WO2005011836A1

WO2005011836A1 - A separation process

Info

Publication number: WO2005011836A1
Application number: PCT/GB2004/003276
Authority: WO
Inventors: James Hanley Clark; Jeffrey James Edwin Hardy; Krzysztof Jakub Milkowski; Francesca Maria Kerton; Andrew John Hunt; Fabien Deswarte
Original assignee: University Of York
Priority date: 2003-07-29
Filing date: 2004-07-29
Publication date: 2005-02-10
Also published as: GB0317752D0

Abstract

The invention relates to a process for separating chemical compounds in which the compounds are separated on the basis of their interactions with a polysaccharide material having a surface area of at least 20m2/g. The polysaccharide material may be, for example, a starch on a cellulose. The method may be a method of chromatography.

Description

A Separation Process

The invention relates to a method of separating chemical compounds using high surface area polysaccharides.

It is often desirable to separate mixtures of chemical compounds for reasons of analysis or purification and many methods of separation are known. One broad class of such methods relies on the individual compounds in a mixture having differing interactions with a separation material. For example, chromatography relies upon the differing interactions of compounds in a mixture to be separated with a stationary material or stationary phase as they are transported past that material in a stream of gas or liquid (the mobile phase). Other methods of the class involve selective adsorption of particular compounds onto a separation media from a mixture, followed by release of the adsorbed compounds in response to a change in conditions, for example, to selectively remove certain gases from a mixed gas stream.

Silica and alumina are well known for use as stationary phases in column and thin-layer chromatography. Cellulose is also used in the chromatographic separation of certain types of compounds. Other materials are also known for use in chromatography but there remains a need for new stationary phase materials having improved properties. For example, there is a need for stationary phases which can separate with a similar efficiency as known materials such as silica but using less polar mobile phases, thereby reducing the need to use polar solvents which are often less environmentally acceptable, are more difficult to evaporate and are more expensive than solvents of lower polarity, such as the hydrocarbons.

Cellulose and starch are biopolymers produced by plants. Cellulose is a linear polymer of D-glucose with β-linkages, whereas starch is composed of a mixture of amylose and amylopectin, which have α-linkages and the latter is also branched. While the chemical composition of cellulose is largely independent of its plant source, the ratio of amylose to amylopectin in starch varies with the origin. Both polymers are non-toxic, naturally abundant and biodegradable and as such represent a vital renewable resource for sustainable development. Some polysaccharides have been used as stationary phases in chromatography. In particular, cellulose has found application in chromatography for the separation of particular types of compounds and there is a range of celluloses commercially available for use in chromatography. Starches have been used to a much more limited extent and there remains a need for improved materials for use in separations.

The invention provides a method of separation in which compounds are separated on the basis of their interactions with a polysaccharide material having a surface area of at least 20m²/g.

The inventors have found that use of polysaccharide materials having a surface area of at least 20m²/g (referred to herein as high surface area or expanded polysaccharides) gives much improved separation efficiency as compared to some known commercially available chromatography grade celluloses.

The polysaccharide materials have been found to be effective at separating a variety of types of compound. The method of the invention therefore permits a range of separations to be carried out using polysaccharide materials prepared from materials originally derived from natural sources.

Moreover, the polysaccharide materials have, in general, been found to have surfaces of lower polarity than the silica commonly used in column chromatography. Accordingly, when used as stationary phases in chromatographic separations, it is often possible to use solvents of lower polarity than would be required if silica were used.

The compounds in the mixture may, for example, adsorb onto the surface of the polysaccharide material with differing affinities, thereby making it possible to separate the compounds, for example, by chromatography or by controlled adsorption and desorption. Typically, a mixture of compounds to be separated is brought into contact with the polysaccharide material and the compounds are then separated, for example, by elution in the case of chromatography or by controlled desorption from the polysaccharide material. The separated compounds may then be detected and/or collected, as desired.

The method of the invention is applicable to a broad range of separation and controlled absorption-release technologies. The method of the invention is especially suitable for chromatography, for example, column chromatography or thin-layer chromatography, in which the polysaccharide materials are used as a stationary phase. Optionally, the method is a method of chromatography in which the mobile phase is a liquid. Typically, the chromatographic method will include the steps of bringing a mixture of compounds to be separated into contact with the polysaccharide material, and eluting with a suitable eluent or eluents. The separated compounds by then be detected and/or collected, as desired.

The polysaccharide materials tend to decompose when exposed to high temperatures, for example, above 150°C, in air for prolonged periods of time and so are not suitable for use in separation techniques involving temperatures above the decomposition temperature of the polysaccharide, for example, high temperature gas chromatography.

Solvents which swell the polysaccharide material to a significant degree, for example, water, dimethylsulfoxide and dimethylacetamide, should not be used in the separation method of the invention or should be used only in amounts that do not cause significant swelling.

In principle, any polysaccharide material having the required surface area may be used in the method of the invention. Starch and cellulose are favoured materials due to their ready availability in a range of forms and their favourable properties. Native starches and celluloses, in general, have surface areas of only several m²/g. Starches and celluloses therefore, in general, need to be treated to increase their surface area to make them suitable for use in the invention. Some methods of treatment to provide high surface area polysaccharides are described in detail below.

The polysaccharide material will typically have a low water content, for example, a water content of less than 15% by weight. In a favoured embodiment, the polysaccharide material is a starch. High surface area starches have been found to be more stable upon storage for extended periods than high surface area cellulose. Preferably, the starch is a retrograded starch having an amylose content of at least 20%, more preferably at least 30% and most preferably at least 60% by weight. It has been found that the higher amylose starches generally give higher surface area materials after treatment than the lower amylose starches and retrograde more rapidly. Moreover, it was noted that, irrespective of the surface area, greater amounts of amylose in the starch tend to give better separation efficiency. Optionally, the starch is a corn starch, a potato starch, a wheat starch or a rice starch. The starch may be a corn starch. The starch may be a modified starch. Modified starches may be readily prepared by methods known to persons skilled in the art (the word 'modified' as used herein means chemically modified to add substituent groups onto the polysaccharide polymer unless another meaning is clear from the context) and starches having a wide range of modifications are commercially available from, for example, National Starch and Chemical Corporation for use in applications such as food. Preferably, however, the starch is not modified. The starch may be modified or unmodified.

One suitable method for preparing high surface area starches for use in the method of the invention involves the steps of i) gelatinising the starch in the presence of water to give a starch/water gel, ii) allowing the starch to retrograde; and iii) exchanging the water in the retrograded starch gel with a water-miscible non-solvent for starch which has a lower surface tension than water, such as ethanol. The method may further involve exchanging the water- miscible non-solvent with one or a series of non-solvents or mixtures thereof with progressively lower surface tensions whereby the subsequent non-solvent or mixture thereof is miscible with the prior non-solvent or mixture thereof. Advantageously, the high surface area polysaccharide is a starch obtainable by that method. Optionally, the method also includes the step of removing the low surface tension non-solvent, for example, by evaporation (typically under reduced pressure) or supercritical drying to leave the high surface area starch. Alternatively, the starch is kept as a slurry in the non-solvent. In another favoured embodiment, the polysaccharide material is a cellulose. High surface area cellulose suitable for use in the method of the invention is obtainable by a method involving the steps of i) swelling the cellulose in a suitable liquid; and ii) exchanging that suitable liquid with a miscible non-solvent for cellulose which has a lower surface tension than the liquid. The method may further involve exchanging the miscible non-solvent with one or a series of non-solvents or mixtures thereof. Advantageously, the high surface area polysaccharide is a cellulose obtainable by the method. The swelling liquid can be a single solvent a mixture of solvents or a solution and the conditions of step i) should be such as to swell the cellulose without dissolving it. Heating the cellulose in water, for example, at 140°C for 40 hours, has been found to swell the cellulose in a suitable way. Microwave heating has been found to give similar results in less time than conventional heating. In a variation on the method, the cellulose can be swollen by sonicating the cellulose suspension in the swelling liquid with ultrasound at a room or an elevated temperature. This also dramatically reduces the preparation time. The non-solvent (or mixture thereof) for cellulose should, in general, be miscible with the swelling liquid and is advantageously volatile, so that it is easily removed from the cellulose by evaporation. The method may also include the step of drying the cellulose to remove the non-solvent. Alternatively, the cellulose may be kept as a slurry in the non-solvent.

The non-solvents used in the above-described methods of preparing high surface area starch and cellulose may be single liquids, mixtures of liquids or supercritical fluids.

The cellulose used in the treatment may be of any type but is preferably microgranular cellulose or fibrous cellulose. After treatment to increase the surface area, it has been found that the initial microgranular or fibrous structure is substantially retained. The high surface area cellulose may be in the form of non-spherical particles.

The storage stability of cross-linked high surface area polysaccharides may be superior to that of an equivalent non-crosslinked high surface area polysaccharide and therefore methods in which the polysaccharide is cross-linked are preferred in some instances. The polysaccharide material may be a modified polysaccharide but is preferably non-modified and, most preferably, has never been modified.

The high surface area polysaccharide material for use in the method of the invention has a surface area of at least 20m²/g, preferably at least 30m²/g, more preferably at least 50m²/g and most preferably at least 75m²/g. The polysaccharide optionally has a surface area in the range of from 20m²/g to 500m²/g. All surface areas mentioned herein refer to surface area as measured by B.E.T.

The high surface area polysaccharide materials prepared by the methods mentioned above have been found to have surfaces of higher polarity than the original starches and celluloses. The high surface area polysaccharide may have a polarity of greater than 0.55, preferably greater than 0.60.

Advantageously, the polysaccharide material has a pore volume of greater than 0.1cm³/g, more preferably greater than 0.2cm³/g and most preferably greater than 0.4cm³/g.

The pore volume distribution of the polysaccharide material also influences the efficiency of separation in a chromatographic method. Preferably, the polysaccharide material will have a narrow pore volume distribution, for example, in the range of from 0.05 to 1.5 ml/g. Short- term exposure of the polysaccharide material to moisture present in the atmosphere can often improve the efficiency of separation by narrowing the pore volume distribution. However, prolonged exposure or exposure to highly humid atmosphere leads to gradual decrease in porosity which progressively reduces the performance of the polysaccharide materials in chromatography and eventually leads to a complete collapse of the expanded structure and essentially total loss of separation ability. The high surface area polysaccharide should therefore generally be stored in dry conditions, for example, in a dry atmosphere or vacuum sealed or under a hydrophobic liquid. The inventors have found that high surface area starches are more resistant to degradation by moisture than high surface celluloses. The polysaccharide material may be in any form appropriate for the particular method of separation. For column chromatography, the polysaccharide material is preferably in the form of particles having an average diameter in the range of 1 to 500 micrometers (μm). The polysaccharide materials tend to form aggregates on storage and it may therefore be advantageous to break up those aggregates, for example, by sonication for a short period of time, prior to use.

In one embodiment, the polysaccharide material is a stationary phase. Preferably, the stationary phase consists essentially of the polysaccharide material. The stationary phase may comprise at least 50% by weight, more preferably at least 90% by weight and most preferably at least 95% by weight of the polysaccharide material.

The invention also provides a method of chromatography in which the stationary phase comprises a polysaccharide having a surface area of at least 20m²/g.

The invention also provides the use of a polysaccharide material having a surface area of at least 20m²/g for the separation of chemical compounds.

The invention also provides a separation apparatus including a separation element which comprises a polysaccharide material having a surface area of at least 20m²/g. In use of the separation apparatus, a mixture of compounds to be separated is brought into contact with the polysaccharide material. The separation apparatus may be, for example, a chromatography apparatus such as a column chromatography apparatus or a HPLC apparatus. The apparatus may be suitable for use in the method of the invention.

The invention also provides a separation element for use in the separation apparatus, the separation element comprising a polysaccharide material having a surface area of at least 20m²/g. The separation element may be, for example, a pre-packed chromatography column or a thin-layer chromatography plate. Examples

Examples of the method of the invention are given below for the purpose of illustration only and with reference to the drawings in which:

Figure 1 is collection of scanning electron micrograms showing the structure of unexpanded starch (top left) and cellulose (top right) and structural changes that are the result of the expansion.

Figure 2 is a comparison between four charts showing GC responses in arbitrary units on the y-axis against fraction numbers on the x-axis for a column chromatography separation of ferrocene, acetylferrocene and diacetylferrocene using expanded com (upper middle chart) and rice starch (lower middle chart) as the stationary phase in comparison to an unexpanded com starch (top chart) and a silica (bottom chart).

Figure 3 shows the comparison in performance for the separation of ferrocenes between corn starches having different amylose contents and different surface areas (the top chart is high amylose expanded com starch, the middle chart is expanded normal com starch and the bottom chart is expanded waxy com starch).

Figure 4 illustrates the results of separation of natural waxes on a starch packed column (lower chart) compared to one packed with silica (upper chart).

Figure 5 shows three charts illustrating the effect of structural collapse on the separation ability of a corn starch in the ferrocene system (the top chart is expanded com starch SA = 120m²/g, the middle chart is semi-collapsed com starch SA = 50m²/g and the bottom chart is fully collapsed corn starch SA ~ 0m²/g).

Figure 6 shows the separation ability in the ferrocene system for celluloses prepared by different methods as well as native cellulose (from top to bottom the charts are for unexpanded cellulose, ethanol exchanged expanded cellulose, multisolvent hexane exchanged expanded cellulose and multisolvent supercritical CO₂ exchanged expanded cellulose).

Figure 7 illustrates the loss of separating ability of cellulose with decreasing surface area and porosity (the top chart is microgranular expanded cellulose SA = 120m²/g, the middle chart is partially degraded microgranular cellulose SA = 45m²/g and the bottom chart is fully degraded microgranular cellulose.

Examples of starches and their use in separations

Example 1

Native corn starch (Sigma-Aldrich Ltd.) was suspended in distilled water (Ratio: lg starch:5mL water). The mixture was heated under reflux at 110°C for 3 hours. The resultant gel was allowed to retrograde in a closed jar for 3 weeks at 5°C. The resultant semi-solid aqua-gel was successively equilibrated by stirring with 25, 50 and 75%) aqueous ethanol. The solid was then filtered to remove most of the water without letting the solid dry. It was then equilibrated with 100% ethanol to dehydrate the solid. The procedure was repeated 3 times until essentially an anhydrous starch suspension in ethanol was obtained. The suspension was dried for 12hrs in a vacuum oven at 50°C to obtain a free-flowing powder. The properties of the resultant material are shown in table 1 and compared to the unexpanded material in table 2. The effect of expansion on structure of starch is shown in Figure 1.

Example 2

The properties of native rice starch (Sigma-Aldrich Ltd.) prepared as for example 1 are shown in table 1 and compared to the unexpanded material in table 2.

Example 3

The properties of native potato starch (Sigma-Aldrich Ltd.) prepared as for example 1 are shown in table 1 and compared to the unexpanded material in table 2.

Example 4

The properties of native wheat starch (Sigma-Aldrich Ltd.) prepared as for example 1 are shown in table 1 and compared to the unexpanded material in table 2.

Example 5

The properties of waxy com starch (Sigma-Aldrich Ltd.) prepared as for example 1 are shown in table 1 and compared to the unexpanded material in table 2.

Table 1: Properties of starches prepared as for examples 1-5.

Obtained with Reichardt's dye based on literature: Macquarrie DJ, Tavener SJ, Gray GW, Heath PA, Rafelt JS, Saulzet SI, Hardy JJE, Clark JH, Sutra P, Brunei D, di Renzo F, Fajula F; NEW JOURNAL OF CHEMISTRY; 23 (7): 725-731, 1999 Table 2: Properties of native unexpanded materials for examples 1-5.

Obtained with Reichardt's dye based on literature: Macquarrie DJ, Tavener SJ, Gray GW, Heath PA, Rafelt JS, Saulzet SI, Hardy JJE, Clark JH, Sutra P, Brunei D, di Renzo F, Fajula F; NEW JOURNAL OF CHEMISTRY; 23 (7): 725-731, 1999

Example 6 High amylose com starch (Hylon Nib National Starch and Chemical Ltd.) was suspended in distilled water (ratio lg starch : 20mL water) and placed in a sealed vessel and heated at 130°C for 48hrs. The resultant gel was allowed to retrograde at 5°C for two days. A solvent exchange and drying was performed as for example 1 and the resultant material had the properties shown in table 3.

Table 3: Properties of expanded high amylose co starch compared to native high amylose corn starch.

" Obtained with Reichardt's dye based on literature: Macquarrie DJ, Tavener SJ, Gray GW, Heath PA, Rafelt JS, Saulzet SI, Hardy JJE, Clark JH, Sutra P, Brunei D, di Renzo F, Fajula F; NEW JOURNAL OF CHEMISTRY; 23 (7): 725-731, 1999

Example 7 A 20 cm long and 1cm in diameter glass column was packed with the expanded starches prepared as for examples 1-6 and used to separate a 30mg (1/1/1 w/w/w) mixture of ferrocene, acetylferrocene and diacetylferrocene in 0.5mL of hexane/dichloromethane (3/2 v/v). The columns were eluted initially with hexane (6 portions of 5mL) then a 95/5%) hexane/acetone mixture (9 portions of 5mL) and finally acetone (7 portions of 5mL). 23 fractions were collected and analyzed by GC. Best results were obtained with rice starch and com starch, while waxy corn starch gave almost no separation owing to its low surface area and negligible amylose content. Results are summarised in Figure 2 and 3 and compared to unexpanded com starch and silica used in analogous systems. Example 8

A 10cm column filled with corn starch prepared as for example 1 and packed as for example 7 was used to separate a lOOmg mixture of ethyl benzoate (EB) and diethyl phthalate (DEP) (1/1 w/w). The mixture was eluted with 100 % hexane, yielding five hexane fractions, of which, the first contained EB, and fractions 2-5 contained DEP. Both components were eluted with hexane with no need for an increase in the polarity of the eluent. For the analogous system using chromatography grade silica, 95:5 hexane: diethyl ether solvent system was needed to elute EB, in the second fraction, while 100% diethyl ether was needed to recover the DEP component after fifth fraction. The diethyl phthalate interactions were therefore stronger with the silica than with starch.

Example 9

A glass column prepared as for example 7 was used to separate natural pigments extracted from spinach (Horowitz, G., J. Chem. Educ. 11, 263-4, 2000). 2.5g frozen spinach (J. Sainsbury Pic) was allowed to thaw and crushed in a pestle and mortar containing lOmL ethanol. The ethanol was removed by filtration and the dehydrated spinach added to 20mL hexane. The solution was stirred for 5 minutes after which the hexane was decanted and concentrated to lmL. This extract was applied to the column. The column was eluted with hexane (4 portions of 5mL), 9/1 hexane/ethyl acetate mixture (11 portions of 5mL) and ethyl acetate (6 portions of 5mL). 21 fractions were collected. The yellow carotenes eluted in fractions 3-6, followed by the green chlorophylls in fractions 9-15 with the yellow xanthophylls finally eluting in fractions 17 onwards. In this study, carotenes were eluted with 100% hexane. On silica columns a more polar 90/10 v/v hexane/ethyl acetate mixture is needed.

Example 10

Natural waxes can be obtained from plant or animal sources. Lanolin was used as a model complex mixture of waxy compounds. Chromatographic separation using expanded starch gave separations comparable to those obtained using silica. The following solvent system was used for both starch and silica: 3 times the column volume of hexane

6 times the column volume of hexane/diethyl ether (99/1) 5 times the column volume of hexane/diethyl ether (95/5)

5 times the column volume of hexane / diethyl ether (92/8)

8 times the column volume of hexane / diethyl ether (85/15)

5 times the column volume of diethyl ether

A TLC method developed Yamashiro¹, was adapted to analyse the fractions from both expanded starch and silica columns. Five fractions were isolated containing different chemical components of the wax. It was noted that expanded starch provided better separation of some components, especially the sterol esters and waxes under the conditions tested. Results are summarised in Figure 4.

Example 11

A glass column prepared as for example 7 was used to separate limonene and carvone from spearmint oil. lOOmg of the spearmint essential oil (Natural by Nature Oils Ltd.) in 2 ml of hexane was applied to the column. The column was eluted with hexane (5mL portions) until limonene had ceased to elute and then carvone was eluted using ethyl acetate . Fractions were collected and analyzed by GC For starch limonene eluted in fractions 3-5 and carvone in fractions 6-9, but for silica 11 fractions were needed whereby limonene eluted in fractions 6-8 and carvone in fractions 10-11.

Example 12

Three columns were packed with the following: (i) expanded corn starch of surface area of 120m²/g, (ii) semi-collapsed corn starch of surface area of 50m²/g and (iii) totally collapsed corn starch material of minimal surface area. The semi-collapsed material was prepared by exposing a sample of the expanded corn starch to a moist atmosphere for a short period whilst the fully collapsed material was made in the same way using a longer exposure. The starches were used to separate ferrocenes as in example 7. With decreasing surface area the separation - li ability of the starch materials decreased until no separation was observed for the essentially collapsed material. Results are summarised in Figure 5.

Comparative Example 13

A glass column packed with the native com starch (unexpanded) was used to separate ferrocenes as for example 7. Similar separations were carried out for the unexpanded wheat, potato, rice and com starches. The native starch materials did not separate the components in the ferrocene system.

Example 14

A sample of expanded com starch (surface area 133.4 m²/g) as in example 1 was sieved and surface areas recorded by BET nitrogen absorption with the following results shown in Table 4. The starch was made up principally of agglomerates of particles.

Table 4: Surface areas of particle size fractions of expanded com starch.

These fractions could all be shown to separate with the same efficiency as for example 7. Example 15

A column packed as for example 7 was used to separate ferrocenes as for example 7. The column was reused several times, being flushed with solvent between separations. The results did not indicate any significant degradation of the column performance after multiple use.

Example 16

Corn starch expanded as for example 1 was stored in a dessicator under vacuum with silica gel desiccant for at least 10 months with no loss in surface area or change in polarity.

Examples of celluloses and their use in separations

Example 17. Preparation of expanded cellulose

Three different types of chromatographic grade cellulose were used as purchased (Sigma- Aldrich): microgranular, fibrous long and fibrous medium.

The cellulose is initially swollen by heating in a sealed thick glass jar in water at 140°C for a period of at least 40hrs with initial concentration of cellulose to water between 5-8 weight percent. The mixture is then allowed to cool to a minimum of 80°C and it is then equilibrated successively with 25, 50, 75 and 100% ethanol and then washed again 3 times with 100% ethanol. The cellulose is then filtered by gravity filtration until most of the solvent is removed but the solid remains as wet slurry. This slurry is then dried under vacuum at 50°C for at least 12 hrs. Material properties are summarised in Table 5. The effect of expansion on the structure of the cellulose is shown in Figure 1. Table 5: Properties of expanded cellulose

Obtained with Reichardt's dye based on literature: Macquarrie DJ, Tavener SJ, Gray GW, Heath PA, Rafelt JS, Saulzet SI, Hardy JJE, Clark JH, Sutra P, Brunei D, di Renzo F, Fajula F; NEW JOURNAL OF CHEMISTRY; 23 (7): 725-731 , 1999

Example 18. Variation of the method - other solvents

Different solvent exchange methods and drying methods can be applied to obtain a range of materials. After equilibration with ethanol the cellulose can be further equilibrated with acetone and then hexane in the same manner as with ethanol and then dried as in example 20 to obtain a material with a surface area measured by BET of 125m²/g.

Example 19. Variation of the method - supercritical CO₂

The ethanol from the equilibrated cellulose described in example 20 can also be removed by equilibrating with liquid carbon dioxide. Such equilibrated solid can be dried by supercritical point drying with very slow release of the pressure to obtain a surface area measured by BET of up to 200m²/g. Material properties are summarised in Table 6.

Table 6: Properties of cellulose exchanged with supercritical CO₂ and dried under supercritical conditions.

Example 20. Variation of the method - microwave heating

Using microwave heating can dramatically reduce the period of heating necessary to swell cellulose. Typically lOmin at full power (300W) is sufficient to aid swelling. The material can then be solvent equilibrated and dried in any of the above-described ways. The resultant materials have the same physical properties as materials heated by conventional methods and treated as in examples 17-19.

Example 21 Variation of the method - expansion using ultrasound

Ultrasound can dramatically decrease the time necessary for swelling the cellulose. The cellulose as described in example 17 is swollen by suspending it in a 50% aqueous ethanol and sonicating for 3hrs at room temperature. The material can then be solvent equilibrated and dried in any of the above-described ways. The resulting material has a surface area of 65m /g and is structurally substantially the same as the above-described materials.

Example 22. Separation of ferrocene, acetylferrocene and diacetylferrocene Ferrocene, acetylferrocene and diacetylferrocene were used as purchased (Aldrich). A slurry of expanded cellulose in hexane was used to prepare a chromatographic column. 30 mg of a mixture of the three components (1/1/1 w/w/w), dissolved in 0.5 mL of hexane/dichloromethane (3/2 v/v) was loaded onto a ~16 cm by 1 cm expanded cellulose column. Compounds were successively eluted by 30 mL hexane, 45 mL hexane/acetone (95/5 v/v), and 35 mL acetone. 22 fractions of 5 mL were collected and analyzed by GC.

Columns were run using different types of expanded cellulose, such as expanded microgranular prepared as in example 20 (SA-120 m²/g, 5g onto a 16 cm by 1 cm column) and expanded fibrous long (SA~75m²/g, 4 g onto a 17 cm by 1 cm column), expanded fibrous medium (SA~60m²/g, 4 g onto a 18 cm by 1 cm column) and expanded microgranular (SA~65m/g, 4 g onto a 11 cm by 1 cm column) prepared as in example 19. All expanded materials gave good separation for the ferrocene system. Separations for microgranular cellulose prepared by methods described in examples 17-19 are compared to native material in Figure 6.

In comparison to starch and silica, expanded cellulose behaves in a similar way to high surface area expanded starch.

Example 23. Separation of ethyl benzoate and diethyl phthalate

Ethyl benzoate and diethyl phthalate were used as purchased (Aldrich). A 5 g slurry of expanded microgranular cellulose in hexane (prepared following the solvent exchange method as explained in example 20, SA~120m²/g) was used to prepare a chromatographic column. A lOOmg mixture of the components (1/1 w/w) was placed on top of a ~16 cm by 1cm expanded cellulose column. The mixture was eluted with 40 mL hexane/diethyl ether (95/5 v/v) and 30 mL diethyl ether successively. 12 fractions of 5 mL each were collected and analyzed by GC. Ethyl benzoate is collected in fractions 4 and 5 and diethyl phthalate is eluted with diethyl ether and collected in fractions from 8 to 12 . This separation is similar to that obtained with silica as described in example 8 for starch. Example 24. Separation of natural pigments

Spinach extract was prepared from frozen spinach (Horowitz, G., J. Chem. Educ. 11, 263-4, 2000). 5 g slurry of expanded microgranular cellulose in hexane (prepared following the solvent exchange method as explained in example 20, SA~120m²/g) was used to prepare a chromatographic column. The concentrated extract (1 mL in hexane) was loaded onto a ~15 cm by 1 cm column. The mixture was eluted with 20 mL hexane/ethyl acetate (90/10 v/v), 55 mL hexane/ethyl acetate (50/50 v/v), and 30 mL ethyl acetate. Four coloured bands were eluted: carotenes (yellow), pheophytins (grey/green), chlorophylls (green) and xanthophylls (yellow). 21 fractions of 5 ml each were collected. The pigments were identified by UV/vis spectrophotometry. The same solvent system used on silica afforded similar results.

Example 25. Separation of limonene and carvone

Spearmint oil was used as purchased as a natural mixture of limonene and carvone. A 5 g slurry of expanded microgranular cellulose in hexane (prepared following the solvent exchange method as explained in example 19, SA~120m²/g) was used to prepare a chromatographic column. 100 mg of spearmint oil was dissolved in 1 mL hexane and loaded onto a -15 cm by 1 cm column. The column was eluted with 25 mL hexane until limonene had ceased to elute and then carvone was eluted using ethyl acetate (35 mL). 11 fractions were collected . Limonene came in fractions 3 and 4 and carvone eluted from fraction 5 tol 1, while for silica limonene was detected in fraction 6-8 and carvone in fractions 10-11.

Example 26. Degraded cellulose material

Expanded cellulose degrades in a humid environment. In order to examine the functionality of partially degraded expanded cellulose, a column packed with partially degraded microgranular cellulose of BET surface area of 45m²/g prepared according to example 19 and treated by exposure to a moist atmosphere was used for the separation of ferrocene, acetylferrocene and diacetylferrocene. The separation of components works very well even at surface areas of below 50m²/g. Results are shown in Figure 7 and are compared with essentially collapsed material.

Example 27. Normal Cellulose (unexpanded)

Unexpanded cellulose was tested for column chromatography in order to compare it with the expanded one. Three types of chromatography grade cellulose i.e. microgranular, fibrous long and fibrous medium (Sigma-Aldrich) were used in the ferrocene system. All the unexpanded materials gave poor separations.

Example 28. Reuse of the material

Columns can be reused. The reuse of columns was tested in the ferrocene system. No significant reduction in separation efficiency was observed.

Claims

1. A method of separation in which compounds are separated on the basis of their interactions with a polysaccharide material having a surface area of at least 20m²/g.

2. A method as claimed in claim 1 in which the method is a method of chromatography and the polysaccharide material is used as a stationary phase.

3. A method as claimed in claim 2 which is a method of column chromatography.

4. A method as claimed in any of claims 1 to 3 in which the polysaccharide material is selected from the group consisting of starches and celluloses.

5. A method as claimed in any of claims 1 to 4 in which the polysaccharide material is a starch.

6. A method as claimed in claim 5 in which the starch is a retrograded starch having an amylose content of at least 20% by weight.

7. A method as claimed in claim 5 or claim 6 in which the starch is a modified or non-modified starch.

8. A method as claimed in any of claims 1 to 4 in which the polysaccharide is a cellulose obtainable by swelling the cellulose in a suitable liquid, and exchanging the suitable liquid with a miscible non-solvent for cellulose which has a lower surface tension than the liquid.

9. A method as claimed in claim 8 in which the cellulose is exposed to ultrasound during the swelling stage.

10. A method as claimed in claim 8 or claim 9 which includes the further step of drying the cellulose.

11. A method as claimed in any of claims 1 to 4, 8, 9 or 10 in which the cellulose is a fibrous cellulose.

12. A method as claimed in any of claims 1 to 11 in which the polysaccharide material is non-modified.

13. A method as claimed in any of claims 1 to 11 in which the polysaccharide material is modified.

14. A method as claimed in any of claims 1 to 13 in which the polysaccharide material has a surface area of at least 30m /g.

15. A method as claimed in any preceding claim in which the polysaccharide material has a pore volume of at least 0.1 cm³/g.

16. A method as claimed in any preceding claim in which the polysaccharide material has an average particle diameter in the range of from 1 to 500 micrometers (μm).

17. A method as claimed in any of claims 1 to 16 in which the polysaccharide material has a pore volume distribution in the range of from 0.05 to 1.5mL/g.

18. A method of chromatography in which the stationary phase comprises a polysaccharide material having a surface area of at least 20m²/g.

19. A method as claimed in claim 18 which is a method of column chromatography.

20. Use of a polysaccharide material having a surface area of at least 20m²/g for the separation of chemical compounds .

21. A separation apparatus including a separation element which comprises a polysaccharide material having a surface area of at least 20m²/g.

22. A separation element for use in the apparatus of claim 2 b the separation element comprising a polysaccharide material having a surface area of at least 20m /g.

23. A separation element as claimed in claim 22 which is a pre-packed chromatography column.

24. A separation element as claimed in claim 22 which is a thin-layer chromatography plate.