A method of chromotagraphic isolation for vitamin E isomers

Abstract: A method of chromatographic isolation of Vitamin E isomers ( α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol, , and δ-tocotrienol) from Vitamin E- comprising compounds, said method including the steps of (a) introducing the substances, which form the source of the Vitamin E isomers, onto selective adsorbents to effect adsorption of Vitamin E isomers and subsequently, (b) effecting an individual desorption of the adsorbed Vitamin E isomers, wherein the adsorption and/or desorption of the Vitamin E isomers are carried out under a supercritical fluid environment.

Description: Field of the Invention The invention relates to a method of chromatographic isolation for Vitamin E isomers, particularly individual Vit E isomers such as α-tocopherol, β-tocopherol, γ-tocopherol δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol and /or the like from Vit E-comprising compounds such as crude palm oil, palm oil products and/or palm oil by-products, vegetable oils and/or the like. Background of the Invention This invention relates to a process of producing isomeric tocols or Vitamin E isomers ( α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol; and α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol) from tocols or Vitamin E comprising compounds. Tocols are Vitamin E and consist of tocopherols (T) and tocotrienols ( (T 3 ) which are also found in vegetable oils in varying quantity ( see Table A ). The tocols present in most vegetable oils are in the form of tocopherols ( α-, β-, γ- and/or δ-tocopherols ). On the other hand, palm oil is unique as the tocols present are mainly in the form of tocotrienols (α-T 3 , γ T 3 and δ- T 3 ). Both of these tocols have shown interesting physiological properties. Tocopherols determined in vegetable oils (mg/kg) Oil α-T β-T γ-T δ-T α-T 3 γ-T3 δ-T 3 Castor 28 29 111 310 - - - Cocoa butter 11 - 170 17 2 - - Coconut - - - 2-4 20 - - Corn 134 18 412 39 - - - Cottonseed 573 40 317 10 - - - Groundnut 169 5 144 13 - - - Linseed - - - - - - - Mustard 75 - 494 31 - - - Olive 93 - 7 - - - - Palm from fibres 1662 - - - 456 485 142 Palm 150 - - - 1127 297 80 Palm 279 - 61 - 274 398 69 Rape 70 16 178 7 - - - Rice bran 324 18 53 - 236 349 - Safflower 477 - 44 10 - - - Sesame 12 6 244 32 - - - Soyabean 116 34 737 275 - - - Sunflower 608 17 11 - - - - Wheat germ 1179 398 493 118 tr - - Both the tocopherols and tocotrienols act as powerful nutritional antioxidants and help to reduce cellular damage due to free radicals arising from the body's normal oxidative energy metabolism or from the action of toxic chemicals and pollutants in our environment. Free radicals have been implicated in aging, chronic degenerative diseases and cancer. Vitamin E is also a natural antioxidants, are present at approximately 600 - 1000 ppm in crude palm oil; the major components being the γ-tocotrienol which has recently been found to have anti-cancer properties besides its known antioxidant activity. Tocotrienols have been found to lower blood cholesterol. A number of patents have been filed on the recovery of Vitamin E from palm oil and they are: i) Production of High Concentration of Tocopherols and Tocotrienols from Palm Oil By-Product. US Patent 5,190,618; ii) Integrated Process for Recovery of Carotenoids and Tocotrienols from oil. US Patent 5,157,132; and iii) Recovery of Carotenoids, Tocopherols, Tocotrienols,and Sterols from esterified Palm Oil GB 2,218,989. The first patent discloses the extraction of Vitamin E from palm fatty acid distillates (PFAD) which involves conversion of a PFAD into methyl esters, distillation of methyl esters followed by ion-exchange chromatography and molecular distillation to get Vitamin E concentrate of more than 90%. The second patent discloses extraction of Vitamin E from crude palm oil (CPO) by converting CPO to methyl esters. Thereafter remove the esters by molecular distillation to yield carotene-rich and vitamin E-rich fractions. The third patent discloses conversion of crude palm oil into methyl esters followed by subjecting the methyl esters in the presence of solvents into chromatographic separation with an adsorbent to yield carotene-rich, vitamin E-rich and sterol-rich fractions. The above three inventions only describe the isolation of vitamin E from palm oil and do not include any work on the isolation of individual vitamin E isomers, which have exhibit important physiological properties. Journal of Chromatography, A. Vol. 679, p 329-34 (1994) and Chemical Analysis (Wersaw) vol. 42, p 245-8 (1997) describe processes for separating tocopherols using supercritical fluid chromatography (SFC). Chromatographic Vol. 39, No. 11/12, December 1994 describes a process for the quantitative analysis of marine oils. GB 2,218,989 describes a method of recovery of tocopherols and tocotrienols from esterified palm oil. The latter document, however, is silent on the use of SFC. Hence, there is a need to provide a separation, particularly an isolation method for Vitamin E isomers which avoids or eliminates the sole use of solvents and consequently, rendering the process "non-hazardous". Summary of the Invention In accordance with the present invention, there is provided a method of chromatographic isolation for Vitamin E (Vit E) isomers as defined in claim 1. In particular, the present invention stipulates the use of supercritical fluid ( such as SC-CO 2 ) in combination with adsorbents ( such as silica gel or C18 reverse phase silica gel) to effect adsorption and/or desorption of Vit E isomers from Vit-E comprising compounds. Most preferably, the present invention is subsequently applied for isolating Vit E isomers from; a) crude vegetable oil esters ( via catalytic alcoholic esterification/trans-esterification of vegetable oils ) alone or by distilling the esters or by distilling the esters followed by saponification to yield vitamin E concentrate, OR b) unsaponifiable matters of vegetable oils and fats ( through saponification process) OR c) Vitamin E concentrate obtained through any/various processes including chromatography ( solvent and supercritical ) as well as supercritical fluid extraction. A main advantage of the invention lies in the use of liquefied gas at supercritical conditions, hence avoiding or reducing the prior requirements for hazardous solvents. Detailed description of the Invention The method of chromatographic isolation of Vitamin E isomers from e.g. crude palm oil (CPO), palm oil products and/or palm oil by-products, vegetable oils and/or Vitamin E-comprising compounds, inclues : (I) Introducing the Vitamin E comprising-compounds onto selected adsorbents to effect adsorption of Vitamin E isomers; and (II) subsequently desorbing the adsorbed Vitamin E isomers wherein, the adsorption/desorption of the vitamin E isomers are carried out under a supercritical fluid environment. Vit E isomers may be isolated from Vit E concentrate as derived through, (i) Esterifying the free fatty acid component of the Vit. E isomers-comprising oils with one or more monohydric alcohols into esters. The esterification may be carried out via employing (a) a solid alkali metal bisulfate, a sulfate acid and/or a strongly acidic-ion exchange resin or any acid as a catalyst; (ii) Trans-esterifying the glyceride components of Vit. E isomers-comprising oils with one or more monohydric alcohols ( C 1 to C 8 into monoesters. The trans-esterification may be carried out via employing a basic catalyst. [Both steps i and ii may be carried out via employing an enzyme e.g. candida rugosa and/or the like effective catalyst]; (iii) Removing the formed esters in step (i-ii) by vacuum distillation or molecular distillation thereby yielding vitamin E concentrate. The esters may be removed via vacuum distillation or molecular distillation. OR Supercritical extraction and/or any chromatographic methods which are effective in extracting Vitamin E concentrate from vegetable oils. Alternatively , the present invention may be applied to unsaponifiable materials as obtained through saponification of vegetable oils. Adsorbents : Selective adsorbent for the adsorption of the Vitamin E isomers comprise silica gel, C18 reverse phase silica gel and/or the like which are equivalently effective in adsorbing Vit E isomers from any Vit E comprising-compounds. Solvents: The solvents employed for the present method comprises; i) supercritical fluid, e.g. SC-CO 2 ( 100% ). Other applicable solvents include propane, propene, ethylene and/or the like; OR ii) supercritical fluid ( e.g. SC-CO 2 ) in combination with an entrainer (solvent such as alcohol and/or the like organic solvent) Operating conditions Individual Vitamin E isomers may be isolated with substantially higher purity (e.g. > 50%) in a single run by varying pressures from 50 kg/cm 2 to 600 kg/cm 2 and temperatures 30°C -100°C. It shall therefore be illustrated, by means of the following examples, that supercritical fluid applied to solid adsorbents (silica gel and/or C18 reverse phase silica gel), provides a very satisfactory means for isolating Vitamin E isomers ( α-T, β-T, γ-T, δ-T and α-T 3 , β-T 3 , γ-T 3 and δ-T 3 ) from CPO (crude palm oil), palm oil products, vegetable oil-and/or the like Vitamin E comprising compounds. The Vitamin E isomers recovered are useful in pharmaceutical compositions, food and health food formulations, dietary supplements and cosmetics formulations. They can also be used as fine chemicals and standard reference materials. Example 1: Isolation of Palm Vitamin E isomers Crude palm oil (containing 600- 1000 ppm of vitamin E) was trans-esterified into methyl esters. The methyl esters were removed through molecular distillation at a temperature of 90-180 degree C followed by saponification to yield a vitamin E concentrate ( > 50% ). Vit. E concentrate (0.01g; concentration: ~50%) was dissolved in organic solvent, such as dichloromethane or ethanol and loaded onto a column (silica gel, with internal diameter 20mm and length 250mm). The Vitamin E concentrate mixture was eluted using a mixture of supercritical carbon dioxide (5ml, 95.2%) and an entrainer, ethanol ( 0.25ml, 4.8%) with a flowrate of 5.25ml/min. The pressure of the supercritical fluid chromatography system was kept constant right through the run at P=180kg/cm 2 , keeping temperature constant at 70°C. The four vitamin E isomers were collected at different time intervals and are shown in Table 1. The presence and concentration of Vitamin E isomer were confirmed by HPLC. Example 2 The procedure of example 1 was repeated except vitamin E concentrate obtained from palm fatty acid distillate was used. The results are shown in Table 2. Example 3 The procedure of example 1 was repeated except palm pressed fiber oil vitamin E concentrate was used. The results are shown in Table 3. Example 4 The procedure of example 1 was repeated except soybean oil vitamin E concentrate was used. The results are shown in Table 4. Example 5 The procedure of example 1 was repeated except corn oil vitamin E concentrate was used. The results are shown in Table 5. Example 6 The procedure of example 1 was repeated except vitamin E from Canola (rapeseed oil) oil was used. The results are shown in Table 6. Example 7 The procedure of example 1 was repeated except vitamin E from olive oil was used. The results are shown in Table 7. Example 8 The procedure of example 1 was repeated except vitamin E from cottonseed oil was used. The results are shown in Table 8. Example 9 The procedure of example 1 was repeated except vitamin E from groundnut oil was used. The results are shown in Table 9. Example 10 The procedure of example 1 was repeated except vitamin E from ricebran oil was used. The results are shown in Table 10. Example 11 The procedure of example 1 was repeated except vitamin E from sunflower seed oil was used. The results are shown in Table 11. Example 12 The procedure of example 1 was repeated except C18 reverse phase silica gel was used as adsorbent instead of silica gel. Other conditions used are : Pressure: 180 kg/cm2, CO2: ethanol ratio 93.75% : 6.25%. The four vitamin E isomers ( alpha-tocopherol, alpha-tocotrienol, gamma-tocotrienol and delta-tocotrienol) were eluted in the same elution order as in example 1 and collected in high purity as in example 1. The same procedures using C18 reverse phase silica gel as adsorbents were repeated for other palm oil products, palm oil by-products and other vegetable oils as mentioned in Examples 2-11. Similar elution order of vitamin E isomers was observed for both types of adsorbents used. Isolation of Palm Vit.E isomers (α-T, α-T 3 , γ-T 3 and δ-T 3 ) Fractions Collection Time (min) Vit.E α-T Vit.E α-T 3 Vit.E γ-T 3 Vit.E δ-T 3 S/M - 12.7 11.4 19 5.8 1 - - - - - 2 41-47 100 - - - 3 47-54 - 100 - - 4 54-61 - - 100 - 5 61-77 - - 99 1 6 77-145 - - 3 97 7 145- - - - - a. S/M : Starting Material (vit. E concentrate ~50%) b. T: tocopherol c. T 3 : tocotrienol d. Conditions: P=180kg/cm 2 , T= 70°C e. SC-CO 2 : ethanol = 5ml:0.25ml (95.2% : 4.8%) Isolation of Palm Vit.E isomers (α-T, α-T 3 , γ-T 3 and δ-T 3 ) from Palm Fatty Acid Distillate Fractions Collection Time (min) Vit.E α-T Vit.E α-T 3 Vit.E γ-T 3 Vit.E δ-T 3 S/M - 22.7 21.4 39 9.8 1 2 41-47 100 3 47-54 100 4 54-61 100 5 61-77 98 2 6 77-145 2 98 7 145- a. S/M : Starting Material (vit. E concentrate ~90%) b. T: tocopherol c. T 3 : tocotrienol d. Conditions: P=180kg/cm 2 , T= 70°C e. SC-CO 2 : ethanol = 5ml:0.25ml (95.2% : 4.8%) Isolation of Palm Vit.E isomers (α-T, α-T 3 , γ-T 3 and δ-T 3 ) from Palm Pressed Fibre Oil Fractions Collection Time (min) Vit.E α-T Vit.E α-T 3 Vit.E γ-T 3 Vit.E δ-T 3 S/M - 42.7 11.4 29 10.8 1 2 41-47 100 3 47-54 100 4 54-61 100 5 61-77 99 1 6 77-145 2.2 97.8 7 145- a. S/M: Starting Material (vit. E concentrate ~90%) b. T: tocopherol c. T 3 : tocotrienol d. Conditions: P=180kg/cm 2 , T= 70° e. SC-CO 2 : ethanol = 5ml:0.25ml (95.2%: 4.8%) Isolation of P Vit.E isomers (α-T, β-T, γ-T and δ-T) from Sovabean Oil Fractions Collection Time (min) Vit.E α-T Vit.E β-T Vit.E γ-T Vit.E δ-T S/M - 11.6 3.4 53.7 17.5 1 2 35-41 100 3 42-47 99 4 48-55 100 5 58-65 100 a. S/M : Starting Material (vit. E concentrate ~80%) b. T: tocopherol c. Conditions: P=180kg/cm 2 , T= 70°C d. SC-CO 2 : ethanol = 5ml:0.25ml (95.2%: 4.8%) Isolation of Vit.E isomers (α-T, β-T, γ-T and δ-T) from Corn Oil Fractions Collection Time (min) Vit.E α-T Vit.E β-T Vit.E γ-T Vit.E δ-T S/M - 23.4 1.8 51.2 3.9 1 2 35-41 100 3 42-47 92 4 48-55 100 5 58-65 92 a. S/M: Starting Material (vit. E concentrate ~80%) b. T: tocopherol c. Conditions: P=180kg/cm 2 , T= 70°C d. SC-CO 2 : ethanol = 5ml:0.25ml (95.2%: 4.8%) Isolation of Vit.E isomers (α-T, β-T, γ-T and δ-T) from Canola Oil Fractions Collection Time (min) Vit.E α-T Vit.E β-T Vit.E γ-T Vit.E δ-T S/M 20 8 58 2 1 2 35-41 100 3 42-47 85 4 48-55 100 5 58-65 80 a. S/M : Starting Material (vit. E concentrate ~90%) b. T: tocopherol c. Conditions: P=180kg/cm 2 , T= 70°C d. SC-CO 2 : ethanol=5ml:0.25ml (95.2%: 4.8%) Isolation of Vit.E isomers (α-T, β-T, γ-T and δ-T) from Olive Oil Fractions Collection Time (min) Vit.E α-T Vit.E β-T Vit.E γ-T Vit.E δ-T S/M - 83 7 1 2 35-41 100 3 42-47 80 4 48-55 5 58-65 a. S/M: Starting Material (vit. E concentrate ~90%) b. T: tocopherol c. Conditions: P=180kg/cm 2 , T= 70°C d. SC-CO 2 : ethanol = 5ml:0.25ml (95.2% : 4.8%) Isolation of Vit.E isomers (α-T, β-T, γ-T and δ-T) from Cottonseed Oil Fractions Collection Time (min) Vit.E α-T Vit.E β-T Vit.E γ-T Vit.E δ-T S/M - 57.3 4 31.7 1 1 2 35-41 100 3 42-47 92 4 48-55 100 5 58-65 85 a. S/M : Starting Material (vit. E concentrate ~90%) b. T: tocopherol c. Conditions: P=180kg/cm 2 , T= 70°C d. SC-CO 2 : ethanol = 5ml:0.25ml (95.2% 4.8%) Isolation of Vit.E isomers (α-T, β-T, γ-T and δ-T) from Groundnut oil Fractions Collection Time (min) Vit.E α-T Vit.E β-T Vit.E γ-T Vit.E δ-T S/M - 42.9 1.5 34.4 3 1 2 35-41 100 3 42-47 80 4 48-55 100 5 58-65 85 a. S/M : Starting Material (vit. E concentrate ~80%) b. T: tocopherol c. Conditions: P=180kg/cm 2 , T= 70°C d. SC-CO 2 : ethanol = 5ml:0.25ml (95.2% : 4.8%) Isolation of Vit.E isomers (α-T, γ-T, α-T3 and γ-T3) from Ricebran Oil Fractions Collection Time (min) Vit.E α-T Vit.E γ-T Vit.E α-T3 Vit.E γ-T3 S/M - 28.2 5 20.6 38.5 1 2 35-41 100 3 42-47 85 4 48-55 100 5 58-65 100 a. S/M : Starting Material (vit. E concentrate ~90%) b. T: tocopherol c. T3: tocotrienol d. Conditions: P=180kg/cm 2 , T= 70°C e. SC-CO 2 : ethanol = 5ml:0.25ml (95.2% : 4.8%) Isolation of Vit.E isomers (α-T, β-T, γ-T and δ-T) from Sunflowerseed Oil Fractions Collection Time (min) Vit.E α-T Vit.E β-T Vit.E γ-T Vit.E δ-T S/M - 80.8 3.3 1.1 1 2 35-41 100 3 42-47 85 4 48-55 85 5 58-65 a. S/M : Starting Material (vit. E concentrate ~80%) b. T: tocopherol c. Conditions: P=180kg/cm 2 , T= 70°C d. SC-CO 2 : ethanol = 5ml:0.25ml (95.2% : 4.8%)

Claims: Claims for the following Contracting State : FR A method of chromatographic isolation of Vitamin E isomers from compounds comprising Vitamin E isomers, said method comprising the steps of: (i) esterifying free fatty acid components of said compounds into esters with one or more monohydric alcohols; (ii) transesterifying glyceride components of said compounds into esters with one or more monohydric alcohols; (iii) introducing the product mixture from steps (i) and (ii) onto selective adsorbents to effect adsorption of Vitamin E isomers; and subsequently (iv) effecting an individual desorption of the adsorbed Vitamin E isomers, wherein the adsorption and/or desorption of the Vitamin E isomers are carried out under a supercritical fluid environment. A method of chromatographic isolation of claim 1, wherein the selective adsorbents include silica gel and/or reverse phase C18 silica gel. A method of chromatographic isolation of claim 1, wherein the adsorption/desorption of the Vit E isomers are carried out under a supercritical fluid environment comprising an effective amount of CO 2 as a solvent. A method of chromatographic isolation of claim 1, wherein the adsorption/desorption of the Vit E isomers are carried out under a supercritical fluid environment comprising an effective amount of a combination of CO 2 and an entrainer. A method of chromatographic isolation of claim 4, wherein the entrainer comprises an alcohol. A method of chromatographic isolation of any one of claims 3-5 wherein the supercritical fluid conditions comprise operating temperatures of T=30-100°C and pressures of P=50-600 kg/cm 2 . A method of chromatographic isolation of Vitamin E isomers according to claim 1, wherein the esters formed in steps (i) and (ii) are removed to form a Vitamin E concentrate, wherein the substances which form the source of the Vitamin E isomers introduced into the selective adsorbents comprise said Vitamin E concentrate. A method of chromatographic isolation of claim 1, wherein: step (i) is carried out via employing a solid alkali metal bisulfate, a sulfate acid and/or a strongly acidic-ion exchange resin as a catalyst; step (ii) is carried out via employing a basic catalyst; or wherein the esterification and/or trans-esterification are carried out via employing an enzyme. A method of chromatographic isolation of claim 1, wherein the esterification and/or trans-esterification comprises the use of one or more C 1 -C 8 alcohols. A method of chromatographic isolation of claim 9 wherein the esterification and/or trans-esterification comprises the use of methanol. A method of chromatographic isolation of claim 5 or 6 wherein the entrainer comprises an alkyl alcohol. A method of chromatographic isolation of Vitamin E isomers according to claim 1 wherein the compounds comprising Vitamin E include crude vegetable oil, vegetable oil products and/or vegetable oil by-products. A method of chromatographic isolation of Vitamin E isomers according to claim 1, said method incorporating additional steps prior to adsorption and desorption stages under the supercritical fluid environment, said additional steps comprising the steps of: (a) removing the esters from the product mixture of steps (i) and (ii) , (b) saponifying the fraction of the product mixture of steps (i) and (ii), which fraction contains the non-esterified components; (c) removing the saponified products, and thereby yielding a fraction containing non-saponifiable products; wherein the substances, which form the source of Vitamin E isomers, introduced onto the selective adsorbents, comprise the fraction containing the non-saponifiable products from step (c) above. A method of chromatographic isolation of Vitamin E isomers according to claim 1, said method incorporating the additional step(s) of concentrating the compounds comprising Vitamin E through any or various processes including chromatography involving the use of the supercritical fluid environment. A method of chromatographic isolation of Vitamin E isomers according to claim 1, said method incorporating the additional step(s) of extracting a Vitamin E concentrate from the compounds comprising Vitamin E through any or various processes including chromatographic isolation involving the use of supercritical fluid environment. A method of chromatographic isolation of Vitamin E isomers according to claim 1, which is employed to separate Vitamin E isomers from soya bean oil, corn oil, canola oil, olive oil, cottonseed oil and/or sunflowerseed oil. A method of chromatographic isolation of Vitamin E isomers according to claim 16, wherein said Vitamin E isomers are separated into α-T, β-T, γ-T and δ-T isomers. A method of chromatographic isolation of Vitamin E isomers according to claim 1, which is employed to separate Vitamin E isomers from groundnut oil. A method of chromatographic isolation of Vitamin E isomers according to claim 18, wherein the Vitamin E isomers are separated into α-T, β-T, γ-T and δ-T isomers. A method of chromatographic isolation of Vitamin E isomers according to claim 1, which is employed to separate Vitamin E isomers from ricebran oil. A method of chromatographic isolation of Vitamin E isomers according to claim 20, wherein the Vitamin E isomers are separated into α-T, γ-T, α-T 3 and γ-T 3 isomers. A method of chromatographic isolation of Vitamin E isomers according to claim 1, which is employed to separate Vitamin E isomers from crude palm oil, palm fatty acid distillate, and/or palm pressed fibre oil. A method of chromatographic isolation of Vitamin E isomers according to claim 22, wherein the Vitamin E isomers are separated into α-T, γ-T 3 , δ-T 3 and α-T 3 isomers. A method of chromatographic isolation of Vitamin E isomers according to claim 5, wherein the alcohol is ethanol. A method of chromatographic isolation of Vitamin E isomers according to claim 1 wherein the supercritical solvent used is propane, propene or ethylene. A method of chromatographic isolation as claimed in claim 1, wherein the esterified and transesterified products of steps (i) and (ii) are removed by vacuum distillation, prior to steps (iii) and (iv). Claims for the following Contracting States : DE, CH, GB A method of chromatographic isolation of Vitamin E isomers from compounds comprising Vitamin E isomers, said method comprising the steps of: (i) esterifying free fatty acid components of said compounds into esters with one or more monohydric alcohols; (ii) transesterifying glyceride components of said compounds into esters with one or more monohydric alcohols; (iii) introducing the product mixture from steps (i) and (ii) onto selective adsorbents to effect adsorption of Vitamin E isomers; and subsequently (iv) effecting an individual desorption of the adsorbed Vitamin E isomers, wherein the adsorption and/or desorption of the Vitamin E isomers are carried out under a supercritical fluid environment, wherein compounds comprising Vitamin E isomers are not crude palm oils and/or palm oil products. A method of chromatographic isolation of claim 1, wherein the selective adsorbents include silica gel and/or reverse phase C18 silica gel. A method of chromatographic isolation of claim 1, wherein the adsorption/desorption of the Vit E isomers are carried out under a supercritical fluid environment comprising an effective amount of CO 2 as a solvent. A method of chromatographic isolation of claim 1, wherein the adsorption/desorption of the Vit E isomers are carried out under a supercritical fluid environment comprising an effective amount of a combination of CO 2 and an entrainer. A method of chromatographic isolation of claim 4, wherein the entrainer comprises an alcohol. A method of chromatographic isolation of any one of claims 3-5 wherein the supercritical fluid conditions comprise operating temperatures of T=30-100°C and pressures of P=50-600 kg/cm 2 . A method of chromatographic isolation of Vitamin E isomers according to claim 1, wherein the esters formed in steps (i) and (ii) are removed to form a Vitamin E concentrate, wherein the substances which form the source of the Vitamin E isomers introduced into the selective adsorbents comprise said Vitamin E concentrate. A method of chromatographic isolation of claim 1, wherein: step (i) is carried out via employing a solid alkali metal bisulfate, a sulfate acid and/or a strongly acidic-ion exchange resin as a catalyst; step (ii) is carried out via employing a basic catalyst; or wherein the esterification and/or trans-esterification are carried out via employing an enzyme. A method of chromatographic isolation of claim 1, wherein the esterification and/or trans-esterification comprises the use of one or more C 1 -C 8 alcohols. A method of chromatographic isolation of claim 9 wherein the esterification and/or trans-esterification comprises the use of methanol. A method of chromatographic isolation of claim 5 or 6 wherein the entrainer comprises an alkyl alcohol. A method of chromatographic isolation of Vitamin E isomers according to claim 1 wherein the compounds comprising Vitamin E include crude vegetable oil, vegetable oil products and/or vegetable oil by-products. A method of chromatographic isolation of Vitamin E isomers according to claim 1, said method incorporating additional steps prior to adsorption and desorption stages under the supercritical fluid environment, said additional steps comprising the steps of: (a) removing the esters from the product mixture of steps (i) and (ii), (b) saponifying the fraction of the product mixture of steps (i) and (ii), which fraction contains the non-esterified components; (c) removing the saponified products, and thereby yielding a fraction containing nonsaponifiable products; wherein the substances, which form the source of Vitamin E isomers, introduced onto the selective adsorbents, comprise the fraction containing the non-saponifiable products from step (c) above. A method of chromatographic isolation of Vitamin E isomers according to claim 1, said method incorporating the additional step(s) of concentrating the compounds comprising Vitamin E through any or various processes including chromatography involving the use of the supercritical fluid environment. A method of chromatographic isolation of Vitamin E isomers according to claim 1, said method incorporating the additional step(s) of extracting a Vitamin E concentrate from the compounds comprising Vitamin E through any or various processes including chromatographic isolation involving the use of supercritical fluid environment. A method of chromatographic isolation of Vitamin E isomers according to claim 1, which is employed to separate Vitamin E isomers from soya bean oil, corn oil, canola oil, olive oil, cottonseed oil and/or sunflowerseed oil. A method of chromatographic isolation of Vitamin E isomers according to claim 16, wherein said Vitamin E isomers are separated into α-T, β-T, γ-T and δ-T isomers. A method of chromatographic isolation of Vitamin E isomers according to claim 1, which is employed to separate Vitamin E isomers from groundnut oil. A method of chromatographic isolation of Vitamin E isomers according to claim 18, wherein the Vitamin E isomers are separated into α-T, β-T, γ-T and δ-T isomers. A method of chromatographic isolation of Vitamin E isomers according to claim 1, which is employed to separate Vitamin E isomers from ricebran oil. A method of chromatographic isolation of Vitamin E isomers according to claim 20, wherein the Vitamin E isomers are separated into γ-T, α-T, α-T 3 and γ-T 3 isomers. A method of chromatographic isolation of Vitamin E isomers according to claim 1, wherein the supercritical solvent used in propane, propene or ethylene. A method of chromatographic isolation of Vitamin E isomers according to claim 5, wherein the alcohol is ethanol. A method of chromatographic isolation as claimed in claim 1, wherein the esterified and transesterified products of steps (i) and (ii) are removed by vacuum distillation, prior to steps (iii) and (iv).

Inventors: BASIRON YUSOF, MAY CHOO YUEN, NGAN MA AH, BASIRON, YUSOF, MAY, CHOO YUEN, NGAN, MA AH
Applicants: MALAYSIAN PALM OIL BOARD
Application year: 2001
Main classification: C07D311
IPC codes: G01N30, C07D311, B01D15