Recent advances in the high performance liquid chromatography of lipids

Prog. LO~ Res. Vd. 2L p~ 5-3~ 1988 Printed ~ G r i t B f i t ~ Aft figh~ rescued

016~2~8~$~00+~ © 1988 Pergamon P ~ ~c

RECENT ADVANCES IN THE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF L I ~ D S V|JAI K. S. SHUKLA Analytical Resear~ & D~elopment Laborat~ies, A ~ h ~ Oliefabr~ A IS, M . ~ Bruunsga~ 2~ ~ 0 . Box 50, DK 8100 A ~ h ~ ~ D ~ m a ~ CONTENTS 5

I. ~ O D U C ~ O N H. H ~ C

S ~

III. ~ c ~

A~ ~

~ O ~ V

S~MS

7 11 11 16 16 24 26 27 28 29 30 31

IV. APP~CA~ONS

A. F ~ y ~ d s 1. ~ M ap#~afions B. ~ f i ~ C. ~ o ~ D. Nmfifion~ ~ d s ~ Anfio~dan~ F. ~ s G. M ~ c ~ a n e o ~ ~ d s H. A ~ m ~ ~ g ~ I. ~ ~ ~ V. M O ~ N ~ e c ~ ~ ~ A. ~ q ~ d c h r o m a t o g r ~ h ~ a s s s ~ ~ B. S ~ I fl~d ~ m ~ o ~ a p h y ~F~ ~. C o s ~ ~ ~

AND ~

6

F ~

O~ HPLC ~ A P P L ~ ~ ~ D S

32 32 32 34 35

~ C ~

35

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

Devdopment of ~gh performance liq~d chrom~ography (HPLC) (or "~gh p ~ u ~ " , "high pficC', "h~h speed" and "modern" ~q~d chrom~ograph~ began ~ the ~ 1960'~ T~s ~ g ~ y e ~ e n t and ~ an~ytic~ ~ch~que util~es the ba~c ~q~d chromatography pfin~ demon~ra~d ~ the ~marka~e work of Martin and Synge ~ 1941~ (for which they won the No~e Prize. The m~or advan~ges ofHPLC ~dude ~gh res~ution~ speed, ve~atilit~ ~ n ~ t i ~ and automatic op~ation, w~ch ~d to Rs acceptance both as a ~ a r c h tod and for routine an~y~s. The supefiofi~ of HPLC over other e~sting an~ytic~ techniques for an~yfing ~ d s is aafibuted to the following: (a) (b)

(c) (d) (e) (f)

HPLC is the m~hod of ch~ce for the nonvo~ti~ thermally ~bile ~gh m~ec~ar w~ght compounds~ for separating these at am~ent ~mper~u~s. HPLC p~o~d~ a means for the de~rm~ation of multiple components ~ a ~n~e an~yfi~ Both aqueous and nonaqueous s a m ~ can be an~yzed without p ~ t ~ m e n t . A v ~ of various sdvents and co~mn pacing m a r r i e s pro~d~ a ~gh deg~e of selectivi~ for spec~c an~yses. Separation times a ~ short. Normally an~ys~ a ~ c o m p ~ d ~ a ~w minutes (high speed)~ or seconds ~uper speed) ~ with excd~nt preston and accuracy. The most impo~ant ~ a t u ~ is that the ~para~d componen~ can easily be collected and recove~d ~om the m o ~ pha~ for fu~her an~y~s or charac~rization by the comp~menmry tech~qu~ such as mass spec~om~ry, nuclear magnetic resonance or ~ a ~ d spectroscopy.

6

V . K . S . Shukla

Because of the confiderab~ growth in the area of HPLC of li~ds since the excd~nt refiew p ~ n ~ d by A i ~ m ~ in 1982, there is a need to summarize recent trends and c o n c e p t u ~ impo~ant apN~ations. The purpo~ of t~s ~ e w is to p ~ n t in a p ~ a ~ e form ~cent devdopmen~ of HPLC in ~ d s . II. H P L C

SYSTEMS

AND

COLUMN

TECHNOLOGY

The ~ch~que of HPLC has grown enormously during the past decade and is now accepted as a m~or tool for the a n a ~ s of ~Nds. HPLC is still one of the ~ t devdoNng areas of an~ytic~ chemistry. The dfi~ng f o r e ~ a ~ n g to this growth has come ~om drastic improvements in HPLC c~umn ~chn~ogy and in~rumenta~on. Reve~ed phase chromatography on o ~ a d e c ~ l a n e bonded phases continues to dom~ate HPLC m o d e , accounting for m o ~ than 65% of the present day applications. ~ ~nce reversed phase c~umns are much ea~er to hand~ and are con~dered the best for the ~paration of a hom~ogous series of compound~ these have been ex~nhve~ em~oyed for the ~paration of finds in our own laboratory. ~s~-~ The most common m~hod of preparing reve~ed phase packing emNoys a sur~ce ~action b~ween a ~l~a support and an appropria~ organochloro~lane (or organo~ k o x ~ mo~fier. The ex~nt to which the bonding reaction approaches c o m p ~ n ~ s (ma~mum coverag~ is an important de~rminant of c~umn qualiff. The ~eric ~ndrance ensues that the b o n i n g reaction is ~ways incomN~e. ~ Some m a n u ~ u ~ apNy a secend ~ z ~ n using trimeth~ch~rosilane as siltation agent to ~move ~sidu~ silan~s. The presence of un~a~ed, a c c ~ e ~lano~ on the silica sur~ce affects c~umn ~a~fiff, re~ntion ~ p r o d u d ~ f f and peak asymm~ry. Because the reaction of wa~r or other reagen~ ~ the m o ~ phase ~ads to the ~ u t i o n of the underling ~l~a, ~ a d ~ g to reduced c~umn fi~tim~ ~ h e m ~ insm~fiff), these accessible ~lanols give rise to mixed retention mecha~sms. Separations on a column fil~d with such packing will occur through an unde~rable mixtu~ of norm~ pha~ and reve~ed phase processes. T~s phenomenon crea~s a m~or p r o t e i n when comparing ~parations ~om one laboratory to another, ~ a ~ n g to serious mi~nterpretations. ~88:°5':°6 Although con~derab~ advances have been made for the ~ p r o d u d ~ f f of ~he c~umn chemistry and t~e ra~al comp~shon ~chno~gy, p e r ~ n is yet to be ach~ved. Although columns with particles as small as 5 ~m have been in routine use for ~ v e r ~ years, research continues towards the devdopment of s m e a r particles, m~t~z:°s Theoretically~ a particle ~ a m ~ e r ~ of about 2 ~m will be optimum for many sepa~ ations. Recent commerci~ availa~liff 3s'~'43m'~:~'~ of 3 ~m partid~ ~d to the deve~pment of ~gh speed fiq~d chromatography (HSLC), w~¢h demands improved m ~ r u m e n t ~ n to ~duce extrac~umn band broade~ng effe~s~ EmNo~ng this ~gh speed sy~em, performance levds up to 450 N a ~ c are ~ t ~ n a ~ ~ thus allowing many isocratic ~parations to be performed in 1-2min with over 10,000 t h e o ~ t ~ p h y s . ~ Be~des offering ~gh speed/high ~s~ufion a n ~ y ~ the HSLC sy~em also ~elds ~wer solvent consumption and reduced gradient cycle times. Several reports ~om the autho¢s hboratory d e m o n s ~ g the uses of 3 ~m ~l~a and ODS partid~ for find analyses have appeared. During the coupe of our work ~ for the ~paration of tri~ycerides u~ng 3 #m ODS-2 partid~, we reafized the need for the ~gh effidency of the c~umn, because many of the tri~yceride components are c o d u ~ d r d a ~ d and need high # a ~ numbers for effident ~parations. We used the approach of generating very ~gh Na~ numbers by seria~y coupling two ~andard c~umns. Thus, it was pos~b~ to ac~eve basel~e ~paration of ~1 the triglycefides of cocoa-but~r (Fig. 1) by ex~n~ng the r ~ n g power of the couNed sy~em. U s u ~ HPLC c~umns packed with 3 ~m particles can ac~eve bet~r separations than those obt~ned for large particle columns ~ ~ss than half the total time ~q~ring very-low-dead v~ume l ~ d chromatograp~c sys~ms. It is ~so worth noth~g lhat sub~anfial time savings can also be obtained during m~hod devdopment due to q~ck

Recent a d ~ n ~ s ~ HPLC of ~ d s

7

(a)

(b)

~6. 1. Comparisonof the ~paration of tfi#y~fidesof cocoabm~r on a 15cm $30DS 2 cdumn (a) and two 15cm cdumns cou#ed ~ ~ries (b).t~ changeovers b~ween mobile phases ~ ad~tion to the shorter anMyfis time and better effi~ency. T~s opens up new capabilities for the routine HPLC Mboratory. The ~ t i m e of 3 ~m columns is apparently the same as 5 #m prodded sperm care ~ taken ~ camful~ clarifying the moMM phase and the s a m # ~ . ~gnificant advances have been made in HPLC ~ r u m e n t a t i o n s based on microproce~or ~chnoMgy) ~ The ev~vement of ~tegrated HPLC sy~ems w~ch contrd ~o~ affecting performance saves confiderabM time and mi~mizes error in routine anMyses. Sample preparation ~ the rate-fimiting step in the automation of HPLC sys~ms. Recent advances in sam#e handling robotics have addressed t~s Hmimtion. Robotics syaems am avMhble for re,aMy and pre~sely performing extractions, filtration and other cleanup procedures prior to injection. ~'2~2 Very reeenfl~ an automated HPLC sam#e inject~n sy~em has been described. ~ During our work, ~ was necessary to mo~fy the av~MMe HPLC ~ g r a ~ d systems accor~ng to our need to optimize ~parat~ns ~ ~pid a n M y ~ a ~ew shared in earlier renews by Aitzetmfiller3 and Hammond. ~ O~en it is desirable during the anMy~s of ~#ds to opera~ at beMw amMent temperatures. To the best of my knowledge, them ~ not a fin#e HPLC sy~em avMhbM w~ch takes ~ t o account the operation below amMent ~mperatums and, at the same tim~ mi~mifing ex~a column band broadening effect ~om the injector and d~ecton III. D E T E C T I O N

SYSTEMS

The progress of HPLC for the an~ysis of ~pids has been restricted due to the hck of a u ~ v e ~ ~nfifive HPLC d~ecton Although there has been confidera~e growth in the devdopment of various detection sy~em~ much romans to be done with respect to the improvements ~ d~ection ~chno~gy for the a n ~ y ~ s of ~ d s . The ~ t r a f i ~ (U.V.) d~ector was one of the e a d ~ and mo~ p o p l a r HPLC detectors used for anfly~ng ~pids. Although most fi~ds in general hck chromophoms w~ch fao~ta~ U.V. d~ecfion but absorb generally in the 190 to 210rim range, t~s short wave~ngth U.V. d~ection ~ more ~nfitive and permi~ the use of grad~n~ but precludes the use of ce~Mn common H#d so~en~ such as chloroform or acetone wMch are opaque in the U.V. region of ~ m ~ . We have recently demonstrated I~ the use of short wavdength U.V. d~ecfion at 220 nm for the quantitative anMyfis of tfi#ycerides. At t~s waveMngth, U.V. absorpt~n of tri#ycerides ~ bafica~y due to the ester C.~---O function of these m~ec~es.

8

V . K . S . ShuEa TABLE 1. Absorbance of Acetonitfile/Tetrahydrofuran (70:30 v/v) Saturated with Dissolved Gases at 20°C ~ 210 W~hout saturation Nitrogen Helium Carbon dioxide Air

0.625 0.551 0.586 0.572 0.673

Wavdenph ~m) 215 218 0.396 0.271 0.350 0.312 0~393

0.252 0.135 0.208 0.150 0.249

220 0.167 0.082 0.124 0.093 0.180

C o m m e r o ~ av~lab~ "HPLC Grade" ~trahydrofuran (THF) has a U.V. cutoff varfing ~om 212 to 220nm depen~ng upon the partic~ar lot recoved. Ac~omtri~ (ACN) cutoff is ~ 190 nm. Thus, a c e ~ n basefine i n s ~ f i f f may occur with inc~a~ng concen~ations of THF, w ~ operating at sho~ wavdengths. Lynch and coworke~ ~ have reposed absorbance baseline in~a~fities caused by chan~ng ~ v e d gas concen~ations and increa~ng ~ m p e r ~ u ~ . We have measu~d ~ the absorbance of the mo~le phase 70:30 ACN/THF~/v) saturated with ~ o g e ~ h~ium, carbon ~ o ~ d e and air. The ~s~ p ~ n ~ d in Table 1 reve~ that d~solved oxygen is the m~n cause of higher absorbance and t~s effect is severe at lower wavden~hs. The ~Nacement of oxygen in the mobile phase by Other ~ o g e ~ hdium or carbon &ox~e renders it useful at ~wer wavdengths. We have con~anfly m~nt~ned a ~asona~e flow (15-20 ml/min) of hdium a~er degas~ng the soNent d ~ a s o ~ c ~ . The very useful prope~y of U.V. de~cfion is that it is a nondestructive ~ch~que and, therefor, a U.V. de~ctor can be coup~d together in series with any other momtofing de~c~ ~ We perform the ~ r u ~ u r ~ ~uddation ~ S L ~ of tfi~ycerid~ ~ unknown otis by fu~her an~yfis of the trapped ~actions and by stop-flow U.V. ~ a n n ~ g u~ng the same vafia~e wavdength d~ector. ~ The devdopment of microproc~sor ~chn~ogy has ~d to a new b~ed of ra~d ~ a n ~ n g U.V. s p e ~ r o m e ~ that permit ~ m d ~ n e o u s mdtiwavdength detection. ~ One of the greatest advan~ges of m~ti-wavden~h mo~tofing is that absorbance rati~ng can be performed and, therefor, solu~ identification and ~scrimination can be ac~eved. The m~or use of a U.V. detector is in the an~y~s of the derivatives of ~ t ~ adds and in phospholipids. Re,active index de~cto~ (diffe~nti~ ~ a c t o m e t e r ~ are proba~y the next most common HPLC detectors and are ~ r ~ss sen~five than U.V. T~s detector is not s ~ e with gra~ent dufion and is very sen~five to ~mperatu~ changes and pressure fluctuations. Thus, it is impos~b~ to ach~ve optimum separationA ~ t ~ Transpo~ flame ~mzation or mofing wi~ d ~ e ~ o r d e f i e d by Maggs and Young ~:°'~:°'::~ proved effective ~ the detection of ~ d s where the use of U.V. and ~ a c t i v e index (RI) mo~tor was not ~ a ~ e . Therefor, it is o~en ~rmed as a "u~vers~ d~ectof'. The duen~ are d e p o s e d on a mo~ng wire or bdt and the v o ~ e solvents ~om the solutes are ~moved ~ an intermediate furnace r e , o n and finally the nonv~ati~ ~p~ is carried through a flame ~ z a t i o n de~ctor (FID) where it is combu~ed and de~c~d. The main advantages of this type of detector are that k c o d d be used with any v~ati~ solvent u~ng gra~ent dution ~chmques, its umvers~ apN~ation for ~ d s and the d~ector ~ s p o n ~ is recti~neafly r d a ~ d to the amount of the ~uting li~d. Pfive~ ~ al. ~49'~ have very successful~ demon~ra~d the use of thor mo~ng bdt-flame ~ z a t i o n detector for the quanti~five a n ~ y ~ s of fi~d da~es and tfiglyceride speoes of vegetab~ oils. The author studied t~s detector in Pfivea's hboratory and found it very eflioent with gra~ent dution, permitting the an~y~s of fi~ds in the ~ w nanogram range by HPLC. However, there are two drawbacks: one, that s a m ~ are destroyed and cannot be used for fu~her a n ~ y ~ s by comp~men~ry ~ c h n ~ u ~ and ~cond, it is ~ttic~t to opera~. It needs to be per~c~d on a commero~ sca~, and this is bong unde~aken by M ~ r o ~ H ~ e r ~ Denmark. Tracor In~rumen~ (Austin, Texa~ have mark~ed a new Tracor model 945 detector incorporat~g a rotating ~ with fibrous qua~z bdt and FID d~ecfion sy~em. Although t~s sys~m was robust and easy to operate, ~ lacked ~ n s i t i ~ and f l e x i b i ~ ~ the s~vent

Recem a d ~ n ~ s ~ HPLC ~ ~ .

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~G. 2. ~ q ~ d chrom~ograph~ ~parat~n of buaer trifly~rides with RI de~cfion. C~umn, Sphefisor~5-ODS 2; d ~ t o L x 1; temperatur~ am~e~; mobile pha~, a~tone/acetonitri~ ~5:35); flow~a~, 1.5mlmin-t. Ada~ed ~om Re£ 163.

sdecfion due to the restriction of uilng only volatile solven~ during the anMysis of trig~cefides. ~ A new concept of opticM detection syaems is devdoping rapidly for the detection of ~pids. Thus, Jorgensen et aL ~ 1 ~ described the devdopment of a nephelometric detector for the seniltive detection of nonpolar ~pids. Recently Macrae et aL u9 described the p o ~ n t i ~ l o f uilng a mass detector for the detection of triglycerides. This mass detector detects solutes on the bails of ~ghVscat~ring after nebul~ation of the d u a ~ and removM of so~ent by evaporation. The prinopM advantages of this d~ector are that ff can be used with gradient dution, can be used at deva~d mmperatures and ~ stable and flee ~om ambient ~mperature effects. A commercial mass detector for HPLC ~ avMMble from Applied Chromatography Sy~ems Ltd. (Cheshire, U.K.) at a cost comparabM to other avMlable HPLC detectors. An excellent comparison of buyer fat tfiglycerides ufing U.V., RI and mass d~ection has been presented by Robinson and Macrae. ~ The chromatograms presented in Figs 2, 3, 4 and 5 cMady reveM the superiority of uilng mass reaction with gradient dution. They dMm that the mass detector should be more senfitive than the FID detector as described earae~ Christie 3° has recently extended the appl~ation of the mass detector for the separation of fipid da~es by HPLC. Stolyhwo and coworke~ l~t~ presented thor excdMnt ~udies on an experimentM ~ght scat~ring detector which util~es a Mser ~ght source. The Varex Corporation (Maryland, U.S.A0 has recently introduced a commerciM Varex light-scat~ring detecto~ Enhanced senfitifity is claimed compared with other univenM detector. This has been confirmed by Macrae et aL ~ls Very recently, Stolyhwo et aL ~ extended the application of thor fighVsca~ering detector for the anMysis of triglycefides. The detection fimits are about 1 ppm, the time constant 100 ms and the cell volume 100 nl. The quantitative anMyils of tri#ycerides carried out without calibrations gave resul~ in excelMnt agreement with data derived from gas chromatography. The anMyfis of a sample of butter fat tfi#ycefides as shown in Fig. 6 shows slightly superior separation as compared to Figs 4 and 5. These resul~ confirm that this senilfive and reliable detector paves the way for new dev~opments in the fidd of ~pid anMyils. Further extensive ~udies are defired before it can be accepted as a universM detector for the HPLC of ~pids. Parris ~ was the first to report the utility ofin~ared O.R.) detector with gradient dution for the separation of triglycerides. The senfififity of the I.R. detector ~ fimflar to that of the RI monitoL There are serious problems in finding the solvent window to monitor and in excessive baseline drift with the change in solvent compofitiom Perhaps the coupling of fourier transform in~ared (FTIR) 9~ will broaden the use of this detecto~ Compared with dasilcal I.R. spectroscopy, FTIR spectroscopy has two mMn advantages: the fignM to noise ratio ~ about 150 times grea~r and throughout the energy per unit time ~ 100-200 times higher.

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V.K.S. ShuEa

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~o. 3. ~q~d c~om~o~ap~c ~ p a r ~ n of buyer ~ y ~ d ~ ~ U.V. d ~ t i o n at 225 nm. Cdumm ~ m o ~ - 5 ~ D s ~ detector, ~02 au~; ~m~r~ur~ am~e~ sdve~ va~em ~om 20% to 100% ethanol ~ a c e ~ ; ~w-ra~ 1 . 5 ~ n - L ~ ofi~n~ chrom~ogram. ~ compu~enhanced c~om~o~am. Ada~ed ~om Re~ 163. Huor~cence ~b~ 113'169has been used ~ ~ d s res~fing ~ ~ g h e r sen~fi~fi~ during r a ~ d ~ a n ~ n g fluorescence detection of H P L C effiuems. The ~ t u r e h o ~ s great p r o ~ s e ~ r liq~d c h r o m a t o ~ - v i ~ u o r o m e ~ l~ as a ~ w e r ~ tool ~ r ~ d an~yfis. Over the past decade, c o n ~ d ~ a ~ e e ~ r t s have been made to produce H P L C - m a s s ~ec~om~er (LC-M~ ~ems. ~ou~ L C - M S has e n o ~ o u s p o ~ ~ r ~pid

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~6.4. ~q~d chrom~ograph~ ~parafion of but~r ~i~y~fides with mass detection. Co~mm Sphefiso~-~ODS ~ d ~ L x 1; pho~m~tipli~ ~ n g , × 2; evapora~r ~ n ~ 30°C; ~r flow, 22 pfi; soNe~ ~ a d ~ ~om 20% to 100% ~hand ~ a~nitdle; flow-rate, 1.5 ml min-i. Ada~ed ~om Re~ 163.

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analyfi~ its implementation has been restricted by practicfl problem~ It will be de~t with in det~l in the ~ a e r pa~ of this renew. Unfogunatd~ the complefity and the high co~ precludes Rs use in routine HPLC. Glod and Kemula ~ demongra~d the use of e~c~okinetic detection for the separation of higher fatty a~ds by reversed phase HPLC. The devdopment of ~se~based detecto~ for chromatography ~ in progres~ and some of the detectors are j u ~ on the verge of acceptance while ot~e~ ~equire fu~her ~xperimental validation. These detecto~ will pro~de the enhanced senfifi~ty, sdectivity and practicality necessary to augment advances in HPLC of fipids. Table 2 summarizes the characteristics of some of the mo~ frequently used detectors in HPLC. IV. A P P L I C A T I O N S

A. F a t t y ~ c i d s

The m~hods for the de~rm~ation of fat~ adds have been ~ e w e d earlier by Lie Ken ~e ~msand Smith. ~ Due to the lack of U.V. absor~ng chromopho~, fat~ adds can be an~yzed dtber ~ c t l y using a s ~ e d~ection sys~m or by ufing derivatization m~hods ~sulting ~ strong U.V.~bsor~ng or fluorescent groups. T~s is done for detection enhancement and Mso for impro~ng the chrom~ograp~c properti~. Pei et aL ~ extended the use of HPLC ~ ~ d s by ac~e~ng the separations of fat~ acids and m ~ h ~ e~e~ on Vydac ~ v e r ~ d phase c~umns. Warthen ~m~used a #Bondpack C~s cdumn and m~hanol/wa~r s~vent sy~em to ~ p a r a ~ m ~ h ~ d ~ d ~ e and o~a~. HPLC offe~ ~ v e r ~ advan~ges over gas-liq~d chromatography (GLC). A m~or advantage is that the mi~er con~tions used in HPLC allow p~ar compounds of b w volatifi~ or compounds s e n ~ v e to heat to be chrom~ographed. T~s ~s~ns the p o s ~ f i ~ that

T ~ s ~ 2. C h ~ f i g ~ s

~ HPLC D e t ~ t o r ~

Characteristics

Ul~a~olet

Re,active ~dex

R~ponse U~ ~ ~ a d ~ m ~ufion Sensiti~ ~ ~vo~e ~m~es

Specific Y~ 5 × | 0 -10 ~/cm~ 5 x 1~

General No 5 X 10-7 (g/cm~) 1~

~n~r ~n~

Transpo~ flame ~zafion

Infra~d

Fluo~scence

Gene~l Y~ 5 X I0 -~ ~/crn~ 1~

Spec~c Yes 10 -6 (g/cm~ 1~

Specific Y~ 10 -~° ~/cra~ I~

12

V . K . S . Shukla

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Recent a d ~ n ~ s ~ HPLC of ~

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~omefizat~n of the fatty add could occur during the an~yfig As the detectors used in HPLC are normally nondestructive, t~s makes the ~action c~lection of the fatty a~ds posfi~ Both norm~ phase and ~ve~ed phase HPLC have been used for the ~so~tion of fat~ adds. Av~dano e t aL ~ ° have ~soNed ~verfl free h ~ y adds on ~ v e r ~ d phase c~umns. Aos~ma 6 has ~ p a r a ~ d flee 13-hydroxyfino~ add and 13-hydroperoxy~n~c add from ~ n ~ c add and ~ n o ~ c add, respectivd~ by HPLC on a porous p~ymer gel and ufing two fatty add separation c~umns in series or a L~hrosorb RP-8 c~umn. W ~ investigating the possibilities for the purification of arac~do~c add, Datta e t al. ~ have recently evdved a relatively q~ck reversed phase HPLC m~hod for the separat~ns of several dcosano~ ac~s. T~s fim~e, r e l i a ~ isocrafic con~anbflow m~hod ena~es the separation of the C20 ~ y adds ~ ~ o ~ c M fl~ds. B~fle e t g~9 have devdoped an HPLC method for the determination of e~entifl tin,tic add and other fimihr ~ngth underivafized ~ t ~ adds ~ margarines. MankC ~ compared the flee h t ~ adds and m ~ h ~ esters of eve~ng primro~ and soyabean oil by GLC and HPLC m~hods. Hira~ e t a~ s2'83 have devdoped HPLC m~hods for the quanti~tion of fatty adds and m ~ h ~ esters using reversed phase chromatography and short wave~ngth U.V. detection at 200-210 nm. B h n c ~ e t aL ~7'63evoNed HPLC m~hods for the quantitative determination of cydopropendc and cydopropano~ fatty acid meth~ esters ufing U.V. d~ecfion at 206 rim. These m~hods gave accurate ~ s ~ and ~ q ~ d ~ss than 1 mg of oil for an anflyfi~ Lovdand e t al. "6 de~ribed a fim~e, conven~nt HPLC m~hod for the an~yfis of cyclopropenoid fat~ adds using an RI detector. A number of derivatives have been prepared for an~y~ng a varify of ~ y adds by HPLC. These derivatives include benzyl, ~ p - ~ o b e n z ~ , g ~ phenacyl, ~s's~'~3~ pb r o m o p h e n a c ~ , " , ~ , ' ~ p - m e t h y l t h i o b e n z ~ 17 p-phenylazophenacyl~ 211 1-napth~amine, ~ pentafluorobenz~ ~ and isatin meth~ e~e~. ~ An alternative approach ~ to prepare fluo~scence derivatives such as the ~naphthac~, ~ 9-diazomethylanthracene, ~° ~bromo~hyl-~7-dim~hoxycoumarine, ~s % a n t h ~ a z o m ~ h a n e (ADAM), ~ %aminophenanth~ne 9: and N - D n ~ h a n d a m ~ e esters. ~

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56

Time (rain)

F [ ~ % A chromatogram showing the ~pical ~parations of phenac~ ~ of ~ t t y acids by HPLC. An~yfis was made on a 250 x &5 mm octadecyl c d u m n ~ a s ~ v e ~ flow-ra~ of Z0 ml min-L The c ~ u m n was run ~ocratically with aceto~tfile/water (80:20) ~ r 25 min then programed to 85:15 ~ 15 min. Peaks: 1 = h u f i ~ 2 = m y f i ~ e ~ ; 3 = tfidecan~ 4 = cis-A~°-pentadecenoic; 5 = fino~n~; 6 = myfiflic; 7 = arac~do~q 8 = trans-Ag-hexadecenoi~ 9 = ~nd~ I0 = pcntadecanoi~ I 1 = all-cis-As, u,mLeicosatrienoic; 12 = pMmifi~ 13 = ~ 14 = ~ M ~ 15 = heptadecanoi~ 16 = ~eafi~ and 17 = cis-A-n~icomnoic. A d a p ~ d ~ o m Re£ 219.

14

V . K . S . ShuMa

I0

8

16:0

4

4

8

12

16

~ 20

24

28 Time

52

36

40

44

48

52

I 56

(min)

F~G. 8. A chrom~ogram showing the ~ p a r ~ m n of ~omeric octadecynoic adds as phenacyl derivatives by HPLC. An~yfis was made on a 250 x 4.5 mm ID o~adec~ co~mn with an ~ocratic s~vent flowqa~ ~ m l m i n -~) of ac~onitri~/wa~r ~ 5 : 2 ~ . Except for p~mim~ (16:~, the numbe~d peaks ~ p r e ~ n t the position of the tri~e bond, relative to the carbox~ group, ~ the acyl hydrocarbon c h i n . Adapted ~om Refi 218.

6

I

4

8

12

16

20

24

28

32

36

40

44

Time ( r a i n )

F~6. 9. A typical chromatogram showing ~solution of some ~omefic c~-octadecenoa~s as phenac~ es~rs by HPLC. An~y~s was made on 250 x &5 mm ID octadec~ column with an ~ocratic flowqate (2.0 ml min- ~) of acetonitfil~water (85: 15). Except for s~ara~ (18:0), the numbered peaks represent the position of the double bond, rdative to the carbox~ group in the acyl hydrocarbon c h i n . Two trans ~omers (3t, 20 were included to show thor relation to the c~ ~omers and the revers~ of the dufion order of the ~anwA 2 ~omer. Adapted from ReL 218.

Recent advances in HPLC of fipids

15

Wood and Led ~9have de~ribed a m~hod for the ra~d preparation of phenac~ and naphthac~ derivatives of h t ~ adds and an~yzcd the~ by HPLC on a C~s m v ~ d phase c~umn at nanogram ~nsitifi~. They examined the quantitative de~rm~ation of c e ~ n seed oils and found that v~ues obt~ned by HPLC compared well with the data obt~ned by GLC. A ~ c ~ chromatogram showing the ~parat~ns of phenac~ esters is shown ~ Fig. 7. They observed that phenac~ derivatives of monoen~c fat~ adds were ~so shown to undergo c s - ~ a n s i s o m e r i z ~ n when expo~d to U.V. ~ght. Wood ~ extended ~s m~hod for the separation of ~omeric o c ~ d e c e n o ~ and o~adecynoa~s. The chromedgrams showing the separation of homeric octadecenoates and oc~decynoa~s are illustrated in Figs 8 and ~ mspectivdy. Them have been ~ v ~ f l ~ u ~ w~ch em~oyed two 60 cm or three 90 cm c~umns for m s , r i n g fat~ ac~s. They all connec~d the same ~pe of c~umns filled with ~entic~ p a c i n g materiMg The anflyfis times for these c~umns were very bng (2-4 h0 with i n c o m ~ e ~parations of the critic~ p ~ such as 14: ~ 20: 4 and 16:1 or 18: 3 and 22: 6. Recently S~o ~ cou~ed a Zorbax C-8 (5 gin) 15 cm x 4.6 mm ID and a L~hrosorb RP-8 (10#m particle 25cm x 4.0mmID) co~mn and ~para~d 12 ~ o b ~ c a l l y rdevant C12-C22 fat~ adds witch 60 min. C o m m i e ~parafions b~ween criticM p~rs 18:3 and 22:6 and 14:0, 20:4 and 16:1 were obt~ned. T~s is the first repo~ d ~ i ~ n g these ~parat~ns. The m~nfion times were Mso ~ g ~ y reproducible. The chrom~ogram show~g the separat~n of s~ndard h t ~ adds ~ d e ~ e d ~ Fig. 10. T~s coupling of two ~ffemnt ~pes of c~umn m s ~ ~ a ~ l e c f i ~ w~ch hdps ~ r ~ n g comp~x m i ~ u r ~ of components. J a i l , s ~ al. ~ reposed an HPLC m~hod ~ r the determination of fat~ acid composition of soybean oil by anMyfing p-bromophenac~ esters. The ~parafion time was Mss than 1 hr. The ms~ts compared very well with the GLC m~hod (TabM ~ without encountering the problems ~vMved with GLC m~hod~ z ~ ~ HPLC wo~d be the m~hod of ch~ce where the~ proMems arise. The HPLC ~parations of ~ e p-bromophenac~ esters of the soybean oil am i~ustra~d ~ Fig. 11. Vioque e t aL 2~ used p-phen~azophenac~ esters for the anMyfis of the fat~ acM derivatives of ~ v ~ M vegemMe oils by HPLC. The duent was aceto~tril~wa~r ~9: l) with a flowqa~ of 1.5 ml/m~ on a reversed phase cCumn. TaMe 4 shows an excdMnt comparison of the GLC and the HPLC m~hod. Very recently, Ban~ and Ansari l~ reposed an HPLC m~hod ~ r the anMyfis of saturated monohydroxy fat~ add mimums contai~ng pofifional ~om~s of various chMn Mn~h~ They recommended a comMnafion of ~ v e r ~ d phase HPLC ~ o w e d by

95

@

,_/~/~/

85

® 2

<

0

L

3O

45

Q I2:0 Q 20:5 QI8:3 @22:6 Q 14:0 Q20:~ Q I6:I ~18:2 ~ 20:3 ~ 16:0 18:1 Q 18:1isome~ Q IS:O

60

Min

~G. I~ HPLC c ~ o m ~ o ~ a m of g a n d a ~ fat~ a~ds. AdaVed from Re~ 170.

16

V. K. S. Shu~a T^nL~ 3. Fatty acid C o m ~ s i t i ~ & ~ y b e ~ ~1 by HPLC and G L ~ s Componem

HPLC (%)

GC (%)

Palmific Steafic Oleic Linoleic Linolenic y-Linolenic Arachidic Behenic

10.38 ___0.18 4.33 ± 0.13 23.18 ___0.14 53.66 + 0.29 6.16±0.11 1.68 ± 0.05 0.36 ± 0.05 0.25 + 0.04

9.65 _ 0.04 4.08 ± 0,02 23.90+ 0.04 55.01 _ 0.03 6.36+_0.02 0.59 _+0.01

*Average of three d~erminations ± standard devotion. Two minor components were unidentified: content 0.42 ± ~01%.

adsorpt~n HPLC for the c o m p ~ ~s~ution of n~urally occurring mi~ur~ of hydroxy fatff adds. 1. P r a ~ a l Applications The HPLC ~chmque has recently been applied to the de~rm~ation of fat~ a~ds ~om a wide range of sources (TaMe 5). A s p e ~ mention coMd be made for the an~yfis of ~ t ~ acid anil~es for the de~rmination of the to~dff of Spa~sh coo~ng ~1 and appl~a~fi~ in anMy~ng Mood and tissue fi~ds for mo~toring metabolic disorders of fi~ds. B. Tr~lyc~ides N~urN ~iNyceride mi~ur~ occurring in vege~Ne N~ and oils are of such c o m p ~ • at no finNe anNyt~N technique can ~sNve HI the m ~ d u N triglycerides that are present. A comN~e duc~ation of triglycefide compo~fion ~ posNNe with the comNnation of argentat~n tNn h y ~ chrom~og~phy (Ag-TLC) and carbon numb~ gas chrom~o~aphy (CN-GC). The cNef ~ d v a ~ a g e s of these ~chNqu~ are that ~ey are ~ o u s and tim~consuming and are not su~ciently ~produNNe ~ r routine anNyfis.

02--

E c

i

L

6

~ IO

8 30

20

4O

9 50

MIn

F~G. 11. HPLC chromatogram of p-bromophenacyl esters of soybean oil fatty adds on a Lichrosorb RP-8 column. Peak identification: l, laurate 0nternal ~andard~ 2, ~no~nate; 3, v-lino~na~; ~ ~noleat~ 5, p~mitat~ 6, oleate; 7, stearate; 8, arachida~; 9, behenate. Adapted ~om Re~ 95.

-%2 51.8 !~6 9.6 . 2.9 %0 1.8 . .

8 :0 10:0 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3

Z8 7.6 51.9 19.5 ~0

b

seed

. . . . 25.5

a

b

5.8 21.1 14.5 51.1

.

.

. .

.

7.0

b

Linseed a

. . . . . . . . . . . . 24A 7.0 . . . . . ~0 5.2 ~5 4.3 5.1 30.5 2%1 18.9 -45.8 44.1 17.9 . . 51.9

*a = GLC; b = HPLC.

a

Co con u t

F atty a d d

Poppy

.

. .

~2 28.9 55~ .

. 9.0

.

a

. ~8

. .

. .

. . 11.0 ~2 49.2 30.9 2.5 .

.

b

Lupinus a

. 10.9 ~5 ~6 49.7 28.5 30.2 55.2 2.7 . . .

.

.

b

Sunflower

. .

7.2 Z3 11~ 79.5 -.

.

.

a

.

.

.

7.7 2.7 1~9 78.7 --

b

Sawflower

.

.

. .

.

.

b

Maize

.

13.2 14.4 . . . Z5 2.6 28.2 28A 54.1 54.5 1.8 . .

.

a

1.2

-

TM

~5 35.8 10.7 .

-49.0

-

--

b

GLC Palm

1.5 46.5 . 4.4 37.2 8.3 .

.

a

TAnL~ 4. C o m p a r i s o n of Analyses of the F a ~ y A d d C o m p o s i t i o n of Vegetable O i ~ by H P L C an d

_

-11.6 1~ 3.8 7%4 ~3 .

_

b

---11.8 1.0 4.8 77.0 5.8

----

Olive

----

a

18

V. K. S.

Shu~a

TABLE5. Applications of HPLC AnMy~s of Fatty A~ds tgt Sam~e Fatty add derivative Re~nce Or~ ~ptococci m-M~hoxyphenac~ 21 Or~ b a c t e r i a m-M~hoxyphenac~ 130 ~-Mycolic adds p-B~phenac~ 203 River water Phenac~ 85 Alkano~m~ Ethan~amides 135 Grins and ~eds p-B~phenacyl 207 Fish oils M~hyl 140 Soybean ~1 p-B~phenac~ 95,124 Coo~ng oil Anil~e 36. 215 Margarine M~hyl 14 Evening primro~ oil M~hyl 124 Various vegetab~ oi~ Phenac~, naphthac~, 211.219 p-Phenylazophenacyl Oil and alk~ reins Free aods 105 n-A~an~ Phenac~ 53 Serum p-B~phenac~ 91 Monkeys M~hyl 177 Bio~c~ ~ds 9-Anthry~zom~hane 170

R e v e ~ e d phase H P L C has been e x ~ n i v d y emNoyed for the ~paration of complex triglycefides m i x t u r ~ during ~ c e n t years. All these ~ p a r a t i o n s were performed e m N o ~ n g nonaqueous soNen~, an M k ~ bonded p h a ~ c N u m n and an RI detection sy~em. Parfis ~ was the fi~t to ~ p o ~ the u t i ~ of an I.R. detector with gradient d u t i o n for the separation of tri~ycerides. The term nonaqueous reversed phase (NARP) was evolved during these ~u~es. Severn papers have appeared ~cenfly on the a p N ~ a t i o n of H P L C for the ~ p a r a t i o n of tfig~cerides in various n ~ u r a l oils such as pMm, 5L~'~48't~ ~ w and Ngh e r u o c a d d rapeseed, ~ ' ~ corn, 5L~ c o t t o n ~ e d , ~ peanut butter, ~6 olive, n ~ s o y b e a ~ 5l'~'~ coconut, 5L~ satflower, ~ sunfloweL ~ hazdnut, ~ c o c o a b u t ~ L ~ ~nseed, t°a~5~ cod ~veL ~ pMm kernd, 8° Mmond, 167 avocado, ~7 butter fat, ~ sheabutter, ~ s~, 157'187 m a n g ~ ~ illipe, ~ l ~ dhupa, ~ mowrah m and Flacourfiaceae seed oil~ ~ Pei e t al. ~ d e m o n ~ r a ~ d the successful application of H P L C to the anNyNs of trig~cefides on Vydac reversed phase cNumns. Plattner e t aL 154 extended tNs ~ c h N q u e for the separation of triNyceride speoes on the bails of chNn ~ n g h and unsaturafion and o b t ~ n e d baseline ~ p a r a t i o n of critic~ p ~ r s of tfi~ycefides c o n t a i ~ n g oleic and p~mitic a n d by the a d ~ f i o n of i l v e r ~ e to the solvent sys~m. The real breakthrough in the a n M y i s of trig~cefides came ~ o m the d a m o n work of E1-Hamdy and PerNns. 5°~ They ~ u ~ e d the interactions of m o N K p h a ~ and c ~ u m n pacNngs in order to optimize the p o ~ n t i N of reversed phase H P L C . They devdoped the concept of theoretical carbon number (TCN) to define s p e ~ ~ p a r a t i o n of unsaturated triNycefid~. One of the ~ c ~ f i e s during the ~ p a r a f i o n of tfiglycerides in H P L C is the '~rificN p N ~ " formation. CfificN p N ~ have been found to have ~enficN behafior in reversed phase c h r o m a t o g r a p h y in spi~ of the Nffe~nces in chain ~ngths, number of d o u b ~ bonds and geometficN configuration. CdticN pNrs are defined as those c o m p o n e n ~ wNch have the same e q u N a ~ n t carbon n u m b e ~ (ECN) ECN = C N - 2n, where C N = a c t u ~ carbon number n = number of d o u b ~ bonds per m o ~ c u ~ . Thus, oleic and p ~ m i t i c a d d s are cfific~ p ~ as they have same ECN = 16, the same is true for ~noleic and myristic a d d ( E C N = 14.0), as wall as for ~ n ~ e n ~ and h u f i c a d d s (ECN = 12.~.

~ t

~n~s

~ HPLC of ~

19

(b)

(c)

~ o . IZ Eff~t & t e m ~ r a t u ~ on the ~paration ~ O y ~ r i d ~ &cocoabutter. a ffi 20°~ b ffi 25°~ c = ~ ° C ~ h u ~ & unpublished w o ~ U j

Tri~ycerdes ~so form critical p~r~ thus t r i t o n ~ 4 : ~ , palmityl~olein (52:2), ~ey~ip~mitin ~0: l) and trpalmifin all have the same ECN of 48:0. Po~tion~ isomers of the tfi~yce~des were ~so c~fic~ p~rs. Tfi0ycerdes contai~ng geom~fical ~omers such as ~ c ~ ) and ~ c (tmns) ~so form c ~ c ~ p ~ . An excellent ~parafion of tfi~yce~de crfic~ p~r~ such as OOO, PO~ PPP and SO0, SOP, SSP and OOO, EEE, was ac~eved during t~s work. 5~ The theo~fic~ carbon number (TCN) can be de~rmined ~om a plot of capadty factor k' vs carbon number of the cor~spon~ng saturated t~ycefides. Thu~ for any saturated tr~yce~de, TCN is equ~ to the actu~ carbon numbe~ The TCN can be c a ~ u ~ d as fofiow~ 3

TCN = ECN - E

U,

1

where U~ ~ the ~ctor de~rmined experimentally for fat~ aods and was found to be as fo~ows: ~ e ~ = ~60--0.65, fin~e~ = 0.70-0.80, d f i d ~ = 0.2, saturated ac~ groups = 0. Thus, the TCN of POL = 4 6 . 0 - ~ (for palmityl)+ ~6 (for d e y l ) + 0.7 (for ~ n o ~ = 46.0 - 1.3 = 44.7.

20

V.K.S. ShuEa

Devdopment of this concept greatly hdps in the identification of unsaturated triglyceride spedes as is demon~ra~d by Phillips and coworke~. ~ The separation of tfi~ycerides according to thor ECN number is ~ron~y influenced by the mobi~ phase compofition and the ~mperature. Thus, Jensen ~ showed improved tfi~yceride separations at subambient ~mperatures and Schulte t~ showed the superior separation po~nti~ of propionitri~ for the separation of tri~ycerides of cocoabut~r. We have ~ud~d t~ the effe~ of ~mperature for the separation of cocoabutter trig~cerides ufing acetonitri~ and tetrahydrofuran mobi~ phase and concluded that 20°C was the best ~mperature for this separation (Fig. 12). Lie Ken Jie ~t5 reposed quantitative aspects of trig~ceride analyfis using differential re~a~ometer. Her~o~ ~ investiga~d the possibilities of ufing short wavdength U.V. detection, These ~udies were followed by others. ~ We have devdoped ~ a simple and direct procedure for the an~yfis of tri~ycerides which demon~ra~s the ~afibility of emplo~ng sho~ wave~ngth U.V. detection for quantitative measurement of tri~ycerides. There are two m~n drawbacks in recording absorption spectra of tri~yceride standards: firstly, a v ~ b l e standards are not chromatograph~ally pure as they possess some unknown impurities and, secondly, they are very expenfive. In order to overcome t~s problem, we have used a chromatographic separation sys~m coup~d with a ~op-flow technique to determine the spectra of indifidu~ pure triglycerides ~om 209-250 nm. Four tfi~yceride standards (OOO, POO, POP, PPP) were chosen for this study. The rdafive mo~cular extinction coettioents of these tri~ycerides were measured using a serial combination of U.V. and RI detectors ufing the same chromatograph~ sys~m. The U.V. detector set at 220 nm was coupled first followed by the RI detector. The fignal hdghts of various tri~ycerides were recorded. The relative mo~cular extinction coeffioents were calculated ~om characteristic response ratios hv~/h~ and the difference in the re~a~Ne index of triglycefides~°~ and the soNenL assuming that the RI fign~ is proportion~ to the concentration of the triglycefides studied. A ~mperature of 60°C was chosen as all the trig~cefides including trip~mitin were liquid at this ~mperature. From the resul~ of the n o r m ~ e d spectra for four different tfig~cerides presen~d in Fig. 13, it is posfib~ to conclude that quantitative ev~uafion of tfi~ycerides is hampered O!

POS

popO~00

~ 005

J

0 210

I

220

~

230

~

2~0

W~ve~ngth (rim)

F~G. 13. U.V. absorbance spectra of differenttfiglycefidesnormalizedfor the same response at 221 nm. Adapted from Re~ 186.

R~t TAB~

6. R d a t i ~ M d ~

Tfi~y~fide ~andard

Refractive ~dex of tri~y~fide ~ 60°C n~

OOO POO POP PPP

1.455 1.451 1.448 1.445

~n~s

21

~ HPLC ~ ~ # ~

Exfin~Mn C o e ~ e n ~ Re,active ~dex of sdvem at 60°C ~ 0 ACN + 30 THF ~

of T f i # y ~ f i d ~ M 220 nm ~

n~-- n~ trig. s~v. 0.110 0.106 0.103 0.100

1.345

hu~

~

Rehfive m~d~ ~xfincfion coe~dent

0.88 0.98 0.97 1.02

0.97 ! .04 1.00 1.02

under 218 nm due to the effect of d o u s e bonds. Howeve~ there appea~ to be no effect of ~ o h ~ d d o u s e bond groups above 218 nm. The ~ s d t of the mo~cdar extinction coefficients pmmnted ~ Ta~e 6 shows an absdu~ ~ffemnce of 5% ~ the mo~c~ar extinction c o e ~ d e n ~ of thee tfig~cefid~. T h e e ~ s ~ deafly show th~ the effect of ~ d ~ e d d o u s e bonds as in OOO do not contribute to the mdecdar extinction coe~dent of t i l d e n at 220 nm and a good quanfitation of tfi~ycefid~ can be ac~eved at 220 nm, thus a v o ~ g the effect of ~ o h ~ d d o u s e bonds. At t~s wave~n~h, U.V. absorption of t ~ y c e f i d e s is baficM~ due to the es~r C---~O function of thee moScOw. The m~n advan~g~ of U.V. d~ection am the ~gh m n f i t i ~ of d~ection and i~ c a p a ~ of gradient elution. We have ~ f i m ~ e d the, by inc~asing detector m n f i t i ~ , 100 ng or ~ss co~d be de~rm~ed. The c o m m i e reparation of tfi~ycefides of cocoabu~er is shown ~ Fig. 14. Five cocoa but~r samp~s w e ~ an~yzed by HPLC and e~sting TLC-GC methods and a ~ compa~d ~ Ta~e 7. Except for some minor ~ffe~nce~ both m~hods produce fimihr ~ s d ~ . The advantages offered by t~s HPLC m~hod a ~ sim~ici~, speed and ease of operation as compared ~ the e~sting TLC-GC m~hod. ~nce HPLC is a nond~tructive a n M ~ M

POS

SOS

POP

PLiS

S0A _

0

~0

20

30

ao

5o

~

~0

B0

A._._,

9O

Time (rain)

F ~ 1~ ~ p a r a t i ~ ~ ~ y ~ d d e s ~ c ~ b m ~ ~ ~ o 1 ~ x ~5 mm ~ ~ b ~0D~ c~umns and a ~ m ~ M ~ h ~ r a n , 73:27 ~ as a m o ~ e phase at 1~ ~ ~n-m; ~ t p ~ u ~ 2000 ~ ~ ~ ; U.V. ~ t ~ f i ~ ~ ~ 0 n ~ A b b ~ i a t i ~ s ~ M = myfi~c ~ P= p ~ ~ S= s ~ ~d; 0 = ~ ~ ~ = ~ ~d; A = ~ c ~d. A ~ ~m ~L l~

0.6 1.2 0.7 0.4 1.1 0.7 1.2 0.6 0.9 0.9

TLC43C HPLC TLC~3C HPLC TLC-GC HPLC TLC~GC HPLC TLC~C HPLC

C ~ b ~

Malay~an

Nigerian

Brazi~an

Sample A

Sample B

2.0 0.5

2.7 0.4

1.5 0.8

2.1 0.5

2.3 1.I

17.0 18.2

15.1 15.0

14.6 14.4

16.6 16.1

15.8 15.1

39.3 39.5

37.2 36.0

33.4 34.5

38.4 40.1

40.1 40.3

23.7 24.5

23.0 24.1

21.2 22.6

23.0 26.8

27.5 29.4

Tfisaturated Monou~ed PPS PSS POP POS SOS

T y ~ of an~y~s

1.0 1.I

0.7 1.2

0.9 0.9

0.7 1.2

1.1 2.2

4.4 3.1

5.9 4.7

7.4 6.7

4.9 2.9

2.7 1.7

3.8 5.1

5.5 6.5

9.6 8.8

3.5 3.6

2.6 3.1

2.0 2.7

2.2 2.7

2.1 2.7

1.8 2.1

1.6 1.1

Tri~ycerid~ ~ o 1 % ) ~ t ~ SOA POO SOO P ~ P

2.8 3.4

2.9 4.5

3.4 4.3

2.7 3.8

2.3 2.9

P~S

2.2 0.9

2.7 2.7

1.6 1.8

3.3 1.7

2.2 2.0

S~S

1.0 trace

1.0 0.9

1.2 1.0

1.0 0.3

0.4 trace

trace 0.3

trace 0.8

2.1 0.8

1.4 0.5

0.7 trace

P ~ y u ~ OOO S~O

TABLE 7. C o m p a r i ~ n of V~ues of T f i ~ y ~ r i d e Compofifion Obt~ned by HPLC and T L C 4 3 C A n ~ y ~ s of 5 S a m ~ C~b~r t~

of

Recent a d ~ n ~ s ~ HPLC of ~

23

~ch~que, it allows the c~Mcfion of pure tfiglycefides to be used as ~andards or for fu~her Menfificafion by com#ementary ~ch~ques. We have ~ud~d I~ the effect of injection s~vent on the ~ p a r a t ~ n of tri#ycefides of pMm ~1. Due to the compMfity of the tfi#ycefides p~sent ~ the mixtur~ ~fferent s~ven~ produce different r e s ~ . Thus, aceton~ pro#o~tfiM and ~propanC produce better chromatograms for p~ar tfi#ycefides and poor peaks for saturated tri#ycefides, while the reverse ~ true for s~ven~ such as THF, chloroform, ~o-o~an~ ~ c ~ o r o m~hane and hexane. We concluded that a 50:50 mixture of acetone/THF produces a fairly ~ce chromatogram for p~ar and nonp~ar tri#ycefide mixtur~ (Fig. 1~. Recent~ P ~ i p s ~ aL ~ described an HPLC m~hod for the quantitative de~rm~ation of tfi#ycefides ~ cocoabu~eL soybean ~1 and o~ve ~1 using an FID detector based on a ~rect proportionMi~ of peak areas. Diffe~nc~ in the ~ s p o n ~ of i n ~ d u M spedes were small and ~ d not ~ a t e the use of ~ s p o n ~ ~ o ~ . RoMnson e t aL ~ ~ u ~ e d the appl~ation of mass detector for quantitative de~rminafion of tfiglycefides and fat~ acM m ~ h ~ esters. The detector ~ s p o n ~ was found to be non,near for tri#ycerides with a detection ~mit of Mss than 1 #g. Sto~hwo et aL ~ demon~ra~d the possibilities of ufing 4~ THF 3~

>

E

200

0 400

300

~ 200

I00

0 400

THF/ocetone 50 :50

~300200100 o 0

~ I0

20

30

~

50

60

70

80

F~. 15. Effect of inject~n s~vents on chromatograms of p~m ~1. (Shu~a and Ni~sen unpublished w o r k ~

24

V . K . S . Shukla

ccc ccc

CGG

--

I0

20

30 E[ution

40 time

50

60

(rain)

~ o . 1~ Separation of tfi#y~fides m Ca~ncoba e ~ a using two 150 x 4.5 mm ID Sphefisorb $ 3 0 D S 2 cdumns and a ~ t o n i t ~ t e t r a h y d r o f u r a n (68:32, v/v) as mobile phase ~ l ~ ml min ~; inlet pr~sure 2500 psi at 2~C; U,V. d ~ f i o n at 220 nm. Abbrefiations: P = palmitic a~d; O = d O c aci& H = hydnocarpic a~d 16:1 c~ C = chaulmoogfic aod 18:l cy; G = g o ~ c ac~ 18:2cy. Ada~ed ~om Reff 187.

a laser light-scattering detector for the quanfitation of tfiglycerides of compMx mixtures with few calibrations. These new detection methodologies hold promise in the quantitafion of ~pids in the future. Quantitation of estolide tfiglycefides in sapium seeds was achieved by HPLC u~ng I.R. detection. ~ Payne-Wahl et al. ~ evolved an HPLC method for the quanfitation of free acids, mono-, di- and triglycerides u~ng an I.R. detector. HPLC has been successfully employed for the analy~s of confectionery fat~ ~.6~t~'~ Te~a-, penta- and hexaacyltfiglycefides have been separated on a #-Bondapak Cts column with an acetonffrile/acetone (2: l) solvent sy~em which separates them according to carbon number and degree of unsaturation, t" We have described an HPLC method 1~ for the separation of triglycerides of Flacourfiaceae seed oils containing cydopentenyl fatty adds (Fig. 16). Two recent repots ~ u attest to the importance of temperature programing in the HPLC of Wiglycerides. The authors claimed improved sdecfivity and reduced analy~s time for complex triglyceride mixtures. SingMton and Paaee ~ showed the use of a pseudo-mobiM phase in decreafing the dufion time of peanut triglycerides in reversed phase chromatography. A new postcolumn reactor detector for the analyfis of triglycerides with high sen~tifity, high sdecfivity and molar response has been evolved, u~ Tfiglycerides duted ~om an HPLC column are hydrolyzed with potas~um hydroxide and the resulting #ycerol ~ oxidized to formaldehyde with periodic a~d. Then formaldehyde is reacted with acetyl acetone in the presence of ammonium acetate to form 3,5-diacetyl-l,4-dihydrolutidine which ~ detected at 410 nm. The detector detec~ 0.1 nmol of trilaurin and ~ves a ~near working range between 0.3 and 60 nmol of trilaurin. "l Takahashi et aL 2°2 have very recent~ described a matrix concept for the identification of triglycerides in reversed phase HPLC. C. Phospholipids

Phosphofipids are the most impo~ant constituents of membranes and they are also technically important because of thor use as emul~fiers. Although HPLC ~ a well

R ~ e n t advan~s ~ HPLC of H # ~

25

estaMished tech~que for the anflysis of fi~d~ then has been fimited progress for anfly~ng complex phospholipid moScOw. Porter and Weenen ~ have refiewed the literature for the HPLC anflyfis of phospholipids and phosph~i~d oxidation products until 198~ t~s was fol~wed by an exhaustive refiew pnsen~d by Aitzetmfiller.~ Geurts Van Kess~ et ak ~ pnsen~d a method for the ~parafion of extracted ~ o ~ c ~ and synthetic phosph~i~ds ufing a Lichrosorb ~ 60 (10gm) c~umn and n-hexane, 2-propan~ and water m~tuns as the duenL Porter et ak ~ anflyzed 10 ~fferent synthetic l e c ~ n s ufing a #-Bondapak C~s reve~ed phase c~umm Crawford et a k " showed the separation of soy phosphafidy~hofine ~to its m~or m~ecular speoes by nver~d phase HPLC and short wave~ngth U.V. detection. Compton and Purdyu improved the efficiency of phosph~i~d separations by deva~d c~umn ~mperatun and mo~fying the mobile phase by strong minerfl adds or ~n-p~fing agent~ An anflyfis of ~ d t ~ n ~ confectionery products was repoged by Hurst and Martin. ~ Rhee and S~n 1~ devdoped a fim~e, ra~d m~hod for the an~yfis of phosphatidylcholine in soy ~dt~n. Chen and Kou :7 npo~ed a ra~d, e~oent HPLC procedure for the ~parafion of p h o s p h ~ s ~ tissue ex~ac~. The separation was accompfished on a micropartic~a~ silva gd co~mn ufing ~ocrafic dution and U.V. d~ecfion at 203 rim. PaRon et a k lo modified Van Kessds m~hod to separate all m~or phosphofi~d dasse~ Ufing rat five~ they have demongra~d that recoveries of phosph~i~d were comO~e and the molec~ar compofitions of the ~ a t e d ~action ~ d not change during chromatography. Hurst and Martin s7 em~oyed the method described earlier for the an~yfis of phosphofipids in soy l e c ~ n by HPLC. A reversed phase HPLC m~hod for the separation of in~fidufl find da~es of phospholipids into subfractions ~ de~ribed) ~ The separation was ac~eved with isocrafic flution ufing Nuc~osil 5 C~s c~umn and a m o ~ phase confisting of m~han~, acetonitfile and w a ~ The compounds wen d~ec~d with a com~nation of a U.V. and a fight ~at~fing mass d~ecto~ w~ch profided quantitative chromatograms reported for the first time. The m~hod ~ s~taMe for an~y~ng phosph~i~ds ~om various ~ o ~ c f l source~ Christie 2~ ~para~d all the m~or ~m#e and com#ex fi#ds in o~y 20 min u~ng a ~rnary gradient s~vent sys~m, a sho~ 10 cm fast speed c~umn 0 Vm sific~ and Applied Chromatography Sys~ms mass detector. The duent was first a gra~ent of isopropano~ chloroform (4: l, v/v) ~to iso-octane ~ order to separate each of the fim#e fipids, and then of ~opropanol/water (1:1, v/~ ~to iso-octane/isopropanol/chloroform (42:40:10, v/v initially) ~ such a way that water con~nt incna~d r~ative to that of the ~o-octane. The separations shown ~ Fig. 17 do not show any baseline dri~ ~ s # ~ of the changes in solvent compofition during the dution program. The compatiMfi~ with grad~nt dudon PE

PC

PI SPH ~

15

I 20

Time (rain)

FIG. 17. Gradient ~udon of rat hea~ fipids (0.3 rag) on a Sphe~sorb 3 # column with gradient ~ufion ~om iso-octane to ~o-octane/chloroform/isopropano~water 02:10:48:&v/v) at a flow-rate of 2 ml rain- L U = unknown component. Adapted ~om Re~ 29.

26

V . K . S . Shu~a

is one of the m~or advantages of the mass detector. Some difficulties were observed during the quantitation because of the fall of the detector responses at low sample concentrations. ~97,~99 Very recently, Christie ~ has modified the sy~em by extending the working ~fe of the columns and impro~ng the separation of the a d d ~ ~pids. A senfitive, r d ~ b ~ method for the quanfitation of tissue phospholipids has been devdoped, ~6 which compares very well with quantitation achieved by the TLC method. Sotirhos et aL ~93 an~yzed soybean phospholipids ufing a silva column, U.V. detection at 210 nm and a mobi~ phase con~sting of hexane, ~opropanol and water. Separations were achieved by keeping the hexane to ~opropanol ratio constant and varying the wa~r content in the dution program. Granular soybean ~dthin as well as its a~ohol extracted ~actions were analyzed, which differed both qualitativdy and quantitafive~. HPLC has been successfully used in the separation of phospholipid da~es in human hea~ ~ and blood platdets) ~ D. N u t r i t i o n M L ~ i d s

Nutritionfl ~ u ~ e s on the m~abofism of arac~don~ add (AA), a C20 p~yunsatura~d fat~ add (eicosano~) to prosta~an~ns (PG), thromboxan~ (Tx), hydroxy ~ y adds (HETE) and ~ukotrienes (LT) are ~ndered by a lack of ~ghly selective, accurate and predse m~hods. Reve~ed pha~ and norm~ pha~ HPLC have been ex~nfivdy em~oyed for flee prosta~and~s ufing RI or sho~ wavdength U.V. detection. Sever~ mo~ficafion reagen~ have been used in order to enhance the ~nsitivi~ and the chromatograp~c properti~ of prosta~an~ns. Van R o a n s et aL ~° have ~ e w e d HPLC of pro~a~an~ns until 1981. Pro~a~an~ns (PGE3, PGF~, PGF~, PGE~, PGE~, PGD~, PGB~ and PGA~) we~ ~ p a r a ~ d ~°° on SuFeko~l LC18 (3 gm) c~umns ufing a gra~ent ef aceto~trile/ 0.0174m H3PO ~ O Z 8 : 6 Z ~ changed to 75:2~. A U.V. detector at 190 nm was em~oyed for de~ction. Inayama et al. ~ described the separation of prosta~an~ns on a Zorbax c~umn ufing hexane/~oxane/wa~r (7~.0:2~.0:0.2) as the m o ~ pha~. Gallon and Barcell~ ~p ~ n ~ d a spedfic senfitive and rdativ~y ~expenfive m~hod for measuring up ~o ~ pro~a~and~s, ~ d u ~ n g the previou~y unmeasura~e PGE~, in ~ o ~ c ~ samples using reversed phase HPLC. Saunders and H o ~ o c k ¢ ~ improved the ex~action procedure for p r o ~ a ~ a n ~ n s and phosphoh~ds for the mafimum recovery and applied the devdoped m~hod for the fim~taneous quantitative recovery of p r o ~ a ~ a n d ~ s and phosph~ipids ~ the same tissue sam~e.

A filver mo~fied, norm~ phase HPLC procedure has been devdoped ~ to ~ s o ~ e ~omers of pro~a~an~n-2Cnaphthac~ e s p y . The filver mottled m o ~ phase ~ s ~ in ~gh chrom~ograp~c s d e c t i ~ . A fim~e, ~ p r o d u d ~ e m~hod ~ for extraction, ~paration and quantification of ~ukotfienes is described. Ad~fication of the sam~es prior to injection into the HPLC ~ s ~ d in improved peak resolution and increased recovery. Leukotrienes were quantified ~4 with nanogram ~nfiti¼~ in lung lavaga~s ufing a C~8 reve~ed pha~ ca~ridge and a p h o t o , o d e array detector which prodded full U.V. spec~a of during compounds. Henke e t aL ~ described a m~hod for the ~parations of PG, HETE and LT in one HPLC run. They used two ~ffe~nt sy~ems, one with ~ #m C~8 r a ~ compr~fion sy~em and the other a norm~ ~ Bm C~ Altex c~umn. The dution time with the radial comp~sfion c~umn (60 min) was shorter than the norm~ Alex cCumn (100 min). However, the Altex c~umn ~lows dution of 1he m~abolites in smaller v~umes, thus ~¼ng ~gber ~ n f i t i ~ with U.V. detection. Separation of all three ~cosan~ds ufing the Altex c~umn is shown in Fig. 18. T ~ s ~ch~que will be of immen~ hdp w ~ ~ud~ng the interactions b~ween the PG synth~a~ and ~poxygenase pathways and the influence of anti-~flamm~ory drugs and other pharmac~o~c~ agents on the two pathway. Thus, Fogh et al. ~ em~oyed a mottled ~ocrat~ sy~em for the detection of ~cosano~s in homogena~s of psoriatic sk~. They used flow programing, ~ w~ch the i~ti~ flow-ra~ was 0.7 ml/min and was increased to 1.2 ml/m~ aRer the dution of ~ukotfienes. T ~ s m~hod produces good ~parat~ns of the produ~s of ~-, 12- and 1$-~poxygenase p~hways of AA m~abolism. c~/~ans

Recent ad~n~s ~ HPLC of ~ d s

27

140

-7 120

I00 %

I00 6"'6b

T

%

:;:i

:i 9: ~ ~ ~ ~

,:,

~ ~o

90

,~8,~ ,

~ ~ ~, ,,

8C

~

~o T

Bo ~

z;;l

'!:,!

so ~

~h~

40

0

80

if,

55%

ioo

~o

4

~ 0_

~ ~ m

~ 2

'

~~

~ 2O

,., . . . . . , ', ~ ....I,.., , I , ,

~';' ~,~

0

I0

~

20

~

30

i:I~

, ,

.I d } "

',

\

~

3o 20

~

H

~ 40

50 Time

60

70

I•l

80

90

r

IO0

{rain)

F~6. 18. Altex Cls, 5 #m, 4.6 x 250 mm c~umn. The chromatogram shows a representativeprofi~ of some tfifiated ( ) Ocosanoid ~andards and u~abeled LTC~, ll4rans-LTC4, LTD,, LTB4 and LTE~(---). Cdumn con~fions we~ 55% m~hand for 28 min, 66% m~hand for 25 min, 77% m~hand for 22 mi~ and 100% m~hand for 25 min. The ~m~nder of the sdvent ~ wa~r/acet~ acid (1:~000~ brought to pH &2 with ammo~um hydroxide.S~ndards were duted from the cdumn ~ a flowof I ml min -~. Fractionswe~ collectedand measuredeveryminute. U.V. mo~tofing was done at 280 nm. Key to numbered peaks: (1) 6-k~o-PGF~, (2) TxB2, (3) PGE~, (4) PGF~, ~--6) PGA~and LTC~,(6b) I l-trans-LTC~, (7) LTD4, (8) LTB~,(9) LTE~,(10) HHT, (I1) I~HETE, (1~ I~HETE, (1~ ~HETE, (1~ AA. Adapted ~om Re£ 79, The advantages of flow programing are that mono-HETEs are duted much earlier compared to normal dution sy~em~ and no baseline dri~ was observed. However, analyfis of Ocosanoids in biologic~ fluids usually requires radioimmunoassay and/or bioassay a~er reversed phase H P L C for the verification of the compounds resolved. Recent nutritional research ~ directed towards unfolding the my~ery behind the benefidal effect of ~cosapentaenoic a d d in myocardi~ infarction. A sen~five H P L C method s9 u~ng 9-anthryldiazomethane has been developed for the separation and determination of ~8,11,14,17-~cosapentaenoic a d d (EPA) ~ o m Cts and C~ fatty adds. The ~mit of detection is about 300 pg, which ~ quite senfitive for ex~nding this methodology for investigating EPA in human blood, serum, plankton body fluid and in aquatic spedes. H P L C u° has been successfu~y used in rapid diagno~s of Adrenoleukodys~ophy (ALD) and Adrenomydoneuropathy (AMN) by a n ~ y ~ n g very long c h i n fa~y adds (24: 0, 25 : 0 and 26:0) specific to these disorders.

E. Antioxidants Sever~ ~ c h ~ q u e s such as c d o f i m ~ r y , spe~rophotom~ry, polarography, papeL t ~ n h y e L GC and H P L C have been widdy used for the de~rmination of a n t i o ~ d a n ~ such as ~5-di-tert-butyl-&hydroxyt~uene (BHT), 3 - ~ - b u ~ P 4 - h y d r o x y a ~ s ~ e (BHA), 2 - ~ r t - b u t y l - 4 - h y d r o x y a n ~ e (BHA) and ~ r t - b u t ~ h y d r o x y q ~ n o n e (TBHQ). H P L C h the most faci~ procedure a v ~ h ~ e with the advantages of sped f i d e , ~ n f i f i f i ~ , accurac~ precision and automation p o s ~ f i e s . The a v ~ h ~ e HPLC m ~ h o d s ~ n e c ~ f i ~ a solvent extraction step w ~ c h m a k ~ m ~ h o d s cumbersome and senfitive to e r r o r . We have recently described a ~ r e c t ~ g h speed ~ q ~ d chromatography (HSLC) m ~ h o d for the quantitative de~rmination of anfio~dan~. The devdoped m ~ h o d enab~s the separations

28

V.K.S. Shukla

F = 2.0 m l / m i n

F=I,O ml / min

F=5.0 m t / m i n

F=40mL/m~n

F~o. 19. HPLC chromatogramof anfioxidantBHT. of BHT, BHA and TBHQ ~ nanogram ~ n s i t i d ~ ~ ~ than a minu~ ufing a 5 cm cyano c~umn (S3CN) and does not ~ q ~ a prior defivafizafion. Figur~ 19-21 ~ u ~ r a ~ the ~ p a r ~ i o n of t h e e anfiofidan~. Indyk and Wo~ard ~ used 30 min for fimflar separations as ~po~ed ~cently.

F. Tocophero~ Numerous m~hods for the an~yfis of tocophcro~ are described ~ the ~terature. Thc~ ~ q ~ d ~ n g an~yfis tim~ with cx~nfivc workup procedure. HPLC has been frequently u ~ d for the de~rrnination of tocophcrols ~:'2~'~ based on silva c~umns and hcxane, ~ p r o p a n ~ m~tu~s. We havC ~ succ~sfuHy applied the concept of HSLC for the F= 1.0 mL/rain

F=20 mt/min

F=5.0 mt/min

F =40 mt/min

~o. 20. HPLC c~oma~am of anfiofidamBHA.

F=5.0 m(/mln

Recent a d ~ n ~ s ~ HPLC of ~

29

I

I

F = 2.0

ml/min

I

F:

3.0

ml/min

o I ~

o

_

i

No. 21. HPLC ehromato~am ~ andoNdan~ BHA and T B H ~

separation of tocophero~ ~ soybeans and p~m oil ufing the cyano cdumn and hexane/ penmnol sdvent sygem. These separat~ns, as shown ~ Figs 22 and 23, are completed wit~n 5 min. No sam~e preparation is necessary, w~ch makes t~s m~hod s~ta~e for the routine application of tocophero~ in va~ous veg~a~e oils. G. Miscellaneous L ~ i d s

Hamilton and Com~ TM described a norm~ phase HPLC method for the separation of neutral ~pid and fatty a~d da~es ufing low wave~ngth detection. These Socratic separations were performed within 30 min. Garfi et aL ~ anMyzed commerci~ sorbitan espy, ~g. from Hamorad, Crod~ Arias, Probiba~cos and Adumin Chemic~s, ufing Lichrosorb RP 18 column and isopropanoUwa~r mixtures as solvent~ Kriiger et al. "~ devdoped a method for the separation and quantita6on of di~ycefides in the picomole 02%

pentonoL in hexone

O 4 % pentono[

0 . 3 % pentono( in hexone

O3% pentono( ~ hexone T¢

in hexone

Zr

~r

~T

~r

4

8 0

I

o

i

I 3

I ~

I 0

I I

Time (rain)

~.

22. HPLC ~ r o m ~ r a m

of t~op~rols ~ s o ~ a n

~ . ~ow-ra~ ~ 0 ~ ~ n - L

I 4

V. K. S. Shukla

3O 0 2%

0 3%

pent~nol in nexgne

pen~ano~ in hexane

0 5% hexone

O 4°/° pentanol in hexane

pen~anot in

yrr ~Tr

~

~

y

rT ~ ~Tr

~ i

I 5

I00

I

4[

8 10

[I

51

6~

~

I

Time (min)

~G. 23. HPLC c~om~o~am of ~cop~roh of palm o~n. F~w-ra~ = 2.0 ~ ~n ~. range a~er derivafizafion with ~ - n a p h t h ~ o c y a n a ~ . This fluorescent m~hod was 10 times more senfitive than hRherto published U.V. d~ection methods for the de~rmination of di~ycefides. Ryan and Honeyman ~ extended the fluorescent hbeling of di~ycerides by reacting them with Dn~ethan~aminephospha~ and achieved much superior resolutions within 20 rain. Sotirhos et al. ~ ~ud~d the o~dative and polymerised decompofition produc~ in commerd~ vegetab~ oils and fats. Brown and Snyder ~9separated conjuga~d dienes from tfi~ycerides of crude soy oil on silva columns. This method fadlitates the ~udy of soy oil during processing. H. A r g e n t a t ~ n

Chromatography

The progress, concerning the use of ~lver ion complexes for the separation of lipids, has been slow due to obvious reasons such as the reprodudbiEty of k' value, elufion of silver ions ~om the column by polar solvent, sen~ti~ty of the silver ion to light and the pos~bifity of ~lver mirror formation on the detector cell windows. However, Scholfi~d's group ~ has been active~ engaged in the application of argentation HPLC to the fipids and has reviewed its applications. Scholfi~d has separated methyl ester ~omers on a modified ~lverqoaded rein by programing column ~mperature f o m 25°C to 70°C. De Jarlais et al. ~ showed that acetonitrile is a powerful ~uent for polyunsaturated esters ~om fully ~lverqoaded ion exchange reins in HPLC. Its use in combination with methanol or acetone ~lows separation of methy~n~in~rrup~d polyunsatura~d esters that are difficult to elute ~om ~lver resin columns. Hsieh et aL ~ ~nificanfly improved the separation of meth~ esters u~ng a ~lver impregna~d s~ica column and dry benzene. They stated that benzene containing traces of water could dissociate Ag-o~fin + complexes more easi~ than dry benzene, thus ~ n g poor separations. Pla~ner ~ ~ud~d the effect of ~iver ions on the selecti~ty of tfig~cerides during reversed phase HPLC. Figure 24 i~ustrates the effect of fiiver ions in improving the separation of tfiglycerides of soybean oil. The resolution of g~ceride ~omers differing only in the pofitions of the acyl group, e.g. 1,3-distearo~, 2-oleolyl~ycerol (SOS) and 1,2-d~tearo~, 3-oleolyl-glycerol (SSO), is not pos~b~ by HPLC on C~8 columns without modifications. This has been achieved on filver ni~ate impregna~d columns at subambient temperatures, n We have very recently ~ separated these ~omers with great success within 2 min ufing different columns packed with fifica particles of varying pore diameters. It was observed that the resolution ach~ved

Recent a d ~ n ~ s ~ HPLC of ~

31

Samp[e : Soybean ale

~

Cotumn: ZorbaxaDS Sotvent60/20/20 / THF/CH2C 2I

~

Asabove

with

Time~ F~o. 2~ Changes in selectivity of column packing for soybean oil triglycerides with the addition of 0.2 N silver ion to the mobile phas. Adapted ~om Re£ 154.

depends upon the pore diameter of the silva employed. Thus, 80 or 100 ~ are probably the best ~ c a p a ~ s during argentation chromatography for obtaining the optimum resolutions (Fig. 25).

I. Preparat&e Chromatography In the broader sense, preparative chromatography is what ~ done to ~olate at Last one fraction of a mixture for further examination ~ (by spectroscopic techniques, other physico-chemical techniques, for synthe~s, commeroalization or for physiological tests in nutritional research, etc). Prep HPLC is, of cours~ preparative chromatography with a

PR POP

PIP

I

I

Time(min) ~ 0 . 25. Separation of tri~yceride ~ome~ ~ h u ~

2

unpuN~hed work~84~

32

V.K.S. Shukla

fiquid as duent and with a high effioency column. Lipidolo~s~ are yet to discover the extreme po~nti~ of ins~umental prep HPLC in s o l i n g thor problems. Scholfidd ~n succeeded in separating 200 mg samples of pure fatty methyl esters from hydrogenated fau for subsequent an~yses by I.R. and U.V. methods. Since then, silver refin chromatographyt ' ~ has been used as a conven~nt prep HPLC procedure for the purification of satura~d, mono-, di- and triunsaturated c~ and ~ans faay add methyl e~ers. However, this procedure was not suitable for the separation of m~tures of re,a-unsaturated ~omers because of poor peak shape and long dution times required for these polyunsatura~d reticules. Lanser and Emken "" extended the application of siNer r e i n chromatography for lhe i~olation of m~hyl arachidonate from ~s isomers using acetonitrile-methanol soNent sy~ems. Basceaa et aL ~ demon~ra~d the capabifities of prep HPLC for the purification of gram quantities of methyl o~a~, methyl fino~ate, m~hyl ~- and v-linolenates and m ~ h ~ r i d n o ~ a ~ ~om appropria~ natural oils in ~ than 30 min. To~wa et aL T M obtained high fields during the purification of e~osapentaenoic add (EPA) and docosahexaeno~ add (DHA) by reversed phase HPLC. They recommended the appl~ation of thor m~hod for the commerd~ production of EPA and DHA as these are expected to produce promi~ng resul~ in hypochole~erole-m~. ant-a~erioschlerot~ and anfi4hrombotic diseases. Christie et al. 3~ isolated milk fatty add derivatives on a Lichrosorb 10 RP 18 column in milligram sere. We ha~e ex~n~vdy employed prep HPLC for the ~tuctur~ d u d d a t o n s of tri~yceride mo~cules during the analy~s of ~ known exotic fats. Van Kessd et aL ~ reposed an HPLC method for the preparative purification of phosphofipids. V. MODERN ASPECTS OF HPLC A. L ~ u M Chromawgraphy-Mass Spectrometry

The coupling of HPLC and MS is one of the mo~ powerful ~ c h ~ q u ~ of mod~n m ~ h o d ~ o g y and is gradually becoming an impo~ant ana~tic~ m~hod, t~ Sever~ approaches have been investiga~d, and mo~ often three ~ r ~ c e types~direct fiq~d introduction, thermospray and the mo~ng belt--are used ~ the magnetic sector as well as quadrupo~ m a ~ s p e e U o m ~ s . A m~or advantage of the ~anspo~ ~ r ~ c e ~ iU capabifity to pro~de the a n ~ y ~ with more than one ~ z a f i o n mode such as e ~ r o n impa~ (EI), chemic~ ion~ation (CI), ~ r desorption, u ~condary~on ma~ specuomeuy~9'~ and ~st ~ o m bombardment~ Kuk~s and cowork~s ~ demon~r~ed the use of the direct l ~ d in~t LC-MS sy~em ~ r the ~paration and ~entification of mo~ n ~ u r ~ t r i a c ~ y c e r ~ s without p~fim~ary purification of the sam~e. They concluded that the mass spec~ome~r can serve as a sen~tive u ~ v ~ s ~ detector of all tri~yceride spedes, in ad~tion to p r o ~ n g ~ r u ~ u r ~ ~formation for ~entificaton purposes. They ex~nded t~s ~ch~que to ~udy ~ ~rtiarybutyl-dim~hyl-sil~ ethos of ~ a c ~ y c e r ~ s u~ng a c e ~ t r i l e and p r o ~ o ~ t r i ~ as eluting s~ven~ in reversed phase chroma~graphy. The developed c ~ r a t i o n ~ o ~ were applied to quantim~ ~ v ~ m i ~ u r ~ of ~ a c ~ y c e r ~ s derived ~om n ~ u r ~ ~ y c ~ phospholi~ds. These ~ s ~ were in excellent agreement with those obtained by capil~ry GLC. An LC-MS an~y~s of the ~mmin E ~om~s in maizegerrn oil is ~po~ed by Van d ~ G ~ e f et al? °9 They found 1h~ decUon impa~ was m o ~ ~n~tive ~ t~s ca~ w ~ u~ng a mo~ng b~t ~ r h c e . The dev~opment of LC-MS is one exam~e of a new ~chnobgy's progresfion from eso~ric hboramry expefimen~ to routine application. B. Supercr~ical Fluid Ch~matography (SFC)

A supercritcal fl~d w~ch possesses the ~ o ~ intermediate b~ween those of a fiq~d and a gas is ~rmed when Me ~ m p ~ a m ~ of a fiq~d is r ~ d above ~s critc~ point and the pressure ~ e ~ b y kept above ~s critcM v~ue. U~ng these fl~ds ~ m o ~ phases

R~ent a d ~ n ~ s ~ HPLC ~ f i # ~

i~

C~00C E ~ C H :CHEH~ CH3

c~(~,~(~~ ~(~)

m~

CH2OOCCiH~gCHC:HC (H~C,H~

I ~ ~H

]

i

I~

~oo~ ~,~l~,~

i ~oi~

i I c~ooclc~l~c~oc~lc~,c~l

,, ~ i

150

33

~,,

~,~ ~ 1

I0

150

0

170

I'~

13

2i0 250 Pressure (otm)

40

80

120

290 160

Time (rain)

~ 6 . 26. Capi~a~ ~ p e ~ f i t ~ fl~d c ~ o m ~ o ~ a p h ~ pro~e of a m i ~ e cf mono~ di- and ~ # y ~ f i d ~ . Numbe~d chmm~ogmp~c peaks a ~ Menfified ~ Ta~e 8. Ada~ed ~om Re~ 216.

in chromatography combined, unique ~atures of both ~quid and gas chromatography (LC and GC) are o[tMned. SFC ~ establishing itself as a compMmentary technique to GC and HPLC. It has the added advantage that ~ ~ compatible with a variety ofde~cfion sys~m~ including FID detection, making ff posfible to quanfitate solutes at Wace Mvds. The fir~ TABLE 8. Identification of C h m m ~ o g a p ~ c ~ g . 2 ~ 16

no. I 2 3 4 5 6 7 8 9 10 ll 12 13 14 15 16 17 18 19 20 21 22 J~LK ~ 1 ~

Compound l-Monocaprylin 1-Monocapfin l-Monolaufin l-Monomyfistin 1-Monopalmifin Dicaprylin Mono~aidin Monoe~osenoin 1,3-Dicapfin Monoerucin 1,3-Dilaufin l~-Dimyristin 1,2-Dipalmitin 1~-Dielaidin Diocosenoin Tfimyfi~olon Dieruon Tdpalmitolein Tfiheptadecanoin Tfidaidin Tfi-I 1-~cosenoin Tfinervonin

~

MW

4.32 218.15 6.80 246.18 10.40 274.21 12.88 302.25 14.72 330.28 20.44 344.26 22.80 356~9 25.80 384.32 32.12 400.32 34.36 412.36 37.60 456.38 44.60 512A4 54.88 568.51 65.88 620.54 69.76 676.60 76.52 716.60 83.80 732.66 88.16 800.69 92.72 848.78 97.72 884.78 1 0 8 . 9 6 968.88 132.96 1137.06

Pea~ ~

~

Form~a C , H2204 Ct3H2~O4 CtsHmO 4 C17H3404 C~9H~O~ CtgH3605 C2~H~O~

C2~H~04 C23H , O5 C25H~O4 C27H~O 5 C31H~O s CasH~sOs C~HnO s C43H~Os C4s HmO6 C47H~ Os Cst H9206 C~H~O6 C~TH~O, C63Ht~O~ C~sH~O~

34

V.K.S. Shuk~

190

2 9 0 hot

F~G. 27. CSFC chromatogram of palm kernd oil on a 10m × 100#m SE-54 column. CoLumn temperature: 170°C~Pressure program: programed from 190bar to 290 bar in 30 min. Adapted ~om Re£ 161. co mmer d ~ ~ r u m e n t was introduced by Hewl~t Packard in 1982, wh~h was based on the modal 1084 B fiq~d chromatograph. T ~ s ~ r u m e n t was ~ r withdrawn ~om the mark~. SFC in~ruments am m a n u f a ~ u ~ d by Browflee Labs (Santa Clara, CA), Computer Chemic~ Sy~ems (Avond~e, PA), Combustion En~neefing (Lewisberg, WV), Lee Soenfific (S~t Lake City, UT) and Sup~x (Pi~sburgh, PA). Although ~ d s ~ck chromopho~s and can be eafi~ de~c~d by FID de~ction cou~ed with SFC, still SFC has not been extensNdy em~oyed for an~yfis of ~ d s . The fi~t appl~ation of SFC in ~ d s was described by Rawdon and Norris, ~ who ~ p a r a ~ d mono-, ~- and tfi~ycefides in a packed C~8 c~umn ufing carbon ~ o f i d e (CO~) or CO:/ m ~ h a n ~ ~9:1) and varia~e wave~ngth U.V. detection. CheWer :8 published the first ~ p o ~ of c a ~ a r y SFC-FID utilization for the an~yfis of ~ycerides. Whi~ and Houck :~6 ~ p a r a ~ d mixed mono-, di- and triglycerides by c a ~ a r y supercritical fl~d chrom~ tography using a 19 m × 100 #m ID fused fil~a ca~Hary c~umn coa~d with 0.25 #m film of DB-5 (95% ~ m ~ h y l / 5 % ~phen~p~ysiloxane), CO2 as m o ~ p h a ~ and FID. The supercfit~al fl~d chromatograp~c pro~e of a mi~ure of 22 mixed ~ycerid~ obtained isothermally at 90°C is shown in Fig. 26. Numbered chromatograp~c ~eaks ~re identified as shown in T a ~ e 8. Very recently Proot et aLI61 have ~udied the resolution of tri~ycerides in ca~Hary SFC as a function of column ~mperatu~. They ~commended the u ~ of ~mperatu~s b~ween 150°C-230°C. The an~yfis of tfiglycerides accor~ng to thor carbon number ~ i~us~ated ~ the examp~ of an~yfis of p ~ m kernd oil contai~ng tri~ycer~es with carbon numbers ~om 28 to 56 (Fig. 27). Vl. CONCLU~ON AND THE FUTURE OF HPLC AS APPLIED TO LIPIDS A~hough HPLC has developed as a m o~ powerful tool in lipid a n a l y ~ its progress has been retarded due to the lack of a universal sen~tive HPLC detector. Light-scattering

R~m

~ s

~ ~

~ ~

35

detecto~ and the new generation of FID based d~ecto~ hdd g ~ promi~ for the future of HPLC as applied to ~#ds. A confid~aMe p r o g ~ ~ fi#d anMyfis ~ y~ to be made by fully ex#ofing lhe posfibi~fies of ~mper~u~ programing ~ u v d o ~ programingn (flow, pressure) and column s w i p i n g ~ch~ques. The m~n breakthrough is expected to be M the a~as of f l u o ~ e n t hbel~g of phosphofi#ds and dg~cefides and ~ the reve~ed phase anMyfis of tfi#ycefid~ and tfi#ycefide h o m e s ~ffefing o~y ~ the pofifions of the ac~ group. Acknowledgements--T~ author ~shes to thank Aarhus ~ ~r ~iding ~fiem ~a~h ~es and ~ m d a t i n g ~vironmem during the p ~ r a t i o n ~ this ~view. The ~ m ~ a n ~ his ~ (nonch~matog~phe0 for gallantly ~ading ~ e d ~ ~ ~ d for her ~ r ~ a r a n ~ . Appreciation ~ ~ n d ~ ~ Saren N i d ~ n ~ r skil~l contribution to the ~ r i m ~ t a l ~ s ~

(Received 12 Apr~ 1987) REFERENCES

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R~t

a d ~ n ~ s ~ HPLC of ~ d s

37

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