Combination of silver ion and reversed-phase high-performance liquid chromatography in the fractionation of herring oil triacylglycerols

Triacylglycerols from North Atlantic herring (Clupea harengus) were separated according to the degree of unsaturation by high performance liquid chromatography (HPLC) in the silver ion mode. Each of the eleven fractions collected was then separated by reversed- phase HPLC, which in these circumstances separated the molecules according to the combined chain- lengths of the fatty acyl residues only. One hundred thirty fractions were obtained for fatty acid analysis. Almost 50% of the triacylglycerol molecules had six or more double bonds in their fatty acyl residues. Saturated-dimonoenes and disaturated- monoenes, 18.9% and 10.4%, respectively, were the most plentiful fractions of the more saturated species. Such a complex mixture of molecules was present that the most abundant subfractions from reversed- phase HPLC represented less than 5% of the total. Indeed, the largest single molecular species [16:0- 22:l- 22:6(n− 3)] represented only 2.8% of the total. These sequential analyses by complementary techniques made it possible to obtain a considerable amount of information on the composition of molecular species, but it was still not possible to identify all components.     

Silver ion high-performance liquid chromatography of the triacylglycerols ofCrepis alpina seed oil

The triacylglycerols ofCrepis alpina oil were characterized because this oil has a high concentration of crepenynic (cis-9-octadecen-12-ynoic) acid, a fatty acid useful in the chemical synthesis of deuterated fats for human metabolism studies. The triacylglycerols were separated from the crude oil by solid-phase extraction. Resolution, quantitation and isolation of the individual triacylglycerols were performed by silver ion high-performance liquid chromatography on a commercial column, an acetonitrile in hexane isocratic mobile phase and flame-ionization detection. Isolated triacylglycerols were identified by capillary gas chromatography of their fatty acid methyl esters. Of the eleven eluted triacylglycerols ofCrepis alpina oil, 85% included 35% tricrepenynoyl, 34% linoleoyldicrepenynoyl and 16% dilinoleoylcrepenynoyl glycerols. Triacylglycerols eluted according to the numbers of alkene and alkyne bonds. Elution times, resolution and quantitation were reproducible over a three-month period. The flame-ionization detector response required no response factors for quantitation of the triacylglycerols present inCrepis alpina oil. The silver ion chromatography system permitted the identification of 95% of the triacylglycerols compared to 70% of the triacylglycerols previously identified with reversed-phase high-performance liquid chromatography.     

The triacylglycerol structure of olive oil determined by silver ion high-performance liquid chromatography in combination with stereospecific analysis

The compositions of positionssn-1,sn-2 andsn-3 of triacylglycerols from “extra-virgin” olive oil (Olea europaea) were determined. The procedure involved preparation of diacyl-rac-glycerols by partial hydrolysis with ethyl magnesium bromide; 1,3-, 1,2- and 2,3-diacyl-sn-glycerols as (S)-(+)-1-(1-naphthyl)ethyl urethanes were isolated by highperformance liquid chromatography (HPLC) on silica, and their fatty acid compositions were determined. The same procedure was also carried out on the five main triacylglycerol fractions of olive oil after separation according to the degree of unsaturation by HPLC in the silver ion mode. Although stereospecific analysis of the intact triacyl-sn-glycerols indicated that the compositions of positionssn-1 andsn-3 were similar, the analyses of the molecular species demonstrated marked asymmetry. The data indicate that the “1-random, 2-random, 3-random” distribution theory is not always applicable to vegetable oils.     

The structure of the triacylglycerols of meadowfoam oil

The triacylglycerols of meadowfoam oil have been resolved by HPLC in the silver ion and reversed-phase modes, and by the two techniques used in a complementary fashion. The fractions obtained were collected and quantified by gas chromatography of their methyl esters in the presence of an internal standard. Silver ion chromatography gave a distinctive resolution in which fractions differing solely in the position and chain-length of a single monoenoic fatty acyl group were resolved, the order of elution being 11−20∶1, 5−20∶1, 13−22∶1, 5−18∶1 and 9−18∶1. Reversed-phase chromatography also gave fractions containing single positional isomers, (11−20∶1<5−20∶1<13−22∶1<5−22∶1), but the pattern was more difficult to discern since fractions containing 22∶2 tended to overlap with those containing 20∶1. The species (5−20∶1)(5−20∶1)(22∶2), (5−20∶1)(5−20∶1)(5−20∶1) and (5−20∶1)(5−20∶1)(13−20∶1) were found to be the most abundant, and together comprised 67% of the total. A small but significant trilinolein fraction was detected and its presence may have biosynthetic implications.     

Separation of monoacylglycerols by high-performance liquid chromatography on nitrile-bonded phase

Monoacylglycerol molecular species, as their di-3,5-dinitrophenylurethane derivatives, were well separated by normalphase high-performance liquid chromatography on nitrilebonded phase. The peaks emerged in the order 20∶0, 18∶0, 16∶0, 18∶1, 16∶1, 18∶2, and 18∶3. The peaks of 1- and 2-monoacylglycerols with the same acyl group showed complete overlapping. This method could be applied to get acyl compositions in the three positions of triacyl-sn-glycerols in their stereospecific analysis. Presented partially at the 83rd AOCS Annual Meeting held in Toronto, Canada, May 10–14, 1992.     

The composition of new zealand milk fat triacylglycerols by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography (HPLC) method was developed for the purpose of analyzing milk fat triacylglycerols by evaporative light scattering detection. With a binary solvent system, comprising dichloromethane and acetonitrile, the method allowed a separation of 61 distinct peaks, on the basis of chainlength and number of double bonds. Triacylglycerols of differing partition numbers were clearly resolved, and the resolution between peaks of the same partition number was high. An argentation thin-layer chromatography method, separating triacylglycerols on the basis of chainlength and degree of unsaturation, provided nine band extracts that were analyzed by HPLC. By using existing fatty acid methyl ester data of these bands, an identity for each HPLC peak has been proposed.     

Stereospecific analysis of triacyl-sn-glycerolsvia resolution of diastereomeric diacylglycerol derivatives by high-performance liquid chromatography on silica

The compositions of positionssn-1, 2 and 3 of triacylglycerols can be determined by partial hydrolysis with ethyl magnesium bromide, derivatization of the total products with (S)-(+)-1-(1-naphthyl)ethyl isocyanate and isolation of the diacyl-sn-glycerol urethane derivatives by chromatography on solid-phase extraction columns containing an octadecylsilyl phase. The diastereomericsn-1,2-and 2,3-diacylglycerol derivatives are separated by high-performance liquid chromatography on silica for determination of their fatty acids by gas chromatography. Each step in the process has been evaluated rigorously. The compositions of all three positions can be calculated with good accuracy from the analyses of these compounds and that of the total triacylglycerols. Although the 1,3-sn-diacylglycerol derivatives can also be isolated easily, they do not give reliable results for the composition of positionsn-2 because acyl migration occurs during their generation. The stereospecific analysis procedure has been applied to some plant and animal triacyl-sn-glycerols of commercial and scientific interest, containing predominantly C₁₆ and C₁₈ fatty acids,i.e. safflower, sunflower, olive and palm oils, tallow, egg and rat adipose tissue. The method is not at present suited to the analysis of more complex triacylglycerols, such as milk fat or fish oils, and problems associated with these are discussed.     

Analysis of the monoenoic fatty acid distribution in hydrogenated vegetable oils by silver-ion high-performance liquid chromatography

A silver-ion high-performance liquid chromatography column (hexane/acetonitrile as solvent, ultraviolet detection) was used to analyze the fatty acid distribution (as fatty acid methyl esters) of a representative sample of hydrogenated oil. Fractions containingcis- andtrans-18:1 isomers were readily separated. The positional fatty acid isomers were separated by rechromatographing these fractions. The elution order and percent compositions were compared with results obtained by gas chromatography. Of the Δ8 to Δ14trans-18:1 isomers, only the Δ8 and Δ9 pair could not be separated. The Δ8 and Δ9cis-18:1 pair also could not be separated, and the Δ10 isomer was poorly separated from this pair. Area percents were comparable to results obtained by gas chromatography.     

Two-step enzymatic synthesis of docosahexaenoic acid-rich symmetrically structured triacylglycerols via 2-monoacylglycerols

Symmetrically structured triacylglycerols (TG) rich in docosahexaenoic acid (DHA) with caprylic acid (CA) at the outer positions were synthesized enzymatically form bonito oil in a two-step process: (i) ethanolysis of bonito oil TG to 2-monoacylglycerols (2-MG) and fatty acid ethyl esters, and (ii) reesterification of 2-MG with ethyl caprylate. Ethanolysis catalyzed by immobilized Candida antarctica lipase (Novozym 435) yielded 92.5% 2-MG with 43.5% DHA content in 2 h. The 2-MG formed were reesterified with ethyl caprylate by immobilized Rhizomucor miehei lipase (Lipozyme IM) to give structured TG with 44.9% DHA content [based on fatty acid composition with caprylic acid (CA) excluded] in 1 h. The final structured lipids comprised 85.3% TG with two CA residues and one original fatty acid residue, 13% TG with one CA residue and two original fatty acid residues, and 1.7% tricaprylolglycerol (weight percent). The amount of TG with two CA residues and one C₂₂ residue (22∶6=DHA, 22∶5, and 22∶4) was 51 wt%. The 1,3-dicapryloyl-2-docosahexaenoylglycerol to 1,2(2,3)-dicapryloyl-3 (1)-docosahexaenoylglycerol ratio (based on high-performance liquid chromatography peak area percentages) was greater than 50∶1. The recovery of TG as structured lipids after silica gel column purification was approximately 71%. Ethyl esters and 2-MG formed at 2 h of ethanolysis could be used to determine the positional distribution of fatty acids in the intial TG owing to the high 1,3-regiospecificity of Novozym 435 and the reduced acyl migration in the system.     

Micro method for stereospecific analysis of triacyl-sn-glycerols by chiral-phase high-performance liquid chromatography

A procedure for micro stereospecific analysis of triacyl-sn-glycerols (TGs) by high-performance liquid chromatography (HPLC) on a chiral column is presented. TGs were partially hydrolyzed with ethyl magnesium bromide, and total products were immediately converted to 3,5-dinitro-phenylurethane derivatives. Each of the 1- and 2-monoacylglycerol (MG) derivatives was isolated by HPLC on a silica column. The 1-MGs were resolved intosn-1 andsn-3 MG fractions by HPLC on a Sumichiral OA-4100 column (Sumitomo Chemical, Osaka, Japan). Fatty acid methyl esters obtained from thesn-1,sn-2 andsn-3 MG fractions were analyzed by gas-liquid chromatography on a capillary column. Analyses of standard TGs showed that, even with 1 mg of sample, accuracy was comparable to that obtained with 100-mg samples. Applying this procedure to the stereospecific analysis of 5 mg of jujube pulp, TGs revealed the positional distribution of the (n-5) series of monounsaturated fatty acids they contained. Honored Student Award Address presented at the 83rd AOCS Annual Meeting held in Toronto, Canada, May 10–14, 1992.     

The effect of fatty acid composition on the acrylation kinetics of epoxidized triacylglycerols

Epoxidized oils, epoxidized triacylglycerols, and epoxidized fatty acid methyl esters were made by reaction with performic acid formed in situ. The extent of epoxidation was ca. 95% for all of the epoxidized samples, as determined by ¹H nuclear magnetic resonance. The epoxidized samples were reacted with an excess of acrylic acid for different reaction times. The acrylation reaction was found to have a first-order dependence on the epoxide concentration for all oils, pure triacylglycerols, and fatty acid methyl esters. However, the rate constant of acrylation was found to depend on the composition of the epoxidized material. The acrylation rate constant for 9,10-epoxystearic acid was 96 L²/(mol²·min). The rate constant of acrylation for the epoxides on 9,10,12,13-diepoxystearic acid was 60 L²/(mol²·min). The acrylation rate constant for the epoxides on 9,10,12,13,15,16-triepoxystearic acid was 50 L²/(mol²·min). Thus, the rate constant of acrylation increased as the number of epoxides per fotty acid decreased. Multiple epoxides per fatty acid decrease the reactivity of the epoxides because of steric hindrance effects, and the oxonium ion, formed as an intermediate during the epoxyacrylic acid reaction, is stabilized by local epoxide groups. These results were used to derive an acrylation kinetic model that predicts rate constants from fatty acid distributions in the oil. The predictions of the model closely match the experimentally determined rate constants.     

High-performance liquid chromatography analysis of peanut phospholipids. II. Effect of postharvest stress on phospholipid composition

Peanuts are harvested in late September, and sometimes the harvest season can extend through most of October. When weather patterns delay harvest, the result may cause an immature crop, curing problems, rain damage, and freeze damage. All of the above stress situations can affect oil quality and flavor of the peanuts by altering phospholipid composition. Such changes are related to refining problems as well as flavor problems. A new high-performance liquid chromatography (HPLC) method was used for the analysis of phospholipids from postharvest stressed peanuts. The concentrations of phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were higher in immature seed when compared to mature seed. A slight increase in concentration was observed for phosphatidylglycerol (PG), and a decrease in phosphatidylinositol occurred in immature peanuts. All phospholipids increased in concentration except PG when peanuts were cured at a high temperature (40°C). When peanut seeds were frozen at −16°C (before curing), a significant increase in concentration was observed for PA and PG, whereas the concentrations of PC and PE decreased to very low levels when compared to the control. Where concentration permitted, molecular species were separated on a reverse-phase column by HPLC.     

Separation of molecular species ofcis- andtrans-triacylglycerols intrans-hardened confectionery fats by silver-ion high-performance liquid chromatography

Silver-phase high-performance liquid chromatography (HPLC) on silver nitrate-loaded silica achieves incomplete separation of major triacylglycerol (TAG) classes present intrans-hardened fats. The “ChromSpher Lipids” silverloaded cation exchange HPLC column has been found to yield good separations oftrans-hardened TAG, with molecular species well resolved. Separations comparable to those previously possible for nonhardened fats are now possible fortrans-hardened fats. The separation is on the basis of number and type (i.e.cis/trans) of double bonds only; the position of the double bond along the acyl group appears not to influence the separation significantly. The analysis of a palm fraction, hardened to a slip melting point of 37°C and chemically randomized, is presented as an example. This technique offers a new approach to understanding and controlling the hydrogenation and processing oftrans-hardened fats.     

Stereospecific analysis of monounsaturated triacylglycerols in cocoa butter

The enantiomeric composition of the monounsaturated triacylglycerols (TG) from cocoa butter was estimated. The monounsaturated TG were separated into three fractions by reversed-phase high-performance liquid chromatography (HPLC), and each fraction was subjected to the stereospecific analysis with chiral-phase HPLC. The results indicated that the major TG consisted of equal amounts of 1-stearoyl-2-oleoyl-3-palmitoyl-sn-glycerol (SOP-sn-TG) and POS-sn-TG (47 mol%), 1,3-distearoyl-2-oleoyl-glycerol (SOS-TG) (33 mol%), and POP-TG (19 mol%). The contents of SOP-sn-TG and POS-sn-TG are 1.30 times that of the POP-TG content, and the SOS-TG content is 1.30² times that of the POP-TG content. The term “priority factor” is proposed for the ratio of the stearoyl group/palmitoyl group, 1:30 at thesn-1 andsn-3 or 1(3)-position. It shows a distinct specificity for particular fatty acids or their Coenzyme A esters in random esterification at each position of the glycerol moiety in the biosynthesis of cocoa butter TG.     

The regiospecific position of 18∶1 cis and trans monoenoic fatty acids in milk fat triacylglycerols

TAG of butterfat were fractionated according to the type and degree of unsaturation into six fractions by silver-ion HPLC. The fractions containing TAG with either cis-or trans-monoenoic FA were collected and fractionated further by reversed-phase HPLC to obtain fractions containing cis TAG of ACN:DB (acyl carbon number:double bonds) 48∶1, 50∶1, and 52∶1 as well as trans 48∶1, 50∶1, and 52∶1. The FA compositions of these fractions were elucidated by GC. The MW distribution of each fraction was determined by ammonia negative-ion CI-MS. Each of the [M-H]⁻ parent ions was fractionated further by collision-induced dissociation with argon, which gave information on the location of cis-and trans-FA between the primary and secondary positions of TAG. The results suggest that the sn-positions of the monoenoic cis-and trans-FA depend on the two other FA present in the molecule. With 14∶0 FA in the TAG molecule, the 18∶1 FA in the sn-2 position are mostly present as cis-isomers. When there is no 14∶0 in the TAG molecule, the trans-18∶1 isomers seem to be more common in the sn-2 position. Also when other long-chain FA are present, the trans-isomers are more likely to be located in the secondary (sn-2) position.     

Determination of molecular species of oil triacylglycerols by reversed-phase and chiral-phase high-performance liquid chromatography

A method is described for the determination of molecular species of oil triacylglycerols. The method is based on the analytical separation of the enantiomericsn-1,2-andsn-2,3-diacylglycerols, derived from triacylglycerols, by high-performance liquid-chromatography (HPLC) on a chiral column containing N-(R)-1-(α-naphthyl)ethylaminocarbonyl-(S)-valinecarbonyl-(S)-valine as stationary phase. Model triacyl-glycerol molecules comprising three known fatty acids were isolated from peanut oil and cottonseed oil by a combination of argentation-TLC and reversed-phase HPLC and submitted to partial chemical deacylation. The derivedsn-1,2(2,3)-diacylglycerols were analyzed and fractionated as 3,5-dinitrophenyl urethane derivatives by reversed-phase HPLC according to chainlength and unsaturation. From thesn-1,2(2,3)-diacylglycerol composition and the diacylglycerolsn-1,2-andsn-2,3-enantiomer composition, the individual molecular species of four peanut oil triacylglycerols and one cottonseed oil triacylglycerol were identified and quantitated. The method can be applied to triacylglycerols of any other oil or fat.     

Soybean oil triacylglycerol analysis by reversed-phase high-performance liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry

Soybean oil triacylglycerols from genetically modified soybean lines were conclusively identified by reversed-phase high-performance liquid chromatography coupled with mass spectrometry with atmospheric pressure chemical ionization. Atmospheric pressure chemical ionization is a soft ionization technique which gives simple spectra for triacylglycerols. Spectral identification of the triacylglycerols was based on the molecular [M+1]⁺ ion and the 1(2)-, 2(3)- and 1(3)-diacylglycerol fragments. Triacylglycerols identified in high-stearic and high-palmitic soybean varieties were quantitated by reversed-phase high-performance liquid chromatography with flame-ionization detection. There was excellent agreement between the fatty acid composition calculated from the triacylglycerol composition and the fatty acid composition obtained by gas chromatography of the transmethylated oils. The oils of the modified soybean varieties, compared to typical soybean oil, contained increased content of triacylglycerols known to be more oxidatively stable, such as linoleoyloleoylstearoyl, linoleoylpalmitoylstearoyl, and linoleoyldipalmitoyl glycerols, and less triacylglycerols like trilinoleoylglycerol, known to decrease oxidative stability. This study showed that the atmospheric pressure chemical ionization technique is suitable for mass spectral identification of neutral molecules, such as triacylglycerols, which do not contain a chargeable functional group.     

Triacylglycerols of winter butterfat containing configurational isomers of monoenoic fatty acyl residues. I. Disaturated monoenoic triacylglycerols

The triacylglycerols of winter butterfat were fractionated according to the type and degree of unsaturation into six fractions by silver ion high-performance liquid chromatography (Ag-HPLC). The acyl carbon number distribution of the triacylglycerols in each fraction was elucidated by reversed-phase HPLC and mass spectrometry (MS). The MS analysis of each fraction gave deprotonated triacylglycerol [M - H]⁻ ions which were produced by chemical ionization with ammonia. The daughter spectrum of each of the [M - H]⁻ ions provided information on its fatty acid constituents. Successful fractionation of triacylglycerols differing in the configuration of one fatty acyl residue by Ag-HPLC was important because geometrical isomers could not be distinguished by the MS system used. In addition to the fatty acid compositions, reversed-phase HPLC analysis demonstrated the purity of the collected fractions: molecules having acis-trans difference were separated nearly to the baseline. Triacylglycerols differing in the configuration of one fatty acyl residue were not equally distributed in relation to their acyl carbon numbers. This indicates that during the biosynthesis of triacylglycerols,cis- andtrans-fatty acids are processed differently. Although the fatty acid compositions of the corresponding molecular weight species of disaturatedtrans- and disaturatedcis-monoenoic triacylglycerols were similar, there may be differences in the amounts of different fatty acid combinations or in the distribution of fatty acids between the primary and secondary glycerol positions. In addition to the main components, it was possible to analyze minor triacylglycerols, such as molecules containing one odd-chain fatty acid, by the MS system used.     

Triacylglycerols of winter butterfat containing configurational isomers of monoenoic fatty acyl residues. II. Saturated dimonoenoic triacylglycerols

Triacylglycerols of Finnish winter butterfat containing one saturated and two monoenoic fatty acyl residues were studied. With silver ion high-performance liquid chromatography (HPLC), molecules were separated according to the difference in the configuration of one fatty acyl moiety. The distribution of the saturatedcis,trans-dimonoenoic and saturatedcis,cis-dimonoenoic triacylglycerols according to their acyl carbon numbers was compared by means of reversed-phase HPLC and tandem mass spectrometry. Furthermore, two examples of the fatty acid composition of a specified molecular weight species were shown. The fatty acid compositions of corresponding saturatedcis,trans-dimonoenoic and saturatedcis,cis-dimonoenoic triacylglycerols were similar; however, there may be differences in the proportions of different fatty acid combinations or in the distribution of fatty acids between primary and secondary glycerol positions.     

Scale-up purification for rutin hyrdrolysates by high-performance counter-current chromatography coupled with semi-preparative high-performance liquid chromatography

The study made the isolation of three constituents from rutin and exemplified how to achieve quercetin separation and quantification in rapid and scale-up processes by high-performance counter-current chromatography (HPCCC). Meanwhile, we also isolated the other two constituents, kaempferol and isorhamnetin, by semi-preparative high-performance liquid chromatography. After systematic optimization of solvent systems, sample concentration and flow rate on analytical Mini-DE centrifuge, the same conditions were scale-up by 50-fold in preparative Midi-DE centrifuge to purify quercetin, and the maximum sample loading achieved 1.65 g. Eventually, we successfully isolated quercetin, kaempferol and isorhamnetin with the purity of 99.80%, 99.84% and 99.95%, respectively. The process, therefore, provided an efficient method of obtaining sufficient quantities of highly purified quercetin, as well as kaempferol and isorhamnetin.