The method of purification of insulin
(57) Abstract:Usage: in the medical industry for more insulin. The inventive method of purification of insulin includes sorption insulinosoderzhaschego raw sulfonic cation exchanger with pre sulfonic cation exchanger with a solution of salt of acetic acid with subsequent zinc deposition and citrate crystallization. Use sulfonation with a grain size of 400-600 μm. Processing sulfonic cation exchanger conduct of 0.15-0.25 M solution of ammonium acetate or sodium acetate at pH 70-7,3. Sorption was carried out from 0,04-0,06 M solution of ammonium acetate or sodium acetate at pH 7.4 and 7.6. Elution conduct of 0.10-0.25 M solution of ammonium acetate or sodium acetate at pH of 6.9 to 7.1. table 1. The invention relates to the medical industry and can be used to obtain preparations of insulin used in the treatment of diabetes mellitus.A known method of purification of insulin by chromatography on anion exchanger Dowex 1X2 and QAE-Sephadex A-25  the Disadvantage of this method is the low yield of purified insulin (40-60% ), as well as use in chromatography of complex water-organic mixtures containing 60% of combustible and toxic components, methanol or etano the percentage recrystallization.In another method of purification of insulin  cation exchange chromatography on slightly acid cation exchanger (amberlite CG 50) is preceded by five additional stages: citrate crystallization, chromatography on Sephadex G-50, planting sodium chloride, isoelectric precipitation and the second citrate crystallization, and the allocation of purified insulin after cation-exchange chromatography involves three stages of planting sodium chloride, isoelectric precipitation and citrate crystallization. The disadvantage of this method, in addition to the complexity and labor intensity is low, the output of insulin (53%), as well as use with cation-exchange chromatography of complex buffer comprising of 0.13 M sodium phosphate, 7M urea and 1% n-butanol.A known method of purification of insulin  (prototype) by sorption insulinosoderzhaschego materials on macroporous sulfonic cation exchanger with a particle size of 20-60 μm, mixed with inert silica filler (particle size of 40-120 μm) in equal volume ratio, followed by washing the mixture of microdisperse and filler 0,20-0,30 M oxymoronical buffer at pH 5,2-6,4 and desorption of insulin of 0.02-0.3 M buffer solution (Tris; phosphate buffer; ammonium acetate) at pH 6.4 and 7.6.To insufficient is nalina after cleaning (64-70% ); high antigenicity of insulin (the content of proinsulin to 0.5%). In the prototype is not specified type of purified porcine insulin or bovine K. R. S.), although different types of insulin may differ materially as output and purification in the same conditions. In addition, the prototype did not specify methods of analysis of the fractional composition, neither the fractional composition of both the original and the purified insulin.Process chromatography in the prototype possible because of the small grain size of the cation only in the presence of the filler. The presence of filler eliminates the possibility of regenerating the resin in the mode of fluidization, because the resin and the filler will delaminate. Thus, the use of filler in the prototype is a necessary measure, since in its absence chromatography will occur with very low velocities.An object of the invention is to eliminate the use of filler in chromatography, the increased output of insulin and reduce its antigenicity.The problem is solved in that the purification of insulin, including sorption insulinosoderzhaschego raw sulfonic cation exchanger with pre sulfonic cation exchanger R is m deposition and citrate crystallization, use sulfonation with a grain size of 400-600 μm, processing sulfonic cation exchanger conduct of 0.15-0.25 M solution of ammonium acetate or sodium acetate at pH 7.0-7,3, sorption was carried out from 0,04-0,06 M solution of ammonium acetate or sodium acetate at pH 7.4 and 7.6, and elution conduct of 0.10-0.25 M solution of ammonium acetate or sodium acetate at pH 6.9 and 7.1.In the described conditions is provided, apparently, the differentiation of the interaction of negatively charged groups of a strong cation exchanger with oppositely charged groups (mainly ammonium and guanidinium arginine residues) shared substances at different pH values and concentrations of acetic acid sodium salt, or ammonium.When using sodium acetate or ammonium acetate in the described conditions, the yield of purified porcine insulin is respectively 90% and 79% of These outputs is significantly more than the release of insulin when cleaning by known methods (50-70%).Fractional composition of the purified insulin pigs and K. R. S. was estimated along with the disc-electrophoresis methods high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). Received data (b) are presented in table 1.As follows from the insulin from 88,7% to >96% in the case of insulin K. R. S. from 84.3 percent up to 95-96% of the content of the main antigenic impurities of proinsulin to insulin pigs is reduced from 4.5% to 0,04-0,05% i.e. reduced by approximately 100 times, and insulin K. R. C. number of proinsulin is reduced approximately 50 times from 4.9 to 0.1% at the same time in the purified insulin significantly reduced the content of desamethasone and other impurities.The method is illustrated by the following examples.Example 1. of 3.9 g of Sodium salt pork insulin, fractional composition of which is presented in the table.1, dissolved in distilled water upon acidification to a pH of 2.6-3,018% hydrochloric acid and the solution diluted to a value of optical density D2776,5-7,0 O. E. (optical unit). To the solution was added sodium acetate at the rate of 0.5 M, 5% sodium hydroxide solution (12,5% ammonium hydroxide solution for examples 2 and 4) establish a pH 7,500,05.The column with sulfonic cation exchanger KU-23 (grain size 400-600 μm), balanced 0.2 M solution of sodium acetate with a pH of 7.2, serves freshly prepared solution of sodium salt pork insulin at a rate of 0.5 Vnumber/h (Vnumberthe column volume), and then with the same speed serves 0.2 M sodium acetate solution with a neutral pH value. Collection inset (after passing the maximum on the curve of elution) when values of D2770,5-0,6 O. E.Fractions containing data disc electrophoresis in polyacrylamide gel of purified insulin, unite, dilute with water to D2773,0-3,5 O. E. and 18% hydrochloric acid to establish a pH of 3.0-3.5. Then add a 10% solution of zinc acetate and precipitated with zinc insulin at pH 6.0 to 6.1. Sediment zinc-insulin is filtered off and crystallized from a citrate buffer in a known manner.Get 3.5 g of crystalline porcine zinc insulin (yield of 89.7%), fractional composition of which is presented in table.1.Example 2. Cleaning 3.6 g of sodium salt pork insulin with the use of ammonium acetate is carried out in conditions analogous to example 1, but for equilibration of the column, dissolution of insulin and subsequent elution instead of sodium acetate using ammonium acetate.After cleaning they receive 2.85 g of crystalline zinc insulin (output 79,2%), fractional composition of which is presented in table. 1.Example 3. Cleaning charge of 40.0 g of sodium salt of insulin bovine K. p. C. ). Cleaning requirements are similar to the conditions described in example 1 (using sodium acetate).After cleaning get to 33.7 g of crystalline zinc-inst of 3.85 g of sodium salt of insulin K. R. S. Cleaning requirements are similar to the conditions described in example 2 (using ammonium acetate).After clearing the receive of 2.81 g of crystalline zinc insulin K. R. C. (yield 73%), fractional composition of which is presented in table. 1. The method of purification of insulin, including sorption insulinosoderzhaschego raw sulfonic cation exchanger with pre sulfonic cation exchanger with a solution of salts of acetic acid and elution of insulin solution of salt of acetic acid with subsequent zinc deposition and citrate crystallization, characterized in that use sulfonation with a grain size of 400-600 μm, processing sulfonic cation exchanger conduct of 0.15-0.25 M solution of ammonium acetate or sodium acetate at pH 7.0-7,3, sorption was carried out from 0,04-0,06 M solution of ammonium acetate or sodium acetate at pH 7.4 and 7.6, and elution conduct of 0.10-0.25 M solution of ammonium acetate or sodium acetate at pH 6.3, and 7.1.
FIELD: chemical engineering; medical engineering.
SUBSTANCE: method involves plotting two chromatograms one of which is based on radioactivity (No 1) and the other one on ultraviolet absorption (No 2) or on radioactivity (No 1) and on fluorescence (No 2) and chromatogram specific relative to ultraviolet absorption (No 3) or relative to fluorescence (No 3). Material quality is estimated to be the more high the more close studied labeled compound peak shape is to trapezoid shape on the third chromatogram.
EFFECT: high accuracy of the method.
FIELD: analytical chemistry, ecology, in particular controlling of environmental air.
SUBSTANCE: claimed method includes aspiration if air sample through chemosorbtive medium, elution of formed dimethylamine salt, eluate closure with alkali, and gas chromatography analysis of gas phase with flame-ionization detection. Dimethylamine salt elution from adsorbent is carried out with 1 cm3 of distillated water; closured with alkali eluate is held in thermostat for 5 min; and as filling in separating chromatography column chromosorb 103, containing 5 % of PEG-20000 and treated with 20 % hexamethyldisilazane solution is used.
EFFECT: method for dimethylamine detection with improved sensibility and accuracy.
FIELD: chemical industry.
SUBSTANCE: during process of taking sample from technological pipe-line, absorption of water vapors and nitrogen oxides (II) and (IV) are conducted simultaneously. For the purpose the chemical agents are used which don't absorb nitrogen oxide and don't react with it. Chromatographic measurement of volume fraction of nitrogen oxide (I) is carried out by means of industrial chromatograph having heat-conductance detector by using column of thickness of 5 m and diameter of 3 mm. The column is filled with polysorbent; temperature of column's thermostat is 20-30 C and temperature of evaporator is 100C. Hydrogen is used as a gas-carrier. Concentrations of nitrogen oxide, measured by the method, belong to range of 0, 05-0, 50% of volume fraction. Method excludes aggressive affect of corrosion-active components on sensitive parts of chromatograph. Method can be used under industrial conditions for revealing factors influencing process of forming of nitrogen oxide at the stage of catalytic oxidation of ammonia and searching for optimal conditions for minimizing effluent of ammonia into atmosphere.
EFFECT: high reproduction; simplification; improved efficiency of operation.
FIELD: oil and gas production.
SUBSTANCE: aim of invention is estimating expectations for oil and gas of oil-source rock areas. For that aim, sampled rock is treated to isolate organic substance soluble in organic solvents, after which organic substance is chromatographed to detect 4-methyldibenzothiophene and 1-methyldibenzothiophene. When ratio of 4- to 1-isomer exceeds 0.9 rock is regarded as ripened.
EFFECT: increased determination reliability and rapidity.
SUBSTANCE: in the method, hard carrier with system of narrow pores and channels is kept under temperature below height of potential barriers for movement of at least one type of separated molecules.
EFFECT: higher efficiency.
FIELD: investigating or analyzing materials.
SUBSTANCE: gas analyzer comprises chromatographic columns, detectors, unit for preparing air mounted inside the thermostat, unit for control and processing signals, member for sampling, switches of gas flows, pump for pumping gas mixture, and separating passages connected in parallel and provided with the check valve interposed between them. Each of the separating passages is made of absorbing and separating chromatographic columns connected in series, and the pump is connected to the input of the gas line through the electric valve. The gas analyzer can be made of two separating passages and low pressure chromatographic columns.
EFFECT: enhanced quality of analyzing.
2 cl, 1 dwg, 1 ex
FIELD: analytical methods.
SUBSTANCE: to determine methyl alcohol in water, sample to be assayed is preliminarily subjected to distillation with sulfuric acid added in amount required to provide its concentration in mixture to be distilled c(1/2 H2SO4) = 0.002 M, while strippings constitute 6-7% of the volume of sample. Stripped liquid is thrice rinsed with hexane or Nefras at 1:1 hexane (Nefras)-to-strippings ratio. Rinsed material is then introduced into packed column filled with diatomite modified with 1,2,3-tris(β-cyanoethoxy)propane having deposited fixed phase thereon, which phase is prepared by way of consecutively keeping glycerol each time for 4 h at ambient temperature, 100°C, 130°C, 160°C, and 200°C, and then for 8 h at 230°C and for 40 h at 200°C under nitrogen bubbling conditions. Calculation of methanol content is performed taking into consideration calibrating coefficient.
EFFECT: enabled determination of small concentrations of methyl alcohol in water with sufficient selectivity and reliability.
2 cl, 2 tbl, 6 ex
FIELD: analytical chemistry.
SUBSTANCE: invention relates to method for quantitative determination of thiotriazoline and pyracetam in complex drugs by high performance chromatography, wherein silicagel with grafted 3-(chlorodimethyl)-propyl-N-dodecylcarbamate having particle size of 5 mum is used as sorbent; and degassed 0.05 M aqueous solution of potassium dihydrophosphate is used as mobile phase. Mobile phase velocity is 1 ml/min, and column temperature is 30°C. Method of present invention makes it possible to determine content of two abovementioned active ingredients simultaneously.
EFFECT: simplified process of sample preparation.
3 ex, 3 tbl
FIELD: biotechnology, in particular content determination of polymer chitosan molecules, chitosan-chitine polymer molecules and molecules of chitosan-protein complex in finished form of chitosan.
SUBSTANCE: claimed method includes application of high performance chromatography column filled with polyvinylbenzene sorbent with refractometer detector. As eluent and for dissolving of chitosan preparation samples acetic acid aqueous solution is used. Chain-length distribution is determined on the base of first chromatography peak, and polymer molecular content is calculated on the base of area of first, second and third chromatography peaks, divided up to zero line and belonging to polymer chitosan molecules, chitosan-chitine polymer molecules and molecules of chitosan-protein complex, respectively. To calculate chain-length distribution of polymer chitosan molecules separately calibration curve is plotted using dextran polymer standards.
EFFECT: new effective method for determination of polymer chitosan molecules in chitosan preparations.
4 cl, 3 dwg
FIELD: the invention refers to laboratory chromatographic devices for conducting high-speed chromatographic analysis.
SUBSTANCE: the express-chromatron has an injector, a chromatographic column located in a thermostat, a detector, an amplifier of the signal of the detector, an analog-digital converter, a control system, a pneumatic system. The column is fulfilled either in the shape of a short capillary column or either in the shape of a polycapillary column. The injector is fulfilled with possibility of introduction of the test for the time of 5-50 ms. The detector and the amplifier of its signal are fulfilled with possibility of ensuring constant time of no worse then 10-3 sec. The analog-digital converter is fulfilled with possibility of ensuring speed of no less then 200 measurements in a second.
EFFECT: ensures conducting high-speed chromatographic analysis.
11 cl, 2 dwg