High Performance Liquid Chromatography comprises a liquid phase (mobile phase) and a solid phase (stationary phase). The mobile phase is pumped through a metal cylinder (column) which contains the stationary phase. Stationary phases can be split into two main groups; normal phase and reverse phase. Normal phase stationary phases are typically made of pure silica and are polar. Reverse phase stationary phases possess a hydro-carbon chain (Cl-C18) bonded onto the silica creating a surface that is non-polar. and are most commonly employed.

Separation on a reverse phase column occurs due to partitioning of analytes between the two phases. The analytes are injected into the flow of mobile phase, reach the column via tubing and are then adsorbed onto the stationary phase. The analytes begin to partition between the mobile and stationary phases as they move further down the column, in the direction of flow, until they elute from the column. After leaving the column they pass out to waste via an absorbance detector which records their response. More soluble (hydrophilic) polar species elute from the column more quickly than more non-soluble (hydrophobic) non-polar species. In some cases hydrophobic non-polar species can remain permanently attached to the hydrophobic stationary phase. High levels of organic solvents are needed to elute these insoluble species. Hydrophilic polar species have a weak adsorption onto the stationary phase. If a mobile phase containing a high level of organic solvent is used these species will immediately elute off the column (unretained) and will not be separated (unresolved). It is therefore difficult to use a single mixture of aqueous and organic solvents to successfully separate mixtures of solutes having widely different solubilities/polarities. Use of a single mixture in a fixed ratio of aqueous- organic solvents is known as isocratic chromatography.

The technique of gradient HPLC has been developed to overcome the difficulty of separating mixtures of water soluble and insoluble compounds. In gradient HPLC the composition of the solvent pumped through the column is continuously varied throughout the separation. This is achieved by the pump being attached to two or more reservoirs of liquid. One reservoir contains a primarily aqueous polar solvent whilst the other contains a solvent containing high levels of organic non-polar solvent. The pump is programmed to draw specific mixing ratios from the two reservoirs. Initially the pump is programmed to pump high levels of the solvent containing low levels of organic solvent. The water soluble compounds are gradually washed off the column and are resolved. The ratio of solvent is altered automatically over time to increase the level of the organic solvent-rich solvent. As the levels of organic solvent in the column are increased the insoluble compounds can be eluted and separated. This movement from a primarily aqueous mobile phase to an organic solvent-rich solvent is termed a gradient. The steepness of the

gradient from low to high organic solvent levels is an important parameter in the method and affects almost every aspect of the separation.

Microemulsions are typically solutions of water which contain nanometre sized suspended droplets of oil. Surfactants are typically added to lower the surface tension between the oil and the water allowing the droplets to form. Often an additional component such as a co-surfactant is added, to further reduce the surface tension of the system. Microemulsions have several industrial uses and their creation and application has recently been reviewed (Kumar, P. and Mittal, K. L. , Handbook of Microemulsion Science and Technology, Marcel Dekker Inc. , New York, 1999, isbn 0-8247-19794).

Microemulsions have been previously utilised in analytical chemistry to achieve resolution of components in sample solutions. They have been used to achieve separations using capillary electrophoresis (Altria, K. D. (2000) J. Chromatogr. A 892,171-186) and high performance liquid chromatography (HPLC) (Berthod, A. , Nicolas, O. and Porthault, M. (1990) Anal. Chem.<BR> <P>62,1402-1407 ; Berthod, A. , Laserna, J. J. and Cerretero, I. (1992) J. Liquid Chromatography 15, 3115-3127; Berthod, A. and De Carvalho, M. (1992) Anal. Chem. 63,2267). However, the use of microemulsions in HPLC was restricted to isocratic separation only and no further work has been performed in this area as the use of microemulsions was not shown to offer advantages over the prevailing technologies. For example, manipulation of microemulsions is well known to be extremely problematic because unless the ratio of oil-water-surfactant is accurate the microemulsion will either not initially form or will decompose into separate oil and water layers, which is extremely undesirable during a sensitive technique such as chromatography.

Furthermore, the lack of resolving power and inability to handle solvents covering a range of solubilities were noted as additional drawbacks.

Surprisingly, in contradistinction to these reports, the inventors of the present invention have developed a novel use of a stable microemulsion complex in place of traditional mobile phases in gradient HPLC to develop an enhanced technique for separation of analytes on a standard HPLC instrument.

Thus, as a first aspect of the present invention we provide a use of a microemulsion complex comprising: (a) a high ionic strength aqueous solution; (b) oil; and (c) one or more surfactants; in analyte separation during gradient high performance liquid chromatography.

It will be appreciated that all references to microemulsion or microemulsion complex within the specification refer to a mixture of components (a), (b) and (c) described above. More specifically, a sample of high ionic strength aqueous solution containing oil in the form of nanometre sized droplets as a consequence of the presence of one or more surfactants.

The ability to form a stable microemulsion is dependent on the surface tension of the oil core.

Examples of oils with a high surface tension include octane and heptane. Examples of oils with a low surface tension include amyl alcohol and ethyl acetate.

The inventors of the present invention have found that where the oil present within the microemulsion has a high surface tension (eg. octane and heptane), the presence of a co- surfactant (such as short chain aliphatic alcohols eg. butanol or propanol) is required and by contrast where the oil present within the microemulsion has a low surface tension (eg. amyl alcohol and ethyl acetate), the presence of a co-surfactant is not required.

It will be appreciated that avoidance of the use of a further organic solvent, such as a co- surfactant is of benefit in terms of reduced costs and environmental impact. Therefore, preferably, the oil will have a low surface tension.

Preferably, the oil will be present within the microemulsion at a concentration of 0.1 to 10% w/w, more preferably around I % w/w.

Preferably, the oil will be immiscible with water.

Preferably, the oil is amyl alcohol or ethyl acetate, more preferably amyl alcohol.

It will be appreciated that the surfactant should be capable of lowering the surface tension between the oil and the water allowing the microemulsion complex to form.

Examples of surfactants which can be used according to the present invention are anionic surfac- tants such as oleic acid, non-ionic surfactants such as sorbitan trioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate (Polysorbate 80), natural lecithin, oleyl polyoxyethylene (2) ether, stearyl polyoxy- ethylene (2) ether, lauryl polyoxyethylene (4) ether, block copolymers of ethylene oxide and of propylene oxide, synthetic lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glyceryl monooleate, glyceryl monostearate, glyceryl monoricinoleate, glyceryl ricinoleate 30 OE, glyceryl ricinoleate 60 OE, cetyl alcohol, stearyl alcohol, polyethylene glycol 400, glyceryl monolaurate or sodium dodecyl sulphate (SDS), or cationic surfactants, such as cetylpyridinium chloride or benzalkonium chloride. Other examples of surfactants include synthetic phosphatides eg. distearoylphosphatidylcholine.

Preferably, the surfactant will be present within the microemulsion at a concentration of 0.5 to 3% w/w, more preferably around 1% w/w.

Preferably a single surfactant will be used.

Preferably, the surfactant is sodium dodecyl sulphate (SDS).

The water phase of the microemulsion complex is provided with a high ionic strength, eg. an ionic concentration of 10 to 500mM. Suitable salts include formic acid, sodium chloride or sodium tetra-borate. The high performance liquid chromatography will employ a gradient of high ionic strength aqueous solution to microemulsion, preferably a gradient of aqueous formic acid, sodium chloride or sodium tetra-borate. The aqueous solution is preferably in the range of 10 to 500mM.

As a further aspect of the present invention we also provide a method of separating a compound from a solution mixture comprising more than one compound which comprises the following steps: (a) application of said solution mixture to a high performance liquid chromatography column; and (b) elution of said compound from said column using gradient high performance liquid chromatography with a microemulsion complex as defined herein above as the mobile phase.

The separation achieved in gradient HPLC is dependent on a number of factors, such as composition of the mobile phase, flow rate, steepness of the gradient profile of the organic solvent, stationary phase coating, additives in the mobile phase, dimensions of the column, particle size of the silica packing, reproducibility of the packing between manufacturers and individual columns, operating temperature and selectivity modification by the use of additives, among others. However, it will be appreciated from the Examples that the advantages of the present invention are that the number of factors which are influential in effective separation are reduced by the use of a stable microemulsion complex according to the present invention.

For example, the technique is both reproducible and repeatable, it is more robust than existing gradient HPLC techniques as it is less impacted by factors such as column-to-column variablilty and temperature effects. The separation process can be conducted using standard HPLC equipment and commercially available reagents and the preparation of the microemulsion involves the use of standard laboratory chemicals and equipment.

The invention is illustrated in accordance with the following Examples: Examples Example 1: Isocratic HPLC separation of neutral compounds using a microemulsion composition An initial experiment was performed to investigate the separation of solutes consisting of mixtures of compounds with differing solubilities by using a range of neutral aromatic compounds and a microemulsion containing SDS-octane-butan-1-ol-borate buffer. The resultant mixture was pumped through a C 18 reversed phase HPLC column under the following experimental conditions:

Microemulsion: 33g SDS, 8. 1g octane, 66. 1g butanol dissolved in 1 litre of 10mM sodium tetra-borate then sonicated for 30 minutes; Chromatography details: 2. 1mm x 5cm long, C18 3pm column, flow rate 0. 5ml/min, 30°C, 240nm; Sample: lOgI injection of neutral aromatic compounds testmix (containing uracil, 2- acetofuran, acetanilide, acetophenone, m-cresol, propiophenone, benzofuran, butyrophenone, valerophenone, hexanophenone, heptanohenone and octophenone) dissolved in 50: 50 MeOH: Water.

The results of this separation may be seen in Figure 1 wherein all compounds were observed to elute from the column in the first 2 minutes. Therefore, this experiment confirmed previous reports that use of microemulsions in isocratic chromatography achieve poor resolution and separation efficiency.

Attempts were made to convert the isocratic separation to a gradient HPLC method by placing water in one of the reservoirs (reservoir A) and the microemulsion in the other reservoir (reservoir B). The pump was programmed initially to pump 100% from reservoir A and then gradually increase the % pumped from reservoir B until the pump delivered 100% of B. This proved unsuccessful as the initial high content of water disrupted the microemulsion structure whilst in the pump. This in turn led to a cloudy two phase suspension being formed in the column which could not produce acceptable chromatography.

Example 2: Gradient HPLC separation using a microemulsion composition (a) Separation of neutral compounds Separation of the same mixture of neutral aromatic compounds used in Example 1 was attempted using a gradient of water and microemulsion. The problems observed in Example 1 with the cloudy two phase suspension were attempted to be overcome by introducing a high ionic strength into the microemulsion, therefore, the experiment in Example 1 was repeated but 500mM NaCI was instead added to the water in reservoir A and the following experimental conditions were used: Microemulsion: 33g SDS, 8. 1g octane, 66. 1g butan-1-ol dissolved in 1 litre of IOmM sodium tetra-borate; Chromatography details: 2. 1mm x 5cm long, C8 3pm column, flow rate 0. 5ml/min, 30°C, 240nm, Reservoir A: 0. 5M NaCI, Reservoir B: Microemulsion, 95% A for 2 mins going to 25% A at 10min hold for 5 minutes; Sample: 21il injection of neutral aromatic compounds testmix (containing uracil, 2-acetofuran, acetanilide, acetophenone, m-cresol, propiophenone, benzofuran, butyrophenone, valerophenone, hexanophenone, heptanohenone and octophenone) dissolved in 50: 50 MeOH: Water.

It was observed that the microemulsion did not decompose into 2 layers (as in Example 1 above) and from Figure 2 it can be seen that the microemulsion produced an efficient separation of the test solutes.

(b) Separation of basic compounds The general applicability of gradient microemulsion liquid chromatography to basic species covering a wide range of solubilities and polarities was investigated using the following experimental conditions: Microemulsion: 33g SDS, 100g of amyl alcohol in llitre of water, dissolved in 1 litre of water with 0.05% formic acid; Chromatography details: A = Water + 0.05% formic acid, B = microemulsion, 0. 5ml/min 2. 1mm 5cm Hypersil C8, 5% A for Imin then 70% at 15min ; 240nm detection.

Sample: 2111 basic compound testmix (containing norepinephine, isoproterenol, atenolol, pindolol, lignocaine, salmeterol, labetalol and bupivacaine) dissolved in 50: 50 MeOH: Water.

The results of this experiment can be seen in Figure 3, wherein it can be seen that efficient and selective separation was obtained.

Example 3: Rapid gradient HPLC separation of neutral compounds using a microemulsion composition Rapid separations were possible using a gradient of a microemulsion complex according to the invention in high performance liquid chromatography as it was possible to employ a very steep gradient over a short time period. For example Figure 4 shows separation of a range of neutral drugs within 4 minutes using a gradient from 5% B to 100% B over a duration of 2 minutes, under the following experimental conditions: Microemulsion: 33g SDS, 100g of amyl alcohol in llitre of water, dissolved in 1 litre of water with 0.05% formic acid; Chromatography details: A = Water + 0.05% formic acid B = microemulsion, 0. 8ml/min, 2. 1mm 5cm Hypersil C8,10% of B then 100% at 3min. Detection at 240nm.

Sample: 2ml injection of neutral compound test mix (containing phenacetine, acetanilide, acetophenone, propiophenone, butyrophenone, valerophenone, hexanophenone, heptanophenone) all dissolved in 50: 50 water: methanol.

The peaks identified in Figure 4 were 0. 60min : phenacetine; 0.84min : acetanilide; 1. 13min : acetophenone; 2. 04min : propiophenone; 2.76min : butyrophenone; 3. 21min : valerophenone; 3. 46min : hexanophenone; 3. 60min : heptanophenone).

Example 4: Gradient HPLC separation of salbutamol using a microemulsion composition Salbutamol (also known as albuterol) is a respiratory pharmaceutical known to have a number of closely associated and related impurities. Therefore, this experiment attempted to separate

salbutamol from a range of closely related impurities using the following experimental conditions: Microemulsion: 33g SDS, 100g of amyl alcohol in llitre of water, dissolved in 1 litre of water with 0.05% formic acid; Chromatography details : A = Water + 0.05% formic acid B = microemulsion, 0. 8ml/min 2. 1 mm 5cm hypersil C8 10% then 100% at 3min.

Sample: 0.2mg/ml salbutamol with 8 related impurities each at a concentration of 0.002mg/ml dissolved in water/MeOH.

The results of this separation experiment can be seen in Figure 5 wherein it can be observed that the technique of microemulsion liquid chromatography can be used to discriminate between structurally similar solutes (such as salbutamol and its related impurities).

Example 5: Reproducibilitv of microemulsion liquid chromatography The reproducibility of the separation achieved by microemulsion liquid chromatography was assessed to ensure that the separation profiles remained consistent across a number of repeated injections using the following experimental conditions: Microemulsion: 33g SDS, 100g of amyl alcohol in llitre of water, dissolved in 1 litre of water with 0.05% formic acid; Chromatography details: A = Water + 0.05% formic acid B = microemulsion, 0. 6ml/min, 2. lmm 5cm Hypersil C8,10% B for 3min then 60% at 15min.

Sample: 2p1 injection of each of above mentioned acidic, basic and neutral test compounds dissolved in 50: 50 water/MeOH.

The results of this reproducibility experiment can be seen in Figure 6 wherein the overlay of the first and 20 injections possess an almost identical profile, confirming the reproducibility of microemulsion liquid chromatography.

Example 6: Effect of temperature upon separation efficiency of microemulsion liquid chromatography Retention times are reduced in conventional HPLC when the temperature is increased as the partitioning onto the stationary phase is reduced. This effect means that a temperature control device (column oven) is often required to maintain a constant temperature to ensure consistent retention times. This experiment therefore investigated the effect of temperature on the separation, which was evaluated over a column oven temperature range of 15°C to 60°C using the following experimental conditions: Microemulsion: 33gSDS, 100gofamylalcoholin llitreofwater, dissolvedin 1 litreofwater with 0.05% formic acid, Chromatography details: A = Water + 0.05% formic acid, B = microemulsion, 0. 5ml/min, 2. 1mm 5cm Hypersil C8,5% A for Imin then 70% at 15min.

Sample: 2gl injection of each of above mentioned acidic, basic and neutral test compounds dissolved in 50: 50 water/MeOH.

The results may be seen in Figure 7, wherein it may be observed that as a consequence of an identical separation profile at both 15°C and 60°C, the separation was largely unaffected by variation in the temperature.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word'comprise', and variations such as'comprises'and'comprising', will be understood to imply the inclusion of a stated integer or step or group of integers but not to the exclusion of any other integer or step or group of integers or steps.