. ANALYSIS OF PROSTANOIDS Gas chromatographic analysis of prostanoids has attracted considerable interest over the last couple of years. '9"33''26 Faced with the problem of very low abundance of these compounds in biological samples in the low nanogram or even picogram range necessitates a specific detection such as mass spectrometry. 5°.82,94 For the same reasons of low abundance, fragmentation in the mass spectrometer should only take place to a very minor extent, and the chemical ionization mode is preferably chosen for this type of analysis. To J.P.L.R. 26/4---B

274

H. Traitler

57

on

Fro. 12. Separationof mono, di- (silylatcd)and triglycerides.For columnand apparatus, see Fig. 8. Chromatographicconditions: 190°C, 1 min iso, 3°C/rainto 230°C,30°C/rainto 300°C, 2°C/min to 330°C, hold. Carrier gas: H2 at 0.75 bar.

increase the sensitivity of the analysis, specific derivatization modes may be combined with negative ion detection. 85,~6° Chromatographic conditions must be carefully chosen to achieve optimum resolution with shortest possible retention times. Short retention times are necessary for minimizing possible thermal degradation of the minute amounts of the various prostanoids in the chromatographic column. Various stationary phases can be used but all require high temperature stability at 270 ° to above 300°C, depending on film thickness, column length and nature of the stationary phase. Two basically different types of polymers may be used: (1) an apolar stationary phase like OV-I or SE-30 and (2) medium polar stationary phase such as Carbowax 20M of the immobilized type to apply the necessary oven temperatures) ~ Also, depending on various degrees of determination, i.e. partial or complete, different columns may be applied. It was shown that methylation of carboxyl groups and silylation of hydroxy groups without methoxymation of possibly present carboxyl groups gives derivatives which can easily be chromatographed on medium polarity immobilized polyethylene glycol columns) 47"15a Separation efficiency is high enough and gives base-line resolution of such prostaglandins as PGEI and PGE2. Figure 13 shows a typical chromatogram of a standard mixture of various prostanoids in the sequence: TxB2, 6-k-F~, PGF~, El and E 2. These types of derivatives are measured mass spectrometrically in a positive ion chemical ionization mode and sensitivity limits in this case are, on the whole, insufficient to be able to detect physiological levels of prostanoids in various biological fluids or tissues. 22 Thus, other ways must be found so as to increase sensitivity. Carboxylic groups can be derivatized as pentafluorobenzylesters, carboxyl groups are methoxymated49 and hydroxy groups trimethyl silylated to give derivatives, which in the mass spectrometer, give one or only a few prominent and intense fragment ions in negative ion chemical ionization mode.l°8

Advances in capillary gas chromatography

275

! .'2a

E,

E2

5K-Ff TXB

I FIG. 13. Separation of prostanoid standards, methylated and silylated (Refs 147, 153). Film thickness: 0.20 mcm. Carrier gas: H2 at 0.80 bar. Chromatographic conditions: 80°C, 2 rain iso, 20°C/rain to 220°C, 3°C/rain to 270°C, hold.

Depending on the prostanoids to be quantified, apolar or medium polar columns can be applied. PGE, and PGE2 can be separated in shorter retention times on an apolar SE-30 stationary phase, whereas TxB: and PGF,~ are better separated on a Carbowax 20M/immobilized.'53 GC/MS analysis of prostanoids, apart from being necessary for avoiding uncontrolled cross-over reactions in radioimmunoassay analyses, is more and more applied routinely for large numbers of samples, and retention times arc an important issue. Reduction of total retention times of above 20 min to below 15 min and thus limiting one analysis cycle at something between 15 and 20 min is a goal which can be achieved by using short apolar columns (ca. 10-15 m) with thin film (0.1-0.2/zm). 4° On-column injection or splitless injection is required to conserve low amounts of prostanoid material in biological samples. On-column injection has some advantages over splitless injection due to its better quantitative reproducibility.64Another means to decrease overall retention times is by choosing an appropriate high boiling solvent." n-Decane gives best results at injection oven temperatures of around 160°C in cold on-column mode. Higher alkanes gives rise to very broad solvent peaks, even at higher oven temperatures (e.g. 200°C) and--a major drawback--arc less reliable as far as impurities are concerned. Decane is an ideal solvent because its evaporation tendency is low, making storage of samples at unchanged concentrations possible even over longer periods of time, i.e. up to 3 months. Quantification of prostanoids is the major issue in this analysis and emphasis has to be placed on reliable methods which give reproducible results. The optimum method is the application of stable isotope standards for each compound to be quantified. Standards are either deuterated (d4) '2 or '80-containing. In most cases, the latter is easier to obtain by enzymatic (transesterase) synthesis but has the disadvantage of being prone to '~2)/180 exchange reactions, thus making quantifications less reliable.4° Primary prostaglandins such as PGE2, PGF~ or primary metabolites such as 6-keto-PGF]~ are available as d4-compounds, whereas thromboxanc B: or PGE~ can be synthesized as '80 products. In the case of PGE,, it is also possible and legitimate to quantify it via PGE:-d4. '~3 Other metabolites such as dinors or tetranors are not commercially available in their deutvrated form and, thus, either lengthy syntheses have to be accepted or quantification is again carried out via another deuterated prostaglandin

276

H. Traitler

such as PGE2. In any case, quantitative linearity must be checked with standard dilution curves consisting of a series of sample mixtures containing unchanged amounts of deuterated or ~sO standards and varying quantities of normal isotope standards. Only this isotope dilution method allows for reliable quantitative results which are safe enough to support or defend a biochemical or nutritional hypothesis. Consideration of the minute amounts of prostanoids to be detected in most of the biological matrices, sometimes as low as l pg per gas chromatographic injection, clearly shows the difficulties encountered on the methodological side of prostanoid analysis. Apart from optimally adapted sampling techniques, which must consider the often very short biological half-life times of cyclooxygenase as well as lipoxygenase products-sometimes below 1 min--the principal issues in this type of analysis are controlled work-up conditions and highly deactivated capillary columns. This latter prerequisite has obviously

1 0 5 M T E * 9 1 3 / 8 / 8 6 NR.I Box: I 100%=954696

Mass r a n g e : -

40-9999

IO0 F

Box : 2

100°/o=605360

Mass range : - 5 2 4

I00%

M a s s range :- 5 2 6

ioo so a Box : 3 ~OO

= 573280

5O 0 Box: 4

oL

I 0 0 % = 173480

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,

Box: I

I00% = 1348

Box

I00%=

100[

:

2

Moss r a n g e : -

298496

612

Mass range : - 614

6K-Fla

1

Box : 3

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D4- 6K-

FI a

I00 I

°°l o

1

60i

I

eo

IO0

r20

FIG. 14. Reconstituted mass chromatogram (NICI-mode) from different prostanoides and their deuterated standards. Column: 1 5 m SE-54 tyl~ column. Carrier gas: I He at i bar. Chromatographic conditions: 160°C, 2 rain iso, 20°C/rain 300°C, 4°C/rain to 320°C, hold.

Advances in capillary gas chromatography

277

been the important issue in all types of lipid analyses by CGC gas chromatography since its very beginning and it has not really changed since. With present physicochemical knowledge in silicon and polymer chemistry, it has only become somewhat easier to solve. XI. N E W T R E N D S

Asking questions which relate to the future trends in CGC may require some sort of prophecy but, on the other hand, some of the trends which have developed over the last couple of years are very likely to be continued in the future. There are three trends which in the next years may be of even greater interest than they are already today: short columns, thin films, and higher maximum oven temperatures for all classes of polarities. All three subjects very obviously aim towards the general theme of higher overall analysis speed at minimum necessary resolution. In routine analyses of known mixtures, this goal is relatively easy to achieve but it can become dangerous in the analysis of complex unknown mixtures where high speed may lead to loss of information and, as a possible consequence, to erroneous hypotheses derived from such analytical results. The borderline between high speed routine and normal velocity specific analysis is very difficult to determine on a general basis and has to be evaluated from case to case. Thus, high resolution CGC in the case of triglycerides may take as long as 60-80 min but can be optimized in such a way that without losing resolution, the total analysis time can be reduced to 15 min. 3s Similarly, the analysis of prostaglandins, prostacyclin and thromboxane may take as long as 25-30 min but can be optimized, with basically the same necessary resolution, so that total retention times drop to below l0 min. 4° Whenever a total profile of a homologous series must be analyzed, ranging from very low (C4) to very high (e.g. C24) molecular compounds, such as FAME acids methyl esters, it becomes obviously more difficult to reduce retention times significantly without losing resolution and, thus, information. It should also be possible here for total FAME analysis from C8-C22 to achieve retention times of close to 5 min and still obtain all the necessary qualitative and quantitative information. Apart from these retention time-related trends, there are other interesting novelties which are on the verge of turning into an applicable tool for the chromatographic community. One of the most interesting developments in this direction is the chromatographic T as proposed by Kaiser et al. which combines columns of different polarity and allows, by flow modulations, the fine-tuning of overall polarity of the chromatographic system to achieve "separation windows" under controlled and reproducible conditions. 93 Other trends concern future developments of stationary phases. The absolutely neutral, i.e. neither acidic nor basic, capillary column is fortunately no longer the issue as it was some 4-5 yr ago and problem-specific stationary phases, so-called tailor-made columns, will be of ever increasing interest. Here the trend goes to either mixed phases of very defined, problem-related polarity or to completely new polymers which will be developed in the future. Larger column diameters of around 0.5 mm have become a widely discussed topic in the last couple of years and these wide-bore columns will probably serve more and more to bring over analysts from packed columns to capillary gas chromatography. This is also one of the aims of the present article.

(Received 30 March 1987) REFERENCES 1. 2. 3. 4. 5. 6.

A ~ , K. and TXMAX,Y. J. Chromatogr. 232, 400--405 (1982). ACKMAN, R. G., B^gLOW, S. M. and ~ , 1. F. J. Chromatogr. Sci. 15, 290-295 (1977). ACKM^~q, R. G. and HOOPER, N. J'. Chromatogr. 12, 131-138 (1974). ALEXANDER,G. and RUT~N, G. A. F. M. Chromatographia 6, 231 (1973). ALEXANDER,G. and RU'rT[N, G. A. F. M. J. Chromatogr. 99, 81 (1974). APPLEn't, A. J. and MAY~£, J. E. O. J. Gas Chromatogr. 5, 266 (1967).

278 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65.

H. Traitler ARRENDALE,R. F., CHAPMAN,G. W. and CHORTYK,O. T. J. Agric. Food Chem. 31, 1334-1338 (1983). BADINGS, H. T., VAN DER POL, J. J. G. and WASSlSK, J. G. Chromatographia 8, 440-448 (1975). BARRETT, C. B., DALLAS, M. S. J. and PADLEY, F. B. Chem. Ind. 1050 (1962). BEYNON, J. H., SAUNDERS,R. A. and WILLIAMS, A. E. Anal. Chem. 33, 221 (1961). BLAIR, I. A. Br. Med. Bull. 39, 223-226 (1983). BLAIR, I. A., BARROW, S. E., WADDELL,K. A., LEWIS, P. J. and DOLLERY, C. T. Prostaglandins 23, 579 (1982). BI,OMBr~G, L., BUIJTEN, J., MARKIDrS, K. and WZNNMAN, T. High Resolut. Chromatogr. Chromatogr. Commun. 4, 578 (1981). Bt~Mnr.~,G, L., BUu'I~N, J., M~a~KID~, K. and W.~rNMAN, T. J. Chromatogr. 239, 51 (1982). BLOMIIERG,L., BuIYrEN, J., MARKIDES, K. and W~.NNMAN, T. High Resolut. Chromatogr. Chromatogr. Commun. 4, 578 (1981). BOHOV, P., BALAZ, V. and HRIVNUK, J. J. Chromatogr. 286, 247-252 (1984). Bucm:mt.o, H. P. and SroPats, E. E. In Biomedical Application of Gas Chromatography, p. 488, Reinhold Publishers, New York, 1962. BURLINGAME,A. L. and JO~IANSON,G. A. Anal. Chem. Ann. Rev, 44, 337R (1972). BYGDEMAN,M. and S~amt.ssoN, B. Clin. Chim. Acta 13, 465 (1966). CAIU,O EI~A STRb'MErCrAZIOr~, High Oven Temperature Cold On-Column Injection. Mega Series Instruction Manual, 5.24-5.26 (1985). Cl-mlSTOPl.mnsoN, S. W. and GLASS, R. L. J. Dairy Sci, 52, 1289 (1969). CLAYES,M., HOVE, C., DUC~TFAU, A. and H~MAN, A. G. Biomed. Mass Spectrom. 7, 544-548 (1980). CRONXN,D. A. J. Chromatogr. 97, 263 (1974). D'At~Nzo, R. P., KOZ~d~K, W. Z. and WADE, R. L. J. Am. Oil Chem. Soc. 59, 292-295 (1982). DA~Er~-~U, R., BERrE, P., Root,~Y, T. and HIsrd~, R. Int. Lab. 69-78 (1979). DANDEI~U, R. and ZEltENNER, E. High Resolut. Chromatogr. Chromatogr. Commun. 2, 351 (1979). DESTY, D. H., GOLOUP, A. and WHYM~, B. H. F. J. Inst. Pet. London 45, 287-298 (1959). DESTY, D. H., HXRESNII,, J. N. and WI-n'MAN, B. H. F. Anal. 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