Title of Invention

A PROCESS FOR HPLC SEPARATION OF ALKYLCYCLOHEXYLOXYLKANOL ISOMER MIXTURES

Abstract

A process for high performance liquid chromatography (HPLC) separation of alkylcyclohexyloxy-alkanol isomer mixtures comprising the steps of: a) providing a chromatography system comprising a chromatographic column packed with a chromatographic mateiral; b) charging the solution of alkylcyclohexyloxy alkanol isomer mixture on the said column; c) separation of the isomers by using a mobile phase which is a mixture of at least two solvents, such that the polarity of the solvent mixture is between 0.1 to 6.0; d) collection of the individual isomers/isomer pairs ; whereby isomers having an useful amber odour are separated from the other isomers.

Full Text

FORM -2 THE PATENTS ACT, 1970 (39 Of 1970) COMPLETE SPECIFICATION (See Section 10) A PROCESS FOR HPLE SEPARATION OF ALKYL CYCLOHEXYLOXYALKANOL ISOMER MIXTURE HINDUSTAN LEVER LIMITED, a company incorporated under the Indian Companies Act, 1913 and having its registered office, at Hindustan Lever House, 165/166, Backbay Reclamation, Mumbai -400 020, Maharashtra, India The following specification particularly describes the nature of the invention and the manner in which it is to be performed. GRANTED 24-8-2004 TECHNICAL FIELD The present invention relates to the separation of the isomers of an alkylcyclohexyloxy alkanol, having intense amber odour, from the other isomers, by High Performance Liquid Chromatography. BACKGROUND TO THE INVENTION Ambergris is a valuable perfume of animal origin and has been in use since antiquity. It is a metabolic product of the sperm whale, Physeter macrocephalus L. Its use is currently restricted due to excessive hunting of the whale as well as the high cost of the material. There has therefore been a search for cheaper, synthetic substitutes for ambergris. Many synthetic amber odorants have been reported and these find extensive use in perfumery compositions for soaps, cosmetics, shampoos and detergents, amongst others. Many amber odorants are synthesised from the easily available sclareol. Several labdane and nordrimane derivatives have also been reported. Certain o- and p-alkylcyclohexanols are reported to possess woody notes and have been widely used in perfume compositions (S. Arctander, 1969). The alkylcyclohexanols can be further reacted to give compounds which are reported to be amber odorants. US 5,194,423 discloses a-(alkylcyclohexyloxy)-β-alkanols as novel compounds having a woody and/or amber odour. An alkylcyclohexanol can be condensed with an epoxide to give the a- (alkylcyclohexyloxy) -β-alkanol. Alternatively, the alkylphenols are condensed with an epoxide, followed by hydrogenation to obtain similar compounds. A mixture of cis and trans isomers is obtained which can optionally be separated by a method like column chromatography. EP 616994 discloses the synthesis of a-(alkylcyclohexyloxy)-p-alkanols by ketal hydrogenolysis. The catalyst is a palladium based catalyst, wherein palladium is at least 50% of the active catalyst. The catalyst may further contain other metals, examples of which are ruthenium, rhodium, platinum and nickel. The prior art does not describe the composition of alkylcyclohexyloxy alkanols and does not reveal which isomer or isomers are respons.ible for the amber note. The separation of the isomers into individual components is also not reported. Therefore there is a need to separate the isomers in as pure a state as possible and to characterise the isomer or isomers responsible for the amber notes of alkylcyclohexyloxy alkanols. BRIEF DESCRIPTION OF THE INVENTION The present invention provides for a rapid and economic process for the separation of the useful isomers of alkylcyclohexyloxy alkanols, having an amber odour, by high performance liquid chromatography. The isomers responsible for the amber odour were identified The isomers responsible for the amber odour may be used in the pure form or as mixtures with other isomers separated by the chromatographic process. The isomers may also be used in other perfume compositions. DETAILED DESCRIPTION OF THE INVENTION Thus, according to a first aspect of the invention, there is provided a process for HPLC separation of alkylcyclohexyloxy-alkanol isomer mixtures comprising the steps of: (a) providing a chromatographic column packed with a chromatographic material (b) charging the solution of alkylcyclohexyloxy alkanol isomer mixture on the said column (c) separation of the isomers by using a mobile phase which is a mixture of at least two solvents, such that the polarity of the solvent mixture is between 0.1 to 6.0 (d) collection of the individual isomers/isomer pairs whereby isomers having an useful amber odour are separated from the other isomers. The general structure of the alkylcyclohexyloxy alkanol is as shown in Figure .1. According to the second aspect of the invention, isomers of alkylcyclohexyloxy alkanols, with a strong and useful amber odour, either in their pure form or as a mixture are provided for. According to a third aspect of the invention, perfume compositions comprising these isomers are provided for. SYNTHESIS OF ALKYLCYCLOHEXYLOXY ALKANOLS The alkylcyclohexyloxy alkanols can be synthesised by any of the methods known in the art. One method is the hydrogenolysis of the cyclic ketals of alkylcyclohexanones as disclosed in EP 616994. Another method is the condensation of an alkylcyclohexanol or an alkylphenol with an epoxide. Alternatively, an alkylphenol can be condensed with an epoxide and further hydrogenated to obtain the alkylcyclohexyloxy alkanol. Such methods are disclosed in US 5,194,423. Alternatively, the hydrogenolysis of cyclic ketals of alkylcyclohexanones may be carried out using nickel as a catalyst. Raney nickel is especially preferred. Raney nickel Type B is further preferred. The pressure is preferably from 100 to 1000 psi, more preferably from 150 to 400 psi and the temperature of hydrogenolysis is preferably from 100 to 250°C, more preferably from 150 to 220°C. Optionally, a solvent may be used in the reaction. Solvents are preferably non-polar or aprotic. Examples of solvents that can be used for hydrogenolysis are isopropyl alcohol, hexane and heptane. The alkylcyclohexyloxy alkanol is present as a mixture of primary and secondary alcohols. When nickel is used as a catalyst, the product mixture contains secondary alcohols. THE SEPARATION PROCESS The mixture of isomers is separated using High Performance Liquid Chromatograph (HPLC). The HPLC technique facilitates separation of a mixture of compounds in solution. The typical high performance chromatography system comprises a solvent delivery module, an injector port, a stainless steel column filled with a stationary phase, a detector and a recorder. The stationery phase can be chiral or non-chiral in nature. Examples of the stationary phase are as given below. The particle size is the diameter of the particle. a) A stationary phase consisting of porous silica particles of particle size from 2 micron to 10 micron. A particle size from 5 to 10 micron is especially preferred. b) A stationary phase wherein octadecyl silane (ODS or Ci8) is chemically bonded to porous silica or ceramic particles. The particle size of the stationary phase is preferably between 3 to 10 micron. c) A stationary phase wherein octyl silane (Cs) is chemically bonded to porous silica or ceramic particles. The particle size of the stationary phase is preferably from 3 to 10 micron. Stationary phases as described in a), b) and c are marketed under the tradenames HypersilR, InertsilR, KromasilR, LiChrosorbR, LiChrospherR, Nova-PakR, NucleosilR, Partisil™, Resolve™, Waters SpherisorbR, SymmetryR, Xterra™ MS, fiBondapak™, Delta-Pak™, PorasilR, YMC-Pack™ ProC18™ and YMC-Pack™Silica. The detector can be chosen from a refractive index detector, evaporative light scattering detector, conductivity detector. Especially preferred is the refractive index detector. The separation process is carried out using a solvent mixture, comprising at least two solvents, as a mobile phase. At least one solvent has to be polar and at least one solvent has to be non-polar. The polarity index of the solvent mixture is preferably between 0.1 to 6.0, more preferably between 1 to 3 and most preferably from 2 to 2.5. The volume ratio (v/v) of the non polar solvents to the polar solvents is preferably from 85:15 to 99.9:0.1, more preferably from 90:10 to 99.9:0.1 and most preferably from 95:5 to 99.9:0.1. Non-limiting examples of non-polar solvents are pentane, n-hexane, n-heptane, n-octane, n-decane or their mixtures thereof. Non-limiting examples of polar solvents include ethyl acetate, propyl acetate, butyl acetate, amyl acetate, ethanol, 1-propanol, isopropyl alcohol, dichloromethane and chloroform and their mixtures thereof. The solution of alkylcyclohexyloxy alkanols, prepared in a suitable solvent mixture is injected in the injector port. The mobile phase is allowed to pass through the injector port and delivers the sample to the column. The alkylcyclohexyloxy alkanols are physically adsorbed on the stationary phase. Depending on the polarity of the isomer (or the isomer pair when a non chiral column is used) , the retention time of each isomer on the stationary phase is different. Since the concentration of mobile phase is very high, the adsorbed isomers get desorbed, thus effecting the separation of constituents in the sample. The desorption time is dependent on the polarity of each isomer. Using a suitable detector, the concentration of each component in the mixture can be estimated. The various isomers or isomer mixtures are collected as their presence is indicated by the detector. IDENTIFICATION OF ISOMERS The purity of the fractions obtained by the above separation process is determined by using conventional chromatographic techniques. Gas chromatography is a particularly preferred.method for determining purity. The purity of the isomers obtained by the separation process is at least 90%. The structure of the isomers is determined by NMR. In particular, 1H and C-13 NMR are suitable techniques for determining the structure. The alkylcyclohexyloxy alkanol is present at. least as a mixture of 06-alkylcyclohexyloxy (3-alkanol and (3-alkylcyclohexyloxy-a-alkanol. The (3- alkylcyclohexyloxy-a-alkanol can be present as four enantiomeric pairs, i.e. eight isomers due to the presence of a minimum of three chiral centers. Similarly, the a-alkylcyclohexyloxy (3- alkanol can also be present as four enantiomeric pairs. Thus, the alkycyclohexyloxy alkanol can exist as sixteen isomers (or eight enationmer pairs). A representative structure of the sixteen isomers of tert-butyl cyclohexyloxy butanol (an example of an alkylcyclohexyloxy alkanol) is given in Figure 2. All sixteen isomers may be separated on a chiral column whereas the mixture may" be separated into the eight enantiomer pairs on a non-chiral column. ODOUR EVALUATION The odour evaluation of the isomers reveals that two isomers, i.e. one enantiomer pair (5 and 6) of figure 2 have distinct, strong amber notes. The enantiomeric pair has an amber odour twice as intense as the reaction product before separation. The other isomers did not have any amber notes or had weak amber notes. For a desirable amber odour,' the two pure isomers may be used. Other HPLC fractions may also be present without significantly changing the odour profile, such that the isomer pair responsible for the amber odour preferably represents at least 3 0% by weight of the total mixture, more preferably 50%. According to yet another aspect of the invention, the isomers responsible for the amber odour may also be included in other perfume compositions. The amount of the isomer pair or its mixtures with other fractions, in such perfume compositions may be from 0.01 to 30%. The said perfume compositions can be used in soaps, detergents, shampoos and skin care compositions amongst others. Having generally understood the invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for illustration only and are not intended to be limiting unless specified otherwise. Reference is made in the Examples to Figures 3 and 4 of the accompanying drawings. Examples Synthesis of tert-butylcyclohexyloxy-butanol (TBCB) 300 g of 2-tert butyl phenol and 5% of a palladium-carbon catalyst was charged into an autoclave. The autoclave was then heated to 100°C and pressurised with hydrogen to 120 psi. The hydrogenation was continued till the amount of tert butyl phenol was less than 0.2% by G.C. The reaction mixture was cooled, the catalyst was removed and the crude product distilled to obtain the 2-tert butyl cyclohexanone. 374 g of tert butyl cyclohexanone, 300 g of 1,2-butanediol, p-toluene sulphonic acid and toluene were taken in a reactor equipped with a Dean Stark apparatus and a reflux condensor. The mixture was heated under reflux and the water continually removed. At the end of 2 0 hours, 7 5% ketal had been formed. Toluene was removed by distillation. The reaction mixture was cooled, washed with water, dried and concentrated to remove toluene. The crude ketal was purified by fractional distillation. The ketal was further hydrogenolysed using a carbon supported ruthenium palladium catalyst. The reaction was continued till the ketal content was less than 0.2%. The tert-butyl cyclohexyloxy-butanol was fractionally distilled to a purity of 99.9% and used for separation. Example 1 Separation of isomers tert-butylcyclohexyloxy-butanol was dissolved in a solvent mixture of 95 parts n-hexane and 5 parts ethyl acetate. The concentration of the solution was 1 mg/ml. A semi-preparative column, E Merck LiChrospher Si 60, was used for the separation. n-hexane and ethyl acetate were taken in a ratio of 95:5 and used as the mobile phase. The flow rate was maintained at 3 ml/min. The tert-butylcyclohexyloxy-butanol solution (2 ml) was injected in the column and the chromatogram recorded. A refractive index detector was used for detection of the isomers. The chromatogram was as shown in Figure 3. Each isomer fraction was manually collected during the elution from the column. Based on the degree of separation of isomers, different regions were selected from the baseline in each peak to collect the fractions in a pure form. Each fraction was collected as given below. Fraction I was collected between 10% of up-slope and 50% of down-slope of peak 1. Fraction II was collected between 80% of up-slope and 10% of down- slope of peak 2. Fraction III was collected between 10% of up-slope and 10% of down-slope of peak 3. Fraction IV was collected between 10% of up-slope and 70% of down-slope of peak 4. Fraction V was collected between 90% and 10% of down-slope of peak 5. Fraction VI, VII and VIII were collected between 10% of up-slope and 10% of down-slope of peak 6,7 and 8. The fractions were concentrated at room temperature by passing dry nitrogen at an approximate flow rate of 3 ml/min. Analysis of fractions The purity of each fraction was checked by Gas Chromatography. A Shimadzu G.C. 14B instrument was used. A DB-FFAP column of 30 m length was used. The temperature range was 80 to 220°C, the hold time was 1 minute and the heating rate was 5 °C/min. GC retention time and concentration of each isomer is shown in Table 1. A qualitative odour evaluation of each fraction was carried out by three trained perfumers and is also indicated in Table 1. The purity of each isomer was more than 98.0%. Table 1 HPLC Fraction No. GC Retention time (min) Fraction content in TBCB {%) Odour Profile 1 13.75 26.2 Bleached, oxidised 2 14.24 30.4 Bleached, oxidised 3 15.94 1.5 No odour 4 15.76 17.9 Strong Amber 5 15.28 16.8 Weak Amber 6 17.46 4.9 Bleached, oxidised 7 16.96 0.6 No odour 8 18.11 1.7 Weak Amber Fraction 4 (comprising enantiomers 5 and 6) was identified as possessing a strong amber odour. Fractions 5 and 8 also exhibited weak amber notes. Example 2 Structure Determination of fractions 1H (200 MHz) and 13C NMR (50 MHz) spectra were recorded for each of the enantiomer pairs obtained by HPLC separation using a Brucker Instrument. Deuterated chloroform was used as the solvent. Table 2 and 3 show the 13C and 1H NMR data for the isomers. Structures were assigned to each of the enantiomer pairs obtained from the HPLC based on these measurements. The numbering system for C and H atoms used for assignment in Table 2 and 3 is indicated below: Table 2: 13C-NMR data of HPLC fractions 1- 8 Carbon 1 2 3 4 5 6 7 8 1' 2' 3' 4' Fraction 1 74.0 50 26 21 19.2 28 32.7 28 70 75.5 21 10 2 74.5 51.5 27.2 21.7 19.7 29 32.8 28.5 71 77 22.4 10 3 71.0 51 26 22 21 28.5 32.7 28 64 75.5 24 9 4 81 50.5 25.8 25 23.5 30.5 32.9 28 70 70.5 24.8 9 5 81 51.5 27 26.2 24.1 31.5 32.9 29.5 71.5 72 26.1 10 6 73 52.5 27.6 22.5 20.5 29.5 32.7 28.8 63.2 77 25.5 10 7 76.8 50.2 26.4 25.0 24.8 22.5 32.2 28.2 68.2 75.5 24.3 10 8 78 51.5 26 25.5 23.5 31.7 29.5 77 25 9.9 Table 3: XH- NMR data of HPLC Fractions 1-8 Fr. No C,-H C2-H C3-H C4-H C5-H C6-H C8-H C1'-H C2'-H C3'-H C4'-H a e a a e a e a e a e a b 1 - 3.76 1.02 1.2 1.75 1.50 1.40 1.12 2.0 0.9 3.23 3.35 3.68 1.48 0.95 2 3.74 1.04 1.22 1.78 1.54 1.41 1.12 2.06 0.94 3.08 3.53 3.64 1.51 0.96 3 - 4.05 1.05 1.25 1.78 1.46 1.35 1.15 2.05 0.94 3.5 3.67 3.45 1.6 0.90 4 3.12 - 1.24 0.92 1.8 1.14 1.64 1.14 1.70 1.15 2.1 0.9 3.15 3.6 3.65 1.48 0.98 5 3.13 - 1.25 0.95 1.82 1.16 1.67 1.15 1.71 1.15 2.15 0.96 3.32 3.43 3.70 1.5 0.98 6 - 4.0 1.05 1.25 1.85 1.3 1.3 1.4 1.4 2.02 0.95 3.58 3.75 3.38 1.6 0.96 7 3.38 - 1.29 1.22 1.73 1.77 1.80 1.24 1.68 1.22 2.01 0.96 3.72 3.74 3.48 1.62 0.96 8 3.4 - 1.3 1.21 1.72 1.8 1.83 1.21 1.72 1.25 2.03 1.0 3.74 3.76 3.43 1.50 0.93 a - axial e-equatorial Figure 4 shows the correlation between the structure and the HPLC fractions. The structures of the isomer pair responsible for the strong amber note was identified to be that of fraction 4 in Figure 4. Example 2 Odor evaluation methodology The isolated enantiomer pair (fraction 4 in Figure 3) was compared with a sample of TBCB by an expert perfumer. The two samples were taken on a strip and then evaluated. The amber odour of the enantiomer pair was found to be twice as intense as that of TBCB. Thus the present invention provides for a process of separation of the useful isomers of alkylcyclohexyloxy alkanols, such isomers having an intense isomer odour. WE CLAIM : 1. A process for high performance liquid chromatography (HPLC) separation of alkylcyclohexyloxy-alkanol isomer mixtures comprising the steps of: a) providing a chromatography system comprising a chromatographic column packed with a chromatographic mateiral; b) charging the solution of alkylcyclohexyloxy alkanol isomer mixture on the said column; c) separation of the isomers by using a mobile phase which is a mixture of at least two solvents, such that the polarity of the solvent mixture is between 0.1 to 6.0; d) collection of the individual isomers/isomer pairs ; whereby isomers having an useful amber odour are separated from the other isomers. 2. A process as claimed in claim 1 wherein the chromatography system used comprises a solvent delivery module, an injector port, a stainless steel column filled with a stationery phase, a detector and a recorder. 3. A process as claimed in claim 2 wherein the stationery phase is chiral or non-chiral in nature. 4. A process as claimed in anyone of claims 2 or 3 wherein said stationery phase used is selected from a) a stationary phase consisting of porous silica particles of particle size from 2 micron to 10 micron preferably a particle size from 5 to 10 micron ; b) a stationary phase wherein octadecyl silane (ODS or C18) is chemically bonded to porous silica or ceramic particles preferably a particle size is between 3 to 10 micron; c) a stationary phase wherein octyl silane (C8) is chemically bonded to porous silica or ceramic particles preferably a particle size of 3 to 10 micron. 5. A process as claimed in anyone of claims 1 to 4 wherein the detector used is selected from a refractive index detector, evaporative light scattering detector, conductivity detector preferably the refractive index detector. 6. A process as claimed in anyone of claims 1 to 5 wherein the separation process is carried out using a solvent mixture, comprising at least two solvents, as a mobile phase. 7. A process as claimed in claim 6 wherein said at least one polar and one non-polar solvent is used. 8. A process as claimed in claim 7 wherein the polarity index of the.solvent mixture is preferably between 0.1 to 6.0, more preferably between 1 to 3 and most preferably from 2 to 2.5. A process as claimed in anyone of claims 7 or 8 wherein the volume ratio (v/v) of the non polar solvents to the polar solvents is preferably from 85:15 to 99.9:0.1, more preferably from 90:10 to 99.9:0.1 and most preferably from 95:5 to 99.9:0.1. 10. A process as claimed in anyone of claims 1 to 9 wherein said non-polar solvents comprise pentane, n-hexane, n-heptane, n-octane, n-decane or their mixtures thereof, preferably polar solvents are selected from ethyl acetate, propyl acetate, butyl acetate, amyl acetate, ethanol, l-propanol, isopropyl alcohol, dichloromethane and chloroform and their mixtures thereof. 11. A process as claimed in anyone of claims 1 to 10 comprising injecting the solution of alkylcyclohexyloxy alkanols prepared in a suitable solvent mixture in the injector port; processing the mobile phase through the injector port and delivering the sample to the column and physically absorbing alkylcyclohexyloxy alkanols on the stationary phase. 12. A process as claimed in anyone of claims 1 to 11 wherein depending on the polarity of the isomer/isomer pair, the retention time of each isomer on the stationary phase is selected. 13. A process as claimed in anyone of claims 1 to 12 wherein the concentration of mobile phase is maintained very high, such that the adsorbed isomers get desorbed, thus effecting the separation of constituents in the sample. 14.A process as claimed in claim 13 wherein the desorption time is dependent on the polarity of each isomer. 15. A process as claimed in anyone of claims 1 to 14 comprising using a suitable detector, to estimate the concentration of each component in the mixture, the various isomers or isomer mixtures being collected as their presence is indicated by the said detector. 16.A process as claimed in anyone of claims 1 to 15 wherein all sixteen isomers of alkylcyclohexyloxy alkanol are separated on a chiral column whereas the mixtures are separated into the eight enantiomer pairs on a non-chiral column. 17. A process as claimed in anyone of claims 1 to 16 comprising subjecting « the isomers thus subjected to odour evaluation. 18. A process for separation of isomers of of alkylcyclohexyloxy alkanol, the isomers thus separated substantially as hereindescribed and illustrated with reference to accompanying examples. Dated this 8th day of November 2001 S. Majumdar Of S. Majumdar & Co. Applicant's Agent

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