Determination of polymer chitosan molecules in chitosan preparations

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

 

The invention relates to biotechnology - production of chitosan, and can be used to determine the method exclusion chromatography of polymer molecules of chitosan blended chitosan-chitin molecules and molecules of chitosan-protein complex in the finished forms of chitosan preparations.

Chitosan is obtained by deacetylation of a natural polymer chitin is a linear aminopolysaccharide coated consisting of N-acetyl-2-amino-2-deoxy-D-glucosamine links allocated from the shells of crustaceans and other natural sources that contain protein to 25-55% [1].

The deacetylation of chitin carry out the processing of a concentrated solution of alkali at elevated temperature. From structural units of chitin cleaved acetyl group, thanks to which chitin is converted into chitosan, which represents a high molecular weight polymer of glucosamine (2-amino-2-deoxy-D-glucose) according to the scheme:

As the deacetylation reaction is accompanied by a simultaneous rupture of part of the glycosidic linkages of the polymer chitin, the resulting chitosan is a polydisperse molecular weight polymer of D-glucosamine containing 5-15% acetamide groups, and the protein content is reduced to values less than 1%, which is present in the form of chitosan-protein complex [1]. So what u need to have methods of determining polymer molecules of chitosan, in which impurity contained heterogeneous molecules with chitosan-chitin chains and chitosan-protein complexes were separate from chitosan chromatographic peaks.

There are various ways of obtaining experimental data needed to calculate the MMP polymer molecules according to the results of exclusion-chromatographic separation of the sample diluted polymer solution [2, s-145].

The disadvantage of this method [2] is that it does not explain how exclusion-chromatographic separation of polymer molecules of chitosan.

Known methods [3, 4] to define the parameters of MMP polymers, which simultaneously defines the hydrodynamic volume of the molecule, the internal viscosity of the solution and the mass of the polymer molecules, allowing to construct a universal molecular mass calibration curve for any type polymers.

A disadvantage of the known methods [3, 4] is that they are applicable only to solutions of polymer molecules having a molecular weight of not less than 2200 daltons, and in the case of MMD definition of polymer molecules of chitosan, interest molecular weight oligomers and polymers chitosan and also to 2200 daltons.

The closest in technical essence and the achieved result for the proposed method selected in the quality of the ve nearest analogue, is the method for determining polymer chitosan molecules using the method of high-performance exclusion chromatography [5].

This method is used a stainless steel column, consisting of sections total length of 5.5 m, an inner diameter of 3.18 mm, filled with grains of the porous glass, graining 37-74 μm, a pore diameter of from 2500 to 40 Å. The porous glass was applied covalently associated glycerole floor to inactivate a group of free silicic acid on the surface of the porous glass. The choice of combinations of the columns was based on the requirement of the allocation of higher molecular weight fractions of chitosan and to obtain a linear calibration curve for dextrans within the required molecular masses. The best combination of pore size glass and lengths of the columns, the joint section 61 cm, was: 2500 Å (61 cm), 1500 Å (121,9 cm), 550 Å (182,9 cm), 250 Å (61 cm), 100 Å (61 cm) and 40 Å (61 cm).

As the elution solvent for dissolving samples drug chitosane and standards of dextran used 2%aqueous solution of acetic acid. In column samples of chitosan were injected with 500 μg (100 μl of 0.5% or 50 µl of 1% solution), and sample standards dextran 250 μg (50 μl of 0.5% solution).

Shared on a column of polymer molecules of chitosan and standards destroy were detected, eluent flowing through the detector - protoco the optical cell differential refractometric detector, the signals which were recorded on the ribbon diagram of the recorder, moving with a speed of 1 cm/min

In separate experiments, using standards of polymers of dextran T-series with the molecular weight of 10000 (T-10) to 2000000 (T-2000) Dalton, received calibration dependence that determines the dependence of the volume retention standards of dextran on their molecular mass (MM). MM duplicate elementary link polymer molecules of chitosan (2-amino-2-deoxy-D-glucose) was approximately equal to MM basic repeating unit of the polymer dextran (D-glucose) and thus gained the calibration dependence for polymer molecules of chitosan. On the basis of the thus obtained calibration based polymer chitosan molecules and the resulting chromatogram of the sample of chitosan, using existing programs expected MMD polymer molecules of chitosan and srednevekovye (MWand srednekislye (MN) molecular weight chitosan.

The disadvantage of the closest analogue is the following:

- not achieved separation of polymer molecules of chitosan on the individual separated to the zero line of the fraction parts of the original preparation of chitosan, the entire region of the chromatogram assigned to one substance - polymer molecules of chitosan without detection of polymer molecules with different chemical is Kim composition, and recognizes only the presence of molecules of chitosan with different MM;

- obtained values of MWand MNare the values for mixed fractions containing both polymer chitosan molecules and polymer molecules with chitosan-chitin chains, and protein-chitosan complexes;

the sorbent has an active centers is a group of free silicic acid on the surface of the porous glass, which is not fully deactivated glycerolipid coating, these groups adsorb the polymer molecules of chitosan, heterogeneous molecules with chitosan-chitin chains and molecules of chitosan-protein complexes, which prevents their separation.

The problem solved by this invention is the determination of polymer molecule chitosan in the chitosan preparations.

The solution of this problem is provided by the fact that in the method of determination of polymer molecules of chitosan in the chitosan preparations, consisting in dissolving in eluent prepared samples of the preparation of chitosan, the introduction of a highly efficient chromatographic column with sorbent for exclusion-chromatographic separation, the partial elution of molecules from the column and the detection of them by passing the sample through a refractometric detector, the signals of which are registered, while for the calculation of MMP polymer molecules of chitosan separately for ucaut calibration curve using standard polymer dextran, as the elution solvent for dissolving samples of the preparation of chitosan use 3-4%aqueous solution of acetic acid, and exclusion-chromatographic separation - chromatographic column Packed with longevity highly crosslinked polymeric sorbent - polydivinylbenzene, while on the chromatogram registriruutsya three chromatographic peak, separated to the zero line and belonging, respectively, of the polymer molecules of chitosan (1 peak), heterogeneous polymer molecules with chitosan-chitin chains (2 peak) and chitosan molecules-protein complex (3 peak). Determination of MMP polymer molecules of chitosan carried out by the first chromatographic peak, and the content of polymer molecules calculate the squares of the first, second and third chromatographic peaks separated to the zero line and belonging, respectively, of the polymer molecules of chitosan mixed chitosan-chitin polymer molecules and the molecules of chitosan-protein complex.

This can be used chromatographic column length of 150 mm, an inner diameter of 3 mm (150 mm × 3 mm), filled longevity highly crosslinked polydivinylbenzene sorbent in the form of monotermicheskih grains with a diameter of 10 microns, characterized by a high separation efficiency.

Example 1. Calibration of the chromatographic column for calculation of MMP polymers the x molecules of chitosan in the chitosan preparations.

Calibration of the chromatographic column made of glass (150 mm × 3 mm), filled monofonicheskie grains with a diameter of 10 μm longevity highly crosslinked polydivinylbenzene (PDVB) sorbent [6, s]prepared by the method of [7], is to establish the dependence between the volume of the holding and the logarithm of molecular mass (MM) standards polymers of dextran in the linear range.

When performing calibration of the chromatographic column must meet the following conditions:

Samples at a concentration of 0.05 wt.% standards of dextran T-series with a molecular weight of 504 Dalton (maltotriose) to 2000000 (T-2000) Dalton prepared in 4%aqueous solution of acetic acid and pre-filtered through a filter with pore size of 0.25 μm.

Chromatographically on a column of standards polymers dextran detects when passing the sample through the optical flow cell of differential refractometric detector RIDK-102 (with a flow-through quartz cuvette with a volume of 10 μl), detector signals recorded using komyuternoy program Achrom" [8] or on the ribbon diagram of the recorder TZ-4620 (speed 6 mm/min).

The volume flow rate of eluent of 4%aqueous solution of acetic acid and 0.2 ml/min Temperature thermostat column, injector, Refractometer - 25°C. the Volume of the dosing loop injector - 10 ál. Sensitivity is refractometric detector RIDK-102 - 4-8×10-7units of refraction.

The calibration is carried out by the sequential introduction using the liquid injector of the chromatograph column PDB sorbent 10 ál of 0.05%aqueous solution of each standard polymer dextran.

After sample introduction, standard polymer dextran in the liquid injector of the chromatograph, the sample from the injector introduces the eluent in column PDB sorbent and after the column passes through the flow cell of the Refractometer, the signals are recorded using the computer or on the ribbon diagram of the recorder in the form of first increasing and then decreasing signal - the so-called chromatographic peak. On chromatographic peak are the maximum values and the corresponding volume passed through the column eluent, which is called the volume retention (Vr, Jr).

Construct a calibration graph of the relationship between volume retention and the logarithm of molecular mass standards polymers dextran presented in figure 1. The chromatographic column made of glass (150 mm × 3 mm), filled longevity highly crosslinked PDB sorbent in the form of monotermicheskih grains with a diameter of 10 μm. 1 - glucose (180 MM), 2 - maltose (342 MM), 3 - maltotriose (504 MM), 4 - dextran T-20 (20000 MM), 5 - dextran T-500 (MM 500000), 6 - dextran T-2000 (MM 2000000). The linear range is 504 MM from up to 2,000,000 daltons.

Example 2. Determine what their polymer molecules of chitosan in the commercial preparation of chitosan. Prepare a 0.05%solution of chitosan in 4%aqueous solution of acetic acid and the sample volume of 10 microns is introduced into a chromatographic column made of glass (150 mm × 3 mm), filled monofonicheskie grains with a diameter of 10 μm longevity highly crosslinked PDB sorbent under the following conditions chromatographic analysis.

Shared on a column of polymer molecules of chitosan, a heterogeneous polymeric molecules with chitosan-chitin chains and chitosan-protein complexes detected by passing the sample through the optical flow cell of differential refractometric detector RIDK-102 with sensitivity - 4×10-7units of refraction, detector signals recorded on the ribbon diagram of the recorder TZ-4620 at a speed chart tape - 6 mm/min

The flow rate of eluent of 4%aqueous solution of acetic acid and 0.2 ml/min Temperature thermostat column, injector, Refractometer - 25°C. the Volume of the dosing loop injector - 10 ál.

Figure 2 presents the obtained chromatogram of polymer molecules of different chemical composition of the commercial preparation of chitosan. In the chromatogram obtained three chromatographic peak, separated to the zero line of the Refractometer. 1 - polymer molecules of chitosan (Vr=value (0.475) ml), 2 - heterogeneous polymer molecules with chitosan-chitin chains (Vr=0,855 ml), 3 - mole the uly chitosan-protein complex (Vr=1,267 ml). These chromatographic conditions allow to determine MMP polymer molecules of chitosan in the preparation of chitosan (see table 1), the content of polymer molecules calculate the squares of the first, second and third chromatographic peaks separated to the zero line and belonging, respectively, of the polymer molecules of chitosan mixed chitosan-chitin polymer molecules and the molecules of chitosan-protein complex.

For the identification of chemical compounds present in the separated chromatographic peaks, i.e. for classification of chromatographic peaks to the polymer molecules of a certain chemical composition, conducting chromatographic separation of the sample preparation of chitosan in the same conditions, but using a UV detector (spectrophotometer Hitachi flow-through quartz cuvette with a volume of 7 μl), measuring the absorption of electromagnetic waves at a wavelength of 235 nm. Other absorption bands in the ultraviolet region of the spectrum in the commercial preparation of chitosan is not detected. Chromatogram obtained using a UV detector at a wavelength of 235 nm, is shown in figure 2 as dashed lines.

Comparison of chromatograms obtained from refraction and ultraviolet detectors, shows that the polymer molecules present vpike 1, do not absorb electromagnetic waves in the ultraviolet region of the spectrum with a wavelength of 235 nm, while the polymer molecules in peaks 2 and 3 absorb in the ultraviolet region of the spectrum with a wavelength of 235 nm. These data allow us to include chromatographic peak 1 to pure chitosan as aliphatic amines do not absorb in the ultraviolet region of the spectrum [9, s].

Polymer molecules in the chromatographic peak 2 absorb in the ultraviolet region of the spectrum at a wavelength of 235 nm is significantly harder than molecules in peak 3, while the areas of these peaks differ slightly. This corresponds to the fact that molecules mediaterranean chitin (N-acetyl-2-amino-2-deoxy-D-glucose) in the preparation of chitosan significantly larger (5-15%)than molecules of chitosan-protein complex (up to 1% protein) [1]. The chitin molecules absorb in the ultraviolet region of the spectrum due to the presence of groups of the peptide bond-CO-NH- [9, s]; molecules of chitosan-protein complex also absorb through peptide linkages that are available in the protein molecule.

Bimodal shape of the chromatographic peak 2 indicates that this peak contains uneven sets of polymer molecules containing linear chains of elementary repeating chains of molecules such as chitosan and chitin. The ratio of the repeating single is tons of chitin and chitosan in different ways for different polymer molecules. Unimodal shape of the peak 3 indicates greater homogeneity of the chemical composition of the molecules of chitosan-protein complexes.

The chromatogram (figure 2, refractometric detector) defined the area of the three chromatographic peaks and calculated the relative content that are equal (in %):

for polymer molecules of chitosan (peak 1)67
for chitosan-chitin polymer molecules (peak 2)17
for molecules of chitosan-protein complexes (peak 3)16

Calculation of MMP polymer molecules of chitosan according to the separation method exclusion chromatography and calibration curve was performed using the computer program "Achrom" [8] and according to the program given in the monograph [10, s-322]. The results of a calculation of MMP polymer molecules of chitosan in the commercial preparation of chitosan are shown in table 1.

Table 1

Data on DFID polymer molecules of chitosan in the commercial preparation of chitosan
iVi, mlhi mmlg MiMi, g/molhi/Mihi·Mi/td>
110,35502726,47863,01·106000
100,396182726,04921,12·1061,61·10-52,08·1070,018
90,434372545,71185,15·1057,18·10-51,91·1070,136
8value (0.475)632175,49973,16·1052,0·10-42,0·1070,262
70,512651545,12061,32·1054,92·10-48,62·1060,239
60,55440894,82026,61·1046,04·10-42,64·1060,147
50,59227494,69985,01·1045,39·10-41,35·106 0,099
40,63311224,3997of 2.51·104of 4.38·10-4was 2.76·1060,0405
30,6747114,10041,26·1045.56mm·10-48,81·1040,0257
20,712443,89987,94·1030,504·10-33,18·1040,0147
10,740003,69985,01·103000
Σ=0,007957Σ=75389900
MW=277169 g/mol, MN=34184 g/mol, MW/MN=8,1

Equations for calculating srednevekovoi (MWand srednekamennogo (MN) molecular weights were as follows:

,,- dispersion of molecular weights.

where: i - number of a fragment of chromatographic the ski peak

Vi is the volume retention, ml;

hi is the height at each 10-second interval chromatographic peak;

Mi - molecular weight polymer molecules of chitosan i;

Wi=hi/Σhi is the proportion of the polymer molecules of chitosan with a given MM in the original sample;

Received DFID polymer molecules of chitosan in the commercial preparation of chitosan is shown in figure 3.

Example 3. Determination of polymer molecules of chitosan in the commercial preparation of chitosan produced under the conditions of example 2, with the difference that for dissolving the sample preparation of chitosan as eluent using 2%aqueous solution of acetic acid. Chromatographic analysis using a refractometric detector shows that in the chromatogram recorded two chromatographic peak. 1 - polymer molecules of chitosan, 2 - heterogeneous polymer molecules with chitosan-chitin chains, the molecules of chitosan-protein complex and low molecular weight chitosan. By area, the second Chromatographic peak is larger than the first, which confirms the presence of the second chromatographic peak of low molecular weight chitosan.

When using as an eluent of 2%aqueous solution of acetic acid by high-performance chromatographic column (150 mm × 3 mm) with PDB sorbent is not achieved reliable determination of MMP chitosan and hetero is i.i.d. polymer molecules with chitosan-chitin chains and molecules of chitosan-protein complex on the column cannot be divided.

Example 4. The method for determining polymer molecules of chitosan in the commercial preparation of chitosan produced under the conditions of example 2, with the difference that for dissolving samples of chitosan as eluent used a 3%aqueous solution of acetic acid. Chromatographic analysis using a refractometric detector shows that the preparation of chitosan released three chromatographic peaks: 1 polymer molecules of chitosan, 2 - heterogeneous polymer molecules with chitosan-chitin chains, 3 molecules of chitosan-protein complex, which are separated on the column to the zero line. This chromatogram also possible to calculate MMP polymer molecules of chitosan in the preparation of chitosan and the content of polymeric molecules of chitosan blended chitosan-chitin polymer molecules and molecules of chitosan-protein complex.

Example 5. The method for determining polymer molecules of chitosan in the commercial preparation of chitosan produced under the conditions of example 2, with the difference that for dissolving samples of chitosan as eluent used 5 and 6%aqueous solution of acetic acid. Chromatographic analysis using a refractometric detector shows that the preparation of chitosan released three chromatographic peaks: 1 polymer molecules of chitosan, 2 - heterogeneous polymer molecules with whom ECOSAN-chitin chains, 3 - the molecules of chitosan-protein complex, which are not separated on the column to the zero line, as the sensitivity analysis is reduced, which is not possible with high accuracy to determine MMP polymer molecules of chitosan and the content of the molecules of chitosan, chitosan-chitin polymer molecules and molecules of chitosan-protein complex.

Sources of information

1. Bykov V.M., Germans SV Raw material sources and methods of obtaining chitin and chitosan. In the book: Chitin and chitosan. Production, properties and application. Edited by Scriabin KG, Vikhoreva GA, Varlamov V.P. M.: Nauka, 2002, p.7-23.

2. Belenky astray freight, Vilenchik LS Chromatography of polymers. - M.: Chemistry, 1978, s-145.

3. U.S. patent No. 3837217 (24.09.1974), NCI 73-61 .1 C. measurement of the molecular weight distribution of polymers.

4. U.S. patent No. 4775943 (04.10.1988), NCI 364-497. Method and apparatus for molecular weight distribution of the parameters of the polymer.

5. ...Wu, W.A.Bough, E.C.Conrad, K.E.Alden. Determination of molecular-weight distribution of chitosan by high-performace liquid chromatography. J. of Chromatography, 128 (1976) 87-99.

6. Yashin YA, Yashin PRODUCED high-performance liquid chromatography. Status and prospects. J. ROS. chem. society for them. D., 2003, t, No. 1, p.64-79.

7. Patent of Russia №2005102875 from 07.02.2005,, MKI G 01 N 15/08. Method of preparation of high-performance speakers with polymeric sorbents for liquid chromatography. /Khabarov V.B. have been, Pronin YA, E the poppies CENTURIES, Buryak, A., Khabarov M.V.

8. GPSMapEdit "Achrom", designed by a leading designer organic chemistry Institute. Ndzinisasa RAS Boitsova VN

9. Chemical encyclopedia. Moscow, 1988, vol. 1, s, 250

10. W.W.Yau, J.J.Kirkland, D.D.Biy. Modem Size-Exclusion liquid chromatograhy. Practice of Gel permleation and Gel Filtration Chromatograhy. New York - Chichester - Brisban - Toronto, 1979. P.77, 318-322, 391-393.

1. The method for determining polymer molecules of chitosan in the chitosan preparations, consisting in the preparation of samples by dissolving the sample preparation of chitosan in an acid solution, the introduction of a highly efficient chromatographic column with sorbent for exclusion chromatographic separation, washing the column eluent is diluted acids, the detection of the partial molecules by passing the sample through a refractometric detector, the signals which are recorded in the form of a chromatogram, the calculation of the molecular mass distribution of polymer molecules of the drug chitosan, and to calculate the molecular mass distribution using the calibration dependence of the values of the logarithm of the molecular mass standards dextran from the volume of their output from the chromatographic column, characterized in that as a sorbent for exclusion-chromatographic separation of polymer molecules of the drug use of chitosan longevity highly crosslinked polydivinylbenzene sorbent and op is adelene molecular mass distribution of polymer molecules of chitosan carried out by the first chromatographic peak, and the content of polymer molecules calculated by the squares of the first, second and third chromatographic peaks separated to the zero line and belonging respectively polymeric molecules of chitosan mixed chitosan-chitin polymer molecules and the molecules of chitosan-protein complex.

2. The method for determining polymer molecules of chitosan in the chitosan preparations according to claim 1, characterized in that use polydivinylbenzene sorbent in the form of monotermicheskih granules with a diameter of 10 microns.

3. The method for determining polymer molecules of chitosan in the chitosan preparations according to claim 1, characterized in that as the elution solvent for dissolution of the drug chitosan use 3-4%aqueous solution of acetic acid.

4. The method for determining polymer molecules of chitosan in the chitosan preparations according to claim 1, characterized in that the identification of chromatographic peaks using ultraviolet and refractometric detectors.



 

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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

Express-chromatron // 2300764

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

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