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Effect of the carbon dioxide modifier on the lipid composition of wool wax extracted from raw wool
Analytica Chimica Acta 477 (2003) 233–242
Effect of the carbon dioxide modifier on the lipid composition of wool wax extracted from raw wool C. Dom´ınguez a , E. Jover b , J.M. Bayona b , P. Erra a,∗ a b
Department of Surfactant Technology, IIQAB-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain Department of Environmental Chemistry, IIQAB-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain Received 2 August 2002; accepted 29 October 2002
Abstract Wool wax or lanolin is a unique substance secreted by sheep and forms a natural protective coating on wool fibres. It is widely used in pharmaceutical and cosmetic formulations. However, different systems of wool wax recovery from scouring liquour provide a dark impurified greasy product. This product has a lipid composition that differs from the wool wax present on wool fibres. The wool wax extraction method from raw wool with pressurised CO2 and different modifiers at constant pressure and temperature was studied. Thin-layer chromatography coupled to an automated flame ionisation detection system (TLC/FID) was used to analyse the different lipid classes present in the collected extracts. Moreover, a detailed structural comparison of the cholesteryl esters and hydroxycholesteryl esters was carried out by means of sub-ambient pressure chromatography mass spectrometry in the electron impact and in the ammonia positive chemical ionisation modes. For comparison, qualitative and quantitative analyses of the lanolin extracted in Soxhlet with dichloromethane and commercial cosmetic lanolin were carried out. Differences in the quantity of wool wax extraction and in the lipid composition of different wool wax extracts were detected by changing the modifier polarity. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Wool wax extraction; Lanolin; Raw wool; CO2 /modifier; TLC/FID; GS–MS; Subcritical CO2
1. Introduction Raw wool contains three main scouring contaminants: solvent soluble substances (referred to as wool grease, wool wax or lanolin), water soluble substances (suint) and insoluble material (mineral dirt) [1,2]. An additional contaminant is the proteinaceous contaminant layer (PCL) [3,4]. This is formed by water-insoluble and water-soluble components with amino acid compositions similar to skin flakes and suint peptides. All these contaminants must be removed before the wool can be mechanically processed. ∗ Corresponding author. Fax: +34-3-2045904. E-mail address: [email protected] (P. Erra).
Wool wet scouring processes are basically extraction systems for removing fibre contaminants. Industrially, scouring processes are carried out in a series of aqueous solutions with surfactant and builder, the wool wax being recovered from the scouring liquor by centrifuging [1]. Although they remove all the wool contaminants providing suitable wool fibres for subsequent processing, large volumes of wastewater with high BOD values are produced. Moreover, both the quantity and the quality of the recovered wool wax are very low. Lanolin is essentially a mixture of esters, diesters, and hydroxy esters of high molecular weight as well as free fatty alcohols and free fatty acids [5–9]. The wax or grease on the wool fibre consists of two fractions—
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an oxidised fraction which is found mainly at the tip and an unoxidised fraction located at the base of the fibre. During scouring the unoxidised fraction is more readily removed and recovered by centrifugation, whereas the oxidised fraction forms complexes with other contaminants in the scouring liquor and is unrecoverable by centrifuging under the conditions existing in a commercial centrifuge [1]. The development of supercritical fluid chromatography has demonstrated that wool wax is soluble in supercritical carbon dioxide [10]. The recovery of wool grease by extraction of raw wool [11–13] and the solubility of wool wax [14,15] with liquid and supercritical carbon dioxide over a limited range of pressures and temperatures have been investigated. Recently, a procedure based on supercritical fluid extraction (SFE) with carbon dioxide (CO2 ) as a solvent has been developed for the determination of wool wax present on raw wool fibres in an attempt to avoid or minimise the use of chlorinated organic solvents [16,17]. Selected extraction variables such as pressure, temperature and percentage of modifier affecting SFE have been optimised [17]. Changes in lipid composition of the extract occur as a function of the different experimental conditions such as pressure, temperature, extraction time, and amount of modifier. In this work, a study of wool wax extraction from raw wool fibre with pressurised carbon dioxide containing different modifiers (acetone, ethanol and methanol) at constant pressure and temperature was carried out. The lipidic composition of the different collected extracts was qualitatively and quantitatively analysed by means of thin-layer chromatography coupled to an automated flame ionisation detection system (TLC/FID). For comparison, the lipidic composition of the wool wax extracted with dichloromethane in a Soxhlet apparatus was included. Dichloromethane removes large amounts of surface residuals and small quantities of internal lipids [16]. The TLC/FID technique is a considerable improvement on the TLC method. In comparison, TLC/FID is a simple technique for separating and identifying compounds present in a mixture, allowing the quantification of the separated compounds [18]. This technique has been employed to identify and to quantitatively determine the composition of wool lipid extracts [19–24].
Gas chromatography coupled to mass spectrometry was used to perform the characterisation of the extract at molecular level. Prior to this characterisation, samples were prefractionated by gel permeation chromatography (GPC). Size exclusion enables to separate high molecular weight compounds from smaller molecules, which allows us to carry out gas chromatographic analysis.
2. Experimental 2.1. Materials and reagents Australian Merino sheared raw wool (φ = 21 m) from local sources. Prior to extraction, the raw wool was homogenised by hand and equilibrated in a conditioned room (24 ◦ C, 60% relative humidity). Commercial lanolin was supplied by Croda (Snaith Goole, UK). Dichloromethane (DCM) (reagent grade) was obtained from Scharlau (Barcelona, Spain), formic acid (85%) from Probus (Badalona, Spain) and chloroform, methanol, n-hexane, diethyl ether and benzene (all for analysis) were supplied by Merck (Darmstadt, Germany). Analytical-grade methanol (M), ethanol (E) and acetone (A) from Merck were used as modifiers. Ethyl acetate and cyclohexane (LiChrosolv grade) and bis-silyl-trifluoroactamide (BSTFA) were obtained from Merck. Palmitic acid behenyl ester (≈99%) (mono-ES), behenyl alcohol (98%) (AL), behenic acid (99%) (FFA) and cholesterol (>99%) (S), supplied by Sigma (St. Louis, MO, USA), and dipalmitic acid hexadecyl ester (di-ES) synthesised in our laboratory were used as standard lipid compounds for TLC/FID lipid analysis. 2.2. Extraction procedures 2.2.1. DCM Soxhlet extraction Raw wool sample (≈3.5 g) was Soxhlet-extracted with DCM (220 ml) for 4 h according to the Woolmark Company, TM 136 (2000). A minimum siphoning rate of 4–5 cycles/h was maintained. The recovered extracts were immediately filtered (GF/F 0.7 m) and concentrated to dryness by rotary evaporation. The extracts were maintained under vacuum overnight in a desiccator over P2 O5 to eliminate moisture traces, and
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finally weighed. The test yielded the total amount of dichloromethane extractable matter. 2.2.2. CO2 /modifier extraction Supercritical fluid extraction (SFE) was performed using a dual-syringe pump (SFC 3000; Carlo Erba, Milan, Italy) for the delivery of carbon dioxide (CO2 , SFE 99.998%, Praxair España, Barcelona, Spain) and modifier as described elsewhere [25]. Extraction cell (6.94 ml, Keystone Scientific) was filled with raw wool sheep sample (3.5 g). The flow-rate as liquid was adjusted by a micrometer forged metering valve (Hoke, Cresskill, NJ, USA) at ca. 1.5–2.0 ml/min measured from LCD of the pump, maintaining the temperature at 100 ◦ C. A total fluid volume of 30 ml was passed through the cell. The extract was collected in an empty screw cap vial (15 ml) with a septum (Supelco, Bellafonte PA, USA) tared previously, with an outlet in order to vent the decompressed CO2 . Three different modifiers (methanol, ethanol and acetone) were tested in order to evaluate their extraction efficiency. Constant pressure and temperature and 20% (v/v) of modifier were selected. The extracts were concentrated to dryness under a gentle stream of nitrogen or maintained at 60 ◦ C in order to eliminate the solvent excess. Collected wool wax extracts were quantified gravimetrically during the extraction process. In an attempt to calculate the fraction percentage values on weight of scoured and dried wool, the wool samples extracted were opened by hand and scoured several times with deionized water in order to remove residual vegetable matter and the suint (soluble salts) respectively, and finally dried and weighed. 2.3. Analytical techniques 2.3.1. Extract analysis by TLC/FID Qualitative and quantitative analyses of the lipid classes present in the collected extracts were performed by TLC coupled to an automated FID system (Iatroscan MK-5; Iatron Laboratories, Tokyo, Japan). The standard compounds or the dry extracts (25–60 mg) were redissolved in chloroform/methanol (2:1, v/v) (5–12 mg/ml). The samples (0.8 l) were spotted onto silica gel-coated Chromarods (type S-III) (Iatron Laboratories) by means of a 2 l precision syringe Hamilton (Bondaluz, Switzerland) coupled to an SES 3202/IS-02 semiautomatic sample
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spotter (Nieder-Olm, Germany). The rods (in sets of 10 mounted semipermanently on stainless steel-racks) were developed consecutively four times using the following mobile phases: (i) 70 ml of chloroform/methanol/water (57/12/0.6, v/v/v) up to 1 cm (twice); (ii) 70 ml of hexane/diethyl ether/formic acid (58/12/0.3, v/v/v) up to 9 cm; (iii) 70 ml of hexane/benzene (35/35, v/v) up to 10 cm. After each development the rods were heated for 5–10 min at 60 ◦ C to dry the remaining solvent, and run through a flame ionisation detector in the Iatroscan by using an air flow of 2.000 ml/min atmospheric air, a hydrogen flow of 160 ml/min (high purity hydrogen, C50) and a scanning speed of 3.0 s/cm. A total scan was performed to identify all the lipid components. Data were processed with the Boreal software version 2.5. An aliquot of the different collected wool wax extracts were redissolved and analysed in duplicate. 2.3.2. Extract analysis by MS 2.3.2.1. Sample preparation prior to MS characterisation. The lipids of lanolin were prefractionated by GPC. About 100 mg of lanolin were weighed and dissolved in 5 ml of ethyl acetate/cyclohexane 1:1. Then the sample was filtered through a 0.45 m nylon membrane filter (Lida, Kenosha, WI, USA). Fractionation was carried out following procedure already described [26]. Next 10 l of the solution were placed in a 2 ml conic vial and 10 l of BSTFA were added. The closed vial was maintained at 70 ◦ C for 1 h and then evaporated to dryness under gentle nitrogen stream. Iso-octane (50 l) was added to the vial to reconstitute the sample and analysed before 48 h to avoid hydrolysis of the trimethylsilyl ether (TMS) group. The main polar constituents of lanolin—free fatty acids, hydroxy acids and diols—were derivatised prior to GC–MS to prevent them from interfering in ester analysis. 2.3.2.2. Instrumental analysis. A sub-ambient pressure CP Sil 8 CB/MS capillary column (5% diphenyl-dimethylpolysiloxane) of 10 m × 0.53 mm i.d. and 0.25 m of film thickness fitted to a deactivated restrictor of 50 cm length and 0.1 mm i.d. at the injection port was obtained from Chrompack (Middelburg, The Netherlands). One microlitre of sample was injected in the splitless mode at 320 ◦ C, activating the injector purge at 90 s from injection. Initial
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column temperature was maintained at 90 ◦ C for 1 min, and then programmed at 10 ◦ C/min to 320 ◦ C, maintaining the final temperature for 20 min (44 min each run). Chromatographic analysis was performed in the constant flow mode at 1.2 ml/min. 2.3.2.3. Mass sprectrometry. For the chemical ionisation mode, a GC 6890A from Agilent Technologies (Palo Alto, CA, USA) coupled to an MS 5973N was used. Quadrupole and transfer line temperatures were maintained at 150 and 280 ◦ C, respectively. Ammonia was used as reagent gas at 12.8 × 10−5 Torr in the positive mode and the ion source was maintained at 200 ◦ C. A GC from Fisons (Manchester, UK), GC 8060 coupled to an MS detector MD 800 in the electron impact mode was used. Transfer line and ion source were maintained at 280 and 230 ◦ C, respectively. Other chromatographic conditions were identical to those reported in the CI section.
modifier, and the yield of wool wax extracted with DCM in a Soxhlet apparatus. The yields were calculated on the weight of scoured and dried wool. The total yield values obtained are in accordance with the modifier polarity. The lowest dipole moment of the acetone exerts a considerable influence on the total yield of the wool wax extracted. Whereas the yields obtained with liquid CO2 /ethanol or CO2 /methanol are similar to the DCM ones, the yield obtained with liquid CO2 /acetone is 50% lower. This could be attributed to a preferential extraction of a given fraction of wool wax with CO2 /acetone or to an additional extraction of other types of compounds (oxidised lanoline fraction, salts, ceramides, protein material) using the other extractant agents tested. The C, H, N elemental analysis and the ash amount (800 ◦ C, 3 h) support these yields. The C, H, N elemental analysis shows a smaller amount of nitrogenated compounds for the CO2 /acetone extract compared with the other extracts. Moreover, the ash analysis value is with CO2 /acetone two orders of magnitude below that of the other extracts (Table 2).
3. Results and discussion 3.2. Qualitative analysis 3.1. Wool wax yield Wool wax is a complex mixture of non-polar and polar lipid compounds. For this reason, by increasing the extractant agent polarity (acetone, ethanol, methanol)—by modifying the Hiderbrand solubility parameter (δ)—an important effect on the solubility of the different lipid compounds present on the wool surface could be expected. Thus, a more selective extraction of the lipid class could be achieved. Table 1 shows the wool wax yield obtained from raw wool with a fixed volume of pressurised CO2 containing 20% of either acetone, ethanol or methanol as Table 1 Yield of wool wax extracted from raw Merino wool with liquid CO2 containing 20% of modifier (methanol, ethanol, acetone) or Soxhlet with DCM Modifier
Dipole moment (D)a
Yield (%)
Methanol Ethanol Acetone DCM
1.7 1.69 1.60 1.60
18.6 22.8 9.4 20.3
a
At 25 ◦ C [27].
The TLC/FID has been used to study the wool internal polar lipids [20], but using these conditions some non-polar lipid compounds could not be resolved and appeared as a single peak [20]. In order to obtain a better resolution, a number of assays were performed with different mobile phase combinations and elution conditions individually and in a mixture of standard lipid compounds indicated above. The best TLC/FID resolution was obtained by developing the rods in accordance with the method indicated in the experimental section. The TLC/FID chromatograms of a standard compound mixture and of the wool wax extracts obtained by different extraction conditions are shown in Fig. 1. The retention time of different Table 2 C, H and N elemental and ash amount of the wool wax extracts obtained under different extraction conditions Extraction method
Ash (%)
C (%)
H (%)
N (%)
CO2 /methanol CO2 /ethanol CO2 /acetone DCM
2.81 1.48 0.029 2.63
72.3 74.8 77.5 75.0
10.7 11.2 11.6 11.0
0.69 0.57 0.09 0.34
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Fig. 1. TLC/FID chromatogram of the standard compound mixture (A), wool wax extracts obtained with CO2 /methanol (B), CO2 /ethanol (C), CO2 /acetone (D), DCM/Soxhlet extraction (E) and commercial lanolin (F). The chromarods were developed using the following mobile phases: (i) 70 ml of chloroform/methanol/water (57/12/0.6, v/v/v) up to 1 cm (twice); (ii) 70 ml of hexane/diethyl ether/formic acid (58/12/0.3, v/v/v) up to 9 cm; (iii) 70 ml of hexane/benzene (35/35, v/v) up to 10 cm. Peak identification is indicated in Table 3.
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Table 3 Retention time (tr ) of lipid compounds identified by TLC/FID of the SFE and DCM/Soxhlet extracts Peak
Lipid class
tr (min)
1 2 3 4 5 6 7 8 9 10
SF Mono-ES U Di-ES TG FFA AL S PL Salts
0.08 0.12 0.15 0.18 0.24 0.27 0.32 0.36 0.38–0.42 0.47
SF, solvent front; mono-Es, monoester; U, unknown compound; di-ES, diesters; TG, tryglicerides; FFA, free fatty acids; AL, free fatty alcohols; S, sterols; PL, polar lipids.
lipid compounds are given in Table 3. According to the retention time of the standard compounds, the peaks 2, 4, 6, 7 and 8 were identified as mono-ES, di-ES, FFA, AL and S, respectively, from left to right in increasing polarity. Peak 1 was identified as solvent front. Peak 5 (TG) was tentatively identified as triglycerides. Further research is in progress in order to clarify this. Peak 9, termed as polar lipids (PL), includes a strong peak and two unresolved peaks. In this peak, we identified the presence of polar lipids such as ceramides, 7-hydroxycholesterol, cholesterol derivatives, and glucoceramides [28]. Peak 10 corresponds to salts, which include cholesterol sulfate (Csulf). These polar lipid compounds are present in internal
wool lipids [20]. Therefore it may be assumed that some internal lipids can be almost partially extracted under our experimental extraction conditions, mainly with CO2 /methanol, CO2 /ethanol and DCM/Soxhlet. 3.3. Quantitative analysis The relative amount of the lipid class compounds extracted from raw wool is plotted in Fig. 2. Clearly, the modifier polarity exerts an influence on both the lipid class extraction and on the amount extracted. The total amount of wool wax extracted increased when the extractant agent polarity was raised (acetone to methanol). Although all extractant agents extract all the different lipid classes, the amount of polar lipids and salts decreased as the polarity of extractant agent was diminished (CO2 /acetone). The lowest extraction of the different lipid classes could be attributed to the wide molecular weight distribution of the lipids present in wool wax. This could suggest a preferential extraction of the lipid compounds with a higher hydrophobic/hydrophilic balance. The total and relative amounts of the different lipid classes extracted with CO2 /ethanol are similar to those obtained with DCM/Soxhlet. The GC/MS technique was used to study the preferential extraction of the different lipid classes in accordance with their molecular weight. Differences between wool wax extracts were pointed out in the electron impact mode using characteristic family ions (m/z = 368 for cholesteryl esters and
Fig. 2. Relative average area value on weight of wool of each lipid class extracted from raw wool by means of CO2 /methanol (CO2 /M), CO2 /ethanol (CO2 /E), CO2 /acetone (CO2 /A) and DCM/Soxhlet.
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Fig. 3. Comparison between cholesteryl esters (m/z = 368) and hydroxycholesteryl esters (m/z = 456) content for different modifier extracts.
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Fig. 4. Comparison between several CO2 /modifier extracts and reference Soxhlet extracts in terms of cholesteryl esters content normalised by C16 OOC22 aliphatic ester. For this, we used the following equation f (n) = area(Cn cholesterol)/area(C15 COOC22 ).
m/z = 456 for silylated hydroxycholesteryl esters). In accordance with TLC/FID analysis, similar cholesteryl ester chromatograms were found for all the extracts. However, large differences were observed for the more polar compounds such as hydroxycholesteryl esters (Fig. 3). The ratio of cholesteryl esters/hydroxycholesteryl esters of CO2 /acetone extract is higher than that of the remaining extracts. On the one hand, the CO2 /ethanol and CO2 /methanol extractant agents gave similar results for reference wool wax extract obtained by the Soxhlet method. On the other hand, CO2 /acetone—acetone being less polar than methanol and ethanol—was not so efficient in the hydroxycholesteryl ester extraction. A detailed study was carried out to characterise molecular cholesteryl esters. Electron impact was not suitable for this analysis owing to the absence of molecular weight information in cholesteryl ester spectra. Therefore chemical ionisation was used as described in an earlier work [29]. Cholesteryl esters of the different extacts were compared on the basis of their acid moiety. Similar patterns were obtained for the different wool wax extracts with monomodal distributions with a maximum for the cholesteryl ester with a C19 acid moiety. It was also interesting to compare the ratio of the extracted cholesteryl esters versus the extracted aliphatic esters for the different extracts. We used the area obtained for the linear aliphatic es-
ter C15 COOC22 as a marker of aliphatic ester content (Fig. 4). A good correlation was observed between extractant polarity and cholesteryl ester aliphatic ester ratio with a lower value for acetone, which is even lower than the DCM reference extract value. Given that ethanol and methanol are more polar, they present the highest results with small differences. Fig. 5 shows the average area value on weight of extract of each lipid class present in the different extracts which are compared with the lipid composition of commercial lanolin. Although each collected extract contains all the lipid classes, the proportion of these varies as a function of the modifier polarity. By increasing the modifier polarity the proportion of polar compounds increases whereas the proportion of non-polar compounds decreases. It may be observed that the CO2 /methanol extract contains a higher proportion of different lipid classes with respect to other extracts, especially polar lipids and salts. Moreover, the CO2 /ethanol extract has a composition similar to that of the DCM extract, with a different proportion of polar lipids and salts. When the modifier polarity decreases (acetone) the proportion of polar lipids and salts falls and the proportion of the other lipid classes shows a slight increase. By contrast, when the modifier polarity increases (methanol) the proportion of polar lipids namely, salts, S, AL and
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Fig. 5. Lipid composition of CO2 /methanol, CO2 /ethanol, CO2 /acetone and DCM/Soxhlet extracts and commercial lanolin.
FFA rises and the proportion of mono-ES, U, di-ES and TG decreases. The commercial lanolin recovered from the scouring bath of raw wool by centrifugation and purification shows a different composition in relation to the extracts obtained by CO2 /modifier and DCM/Soxhlet. Whereas the proportion of mono-ES and di-ES is clearly higher, the proportion of polar lipids and salts is lower, especially in salts. However, the proportion of TG and AL is similar. It should be pointed out that neither U nor FFA are present in the commercial lanolin.
was shown by cholesteryl esters/hydroxycholesteryl esters. The presence of some polar lipids belonging to internal lipids suggests a partial extraction of these under the experimental conditions employed.
Acknowledgements This research was supported by “Comisión Interministerial de Ciencia y Tecnolog´ıa (CICYT)” project no. 2FD97-0509. Authors are grateful to Praxair España, S.A. and Peinaje del r´ıo Llobregat, S.A.
4. Conclusions References SFE is a suitable methodology for wool wax extraction from raw wool. The wool wax composition could be easily modified by increasing the polarity of the organic modifier. The total amount of wool wax extracted from raw wool fibres is increased by raising the modifier polarity. The CO2 /ethanol and CO2 /methanol extractants provide wool wax extract yields similar or higher, respectively, to the those obtained by dichloromethane extraction. Owing to the complexity of the composition of raw wool wax, it was not possible to extract a given class of lipidic compounds by liquid CO2 /modifier. However, some enrichment in certain lipidic classes could be obtained as a function of the modifier polarity, as
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[21] C. Dom´ınguez, J.M. Bayona, P. Erra, in: Proceedings of the International Textile Congress, Terrassa, vol. 1, 2001, p. 106. [22] C. Dom´ınguez, F. Garde, R. Alzaga, J.M. Bayona, P. Erra, in: Proceedings of the 10th International Wool Textile Research Conference, Aachen RW-8, CD-Rom, 2000. [23] C. Dom´ınguez, J.M. Bayona, L. Coderch, P. Erra, D. Palet AATCC Rev. 7 (2001) 6. [24] J. Fonollosa, M. Mart´ı, A. de la Maza, J.L. Parra, L. Coderch, J. Planar Chromatogr. 13 (2000) 119. [25] J. Dachs, R. Alzaga, J.M. Bayona, Anal. Chim. Acta 286 (1994) 319. [26] E. Jover, J.M. Bayona, J. Chromatogr. A 950 (2002) 213. [27] R.C. Weast (Ed.), CRC Handbook of Chemistry and Physics, 67th ed., CRC Press, Boca Raton, FL, 1986/1987. [28] C. Dom´ınguez, J.M. Bayona, P. Erra, unpublished results. [29] E. Jover, Z. Moldova, J.M. Bayona, J. Chromatogr. A, in press.
Effect of the carbon dioxide modifier on the lipid composition of wool wax extracted from raw wool C. Dom´ınguez a , E. Jover b , J.M. Bayona b , P. Erra a,∗ a b
Department of Surfactant Technology, IIQAB-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain Department of Environmental Chemistry, IIQAB-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain Received 2 August 2002; accepted 29 October 2002
Abstract Wool wax or lanolin is a unique substance secreted by sheep and forms a natural protective coating on wool fibres. It is widely used in pharmaceutical and cosmetic formulations. However, different systems of wool wax recovery from scouring liquour provide a dark impurified greasy product. This product has a lipid composition that differs from the wool wax present on wool fibres. The wool wax extraction method from raw wool with pressurised CO2 and different modifiers at constant pressure and temperature was studied. Thin-layer chromatography coupled to an automated flame ionisation detection system (TLC/FID) was used to analyse the different lipid classes present in the collected extracts. Moreover, a detailed structural comparison of the cholesteryl esters and hydroxycholesteryl esters was carried out by means of sub-ambient pressure chromatography mass spectrometry in the electron impact and in the ammonia positive chemical ionisation modes. For comparison, qualitative and quantitative analyses of the lanolin extracted in Soxhlet with dichloromethane and commercial cosmetic lanolin were carried out. Differences in the quantity of wool wax extraction and in the lipid composition of different wool wax extracts were detected by changing the modifier polarity. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Wool wax extraction; Lanolin; Raw wool; CO2 /modifier; TLC/FID; GS–MS; Subcritical CO2
1. Introduction Raw wool contains three main scouring contaminants: solvent soluble substances (referred to as wool grease, wool wax or lanolin), water soluble substances (suint) and insoluble material (mineral dirt) [1,2]. An additional contaminant is the proteinaceous contaminant layer (PCL) [3,4]. This is formed by water-insoluble and water-soluble components with amino acid compositions similar to skin flakes and suint peptides. All these contaminants must be removed before the wool can be mechanically processed. ∗ Corresponding author. Fax: +34-3-2045904. E-mail address: [email protected] (P. Erra).
Wool wet scouring processes are basically extraction systems for removing fibre contaminants. Industrially, scouring processes are carried out in a series of aqueous solutions with surfactant and builder, the wool wax being recovered from the scouring liquor by centrifuging [1]. Although they remove all the wool contaminants providing suitable wool fibres for subsequent processing, large volumes of wastewater with high BOD values are produced. Moreover, both the quantity and the quality of the recovered wool wax are very low. Lanolin is essentially a mixture of esters, diesters, and hydroxy esters of high molecular weight as well as free fatty alcohols and free fatty acids [5–9]. The wax or grease on the wool fibre consists of two fractions—
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an oxidised fraction which is found mainly at the tip and an unoxidised fraction located at the base of the fibre. During scouring the unoxidised fraction is more readily removed and recovered by centrifugation, whereas the oxidised fraction forms complexes with other contaminants in the scouring liquor and is unrecoverable by centrifuging under the conditions existing in a commercial centrifuge [1]. The development of supercritical fluid chromatography has demonstrated that wool wax is soluble in supercritical carbon dioxide [10]. The recovery of wool grease by extraction of raw wool [11–13] and the solubility of wool wax [14,15] with liquid and supercritical carbon dioxide over a limited range of pressures and temperatures have been investigated. Recently, a procedure based on supercritical fluid extraction (SFE) with carbon dioxide (CO2 ) as a solvent has been developed for the determination of wool wax present on raw wool fibres in an attempt to avoid or minimise the use of chlorinated organic solvents [16,17]. Selected extraction variables such as pressure, temperature and percentage of modifier affecting SFE have been optimised [17]. Changes in lipid composition of the extract occur as a function of the different experimental conditions such as pressure, temperature, extraction time, and amount of modifier. In this work, a study of wool wax extraction from raw wool fibre with pressurised carbon dioxide containing different modifiers (acetone, ethanol and methanol) at constant pressure and temperature was carried out. The lipidic composition of the different collected extracts was qualitatively and quantitatively analysed by means of thin-layer chromatography coupled to an automated flame ionisation detection system (TLC/FID). For comparison, the lipidic composition of the wool wax extracted with dichloromethane in a Soxhlet apparatus was included. Dichloromethane removes large amounts of surface residuals and small quantities of internal lipids [16]. The TLC/FID technique is a considerable improvement on the TLC method. In comparison, TLC/FID is a simple technique for separating and identifying compounds present in a mixture, allowing the quantification of the separated compounds [18]. This technique has been employed to identify and to quantitatively determine the composition of wool lipid extracts [19–24].
Gas chromatography coupled to mass spectrometry was used to perform the characterisation of the extract at molecular level. Prior to this characterisation, samples were prefractionated by gel permeation chromatography (GPC). Size exclusion enables to separate high molecular weight compounds from smaller molecules, which allows us to carry out gas chromatographic analysis.
2. Experimental 2.1. Materials and reagents Australian Merino sheared raw wool (φ = 21 m) from local sources. Prior to extraction, the raw wool was homogenised by hand and equilibrated in a conditioned room (24 ◦ C, 60% relative humidity). Commercial lanolin was supplied by Croda (Snaith Goole, UK). Dichloromethane (DCM) (reagent grade) was obtained from Scharlau (Barcelona, Spain), formic acid (85%) from Probus (Badalona, Spain) and chloroform, methanol, n-hexane, diethyl ether and benzene (all for analysis) were supplied by Merck (Darmstadt, Germany). Analytical-grade methanol (M), ethanol (E) and acetone (A) from Merck were used as modifiers. Ethyl acetate and cyclohexane (LiChrosolv grade) and bis-silyl-trifluoroactamide (BSTFA) were obtained from Merck. Palmitic acid behenyl ester (≈99%) (mono-ES), behenyl alcohol (98%) (AL), behenic acid (99%) (FFA) and cholesterol (>99%) (S), supplied by Sigma (St. Louis, MO, USA), and dipalmitic acid hexadecyl ester (di-ES) synthesised in our laboratory were used as standard lipid compounds for TLC/FID lipid analysis. 2.2. Extraction procedures 2.2.1. DCM Soxhlet extraction Raw wool sample (≈3.5 g) was Soxhlet-extracted with DCM (220 ml) for 4 h according to the Woolmark Company, TM 136 (2000). A minimum siphoning rate of 4–5 cycles/h was maintained. The recovered extracts were immediately filtered (GF/F 0.7 m) and concentrated to dryness by rotary evaporation. The extracts were maintained under vacuum overnight in a desiccator over P2 O5 to eliminate moisture traces, and
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finally weighed. The test yielded the total amount of dichloromethane extractable matter. 2.2.2. CO2 /modifier extraction Supercritical fluid extraction (SFE) was performed using a dual-syringe pump (SFC 3000; Carlo Erba, Milan, Italy) for the delivery of carbon dioxide (CO2 , SFE 99.998%, Praxair España, Barcelona, Spain) and modifier as described elsewhere [25]. Extraction cell (6.94 ml, Keystone Scientific) was filled with raw wool sheep sample (3.5 g). The flow-rate as liquid was adjusted by a micrometer forged metering valve (Hoke, Cresskill, NJ, USA) at ca. 1.5–2.0 ml/min measured from LCD of the pump, maintaining the temperature at 100 ◦ C. A total fluid volume of 30 ml was passed through the cell. The extract was collected in an empty screw cap vial (15 ml) with a septum (Supelco, Bellafonte PA, USA) tared previously, with an outlet in order to vent the decompressed CO2 . Three different modifiers (methanol, ethanol and acetone) were tested in order to evaluate their extraction efficiency. Constant pressure and temperature and 20% (v/v) of modifier were selected. The extracts were concentrated to dryness under a gentle stream of nitrogen or maintained at 60 ◦ C in order to eliminate the solvent excess. Collected wool wax extracts were quantified gravimetrically during the extraction process. In an attempt to calculate the fraction percentage values on weight of scoured and dried wool, the wool samples extracted were opened by hand and scoured several times with deionized water in order to remove residual vegetable matter and the suint (soluble salts) respectively, and finally dried and weighed. 2.3. Analytical techniques 2.3.1. Extract analysis by TLC/FID Qualitative and quantitative analyses of the lipid classes present in the collected extracts were performed by TLC coupled to an automated FID system (Iatroscan MK-5; Iatron Laboratories, Tokyo, Japan). The standard compounds or the dry extracts (25–60 mg) were redissolved in chloroform/methanol (2:1, v/v) (5–12 mg/ml). The samples (0.8 l) were spotted onto silica gel-coated Chromarods (type S-III) (Iatron Laboratories) by means of a 2 l precision syringe Hamilton (Bondaluz, Switzerland) coupled to an SES 3202/IS-02 semiautomatic sample
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spotter (Nieder-Olm, Germany). The rods (in sets of 10 mounted semipermanently on stainless steel-racks) were developed consecutively four times using the following mobile phases: (i) 70 ml of chloroform/methanol/water (57/12/0.6, v/v/v) up to 1 cm (twice); (ii) 70 ml of hexane/diethyl ether/formic acid (58/12/0.3, v/v/v) up to 9 cm; (iii) 70 ml of hexane/benzene (35/35, v/v) up to 10 cm. After each development the rods were heated for 5–10 min at 60 ◦ C to dry the remaining solvent, and run through a flame ionisation detector in the Iatroscan by using an air flow of 2.000 ml/min atmospheric air, a hydrogen flow of 160 ml/min (high purity hydrogen, C50) and a scanning speed of 3.0 s/cm. A total scan was performed to identify all the lipid components. Data were processed with the Boreal software version 2.5. An aliquot of the different collected wool wax extracts were redissolved and analysed in duplicate. 2.3.2. Extract analysis by MS 2.3.2.1. Sample preparation prior to MS characterisation. The lipids of lanolin were prefractionated by GPC. About 100 mg of lanolin were weighed and dissolved in 5 ml of ethyl acetate/cyclohexane 1:1. Then the sample was filtered through a 0.45 m nylon membrane filter (Lida, Kenosha, WI, USA). Fractionation was carried out following procedure already described [26]. Next 10 l of the solution were placed in a 2 ml conic vial and 10 l of BSTFA were added. The closed vial was maintained at 70 ◦ C for 1 h and then evaporated to dryness under gentle nitrogen stream. Iso-octane (50 l) was added to the vial to reconstitute the sample and analysed before 48 h to avoid hydrolysis of the trimethylsilyl ether (TMS) group. The main polar constituents of lanolin—free fatty acids, hydroxy acids and diols—were derivatised prior to GC–MS to prevent them from interfering in ester analysis. 2.3.2.2. Instrumental analysis. A sub-ambient pressure CP Sil 8 CB/MS capillary column (5% diphenyl-dimethylpolysiloxane) of 10 m × 0.53 mm i.d. and 0.25 m of film thickness fitted to a deactivated restrictor of 50 cm length and 0.1 mm i.d. at the injection port was obtained from Chrompack (Middelburg, The Netherlands). One microlitre of sample was injected in the splitless mode at 320 ◦ C, activating the injector purge at 90 s from injection. Initial
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column temperature was maintained at 90 ◦ C for 1 min, and then programmed at 10 ◦ C/min to 320 ◦ C, maintaining the final temperature for 20 min (44 min each run). Chromatographic analysis was performed in the constant flow mode at 1.2 ml/min. 2.3.2.3. Mass sprectrometry. For the chemical ionisation mode, a GC 6890A from Agilent Technologies (Palo Alto, CA, USA) coupled to an MS 5973N was used. Quadrupole and transfer line temperatures were maintained at 150 and 280 ◦ C, respectively. Ammonia was used as reagent gas at 12.8 × 10−5 Torr in the positive mode and the ion source was maintained at 200 ◦ C. A GC from Fisons (Manchester, UK), GC 8060 coupled to an MS detector MD 800 in the electron impact mode was used. Transfer line and ion source were maintained at 280 and 230 ◦ C, respectively. Other chromatographic conditions were identical to those reported in the CI section.
modifier, and the yield of wool wax extracted with DCM in a Soxhlet apparatus. The yields were calculated on the weight of scoured and dried wool. The total yield values obtained are in accordance with the modifier polarity. The lowest dipole moment of the acetone exerts a considerable influence on the total yield of the wool wax extracted. Whereas the yields obtained with liquid CO2 /ethanol or CO2 /methanol are similar to the DCM ones, the yield obtained with liquid CO2 /acetone is 50% lower. This could be attributed to a preferential extraction of a given fraction of wool wax with CO2 /acetone or to an additional extraction of other types of compounds (oxidised lanoline fraction, salts, ceramides, protein material) using the other extractant agents tested. The C, H, N elemental analysis and the ash amount (800 ◦ C, 3 h) support these yields. The C, H, N elemental analysis shows a smaller amount of nitrogenated compounds for the CO2 /acetone extract compared with the other extracts. Moreover, the ash analysis value is with CO2 /acetone two orders of magnitude below that of the other extracts (Table 2).
3. Results and discussion 3.2. Qualitative analysis 3.1. Wool wax yield Wool wax is a complex mixture of non-polar and polar lipid compounds. For this reason, by increasing the extractant agent polarity (acetone, ethanol, methanol)—by modifying the Hiderbrand solubility parameter (δ)—an important effect on the solubility of the different lipid compounds present on the wool surface could be expected. Thus, a more selective extraction of the lipid class could be achieved. Table 1 shows the wool wax yield obtained from raw wool with a fixed volume of pressurised CO2 containing 20% of either acetone, ethanol or methanol as Table 1 Yield of wool wax extracted from raw Merino wool with liquid CO2 containing 20% of modifier (methanol, ethanol, acetone) or Soxhlet with DCM Modifier
Dipole moment (D)a
Yield (%)
Methanol Ethanol Acetone DCM
1.7 1.69 1.60 1.60
18.6 22.8 9.4 20.3
a
At 25 ◦ C [27].
The TLC/FID has been used to study the wool internal polar lipids [20], but using these conditions some non-polar lipid compounds could not be resolved and appeared as a single peak [20]. In order to obtain a better resolution, a number of assays were performed with different mobile phase combinations and elution conditions individually and in a mixture of standard lipid compounds indicated above. The best TLC/FID resolution was obtained by developing the rods in accordance with the method indicated in the experimental section. The TLC/FID chromatograms of a standard compound mixture and of the wool wax extracts obtained by different extraction conditions are shown in Fig. 1. The retention time of different Table 2 C, H and N elemental and ash amount of the wool wax extracts obtained under different extraction conditions Extraction method
Ash (%)
C (%)
H (%)
N (%)
CO2 /methanol CO2 /ethanol CO2 /acetone DCM
2.81 1.48 0.029 2.63
72.3 74.8 77.5 75.0
10.7 11.2 11.6 11.0
0.69 0.57 0.09 0.34
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Fig. 1. TLC/FID chromatogram of the standard compound mixture (A), wool wax extracts obtained with CO2 /methanol (B), CO2 /ethanol (C), CO2 /acetone (D), DCM/Soxhlet extraction (E) and commercial lanolin (F). The chromarods were developed using the following mobile phases: (i) 70 ml of chloroform/methanol/water (57/12/0.6, v/v/v) up to 1 cm (twice); (ii) 70 ml of hexane/diethyl ether/formic acid (58/12/0.3, v/v/v) up to 9 cm; (iii) 70 ml of hexane/benzene (35/35, v/v) up to 10 cm. Peak identification is indicated in Table 3.
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Table 3 Retention time (tr ) of lipid compounds identified by TLC/FID of the SFE and DCM/Soxhlet extracts Peak
Lipid class
tr (min)
1 2 3 4 5 6 7 8 9 10
SF Mono-ES U Di-ES TG FFA AL S PL Salts
0.08 0.12 0.15 0.18 0.24 0.27 0.32 0.36 0.38–0.42 0.47
SF, solvent front; mono-Es, monoester; U, unknown compound; di-ES, diesters; TG, tryglicerides; FFA, free fatty acids; AL, free fatty alcohols; S, sterols; PL, polar lipids.
lipid compounds are given in Table 3. According to the retention time of the standard compounds, the peaks 2, 4, 6, 7 and 8 were identified as mono-ES, di-ES, FFA, AL and S, respectively, from left to right in increasing polarity. Peak 1 was identified as solvent front. Peak 5 (TG) was tentatively identified as triglycerides. Further research is in progress in order to clarify this. Peak 9, termed as polar lipids (PL), includes a strong peak and two unresolved peaks. In this peak, we identified the presence of polar lipids such as ceramides, 7-hydroxycholesterol, cholesterol derivatives, and glucoceramides [28]. Peak 10 corresponds to salts, which include cholesterol sulfate (Csulf). These polar lipid compounds are present in internal
wool lipids [20]. Therefore it may be assumed that some internal lipids can be almost partially extracted under our experimental extraction conditions, mainly with CO2 /methanol, CO2 /ethanol and DCM/Soxhlet. 3.3. Quantitative analysis The relative amount of the lipid class compounds extracted from raw wool is plotted in Fig. 2. Clearly, the modifier polarity exerts an influence on both the lipid class extraction and on the amount extracted. The total amount of wool wax extracted increased when the extractant agent polarity was raised (acetone to methanol). Although all extractant agents extract all the different lipid classes, the amount of polar lipids and salts decreased as the polarity of extractant agent was diminished (CO2 /acetone). The lowest extraction of the different lipid classes could be attributed to the wide molecular weight distribution of the lipids present in wool wax. This could suggest a preferential extraction of the lipid compounds with a higher hydrophobic/hydrophilic balance. The total and relative amounts of the different lipid classes extracted with CO2 /ethanol are similar to those obtained with DCM/Soxhlet. The GC/MS technique was used to study the preferential extraction of the different lipid classes in accordance with their molecular weight. Differences between wool wax extracts were pointed out in the electron impact mode using characteristic family ions (m/z = 368 for cholesteryl esters and
Fig. 2. Relative average area value on weight of wool of each lipid class extracted from raw wool by means of CO2 /methanol (CO2 /M), CO2 /ethanol (CO2 /E), CO2 /acetone (CO2 /A) and DCM/Soxhlet.
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Fig. 3. Comparison between cholesteryl esters (m/z = 368) and hydroxycholesteryl esters (m/z = 456) content for different modifier extracts.
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Fig. 4. Comparison between several CO2 /modifier extracts and reference Soxhlet extracts in terms of cholesteryl esters content normalised by C16 OOC22 aliphatic ester. For this, we used the following equation f (n) = area(Cn cholesterol)/area(C15 COOC22 ).
m/z = 456 for silylated hydroxycholesteryl esters). In accordance with TLC/FID analysis, similar cholesteryl ester chromatograms were found for all the extracts. However, large differences were observed for the more polar compounds such as hydroxycholesteryl esters (Fig. 3). The ratio of cholesteryl esters/hydroxycholesteryl esters of CO2 /acetone extract is higher than that of the remaining extracts. On the one hand, the CO2 /ethanol and CO2 /methanol extractant agents gave similar results for reference wool wax extract obtained by the Soxhlet method. On the other hand, CO2 /acetone—acetone being less polar than methanol and ethanol—was not so efficient in the hydroxycholesteryl ester extraction. A detailed study was carried out to characterise molecular cholesteryl esters. Electron impact was not suitable for this analysis owing to the absence of molecular weight information in cholesteryl ester spectra. Therefore chemical ionisation was used as described in an earlier work [29]. Cholesteryl esters of the different extacts were compared on the basis of their acid moiety. Similar patterns were obtained for the different wool wax extracts with monomodal distributions with a maximum for the cholesteryl ester with a C19 acid moiety. It was also interesting to compare the ratio of the extracted cholesteryl esters versus the extracted aliphatic esters for the different extracts. We used the area obtained for the linear aliphatic es-
ter C15 COOC22 as a marker of aliphatic ester content (Fig. 4). A good correlation was observed between extractant polarity and cholesteryl ester aliphatic ester ratio with a lower value for acetone, which is even lower than the DCM reference extract value. Given that ethanol and methanol are more polar, they present the highest results with small differences. Fig. 5 shows the average area value on weight of extract of each lipid class present in the different extracts which are compared with the lipid composition of commercial lanolin. Although each collected extract contains all the lipid classes, the proportion of these varies as a function of the modifier polarity. By increasing the modifier polarity the proportion of polar compounds increases whereas the proportion of non-polar compounds decreases. It may be observed that the CO2 /methanol extract contains a higher proportion of different lipid classes with respect to other extracts, especially polar lipids and salts. Moreover, the CO2 /ethanol extract has a composition similar to that of the DCM extract, with a different proportion of polar lipids and salts. When the modifier polarity decreases (acetone) the proportion of polar lipids and salts falls and the proportion of the other lipid classes shows a slight increase. By contrast, when the modifier polarity increases (methanol) the proportion of polar lipids namely, salts, S, AL and
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Fig. 5. Lipid composition of CO2 /methanol, CO2 /ethanol, CO2 /acetone and DCM/Soxhlet extracts and commercial lanolin.
FFA rises and the proportion of mono-ES, U, di-ES and TG decreases. The commercial lanolin recovered from the scouring bath of raw wool by centrifugation and purification shows a different composition in relation to the extracts obtained by CO2 /modifier and DCM/Soxhlet. Whereas the proportion of mono-ES and di-ES is clearly higher, the proportion of polar lipids and salts is lower, especially in salts. However, the proportion of TG and AL is similar. It should be pointed out that neither U nor FFA are present in the commercial lanolin.
was shown by cholesteryl esters/hydroxycholesteryl esters. The presence of some polar lipids belonging to internal lipids suggests a partial extraction of these under the experimental conditions employed.
Acknowledgements This research was supported by “Comisión Interministerial de Ciencia y Tecnolog´ıa (CICYT)” project no. 2FD97-0509. Authors are grateful to Praxair España, S.A. and Peinaje del r´ıo Llobregat, S.A.
4. Conclusions References SFE is a suitable methodology for wool wax extraction from raw wool. The wool wax composition could be easily modified by increasing the polarity of the organic modifier. The total amount of wool wax extracted from raw wool fibres is increased by raising the modifier polarity. The CO2 /ethanol and CO2 /methanol extractants provide wool wax extract yields similar or higher, respectively, to the those obtained by dichloromethane extraction. Owing to the complexity of the composition of raw wool wax, it was not possible to extract a given class of lipidic compounds by liquid CO2 /modifier. However, some enrichment in certain lipidic classes could be obtained as a function of the modifier polarity, as
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