Method for determining substitution degrees of three hydroxypropylated polysaccharides by infrared spectroscopy

Abstract

The invention provides a method for simultaneously determining the substitution degrees of hydroxypropyl chitosan, hydroxypropyl mannan and hydroxypropyl starch by utilizing an infrared absorption spectrometry. Comprising the following steps: (1) drying: drying hydroxypropylated polysaccharide samples to remove moisture, wherein each hydroxypropylated polysaccharide sample is prepared through a polysaccharide hydroxypropylation reaction and purification; (2) establishment of a detection model: establishing a regression model with good linearity for a substitution degree Y obtained by nuclear magnetic resonance according to a ratio X of a probe peak to a reference peak in an infrared spectrum; and determining the infrared spectrogram of a to-be-detected hydroxypropylated polysaccharide sample, substituting the corresponding ratio into the linear regression model, and calculating to obtain the substitution degree Y of the to-be-detected hydroxypropylated polysaccharide sample. The method creatively finds that B2970-4 serves as a general probe peak, the substitution degrees of the three hydroxypropyl polysaccharides can be measured at the same time, the method is not affected by the deacetylation degree, temperature and moisture of the raw material chitosan, the detection cost is relatively low, precise weighing is not needed, and operation is easy, convenient and rapid.

Description

Technical Field

[0001] The present invention belongs to the research and application field of the detection method of the degree of substitution of hydroxypropylated polysaccharides, and particularly relates to a method for determining the degree of substitution of hydroxypropyl chitosan, hydroxypropyl mannan and hydroxypropyl starch by infrared absorption spectroscopy, which is not affected by the degree of deacetylation, temperature and moisture of the raw material chitosan. Background Art

[0002] The solubility of chitosan, mannan and starch depends not only on the molecular weight, but more importantly on the degree of substitution of the substituents in the polysaccharide structure. For example, the average number of substituents contained in each glucosamine molecule or N-acetylglucosamine molecule in the chitosan molecule can be represented by MS, and is calculated by the ratio of the total number of moles of the substituent groups in the polysaccharide derivative molecule to the total number of moles of the sugar residues in the molecule. By using different preparation methods, the degree of substitution of the obtained polysaccharide derivative products is different, which will directly affect the physical and chemical properties and applications of the products, such as antibacterial property, solubility, ionic conductivity, etc.; it will also affect subsequent structural modification or graft copolymerization, etc.

[0003] At present, the methods for determining the degree of substitution of hydroxypropyl chitosan include elemental analysis method (Zhang Yongqin, Zhang Kun. Simultaneous determination of the degree of substitution and moisture of hydroxypropyl chitosan by elemental analysis method [J]. Chinese Journal of Analysis Laboratory, 2014, 33(08): 978-980.), nuclear magnetic method (Wang P, Lv X, Zhang B, et al. Simultaneous determination of molar degree of substitution and its distribution fraction, degree of acetylation in hydroxypropyl chitosan by 11H NMR spectroscopy[J]. Carbohydrate Polymers, 2021, 263, 117950.), infrared method (Zhang Yongqin, Zhang Kun, Zhang Jie, et al. A method for determining the degree of substitution of hydroxypropyl chitosan: China, CN201510575798.7[P]. 2016-02-03; Zhang Yongqin, Zhang Jie, Zhang Kun, et al. A method for determining the degree of substitution of hydroxypropyl chitosan: China, CN201510575794.9[P]. 2016-01-27; Zhang Yongqin, Zhang Kun, Zhang Jie, et al. A method for determining the degree of substitution of hydroxypropyl chitosan: China, CN201510575770.3[P]. 2015-2-30; Zhang Yongqin, Zhang Kun, Zhang Jie, et al. A method for determining the degree of substitution of hydroxypropyl chitosan: China, CN201510575806.8[P]. 2015-2-6; Zhang Yongqin, Zhang Jie, Zhang Kun, et al. A method for determining the degree of substitution of hydroxypropyl chitosan: China, CN201510575791.5[P]. 2015-2-9; Zhang Yongqin, Zhang Jie, Zhang Kun, et al. A method for determining the degree of substitution of hydroxypropyl chitosan: China, CN201510575792.X[P]. 2015-2-2.), spectrophotometry (Zhang Yongqin, Zhang Kun, Liu Fusheng, et al. A method for determining the degree of substitution of hydroxypropyl chitosan: China, CN201410841212.2[P]. 2015-06-10), etc.

[0004] The elemental analysis method only uses the mass ratio of C and N in the sample and compares the measured value of H% with the calculated value. The parameters are single and not affected by the moisture content. It is only not applicable to samples with unknown or medium and low degrees of deacetylation. Therefore, this method is often used for the determination of the degree of substitution of samples with known and high degrees of deacetylation; 1The advantages of the ¹H NMR method are that it is easy to operate, requires less sample, does not need to accurately weigh the sample mass, does not require a known degree of acetylation, and can also determine the position and quantity of H in the chemical structure. However, the reagents used in this method are expensive, the equipment requirements are high, and the sample must be water-soluble. Moreover, the detection costs of the above two methods are relatively high, and they are not suitable for large-scale routine detection by hydroxypropyl chitosan production enterprises and research institutions. The infrared method does not require accurate weighing and dissolving of samples, is simple and fast to operate, and has a relatively low measurement cost. Therefore, it is very suitable for production quality control and application research. However, the currently known infrared method reported by Zhang Yongqin et al. can only measure the degree of substitution of hydroxypropyl chitosan, with a single detection object. Moreover, this method is relatively susceptible to the deacetylation degree, temperature, and moisture of the raw material chitosan, and its universality is relatively low, and it cannot be directly applied to the determination of the hydroxypropyl substitution degree of other polysaccharides. The reagents and instruments used in the spectrophotometry method are relatively simple, and the detection cost is relatively low. However, the determination conditions are relatively harsh, requiring precise reaction of hydroxypropyl chitosan with concentrated sulfuric acid in a water bath at 100 °C for 3 min (Xie Linlin, Xu Ningning, Cao Huan, et al. Preparation and antibacterial properties of hydroxypropyl chitosan biguanide hydrochloride iodine film [J]. New Chemical Materials, 2016, 44(8): 186-189.). It is relatively difficult to control the reaction conditions, and operating an experiment containing concentrated sulfuric acid under the condition of a 100 °C water bath is quite dangerous, not to mention large-scale operations.

[0005] The spectrophotometry method can also be used to determine the degree of substitution of hydroxypropyl starch and hydroxypropyl mannan, such as the national standard "GB / T 40998-2021 Determination of hydroxypropyl content in modified starch - Spectrophotometry method". Therefore, it will also face the same problems. In addition, gas chromatography can be used to determine the degree of substitution of hydroxypropyl mannan (Wu Yanan, Chen Xiangyu, Wang Tengbin, Guo Youli, Xu Wenting, Lü Xingshuang,... & Zhang Yongqin. (2023). Determination of molar substitution degree of hydroxypropyl konjac glucomannan by gas chromatography. Journal of Food Safety & Quality, 14(12).). However, the sample preparation of this method is cumbersome, and the reagents and consumables used are expensive. In particular, this method measures the hydroxypropyl groups decomposed from the polysaccharide sample through the reaction, and whether the reaction is complete will directly affect the measurement result. After calibration by the NMR method, the calibration coefficient is 1.159-1.164. So far, there is no relevant infrared method for determining the degree of substitution of hydroxypropyl starch and hydroxypropyl mannan.

[0006] The detection cost of the infrared absorption spectroscopy method is relatively low, and it does not require precise weighing and dissolving of chitosan samples, and is simple and fast to operate. Therefore, this method is of great significance for the quality control and applied research and development of hydroxypropyl polysaccharide production enterprises, rapid detection by quality inspection departments, and scientific research in scientific research institutions. Summary of the Invention

[0007] To solve the deficiencies of the above-mentioned existing technologies, the primary objective of the present invention is to provide a simple, rapid, accurate, and low-cost detection method that is not affected by conditions such as the deacetylation degree, moisture, and temperature of the raw material chitosan, and can detect the substitution degrees of hydroxypropyl chitosan, hydroxypropyl mannan, and hydroxypropyl hydroxypropyl starch.

[0008] The present invention is achieved through the following technical solutions:

[0009] A method for determining the substitution degrees of three hydroxypropylated polysaccharides by infrared spectroscopy, wherein the three hydroxypropylated polysaccharides include hydroxypropyl chitosan, hydroxypropyl mannan, and hydroxypropyl starch, and the method for determining the substitution degrees of the three hydroxypropylated polysaccharides by infrared spectroscopy comprises the following steps:

[0010] (1) Drying:

[0011] Dry the hydroxypropylated polysaccharide samples to remove moisture, wherein each hydroxypropylated polysaccharide sample is prepared through a polysaccharide hydroxypropylation reaction and purification, and is derived from a polysaccharide raw material with a known acetylation degree;

[0012] (2) Establishing a detection model:

[0013] a. Use an infrared absorption spectrometer to obtain the infrared spectra of the dried hydroxypropylated polysaccharide samples with different substitution degrees in step (1);

[0014] b. Use the application software of the infrared absorption spectrometer to obtain a series of integral values of peak heights or peak areas as reference peaks from the infrared spectra of the hydroxypropylated polysaccharide samples obtained in step a, and the integration method for the reference peaks is as follows:

[0015] ① Vibration peaks for integration:

[0016] O-H stretching vibration peak or N-H stretching vibration peak at 3400 - 3460 cm -1 (Peak No. 1), methyl C-H asymmetric stretching vibration peak at 2968 - 2972 cm -1 methylene C-H asymmetric stretching vibration peak at 2910 - 2940 cm -1 (Peak No. 3), methyl and methylene C-H symmetric stretching vibration peak at 2860 - 2900 cm -1 methyl C-H asymmetric bending vibration and methylene bending vibration peak at 1450 - 1465 cm -1 methylene C-H bending vibration peak at 1410 - 1425 cm -1 (Peak No. 6), methyl C-H symmetric bending vibration and methylene bending vibration peak at 1370 - 1385 cm -1 (Peak No. 7), methylene C-H bending vibration peak at 1320 - 1335 cm-1 (Peak No. 8), C-O-C stretching vibration peak at 1145 - 1160 cm -1 (Peak No. 9), the entire spectral band composite vibration peak including C-O stretching vibration peak with hydroxyl group and C-O-C stretching vibration peak at 820 - 1235 cm -1 (Peak No. 10);

[0017] ② Baseline for integration

[0018] The baseline is selected from the valley tangent lines of the entire spectral band at 2200 - 3990 cm -1 to form baseline (BL0), the baseline is selected from the valley tangent lines of the entire spectral band at 1800 - 3990 cm -1 to form baseline (BL1), the baseline is selected from the valley tangent lines of the entire spectral band at 2995 - 3850 cm -1 to form baseline (BL2), the baseline is selected from the valley tangent lines of the entire spectral band at 2300 - 3070 cm -1 to form baseline (BL3), the baseline is selected from the valley tangent lines of the entire spectral band at 2945 - 3020 cm -1 to form baseline (BL4), the baseline is selected from the valley tangent lines of the entire spectral band at 1210 - 1570 cm -1 to form baseline (BL8), the baseline is selected from the valley tangent lines of the entire spectral band at 1430 - 1520 cm -1 to form baseline (BL9), the baseline is selected from the valley tangent lines of the entire spectral band at 1345 - 1400 cm -1 to form baseline (BL10), the baseline is selected from the valley tangent lines of the entire spectral band at 820 - 1235 cm -1 to form baseline (BL11);

[0019] ③ Integration method

[0020] There are three integration methods: R, B, and C. R represents the maximum peak height under the baseline. If the spectral peak is not prominent, R is the peak height at the specified wave number under the baseline. If the spectral peak at the specified wave number is not the highest peak within the spectral band, R is the peak height at the specified wave number under the baseline; both B and C represent the area between the spectral band and the baseline. Among them, the start and end points of the integration of area B are the two valleys of the spectral band, which are the same as the start and end points of the baseline. The start and end points of the integration of area C are less than those of the baseline, and it is only the area of the required absorption peak under this line. Taking the infrared spectrum of hydroxypropyl chitosan as an example, R 3440-1 is the integral value of the peak height where the highest point perpendicular to the wave number abscissa of Peak No. 1 intersects BL1; R 3440-2 is the integral value of the peak height where the highest point perpendicular to the wave number abscissa of Peak No. 1 intersects BL2; C 2970-3 is the integral value of the peak area formed by the two valley endpoints where the perpendicular to the wave number abscissa of Peak No. 2 intersects BL3 respectively, and B2970-4 Namely, it is the integral value of the peak area where the entire spectral band of Peak 2 is surrounded by BL4, R 2922-3 Namely, it is the integral value of the peak height where the highest point perpendicular to the wavenumber abscissa of Peak 3 intersects BL3, R 2875-3 Namely, it is the integral value of the peak height where the highest point perpendicular to the wavenumber abscissa of Peak 4 intersects BL3; for others, R 2970-1 , R 2922-1 , R 2875-1 , R 2970-3 , R 2970-4 And so on, R 1458-8 It is the integral value of the peak height where the highest point perpendicular to the wavenumber abscissa of Peak 5 intersects BL8; R 1415-8 It is the integral value of the peak height where the highest point perpendicular to the wavenumber abscissa of Peak 6 intersects BL8, R 1378-8 It is the integral value of the peak height where the highest point perpendicular to the wavenumber abscissa of Peak 7 intersects BL8, R 1328-8 It is the integral value of the peak height where the highest point perpendicular to the wavenumber abscissa of Peak 8 intersects BL8, C 1378-8 It is the integral value of the peak area formed by the two endpoints of the wave trough perpendicular to the wavenumber abscissa of Peak 7 intersecting BL8 respectively, B 1458-8 It is the integral value of the peak area where the entire spectral band of Peak 5 is surrounded by BL8, B 1378-10 It is the integral value of the peak area where the entire spectral band of Peak 7 is surrounded by BL10, R 1378-10 It is the integral value of the peak height where the highest point perpendicular to the wavenumber abscissa of Peak 7 intersects BL10, R 1152-11 It is the integral value of the peak height where the highest point perpendicular to the wavenumber abscissa of Peak 9 intersects BL11, B 1080-11 It is the integral value of the peak area where the entire spectral band of Peak 10 is surrounded by BL11, R 1080-11 It is the integral value of the peak height where the highest point perpendicular to the wavenumber abscissa of Peak 10 intersects BL11, and so on for others;

[0021] c. Using B 2970-4 as the probe peak; calculating the reference peak by the integration method in step b;

[0022] Calculating the ratio of the probe peak B 2970-4 to the reference peak respectively, denoted as X;

[0023] When the hydroxypropylated polysaccharide is hydroxypropyl chitosan, the reference peak is selected from R 3440-1 , R 2922-1 , R 2875-1 , R 3440-2 , R 2922-3 , R 2875-3 , R 1415-8 , R 1378-8 , C1378-8 , R 1328-8 , R 1378-10 , B 1378-10 , R 1152-11 , R 1080-11 , C 1152-11 and B 1080-11 ;

[0024] When the hydroxypropylated polysaccharide is hydroxypropyl mannan, the reference peaks are selected from R 2970-1 , R 2921-1 , R 2880-1 , R 2970-3 , R 2921-3 , R 2880-3 , R 1458-8 , C 1458-8 , R 1413-8 , R 1376-8 , C 1376-8 , R 1328-8 , R 1458-9 , B 1458-9 , R 1152-11 , R 1058-11 , C 1152-11 , B 1058-11 , R 1413-8 , R 3423-1 , R 3423-2 ;

[0025] When the hydroxypropylated polysaccharide is hydroxypropyl starch, the reference peaks are selected from R 2970-1 , R 2930-1 , R 2882-1 , R 2970-3 , R 2930-3 , R 2882-3 , R 1459-8 , C 1459-8 , R 1414-8 , R 1376-8 , C 1376-8 , R 1332-8 , R 1459-9 , B 1459-9 , R 1153-11 , R 1089-11 , C 1153-11 , B 1089-11 ;

[0026] d. Measure the degree of substitution Y of the hydroxypropylated polysaccharide sample in step a by 1 the HNMR method;

[0027] e. Establish a regression model with good linearity for a set of ratios X obtained by any method in step c and a set of degrees of substitution Y of the hydroxypropyl chitosan sample obtained in step d, that is, Y = kX + b, so as to obtain the specific values of k and b in the model;

[0028] (3) Degree of substitution calculation:

[0029] Dry the sample of hydroxypropylated polysaccharide to be tested according to the method in (1), obtain the infrared spectrum from step a in (2), obtain the ratio X according to step c in (2), and substitute it into the linear regression model obtained in step e in (2), then the degree of substitution Y of the sample of hydroxypropylated polysaccharide to be tested can be calculated.

[0030] Furthermore, the method for drying the sample of hydroxypropylated polysaccharide is to dry it at 80 °C for 4 - 5 hours and at 105 °C for 1.5 - 2 hours, and after taking out the sample, place it in a desiccator containing silica gel for standby.

[0031] Furthermore, the method for drying the sample of hydroxypropylated polysaccharide is freeze-drying or vacuum drying.

[0032] Furthermore, the infrared absorption spectrometer is a Fourier transform infrared spectrometer, and the application software of the infrared absorption spectrometer is OMNIC software.

[0033] Furthermore, the detection conditions of the infrared absorption spectrometer are: scanning range 400 - 4000 cm -1 , scanning 64 times, and the resolution is 4 cm -1 .

[0034] Furthermore, perform background detection on the infrared spectrum of the obtained sample of hydroxypropylated polysaccharide and perform background subtraction processing.

[0035] Furthermore, when the hydroxypropylated polysaccharide is selected from hydroxypropyl chitosan and the reference peaks are selected from R 2875-3 and R 2922-3 , the method for determining the hydroxypropylated polysaccharide does not include step (1) drying.

[0036] Furthermore, when the hydroxypropylated polysaccharide is selected from hydroxypropyl mannan and the reference peaks are selected from R2884 - 1, R2884 - 3, R2922 - 3, the method for determining the hydroxypropylated polysaccharide does not include step (1) drying.

[0037] The present invention creatively discovers that using B 2970-4 as a general probe peak, the degree of substitution of three kinds of hydroxypropyl polysaccharides can be simultaneously determined. Compared with the prior art, the present invention has the following advantages:

[0038] (1) It is not affected by the degree of deacetylation, temperature and moisture of the raw material chitosan;

[0039] (2) It has a good linear relationship, high precision, and can simply, quickly and conveniently detect the degree of substitution of hydroxypropyl chitosan, hydroxypropyl mannan and hydroxypropyl starch;

[0040] (3) There is no need for precise weighing, dissolution and other steps of the sample, which is applicable to the quality control of hydroxypropyl chitosan products by production enterprises, applied research and development, and sample detection by quality inspection institutions.

[0041] (4) It can be used to identify whether the sample is pure or deteriorated. Description of the Drawings

[0042] Figure 1 It is a schematic diagram of the spectral attribution and baseline drawing method of the infrared spectra of chitosan (CS1) and hydroxypropyl chitosan (HPCS-10).

[0043] Figure 2 It is a schematic diagram of the selection method of baselines BL-3, BL-4, BL-9 and BL-10 in the infrared spectrum of hydroxypropyl chitosan.

[0044] Figure 3 For the infrared spectrum of hydroxypropyl chitosan, C 1378-8 and B 1080-11 Integration method.

[0045] Figure 4 It is the infrared spectrum, spectral attribution and baseline selection method of low-substitution-degree hydroxypropyl starch (bottom) and high-substitution-degree hydroxypropyl starch (top).

[0046] Figure 5 It is a schematic diagram of the selection method of baselines BL-9 and BL-10 in the infrared spectrum of hydroxypropyl starch.

[0047] Figure 6 For the infrared spectrum of hydroxypropyl starch, B 2970-4 / R 2930-1 、R 1089-11 Integration method.

[0048] Figure 7 It is the spectral attribution and baseline selection method of the infrared spectra of mannan (bottom), low-substituted hydroxypropyl mannan (middle) and high-substituted hydroxypropyl mannan (top).

[0049] Figure 8 It is a schematic diagram of the selection method of baselines BL-0, BL-1, BL-3 and BL-4 in the infrared spectrum of hydroxypropyl mannan.

[0050] Figure 9 It is a schematic diagram of the selection method of baselines BL-9, BL-10 and BL-12 in the infrared spectrum of hydroxypropyl mannan.

[0051] Figure 10 It is the nuclear magnetic resonance spectra of CS1, HPCS-1, 3, 5, 7, 9, 11.

[0052] Figure 11Blind screening radar chart of the detection peak and reference peak of hydroxypropyl chitosan.

[0053] Figure 12 Effect of hydroxypropylation temperature on MS determination.

[0054] Figure 13 Effect of residual acid on MS determination.

[0055] Figure 14 Effect of trace moisture in HPCS samples on mass spectrometry analysis.

[0056] Figure 15 Effect of DA on MS determination.

[0057] Figure 16 Regression line of hydroxypropyl chitosan MS: from left to right and downwards, including R 3440-1 ,R 2922-1 ,R 2875-1 ,R 3440-2 ,R 2922-3 ,R 2875-3 ,R 1415-8 ,R 1378-8 ,C 1378-8 ,R 1328-8 ,R 1378-10 ,B 1378-10 ,R 1152-11 ,R 1080-11 ,C 1152-11 and B 1080-11 .

[0058] Figure 17 Regression line of hydroxypropyl mannan MS: from left to right and downwards, including R 2921-0 ,R 2880-0 ,R 2921-1 ,R 2880-1 ,R 2921-3 ,R 2880-3 ,B 1376-10 ,C 1376-8 ,R 1376-8 ,R 1152-11 ,B 1058-11 ,R 1058-11 ,C 1152-11 ,R 1413-8 ,R 1328-8 ,R 2970-1 and C 2970-1 .

[0059] Figure 18 Schematic diagram of integral for determining the degree of substitution of HPKGM by FTIR.

[0060] Figure 19 Infrared spectrum of HPKGM.

[0061] Figure 20 The peak of the CH asymmetric stretching vibration of the methyl group in the infrared spectrum of HPKGM is 2970 cm -1 The integral ratio value of 1 Linear regression curve of HNMR and MS measurements.

[0062] Figure 21 Infrared spectrum of HPS.

[0063] Figure 22 B 2970-5 / R 2930-3 and 1 Linear regression curve of the two MS measurements measured by HNMR.

[0064] Figure 23 Regression line for hydroxypropyl starch MS: from left to right and downward, including R 2970-1 ,R 2930-1 ,R 2882-1 ,R 2930-3 ,R 2882-3 ,R 1459-8 ,C 1459-8 ,R 1414-8 ,R 1376-8 ,C 1376-8 ,R 1332-8 ,R 1459-9 ,R 1153-11 ,R 1089-11 ,C 1153-11 and B 1089-11 . DETAILED DESCRIPTION

[0065] The present invention is further described in detail below in conjunction with examples, but the implementation mode of the present invention is not limited to these examples, and any changes or equivalent substitutions that do not deviate from the concept of the present invention are within the protection scope of the present invention.

[0066] Example 1

[0067] Reagents: Chitosan (CS-1, CS-2, CS-3) with acetylation degree of 15%, 1% and 8.8% were purchased from Shandong Laizhou Haili Biological Products Co., Ltd.; potassium bromide was purchased from Aladdin Reagent (Shanghai) Co., Ltd.; deuterium oxide (D 2 O, 99.9%, w / w), deuterium chloride (DCl, ≥ 99.5atom%D; 20%, w / w in D 2 O) was purchased from Adamas Reagent Co., Ltd. Ultrapure water was used throughout the experiment.

[0068] Instrument: The present invention is applicable to various Fourier transform infrared spectrometers. The instrument used in the present invention is IS10 Fourier transform infrared spectrometer, Thermo Fisher Scientific (China) Co., Ltd.; the nuclear magnetic resonance hydrogen spectroscopy method (1 The degree of deacetylation of chitosan and the degree of substitution of hydroxypropyl chitosan were determined by 1H NMR at the testing center (national-level open laboratory) of the Institute of Bioenergy, Chinese Academy of Sciences, which is certified by the National Metrology Bureau. The instrument used was a JNM-ECZ600R / S1 nuclear magnetic resonance spectrometer, manufactured by JEOL Ltd., Japan. The conditions were as follows: 1 1H: 600.20211 MHz; 13 13C: 150.92092 MHz. The ambient temperature was 25 °C, and the chemical shift was based on TMSP (δ 0 ppm). The acquisition time (AQ) was adjusted to 1.09052 s, while the relaxation time (D1) was adjusted to 5.0 s. A total of 32 scans were acquired.

[0069] (1) Preparation and purification of hydroxypropyl chitosan:

[0070] 10 g of CS-1 was mixed with 150 mL of NaOH solution (33%, weight ratio), degassed under vacuum until no bubbles were generated, and then frozen at -18 °C for more than 8 days. After thawing and filtration, an alkalized chitosan cake was obtained, which was transferred to a three-necked flask, and 150 mL of isopropanol was added. It was vigorously stirred to break it up. Propylene oxide was slowly added dropwise from a separatory funnel, and the hydroxypropylation reaction was carried out under reflux condensation. The product was filtered through a vacuum filter, washed repeatedly with absolute ethanol, dissolved in deionized water, the pH value was adjusted with 2 mol / L HCl, dialyzed, and freeze-dried. The obtained HPCS product was stored in a refrigerator at 4 °C.

[0071] The hydroxypropylation reaction conditions, degree of substitution (MS), and degree of deacetylation (DA) of CS and HPCS samples are shown in Table 1.

[0072] (2) Drying the hydroxypropyl chitosan sample to remove moisture;

[0073] The chitosan and hydroxypropyl chitosan samples and KBr were dried at 80 °C for 4 h, then heated to 105 °C and dried for another 1.5 h. After drying, the lid of the centrifuge tube was quickly closed and placed in a desiccator containing color-changing silica gel for standby.

[0074] (3) Establishing a detection model:

[0075] a. Under an infrared lamp irradiation environment, the dried sample was mixed with dried KBr (1 - 2%, w / w), ground, and pressed into a transparent tablet. The spectrum was recorded using a Fourier transform infrared spectrometer (Nicolet IS10, Thermo Scientific, USA), scanned 64 times, and the scanning step was 4 cm -1, using a KBr background sheet as a blank, obtain the infrared spectra of the dried samples of hydroxypropyl chitosan with different degrees of substitution in step (1) (hereinafter collectively referred to as samples) using an infrared absorption spectrometer.

[0076] b. Using the application software of the infrared absorption spectrometer, obtain a series of integral values of peak heights or peak areas from the infrared spectra of the hydroxypropylated polysaccharide samples obtained in step a: using the peak area formed by the two valleys of the methyl characteristic peak at 2968 - 2972 cm -1 from 2952 - 3007 cm -1 as the left and right endpoints to form a baseline (BL4) as the probe peak, denoted as B 2970-4 ;

[0077] There are various methods for selecting the reference peak. For example, it can be selected from the O - H and N - H stretching vibration peaks at 3400 - 3460 cm -1 (peak 1) the peak height value formed on the baseline (BL1) formed between the two valleys of this peak from 2200 - 3990 cm -1 is denoted as R 3440-1 ; or, the O - H and N - H stretching vibration peaks at 3400 - 3460 cm -1 (peak 1) the peak height value formed on the baseline (BL2) formed between the two valleys of this peak from 2995 - 3850 cm -1 is denoted as R 3440-2 ; or, the methylene characteristic peak at 1413 - 1417 cm -1 the peak height value formed on the baseline (BL8) formed between the two valleys of this peak from 1210 - 1570 cm -1 is denoted as R 1415-8 ; or the methylene and methine characteristic peaks at 2873 - 2877 cm -1 the peak height value formed on the baseline (BL3) formed between the two valleys of this peak from 2300 - 3070 cm -1 is denoted as R 2875-3 ;

[0078] Fourier transform infrared spectroscopy (FT - IR) is a widely accepted analytical method for determining the structure of compounds because of its low cost, environmental friendliness, and small sample volume for analysis. However, its complex and overlapping absorption peaks make it very difficult to achieve independent quantitative determination. In the Fourier transform infrared spectrum of HPCS, compared with the raw material chitosan, the newly emerging absorption peaks at 2970 cm -1 , 1458 cm -1 are the asymmetric stretching and asymmetric bending vibration peaks of hydroxypropyl methyl C - H. Other peaks related to methyl (symmetric stretching at 2878 cm -1 , symmetric bending at 1378 cm -1 ) and methylene (2922 cm-1 Asymmetric stretching at 1415 cm -1 The absorption peaks related to the scissor bending) C-H vibration at 1660 cm overlap partially with the chitosan peaks. -1 1598 cm -1 and 1328 cm -1 The absorption peaks at 1152 cm belong to the amide I band (C=O stretching vibration), amide II band (N-H bending vibration), and amide III band (C-N stretching vibration), respectively. -1 The peak at 1080 cm is attributed to the C-O-C stretching vibration, -1 and 1034 cm -1 The peaks at are related to the C-O stretching vibrations of primary and secondary hydroxyl groups, respectively. Correspondingly, the O-H bending vibrations of hydroxyl groups overlap with the C-H bending at 1415 cm -1 and the C-N stretching at 1328 cm -1 Obviously, the integral values of the overlapping peaks make the infrared spectrum too complex to establish an MS determination method using only Fourier transform infrared spectroscopy. Therefore, an accurate method must be introduced as a reference standard.

[0079] Calculate the ratio of the probe peak B 2970-4 to the reference peak, denoted as X;

[0080] c. Using 1 The degree of substitution Y of the hydroxypropylated polysaccharide sample in step a was measured by HNMR method;

[0081] Take the dried HPCS sample (6 mg), then add DCl / D 2 O (180 μL DCl and 420 μL D 2 O), mix until dissolved, transfer to a labeled NMR tube, and seal for use. It should be noted that chitosan and low-substituted HPCS (MS ≤ 0.5) are insoluble in concentrated DCl solution. Therefore, they are dissolved in a diluted DCl solution (15 μL DCl and 585 μL D 2 O) and measured at 70 °C to avoid interference from the HOD peak during measurement.

[0082] The DA and MS of HPCS are calculated as follows

[0083]

[0084] where I 1-D 、I 9-N 、I Ac are the integrals of H 1-D 、H 9-N 、H Ac respectively, and I 9 is H9-N and H 9-O The sum of the integrals.

[0085] Table 1 Hydroxypropylation reaction conditions of HPCS samples and their MS and DA

[0086]

[0087] (a) The numbers or symbols of the samples represent HPCS numbers or HPCS symbols. For example, 1 is HPCS-1, representing sample 1, and A represents HPCS-A, representing sample A.

[0088] (b) HPCS-8-1, HPCS-9-1, and HPCS-10-1 are undried samples of HPCS-8, HPCS-9, and HPCS-10.

[0089] Data analysis: In the NMR spectra of HPCS samples, compared with chitosan, new absorption peaks related to hydroxypropyl appeared, including methyl proton peaks (H 9-N and H 9-O , at 1.26 ppm and 1.16 ppm respectively); methylene proton peaks (H 7-N , H 7-O and the re-substitution peaks in the range of 3.3 - 3.6 ppm); methyl proton peaks (H 8-N , H 8-O and the re-substitution peaks at 4.06, 4.04, and 3.59 ppm). These peaks overlap with each other, and only H 9 (including H 9-N and H 9-O ) can be used as a reference peak for MS determination. In terms of reference peaks, H 1 showed three independent peaks, including H 1-N (5.11 ppm), H 1-D (4.89 ppm), and H 1-AC (4.83 ppm), meeting the requirements of MS determination. Therefore, in Fourier transform infrared analysis, 1 The 1H NMR method can be used as a reliable reference standard for accurately determining the MS of HPCS.

[0090] The corresponding infrared spectral data were processed using OMNIC software. The band intensity and band area were measured according to the baseline, as Figure 1 shown. The analysis results were statistically analyzed using the data analysis add-in function of Microsoft Excel software (version 2016) to find the 1 MS values measured by the 1H NMR method and the corresponding ratios obtained from Fourier transform infrared spectra (probe peak integral / reference peak integral, I P-B / I R-B) The linear regression relationship between them. In the infrared spectrum, the baseline graph and the corresponding peak heights (denoted by R) and peak areas (denoted by B or C) measurement results are as Figures 1-3 shown. The NMR spectrum of HPCS is as Figure 10 shown.

[0091] d. For a set of ratios X obtained by any of the methods in step b and a set of degrees of substitution Y of the hydroxypropyl chitosan samples obtained in step c, establish a regression model with a good linear relationship, that is, Y = kX + b, so as to obtain the specific values of k and b in the model;

[0092] (3) Degree of substitution calculation: Dry the sample of the hydroxypropylated polysaccharide to be measured according to the method in (2), obtain the infrared spectrum from step a in (3), obtain the ratio X according to step b in (3), and substitute it into the linear regression model obtained in step d in (3), then the degree of substitution Y of the sample of the hydroxypropylated polysaccharide to be measured can be obtained by calculation.

[0093] Research and selection of integration methods: The design of the probe peak, reference peak and their integration methods plays a crucial role in accurately measuring the MS in HPCS samples. In the Figure 1 shown Fourier transform infrared spectrum of HPCS, three integration methods of R, B and C are used for measurement. The C-H stretching vibration and bending vibration peaks of hydroxypropyl methyl at 2970 cm -1 and 1458 cm -1 can be determined as the probe peaks. According to Figure 1 , taking the 2970 cm -1 peak as an example, it can be characterized by the peak intensity (denoted as R 2970 ) and area (denoted as C 2970 ). These two parameters can be measured according to the baselines BL1, BL2 and BL3 using any possible combination. Regarding the baseline BL4 specifically pointing to the 2970 cm -1 peak, there are two calculation methods: the peak area B 2970-4 and the peak intensity R 2970-4 .

[0094] However, in terms of the reference peak, it becomes challenging to determine which peak can be used as the reference peak because it is difficult to find a peak that has nothing to do with hydroxypropylation. The peak at 1660 cm -1 is attributed to the C=O stretching of the amide I band and has nothing to do with hydroxypropylation. However, during the hydroxypropylation process in a highly alkaline environment under heating conditions, deacetylation will inevitably occur. In addition, this C=O stretching is also affected by the H-O-H bending of water (1650 cm -1 ). Therefore, we have to design multiple integration methods entirely based on the convenience of measuring the integral value of the peak for subsequent screening and optimization.

[0095] As Figure 1 shown, we designed 11 baselines to correspond to these spectral bands. Therefore, although we still name the integral values of the selected peaks as reference peaks, they are not the integral values of truly constant peaks. We assume that the integral growth rate of the reference peaks is lower than that of the probe peaks. Therefore, we use the least squares method to establish a linear regression model between the MS obtained by 1 H NMR and the ratio (I P-B / I R-B ) measured from the Fourier transform infrared spectrum. Therefore, Figure 11 shows the linear regression results obtained by combining any one of the 14 probe peak methods with any one of the 31 reference peak methods, which correspond to different raw materials CS-1 and CS-2 respectively. We found that 143 linear regressions met the requirement of R 2 > 0.95.

[0096] According to the traditional basic measurement theory, FTIR cannot be used for the MS determination of HPCS samples because after propylation, the C-H stretching vibrations and C-H bending vibrations of methyl and methylene groups overlap with the corresponding peaks of chitosan itself, and it is difficult to find an unaffected constant peak as a reference peak. So far, it is reasonable that there is still a lack of relevant publicly reported infrared determination methods. In fact, compared with the integration of other peaks, the C-H asymmetric stretching vibration and asymmetric bending vibration of methyl show obvious growth trends and are expected to be used for the accurate determination of the MS value of HPCS samples.

[0097] Study on the linear range: Regarding the lower limit of the working range, B 2970-4 is more superior to C 2970 and R 2970 because it is easy to measure. A similar situation also applies to the probe peak at 1458 cm -1 . As we expected, most linear regressions tend to the integration methods such as B 2970-4 , B 1458-9 .

[0098] Effect of propylation temperature on MS determination: According to the traditional basic measurement theory, FTIR cannot be used for the MS determination of HPCS samples because after propylation, the various C-H stretching and bending vibration bands of the methyl, methylene and methine groups of propyl overlap with the vibration bands of chitosan itself, making it difficult to find an unaffected constant peak as a reference. This can explain the reason for the lack of relevant literature reports so far. In fact, compared with the integral values of other peaks, as the MS of HPCS increases, the integral area of the methyl C-H vibration shows a higher increase rate. As seen from Figure 12 c, when the ordinate selects B 2970-4 / R3440-1 When it comes to this, the hydroxypropylation temperature has no influence on MS measurement.

[0099] Effect of residual acid on MS measurement: After hydroxypropylation, the strongly basic mixture must be neutralized to a certain pH level. Under this pH condition, the residual base is completely consumed, and all free amino groups in the HPCS molecule are in the -NH 2 state, rather than -NH 3 + state. The traditional method is to adjust the pH to neutral, resulting in the coexistence of -NH 2 and -NH 3 + groups to varying degrees. It is worth noting that, as Figure 13 shown in a, the C=O stretching vibration (1630 cm 3 + -1) and N-H bending vibration (1535 cm -1 -1) of -NH -1 (HPCS-1 to 5) are significantly different from the C=O stretching vibrations (1660 cm 2 -1 and 1598 cm -1 -1) of the -NH -1 group (HPCS-6). Correspondingly, a strong broad band appears in the range of 3000–2800 cm -1 , interfering with the accurate measurement of MS. Even thorough dialysis cannot solve this problem. Therefore, even trace amounts of residual -NH 3 + will cause imperceptible deformation of the absorption band, resulting in measurement errors, as Figure 13 shown in b and 13c. Among many reference peaks and their corresponding integration methods, the ratios of B 2970-4 / R 3400-1 and B 2970-4 / R 3440-2 are least affected by acid interference.

[0100] Effect of trace moisture in HPCS samples on mass spectrometry analysis: FTIR spectra are easily affected by moisture, so drying is necessary during sample preparation. As Figure 14 shown in a, due to the superposition of the H-O-H stretching vibration and bending vibration of water, the bands of undried samples (HPCS-8-1, 9-1, and 10-1) at 1660 cm -1 and 3440 cm -1 are significantly broader than those of dried samples (HPCS-8, 9, and 10). Therefore, when measured based on BL1 (B 2970-4 / R 2922-1 ), the bands adjacent to the 3440 cm -1 band (2970 cm-1 and 2922 cm -1 ) is also affected by it, such as Figure 14 as shown in Fig. b. In addition, in the presence of trace moisture, the C-OH stretching vibration band in the range of 1140 cm -1 to 1000 cm -1 broadens. BL3 is the best choice to avoid moisture interference, especially B 2970-4 / R 2875-3 (p = 0.620), as shown in Figure 14 Fig. c. There is no significant difference between the drying process and the non-drying process. Similar situations also include B 2970-4 / R 2922-3 (p = 0.398).

[0101] Effect of DA on MS measurement: During the etherification reaction, the acetyl groups attached to the C 2 -NH 2 on chitosan can be partially removed under high-temperature alkaline conditions. As shown in Table 1 and Figure 15 Fig. a, the change in DA from CS-1 to HPCS-12 (0.139 to 0.037) and from CS-2 to HPCS-24 (0.027 to 0.003) shows a similar trend. Therefore, as shown in Figure 15 Fig. b, the integration methods related to amide I (C=O stretching, 1660 cm -1 ) and amide II (N-H bending, 1598 cm -1 ) bands are not applicable to the determination of HPCS samples in a wide DA range (0.003–0.139, see Table 1). Linear regression can only be constructed based on two independent DA ranges, namely 0.037–0.139 and 0.027–0.003. Integration related to the reference peak at 2922 cm -1 ; 2875 cm -1 ; 1415 cm -1 ; 1378 cm -1 ; 1152 cm -1 ; 1080 cm -1 meets the requirements of MS measurement. Surprisingly, as shown in Figure 15 Fig. c, R 3440-1 and R 3440-2 are also suitable reference peaks for measurement. It should be noted that the change in DA may lead to an increase in symmetric and asymmetric N-H stretching vibrations around 3440 cm -1 .

[0102] Method Validation: A comprehensive study of the integration method shows that MS determination requires generality, accuracy, robustness, and simplicity. Considering that it can be very challenging to predict the DA of samples before testing, there is an urgent need for a broad-spectrum integration method that does not require prior knowledge of the DA and MS ranges. Therefore, we used 20 HPCS samples with broad-spectrum DA values (ranging from 0.003 to 0.139) as the research objects, and used the least squares method to perform linear regression on the MS values measured by NMR and the infrared integration ratio, resulting in 16 linear regression equations (R 2 > 0.98), whose MS values have a wide MS range from 0.17 to at least 1.22, as shown in Figure 16 and Table 2. Under these conditions, another five HPCS samples (HPCS-A, B, C, D, and E) were used for method validation. By measuring the spectra of each HPCS sample and calculating according to the corresponding 16 linear regression equations, 16 MS results were obtained. Among these results, the recovery rates and relative standard deviations of HPCS-A, HPCS-B, HPCS-C, and HPCS-E were 98.5 ± 9.4%, 97.9 ± 10.0%, 100.0 ± 6.3%, and 95.0 ± 3.4% respectively, and the recovery rates of parallel samples were 98.2 ± 7.4% and 103.6 ± 6.2%.

[0103] Table 2 Method Validation

[0104]

[0105]

[0106] Example 2

[0107] Preparation of Hydroxypropyl Starch: Weigh 10 g of starch in a 100 mL flask. Take 20 mL of 20% NaOH solution and 150 mL of 70% isopropanol and mix them well in a three-necked flask. Add 10 g of starch to the mixture spoon by spoon, stirring while adding, and alkalize for one hour. Place the three-necked flask in an oil bath and set the temperature of the oil bath. The three outlets of the three-necked flask are respectively connected to a condenser, a stirrer (300 r), and a separatory funnel containing 70 mL of propylene oxide. Add propylene oxide drop by drop and set the hydroxypropylation time. Stop stirring to terminate the reaction to obtain the crude hydroxypropylated starch (HPS). Set the hydroxypropylation temperature and time as shown in Table 3.

[0108] Table 3 Starch Hydroxypropylation Temperature and Time

[0109]

[0110] Pour the obtained crude HPS onto the filter paper on the Buchner funnel, remove the solution part, and retain the filter cake. Wash it with ethanol, remove the filtrate, dissolve it in water, adjust the pH to neutral, place the solution in a dialysis bag, and dialyze it with ultrapure water until the conductivity is below 4. HPS8 was purchased from Shanghai Zhixi Chemical Industry.

[0111] Characterization of hydroxypropyl starch by NMR method and determination of its degree of substitution: The sample was dried as described by the infrared method. Dissolve the dried sample in D 2 O / DCl. The sample was tested with a JNM-ECP600 nuclear magnetic resonance spectrometer (JEOL, Japan), and the conditions were 1 H: 600.20211 MHz; ambient temperature 25 °C, and the chemical shift was based on TMSP (δ 0 ppm). The acquisition time (AQ) was adjusted to 1.09052 s, while the relaxation time (D1) was adjusted to 5.0 s, and a total of 32 scans were acquired. Degree of substitution MS formula:

[0112]

[0113]

[0114] where: I H1 , I H9 , I H2-8 are the integrals of the H 1 , H 9 , H 2-8 peaks respectively. The results are as follows:

[0115] Table 4 Degree of substitution of hydroxypropyl starch

[0116]

[0117] Characterization of hydroxypropyl starch by infrared method: Place the sample in an oven, dry it at 80 °C for 2 h, then at 105 °C for 2.5 h, seal it, and cool it. Under dry and room temperature conditions, mix the dried sample with KBr and press it into a tablet. The FT-IR spectrum was measured using a Fourier transform infrared spectrometer (Nicolet NicoletIS10, ThermoFisher). The spectrum was scanned 64 times in the range of 400 - 4000 cm -1 .

[0118] Analysis of the infrared spectrum, such as Figure 4 : The main absorption peaks of starch are the O-H stretching vibration at 3423 cm -1 , the C-H stretching vibration (methylene and methylene) at 2930 cm -1 , and the -1The OH bending vibration (methylene and methine) at 2970 cm-1 is different from that of raw starch. The main difference between hydroxypropyl starch and raw starch is the methyl group on the hydroxypropoxy group. -1 A new absorption peak is added at 1459cm, which is the methyl absorption peak of hydroxypropoxy (CH stretching vibration). -1 1376cm -1 The asymmetric bending vibration and symmetric bending vibration of the CH of the methyl group are at 2970 cm-1. -1 There is basically no absorption, HPS4 has a slight absorption peak, and HPS5-HPS8 has a clear absorption peak. This is because the reaction time of the first few samples is short and the degree of hydroxypropylation is low, so the absorption peak is not obvious, while the reaction time of the latter few samples is long, the degree of hydroxypropylation is high, and the absorption peak is obvious. As the reaction temperature increases and the reaction time becomes longer, the intensity of the hydroxypropylated methyl absorption peak increases.

[0119] Determination of substitution degree of HPS by FTIR method: The FTIR spectrum of HPS also shows dynamic changes with the substitution degree. The determination of substitution degree of HPS by FTIR is to select appropriate reference peak (RB), probe peak (PB) and baseline (BL), then calculate the value of PB / RB, and compare the obtained ratio with 1 The substitution degree measured by HNMR was combined with the substitution degree measured by FTIR to study the substitution degree of HPKGM. The baseline used for FTIR determination of the substitution degree of HPS is as follows: Figure 4 , Figure 5 , Figure 6 The baselines are numbered BL1-BL14, and the FTIR baseline must be strictly tangent to the two selected points. The FTIR determination of the HPS substitution degree integral is shown in the figure below. Figure 18 R represents the maximum peak height under the baseline. If the maximum peak height cannot be identified, R is the peak height under the specified wave number, such as R 2970-3 , then the wave number is 2970cm -1 The integral value of the peak height at the FTIR spectrum. B and C both represent the area between the baseline and the bottom of the FTIR spectrum, where the starting and ending points of the integration of area B are the same as the starting and ending points of the baseline, and the starting and ending points of the integration of area C are smaller than the starting and ending points of the baseline, which is only the area of ​​the required absorption peak under the baseline. Table 5 shows the probe peak and reference peak used in FTIR determination of HPS substitution degree.

[0120] Table 5 Probe peak and reference peak used for FTIR determination of starch (HPS) substitution degree

[0121]

[0122] The FTIR spectra of each HPS are shown in Figure 21As shown, different probe peaks and reference peaks are selected for integration of different samples by the above integration method, and the integration ratio of the probe peak to the reference peak is calculated, and the ratio is compared with 1 MS measured by HNMR 1 Combined, the linear correlation coefficient between the two is obtained, thus obtaining the integral ratio and MS 1 or MS 2 The linear relationship between FTIR (B 2970-4 / R 2930-3 ) and MS 1 Linear relationship diagram Figure 22 After screening, R 2 The PB / RB values ​​greater than 0.95 are listed in Table 6. The results show that when the -1 When is the probe peak, 1 There are multiple linear relationships between HNMR methods, the reference peak and the corresponding integration method (R 2 >0.98) has B 2970-4 / R 2970-1 , B 2970-4 / R 2930-1 , B 2970-4 / R 2882-1 , B 2970-4 / R 2970-3 , B 2970-4 / R 2930-3 , B 2970-4 / R 2882-3 , B 2970-4 / R 1459-8 , B 2970-4 / C 1459-8 , B 2970-4 / R 1414-8 , B 2970-4 / R 1376-8 , B 2970-4 / C 1376-8 , B 2970-4 / R 1332-8 , B 2970-4 / R 1459-9 , B 2970-4 / B 1459-9 , B 2970-4 / R 1153-11 , B 2970-4 / R 1089-11 , B 2970-4 / C 1153-11 , B 2970-4 / B 1089-11 , R 2970-3 / R 1414-8 , C 2970-3 / R 3423-1 , C 2970-3 / R 3423-2 , R2970-1 / R 1414-8 This method brings convenience to the determination of the degree of substitution of HPS.

[0123] Table 6 Linear relationship between FTIR PB / RB and 1 MS of 1H NMR of hydroxypropyl starch 2 between

[0124]

[0125]

[0126] Example 3

[0127] Preparation of hydroxypropyl mannan (HPKGM): First, mix a certain concentration of NaOH solution and a certain concentration of ethanol solution evenly, and add it to a three-necked flask containing 10 g of mannan (KGM). Add the NaOH and ethanol mixed solution to the three-necked flask, quickly stir to make KGM evenly dispersed, and then place it at room temperature for alkalization for 30 min. After the alkalization is completed, transfer the three-necked flask to a water bath, slowly drop propylene oxide while stirring to make propylene oxide fully contact with KGM, and filter to terminate the reaction. The specific preparation conditions are shown in Table 7.

[0128] Table 7 Preparation conditions of HPKGM

[0129]

[0130]

[0131] Separation and purification of HPKGM: Immerse the product in ethanol first, ultrasonicate, stir, and filter, repeat three times to ensure the removal of impurities. Dissolve the filter cake in water, adjust the pH value to neutral with hydrochloric acid, dialyze until the conductivity of the solution is equal to that of distilled water, concentrate under reduced pressure, freeze-dry to obtain HPKGM, seal it in a vacuum, and store it at 4 °C.

[0132] FTIR structural characterization: Dry the KGM and HPKGM samples at 80 °C for 4 h and at 105 °C for 2 h to remove the moisture in the samples. After cooling, mix with KBr, grind it into extremely fine powder in a mortar, press tablets, and detect with an infrared spectrometer. The detection conditions are step 4 cm -1 , scanning times 64 times, scanning range 4000 - 400 cm -1 . The results are as Figure 7 shown.

[0133] NMR structural characterization:

[0134] Preparation of KGM and HPKGM sample solutions (30 mg / mL): 30.0 mg of KGM and HPKGM samples were respectively mixed with 1 mL of NaOH or HCl solution, sealed, and allowed to stand until dissolved. To explore the sample preparation method suitable for NMR measurement, the effects of acid and base concentrations and standing temperature on the apparent solubility and viscosity of KGM and HPKGM samples were investigated respectively.

[0135] Obtaining of KGM and HPKGM NMR spectra: The samples were tested using a JNM-ECP600 nuclear magnetic resonance spectrometer (JEOL, Japan), and the conditions were 1 H: 600.20211 MHz, ambient temperature 25 °C, and the chemical shift was based on TMSP (δ 0 ppm). The acquisition time (AQ) was adjusted to 1.09052 s, while the relaxation time (D1) was adjusted to 5.0 s, and a total of 32 scans were acquired.

[0136]

[0137] where: I H9 、I H2-8 are the integrals of the H 9 、H 2-8 peaks respectively.

[0138] FTIR characterization of KGM and HPKGM: The FTIR spectra of KGM, L-HPKGM, and H-HPKGM further illustrate the structural changes of KGM and its hydroxypropylated derivatives L-HPKGM and H-HPKGM, as Figure 7 shown. Compared with the KGM spectrum, there is no acetyl C=O stretching vibration (1734 cm -1 ) in the HPKGM spectrum, which has been removed during the hydroxypropylation process, and is replaced by the characteristic absorption peaks of hydroxypropyl, such as the C-H asymmetric stretching vibration of the methyl group of hydroxypropyl at 2970 cm -1 , the asymmetric bending vibration at 1458 cm -1 and the symmetric bending vibration at 1376 cm -1 . Hydroxypropylation significantly increases the ether bond C-O-C stretching vibration at 1152 cm -1 , gradually weakens the C-O stretching vibration of the primary hydroxyl group at the C 6 position at 1030 cm -1 , and is gradually replaced by the enhanced C-O stretching vibration of the secondary hydroxyl group at 1058 cm -1 .

[0139] Determination of substitution degree of HPKGM by FTIR method: The FTIR spectrum of HPKGM also shows dynamic changes with the change of substitution degree. The determination of substitution degree of HPKGM by FTIR is to select appropriate reference peak (RB), probe peak (PB) and baseline (BL), then calculate the value of PB / RB, and compare the obtained ratio with 1 The substitution degree measured by HNMR was combined with the FTIR method to study the substitution degree of HPKGM. Here, the MS and FTIR ratios were used for combination. The baseline used for FTIR determination of the substitution degree of HPKGM is as follows: Figure 7 , Figure 8 , Figure 9 The baselines are numbered BL1-BL11, and the FTIR baseline must be strictly tangent to the two selected points. The FTIR determination of the HPKGM substitution degree integral is shown in Figure 18 R represents the maximum peak height under the baseline. If the maximum peak height cannot be identified, R is the peak height under the specified wave number, such as R 2970-3 , then the wave number is 2970cm -1 The integral value of the peak height at the FTIR spectrum. B and C both represent the area between the baseline and the bottom of the FTIR spectrum, where the starting and ending points of the integration of area B are the same as the starting and ending points of the baseline, and the starting and ending points of the integration of area C are smaller than the starting and ending points of the baseline, which is only the area of ​​the required absorption peak under the baseline. Table 8 shows the probe peak and reference peak used in FTIR determination of the degree of substitution of HPKGM.

[0140] Table 8 Probe peaks and reference peaks used in FTIR determination of HPKGM substitution degree

[0141]

[0142] The FTIR spectra of each HPKGM are as follows: Figure 19 As shown, the above integration method is used to integrate different probe peaks and reference peaks of different samples, calculate the integration ratio of the probe peak to the reference peak, and compare the ratio with 1 The MS measured by HNMR was combined to obtain the linear correlation coefficient between the two, thereby obtaining the linear relationship between the integral ratio and MS. 2970-5 / C 2970-1 ) and MS linear relationship diagram as shown in Figure 20 After screening, R 2 The PB / RB values ​​greater than 0.95 are listed in Table 9. The results show that when the -1 When is the probe peak, 1 There are multiple linear relationships between HNMR methods, and the reference peaks and corresponding integration methods are B 2970-4 / C 2970-0 , B 2970-4 / R 2880-0 , B 2970-4 / R 2970-1 , B 2970-4 / C 2970-1 , B 2970-4 / R 2921-1 , B 2970-4 / R 2880-1 , B 2970-4 / R 2921-3 , B 2970-4 / R 2880-3 , B 2970-4 / C (1328+1255)-8 , R 2970-4 / R 2880-1 , R 2970-4 / R 2880-1 and R 2970-4 / R 1376-8 At the same time R 2925-0 , R 2970-0 , R 1376-8 , R 1152-11 , R 2970-1 and R 2880-0 The ratio with some reference peaks also has a good linear relationship and can also be used to calculate the degree of substitution. This method brings convenience to the determination of the degree of substitution of HPKGM.

[0143] Table 9 FTIRPB / RB and 1 Linear relationship between H NMR

[0144]

[0145]

[0146] Effect of moisture on the substitution degree of HPKGM determined by FTIR method: HPKGM contains a large number of hydroxyl groups, which are hydrophilic groups. Therefore, HPKGM has a certain degree of water absorption. In actual operation, it is difficult to avoid the interference of moisture on the substitution degree determination. In order to explore the effect of moisture on the substitution degree of HPKGM determined by FTIR method, low, medium and high substitution degree HPKGM samples HPKGM-1, HPKGM-4 and HPKGM-12 were taken respectively, and tested directly without drying. Then their integral values ​​were calculated respectively, and compared with MS 1 Lian Li R 2 The results are shown in Tables 10 and 11. After HPKGM-1, HPKGM-4 and HPKGM-12 absorbed water, all the integrals increased, as shown in Table 10.

[0147] Table 10 Effect of moisture on HPKGM FTIR integral

[0148]

[0149]

[0150] After replacing the integral values of HPKGM-1, HPKGM-4, and HPKGM-12 after water absorption with the integral values of the dry ones, calculate the slope, intercept, and R of the regression curve with MS 1 of the regression curve 2 It was found that, taking the absorption peak at 3423 cm -1 as the reference peak, the slope, intercept, and R of the regression curve with MS 1 of the regression curve 2 changed greatly. This is because the strong absorption at PB / RB corresponds to the stretching vibration of -OH, and the stretching vibration of -OH in water is also at this position, so the variation range is large. At the same time, a regression curve of PB / RB with an R 2 greater than 0.99 and almost unaffected by moisture was also found. There was no significant difference between the dry and water-containing regression curves of PB / RB that was not affected by moisture. The relationship between PB / RB that was not affected by moisture and MS 1 is as Figure 11 shown, where the maximum value of R 2 can reach 0.9972, and it can be directly used for the determination of the degree of substitution of HPKGM without considering the influence of moisture on it.

[0151] Table 11 Influence of moisture on the regression curve of HPKGM and analysis of significant differences

[0152]

[0153] The above experimental conclusions are as follows:

[0154] (1) When taking 2970 cm -1 as the probe peak, there are multiple linear relationships with 1 the HNMR method. The reference peak and the corresponding integration methods have B 2970-4 / C 2970-0 ,, R 2970-4 / R 2921-0 , B 2970-4 / R 2880-0 , B 2970-4 / R 2970-1 , B 2970-4 / C 2970-1 , B 2970-4 / R 2921-1 , B 2970-5 / R 2880-1 , B 2970-4 / R 2921-3 , B 2970-4 / R 2880-3 , B 2970-4 / C (1328+1255)-8 , R 2970-4 / R 2880-1 and R 2970-4 / R1376-10 Their R 2 values are all greater than 0.99;

[0155] (2) The absorption peak at 3423 cm -1 is greatly affected by moisture. The ratio of its probe peak to this reference peak varies significantly due to different moisture contents. Generally, the sample needs to be thoroughly dried under the same dry conditions before it can be used for sample testing.

[0156] (3) In addition, four probe peak / reference peaks that are not affected by moisture are found. They are B 2970-4 / R 2880-0 , B 2970-4 / R 2880-1 , B 2970-4 / R 2880-3 and B 2970-4 / R 1376-8 . Among them, some R 2 can even reach 0.9972. This method is simple to operate, without the need for weighing or drying, and the testing cost is lower than that of nuclear magnetic resonance hydrogen spectroscopy. It can be used for the routine determination of this type of product.

Claims

1. A method for determining the degree of substitution of three hydroxypropylated polysaccharides by infrared spectroscopy, characterized in that: The three hydroxypropylated polysaccharides include hydroxypropyl chitosan, hydroxypropyl mannan and hydroxypropyl starch, and the method for determining the degree of substitution of the three hydroxypropylated polysaccharides by infrared spectroscopy includes the following steps: (1) Drying: drying hydroxypropylated polysaccharide samples to remove water, wherein each hydroxypropylated polysaccharide sample is prepared by polysaccharide hydroxypropylation reaction and purification and is derived from a polysaccharide raw material with a known degree of acetylation; (2) Establish a detection model: a. using an infrared absorption spectrometer to obtain infrared spectra of the dried hydroxypropylated polysaccharide samples with different degrees of substitution in step (1); b. Using the application software of the infrared absorption spectrometer, a series of peak heights or peak area integral values ​​are obtained from the infrared spectrum of the hydroxypropylated polysaccharide sample obtained in step a as reference peaks. The integration method of the reference peak is: ① Vibration peaks used for integration: OH stretching vibration peak or NH stretching vibration peak 3400-3460cm -1 (Peak 1), methyl CH asymmetric stretching vibration peak 2968-2972cm -1 (Peak 2), methylene CH asymmetric stretching vibration peak 2910-2940cm -1 (Peak 3), methyl and methylene CH symmetric stretching vibration peaks 2860-2900 cm -1 (Peak 4), methyl CH asymmetric bending vibration and methylene bending vibration peak 1450-1465cm -1 (Peak 5), CH bending vibration peak of methylene 1410-1425cm -1 (Peak 6), methyl CH symmetric bending vibration and methylene bending vibration peak 1370-1385cm -1 (Peak 7), CH bending vibration peak of methylene 1320-1335cm -1 (Peak 8), COC stretching vibration peak 1145-1160cm -1 (Peak 9), including the CO stretching vibration peak of the hydroxyl group and the COC stretching vibration peak, the entire band composite vibration peak 820-1235cm -1 (Peak 10); ②Baseline for integration The baseline is selected from the entire spectral band 2200-3990cm -1 The baseline (BL0) is formed by the tangent line of the trough of the wave, and the baseline is selected from the entire spectral band 1800-3990cm -1 The baseline (BL1) is formed by the tangent line of the trough of the wave, and the baseline is selected from the entire spectral band 2995-3850cm -1 The baseline (BL2) is formed by the tangent line of the trough of the wave, and the baseline is selected from the entire spectral band 2300-3070cm -1 The baseline (BL3) is formed by the tangent line of the trough of the spectrum, and the baseline is selected from the entire spectral band 2945-3020cm -1 The baseline (BL4) is formed by the tangent line of the trough, and the baseline is selected from the entire spectral band 1210-1570cm -1 The baseline (BL8) is formed by the tangent line of the trough, and the baseline is selected from the entire spectral band 1430-1520cm -1 The baseline (BL9) is formed by the tangent line of the trough of the spectrum, and the baseline is selected from the entire spectral band 1345-1400cm -1 The baseline (BL10) is formed by the tangent line of the trough of the spectrum, and the baseline is selected from the entire spectral band 820-1235cm -1 The baseline (BL11) is formed by the tangent line of the trough; ③Integral method There are three integration methods: R, B, and C. R represents the maximum peak height under the baseline. If the spectrum peak is not prominent, R is the peak height of the specified wave number under the baseline. If the spectrum peak of the specified wave number is not the highest peak in the spectrum band, R is the peak height of the specified wave number under the baseline. B and C both represent the area between the spectrum band and the baseline. The starting and ending points of the integration of area B are the two troughs of the spectrum band, which are the same as the starting and ending points of the baseline. The starting and ending points of the integration of area C are smaller than the starting and ending points of the baseline, which is only the area of ​​the required absorption peak under the line. Taking the infrared spectrum of hydroxypropyl chitosan as an example, R 3440-1 That is, the integral value of the peak height of peak 1 perpendicular to the wave number horizontal axis intersecting BL1; R 3440-2 That is, the integral value of the peak height of peak 1 perpendicular to the wave number horizontal axis intersecting BL2; C 2970-3 That is, the integral value of the peak area formed by the two end points of the trough of peak 2 perpendicular to the horizontal coordinate of the wave number intersecting at BL3, B 2970-4 That is, the integral value of the peak area of ​​the entire spectrum of peak 2 surrounded by BL4, R 2922-3 That is, the integral value of the peak height of peak 3 perpendicular to the wave number horizontal axis intersecting BL3, R 2875-3 That is, the integral value of the peak height of peak 4 perpendicular to the wave number horizontal axis intersecting BL3; other R 2970-1 , R 2922-1 , R 2875-1 , R 2970-3 , R 2970-4 By analogy, R 1458-8 R is the integral value of the peak height of peak 5 perpendicular to the wave number horizontal axis and intersecting the highest point at BL8; 1415-8 R is the integral value of the peak height of peak 6 perpendicular to the wave number horizontal axis and intersecting the peak height at BL8. 1378-8 R is the integral value of the peak height of peak 7 perpendicular to the wave number horizontal axis and intersecting the peak height at BL8. 1328-8 C is the integral value of the peak height of peak 8 perpendicular to the wave number horizontal axis and intersecting BL8. 1378-8 B is the integral value of the peak area formed by the two end points of the trough of peak 7 perpendicular to the horizontal coordinate of the wave number intersecting at BL8. 1458-8 is the integral value of the peak area of ​​the entire spectral band of peak 5 surrounded by BL8, B 1378-10 is the integral value of the peak area of ​​the entire spectrum of peak 7 surrounded by BL10, R 1378-10 R is the integral value of the peak height of peak 7 perpendicular to the wave number horizontal axis and intersecting the peak height at BL10. 1152-11 B is the integral value of the peak height of peak 9 perpendicular to the wave number horizontal axis and intersecting the peak height of BL11. 1080-11 is the integral value of the peak area of ​​the entire spectral band of peak 10 surrounded by BL11, R 1080-11 It is the integral value of the peak height of peak 10 perpendicular to the wave number horizontal axis and intersecting the highest point at BL11, and the others are similar; c.B 2970-4 as the probe peak; calculate the reference peak using the integration method in step b; Calculate probe peak B separately 2970-4 The ratio to the reference peak is denoted as X; When the hydroxypropylated polysaccharide is hydroxypropyl chitosan, the reference peak is selected from R 3440-1 ,R 2922-1 ,R 2875-1 ,R 3440-2 ,R 2922-3 ,R 2875-3 ,R 1415-8 ,R 1378-8 ,C 1378-8 ,R 1328-8 ,R 1378-10 ,B 1378-10 ,R 1152-11 ,R 1080-11 ,C 1152-11 and B 1080-11 ; When the hydroxypropylated polysaccharide is hydroxypropyl mannan, the reference peak is selected from R 2970-1 , R 2921-1 , R 2880-1 , R 2970-3 , R 2921-3 , R 2880-3 , R 1458-8 , C 1458-8 , R 1413-8 , R 1376-8 , C 1376-8 , R 1328-8 , R 1458-9 , B 1458-9 , R 1152-11 , R 1058-11 , C 1152-11 , B 1058-11 , R 1413-8 , R 3423-1 , R 3423-2 ; When the hydroxypropylated polysaccharide is hydroxypropyl starch, the reference peak is selected from R 2970-1 , R 2930-1 , R 2882-1 , R 2970-3 , R 2930-3 , R 2882-3 , R 1459-8 , C 1459-8 , R 1414-8 , R 1376-8 , C 1376-8 , R 1332-8 , R 1459-9 , B 1459-9 , R 1153-11 , R 1089-11 , C 1153-11 , B 1089-11 ; d. 1 The degree of substitution Y of the hydroxypropylated polysaccharide sample in step a is measured by HNMR method; e. Using a set of ratios X obtained by any method in step c to a set of substitution degrees Y of the hydroxypropyl chitosan sample obtained in step d, a good linear regression model is established, ie, Y = kX + b, so as to obtain specific values ​​of k and b in the model; (3) Calculation of degree of substitution: The hydroxypropylated polysaccharide sample to be tested is dried according to the method in (1), and the infrared spectrum is obtained from step a in (2). The ratio X is obtained according to step c in (2), and is substituted into the linear regression model obtained in step e in (2), and the degree of substitution Y of the hydroxypropylated polysaccharide sample to be tested can be obtained by calculation.

2. The method for determining the degree of substitution of three hydroxypropylated polysaccharides by infrared spectroscopy according to claim 1, characterized in that: The method for drying the hydroxypropylated polysaccharide sample is to dry it at 80°C for 4-5 hours and at 105°C for 1.5-2 hours, and then take out the sample and place it in a desiccator filled with color-changing silica gel for later use.

3. The method for determining the degree of substitution of three hydroxypropylated polysaccharides by infrared spectroscopy according to claim 1, characterized in that: The method for drying the hydroxypropylated polysaccharide sample is freeze drying or vacuum drying.

4. The method for determining the degree of substitution of three hydroxypropylated polysaccharides by infrared spectroscopy according to claim 1, characterized in that: The infrared absorption spectrometer is a Fourier transform infrared spectrometer, and the application software of the infrared absorption spectrometer is OMNIC software.

5. The method for determining the degree of substitution of three hydroxypropylated polysaccharides by infrared spectroscopy according to claim 1, characterized in that: The detection conditions of the infrared absorption spectrometer are: scanning range 400-4000cm -1 , scanned 64 times, with a resolution of 4cm -1 .

6. The method for determining the degree of substitution of three hydroxypropylated polysaccharides by infrared spectroscopy according to claim 1, characterized in that: The infrared spectrum of the obtained hydroxypropylated polysaccharide sample was subjected to background detection and background subtraction processing.

7. The method for determining the degree of substitution of three hydroxypropylated polysaccharides by infrared spectroscopy according to any one of claims 1 to 6, characterized in that: When the hydroxypropylated polysaccharide is selected from hydroxypropyl chitosan, and the reference peak is selected from R 2875-3 and R 2922-3 When the method for determining hydroxypropylated polysaccharides does not include step (1) drying.

8. The method for determining the degree of substitution of three hydroxypropylated polysaccharides by infrared spectroscopy according to any one of claims 1 to 6, characterized in that: When the hydroxypropylated polysaccharide is selected from hydroxypropyl mannan, and the reference peak is selected from R 2884-1 ,R 2884-3 ,R 2922-3 When the method for determining hydroxypropylated polysaccharides does not include step (1) drying.

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