INSTRUMENTATION. HPLC course covers general, specialized uses

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1 825 HPLC course covers general, specialized uses New and experienced users of high pressure liquid chromatography (HPLC) learned more about the applications of HPLC to lipid analysis at the AOCS shari course "HPLC of Lipids," held May in Bloomingdale. Illinois. The course detailed essential principles of HPLC along with general and specialized uses in lipid analysis. Participants were able to meet with representatives from different manufacturers of HPLC systems and see the equipment in operation. The shan course was led by chairperson Vijai Shukla of the International Food Science Center in Lystrup, Denmark, and co-chairperson Robert Moreau of the United States Department of Agriculture (USDA) Eastern Regional Research Center in Philadelphia. Tips for beginners Participants and speakers alike had good advice to offer to the beginning user of HPLC techniques. Shukla, who has advised many students and guest researchers in the use of HPLC, stressed the importance of "hands on" experience. He suggested reading a basic book on HPLC. then experimenting. "People are afraid of the instrumentation:' he said. "Building up your own experience leads you to the best solutions for your problems." In his opinion. there is no substitute for experirnenreuon: "If students have a problem. I do not help them if I just tell them the answer; if I give them the answer. the next lime they do not know how to find the answers themselves:' Shukla stressed the importance of maintaining a logbook of what has been tried as a way 10 address and ultimately solve the problems that arise. Geoffrey COlt. a research director at Prochrom Inc.. suggested that the researcher find a good course to attend, one which offers practical experience in addition to lectures. Cox regularly teaches HPLC techniques at conferences in Europe. Brian Mess. demonstrating equipment for Gilson Medical Electronics This summary of lilt' 199/ AOCS Short Course 011 IIPLC of Upids was prepared by INFORM newswriter Karen DOlson. Inc., said that the specific system used was not that critical, but that "it's what column you use. and what's in the column, that determines your separation." He suggested that new HPLC users see what others have tried. then try to determine what will work for the unique needs of their analysis. Throughout the presentations, the researchers gave evidence of the fact that HPLC. although powerful. is not a "cookbook" method. It is a complicated technique which, when understood and carefully controlled, can yield accurate, in-depth analysis of lipid components. HPLC principles Moreau provided an opening overview of HPLC principles. Whimsically defining HPLC as a "High Priced Lipidological Contraption," Moreau also described it as "Ihe most recently developed technology." HPLC is a highly efficient and fast analytical technique for the analysis of a variety of compounds. he said. In the past most HPLC applications for lipids have been useful only for qualitative separations. but now HPLC is emerging as an excellent technique for quantitative analysis. Moreau said. The purpose of chromatography is to effect the separation of individual components of a mixture into distinct peaks as these components pass through a column packed with an inert ~ So/YenI(S) (Mobile Phase) ''''... FIG. I, Ftow chart or HPLC system. substance. Moreau said. The researcher can select operating conditions to elute the components from the column at different times. causing each component. identified as a peak on a chromatogram. to be completely separated. or resolved. from other components. Sample preparation is a critical step. Moreau said. For lipid analysis. samples are usually extracted with nonaqueous solvents. The samples must be filtered to remove any insoluble material which remains after extraction. If a researcher is interested in a particular lipid subclass. solidphose extracricn may be used to isolate the subclass before applying the sample to the column. Moreau presented a diagram (shown in Fig. I) of the six major units of a modern HPLC system. and discussed the functions of each. Sotvems: Also known as the "mobile phase." solvents are the media Ihot carry the test mixture through the column. "The right combination of column and mobile phase is absolutely critical to a good separation of components." Moreau said. Common solvents are n-bexane. chloroform. acetonitrile, methanol. and water. Solvents used for HPLC must be very pure, Moreau said. This is very important for the health of the HPLC equipment and the integrity of the resulting data, he said. Solvents specifically developed for HPLC are available, but Moreau advised that even these be treated to remove fine particulates. traces of water. and dissolved gasses. When the same solvent or solvent mixture is used for an entire HPLC f-+ "'""" f-+ R+ - ~Valve (Sralionary Phase) Oetedor{5} "1 INFORM, Vol. 2, no. 9 (September 1991)

2 826 separation, it is called an Isocranc separation. A mixture of solvents. for example acetone and acetonitrile. often gives better separations than single solvents. Separation of complex mixtures is usually more effeclive when the researcher is using gradients of solvent mixtures in which a mixing system blends 2-4 solvents during the separation, delivering a precise gradient. Pump: Piston-type pumps arc used in HPLC separations 10 inject the sample into the separation column. The pumps operate at high pressure, up to 6000 psi; variations in pressure can cause a problem called "pulsing." Many pumps have built-in pulse dampers 10 provide a smoother. more pulse-free solvent delivery. Two-piston pumps also are used for this purpose. Sample valve: Because of the pressure at which HPLC systems operate. a sample valve is required to introduce the sample into the flow of the mobile phase. Generally, the researcher will introduce the sample into a loop of fixed volume. and subsequently introduce it into the system by manually turning a valve. Some systems have an autosampler which injects the sample into the column as programmed. Column: The "stationary phase" or "bonded phase:' on the surface of the column packing material. is the surface that actually effects separation of components. This sepaniou is often accomplished based on polarity. in which the stationary phase can be either polar or nonpolar and retains components according to their polarity. Adsorption chromatography. also called "normal phase chromatography:' is based on silica gel. used with a nonpolar mobile phase. In this system. polar components will elute from the column behind the nonpolar components, which will be retained on the column by interaction with the silica gel. "Reverse phase chromatography," in which the polarities of the two phases are the opposite of those used with silica gel. uses a polar solvent in combination with a nonpolar stationary phase. Often a nonpolar ligand. such as CSt is covalently attached to silica gel to create a nonpolar stationary phase. bonded Other types of columns are also used for component separation. Ion exchange chromatography utilizes a column in which a charged group. such as a sulfonic acid or quaternary ammonium group. is covalently bound to silica gel. Although this method has not been widely used to chromatograph lipids. some studies have used ion exchange to separate phospholipids. Chiral phase chromatography is a method for separating optical isomers, using a specialized bonded phase column. This method will be discussed in detail later in this article. Detector: The detector is able to detect and quantify solutes as they elute from the column. Detectors must be able to recognize low levels of solutes often dissolved in large volumes of mobile phase solvents. The output signal from the detector usually is monitored by some type of recorder. Some detectors are destructive. that is, they destroy the sample. while others are nondestructive and allow the sample to be recovered after it passes through the column. Integrator: The integrator plots signal output over time and calculates the area under the sample peak. which under ideal conditions is proportional to sample mass. A strip chart recorder can provide a hard copy of the data being recorded and calculated by the integrator. Detectors Moreau continued his description of HPLC basics with an elaboration on the detectors available for use in TWoport!ciponb at the IIhoft COUrt. examine component. of on HPLC syst.m. HPLC of lipids. In order for the HPLC detector to "see" and quantify analyles. the mobile phase should either not be recognized by the detector or should produce a small but constant background level of signal output, called a baseline. To calibrate the system. sample standards are injected daily; peak areas of analytes can then be convened to units of mass. Types of commonly used detectors were described by Moreau as below. Ultraviolet (UV) detectors: The UV detector is the most common type found in analytical laboratories. Advantages of the UV detector are that it is relatively inexpensive. can be used with solvent gradients, and is insensitive to changes in now and temperature. Disadvantages are that some lipids have lillie or no UV absorption. making them difficult to chromatograph; absorption is often proponionalto the degree of unsaturalion of the lipid sample; and certain common solvents, such as chloroform, are strongly UV-absorbing and therefore cannot be used in this system. Some investigators use diode array detectors. which collect data from the entire UV range rather than one wavelength only. The diode array detector is less sensitive than variable UV. but it allows the researcher to build a "library" of spectra of known samples. against which unknowns may be compared. Refractive index (RI) detectors: This type of detector is the second most common type in use with HPLC of lipids. It operates by measuring the INFORM, Vol. 2. no. 9 (September 1991)

3 refractive index of the solvent plus the sample and compares that with the refractive index of the solvent alone. An advantage of using the RI detector is that the response is not restricted to a chromophore; thus the RI detector is called a "universal detector." However. it is sensitive to changes in temperature and flow rate. gives a high level of "noise:' and cannot be used with solvent gradients. The major drawback of the RI detector is that it is limited to isocratic separations. Fluorescence detectors: Fluorescence detectors measure emission of light when a sample is irradiated with UV light. This highly sensitive technique is useful with chlorophyll and other pigments and with lipids that fluoresce. The fluorescence detector has low minimum limits of detection and can be used in solvent gradient analysis. Because few lipids naturally possess fluorescence. a f1uorophore can be attached to some lipids. Svaporanve light scattering detectors (ELSD): These detectors are sometimes referred to as "mass" detectors. The mobile phase is rapidly evaporated in an evaporative chamber by a nebulizer. which mixes the eluant stream with a large amount of gas, usually nitrogen or air. The more volatile mobile phase is converted into a gas. while the solute remains in droplets. These particles. when directed through a light source. will scatter the light in proportion to their mass. The ELSO system is relatively easy to use. provides a very stable base line, and requires only routine maintenance. Although the mass-to-area response is linear from 10 to 200 micrograms. quantitative analysis is somewhat limited with less than 10 micrograms of lipid. An important disadvantage of the ELSO is-that it canner be used with volatile components. If the analyte is even slightly volatile. it will be evaporated along with mobile phase solvents and will either remain undetected or will produce a weak signal. Moreau reported that he and his coworkers found that palmitic acid. not usually considered to be volatile. was being volatilized by their ELSO system: lowering the temperature of Ihe detector allowed them 10 detect this fatty acid accurately. Flame ionization detectors (FID): In this system. the components are burned in a very hot flame and resulting carbon ions are measured by electrodes placed near the flame. An advantage of the flo is that the ratio of mass to peak area is very linear in the range of I to 200 micrograms of lipid. A disadvantage is that a great effort is required to maintain the FID system. Furthermore. volatile analytes are not quantified accurately by the flo. Neutral lipids Shukla spoke to short course participants on using reverse phase HPlC techniques to separate and analyze neutral lipids. Complex mixtures of lipids can be very complex. indeed, Shukla said. and reverse phase HPlC has proven very useful in the analysis of natural triglyceride mixtures. Shukla cited a recent survey conducted by LeGe magazine. which found that 61% of the persons they surveyed used reverse phase chromatography for lipid analysis. Unusual lipids. such as those with a high level of long-chain unsaturated fatty acids. can be evaluated with reverse phase HPLC. There are a number of ways for things to go wrong in HPlC analysis. Shukla said. Certain physical effects can alter the success of an analysis. The velocity of mobile phase flow is important in effecting separation. and column length and particle size will also affect the efficiency of the system. Column selection is very important to the success of an analysis. he said. Not all reverse phase columns are the same. Because silanol groups in the column can sometimes interfere with analysis. Shukla recommended that these groups be "capped" when a new column is conditioned in order to get a highly reproducible column. "This is the kind of column you want 10 work with;' Shukla said. Chemical effects are also important to good separation. These include the compositions of the mobile and stationary phases. Because of the complexity of triglyceride molecules. with fatty acids that are saturated. unsaturated. polar. and nonpolar. a solvent system. that combines several solvents TheVarex HPLC Universal Detector REVEALS -UNIVERS.lL... vm" crca L I, = ~SoIutes.~WIth Muldple Soh'ent Gradlenu Detects carbobydrates, Uplds, 'Diglycerides. Polymers, ADd More. Unrivalled Stability 827 Call ForYourFREE Video ~ Burtoosvi11e. MD SEE US AT E A S NOV SOMERSET, NJ

4 828 is most effective. and sensitivity can be improved by alterations in the solvent system. Temperature also has a great influence on triglyceride analysis: some separations must be completed ill less than 200C to be efficient. None of the available HPLC systems is designed for use below ambient temperature. so a water bath is sometimes required to achieve a cooler column temperature. "Reverse phase HPLC can be adapted to the level of precision you need." Shukla said. Triglycerides, diglycerides Shukla also spoke on the analysis of triglycerides and diglycerides. In addition to information on the theory behind HPLC separation. Shukla had practical tips for successful analysis. Methodology should be quick and reproducible. he said. The baseline is important because it describes the ratio of signal to background noise. Shukla said. If noise is too high. the signal recorded will not be as precise as possible. To dampen noise. he suggested putting the column in a jacket at 60 C and then operating at a lower pressure. "Once you've developed the right combination of columns. you must work with the temperature to achieve the best separation:' Shukla said. To safeguard the column. he suggested the use of guard columns. which filter the sample before it is injected into the column. However. guard columns often decrease efficiency. Shukla also described new systems in use for analysis of specific types of compounds. Isomers of synthetic antioxidants. used to protect oils from oxidation. may be analyzed in less than three minutes using a high-speed system. he said. Superspeed liquid chromatography has certain advantages over conventional systems in that it permits faster separation, increased resolution. decreased solvent use. and decreased gradient cycle time while not compromising precision. Size exclusion chromatography has been used with fish oils to separate polymeric material from the fatty acids. di- and triglycerides. Lipid class separations William Christie. of the Hannah aidchibiie. right. toik, to portic~ts short coune. at the Research Institute in Ayr. Scotland, spoke on lipid class separations. "There's a lot of magic and mystery, and not a lot of science. connected with the use of silica:' Christie said. Although adsorption chromatography on silica gel has been used to separate neutral. or "simple:' lipids. there have not been numerous publications on the subject. Christie attributed this to the fact that it is difficult to obtain reproducible retention times because of the nature of the adsorbent surface. The problem with reproducibility when using silica is related to the water of hydration bound to the column. Loosely bound water can have a marked effect on the reproducibility of separations, especially of nonpolar lipids. because it is readily removed. HPLC separation of simple lipids is dependent on the column. the detector, and the mobile phase. Christie said. He identified three key items in an analysis: (a) getting the separation right; (b) proper detection, and (c) proper quantification. Using a silica column. separation may be based on chain length. degree of saturation. or absorption. Different stationary phases are useful for different purposes. he said, and changing the stationary phase provides a great opportunity for changing the selectivity of the system. Changing the solvent also can greatly alter the separation. Christie said. Silver ion chromatography Christie also spoke on silver ion chromatography. This analysis is based on the principle that silver ions interact with double bonds in the fatty acid carbon chain, reversibly forming polar compounds; elution with a solvent will give different separations according to the degree of unsaturaucn. posilion of double bonds, and configuration. The greater the number of double bonds, the stronger the compound will complex with silver. Fully saturated lipids. which don't form complexes. will migrate to the top of a thin-layer chromatography plate; lipids with one monoenoic fatty acyl residue will migrate less, followed by those cornponenrs with increasing unsaturation. This technique is used most often for the fractionation of molecular species of triglycerides. The silver ion procedure is useful also for analysis of unknown samples. the identification of which then may be refined using reverse phase HPLC. Because the technique is based on a single molecular principle. identification of components "is almost intuitive," Christie said. This is in contrast to HPLC. in which components are identified based on chain length and number of double bonds in the fatty acid residues and intuitive identification of peaks is not generally possible. There are some drawbacks to silver ion procedures. however. including messiness and potential for sample contamination by silver ions. Because the equipment is simple. inexpensive and available in m3ny laboratories, Christie predicted that it probably will continue 10 be widely used. There have been many attempts to adapt silver ion chromatography to HPLC systems. Christie said. Researchers generally will introduce silver nitrate into HPLC-grade silica gel, and then pack it into columns. lsocratic and gradient elution procedures have been developed. using FlO. which yield excellent resolution and quantification of fats-confectionery fats for example. Chromatograms from this type of analysis are relatively easy to interpret. in contrast with those from reverse phase HPLC. Application of silver ion chromatography to highly unsaturated fats has not been widespread: Christie eunbotcd this to the problems with contamination of the samples and the detector from the continuous elution of silver ions. INFORM. Vol. 2. no. 9 (September 1991)

5 Phospholipids Amis Kuksis, of the Banting and Best Department of Medical Research at the University of Toronto, addressed short course participants on the use of HPLC for analysis of phospholipids. Numerous molecular species make up the group of lipids classified as phospholipids. Because the properties of the phospholipids depend on the nature of component molecular species, there is much interest in determining positional distribution and molecular association of fatty chains in these classes. The molecular species composing a phospholipid can be determined using chromatography 10 separate either the intact molecules or derived moieties. The most extensive separations are obtained with reverse phase HPLC on Cu columns, Kuksts said. Because phospholipids do not possess strongly UV-absorbing or fluorescent groups, sensitive detection of the resolved component can be a problem: light scattering detectors and name ionization detectors are useful in some cases. Some phospholipids. such as phosphatidylethanolamine and phospbatidylserine, can be convened to Uv-absorbing dinitrophenyl or triniuophenyt derivatives. On reverse phase HPLC, these derivatives are then separated into individual molecular species and quantitated by UV absorption of the attached chromophore. Retention times will be affected by double bonds at different positions and in different configurations. An alternate method for resolution of molecular species of phospholipids is removing the polar head groups and separating the remaining neutral lipid moieties. This technique allows a researcher to prepare Uv-abscrblng or fluorescent derivatives for quaruttation. Certain derivatives possess better chromatographic properties than the intact phospholipids. In general. the species are resolved in order of decreasing unsaturauon and increasing chain length. Reliable identification of molecular species requires either peak collection and analysis of the fatly acids or online liquid chromatography/mass spectroscopy (LC/MS) examination of the peaks, Kuksis said. Only tentative peak assignments can be made on the basis of relative elution times. To resolve the molecular species of intact phospholipids, a first step is to isolate the pure phospholipid classes. The first HPLC resolution of molecular species of intact phospholipids was obtained for sphingomyelins by elution with an Isocrauc methanolaqueous phosphate buffer system and detecting with short wave UV. Kuksis said. The peaks were seen to emerge in order of decreasing number of double bonds and Increasing chain length. However, peak assignment is 'It almost seems as If supercritical fluid chromatography was designed with lipid analysis in mind.' difficult on basis of chromatographic retention time alone because most natural sphingomyelin contain several sphenoid moieties, he said. More elaborate solvent systems allow for resolutions of the molecular species of intact glycerophospholipids. A major shoncoming of this method is the inability to resolve certain subclasses of phospholipids. Detection systems which don', rely on the use of chromophores permit a wider selection of derivatives to be employed. and also allow the use of gradient solvent systems. Supercritical fluid chromatography Ellen Derrico of Dicnex Corporation spoke on the equipment and principles involved in supercritical fluid chromatography (SFC). When a gas or liquid is heated above its critical point and pressurized above its critical pressure, it becomes a supercritical Fluid, Derrico said. Such fluids possess many unique properties that are useful in chromatography. She said that as a liquid reaches its critical point. the solubility of many solutes can be as much as 100 times greater in the supercriticaj state. Advantages of this technique are a decrease in sample preparation time, extraction at low temperatures, which is important for unstable compounds. and no need for organic solvents. SFC is adaptable to a variety of detectors. William Am, of the University of Illinois Department of Food Science, spoke on the application of supercritical fluid chromatography to lipids. Fats, oils, and mosi of their derivatives are very soluble in supercritical carbon dioxide. Anz said. "It almost seems as if supercrttlcal fluid chromatography was designed with lipid analysis in mind... he said. SFC has several advantages over gas chromatography (GC) and HPLC. Artz said. Detectors which lire compatible with either HPLC or GLC can be adapted for use with SFC. For certain types of compounds. the SFC technique provides much improved resolution over HPLC. Often, the 'derivalivization required for GC analysis can be avoided with SFC. The technique is also useful for separating compounds that normally would decompose at the temperatures required for GC analysis. SFC is particularly well suited for separation of oligomers and for samples of high molecular weight compounds. The appropriate sample solvent for SFC use is one that readily solubilizes the analytes and causes only minimal interference with the separation. Kuksis said. Water must be removed from the sample before analysis, he said. although on-line supercrtucal fluid extraction (SFE) can be used to bypass lyophilization steps. Off-line SFE also holds considerable promise. Kuksis said. Although many of the same constraints hold for SFE as for normal solvent extractions. i.e.. that the analyte must be soluble in the extracting fluid and the particle size must be as small as possible. most analytes appear to be extractable within a much shorter lime frame and with much less solvent than is required with classical extraction systems. Kuksis described techniques for enhancing SFE, such as co-sonication or derivativization during extraction. addition of a modifier to the sample matrix, or addition of tertiary modifiers. A modifier changes the polarity INFORM, Vol. 2, no. 9 (September 1991)

6 832 of the supercritical Fluid and thereby increases the range of polar compounds that can be extracted. In selecting the supercritical Fluid 10 use, Kuksis suggested that the researcher consider the critical pressure and temperature of the Fluid. its dipole moment, possible chemical interactions with the stationary phase and analytes. compatibility with detector systems, seals, tubing and pumps, environmental and safety considerations. COSI and available quality. Carbon dioxide is the most common mobile phase, preferred because it is nontoxic, nonflammable, is very compatible with nearly all detectors, has a low critical temperature and pressure, is easily purified and readily mixes with a variety of modifiers. Although nitrous oxide has similar characteristics, it has a deleterious effect on some polyunsaturated lipids because of its actions as an oxidizer. Xenon is also a good choice, he said, but it is too expensive for common use. Nonpolar stationary phases are the best choice for separation of fatty acids or their derivatives based on number of carbons, Kuksis said. Polar stationary phases are useful to separate triglycerides or fatty acids with the same carbon number but a different number of double bonds; separation is accomplished on the basis of double bonds or particular functional groups. HPLC-mass spectrometry Kuksis also spoke on HPLC-mass spectrometry (MS). Due to its Simpler operation and wider range of applicability, reverse phase HPLC has largely replaced polar capillary GC as the method for determining the molecular species of both neutral and polar glycerolipids. and normal phase HPLC has replaced thin-layer chromatography (TLC) as the method of isolating lipid classes, Kuksis said. Nevertheless. sensitive detection of solutes in the column effluents continues to be a problem in HPLC analysis of glycerolipids, he said. because these do not possess UV-absorbing or fluorescent chromophores for easy spectrophotometric assay. Peak identification remains tentative when based on retention times alone, but HPLC in combination with MS pro- AnnIe Anderson, of the Intemotlonol Food SClenee Center In Aamul, Oenmorlt, talkllo a Ihort course participant about the HPlC sys1em she Is demomhatlng. vides both sensitive detection and unequivocal identification of all lipid species, Kuksis said. Solid-phase extraction Lydia Nolan of Supelco Inc. spoke on solid-phase extraction (SPE) of lipids. SPE allows for the rapid and simultaneous preparation of multiple samples for analysis. she said. The samples are typically more concentrated and have less interferences than samples prepared by other techniques. Separation of vegetable oils is a common SPE application. Other applications include concentration of trace compounds from sample solutions and removal of impurities. The most popular SPE packing for preparing lipid samples is silica gel. With silica. lipid fractions can be separated on the basis of their polarity, as progressively more polar wash solvents arc used to obtain fractions containing analytes which are increasingly more polar. SPE can also be used to remove polar interferences from nonpolar sample solutions prior to analysis. Nolan said that the power of SPE comes from the fact that ir can separate compounds of interest from impurities: the compounds of interest are retained by the packing but the impurities are not. Wash solutions are employed which are just strong enough to remove impurities but weak enough to leave the compounds of interest behind on the packing. Analytes can be fractionated on the basis of polarity by carefully changing the polarity of successive wash solutions. Finally, selective elution of the desired compounds can be obtained by choosing an eluting solvent that only removes the compounds of interest and leaves behind any more strongly retained impurities. Solvent strength is a critical factor in analysis with SPE, Nolan said. If the polarity of the analytes and the impurities are sufficiently different, appropriate packing and solvents can be easily selected to allow either the analytes or the impurities to be retained on the packing while the other passes through the extraction tube unretained. SPE operates on the same principles as liquid chromatography for purification and/or concentration of a sample, Nolan said. Some benefits of SPE include: reduced use of solvent, shorter sample preparation times, and higher analyte recoveries. To develop successful SPE methods, the analyst should have an idea of the relative polarities of functional groups of the packings being tested, the solutions used. and the various compounds in the sample solution, Nolan suggested. By understanding INFORM, Vol. 2. no. 9 (September 1991)

7 - 833 how the different functional groups may interact, the researcher can determine how to manipulate the method to accomplish the desired level of extraction setectivuy,, SPE guides from various sources con lain information that can be helpful to the researcher in developing new procedures and understanding these concepts, Nolan said; she also offered other hints in methods development: Packing selection: For initial development of a method. a packing should be chosen which best matches the polarity of the compound to be retained. For example. sample solutions containing nonpolar solvents or nonpolar solvent mixtures require the use of normal phase adsorbents. such as silica gel. In some cases the use of polar bonded phases may also be effective for normal phase solutions. When using polar adsorbents, components to be retained on the packing would have to be somewhat polar in nature also. If the sample solution is aqueous or an aqueous/organic mixture, a reverse phase packing type would be a better choice if the compounds to be extracted are relatively nonpolar and have limited solubility in the sample solution. Nolan said. Pocking conditioning; Before a sample solution is added to an exrraclion tube, the tube packing should be conditioned with an appropriate solvent or solvents. This conditioning will equilibrate the packing so it is prepared to receive the sample and will facilitate extraction of the desired compounds. The polarity of the final conditioning solution should differ greatly from the packing polarity, Nolan suggested. Conditioning solutions for reverse phase packings typically consist of first a water miscible, organic solvent. followed by an aqueous solution. Matrix adjustment: Sometimes the polarity of the sample matrix must be adjusted to make it as unlike the packing as possible without precipitating the compounds of interest. Addition of acid or base to the sample may be necessary to eliminate or enhance ionization of various sample components. This is particularly useful when compounds with ionizable groups are to be extracted on nonpolar packings. Sample addition: In developing a new method, it is generally useful to analyze the effluent from the tube following sample addition to check for unintentional loss of the retained compounds. Tube capacity for a given analyte usually varies from application to application depending on the polarity of the analyte, analyte matrix composition, and packing type. Nolan said that sample flow rate can also affect the capacity of a given packing, and whenever possible the sample should pass through the packing at a slow, drop-wise rate. Packing washes: It is important to remove any matrix interferences that may remain on the packing following sample addition. Several milliliters of sample solvent can be used to remove residual, unrerained impurities. Nolan warned that wash solutions can be a major source of sample loss because of the sensitivity of the sample to solution strength. She suggested that when multiple wash solutions are used that the solution most unlike the compound of interest should be used first. Air-drying the tube packing between the packing wash and sample elution will remove residual solvent and increase recoveries, she said. Analyte elution: Compounds of interest are eluted with solutions in which they are freely soluble, In the ideal situation, selective elution will use a solvent that is strong enough to remove the compound of interest but stili weak enough to leave any more strongly retained impurities on the packing. Nolan identified several problems that consistently cause problems with development of methods for SPE. Frequently the incorrect packing is used to extract a particular sample. A packing may have good capacity for the compound of interest but not allow the compound to be recovered from that particular packing. The packing may also pick up an impurity that is difficult to remove and co-elutes from the packing with the compound of interest. Matrix impurities may also coextract with the analytes. Variations in tube performance may occur because the packings are not properly conditioned or not allowed to dry before sample addition. Adding too much sample and overloading the packing is a frequent problem, she said. Wash solutions that are too weak or too strong can result in an impure extract or low recoveries, respectively. Finally, if the eluting solution is too weak, or not enough is used, low recoveries will result. The use of SPE for the preparation of lipid samples is an attractive alternative 10 liquid/liquid extraction, Nolan said. Using this technique, samples can be fractionated into various classes based on the differing polarities of components, may be concentrated for trace analysis, or may be purified. Understanding the interaction of functional groups of the adsorbent, sample, and solvents is necessary to successfully develop SPE procedures. Other topics Heikki Kallio presented a new method for stereochemical analysis of triglycerides. This method, based on ammonia-negative ion tandem mass spectrometry. distinguishes between the fatty acyl groups at the sn-2 and sn- 1/3 positions of the individual triacylglycerol molecules. The aim of the study was to develop and apply methods which give information about individual TAG molecules and data concerning the stereospecific position of $11 2 in these molecules. Applications of the method include on-line process control method in edible oil processing, dairy, pharmaceutical, and fine chemical industries. Toru Takagi of Hokkaido University in Hakodate, Japan, presented a method for chiral chromatography of lipid enantiomers. Bnenuomers are compounds that are mirror images of each other; these compounds have the same physical properties and hence are not resolved from each other by common methods of chromatography. The use of a chiral stationary phase allows for resolution of these compounds. Applications of chiral chromatography include examination of optical impurities in lipid standards and reagents, analysis of the composition of natural lipid mixtures. and preparative enantiomer resolution for synthesis of lipid enantiomers. 0 INFORM. Vol. 2. no. 9 (September 1991)