Gas chromatographic characterization of vegetable oil deodorization distillate

Transcription

1 Journl of Chromtogrphy A, 921 (2001) locte/ chrom Gs chromtogrphic chrcteriztion of vegetle oil deodoriztion distillte, c T. Verleyen *, R. Verhe, L. Grci, K. Dewettinck, A. Huygheert, W. De Greyt Deprtment of Orgnic Chemistry, University of Ghent, Coupure Links 653, 9000 Ghent, Belgium Deprtment of Food Technology nd Nutrition, University of Ghent, Coupure Links 653, 9000 Ghent, Belgium c De Smet Engineering, Prins Boudewijnln 265, Edegem, Belgium Received 14 Decemer 2000; received in revised form 9 April 2001; ccepted 17 April 2001 Astrct Becuse of its complex nture, the nlysis of deodorizer distillte is chllenging prolem. Deodorizer distillte otined from the deodoriztion process of vegetle oils consists of mny components including free ftty cids, tocopherols, sterols, squlene nd neutrl oil. A gs chromtogrphic method for the nlysis of deodorizer distillte without sponifiction of the smple is descried. After concise smple preprtion including derivtiztion nd silyltion, distillte smples were injected on column t 608C followed y grdul increse of the oven temperture towrds 3408C. The temperture profile of the oven ws optimized in order to otin seline seprtion of the different distillte components including free ftty cids, tocopherols, sterols, squlene nd neutrl oil. Good recoveries for d-tocopherol, -tocopherol, stigmsterol nd cholesteryl plmitte of 97, 94.4, 95.6 nd 92%, respectively were otined. Repetility of the descried gs chromtogrphic method ws evluted y nlyzing five replictes of soyen distillte. Tocopherols nd sterols hd low reltive stndrd devitions rnging etween 1.67 nd 2.25%. Squlene, mono- nd dicylglycerides hd higher reltive stndrd devitions rnging etween 3.33 nd 4.12%. Severl industril deodorizer distilltes otined from chemicl nd physicl refining of corn, cnol, sunflower nd soyen hve een nlyzed for their composition Elsevier Science B.V. All rights reserved. Keywords: Vegetle oils; Sterols; Tocopherols; Vitmins; Ftty cids 1. Introduction over the yers. Historiclly, deodorizer distillte used to e sold for its sterol content, s phytosterols hve Deodorizer distillte otined from the deodoriz- een used s strting mterils or s intermedites tion process of vegetle oils is complex product for the mnufcturing of phrmceuticl drugs nd consisting of mny components including free ftty steroids for medicinl purpose [4]. Recently n cids, tocopherols, sterols, squlene nd neutrl oil incresing interest in phytosterols is oserved s [1 3]. The vlue of deodorizer distillte hs vried phytosterols re used in functionl foods (e.g., mrgrines, etc.) for their potentil to inhiit intestinl cholesterol dsorption nd serum low-density *Corresponding uthor. Tel.: ; fx: lipoprotein (LDL) lowering cpcities [5,6]. Tody, E-mil ddress: tom.verleyen@rug.c.e (T. Verleyen). the vlue of the distillte is primrily determined y / 01/ $ see front mtter 2001 Elsevier Science B.V. All rights reserved. PII: S (01)

2 278 T. Verleyen et l. / J. Chromtogr. A 921 (2001) the tocopherol content. Tocopherols re nturl t 48C for mximum of 4 months until nlysis. ntioxidnts, which re vlued for their ntioxidtive The free ftty cid (FFA) content of the smples ws nd vitmin E ctivity [7]. As the economicl vlue determined y titrtion ccording to the AOCS of the distillte depends on its composition nd Officil Method C 5-40 [9] nd expressed s oleic content of tocopherols nd sterols, n ccurte cid (C 18:1). quntifiction of these nd other minor components Anlyticl-grde solvents were purchsed from present in the distillte is primordil. Merck (Drmstdt, Germny). All nlyticl-grde Becuse of its complex nture, the nlysis of reference sustnces, squlene (2,6,10,15,19,23-hexdeodorizer distillte is chllenging prolem. Few methyl-2,6,10,14,18,22-tetrcoshexene), stigmnlyticl techniques re ville to provide sterol (3-hydroxy-24-ethyl-5,22-cholestdiene), detiled nlysis of deodorizer distillte [1,8,20,21]. cholesteryl sterte (5-cholesten-3-yl-octdecno- The AOCS Ce3-74 Officil Method [9] descries te), monocylglycerol olein, dicylglycerol olein method for the determintion of tocopherols nd nd tricylglycerol olein, etulinol (lup-20[29]-enesterols in soy sludges y gs liquid chromtogrphy 3,28-diol) were purchsed from Sigm (Bornem, using pcked glss column. After long smple Belgium) nd were t lest 96% pure. A tocopherol preprtion, which involves sponifiction of the kit consisting of -, -, g- nd d-tocopherols ws distillte nd extrction of the unsponifiles, otined from Merck. tocopherols nd sterols re trnsformed into their The internl stndrd heptdecnyl sterte (HDS) utyrte esters nd re roughly seprted y gs ws prepred y condenstion of heptdecnol nd chromtogrphy (GC). The more recently pulished steroyl chloride oth purchsed from Aldrich (Bor- AOCS Ce7-87 recommended prctice [9] nlyzes nem, Belgium). Heptdecnol (50 g) ws weighed the totl tocopherol content in the deodorizer dis- into 1-l flsk nd steroyl chloride (30 g) ws tillte y cpillry GC on n polr column. After dded under slow gittion. The mixture ws disconcise smple preprtion, which consists of solved in hot ethnol (500 ml), cooled to room silyltion, short temperture profile is pplied temperture, crystllized during the night nd filwhich seprtes d-, / g- nd -tocopherols. In this trted. HDS crystls were crystllized twice from method d-, / g- nd -tocopherols re seprted ut ethnol (500 ml). due to the short temperture profile other components present in the deodorizer distillte re not well 2.2. Anlysis of non-sponified deodorizer distillte resolved. The ojective of the reserch ws to introduce Deodorizer distillte smples ( g of dry method comining seline seprtion of the nd homogeneous distillte) were directly weighed in different components present in deodorizer distillte screw-cpped test tue. Deodorizer distillte sm- (free ftty cids, squlene, tocopherols, free nd ples were derivtized in order to increse the voltiliesterified sterols nd neutrl oil) within resonle ty of the components. After dissolving the smple in nlysis time of mximlly 1 h y optimiztion of 0.5 ml of pyridine nd ddition of 1 ml N,O-isthe temperture profile. (trimethylsilyl)trifluorcetmide (BSTFA) contining 1% trimethylchlorosilne (TMCS) solution s derivtizting nd silylting gent, the test tue ws 2. Experimentl plced in n oven t 708C for 20 min for completion of the silyltion. Afterwrds 1 ml of HDS solution 2.1. Mterils with known concentrtion (6400 mg HDS/ 100 ml chloroform) ws dded s internl stndrd nd the Deodorizer distilltes originting from industril- smple ws further diluted with 8 ml of chloroform. nd lortory-scle deodoriztion of severl vege- After trnsferring 15 ml of derivtized smple to tle oils (soyen, cnol, sunflower nd corn oil) vil nd dilution with 1.5 ml of chloroform the were otined from Extrction De Smet (Edegem, smple ws redy for injection. After derivtiztion Belgium). All smples were stored in the refrigertor ll smples were nlyzed within 6 h of preprtion.

3 T. Verleyen et l. / J. Chromtogr. A 921 (2001) Anlysis of sponified deodorizer distillte distillte followed y n optimized GC temperture smples profile. Compring their GC retention times with stndrds ws performed for the identifiction of the Deodorizer distillte smples (0.7 g) were different peks. Retention times nd reltive retensponified with 10 M KOH (5 ml) in 45 ml ethnol, tion times using HDS s internl stndrd for the together with 5 mg etulinol s internl stndrd to identified components re presented in Tle 1. quntify losses during sponifiction nd extrction. Chromtogrms of non-sponified soyen nd The solution ws heted for 30 min t 708C. After rpeseed distillte re shown in Figs. 1 nd 2. sponifiction, 100 ml wter ws dded nd the Upon optimiztion of the procedure the seprtion unsponifiles were extrcted twice with 100 ml of two criticl pirs ws oserved. Within the tested diethyl ether. The comined diethyl ether frctions temperture profiles, co-elution of the - nd g- were wshed twice with 20 ml of 0.5 M KOH, four tocopherols ws lwys found (Fig. 2). However, times with 20 ml wter nd dried over nhydrous this ws expected s due to their similr chemicl sodium sulfte. The residue otined fter evpor- structures, seprtion etween the - nd g- tion ws derivtized ccording to the procedure tocopherols homologues is hrdly chieved y GC descried in Section 2.2. [1,10,11]. Bseline seprtion of the - nd g- tocopherols cn only e otined y norml-phse 2.4. Cpillry column gs liquid chromtogrphy high-performnce liquid chromtogrphy [11 14]. Actully this incomplete seprtion ws not regrded Seprtions were performed on Hewlett-Pckrd s prolem s -tocopherol is only present in most 5890 series gs chromtogrph with cold on-column nturl vegetle oils, nd consequently in deodorinjection on cpillry column CP-Sil 8 CB Low izer distillte, in very low concentrtions [15 17]. leed/ MS 15 m30.25 mm, 0.1 mm (Chrompck, Another pir difficult to seprte ws -tocopherol Middelurg, The Netherlnds) with detection y nd rssicsterol (Fig. 1). By pplying the proposed flme ioniztion detection. temperture profile pek resolution of 0.75 ws A temperture progrm with injection t 608C, otined for the seprtion etween -tocopherol nd rising t 308C/ min to 1408C nd continuing the oven rssicsterol. This resolution fctor is not completeheting t speed of 58C/min to 2358C with 7 min ly perfect, s resolutions ove 0.9 re necessry for hold nd further heting t 158C/ min to 3408C with mking good quntifiction possile. No further 15 min hold, ws found optiml for good seprtion of the different components. The detector temperture ws set t 3608C. Helium ws used s Tle 1 Retention times (t R) nd reltive retention times (RRTs) of crrier gs t pressure of 41.3 kp. The peks were identified components present in deodorizer distillte computed using HP 3396 series II integrtor. t R (min) RRT Monocylglycerides Results nd discussion Squlene d-tocopherol Tocopherol Anlysis of non-sponified deodoriztion g-tocopherol distilltes: optimiztion of seprtion -Tocopherol Brssicsterol Literture references nlyze the tocopherol nd Cmpesterol Stigmsterol sterol content of deodorizer distillte fter lori- Sitosterol ous smple preprtion procedure including sponifi- Heptdecnyl sterte (HDS) ction nd derivtiztion. In this reserch, seline Dicylglycerides seprtion of the different components present in Steryl esters vegetle oil deodorizer distillte ws chieved y Tricylglycerides direct derivtiztion of non-sponified deodorizer Retention time reltive to HDS.

4 280 T. Verleyen et l. / J. Chromtogr. A 921 (2001) Fig. 1. Chromtogrm of non-sponified rpeseed deodorizer distillte nlyzed y gs chromtogrphy. Time scle in min. reserch ws undertken to improve this seprtion, cholesteryl sterte ws used to quntify the whole s rssicsterol is only present in rpeseed or cnol steryl ester group. Monooleylglycerol, dioleylcultivrs. glycerol nd trioleylglycerol re the stndrds used for the response fctors of mono-, di-, tricyl Determintion of response fctors glycerides. The response fctors determined for the different stndrds re listed in Tle 2. All response Quntifiction of the different components present fctors were determined fivefold nd showed good in deodorizer distillte ws done y use of n repetility s indicted y the low stndrd deviinternl stndrd. HDS ws selected s internl tion (SD) nd reltive stndrd devition (RSD) stndrd s no components eluted in this region. rnging etween 3.5 nd 4.6%. A good stility of Response fctors (RFs) were determined y ccu- the response fctors over period of severl months rtely weighing mg of pure stndrd com- ws oserved. An ccurte determintion of the ponent into test tue, followed y the derivtiztion response fctors is necessry for otining good procedure descried. quntifiction of the different distillte components. Response fctors were determined for - nd d- tocopherols. The men of these response fctors ws 3.3. Recovery used s the estimted response fctor for - nd g-tocopherols. Stigmsterol ws selected for the In order to exclude n incorrect quntifiction of determintion of the sterol response fctor nd ws deodorizer distillte smples the recovery of the used for ll free sterols. The response fctor of nlysis method ws evluted. A synthetic dis-

5 T. Verleyen et l. / J. Chromtogr. A 921 (2001) Fig. 2. Chromtogrm of non-sponified soyen deodorizer distillte nlyzed y gs chromtogrphy. Time scle in min. tillte ws prepred y dissolving d- nd - grphic dt. Upon nlyzing the distilltes good tocopherols, stigmsterol nd cholesteryl sterte in recoveries of 94 97% for the tocopherol nd sterol free ftty cid mtrix, which consisted of oleic cid contents were found (Tle 3). A perfect recovery (C 18:1). Three distilltes with different tocopherol ws expected due to the miniml smple preprtion. nd sterol content were prepred. Tocopherol nd The current recovery levels for tocopherol nd sterol sterol concentrtions re expressed s g/ 100 g of concentrtion re in ccordnce with literture redistillte for oth grvimetric nd gs chromto- ports [10,11,18]. A lower ut still cceptle recovery of 92% ws oserved for cholesteryl sterte. Lechner et l. [10] lso reported lower nlyte Tle 2 recovery fctor for steryl esters. This lower nlyte Response fctors (RFs) otined of stndrds reltive to heptderecovery for steryl esters ws explined y degrcnyl sterte (HDS) s internl stndrd dtion on the GC column due to the high temperture RF men SD RSD (%) pplied in GC. Monocylglycerides Squlene d-tocopherol Repetility -Tocopherol Stigmsterol Repetility of the descried gs chromtogrph- Dicylglyceride ic method ws evluted y nlysing five replictes Cholesteryl plmitte of soyen distillte including derivtiztion pro- Tricylglycerides cedure (Tle 4). Only identifile peks were tken Response fctor clculted s men of five replictes. into considertion nd concentrtion ws expressed

6 282 T. Verleyen et l. / J. Chromtogr. A 921 (2001) Tle 3 Recovery of d- nd -tocopherols, stigmsterol nd cholesteryl plmitte spiked t three concentrtion levels in oleic cid (C 18:1) Concentrtion (g/ 100 g distillte) Recovery (%) Distillte A Distillte B Distillte C Men SD RSD Grv GC Grv GC Grv GC (%) d-tocopherol Tocopherol Stigmsterol Cholesteryl plmitte Grv, concentrtion clculted on grvimetric sis. GC, concentrtion determined y GC nlysis. The tocopherol nd sterol content of deodorizer distillte strongly depends on the initil free ftty cid content nd the pplied deodoriztion process conditions including temperture, stem nd pressure [4]. The quntittive composition of the different distilltes otined from the deodoriztion of soy- en, corn, sunflower nd rpeseed oil is listed in Tle 5 nd is lrgely ffected y the refining technique (chemicl or physicl). Results re ex- pressed s g/100 g of oil. In the physicl refining (numers 3, 4, 5, 6) free ftty cids re vporized during the deodoriztion process. Consequently the s g/ 100 g of distillte. Tking into ccount the short smple preprtion good repetility ws oserved with similr RSDs to nother nlyticl report on tocopherol nd sterol quntifiction [11]. The four tocopherols nd different sterols hd similr RSDs rnging etween 1.67 nd 2.25%. Only for - tocopherol higher RSD ws otined, proly due to the incomplete seprtion etween - nd g- tocopherols. Squlene, mono- nd dicylglycerides hd higher ( %) ut still cceptle RSD vlues Overview of different deodoriztion distillte distillte contins minly free ftty cids (.80%) smples with only smll mounts of unsponifiles (5 10%) nd is usully sold s source of technicl ftty Tocopherols nd phytosterols were identified s cids. In the chemicl refining (numers 1, 2, 7, 8) the most importnt minor components nd were present in vrying concentrtions in the distillte. free ftty cids re neutrlized y custic solution nd wshed out of the oil efore deodoriztion. Deodorizer distilltes otined from chemicl refining oviously hve lower free ftty cid content Tle 4 (30 50%) nd higher level of unsponifiles Repetility of the quntittive gs chromtogrphic nlysis of soyen deodorizer distillte otined y repeted nlysis (n55) rnging etween 25 nd 33% [19]. Due to this higher tocopherol nd sterol content deodorizer distilltes Men 6SD RSD (g/100 g distillte) (%) otined from chemicl refining re good source of vitmin E nd phytosterols. Especilly the dis- Monocylglycerides tillte of chemiclly refined soyen oil is n ttrc- Squlene d-tocopherol tive source of tocopherols nd sterols [4]. For every -Tocopherol type of oil, sterol-to-tocopherol rtio exists which g-tocopherol is chrcteristic. Monitoring the sterol over -Tocopherol tocopherol rtio is wy to determine if degrdtion Cmpesterol is occurring during processing or storge of the Stigmsterol Sitosterol distillte [4]. Dicylglycerides Steryl esters Anlysis of sponified deodoriztion distilltes Tricyglycerides Men of five replictes. Distillte smples cn lso e nlyzed y

7 T. Verleyen et l. / J. Chromtogr. A 921 (2001) Tle 5 Composition of non-sponified deodorizer distilltes nlyzed y quntittive gs chromtogrphy Soyen Corn Sunflower Rpeseed Squlene d-tocopherol nd nd Tocopherol nd nd g-tocopherol Tocopherol Brssicsterol nd nd nd nd nd nd nd Cmpesterol Stigmsterol nd 0.01 Sitosterol Other sterols nd nd nd nd nd nd nd Steryl esters nd Monocylglycerides Dicylglycerides Tricylglycerides FFA (C 18:1) Concentrtions expressed s g/ 100 g of distillte. Sum of D5 vensterol, D7 vensterol nd D5 stigmsterol. nd, Not detected. Fig. 3. Chromtogrm of sponified soyen deodorizer distillte nlyzed y gs chromtogrphy. Time scle in min.

8 284 T. Verleyen et l. / J. Chromtogr. A 921 (2001) Tle 6 Comprison etween sponified nd non-sponified deodorizer distillte composition nlyzed y quntittive gs chromtogrphy nd expressed s g/ 100 g distillte Rpeseed Rpeseed Soyen Non-sponified Sponified Non-sponified Sponified Non-sponified Sponified Squlene d-tocopherol Tocopherol 0.18 nd nd nd 0.27 nd g-tocopherol Tocopherol Brssicsterol nd nd Cmpesterol Stigmsterol Sitosterol Steryl esters nd 2.47 nd Monocylglycerides, dicylglycerides nd tricylglycerides could not e detected nymore fter sponifiction. preceding sponifiction, s explined in Section 2.3, umn GC s it gives good seprtion of the different followed y the sme derivtiztion procedure nd distillte components nd perfect result is otined gs chromtogrphic nlysis procedure. Sponific- with miniml smple preprtion. In further reserch tion of distillte smples neutrlizes free ftty cids the reltionship etween the deodoriztion process nd hydrolyses neutrl oil nd esterified minor condition nd the composition of the deodorizer components. As expected, free ftty cids, mono-, distillte will e studied. di-, tricylglycerides could not e detected nymore in the gs chromtogrm fter sponifiction s shown in Fig. 3. Sometimes prt of the components References eluting in the steryl esters region remined present fter sponifiction. Chromtogrms otined fter [1] C. Mrks, J. Am. Oil Chem. Soc. 65 (1988) sponifiction showed much more interferences of [2] S. Rmmurthi, A. McCurdy, J. Am. Oil Chem. Soc. 70 smll components ppering in the tocopherol nd (1993) 287. sterol region. Proly this cn e explined y n [3] S. Ghosh, D.K. Bhttchryy, J. Am. Oil Chem. Soc. 73 (1996) enrichment of some tocopherol nd sterol oxidtion [4] L. Wlsh, L. Winters, G. Gonzlez, Inform 9 (1998) 78. products during sponifiction. [5] R. Tilvis, T. Miettinen, Am. J. Clin. Nutr. 43 (1986) 92. A comprison etween the nlysis method with- [6] J.A. Weststrte, G.W. Meijer, Eur. J. Clin. Nutr. 52 (1998) out preceding sponifiction nd the method includ ing preceding sponifiction ws mde for some [7] A. Kml-Eldin, L. Appelqvist, Lipids 31 (1996) 671. [8] D. Feeter, J. Am. Oil Chem. Soc. 51 (1974) 184. deodorizer distillte smples (Tle 6). The [9] Officil Methods nd Recommended Prctices, 4th ed., sponified distilltes hve tocopherol content which Americn Oil Chemists Society, Chmpign, IL, is rther similr to the stright nlysis indicting [10] M. Lechner, B. Reiter, E. Loreer, J. Chromtogr. A 857 tocopheryl esters re hrdly present in the distillte. (1999) 231. The sterol content of sponified deodorizer distillte [11] W. De Greyt, V. Petruskite, M. Kellens, A. Huyhgeert, Fett/ Lipid 100 (1998) 503. smples is significntly higher due to hydrolysis of [12] A. Kml-Eldin, S. Gorgen, J. Pettersson, A.M. Lmpi, J. steryl esters during sponifiction. Chromtogr. A 881 (2000) 217. [13] S.L. Aidi, T.L. Mounts, J. Chromtogr. A 782 (1997) 25. [14] E. Gimeno, A.I. Cstellote, R.M. Lmuel-Rvenots, M.C. 4. Conclusion de l Torre, M.C. Lopez-Str, J. Chromtogr. A 881 (2000) 251. In conclusion, dvntge is given to n nlysis [15] A.J. Speek, J. Schrijver, W.H.P. Scheurs, J. Food Sci. 50 (1985) 121. non-sponified deodorizer distillte y cpillry col- [16] U. Coors, Ft Sci. Technol. 93 (1991) 519.

9 T. Verleyen et l. / J. Chromtogr. A 921 (2001) [17] M. Blz, E. Schulte, H.P. Thier, Ft Sci. Technol. 94 (1992) [20] M.V. Ruiz-Mendez, G. Mrquez-Ruiz, M.C. Dorgnes, 209. Grss Aceites 46 (1995) 21. [18] F. Ulerth, H. Reich, W. Kneifel, Ft Sci. Technol. 94 (1992) [21] S. Rmrmurthi, A.R. McCurdy, J. Am. Oil Chem. Soc (1993) 287. [19] W. De Greyt, M. Kellens, in: W. Hmm, R.J. Hmilton (Eds.), Edile Oil Refining, Sheffield Acdemic, Sheffield, 2000.