High temperature capillary gas liquid chromatography of triacylglycerols and other intact lipids

Prog. Lipid Res. Vol. 27, pp. 107-133, 1988 Printed in Great Britain. All rights reserved

0163-7827/88/$0.00 +0.50 © 1988 Pergamon Press pie

HIGH TEMPERATURE CAPILLARY GAS LIQUID CHROMATOGRAPHY OF TRIACYLGLYCEROLS AND OTHER INTACT LIPIDS PI~,EMYSL MAREg

Lipid Laboratory, Faculty of Medicine, Charles University, U nemocnice 2, 128 08 Prague 2, Czechoslovakia CONTENTS I. INTRODUCTION

107

II. HISTORICAL A. Triacylglycerols B. Steryl esters C. Wax esters D. Polar lipid derivatives E. Mixtures of intact lipids

108 108 109 109 l l0 ll0

III. GLC ANALYSIS A. General requirements 1. Instruments 2. The injection technique 3. Columns (a) Materials and dimensions (b) Stationary phases 4. Carrier gas 5. Other analytical conditions B. Qualitative analysis I. Triacylglycerols 2. Steryl esters 3. Wax esters 4. Diradylglycerols 5. Optimization of conditions and column efficiency C. Quantitative analysis 1. Triacylglycerols 2. Steryl esters 3. Wax esters 4. Diradylglycerols 5. Common problems of quantification of intact lipids 6. Practical analysis of intact lipids by capillary GLC (a) Sample preparation (b) Optimization of the analytical conditions (c) Column calibration (d) Evaluation of chromatograms (e) Reproducibility of the results (f) Accuracy of the results

I10 l l0 I l0 l l0 ll2 112 I12 ll2 ll3 113 114 116 116 117 118 118 119 122 123 123 123 123 123 124 124 125 125 126

IV. FUTURE POTENTIAL A. Capillary GLC B. Comparison of capillary GLC with other methods

127 127 127

V. TYPICAL APPLICATIONS A. Triacylglycerols B. Steryl esters C. Wax esters D. Diradylglycerols E. Lipid profiles F. Conclusions

128 128 129 130 130 131 132

REFERENCES

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

The analysis of intact lipids by gas liquid chromatography (GLC) was always one of the most difficult applications of this technique, because of both the physical and chemical properties of intact lipids, especially their low volatility and the frequent thermal instability of the unsaturated substances. In spite of the considerable progress made in instrumental GLC and in the technology and quality of capillary columns in recent years, all the problems encountered in the analysis of intact lipids have not yet been solved. These 107

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P. Mareg

include losses and discrimination of substances during injection, losses during separation on the column and also the stability of the stationary phase under conditions that can still be considered as borderline in GLC. Simultaneously, it is necessary to bear in mind that the information obtained in the analysis of intact lipids is irreplaceable for triacylglycerols (TAG), diradylglycerols and wax esters (WE). The analysis of fatty acids can never yield sufficient information on the original combination of the components in the lipid molecule. Thus, the analysis of intact lipids is especially attractive and has been the subject of considerable effort for more than two decades, in spite of the above-mentioned difficulties. It must also be recalled in the introduction to this review that all the intact lipids were analyzed on packed columns before attempts were made to analyze them on capillary columns. The switch to capillary columns was necessitated by the need for better separation of the sample components. The development of other chromatographic methods, primarily high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) has led to a number of successful applications of these techniques for the analysis of intact lipids. Nonetheless, GLC on fused silica capillaries with immobilized stationary phases has retained a dominant position, especially for the analysis of TAG, yielding information on the structure of these important natural substances that could formerly be obtained only by combinations of chromatographic techniques. This work provides a review of the most important applications of GLC of intact lipids and gives a detailed description of the problems encountered in the quantitative analysis of these substances, which are not always considered in sufficient detail in the literature. A recent review by Traitler on capillary GLC appeared in 1987.71a

II. HISTORICAL A. Triacylglycerols

Novotn~, et al. 6~ first described the practical analysis of TAG and other lipids on a glass capillary column in 1972. The authors analyzed cholesteryl esters (CE), TAG and WE on the basis of the number of carbons using a glass capillary coated with DEXSIL 300 GC. This carborane-type stationary phase permitted use of temperatures up to 350°C, which had so far been impossible using capillary columns. The use of these temperatures is necessary for the elution of intact lipids from column. It should be recalled here that the formerly used silicon polymers, such as OV-1, could not be used on capillary columns at temperatures employed on packed columns for the analysis of intact lipids with considerable success for a number of years. 34'3sm In 1979, Grob e t al. ~5 described the preparation of high-temperature glass capillaries. In the same year, Schmid e t al. 6v described the analysis of butter TAG using the GC/MS technique. In 1979, Monseigny et al. 54 also published an important study of the problems encountered in the quantitative analysis of TAG on glass capillaries, including a number of applications. The temperature limit of 370°C, given in this paper, is worth noting and was attained through an i n - s i t u polymerization of polysiloxane stationary phase. Especially in recent years, fused silica capillaries, first described in 19793 have also become important for the analysis of intact lipids. Lipsky et al. 45 have greatly contributed to the wide application of these columns. The first indications of the separation of TAG according to unsaturation can be found in the work of Grob and Grob) 4 Grob e t al. 2s published another paper one year later documenting progress made in the separation of TAG on the basis of unsaturation using a glass capillary. Traitler and Pr6vot 72 considered this problem in detail and published a paper in 1982 describing the separation of these substances on the basis of the number of unsaturated fatty acids. However, the possibility of separation of TAG according to unsaturation is accompanied by problems connected with loss of these substances during

High temperaturecapillaryGLC

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analysis. Grob ~s employed an experimentally original method for determining loss of higher unsaturated TAG; he found that triolein is lost on a nonpolar glass capillary primarily as a result of thermal degradation and, to a lesser degree, through polymerization. Improvement of the separation on the basis of unsaturation necessarily led to problems connected with identification of the separated fractions. One approach, involving ozonization, was described in 1982 by Geeraert and DeSchepper. 7 The method is based on the splitting of the double bond in the unsaturated fatty acid using ozone, decreasing the carbon number of the given TAG. Development of the technology of capillary columns has led to increasing instrumental requirements, especially improved injector design. The originally employed "cold oncolumn" injector was found to be less useful for the analysis of TAG, because it requires a low initial column temperature, depending on the type of solvent used. Geeraert et al. s'~2 described a modification of this injector, a moveable cold on-column injector. Another version that can be employed for the analysis of substances with low volatility is the programmed-temperature on-column injector described by Hinshaw and Feinstein. 29 Recently, fused silica columns have been used increasingly. Geeraert and Sandra 9 first used these columns with a "polarizable" methylphenylsilicone stationary phase for the analysis of TAG in 1984. These authors 1°'~ have described the analysis of vegetable and animal fats using this type of column. It was possible to separate substances differing by a single double bond with the same number of carbon atoms. Quantitative aspects of the separation of TAG on nonpolar fused silica capillaries were studied in detail by Mareg and Hu~ek. 5° No similar study has yet been published dealing with columns with polar stationary phases; experience49 has, however, indicated that attention must be paid to possible loss of higher unsaturated TAG. Some of these results will be discussed where appropriate in this work.

B. Steryl Esters

So far, considerably less attention has been paid to the analysis of intact steryl esters by capillary GLC, compared to the analysis of TAG. The above-mentioned authors 61 described the first separation of CE in 1972. They separated CE of short and medium-chain saturated fatty acids using a glass capillary with a nonpolar stationary phase. The greater sensitivity compared to analysis on packed columns69 led to increased interest in the capillary analysis of intact CE a number of years later. In 1983 and 1984, Smith described the analysis of CE using nonpolar and polar capillaries and splitless or on-column injection techniques. 69.7°Work has also been published describing attempts to separate and identify complex mixtures of steryl esters using the gas chromatography/mass spectrometry (GC/MS) combination and nonpolar capillaries for substance separation. 47'73 Finally, unpublished results obtained in the author's laboratory using a fused silica capillary with a polarizable stationary phase indicate that the analysis of intact CE could, in some cases, replace the currently employed analysis of the CE fatty acids.

C. W a x Esters

Similarly to the other intact lipids, WE were first separated using glass capillaries with nonpolar stationary phase, 47 with separation based on the number of carbons and the vapour pressure. Dewitt et al. 5 employed an efficient capillary with a nonpolar stationary phase to separate WE according to the chain length and total number of double bonds. Itabashi and Takagi 32 in 1984 described the separation of WE with 28-44 carbon atoms on a glass capillary with a polar SP-2340 stationary phase. Under these conditions, it was possible to separate a number of substances according to the position of the ester bond in the molecule at a constant number of carbon atoms. In this work, the authors also considered optimization of conditions for separation of saturated and unsaturated WE.

110

P. Mare~ D. Polar Lipid Derivatives

There are a number of reasons why polar complex lipids, including phospholipids (PL) cannot be analyzed by GLC directly. However, the structures of a number of PL can be studied on the basis of analysis of diradylglycerols. In 1982, Myher and Kuksis described the separation of diacylglycerol derivatives according to the carbon number and degree of unsaturation using a polar capillary?5 Two years later, the same authors 57described the separation of alkenylacylglycerols on the same type of column. Both these works contributed to the study of the structure of natural PL. A paper published by a group of Italian authors ~ is also interesting and describes a capillary GLC study of the pyrolysis products of cow's milk PL. This work is based on the fact that thermal decomposition of the individual classes of PL using a split type injector yields characteristic pyrolytic products indicating the presence of the individual PL in the mixture. E. Mixtures of Intact Lipids Capillary GLC can be used to separate both the individual classes of intact lipids as well as mixtures. A typical example is the analysis of the lipid profile of blood lipids, including a number of lipid classes. In 1981, Kuksis et al. 38 published what was probably the first capillary analysis of a lipid profile. In 1983, Lercker42 described the separation of fatty acids, cholesterol, CE, diacylglycerols and TAG on a glass capillary coated with SE-52. Myher and Kuksis58 employed a fused silica column with a chemically bonded nonpolar stationary phase to analyze the total lipid profile of human blood lipids. From an analytical point of view, the separation of bees wax published in 198444 is also interesting. No paper has yet been published describing analysis of the lipid profile on a polar capillary. It has been found in the author's laboratory 49 that analyses carried out on polarizable columns exhibit overlapping of the CE fractions and TAG, with the formation of critical pairs.

IIl. GLC ANALYSIS A. General Requirements 1. Instruments The analysis of intact lipids using capillary GLC can be carried out using a good-quality instrument equipped with a temperature programmer. A multilinear temperature programmer is useful, although not essential. Electronic compensation of the baseline drift caused by the temperature program is also useful. The other technical instrumental parameters, such as optimal oven design, the quality of the gas pressure and flow regulation, which should now automatically be part of good-quality commercial instruments, will not be considered here. However, it should be recalled that these factors can affect the results obtained in the analysis of intact lipids. 2. The Injection Technique The technique of sample injection is one of the important factors affecting the results of analysis of intact lipids by capillary GLC. The technique of sample injection has been studied most often in connection with TAG analysis, which has so far received the greatest attention. In 1979, Grob 17published a detailed study of the effect of the injection technique on TAG recovery. He demonstrated that the injection techniques based on sample vaporization in the injector are not suitable for TAG, as discrimination of the less volatile components occurs. This discrimination is a result not only of the actual vaporization process in the heated part of the injector, but also occurs during vaporization of the sample from the microsyringe needle during injection or prior to injection. It was found that the content of nonvolatile impurities in the injector resulting from previous analyses can also

High temperature capillary GLC

111

affect the injection process through adsorption of part of the sample by impurities deposited on the injector walls. All these processes are dynamic in character and are affected by physical factors. In addition to the injector geometry, the carrier gas flow-rate can greatly affect sample loss and discrimination of less volatile components, as was demonstrated by Mareg and Hu~ek. 5°These authors carried out a detailed study of the loss of higher TAG during various phases of the analytical process. Finally, thermal decomposition during the vaporization process can be an important factor affecting changes in the sample composition during injection; this factor is especially important for unsaturated substances. Grob 17 studied various TAG and demonstrated that the optimal sample injection technique for analysis of these and similar substances is cold on-column injection. In this injection technique, the vaporization process is eliminated in the injector, avoiding all the above complications. The sample is injected in liquid form at the beginning of the column and is carried into the column to a distance of 10-60 cm in the form of "plug" by the carrier gas; this distance depends on the sample volume and the column diameter. The plug enters the column and forms a fairly thick layer on the internal wall, from which the solvent is first vaporized, followed by the other sample components as the temperature is increased. This mechanism is greatly simplified here, and other physical factors are also important, as described in detail by Grob in 1980-1983.19'2°'27 Under certain conditions, peak broadening can occur. In addition, cold on-column injection involves specific problems, including contamination of the stationary phase in the column by nonvolatile impurities entering the column with the sample. This factor can negatively affect the dynamics of the separation process, again leading to broadening of the peak for the separated substances. 22 Contamination of the analytical column can be prevented in the cold on-column technique either by efficient sample purification or, preferably, by placing a one-to-several meter long fused silica deactivated tubing without a stationary phase prior to the column. This modification, known as a "retention gap", is employed here to remove nonvolatile impurities from the sample during the cold on-column injection technique. In this modification, a deposit of nonvolatile components does not contaminate the stationary phase but can, on the other hand, lead to sorption of part of the sample in the precolumn. Removal of this deposit by washing of the precolumn is not usually possible. As the precolumn is connected to the analytical column by a demountable connector, it is usually a simple matter to cut off the contaminated part of the precolumn, or change it for a new one. The requirement of liquid sample transfer, which is the main characteristic of cold on-column injection is, however, connected with a limited initial temperature of the analytical column, primarily determined by the boiling point of the solvent used.16 The low initial temperature of the column results in lengthening of the time required for carrying out the analysis, with an increase in the retention time of the substance in the column and prolonged thermal exposure. These factors can lead to increased losses during the separation process, especially of higher unsaturated lipids. Thus, it is understandable that attempts have been made to eliminate this disadvantage of the cold on-column technique by various modifications of the injector design. These include cooling of the inlet part of the column during the injection period or the use of an independently thermostatted inlet part of the column. 25 The best solution of problems encountered in the cold on-column technique with high initial column temperature would appear to be the "moveable on-column" injector proposed and tested by Geeraert e t al. 8 The principle of its function is based on the possibility to move up (out of the oven) the injector and inlet part of the column to obtain natural cooling before the injection. After completion of the injection, when most of the solvent has been removed by the carrier gas, the injector with the inlet part of the column is moved down into the oven, where the remaining sample components are vaporized at a high temperature. Similar to the classical cold on-column technique, the sample is vaporized from the column, not from the injector insert. If the injector is not moved up (out of the oven) before the injection, then it can act as an ordinary on-column injector, with all the disadvantages described above. The injection of a liquid sample by the on-column technique is connected with further problems of a physical nature, such as

112

P. Mareg

back pressure during solvent evaporation, etc. Thus, considerable attention has been paid in recent years to the technique of sample injection in capillary GLC and to the study of the processes involved. 2~'23"26 A further approach fulfilling the requirement of liquid transfer of the sample at column temperatures greatly exceeding the boiling point of the solvent used involves application of a programmed-temperature vaporizer. This injector has been used successfully for the analysis of various natural TAG and the results were published in 1986.30 Basically, this is a split/splitless type of injector, which is maintained at a temperature close to the boiling point of the solvent used prior to sample injection, ensuring liquid sample transfer. After the solvent is removed by a stream of carrier gas, the injector is rapidly heated (at a velocity of the order of tens of °C/s), with sample vaporization. It follows from the description of the function of this injector that liquid sample transfer is ensured in the first phase, eliminating discrimination of higher components during sample transfer; however, the next step involving vaporization of the sample inside the injector is similar to that in classical split/splitless injectors. However, the quantitative results for model sample mixtures including TAG were comparable with those obtained using the cold on-column technique. 3°'31 The manufacturer (Perkin-Elmer) has stated that this injector approaches most closely the ideal of a universal capillary injector. However, only two papers have so far been published 3°,3~where this injector was employed for TAG analysis. Its advantages and disadvantages will be found in practice in the analysis of lipids with various compositions.

3. Columns

(a) Materials and dimensions. Intact lipids can be analyzed using both glass and fused silica columns. In the last few years, quartz capillaries have been increasingly employed in combination with a chemically-bonded stationary phases. The column length usually varies from 5 to 25 meters, with an internal diameter of 0.2 to 0.32 mm. When longer columns are used, greater losses must be expected, especially of high molecular weight and highly unsaturated lipids. (b) Stationary phases. Lipid analysis was originally carried out on columns with a nonpolar stationary phase, usually of the methylsilicone type. The short column lifetime and high drift of the baseline at the elevated temperatures required for the elution of intact lipids were later eliminated by immobilization of the stationary phase. In the second half of the eighties, fused silica columns appeared with chemically-bonded polarizable stationary phases of the methylphenylsilicone type. Columns with these stationary phases can now be obtained from two companies (Chrompack and Quadrex). These stationary phases have a temperature limit of about 360°C and this limit will probably be extended 6 for additional polar stationary phases. Steryl esters, diacylglycerols and wax esters have been successfully analyzed on cyanosiloxane-type stationary phases, such as SP-2330 or SP-2340. 6'37'57 Columns with this type of immobilized stationary phase are not yet commercially available. The optimal thickness of the layer of stationary phase considering the actual elution temperatures for intact lipids would appear to be between 0.10 and 0.12 ~ M.

4. Carrier Gas

As capillary GLC develops, hydrogen is being increasingly employed as a carrier gas. Compared with nitrogen, which is considered to be the optimal carrier gas for GLC on packed columns, hydrogen has important advantages as a carrier gas in capillaries. An exhaustive review of the advantages of hydrogen over nitrogen as a carrier gas for capillary GLC is given in the paper by Grob and Grob. ~4 The most important advantage of hydrogen in the analysis of intact lipids is the lower elution temperatures compared to both nitrogen and helium, leading to shorter analysis times. The decrease in the elution temperature and shorter analysis time lead to lower thermal degradation and thus

High temperature capillary GLC

113

decreased loss of the sensitive unsaturated lipids during the analysis. In addition, the lower elution temperatures and shorter analysis time lead to increased effective column lifetimes, expressed in terms of the number of analyses carried out. At a given maximal column operating temperature, thermostable substances with higher molecular weights can be analyzed. When using hydrogen, the efficiency of the column is less dependent on the carrier gas linear velocity than with helium or nitrogen, permitting use of higher cartier gas flow-rates. Combined with a faster temperature program, this leads to improved sensitivity, as less substance is required to yield a peak of the same height. The disadvantages of hydrogen include a possible danger of explosion because of leakage into the oven during operation. Unfavourable hydrogenation of unsaturated CE has also been described: 8 However, this phenomenon has not been found by any other authors and can be considered as a property of the particular column or stationary phase in combination with hydrogen as a carrier gas, rather than a characteristic of hydrogen carrier gas alone.S° For attaining optimal lifetimes of capillary columns, it is necessary to completely remove traces of oxygen and moisture from the carrier gas, whatever its type. The flow rates of carrier gas in the analysis of intact lipids depend on the column diameter, carrier gas type and other factors. In general, rates from 1 to 10 ml/min are employed. The carrier gas flow rate or, more precisely, its linear velocity, is an important quantitative factor, especially in the analysis of TAG, as demonstrated in earlier works. 5°'54This factor will be considered in more detail in the section on quantitative analysis.

5. Other Analytical Conditions In addition to the analytical conditions discussed above, the temperature program (rate and shape) plays an important role. It has been demonstrated on fused silica capillaries with a nonpolar stationary phase that the temperature program rate affects the TAG elution temperature and separation efficiency. 5° For example, the elution temperature of triarachidin at a temperature program rate of 2 K/min is 315.8°C, while this value equals 338.2°C at a rate of 8 K/min. In addition, at a constant temperature program rate, the TAG separation efficiency decreases with increasing molecular weight. 5° Thus, a multilinear temperature program must be employed to maintain a constant separation efficiency. It thus follows that it is important to select an optimal temperature program considering the sample composition and purpose of the analysis. The temperature program rate must also be selected in combination with the initial and final temperatures and the initial and final times. These values are connected primarily with the sample composition, the length and type of column, the stationary phase used and the thickness of the layer and, finally also the type of carrier gas.

B. Qualitative Analysis Detailed qualitative analysis of a sample using any type of separation technique, i.e. also capillary GLC, assumes complete separation of the sample and subsequent identification of the individual components. In spite of all the progress made in the analysis of intact lipids in the last few years, this analysis almost always results in group separation rather than separation of the chemical individuals. This is a result of the great complexity of natural mixtures of intact lipids. Analysis on columns with nonpolar stationary phases is based on the vapour pressure differences between the sample components, so that roughly a molecular weightdistribution chromatogram is obtained and unsaturation can hardly contribute to the component retention. On polarizable stationary phases, there is a substantial contribution of the double bonds to the retention, resulting in separation according to the carbon number and unsaturation. In some cases, components having the same number of carbons and double bonds, but differing in fatty acid composition, are partially separated. This general concept differs for the individual lipid classes, and thus the analysis of TAG, steryl esters, WE and diradylglycerols will be discussed separately. JPLR 27/2~C

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P. Mareg

1. TriacylglYcerols The separation of TAG on the basis of the carbon number (total number of carbon atoms in all the acyl groups) can be achieved using a short capillary with a nonpolar stationary phase. 38'4z'5° Medium-length (about 10m) nonpolar columns yield partial separations of unsaturated TAG. The unsaturation becomes important on nonpolar columns with high efficiency, but the separation is not based on the number of double bonds, but rather on the number of unsaturated fatty acids in the TAG molecule. 72 Similarly, when the TAG molecule contains fatty acids with very different chain lengths, a certain degree of separation can be observed on nonpolar columns? ° Columns with polarizable stationary phases can be employed to separate TAG also on the basis of the number of double bonds and, in some cases, according to the fatty acid composition.9-'j The separation of TAG on polarizable columns involves identification problems. It should be borne in mind that unsaturated substances are eluted prior to saturated ones on nonpolar columns, while, on polar or polarizable columns, the interaction with the n-electrons of the double bonds leads to retardation of unsaturated substances, as can be seen in Fig. 1. This retardation is dependent on the number of double bonds and also on the type of fatty acid. Identification can only rarely be carried out using pure substances, as most separated TAG are not available in the pure state. Preliminary identification of the individual fractions can be based on analysis of oils and fats with known composition, literature data, data obtained by other methods, etc. ~° The retention characteristics of the individual fractions are also important for their identification. There are proportional differences in the retention times between SSS, SOS,

o0o iLL +

SSS

000

tntntn

SSS

ttl

(A)

,

i

7

l'O (rain)

23

I

26 (rain)

I

(B)

29

FIG. I. Separation of a model mixture of saturated and unsaturated TAG on nonpolar (A) and polarizable (B) capillaries. Analytical conditions (column A): instrument, Hewlett-Packard model 5730A (Hewlett-Packard, Palo Alto, CA, U.S.A.); column, fused silica, CP-Sil 5 CB, 5 m x 0.32 mm, stationary phase layer thickness 0.12 #m (Chrompack, Middelburg, The Netherlands); injection, splitless; carrier gas, H2, linear velocity 140cm/s; temperature program, 260-340°C, 8 K/min; sample, 1 #1 in undecane containing the same weight proportions of each component. Analytical conditions (column B): instrument, Pye-Unicam model 4900 (Pye-Unicam, Cambridge, England); column, armoured fused silica, 25 m × 0.25 mm, stationary phase TAP, layer thickness 0.I pm (Chrompack, Middelburg, The Netherlands); injection, cold on-column; carrier gas, H 2, linear velocity 120 cm/s; temperature program, 60°C isothermal for 2 min, then 60-335°C, 25 K/min, then 335-350°C, 2 K/min, 350°C isothermal for 2 min; sample, 0.2#1 in hexane, composition was the same as on column A. Peak designation: SSS--tristearoylglycerol, OOO--trioleoylglycerol, LLL--trilinoleoylglycerol, LnLnLn--trilinolenoylglycerol.

115

High temperature capillary GLC TAm~ I. The Differences in the Retention Times (ART) for various TAG Fractions Separated on a Polarizable Capillary Fraction

ART

Fraction

ART

Fraction

A RT

SSS SOS SO0 000

0'18a 0.18 0.18

000 OLO OLL LLL

0.38 0.37 0.39

PPS PPO POO PLO

0.18 -0.37

avalues in min. Analytical conditions were the same as in Fig. 9. S = stearic acid, O = oleic acid, L = linoleic acid, P = palmitic acid.

SOO and OOO, between SOO, SLO and SLL or OOO, OLO, OLL and LLL. Practical example of these differences is given in Table 1. Final and complete identification can be carried out using GC/MS, but this generally accepted identification technique has not yet been described in connection with polarizable capillary columns. On the other hand, the GC/MS of TAG using nonpolar columns has been already published repeatedly. 63,67,73 Of the chemical methods, ozonization 6 or hydrogenation" can be employed for TAG identification. The basis for the use of ozonization for identification of TAG fractions has already been discussed here. Special note should be made of the application of multidimensional chromatographic techniques, especially the GLC and AgNOa-TLC combination. In this procedure, the original sample is separated on an impregnated thin layer chromatography (TLC) layer according to the degree of unsaturation and the isolated TLC fractions as well as the original sample are analyzed by GLC on a polarizable capillary column. Comparison of the chromatograms of the individual TLC fractions with that of the original sample then facilitates identification of the components in the original sample. Identification of the TAG fractions is connected with nomenclature problems. Various symbols are employed to designate the individual TAG, mostly based on abbreviations of the trivial names of fatty acids or on a combination of the number of carbons and number of double bonds. Table 2 gives examples of various designations of TAG and their fractions. Separation of the positional isomers of TAG* has not yet been described on any type of capillary column.

TABLE 2. Designation of TAG Fractions Separated by Capillary GLC and Examples of Use Designation

Application

Capillary

54

Separation according to the total carbon number

Short nonpolar

SUU a

Separation according to the carbon number and number of unsaturated fatty acids

Efficient nonpolar

54:3 SLOb

Separation according to the carbon number and number of double bonds

Polarizable

as = saturated fatty acid, U = unsaturated fatty acid. bs = stearic acid, L = linoleic acid, O = oleic acid. The use of the same abbreviation (S) for saturated fatty acids and stearic acid cannot be confusing in practice, because each system of designation is used separately in different cases.

*i.e. substances differing only in the position of the fatty acids on the glycerol skeleton, such as SOS and SSO or SLO and SOL, etc.

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P. Mare~

2. Steryl Esters Steryl esters can be separated on a nonpolar capillary according to the number of carbons or on polarizable and polar columns according to the number of carbons and fatty acid double bonds. No paper has yet been published describing the separation of intact steryl esters on the basis of fatty acid positional or geometric isomers. The identification is not a problem in some simple cases, where pure compounds are usually available, for example the c o m m o n cholesteryl esters (CE). The situation becomes more complex for mixtures of natural origin where higher numbers of sterols and fatty acids appear. In this case, G C / M S identification becomes necessary. 73 The individual esters are designated either according to the number of carbons or simply by the abbreviated listing of the fatty acids; nomenclature based on the total number of carbons and of double bonds in the fatty acid molecule can also be employed. Table 3 lists the nomenclature for CE fractions, giving the individual types of symbols and their use with the example of cholesteryl linoleate. TABLE3. Designation of CE Fractions Separated by Capillary GLC and Examples of Use Designation Application Capillary 45 Separationaccording to Nonpolar the total carbon number 45 : 2 Separationaccording to Polarizable 18:2 the carbon number and number of double bonds

3. W a x Esters Wax esters (WE) are separated on a nonpolar capillary according to the total number of carbons and double bonds, as can be seen in Fig. 2. Similar to T A G , the substances are eluted according to decreasing unsaturation, i.e. the substance with the greatest number of double bonds for a given number of carbon atoms is eluted first. The fractions are most often designated using a symbol consisting of the total number of carbons and the total number of double bonds after a colon, for example, 32:2. It is difficult to separate WE

o

o

?.

i

II o

.': "..,

o

o

o ,.

-~

I

I

IO

20

~.

I

30

.:..

o

o - o . . . . . .

;;

;

oo

t

40

rnm

FIG. 2. Analyis of wax esters from Chlorella kessleri. Analytical conditions as given by l~ezanka and Podojil.64Peaks are designated by total number of carbons and double bonds. Reprinted with kind permission of Elsevier Science Publishers and the authors.

High temperaturecapillaryGLC

117

¢

t

149

15B

151 t, i m e ( m i n )

FIG. 3. Separation of peak 46:1 from Fig. 2 during isothermal analysis. Analytical conditions as given by Rezanka and Podojil. ~ Peak designation: (A) 26:1-20:0 (acid-alcohol); (B) 28:1-18:0; (C) 30:1-16:0. Reprinted with kind permission of Elsevier Science Publishers and the authors.

with identical numbers of carbons and double bonds, i.e. according to the position of the ester bond; this separation has been partly achieved on a nonpolar column under isothermal conditions at efficiency of about 100,000 theoretical plates, 64 as can be seen in Fig. 3. Identification was, in this case, carried out using GC/MS. A partial separation of the isomers of saturated wax esters according to the position of the ester bond differing by four carbons (for example, combination of 10:0-18:0 was separated from the combination 14:0-14:0)* was achieved using a polar capillary) 2 The closer the position of the ester bond to the center of the wax ester molecule, the more difficult the separation becomes. The authors observed a dependence of the separation on the column temperature during isothermal analysis. The same authors 32achieved complete separation of unsaturated WE on a polar column according to degree of unsaturation, and partial separation according to the position of the double bond, for a model mixture of WE prepared by transesterification of linseed oil or fish oil with hexadecanol. The separation was once again temperature-dependent. The separation of the isomers of unsaturated WE formed by various alcohols and fatty acids is temperature-dependent and is obviously also dependent on the position of the ester bond and double bonds in the molecule. It is, of course, more difficult than the above discussed separation. Because of the greater number of possible combinations of alcohols and fatty acids, critical pairs are formed with different numbers of carbons and double bonds that are very difficult to separate. A change in the temperature of isothermal analysis was only partly successful in solving this problem. 32

4. Diradylglycerols This group of substances has received increased attention recently as the separation and identification of diradylglycerols permits study of the structure of phospholipids, from which diradylglycerols are prepared by dephosphorylation for this purpose. It should be recalled here that HPLC is used far more often for the analysis of phospholipids than is GLC. Primarily polar capillaries are now used in GLC, as analysis on nonpolar columns does not yield satisfactory separation. When a cyanoalkylsiloxane-type stationary phase is employed, diacylglycerols, alkenylacylglycerols and alkylacylglycerols can be separated following derivatization according to the number of carbons and double bonds. The separated compounds can be identified using the pure substances where available. Preliminary separation has been found useful for more complex samples; this is carried *The symbolsemployedfor designationof the compositionof wax esters indicatethe number of carbons and double bonds in the alcohol and fatty acid molecules.The first number corresponds to the alcohol.

118

P. Mare~

out on the basis of the degree of unsaturation using AgNO3-impregnated TLC. Similarly, identification using GC/MS has been employed successfully for the analysis of these substances. 57 The fractions are designated using a similar scheme as for wax esters, i.e. using nomenclature giving the number of carbons and number of double bonds for both radyl groups. 5. Optimization of Conditions and Column Efficiency

Analysis of complex natural mixtures requires highly efficient capillary columns. The column efficiency depends on various factors. The best known is the linear velocity of the carrier gas, and the type of carrier gas and temperature program are also important. All these factors must be optimized to attain the most efficient separation of the sample. For polar or polarizable capillaries, the separation is also affected by changes in the polarity of the column as a function of the temperature. For publication purposes or in comparison of columns, the efficiency should be expressed in some suitable manner. As the classical expression for the column efficiency based on the height of a theoretical plate is not practical in TAG analysis,48 an expression has been introduced for the separation efficiency in this type of analysis in terms of the AC value*. 46 This means of expressing the separation efficiency was introduced for nonpolar columns for separation according to the carbon number. However, it is no longer practical for separation of substances with the same number of carbons on the basis of the number of double bonds. Grob and Grob 24 proposed the use of a different means of expressing the separation efficiency useful also under the temperature program conditions, i.e. the separation number (trennzahl), TZ: TZ -

tR2 -

tRi

1

(1)

Wl0 5 -1- W205

where t ~ : - tR~ is the distance between two peaks and w~o5, w2o, are the peak widths at half-height. For preliminary identification of substances under the conditions of the temperature program, Saxton65 proposed a value termed the "emergence temperature", which, in contrast to relative retention times or Kovhts indices, is not temperature-dependent. The emergence temperature is specific for the substance and the stationary phase and is not dependent on the analytical conditions, assuming they are standardized using a reference substance. Standardization of the analytical conditions is understood to be attainment of a constant emergence temperature for the reference substance. This approach to tabulation of data for preliminary identification has not yet been used for intact lipids, although it could be useful where the pure substances are not available. The emergence temperature is defined by the relationship: 65 T~x = Te,~+ r(tR_~ - t~),

(2)

where T~ is the emergence temperature, x corresponds to the studied substance, s corresponds to the reference substance, r is the rate of the temperature program and tR is the retention time. Definite identification of intact lipids can be carried out by the GC/MS technique, in the same way as for other substances separated by GLC. This approach has the advantage that even substances that are incompletely separated can often be identified. C. Quantitative Analysis

Quantitative analysis in gas chromatography is generally based on the relationship between the mass or concentration of the analyzed substance and the detector response. *AC corresponds to the minimal difference in the number of carbons for two subsequently eluted T A G separated to the baseline.

High temperature capillary GLC

119

For the flame ionization detector, which is used almost exclusively for the analysis of intact lipids, this is the dependence of the ionization current on the input of "effective" carbons to the detector, expressed by the relationship: 6°

Ri = Cot(~, Cef)i(dN,/dt ),

(3)

where Ri is the detector response for substance i, (ECef)i is the sum of effective carbon atoms in component i, dNi/dt is the molar velocity of effluent input into the detector, C is a proportionality constant and ,t is the degree of efficiency of ionization. Constants C and ~ are defined for the given detector and working conditions. Equation 3 describes the response of the detector in its dependence on the substance input and thus does not include possible loss of substance during the individual stages in the sample analysis, which is important in quantitative measurement. These losses, which are not always negligible for intact lipids, can be divided into two groups. The first includes losses during sample preparation and the second those incurred during the individual phases of the analysis, i.e. during injection and separation on the column. Losses during sample preparation can usually be eliminated using the internal standard method, which will be considered in greater detail below. Losses during analysis can sometimes become serious for intact lipids and are compensated for by using correction factors determined by analysis of pure substances or standards. The problems involved in quantification are rather different for the individual classes of intact lipids and will thus be discussed separately.

1. Triacylglycerols In 1979, Monseigny et air published the first paper dealing with the quantitative analysis of TAG on capillary columns. In 1985, Mare~ and Hu~ek 5° published a detailed study of the quantitative GLC of saturated TAG on fused silica capillaries with a chemically bonded nonpolar stationary phase. They demonstrated that the recovery of higher saturated TAG using a short column is affected by the following factors: injection technique, carrier gas flow rate, weight and molecular weight of the substance, column quality, etc. All these factors can be considered constant during a given analysis except for the sample component weight and molecular weight (i.e. sample composition and concentration). Figure 4 depicts a typical dependence of the recovery of higher saturated and unsaturated TAG on the weight of the analyzed substance on a capillary with a polarizable stationary phase. It can be seen from Fig. 4 that the shape of the dependence for saturated and unsaturated TAG is somewhat different. The recovery of higher saturated homologues depends more on the weight than for substances with fewer carbons. Similarly, the dependence increases with increasing number of double bonds. The effect of the weight of the substance on the change in the recovery can be clearly seen in Fig. 5, depicting the increase in the recovery of the given TAG in dependence on the increased weight of this substance. The dependence of TAG recovery on the weight of the analyzed substance was described 12 years ago by Kuksis et al. using packed columnsfl These dependences were studied in detail in subsequent years in connection with clinical applications of neutral lipid profiles, first on packed columns 5~-53 and later on capillaries. 5° In comparison of polarizable and nonpolar capillaries considering the dependence of the recovery on the weight of substance, it is important that, in spite of its relative length, the polarizable column yields very good, reproducible recoveries of higher saturated TAG in the low weight region. However, losses of higher unsaturated substances are greater than on shorter nonpolar capillaries, although quite reproducible. The assumption of constant recovery affecting factors during the analysis is not completely fulfilled during temperature programming, especially when hydrogen is employed as a carrier gas. In many contemporary commercial gas chromatographs, the

120

P. Mare~

100 SSS 000

80

AAA

o~60 J - 40

LLL

20

0

I

I

I

I

I

10

30

50

70

90

iniected (ng)

amount

FIG. 4. The dependence of the relative recovery of higher saturated and unsaturated TAG on the weight of the substance on a polarizable capillary (recovery of tripalmitoylglycerol = 100%). Analytical conditions as for Fig. 1, column B. Designation of TAG as in Fig. I, except that AAA = triarachidoylglycerol. Samples contained 10-100 ng of each component in 0.2/~1 of hexane.

hydrogen flow rate significantly decreases with increasing temperature, as pointed out by Davies in 1984.4 The carrier gas flow rate, or precisely, its linear velocity, affects TAG recovery, as already mentioned. The dependence of this type for higher saturated and unsaturated TAG obtained again on the polarizable column is depicted in Fig. 6. This shows the slight dependence of the recovery of higher saturated TAG and TAG with a lower number of double bonds on carrier gas linear velocity, while the recovery of TAG with six double bonds is more dependent on the above mentioned parameter. This is more evident also in Fig. 7, depicting the dependence of the change in the recovery of the given TAG on increasing linear velocity of the carrier gas.

100

60

AAA

g 40

20

o

sss 000 I

I

10

30

I

I

I

50

70

90

amount ifliected (ng)

FIG. 5. The percent increase in the recovery of selected TAG as a function of increased substance weight (recovery for 10 ng of each component is taken as calculation base). Analytical conditions and curve designation as in Fig. 4.

121

High temperature capillary GLC 100 SSS 000

80

o~ 60 w AAA = 40

LLL

m

20

0

I

80

I

I

l

100 120 carrier 8as linear velocity (cm/sec)

140

F10. 6. Dependence of the relative recovery of higher saturated and unsaturated TAG on the carrier gas linear velocity on a polarizable capillary (recovery of tripalmitoylglycerol = 100%). Analytical conditions and designation of curves as in Fig. 4. The samples contained identical amounts (20 ng) of each substances in 0.2 #1 of hexane. The carrier gas linear velocity varied from 85 to 140 cm/s. The column quality is a further i m p o r t a n t factor affecting the T A G recovery. The concept o f column quality includes not only the separation efficiency, but also factors affecting the quantitative analysis, such as inertness o f the tubing, content, homogeneity o f the layer, chemical composition and the degree o f c o n t a m i n a t i o n o f the stationary phase during the column lifetime. The column length also affects the recovery for a given substance. As the column length increases, the losses o f m o r e unsaturated T A G increase rapidly. F o r example, the loss o f trilinolein on a 25 m long column with a polarizable stationary phase was 5 0 - 8 0 % , depending on the sample weight and linear velocity of the carrier gas. T o retain a good separation capability, a lower carrier gas linear velocity was selected on a polarizable 100

ttt/

80 ~ 60

/

/

/

/

/

/

/ /

/

u 40

/

/ / /

2O

/

/

/

/

I

80

I

AAA __JOg._ $$$ I

100 120 carrier gas lieear velocity fcm/secl

I

140

FZG.7. The percent increase in the recovery of selected TAG in dependence on the increasing linear velocity of the carrier gas (recovery at 85 cm/s = base) Analytical conditions and curve designation as in Fig. 6.

122

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column, compared to that on a nonpolar capillary. On the other hand, surprisingly good recoveries of high saturated TAG, also at a very small amounts, were obtained on a polarizable column. This fact is probably a result of the more favourable interaction of the polarizable stationary phase with the separated TAG than for the nonpolar phase• The results obtained suggest that there are two mechanisms of TAG loss during analysis on capillary columns.49 For saturated TAG, losses probably occur through a mechanism of reversible saturation of the stationary phase by the solute; for unsaturated substances. losses probably occur primarily through thermal degradation or polymerization.6-~8Losses of unsaturated substances can be decreased by shortening the analysis time, which is in good agreement with the above suggested mechanism. More detailed investigation is required for clarification of these processes. Weight or molar correction factors46 for compensation of losses of TAG during the analysis were introduced 22 years ago; these factors are defined as the ratio of the weight or molar percent of substance in the mixture to the percent peak area for the given component: fw -

weight % area % '

fm =

mole % area %

(4)

The dependences measured originally on packed columns are valid in modified form also for quantitative analysis of TAG on capillaries, as has been demonstrated repeatedly. ~8,50,54 Because of the separation capabilities of polarizable capillaries, it is necessary to determine correction factors for fractions that are not available in the pure state. This problem has, in practice, not been encountered in analyses on nonpolar columns, where correction factors were measured either using synthetic substances 5° or using a natural mixture with known composition.37 In one of his more recent papers, Geeraert 6 has stated that the correction factors for most fractions of TAG except for highly unsaturated substances are almost equal to one and that no special attention need be paid to this subject. Where losses of TAG are encountered, they are reproducible, as he demonstrated on the analysis of corn oil. The reproducibility of the losses of unsaturated TAG was also confirmed in our laboratory. However, in precise quantitative analysis, factors affecting losses must also be considered, so that the recovery and reproducibility of losses of high and more unsaturated components should be checked in each case. It follows from the results published by Geeraert 6 and from our results that TAG containing highly unsaturated fatty acids should not be analyzed on capillary columns. Experience gained from quantitative analysis of TAG on capillary columns has indicated that the values of the correction factors for higher and unsaturated TAG are affected by the following factors: the quality and length of the column, the type and content of the stationary phase, injection technique, analytical conditions, the chemical character of the substances (molecular weight, degree of unsaturation) and the weight of component and sample analyzed. These factors should be borne in mind and, where possible, their effects should be minimized. Where the pure substances are available, experimental values for the correction factors should be determined and employed in the analysis of unknown samples. These samples should then be analyzed under conditions that are identical with those under which the correction factors were determined. Where correction factors for a given substance cannot be determined by direct measurement, which often occurs in analysis on polarizable capillaries, then approximate values should at least be determined using close homologues through interpolation in the required interval of carbon numbers or number of double bonds. This approach, which is certainly better than a simple unverified assumption that the correction factors equal one under all conditions, will be considered in greater detail later. 2. Steryl Esters Because of their much lower molecular weight and lower unsaturation compared to TAG, the losses of steryl esters during injection and separation are much lower. This is,

High temperature capillary GLC

123

however, not true for esters containing highly unsaturated fatty acids, such as arachidonic or eicosapentaenoic acids, for which considerable losses have been found on polarizable capillaries. Smith 7° has described very reproducible yields of cholesteryl esters (CE) with up to three double bonds during on-column injection into polar or nonpolar columns. The required correction factor values can readily be obtained for CE which are frequently available in pure form. In addition, direct analysis of intact CE is not often carried out and its application is useful only when very small amounts of material are available, insufficient for transesterification and isolation of the fatty acid methyl esters, which is the usual analytical procedure for CE. Mixed steryl esters have been analyzed primarily to identify the individual substances by the GC/MS technique. 47'73 Quantification is then usually carried out on the basis of MS-data alone.

3. Wax Esters Similarly to steryl esters, there are various purposes for the application of capillary columns for wax esters (WE). No specific problems have been described for the quantitative analysis of WE, as for TAG. 5'32'64 Because of their molecular weights, the losses of these substances on capillary columns should not be too large. Itabashi and Takagi, 32 who described the analysis of WE containing highly unsaturated fatty acids, do not describe any problems connected with quantification of these substances. Described methods of quantitative analysis are based either on uncorrected flame ionization detector (FID) responses or on the relative intensities of selected ions in the mass spectra.

4. Diradylglycerols The GLC of diradylglycerol derivatives is carried out primarily to study phospholipid structure. Myher and Kuksis57have stated that the separated substances can be quantified simply on the basis of the uncorrected responses of the flame ionization detector. The problem of correction factors and their dependences has not been described in the literature. The molecular weights of diradylglycerol derivatives are significantly lower than those of TAG. Pure substances are available only in some cases for direct response measurements and for control of the recovery of the individual fractions.

5. Common Problems of Quantitation of Intact Lipids It follows from published descriptions of the quantitative analysis of intact lipids that the greatest attention has so far been paid to TAG. This is a result of their natural occurrence and biological importance, and also because precise quantitative analysis is still difficult. Because a wide range of intact lipids is not available in pure form, precise determination of correction factors for large number of important compounds that can be separated by contemporary capillary GLC remains a problem. In 1985, Scanlon and Willis 66 published a study of the possibility of calculating the relative FID response for substances that are not available in pure form on the basis of the number of effective carbons in the molecule. However, this approach cannot be employed when there are losses of substances during the analytical process, i.e. this method cannot be employed for higher and more unsaturated TAG. It would first be necessary to verify this procedure experimentally for other intact lipids.

6. Practical Analysis of Intact Lipids by Capillary GLC (a) Sample preparation. It is not the purpose of this review to provide a detailed procedure for the quantitative analysis of the individual classes of intact lipids. However, attention should be paid to some specific problems involved in the application of capillary columns. The greatest attention should be paid to sample purity. It should be realized that

124

P. Mare~

a capillary column contains 100-1000 times less stationary phase than a packed column, so that the danger of contamination by impurities from the sample is much greater. Contamination of the stationary phase can result in a decrease of the column efficiency and other unfavourable effects. The on-column injection technique is a main factor affecting the danger of contamination of the stationary phase, resulting in deposition of all the impurities from the sample in the column. Thus, it is obvious that high purity of the sample is essential for maintaining the column lifetime. Sufficient sample purity can be achieved, for example, by employing a suitable separation technique immediately prior to sample analysis. Losses necessarily occur during isolation of intact lipids from biological materials. In order to control these losses, the internal standard method is mostly used; this substance should be added to the sample at the beginning of the isolation. The choice of a suitable standard is also important as it must not interfere with any component of the analyzed sample. The purpose of the analysis must also be considered in the choice of the internal standard. For example, if it is required to determine only the relative contents of the individual fractions without determination of the lipid class content, the addition of the internal standard at the beginning of sample preparation is not mandatory. On the other hand, if the analysis purpose is to determine not only the composition of the lipid class, but also the content in the original sample, then it becomes necessary to add a suitable internal standard as soon as possible during the isolation procedure. (b) Optimization of the analytical conditions. The optimization of the analytical conditions in the analysis of intact lipids is understood to entail not only attainment of optimal separation efficiency considering the sample composition, but also optimization of the recovery of the separated substances. This requirement is in opposition to that of optimal separation, as can be seen from Fig. 8. It is also obvious from this dependence that optimization in the capillary GLC of intact lipids is not simply a compromise between the separation efficiency and the length of the analysis, but between the efficiency and the recovery of higher or more unsaturated lipids. In addition to the carrier gas linear velocity, it is necessary to optimize the initial temperature and the temperature program rate. All these parameters should be selected in relation to the sample composition. (c) Column calibration. The actual calibration procedure is dependent on the availability of the pure substances or a natural mixture with a defined composition. 37 If a suitable

100 ooo

80

6

5

60

4

40

3~

20

t

0 J 80

2

~

1 i 100

I 120

i 0 140

carrier gas linear velocitv (cm/sec) FIG. 8. The dependence of the relative recovery and separation efficiency (TZ) of higher saturated and unsaturated TAG on the carrier gas linear velocity on a polarizable capillary. Analytical conditions and designation of substances as in Fig. 6.

High temperature capillary GLC

125

TABLE 4. Available TAG and Possible System of Correction Factor Determination by Linear Interpolation Double bond number 0 1 2 3 4 5 6

Carbon number 42

44

46

48

50

52

54

56

58

60

MMM i 42: I b 42:2 MoMoMo

MMP 44: 1.~ 44:2? 44:3

MPP 46: 1? 46:2? 46: 3

PPP 48: l 48:2 PoPoPo

PPS PPO 50:2? 50: 3

PSS 52:1 POO 52: 3

SSS SSO SO0 OOO 54:4 54:5 LLL d

56:0 56: 1? 56:2? 56: 3

58:0 58: 1? 58:2? 58 : 3

AAA 60: l 60:2 EiEiEi

aAvailable compounds with defined chemical structure; correction factors can be obtained by direct measurement. bFractions for which the approximative correction factor can be obtained by linear interpolation. CFractions for which the approximative correction factor cannot be obtained by linear interpolation. OThe application of this system for more unsaturated fatty acid is not recommended. The structure of TAG fractions is expressed by the number of carbons and double bonds only, because of possible structural difference of each fraction, i.e. for example fraction 52:3 can have the following composition: PLO, PoOO, MLEi etc. Mo = myristoleic acid, A = arachidic acid, Ei = eicosenoic acid; other fatty acid abbreviations as in Figs 9 and 10.

calibration mixture or pure substances can be obtained, calibration is carried out with maintenance of all the usual conditions for quantitative GLC. Where some components only of the analyzed spectra are available, a common situation in the capillary analysis of TAG using a polarizable column, the factors obtained indirectly by interpolation between available substances can be employed. This procedure is illustrated in Table 4, from which it can be seen that, for example, the correction factor values for unavailable TAG with carbon number 54 and four or five double bonds (OOL and OLL) can be determined approximately using the corresponding factors for triolein and trilinolein by linear interpolation. The procedure is the same for the other examples given in Table 4. This approximate approach will certainly lead to smaller errors than the unverified assumption that the correction factors equal one. The correction factors can also be calculated using a mixture of natural TAG with a known composition determined, for example, by HPLC. (d) Evaluation ofchromatograms. The automation of evaluation of chromatograms using microcomputers, which are now commonly used in GLC, has made a great contribution to practical quantitative analysis. However, it is necessary in the analysis of complex natural samples to control the integration of the chromatographic curve and to make adjustments where necessary, j° A number of data systems and some integrators are fitted with software for electronic reintegration of chromatograms, permitting also, in some cases, correction of the integration of the individual peaks or sections of the chromatogram, without it being necessary to repeat the analysis. Nonetheless, the error in the evaluation appearing in the results of the analysis is dependent on the relative content and on the completeness of the separation of each component. (e) Reproducibility of the results. The reproducibility of the results of any analytical process is dependent on the reproducibility of all the operations. The following steps participate in the reproducibility of the GLC of intact lipids: sample preparation, injection into the capillary column, the actual separation and the integration of the individual components of the sample. The sample preparation for all natural substances depends on the type of matrix from which the sample is isolated, on the content and on the chemical character of the analyzed substances, as well as on further sample components, on the size of the sample that is available and on other factors. In practice, each type of analysis has its own specific characteristics. It is outside the scope of this review to discuss the individual cases separately. Greater attention will be paid here to the reproducibility of the actual separation process. Similar to all chromatographic separations, the reproducibility of the separation of intact lipids is affected primarily by losses. Wherever there are only reproducible losses during the separation, high reproducibility is obtained even in the analysis of intact lipids on capillary columns, comparable to that with the capillary analysis

126

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of volatile substances. Such a situation is encountered in the analysis of saturated TAG? ° The analysis of unsaturated lipids is more complex. Nonetheless, the results obtained so far n'49 indicate that the losses of unsaturated TAG are sufficiently reproducible when there are up to six double bonds in the molecule. However, the magnitude and reproducibility of the losses are not determined only by the total number of double bonds in the molecule of intact lipids, but also by the number of double bonds in the individual fatty acids, which should not be greater than three. In addition to the degree of unsaturation, the absolute weight of substance affects the magnitude and reproducibility of losses of intact lipids. This factor is limited by two variables: the total mass of the analyzed sample and the relative content of the substance in it. The total permitted weight of the analyzed sample depends on the column capacity, which is dependent on the stationary phase layer thickness and the inner diameter of the capillary. The interrelationships between these factors are generally valid in capillary GLC and will not be discussed in detail here. The reproducibility of the integration also depends on the relative content of the component; this factor is again generally valid in GLC. This is also true of the degree of separation. Losses during the separation, or more specifically their magnitude and reproducibility, can differ for different columns of a single type from a single manufacturer, and vary during the lifetime of the column. Thus, the reproducibility of the results should be controlled regularly during the lifetime of the column. (f) Accuracy of the results. The accuracy of the results of the analysis of intact lipids by GLC can be controlled indirectly by comparison with the results of analysis of the given sample by some other analytical method. A typical example is comparison of the results of the analysis of TAG on a polarizable capillary and HPLC. The accuracy of the results of the analysis of TAG on both packed and capillary columns can be controlled using a method consisting of comparison of the results of analysis of intact TAG and their fatty acids. 4s The procedure is based on the assumption that the average carbon number of the fatty acids equals one-third of the average carbon number of the TAG, when all the fractions of intact TAG are completely eluted from the chromatographic column. This assumption can be expressed mathematically in terms of the following equation: (mvA~'CvA~)= 1/3 ~ (mTG,'Cm,),

(5i

where mFai and rnTc~are the mole percent of the given fatty acid or TAG fraction and CFA~ and Cmi are the number of carbon atoms in the fatty acid or TAG fraction, respectively. A simplified form of eqn. 5 based on the equality between the content of a given fatty acid found by analysis of the intact TAG and the analysis of the fatty acids can be employed only for the separation of TAG on a polarizable capillary, or more generally, when TAG are separated according to the carbon numbers and unsaturation. Mathematically, this can be expressed by eqn. 6; mFAi = Y~ a/3 rnvGi,

(6)

where tuFAi corresponds to the mole percent of a given fatty acid found by analysis of the TAG fatty acids, Za/3 rnxG~is the sum of molar contents of the given fatty acid in all the TAG fractions in which this fatty acid occurs, and a is the number of acyls of the given fatty acid in TAG fraction i. This control can be carried out stepwise for all the fatty acids detected. In the application of both equations, i.e. eqn. 5 and eqn. 6, a result in which the right-hand side of the equation corresponds to 95-105% of the left-hand side can be considered satisfactory. Such checking of the TAG recovery can serve in practice as a sensitive indicator of losses during the analysis. Modified equations can be derived also for other intact lipids. Unfortunately, these mathematical procedures cannot replace the direct measurement of the correction factors using pure compounds. The reason is that a mathematical procedure permits calculation of only the sum of losses, and losses of the individual fractions cannot be estimated.

High temperaturecapillaryGLC

127

IV. FUTURE POTENTIAL

A. Capillary GLC It is clear that progress in the technology of polarizable capillaries allowing the use of temperatures over 360°C is important for the analysis of intact lipids. Although applications of these columns to TAG analysis only have been published in the literature, their use for the separation of other intact lipids will provide useful information on the structure of these substances that cannot be obtained by other analytical methods. Further information will also be obtained from the combination of gas chromatography with mass spectrometry (GC/MS), primarily in the definite identification of separated fractions of intact lipids. The application of multidimensional chromatographic techniques, such as the AgNO3-TLC/GLC or HPLC/GLC combination is a potential source of new information on the composition of intact lipids. The study of the effect of various parameters on the recovery of these substances during separation by capillary GLC will refine the quantification of intact lipids, especially some TAG fractions. In spite of the great advantages provided by polarizable or polar capillaries for the further development of the analysis of intact lipids, a realistic approach must be adopted towards the capabilities in the development of biological applications of gas chromatography at high temperatures. The increase in the column polarity and thermal stability to the present values permitted the separation of lipids according to their degree of unsaturation and, in some cases, according to their fatty acids, but led to problems connected with the thermal stability of the unsaturated lipids during the analysis. Although it is now believed, based on experimental results, 6:8 that losses of unsaturated lipids are significantly affected by the activity of the column or stationary phase, it must be assumed that improvements in the quality of the column and of the stationary phase can only partly solve this problem. The lower thermal stability of unsaturated substances is a result primarily of their chemical properties and it must thus be expected that even short-term exposure to temperatures of 300°C and higher can lead to thermal degradation of part of the unsaturated lipids. Consequently, it would not be realistic to assume that capillary GLC will become the method of choice for the analysis of highly unsaturated intact lipids.

B. Comparison of Capillary GLC with Other Methods Supercritical fluid chromatography (SFC) can be employed to separate TAG at much lower temperatures than those employed in GLC. Compared to GLC, this technique has the advantage of almost complete elimination of losses of unsaturated substances and higher saturated homologues resulting in highly reproducible quantitative data. In addition, the apparatus can be obtained by a relatively simple, reversible modification of the gas chromatograph, although commercial equipment is also available. The detector can be an ordinary FID employed for GLC. So far, results for the separation of TAG are not comparable with those obtained using capillary GLC on polarizable columns, 74 but the technique has undergone considerable development in the last two years. At present, separations by SFC are carried out using narrow bore quartz capillaries with an i.d. of 0.1 mm with an immobilized nonpolar or polar stationary phase. The separation by SFC is similar to that by GLC, i.e. the substances are separated on nonpolar columns according to the number of carbons and on polar columns according to the number of carbons and the number of double bonds. TM In addition to TAG, mono- and diacylglycerols have been separated by SFC. 62 HPLC is a further competitive technique, and has been applied repeatedly for the analysis of intact lipids) 7 It is very difficult to briefly evaluate the contemporary advantages and disadvantages of GLC and HPLC for the analysis of intact lipids. The greatest advantage of HPLC is again the elimination of losses of sensitive unsaturated substances and higher homologues. This advantage is, however accompanied by other problems, including less sensitive detection, more difficult identification and longer analytical times.

128

P. Mare~

A completely suitable detector for the analysis of intact lipids by H P L C has not yet been found, except for the H P L C / M S combination. The relatively low sensitivity differential refractometer cannot accept gradient elution and c o m m o n U.V. detectors are not suitable for lipids because of the lack of chromophores in the molecule. Thus, a number of various modifications of the F I D detector have appeared in the literature over the years, based on solution of the problem of transport of the liquid sample to the detector, removal of the solvent and vaporization of the analyzed substances. In addition, the identification of the separated substances is more difficult in the H P L C of intact lipids, as the individual intervals of carbon numbers overlap as a result of the strong effect of polarity on the separation in reversed-phase systems. The H P L C / M S combination solves the problems connected with the detection of analyzed substances and their identification. The only serious problem connected with this approach is the complex instrumentation and its high price. v. TYPICAL APPLICATIONS

A. Triacylglycerols The most frequent applications o f the capillary analysis of intact lipids lie in the study of raw materials and products of the fats and oils industry. Capillary G L C has been used in number of cases for the analysis of b u t t e r 10-12"14'31"33"36'59"67"72 o r vegetable oils 6'7'9-12"22'31'36'54'61'72'73 and animal fats. 6'10'11'36 Analyses have been carried out on nonpolar 7'12"]4'22"33"36'54'59'61and polarizable 6,9-1~'31 capillaries. Figure 9 depicts the separation of palm oil on a polarizable capillary. The analysis of intact T A G in biological and clinical research has not been used as extensively. This type of application includes the work of l~ezanka et a / . , 63 who analyzed

12

li

3

5

1

r

17

1

I

21

?

i

13 171 19

I

(rain)

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i

1

29

FIG. 9. Analysis of palm oil on a polarizable capillary. Analytical conditions: temperature program, 60°C isothermal for 2 min, then 60-320°C, 25 K/min, then 320-350°C, 2 K/min, 350°C isothermal for 2 min. Other analytical conditions as in Fig. lB. Sample: 0.2/~1 of 0.5% solution in hexane. Peak designation: I--MPP, 2--PPP, 3--MOP, 4--MLP, 5--PPS, 6--POP, 7--MOO, 8--PLP, 9--MLO, 10---PSS, II--POS, 12--POO, 13--PLS, 14---PLO, 15--PLL, 16---SOS, 17--SOO, 18----OOO, 19--SLO, 20---OLO. M = myristic acid, P ---palmitic acid, S = stearic acid, O = oleic acid, L = linoleic acid.

High temperature capillary GLC

129

TAG in green freshwater algae, or of Cranwell et al? in which the authors studied the composition of TAG in freshwater plankton. One of the few clinical applications of the analysis of intact TAG is the work of Kuksis and Myher, 36 which gives the spectrum of intact TAG of human blood plasma on a polar capillary. Figure 10 depicts a similar spectrum. So far, applications have been concerned with study of the structure of natural TAG. It can be seen from Figs 9 and 10 that even on polarizable capillaries, in spite of the relative complexity of the spectra of the separated substances, only a group separation is obtained without differentiation of the position of fatty acids on the glycerol skeleton. Combination of this technique with MS can yield more detailed information. The GC/MS of triacylglycerols has not appeared very often in the literature, and so far all the published results have been achieved on nonpolar capillaries. 2'54'63'73No application of polarizable capillaries in combination with MS has yet been described, although such a work would certainly provide useful information on TAG structure. B. S t e r y l Esters

One of the first applications to the analysis of intact cholesteryl esters (CE) on polar and nonpolar columns was the work of Smith, 69'7°dealing with the study of experimental atherosclerosis in rabbits. The author employed capillary GLC for the study of the composition of CE in atheromatic aorta plaques. Another application of the capillary profile of steryl esters was described for the study of the organic components of sea sediments. 73 The authors employed the GC/MS technique for identification of the separated substances. In 1985, Cranwell et al. z described further application of GC/MS to the study of the steryl esters of sea plankton. Figure 11 depicts the separation of human blood plasma CE on nonpolar and on polarizable capillaries. It can also be seen from Fig. 11 that there are considerable losses of cholesteryl arachidonate and other polyenoic CE, indicating that a capillary length of 12

14 9

,;

i'9

,,[13

2's (min)

FIG. 10. Analysis of TAG of human blood plasma on a polarizable capillary. Analytical conditions were the same as in Fig. 9. Peak designation (tentative identification according to the literature data ~ and the retention behaviour): I--MPP, 2 - - M O M + M P o P , 3---PPP, 4---PPoP, 5----MPoO + MLP, 6--PPS, 7--POP, 8--MOO, 9--PPoO + PLP, 10---PPoL + ?, 11--POS, 12--POO, 13--PLS, 14--PLO, 15--PLL+PoOL, 16--SOS, 17--SOO, 18-----OOO, 19---SLO, 20--OLO, 21--OLL. Po = palmitoleic acid, other fatty acid abbreviations se¢ Fig. 9. JPLR 27;2--D

130

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3+4

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9

I

(A)

. 8

12 (min)

16

16

.

.

.

. 19

.

(B) 22

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FIG. 11. Analysis of cholesterylesters of human blood plasma on nonpolar (A) and polarizable (B) capillaries. Analyticalconditions (column A): as in Fig. 1A except that temperature program: 180-340°C, 8K/rain. Analytical conditions (column B): as in Fig. 1B except that temperature program: 60°C isothermal for 2 min, then 60-320, 25 K/min, then 320-350°C, 2 K/min., 350°C isothermal for 2 rain. Fraction designation: 1--41 :0, 2--42:0, 343:0, 4--43 : l, 5~44: 0, 6--45 :0, 7-~15:1, 84-45:2, 9~7:4.

about 25 m is not suitable for the analysis of polyenoic CE. When the sample contains CE with fatty acids containing up to three double bonds, then the analysis of intact CE can replace the analysis of the fatty acids of CE. C. W a x Esters

As mentioned above, Wakeham and Frew 73 used capillary GLC to study CE from sea sediments and also wax esters (WE) of the same source. Dewitt et al. 5 describes the analysis of bacterial WE by capillary G L C on a nonpolar fused silica column. Similarly, l~ezanka and Podoji164 employed a nonpolar capillary to study WE from freshwater green algae. In all these works, the separated substances were identified by the GC/MS technique. Graille et al. 13 employed a nonpolar capillary to analyze the composition of Jojoba wax. D. Diradylglycerols

The importance of the study of the structure of phospholipids has increased in recent years. One way of obtaining information on the combination of fatty acids on the glycerol skeleton is analysis of phospholipid derivatives. H P L C is used more often for this purpose than capillary GLC, but the literature contains several works dealing with this problem in connection with GLC. Almost all of these papers were published by Myher and Kuksis, a6'37'4°'56-5~and deal with a number of applications of polar and nonpolar capillaries for the analysis of the products of the dephosphorylation of vegetable phospholipids or the products of partial T A G hydrolysis. For example, the authors studied the structure of soya phosphatidylinosito136 and diacylglycerols of various vegetable oils. 4° Kuksis et al.

High temperature capillary GLC

131

described the application of polar capillaries in the analysis of various phospholipids of a n i m a l tissues 36'37'57 and of nonpolar columns for the study of the structure of phospholipids in human blood plasma. 58 Caboni et aL I analyzed the composition of the pyrolytic degradation products of various classes of phospholipids of cows milk.

E. Lipid Profiles

The most difficult task encountered in the analysis of intact lipids involves the determination of lipid profiles. In contrast to the applications described so far, this corresponds to the analysis of groups of different lipids rather than study of the structure of a single lipid class. The greatest attention has been paid to the determination of human blood lipid profiles. In 1984 and 1986, Myher and Kuksis described the application of nonpolar capillaries to the analysis of the total lipids profile of human blood plasma. 5s Other authors have described similar studies. 42'43'~ Kuksis et aL 39 employed the capillary profile of the total lipids of human blood plasma for diagnosis of phytosterolemia. Limsathayourat and Melchert 44 described analysis of the total lipids in bee's wax on a nonpolar capillary. The authors separated hydrocarbons, fatty acids, mono-, di- and triacylglycerols, wax esters and cholesteryl esters in a single analysis on a 9 m long column. All the analyses of lipid profiles described so far have a common feature in that they were carried out on nonpolar capillaries. Polarizable capillaries have been found to be less suitable for the analysis of the lipid profile of human blood lipids, as overlapping occurs between CE and TAG fractions as a result of separation of lipids both according to degree of unsaturation and to the number of carbons, as can be seen in Fig. 12. It is apparent from this figure that, prior to the application of polar or polarizable capillaries for the analysis of intact lipid profiles, which have been successfully achieved on a nonpolar capillary, it is necessary to consider the formation of critical pairs, such as are found in human blood CE and TAG.

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CE I

U

¢o

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TAG

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FIG. 12. Part of the profile of neutral lipids of human blood plasma on polarizable capillary. Analytical conditions as in Fig. 11.

132

P. Mare~ F. Conclusions

T h i s b r i e f review o f the a p p l i c a t i o n s o f c a p i l l a r y c o l u m n s for the a n a l y s i s o f i n t a c t lipids i n d i c a t e s the b r o a d possibilities p r o v i d e d b y G L C in this field for the a n a l y s i s o f n a t u r a l s u b s t a n c e s . W h e r e v e r precise q u a n t i f i c a t i o n o f the s e p a r a t e d s u b s t a n c e s is necessary, especially o f h o m o l o g u e s w i t h h i g h m o l e c u l a r w e i g h t o r a h i g h e r n u m b e r o f d o u b l e b o n d s , s u i t a b l e c o n t r o l o f the r e c o v e r y o f the given s u b s t a n c e s m u s t be c o n s i d e r e d to a v o i d u n n e c e s s a r y errors. C a p i l l a r y G L C o f i n t a c t lipids r e m a i n s o n e o f the m o s t difficult a p p l i c a t i o n s o f this m e t h o d , b u t it c a n , o n the o t h e r h a n d , p r o v i d e i m p o r t a n t o r i g i n a l i n f o r m a t i o n o n the s t r u c t u r e o f i n t a c t iipids. ( R e c e i v e d 1 A u g u s t 1987)

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