BOCIAN, Szymon (Otmianowo 29, PL-87- 581 Boniewo, PL)
BUSZEWSKI, Bogusław (ul. Konwaliowa 4, Stary Toruń, PL- 87-134 Zławieś Wielka, PL)
BOCIAN, Szymon (Otmianowo 29, PL-87- 581 Boniewo, PL)
| We claim: 1. A method of manufacturing selective alkyl-phosphate stationary phases for liquid chromatography of the general formula 1, wherein alkyl dichlorophosphate obtained in a reaction of alcohol dissolved in a non-polar solvent with phosphorus(V) trichloride oxide dissolved in a non-polar organic solvent undergoes a reaction with silica gel previously modified with aminosilane in the presence of an activator. 2. A method according to claim 1, wherein alkyl dichlorophosphate is obtained in the reaction of alcohol with phosphorus (V) trichloride oxide. 3. A method according to claim 1, wherein triethylamine is used as an activator and organic toluene - as a solvent. 4. A method according to claim 1, wherein an alcohol to phosphorus (V) trichloride oxide ratio is in the range between 1:2 and 4:2. 5. A method according to claim 1, wherein the product is purified from an excess of substrates on a filter funnel with a sintered disc of G4 porosity by rinsing with toluene, methanol, and n-hexane, respectively. |
The object of the present invention is the method of selective alkyl-phosphate phases functioning as column packing manufacture for liquid chromatography and related techniques.
From the medical and pharmaceutical point of view, alkyl-phosphate stationary phases can imitate biological membranes. They are important for predicting properties of newly developed pharmaceuticals, examining their transport through biological membranes as well as interacting with those membranes.
The aim of the invention is to develop the synthesis of selective, stable, and reproducible column packing for liquid chromatography applied in reverse phases systems as well as liquid-solid extraction columns used for separating organic compounds of varied polarities. The advantage of the method is high determination selectivity.
According to the invention, the method is based on manufacturing alkyl-phosphate stationary phases designed for liquid chromatography with the general formula 1 in which silica gel, previously modified with aminosilane, undergoes a reaction with a alkyl-phosphate ligand in an aprotic organic solvent, favourably in toluene, in the presence of triethylamine as an activating agent. The alkyl-phosphate ligand is obtained in the reaction of alcohol with phosphorus(V) trichloride oxide. The sorbent obtained is purified from the excess of substrates on a filter funnel with a sintered disc of G4 porosity. The sorbent is rinsed with toluene, methanol, and n-hexane, respectively. As a result, a hydrophobic stationary phase containing a phosphate group and residual amine groups is received, owing to which chromatographic material gains biological membrane properties.
In formula 1, R denotes an alkyl chain.
By changing the length of an alkyl chain, alkyl-phosphate stationary phases of different hydrophobicity can be obtained. Chromatographic properties of these phases enable analysing compounds of varied polarities, of both acidic and basic character as well as, due to controlled hydrophobicity, non-polar compounds. It results from the structure of the stationary phase containing an alkyl chain and a phosphate group. The phosphate group can dissociate to gain the negative charge which is related to the pH of a mobile phase. The above mentioned stationary phase properties make it applicable also in electro- chromatography or solid phase extraction.
The properties of the packing described allow isolating substances such as medicines or narcotics from natural matrices such as blood and urine or pesticides residue from food products.
The method based on the invention was exemplified below. The examples, however, do not limit the range of applications protected by the patent.
An elemental analysis, an analysis of infrared and nuclear magnetic resonance spectra confirmed the existence of the functional groups expected on the surface of silica gel
Example 1.
In a reactor that prevents reactants from contacting the surrounding, a 5g sample of silica gel modified with y-aminopropyl ligands is dried in vacuum 10 '3 MPa at temperature 120°C for 6 hours in oil bath.
The alkyl-phosphate ligand is obtained by stirring a solution of 0.8 ml (9 mmol) phosphorus(V) trichloride oxide (POCI3) in 10 ml anhydrous toluene and 1.7 ml (9 mmol) octanol dissolved in 10 ml anhydrous toluene, for 120 minutes, with a magnetic stirrer.
Subsequently, the solution containing alkyl-phosphate ligands and 4 ml (30 mmol) triethylamine is added to the reactor with dried, modified silica gel. The reactions are carried out at 100°C for 18 hours. The reaction product is separated from the mixture obtained as a result of the reaction on a sintered glass G4. Then, it is rinsed with toluene, methanol, and hexane, respectively, and finally dried.
Example 2.
In a reactor that prevents reactants from contacting the surrounding, a 5g sample of silica gel modified with γ -aminopropyl ligands is dried in vacuum 10 "3 MPa at temperature 120°C for 6 hours in oil bath.
The alkyl-phosphate ligand is obtained by stirring a solution of 0.8 ml (9 mmol) phosphorus (V) trichloride oxide (POCI3) in 10 ml anhydrous toluene and 2.4 ml (9 mmol) octadecanol dissolved in 10 ml anhydrous toluene, for 120 minutes, with a magnetic stirrer.
Subsequently, the solution containing alkyl-phosphate ligands and 4 ml (30 mmol) triethylamine is added to the reactor with dried, modified silica gel. The reactions are carried out at 110°C for 18 hours.
The reaction product is separated from the mixture obtained as a result of the reaction on a sintered glass G4. Then, it is rinsed with toluene, methanol, and hexane, respectively, and finally dried.
According to Example 1 and 2, the application of alkyl-phosphate phases for selected organic compounds determination (e.g. alkyl benzene derivatives, polycyclic aromatic hydrocarbons, or beta-adrenolytic drugs) allows for their complete separation. Typical mobile phases used in reverse phases systems in liquid chromatography can be applied as an eluent.