WO2014072862A1

WO2014072862A1 - Modified chitosan for argentation chromatography

Info

Publication number: WO2014072862A1
Application number: PCT/IB2013/059617
Authority: WO
Inventors: Moegamat Anwar Jardine
Original assignee: University Of Cape Town
Priority date: 2012-10-24
Filing date: 2013-10-24
Publication date: 2014-05-15
Also published as: WO2014072862A9

Abstract

A solid support or resin for argentation chromatography is provided, comprising a chitosan polymer which has been sulfonated and loaded with silver. The solid support can be used for chromatographically separating saturated fatty acids from unsaturated fatty acids in a mixture, and optionally also for separating trans and cis unsaturated fatty acids. The chitosan polymer has sulfonate groups complexed to silver ions. Suitable sulfonate groups include RSO 3 -, where R can be an alkyl group, an aryl group or a benzoyl group. More particularly, the sulfonate groups are -(CH₂) _n SO₃ -, where n = 0, 1, 2 or 3; or (C=O)RSO₃ -, where R is an aryl group. The fatty acids can be fatty acid methyl esters (FAMEs). The chitosan polymer can be formed from naturally occurring chitosan or from a chitosan derivative, such as any one of sulfonamide chitosan, 2-N-sulfopropyl chitosan, 2-N- sulfobenzamido chitosan, 6-deoxy-amino chitosan, sulfonamide-6-deoxy 6- amino chitosan, 6-deoxy-2,6-bis-[sulfopropyl] chitosan, and 6-deoxy-2,6-bis- [sulfobenzamido] chitosan.

Description

MODIFIED CHITOSAN FOR ARGENTATION CHROMATOGRAPHY

FIELD OF THE INVENTION

This invention relates to modified chitosan for use as solid support in argentation chromatography.

BACKGROUND TO THE INVENTION

Marine and plant oils contain both saturated and unsaturated fatty acid methyl esters (FAMEs), both of which are of economic value once separated. Unsaturated fatty acids such as omega 3 and 6 fatty acids are of nutritional value, while saturated fatty acids have a cosmetic value and serve as good feedstock for biodiesel.

There are two types of unsaturated fatty acids: trans fatty acids and cis fatty acids. Trans fatty acids have been known to increase the risk of coronary heart disease and, as a result, there is considerable interest in separating trans fatty acids from cis fatty acids.

There are many technologies which claim selective separation for saturated and unsaturated fatty acids, for example, liquid-liquid extraction using a variation of solvents and temperatures (US 3,755,385); selective liquid extraction of urea complexed fatty acids (US 8,003,813); and argentation chromatography using solid supports such as styrene-divinyl benzene sulfonate resins (US 4, 189,442 and US 4,305,882), zeolites (US 4,210,594) or alumina (US 8,088,710).

Argentation is a silver ion liquid chromatography technique broadly used for the separation of saturated and unsaturated fatty acids. Ion exchange columns can be prepared by integrating silver ions, usually as silver nitrate, onto the stationary phase, where the silver can react with unsaturated components of fatty acids to form weak polar complexes. Alternatively, silver ions can be incorporated into the mobile phase or a cation-exchange column can be converted into a silver ion column. Separation of the fatty acids is based on the position, configuration and number of double bonds, as well as the chain length, of the fatty acids.

However, bulk separation of oils containing fatty acids using argentation chromatography has not been described. The only commercially available columns for the process of argentation are Discovery™ SPE columns (available from Sigma-Aldrich), which contain a p-propyl benzenesulfonic acid group bonded to a silica solid support in which the acid hydrogens have been exchanged with silver ions (Ag⁺). These columns have been used to separate saturated and unsaturated fatty acids, and c/^'s and trans unsaturated fatty acids, but only on an analytical scale, and it would be too expensive to use these columns to separate fatty acids for preparative purposes.

There is thus a need for an alternative method of separating saturated and unsaturated fatty acids.

SUMMARY OF THE INVENTION

According to a first embodiment of the invention, there is provided a chromatography resin comprising a sulfonated or sulfonamide chitosan polymer complexed with silver ions.

The chitosan polymer may be formed from a chitosan derivative. The sulfonated chitosan polymer may comprise a sulfonate group on at least a portion of the amino groups in the chitosan polymer. The silver ions may be complexed to the sulfonate groups.

The sulfonate group may be RS0₃ ^~, where R is an alkyl group, an aryl group or a benzoyl group. Preferably, the sulfonate group is -(CH₂)_nS0₃ ^~, where n = 0, 1 , 2 or 3, and more preferably the sulfonate group is (C=0)RS0₃ ^~, where R is an aryl group.

The sulfonated chitosan polymer may be selected from sulfonamide chitosan, 2-N- sulfopropyl chitosan, 2-/V-sulfobenzamido chitosan, 6-deoxy-amino chitosan, sulfonamide-6-deoxy 6-amino chitosan, 6-deoxy-2,6-£>/s-[sulfopropyl] chitosan, and 6-deoxy-2,6-£>/s-[sulfobenzamido] chitosan. Preferably, the sulfonated chitosan polymer is 2-/V-sulfobenzamido chitosan.

At least about 5 mmol/g silver may be complexed to the sulfonated chitosan polymer. More preferably, at least about 7.5 mmol/g silver is complexed to the sulfonated chitosan polymer, and even more preferably at least about 10 mmol/g silver is complexed to the sulfonated chitosan polymer.

The resin may be suitable for use in argentation chromatography, such as for separating saturated fatty acids from unsaturated fatty acids and/or for separating trans unsaturated fatty acids from c/^'s unsaturated fatty acids. The saturated and unsaturated fatty acids may be fatty acid methyl esters (FAMEs).

The saturated fatty acids and unsaturated fatty acids may be from a marine oil or citrus oil and other vegetable oils. The marine oil may be selected from fish oil, salmon oil, krill oil and cod liver oil, the vegetable oil may be selected from olive oil, grapeseed oil, coconut oil, canola oil, sunflower oil, avocado oil, sesame seed oil and other nut and seed oils and the citrus oil may be selected from orange oil and grapefruit oil. According to a second embodiment of the invention, there is provided a method for separating saturated and unsaturated fatty acids from a mixture, the method comprising:

a) loading the mixture on a bed of a chromatography column with a resin comprising an argentated sulfonated chitosan polymer, whereby the unsaturated fatty acids are selectively adsorbed onto the resin;

b) eluting the saturated fatty acids through the resin with a solvent; c) collecting the saturated fatty acids as they are eluted from the resin; d) eluting the unsaturated fatty acids through the resin with a solvent which separates the unsaturated fatty acids from the resin; and

e) collecting the unsaturated fatty acids as they are eluted from the resin.

The method may further comprise the steps, between steps (c) and (d), of:

i) eluting trans unsaturated fatty acids through the resin with a solvent which selectively releases the trans unsaturated fatty acids from the resin while the cis fatty acids remain adsorbed to the resin, and

ii) collecting the trans unsaturated fatty acids as they are eluted from the resin,

in which case in steps (d) and (e) the unsaturated fatty acids which are eluted and collected, respectively, are cis fatty acids.

The solvents may be hexane, acetone or acetonitrile, or any combination thereof.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be described, by way of example only, with reference to the figures in which:

Figure 1 shows fractionation of fatty acids by means of argentation. 1 : Loading of fatty acid mix onto column, 2: Retention of unsaturated fatty acid and elution of saturated fatty acid, 3: Further retention of c/^'s unsaturated fatty acid and elution of trans unsaturated fatty acid;

Figure 2 shows a ¹H NMR spectrum of chitosan (1 ) in 2 % TFA/D₂0;

Figure 3 shows a ¹H NMR spectrum of 2-/V-sulfopropyl chitosan (3) in D₂0/DCI;

Figure 4 shows a ¹ H NMR spectrum of sodium 2-/V-sulfobenzamido chitosan (4) in 2 % TFA/D₂0;

Figure 5 shows a ¹ H NMR spectrum of 6-deoxy-2,6-£>/s[sulfopropyl] chitosan (7)

Figure 6 shows a ¹ H NMR spectrum of 6-deoxy-2,6-£>/s[sulfobenzamido] chitosan

(8) in D₂0;

Figure 7 shows IR spectra of (a) LMW chitosan (1 ), (b) 2-/V-sulfopropyl chitosan

(3) and (c) 2-/V-sulfobenzamido chitosan (4);

Figure 8 shows IR spectra of A: (a) 6-deoxy-6-amino chitosan (5), (b) 6-deoxy- 2,6-£>/s[sulfopropyl] chitosan (7), (c) 6-deoxy-2,6-£>/s[sulfobenzamido] chitosan (8), and B: (d) 2-/V-sulfonamide chitosan (2) and (e) 6-deoxy 2,6-sulfonamido chitosan (6); Figure 9 shows PXRD profiles of (a) chitosan (1 ), (b) 6-deoxy-6-amino chitosan

(5), (c) (i) 2-/V-sulfopropyl chitosan (3) (ii) Ag-2-/V-sulfopropyl chitosan, (d) (i) 2-/V-sulfobenzamido chitosan (4), (ii) Ag-2-/V-sulfobenzamido chitosan, (e) (i) 6-deoxy-2,6-£>/s[sulfopropyl] chitosan (7), (ii) Ag-deoxy- 2,6-£>/s[sulfopropyl] chitosan and (f) (i) 6-deoxy-2,6-£>/s[sulfobenzamido] chitosan (8) and (ii) Ag-6-deoxy-2,6-£>/s[sulfobenzamido] chitosan; Figure 10 shows TEM images and size distributions of (a) Discovery™ SPE column, (b) 2-/V-sulfobenzamido chitosan (4) and (c) 6-deoxy-2,6- £>/s[sulfopropyl] chitosan (7); and Figure 1 1 shows bar chart representations of (a) crude FAME and FAME separation on columns containing (b) Discovery™ SPE column, (c) 2- /V-sulfopropyl chitosan (3) and (d) 2-/V-sulfobenzamido chitosan (4).

DETAILED DESCRIPTION OF THE INVENTION

A solid support or resin for argentation chromatography is provided, comprising a chitosan polymer which has been sulfonated and loaded with silver (Ag+) ions. The solid support can be used for chromatographically separating saturated fatty acids from unsaturated fatty acids in a mixture, and optionally also for separating trans unsaturated fatty acids from c/^'s unsaturated fatty acids. The mixture is typically a natural oil such as a marine, citrus or vegetable oil.

Although chitosan itself has been extensively exploited in the biomedical, food, pharmaceutical and chemical industries due to its favourable antimicrobial, antibacterial and haemostatic properties, derivatives of these types of biodegradable natural polymers have hitherto not been evaluated as solid supports for argentation chromatography. The chitosan polymer has sulfonate groups on at least some of its amino groups, and these are complexed to silver ions. Suitable sulfonate groups include RS0₃ ^", where R can be an alkyl group, an aryl group or a benzoyl group. More particularly, the sulfonate groups are -(CH₂)_nS0₃ ^", where n = 0, 1 , 2 or 3; or (C=0)RS0₃ ^~, where R is an aryl group.

The fatty acids can be fatty acid methyl esters (FAMEs). The chitosan polymer can be formed from naturally occurring chitosan or from a chitosan derivative, such as any one of sulfonamide chitosan [2], 2-/V-sulfopropyl chitosan [3], 2-/V-sulfobenzamido chitosan [4], 6-deoxy-amino chitosan, sulfonamide-6-deoxy 6-amino chitosan [6], 6-deoxy-2,6-£>/s-[sulfopropyl] chitosan [7], and 6-deoxy-2,6-£>/s-[sulfobenzamido] chitosan [8], shown below. Preferably, the chitosan polymer is 2-/V-sulfobenzamido chitosan [4].

The chitosan derivative can be a 6-amino 6-deoxy chitosan polymer with a linker provided on at least some of the amine groups of the polymer, as described in copending patent application WO201 1/083360, the contents of which are expressly incorporated herein. The linker is introduced as a heterobifunctional moiety which is capable of covalent coupling with the amino functional groups of the polymer on one end and a functional molecule on the other end.

The degree of retention of unsaturated molecules is dependent on the loading of Ag⁺ ions on the solid support. Sulfonation of the chitosan increases sites on the chitosan for complexing silver, and thus the solid support of the present invention can comprise a higher loading of silver compared to solid supports used in other argentation chromatographic processes (the polymers investigated herein had up to 10x more loaded Ag⁺ compared to the Discovery® SPE column which was used as a benchmark). Thus, the silver loading can be at least 3 mmol/g, although this is more preferably at least 5 mmol/g, even more preferably at least 7.5 mmol/g, and most preferably at least 10 mmol/g. Numerous methods are available for the synthesis of sulfated and sulfonated chitosan, and these methods will be readily apparent to a person skilled in the art.

A mixture of saturated and unsaturated fatty acids can be loaded onto a column containing the sulfonated chitosan polymer of the invention. The saturated fatty acids will be adsorbed onto the silver ions of the solid support, and a suitable solvent can be used to elute the saturated fatty acids from the column, and these saturated fatty acids can be collected. A different solvent can then be used to release the unsaturated fatty acids from the solid support and to elute them form the column. The solvents can be hexane, acetone or acetonitrile, or any combination thereof. If desired, trans unsaturated fatty acids can be separated from cis unsaturated fatty acids when eluting the unsaturated fatty acids.

An example of such a process is depicted in Figure 1 . A mixture of fatty acids is loaded onto a column in step 1 . Fractionation occurs by means of retention of unsaturated fatty acids on the column containing the solid support polymer of the invention and the saturated fatty acids are eluted (step 2). This is followed by further retention of cis unsaturated fatty acids and elution of trans unsaturated fatty acids (step 3). The invention will now be described in more detail with reference to the following non-limiting examples.

Examples

Various sulfonamides and sulfonated chitosan polymers as well as 6-deoxy-6- amino chitosan derivatives were synthesised. Silver was loaded onto these polymers and they were fully characterized using FT-IR, EA, TEM and ICP-MS analyses. The silver loaded chitosan derivatives were used as a solid support for argentation and tested against the only commercially available column, the Discovery™ column, which was used as a benchmark.

Low molecular weight (LMW) chitosan (1 ) was used to synthesize a range of sulfonate and sulfonamide chitosan derivatives (2) - (8). A synthetic pathway is shown below:

In this scheme, LMW chitosan (1 ) is used to synthesize sulfonamide chitosan (2), 2-/V-sulfopropyl chitosan (3), 2-/V-sulfobenzamido chitosan (4), 6-deoxy-amino chitosan (5), sulfonamide-6-deoxy 6-amino chitosan (6), 6-deoxy-2,6-£>/s- [sulfopropyl] chitosan (7) and 6-deoxy-2,6-£>/s-[sulfobenzamido] chitosan (8).

The sodium derivatives of these polymers were obtained through either stirring in 5 M NaOH or suspending the polymer in H₂0 and dialyzing against saturated NaHC0₃. Silver was loaded onto polymers by the addition of AgN0₃ solution.

Polymer analysis

¹ H NMR Spectroscopy

LMW chitosan (1): In the ¹ H NMR spectrum of chitosan, shown in Figure 2 of the accompanying drawings, the broad signal at 4.56 ppm was assigned to the anom/eric proton, H-1 . Resonances between 3.59 ppm and 3.40 ppm are due to protons H-3 to H-6 of the pyranose ring, while H-2 resonates at 2.87 ppm. The presence of acetylated units (15 %) of chitosan is confirmed by the signal at 1 .75 ppm.

2-N-sulfopropyl chitosan (3): The ¹ H NMR spectrum of (3) reveals a signal at

4.7 ppm which was assigned to H-1 (Figure 3). A multiplet between 3.5 and

3.8 ppm corresponds to the pyranose protons H-3 - H-6 while H-2 resonates at 3.2 ppm. The methyl groups of the propyl moiety resonate at 2.9, 2.8 and 1 .9 ppm, respectively. The ¹ H NMR spectrum obtained agrees well with that presented in the literature.

2-N-sulfobenzamido chitosan (4): Comparing the ¹ H NMR spectrum of (1 ) with (4) (Figure 4), additional signals can be observed due to the presence of the aromatic functional group. This can be seen by the resonance at 7.92 ppm which was assigned to H-10. Aromatic protons H-7 and H-8 resonate at 7.63 ppm, while H-9 resonates at 7.57 ppm. The ¹ H NMR spectra results obtained in here agrees with data presented for a similar structure by Crini et al.

6-Deoxy-2,6-bis[sulfopropyl] chitosan (7): Similar resonances were observed for compound (7) as that obtained for compound (3) with additional broadening of resonances (Figure 5). In addition, residual /V-phthaloyl absorbances were observed due to incomplete deprotection.

6-Deoxy-2,6-bis[sulfobenzamido] chitosan (8): Similar but broad absorbances were observed as obtained for compound (4) (Figure 6).

FT-IR analysis

LMW chitosan (1): Figure 7a shows the FT-IR spectrum of chitosan and its sulfonated derivatives. The FT-IR spectrum of chitosan reveals the characteristic bands of chitosan at 3456, 1669, 1421 and 1070 cm^"1.

2-N-sulfonamide chitosan (2): The FT-IR spectrum of (2) reveals additional absorption bands at 1421 , 1 154 and 616 cm^"1 due to the presence of RS0₃H group. These bands corresponds to the S=0 asymmetric and symmetric stretching vibrations, as well as the S-0 symmetric stretch, respectively (Figure 8d).

2-N-sulfopropyl chitosan (3): Similarly, the sulfonic acid group present on (3) can be confirmed by bands at 1367, 1 183 and 740 cm^"1 (Figure 7b). The data obtained was in close agreement with Jung et al.

2-/V-sulfobenzamido chitosan (4): The FT-IR spectrum of (4) (Figure 7c) reveals bands at 1397, 1080 and 706 cm^"1 due to the presence of RS0₃H. The aromatic group produced bands at 835 and 631 cm^"1, due to the C=C stretch and C-H out- of-plane bend of an aromatic ring. 6-Deoxy-6-amino chitosan (5): Characteristic bands of (5) can be seen at 3478, 1674, 1583, 1 179 and 1028 cm^"1 and agrees with literature values (Figure 8a).

Sulfonamide-6-deoxy 6-amino chitosan (6): The RS0₃H produced absorption bands at 1067 and 712 cm^"1.

6-Deoxy-2,6-bis[sulfopropyl] chitosan (7): The presence of the sulfonic group is confirmed by the bands at 1393 cm^"1 , 1043 cm^"1 and 736 cm^"1 (Figure 8b). 6-Deoxy-2,6-bis[sulfobenzamido] chitosan (8): The presence of a sulfonated group was confirmed by the bands at 1332 cm^"1 , 1090 cm^"1 and 697 cm^"1. Evidence of the presence of an aromatic ring are indicated by the bands at 1667 cm^"1 and 743 cm^"1 due to the C=C stretch and C-H out-of-plane bend of an aromatic ring, respectively (Figure 8c).

Powder X-ray Diffraction (PXRD)

The XRD patterns of different polymers are shown in Figure 9. The chitosan (1 ) and 6-deoxy-6-amino chitosan (4) showed two peaks at 2Θ of 9° and 20° contributed respectively by hydrated and anhydrous chitosan crystals. The diffractograms of sulfonated polymers were superimposed on the same graph as the silver loaded version of the same polymer. The peak patterns appeared very similar for all polymers except the 6-deoxy-2,6-£>/s[sulfobenzamido] chitosan (8), which showed a major reduction in peak intensity at the 2Θ of 9° and 20 °. The corresponding crystalline region of chitosan almost disappeared. The Ag-2-/V- sulfobenzamido chitosan (4), Ag-deoxy-2,6-£>/s[sulfopropyl] chitosan (7) and Ag-6- deoxy-2,6-£>/s[sulfobenzamido] chitosan (8) had a well-defined characteristic diffraction peak at 30°, and weak peaks at 45^Q and 58^Q, respectively, which corresponded to (1 1 1 ), (200) and (220) planes of face centred cubic (fee) crystal structure of metallic silver, indicating the presence of Ag nanoparticles in the polymer. TEM and ICP-MS analysis

The loading of silver onto modified chitosan polymers was confirmed by transmission electron microscopy (TEM) and inductively coupled mass spectroscopy (ICP-MS) analysis. The TEM images and size distributions of selected polymers can be seen in Figure 10.

The size distribution of the chitosan polymers (Table 1 ) were generally found to be broader compared to the Discovery™ polymer. The TEM analysis shows that the Discovery™ column has uniform particles with an average size of 6.44 nm and a narrow size distribution. The smaller size and uniform distribution of the Discovery™ column and polymers (4) and (7) particles may influence retention of saturated and unsaturated FAME. Table 1 : Polymers and their particle size distribution

ICP-MS analysis (Table 2) revealed that 2-/V-sulfobenzamido chitosan (3) contains the highest loading of silver with 10.2 mmol/g silver. The expected higher loading of silver on (5) and (6) due to the presence of additional Ag⁺ complexation sites was not observed by ICP-MS analysis. The lower yield of silver obtained for these polymers could be as a result of its low solubility. This means that the silver loaded onto the polymers was not fully released in HN0₃ during preparation for ICP-MS analysis or the additional silver was not loaded in the first instance. The commercially available, Discovery™ polymer contains 1 .19 mmol/g of silver which is much lower than the polymers synthesized. Table 2: ICP-MS analysis of silver complexed to modified chitosan polymers

GC-MS analysis

Marine fish oils are unique in the variety and degree of unsaturation of their fatty acid composition. The data presented below indicate the composition of the methyl esters of the crude marine fish oil analysed (Figure 1 1 a). The marine oil used was PUFA No.1 (Marine Source), Sigma analytical standard. All unsaturated components were found to be cis unsaturated.

Therefore, due to the absence of trans fatty acids, separation between trans and cis components was not studied. The separation between saturated and unsaturated fatty acids was, however, evaluated. Of the data collected, 67% could be conclusively identified. The other 33% could not be unambiguously identified using the NIST database. The crude marine oil includes 34% saturated components of those fatty acids identified, which include C14:0 and 016:0. The remaining 66% are unsaturated and the major fatty acids are C16:1 n7, 018:1 , C20:5n3 and C22:6n3 identified using the NIST database.

Table 3: Identification of FAME of crude marine fish oil

Rt M⁺ Chain Fatty acid methyl ester

18.27 242 14:0 Tetradecanoic acid

20.75 270 16:0 Hexadecanoic acid 21 .05 268 16:1 7- Hexadecanoic acid

23.12 296 18:1 9- Octadecenoic acid

25.05 324 20:1 11 - Eicosanoic acid

26.87 31 6 20:5n3 5,8, 11 ,14,1 7-Eicosapentanoic acid

30.02 342 22:6n3 4,7, 10,1 3, 1 6, 19- Docosahexanoic acid

The solvent elution programme for the Discovery™ Ag-ion SPE column was designed to be compatible with a relatively non-polar stationary phase (polymerically bonded benzenesulfonic acid) and was preconditioned with acetone (4 ml.) followed by hexane (4 ml_). Elution occurred with hexane: acetone (96:4; 6 ml_), followed by hexane: acetone (90:10; 4 ml.) and finally acetone (4 ml_).

The stationary phase of the columns prepared according to the invention are polar and of natural origin. When compared to the commercial stationary phase, a much stronger interaction with FAMEs was expected for the stationary phase of the invention. Hence, the elution gradient was varied to attain maximum retention of fatty acids. Due to the increased hydrophobicity of chitosan polymers (3), (4), (7) and (8), the solvent gradient was further modified to include acetonitrile. Columns containing (1 ), (2), (5) and (6) as a solid support were preconditioned as described above. This was followed by loading of FAME and hexane (4 ml_). Followed by hexane: acetone (98:2, 6 ml_), hexane:acetone (92:8, 6 ml_), hexane:actone (90:1 0, 4 ml_), hexane:acetone (60:40), hexane :acetone (50:50,

4 ml_), acetone (100, 6 ml_).

Columns containing (3), (4), (7) and (8) were preconditioned as the Discovery™ column. This was followed by hexane: acetone (96:4; 5 ml_), followed by hexane: acetone (90:1 0; 5 ml_), acetone (5 mL) and finally acetone:acetonitrile (94:6,

5 mL).

Evaluating the separation of fatty acids obtained from the Discovery™ SPE column (Figure1 1 b), it is evident that almost complete separation could be observed between saturated and unsaturated FAME. In the first fraction 89% of the fatty acids collected, contained saturated fatty acids, while only 1 1 % was due to unsaturated fatty acids. The second fraction contained 89% unsaturated and only 1 1 % saturated fatty acids.

All FAMEs of interest were eluted from the Discovery™ SPE column after 2 fractions. The most promising results were obtained from 2-/V-sulfobenzamido chitosan (4). Good retention of unsaturated FAME is observed and this can be seen by the low content of unsaturated FAME within the early fraction. The increased separation of saturated and unsaturated FAME can be attributed to the highest loading of silver observed from ICP-MS analysis. Also, the chemical structure of this polymer resembles that of the Discovery™ polymer, which may contribute to the increased interaction of the polymer to unsaturated FAME. With regard to the other columns, retention of both saturated and unsaturated FAME was observed with little separation. The use of native chitosan (1 ) as a solid material for SPE chromatography demonstrated poor separation but notable retention of FAME. The complexation may be due to weak Van der Waals interactions in the absence of the electrostatic interactions.

Due to the increased loading of Ag⁺ expected on 6-deoxy-2,6-amino chitosan derivatives (5)-(8) an increased separation of FAME was expected. The increased loading of Ag⁺ was only observed for (6) by ICP-MS analysis which can explain the increased retention of unsaturated FAME as compared to (2).

Citrus oils comprise of mixtures of mono- and di-unsaturated terpenoids, including oxygenated varieties in the form of epoxides, alcohol, aldehydic or ketonic constituents. Partial fractionation of standard mixtures, including some of the aforementioned essential oils was demonstrated by thin layer chromatography and subsequent staining of such mixtures.

Discussion 6-Deoxy-6-amino chitosan was synthesised as well as its sulfonated and sulfonamide derivatives to produce additional sites for the complexation of silver, thus allowing for a greater interaction with unsaturated fatty acid methyl esters (FAMEs) and hence, increasing the separation of FAME. The potential of Ag⁺ loaded chitosan derivatives of the invention to separate saturated and unsaturated FAME has been demonstrated against the benchmarked Discovery™ SPE column. Overall, good retention of unsaturated FAMEs was achieved. The best results were obtained from a 2-/V-sulfobenzamido chitosan (4) column. This could be of a result of its high silver loading and possibly also its similar structure to the p-propyl benzenesulfonic acid Discovery™ column.

Chitosan based argentation columns of the present invention can be recycled and reused at least twice. They are derived from a renewable waste material and cost a fraction of the price to manufacture. Although no attempt was made to reduce the particle size of the chitosan based polymers, it is anticipated that doing so will lead to an increase in column efficiency.

The modified chitosan polymer is versatile due to the presence of reactive amino and hydroxyl groups, and it is relatively inexpensive, readily available and biodegradable. Use of these biodegradable polymers on a bulk scale would contribute to environmentally friendly processing methods (after the extraction of residual silver, the material can be used as an organic fertiliser). It will be appreciated that there are numerous modifications and variations of the embodiments of the invention that would be obvious to a person skilled in the art which are deemed to be within the scope of the invention, the nature of which are to be determined from the above description and the examples.

Claims

A chromatography resin comprising a sulfonated chitosan polymer complexed with silver ions.

A chromatography resin according to claim 1 , wherein the chitosan polymer is formed from a chitosan derivative.

A chromatography resin according to either of claims 1 or 2, wherein the sulfonated chitosan polymer comprises a sulfonate group on at least a portion of the amino groups in the chitosan polymer.

A chromatography resin according to claim 3, wherein the silver ions are complexed to the sulfonate groups.

A chromatography resin according to either of claims 3 or 4, wherein the sulfonate group is RS0₃ ^~, where R is an alkyl group, an aryl group or a benzoyl group.

A chromatography resin according to either of claims 3 or 4, wherein the sulfonate group is -(CH₂)nS0₃ ^~, where n = 0, 1 , 2 or 3.

A chromatography resin according to either of claims 3 or 4, wherein the sulfonate group is (C=0)RS0₃ ^~, where R is an aryl group.

A chromatography resin according to any one of claims 1 to 7, wherein the sulfonated chitosan polymer is selected from:

sulfonamide chitosan,

2-/V-sulfopropyl chitosan,

2-/V-sulfobenzamido chitosan,

6-deoxy-amino chitosan,

sulfonamide-6-deoxy 6-amino chitosan, 6-deoxy-2,6 >/^'s-[sulfopropyl] chitosan, and

6-deoxy-2,6-£>/s-[sulfobenzamido] chitosan.

9. A chromatography resin according to claim 8, wherein the sulfonated chitosan polymer is 2-/V-sulfobenzamido chitosan.

10. A chromatography resin according to any one of claims 1 to 9, wherein at least about 5 mmol/g silver is complexed to the sulfonated chitosan polymer.

1 1 . A chromatography resin according to any one of claims 1 to 10, wherein at least about 7.5 mmol/g silver is complexed to the sulfonated chitosan polymer.

12. A chromatography resin according to any one of claims 1 to 1 1 , wherein at least about 10 mmol/g silver is complexed to the sulfonated chitosan polymer.

13. A chromatography resin according to any one of claims 1 to 12, which is suitable for use in argentation chromatography.

14. A chromatography resin according to any one of claims 1 to 13, which is suitable for separating saturated fatty acids from unsaturated fatty acids.

15. A chromatography resin according to claim 14, which is suitable for separating trans unsaturated fatty acids from cis unsaturated fatty acids.

16. A chromatography resin according to either of claims 14 or 15, wherein the saturated and unsaturated fatty acids are fatty acid methyl esters (FAMEs).

17. A chromatography resin according to any one of claims 1 to 16, wherein the saturated fatty acids and unsaturated fatty acids are in a marine oil, selected from fish oil, salmon oil, krill oil and cod liver oil.

18. A chromatography resin according to any one of claims 1 to 16, wherein the saturated fatty acids and unsaturated fatty acids are in a vegetable oil selected from olive oil, grapeseed oil, coconut oil, canola oil, sunflower oil, avocado oil, sesame seed oil and other nut and seed oils or citrus oil, selected from orange oil and grapefruit oil.

19. A method for separating saturated and unsaturated fatty acids from a mixture, the method comprising:

a) loading the mixture onto a chromatography column with a resin comprising an argentated sulfonated chitosan polymer, whereby the unsaturated fatty acids are selectively adsorbed onto the resin;

b) eluting the saturated fatty acids through the resin with a solvent; c) collecting the saturated fatty acids as they are eluted from the resin;

d) eluting the unsaturated fatty acids through the resin with a solvent which separates the unsaturated fatty acids from the resin; and

e) collecting the unsaturated fatty acids as they are eluted from the resin.

20. A method according to claim 19, which further comprises the steps, between steps (c) and (d), of:

(i) eluting trans unsaturated fatty acids through the resin with a solvent which selectively releases the trans unsaturated fatty acids from the resin while the cis fatty acids remain adsorbed to the resin, and (ii) collecting the trans unsaturated fatty acids as they are eluted from the resin,

and in steps (d) and (e) the unsaturated fatty acids which are eluted and collected, respectively, are c/^'s fatty acids.

21 . A method according to either of claims 19 or 20, wherein the resin is a chromatography resin of any one of claims 1 to 12.

22. A method according to any one of claims 19 to 21 , wherein the mixture is a marine oil selected from fish oil, salmon oil, krill oil and cod liver oil.

23. A method according to any one of claims 19 to 21 , wherein the mixture is a vegetable oil selected from olive oil, grapeseed oil, coconut oil, canola oil, sunflower oil, avocado oil, sesame seed oil and other nut and seed oils or citrus oil, selected from orange oil and grapefruit oil.

24. A method according to any one of claims 19 to 23, wherein the fatty acids are fatty acid esters.

25. A method according to any one of claims 19 to 24, wherein the fatty acids are fatty acid methyl esters.

26. A method according to any one of claims 19 to 25, wherein the solvents are hexane, acetone or acetonitrile, or any combination thereof.