CN105457503A - Preparation and application of chlorogenic acid molecular imprinting chitosan membrane - Google Patents

Abstract

The invention discloses a preparation method of chlorogenic acid molecularly imprinted chitosan film: dissolving chitosan and chlorogenic acid in an acetic acid solution with a volume fraction of 2%, filtering with gauze after sufficient stirring, and defoaming the filtrate to obtain cast Membrane liquid; the mass ratio of chitosan and chlorogenic acid is 10:1, and the volume consumption of described acetic acid solution is counted as 40mL/g by the mass of chitosan; Put into 0.5mol. Soak in L ^-1 sulfuric acid solution for 24h to carry out cross-linking reaction, and then elute with eluent to prepare chlorogenic acid molecularly imprinted chitosan membrane. The chlorogenic acid molecularly imprinted chitosan membrane provided by the invention can be used to adsorb chlorogenic acid, and the preparation method is simple, easy to operate, good in mechanical strength, stable and acid-resistant, and can be directly applied without cumbersome preparation processes such as crushing and grinding. .

Description

Preparation and application of a chlorogenic acid molecularly imprinted chitosan film

technical field

The invention belongs to the field of preparation technology of adsorption materials, and relates to a method and performance research for preparing chlorogenic acid molecularly imprinted chitosan film by using molecular imprinting technology and phase inversion method.

Background technique

Chlorogenic acid is a carboxyphenolic acid composed of caffeic acid and quinic acid, which is a plant polyphenol. The content in Eucommia ulmoides, honeysuckle, coffee, chrysanthemum and other plants is very high; in addition, potatoes, carrots, spinach, apples and other vegetables and fruits also contain chlorogenic acid. Since chlorogenic acid is the main active ingredient in many traditional Chinese medicines and fruits and vegetables, it has a variety of biological activities, such as: cardiovascular protection, anti-oxidation, lipid-lowering and hypoglycemic effects. The active ingredients of traditional Chinese medicine have complex structures, various types, instability and low content, which lead to difficulties in the separation and purification of active ingredients in traditional Chinese medicine. Therefore, chlorogenic acid, which is an active ingredient in traditional Chinese medicine, is also difficult to separate and purify. At present, the methods for extracting and purifying chlorogenic acid include water extraction and alcohol precipitation method, water extraction lime milk precipitation method, alcohol extraction lead salt precipitation method, ultrasonic method, polyamide column chromatography, enzymatic method, supercritical method and macroporous method. Resin adsorption method, etc., but these methods have disadvantages such as low extraction rate, low selectivity, large damage to chlorogenic acid, small processing capacity, long process time, low product purity, poor safety and sanitation, and high cost.

Due to the advantages of molecular imprinting technology in preparation and specificity in recognition, it provides a method reference for the identification, analysis, separation and purification of components with low content and high efficacy in natural products. Although the traditional method of preparing molecularly imprinted polymers has a simple polymerization process, the follow-up processing is cumbersome and time-consuming, resulting in serious product loss, irregular shape and poor dispersion. The molecularly imprinted membrane, which has the dual characteristics of molecular imprinting technology and membrane separation technology, has a high retention rate of molecularly imprinted holes because it does not need to be pulverized and milled.

Chitosan is a natural polymer material with excellent performance. It has the advantages of good biocompatibility, biodegradability, non-toxicity, and low price. Because the molecule is rich in hydroxyl and amino groups, it can be combined with chlorogenic acid. The hydroxyl and carboxyl groups form hydrogen bonds and ion pairs. However, chitosan is prone to swelling in aqueous solution, which greatly limits its application. Through the reaction of functional groups such as aldehyde group and epoxy group with the amino group and hydroxyl group in chitosan, the crosslinking of chitosan can be realized, and its swelling property can be improved. Currently commonly used cross-linking agents include glutaraldehyde and epichlorohydrin, but due to their high degree of cross-linking, the cross-linked film is relatively brittle.

Contents of the invention

The purpose of the present invention is to provide a chlorogenic acid molecularly imprinted chitosan film and its preparation method and application. The present invention utilizes molecular imprinting technology, uses chlorogenic acid as a template molecule, chitosan as a film-forming material, and sulfuric acid as a cross-linked membrane. The chlorogenic acid molecularly imprinted chitosan film prepared by the phase inversion method, the chlorogenic acid molecularly imprinted chitosan film prepared by the present invention can effectively adsorb chlorogenic acid and separate chlorogenic acid and its structural analog coffee A mixture of acids, the separation effect is better than the corresponding blank membrane.

The technical scheme adopted in the present invention is:

A method for preparing a chlorogenic acid molecularly imprinted chitosan film, the method comprising the steps of:

(1) Prepare casting solution: dissolve chitosan and chlorogenic acid in the acetic acid solution of volume fraction 2%, filter with gauze after fully stirring, and the filtrate is defoamed to obtain casting solution; the chitosan and green The mass ratio of former acid is 10:1, and the volume consumption of described acetic acid solution is 40mL/g by the mass of chitosan;

(2) Pour the casting solution prepared in step (1) onto a clean glass plate placed horizontally, use a scraper to scrape the film, and dry to obtain a transparent film with a thickness of 60 μm;

(3) Soak the transparent film prepared in step (2) in a 0.5 mol·L ^-1 sulfuric acid solution for 24 hours to carry out the crosslinking reaction, and elute the crosslinked film with an eluent, and the elution The agent is a mixed solution of ethanol, acetic acid, and water with a volume ratio of 2:3:7, and then the excess acetic acid on the membrane surface is washed away with a 20% ethanol solution by volume fraction, and dried to obtain a chlorogenic acid molecularly imprinted chitosan membrane.

In the step (1), the stirring is generally performed under magnetic stirring at 60° C. for 6 hours.

The degassing is generally performed by vacuum degassing.

In the step (2), the drying temperature is generally 60° C., and the drying time is generally 12 hours.

In the step (3), the cross-linked membrane is eluted with an eluent, the chlorogenic acid component in the eluent is detected, and eluted until no chlorogenic acid is detected in the eluent.

The present invention adopts sulfuric acid to carry out cross-linking, and its cross-linking equation is as follows:

The present invention also provides the application of the chlorogenic acid molecularly imprinted chitosan film in adsorbing chlorogenic acid.

The invention also provides the application of the chlorogenic acid molecularly imprinted chitosan membrane in adsorption separation of chlorogenic acid and its structural analog caffeic acid.

The present invention has the following beneficial effects: the chlorogenic acid molecularly imprinted chitosan membrane provided by the present invention has the dual characteristics of molecular imprinting technology and membrane separation technology:

(1) The specific binding sites and functional groups on the molecularly imprinted chitosan membrane of chlorogenic acid can realize the specific recognition of the analyte;

(2) Compared with traditional molecularly imprinted polymers, the molecularly imprinted membrane of the present invention has a simple preparation method, is easy to operate, has good mechanical strength, is stable and acid-resistant, and can be directly applied without cumbersome preparation processes such as crushing and grinding.

Description of drawings

Fig. 1 is the schematic diagram of the reaction for preparing chlorogenic acid molecularly imprinted chitosan film according to the present invention.

Figure 2 is a scanning electron microscope photo of a chitosan blank film and a chlorogenic acid molecularly imprinted chitosan film. Figure 2 A on the left is a chitosan blank film, and B on the right is a chlorogenic acid molecularly imprinted chitosan membrane.

Figure 3 is the infrared spectrogram of chitosan blank film and chlorogenic acid molecularly imprinted chitosan film, in which Figure 3 A is the uncrosslinked chitosan blank film and uncrosslinked chlorogenic acid molecularly imprinted shell The infrared spectrum of the polysaccharide film, B is the infrared spectrum of the chitosan blank membrane before and after crosslinking; C is the infrared spectrum of the chlorogenic acid molecularly imprinted chitosan membrane before and after the template molecule chlorogenic acid is eluted after crosslinking picture.

Fig. 4 is the thermogravimetric analysis diagram of chitosan blank film and chlorogenic acid molecularly imprinted chitosan film.

Fig. 5 is the swelling curve of chitosan blank film and chlorogenic acid molecularly imprinted chitosan film.

Fig. 6 is a dynamic adsorption curve of chlorogenic acid on a chitosan blank film and a chlorogenic acid molecularly imprinted chitosan film.

Figure 7 is the static adsorption curve and Scatchard curve of chitosan blank membrane and chlorogenic acid molecularly imprinted chitosan membrane to chlorogenic acid, wherein the upper right small picture in Figure 7 is the chitosan blank membrane and chlorogenic acid molecularly imprinted The static adsorption curve of chitosan film to chlorogenic acid, the lower left big picture in Fig. 7 is the Scatchard curve.

Fig. 8 is a curve diagram of a single-component solution permeation experiment performed on a chitosan blank membrane and a chlorogenic acid molecularly imprinted chitosan membrane.

Fig. 9 is the HPLC analysis diagram of the mixed solution permeation experiment carried out by the chitosan blank membrane and the chlorogenic acid molecularly imprinted chitosan membrane, wherein the (a) diagram of Fig. 9 is the HPLC analysis diagram of the raw material solution, and the (b) diagram is green The HPLC analysis graph of the permeate obtained in the mixed solution permeation experiment of the original acid molecularly imprinted chitosan membrane, (c) is the HPLC analysis graph of the permeate obtained by the mixed solution permeation experiment of the chitosan blank membrane.

In Figure 9: C represents chlorogenic acid; D represents caffeic acid.

detailed description

The technical solutions of the present invention will be further described below with specific examples, but the protection scope of the present invention is not limited thereto.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1, Preparation of Chlorogenic Acid Molecularly Imprinted Chitosan Membrane

(1) Preparation of casting solution: Dissolve 1.25g of chitosan and 0.125g of chlorogenic acid in 50mL of acetic acid aqueous solution with a volume fraction of 2%, stir magnetically at 60°C for 6h, filter with gauze to remove insoluble matter, Vacuum defoaming overnight to obtain a casting solution.

(2) Film preparation: pour the film casting solution prepared in (1) above on a clean glass plate placed horizontally, use a scraper to scrape the film, and then dry it in an electric heating constant temperature blast drying oven at 60°C for 12 hours, namely A smooth and transparent film was obtained with a thickness of 60 μm. This membrane is an uncrosslinked chlorogenic acid molecularly imprinted chitosan membrane, denoted as uncrosslinked C-MIM

(3) Soak the film in (2) above for 24 hours in a sulfuric acid solution of 0.5 mol L ^-1 for cross-linking reaction, and use ethanol-acetic acid-water solution (volume ratio 2/3/7 ) repeatedly eluted to remove chlorogenic acid, and the eluent was detected by high performance liquid chromatography until no chlorogenic acid was detected in the eluent, then the excess acetic acid on the membrane surface was washed away with a volume fraction of 20% ethanol solution, and dried , that is, the molecularly imprinted chitosan membrane of chlorogenic acid is obtained, which is denoted as C-MIM.

The preparation of comparative example 1 chitosan blank film

(1) The preparation method of chitosan blank film is: dissolve 1.25g chitosan in 50mL acetic acid solution with a volume fraction of 2%, stir magnetically at 60°C for 6h, filter with gauze, and vacuum defoam the filtrate to obtain cast Membrane fluid;

(2) Pour the casting solution on a clean glass plate placed horizontally, scrape the film with a scraper, and then dry it in an electric constant temperature blast drying oven at 60°C for 12 hours to obtain a smooth and transparent film with a thickness of 60 μm. This is an uncrosslinked chitosan blank film, denoted as uncrosslinked N-MIM

(3) Soak the film in (2) above in 0.5mol L ^-1 sulfuric acid solution for 24h to carry out the cross-linking reaction, wash the cross-linked film with 20% ethanol solution by volume fraction to remove the sulfuric acid on the film surface, That is, a chitosan blank film is obtained, which is denoted as N-MIM.

The characterization of the chlorogenic acid molecularly imprinted chitosan film prepared in embodiment 2 and embodiment 1

(1) SEM analysis

The microstructures of the chitosan blank film and the chlorogenic acid molecularly imprinted chitosan film were characterized by scanning electron microscopy, and the results are shown in Figure 2 (left and right), respectively.

It can be known from Figure 2 that the surface of the chitosan blank film in picture A on the left is relatively smooth and compact, with slightly granular protrusions; the surface of the chitosan film imprinted with chlorogenic acid molecules in picture B on the right is rough and loose. , this is because a molecularly imprinted structure is formed on the imprinted membrane, which facilitates the contact between the substrate and the binding site, thus increasing the adsorption capacity of the membrane.

Considering the imprinting conditions and the structural characteristics of known raw materials, the synthesis principle of chlorogenic acid molecularly imprinted chitosan membrane is shown in Figure 1. Figure 1 shows the template molecule chlorogenic acid with hydroxyl and carboxyl groups and the template molecule with amino and hydroxyl groups. The film-forming material chitosan is pre-polymerized by ion pairing and hydrogen bonding, and then washed repeatedly with ethanol-acetic acid-water solution (volume ratio 2/3/7) to remove the template molecule chlorogenic acid to form a cavity structure. Molecularly imprinted membrane, which can be used for the adsorption of chlorogenic acid and its structural analogues.

(2) Infrared spectral analysis

The chitosan blank film and chlorogenic acid molecularly imprinted chitosan film were analyzed by infrared spectrometer, and the results are shown in A, B, and C of Figure 3, respectively.

Figure 3 A is the infrared spectrum of the uncrosslinked chitosan blank film and the uncrosslinked chlorogenic acid molecularly imprinted chitosan film. The chitosan blank film has a typical chitosan structure, and there are obvious characteristic peaks of polysaccharide structure. Among them, the absorption peak at 1062cm ^-1 is the COC stretching vibration peak; 896cm ^-1 and 1151cm ^-1 are the characteristic absorption peaks of glycosidic bonds in chitosan; 1323cm ^-1 is the CO stretching vibration peak; 1407cm ^-1 CH ₂ deformation vibration absorption peak at 1548cm ^-1 NH deformation vibration peak at 1632cm ^-1 chitosan residual NH ₂ CO absorption peak. Compared with the blank chitosan membrane, new peaks appeared at 858cm ^-1 , 812cm ^-1 , and 763cm ^-1 on the chlorogenic acid molecularly imprinted chitosan membrane, which were the out-of-plane CH on the benzene ring in chlorogenic acid Bending the vibration peak, it can be seen that the combination of chlorogenic acid and chitosan occurred. In addition, the intensity of some absorption peaks changed, for example, the NH ₂ CO absorption peak at 1629 cm ^-1 in the chlorogenic acid molecularly imprinted chitosan film and the stretching vibration absorption peak of the C=C double bond in chlorogenic acid were enhanced , proving that the imprinted molecule chlorogenic acid has been combined with chitosan or a new NH ₂ CO is formed between -COOH on chlorogenic acid and -NH ₂ on chitosan, a film-forming material. 1263cm ^-1 is the absorption peak of phenol-OH stretching vibration, which is significantly stronger than that of the chitosan blank film, which proves that the imprinted molecule chlorogenic acid has been combined with chitosan.

Figure 3 B is the infrared spectrum of the chitosan blank film before and after crosslinking, both of which have obvious chitosan structure. The absorption peak at 3350cm ^-1 is the NH stretching vibration peak in the primary amine, which disappears after crosslinking, and a new peak of NH ₃ ⁺ appears at 2080cm ^-1 , which all indicate that it is the crosslinking of sulfuric acid and chitosan Ammonium salts were formed; moreover, the peak at 1151 cm ⁻¹ for glycosidic linkages disappeared after crosslinking because of hydrolysis of glycosidic linkages in sulfuric acid.

Figure 3 C is the infrared spectrum of chlorogenic acid molecularly imprinted chitosan membrane before and after elution after crosslinking, same as the chitosan blank membrane, the peaks at 3350cm ^-1 and 1151cm ^-1 after crosslinking Disappeared, and a new peak appeared at 2080cm ^-1 at the same time, which was the result of ammonium salt formed after sulfuric acid and chitosan were cross-linked. In addition, after elution of chlorogenic acid molecularly imprinted chitosan membrane, the phenol-OH stretching vibration absorption peak at 1263cm ^-1 was significantly weakened, and the out-of-plane bending of CH on the benzene ring at 858cm ^-1 , 812cm ^-1 , and 763cm ^-1 The vibration peaks disappeared, which indicated that part of the chlorogenic acid was eluted.

(3) Thermogravimetric analysis

The chitosan blank film and chlorogenic acid molecularly imprinted chitosan film were subjected to spectral analysis with a microcomputer differential thermal analyzer, and the results are shown in Figure 4.

It can be seen from Figure 4 that both have mass volatilization between 30 and 200 ° C, which is the loss of volatile components in the film, mainly water, ethanol, acetic acid and unreacted sulfuric acid, and chitosan The blank membrane lost more weight than the chlorogenic acid molecularly imprinted chitosan membrane. Between 200°C and 250°C, the film mass volatilizes rapidly, and the curve drops sharply, which is mainly because the film-forming material chitosan is burned and decomposed. Between 250°C and 800°C, the film mass volatilizes slowly and almost reaches a complete combustion state, but the difference in the differential thermal analysis diagram is obvious, which may be because the structural order of the chlorogenic acid molecularly imprinted chitosan film is better than that of the chitosan blank film , so the heat resistance is higher than that of the blank film. In conclusion, the weight loss rates of chlorogenic acid molecularly imprinted chitosan membrane and chitosan blank membrane were 65.6% and 73.6%, respectively, and the thermal stability was better.

Embodiment 3, research on the performance of chlorogenic acid molecularly imprinted chitosan membrane

(1) Study on swelling properties of chlorogenic acid molecularly imprinted chitosan film

Place the chitosan blank film and the chlorogenic acid molecularly imprinted chitosan film in an electric constant temperature blast drying oven at 60°C for about half an hour, take them out and weigh them. Soak in ethanol aqueous solutions with different volume fractions at room temperature until its mass no longer changes, place the membrane between two layers of absorbent filter paper to wipe off the liquid on the surface of the membrane, weigh it, and calculate the swelling degree (DS) of the membrane by the following formula out:

$\mathrm{<span\; class="notranslate">\; D.</span>}\mathrm{<span\; class="notranslate">\; S</span>}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; m</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; m</span>}}{\mathrm{<span\; class="notranslate">\; m</span>}}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; 100</span>}\mathrm{<span\; class="notranslate">\; \%</span>}$

Among them, _m is the mass of the dry film, and m1 is the mass of the film after swelling.

The swelling curves further drawn from the experimental data are shown in Fig. 5. The experimental results show that the swelling performance of each membrane changes with the volume fraction of ethanol solution. On the one hand, with the increase of the volume fraction of ethanol solution, the swelling rate of chlorogenic acid molecularly imprinted chitosan membrane and chitosan blank membrane decreased, and the swelling performance improved, which was mainly due to the swelling degree of chitosan in water. Larger, and strong resistance to ethanol. On the other hand, compared with the chitosan blank film, the swelling performance of the chlorogenic acid molecularly imprinted chitosan film is better, which is mainly because the interaction between chitosan and chlorogen during the formation of the chlorogenic acid molecularly imprinted chitosan film Acids bind due to hydrogen bonding and ion pairing, thereby limiting the swelling behavior of chitosan.

(2) Study on the dynamic adsorption performance of chlorogenic acid molecularly imprinted chitosan film

Weigh 11 copies of 9 mg dried chitosan blank membrane and chlorogenic acid molecularly imprinted chitosan membrane, respectively, and place them in 50 mL conical flasks, and label each conical flask with 1, 4, 8, 12 , 20, 30, 40, 50, 60, 75, 90min, then add 40mL of 20mg·L ^-1 chlorogenic acid 20% ethanol solution to each Erlenmeyer flask, and place it in a desktop constant temperature oscillation box at 30°C for constant temperature oscillation adsorption . Then, use a pipette gun to sample the solution in the Erlenmeyer flask with the label at the specified time, measure the absorbance at 327nm (UV-vis) by a UV-visible spectrophotometer, further calculate the concentration according to the standard curve, and then according to the following Calculate the adsorption capacity at each time. The dynamic adsorption curve drawn according to the experimental data is shown in Fig. 6.

Calculate the adsorption capacity according to the following formula

${\mathrm{<span\; class="notranslate">\; Q</span>}}_{\mathrm{<span\; class="notranslate">\; t</span>}}<span\; class="notranslate">\; =</span>\frac{<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; 0</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; t</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; V</span>}}{\mathrm{<span\; class="notranslate">\; m</span>}}$

Among them, Q _t is the adsorption capacity at time t (μmol·g ^-1 ), C ₀ and C _t are the adsorption concentrations (mmol·L ^-1 ) at the initial time and time t, respectively, m is the mass of the adsorption film, and V is solution volume.

The dynamic adsorption curves further drawn from the experimental data are shown in Fig. 6. The experimental results showed that when a 9 mg membrane was adsorbed in 40 mL of 20 mg·L ^-1 chlorogenic acid in 20% ethanol solution, the initial adsorption rate of the chlorogenic acid molecularly imprinted chitosan membrane was faster than that of the chitosan blank membrane. The main reason is that there are specific adsorption sites on the chitosan membrane imprinted with chlorogenic acid molecules, that is, after the imprinted molecules are eluted, the imprinted membrane still retains binding sites that can completely match the imprinted molecules. At the same time, the adsorption equilibrium time of the chlorogenic acid molecularly imprinted chitosan membrane was about 40 minutes, which was longer than that of the chitosan blank membrane, so the adsorption capacity of the chlorogenic acid molecularly imprinted chitosan membrane was larger.

(3) Study on static adsorption performance of chlorogenic acid molecularly imprinted chitosan film

Weigh 10 copies of 9 mg dried imprinted membrane and 10 blank membranes, place them in 50 mL Erlenmeyer flasks respectively, add 40 mL of chlorogenic acid 20% ethanol standard solution with concentrations of 0.1-5.0 ^{mmol·L -1} respectively, place in Shake at a constant temperature of 30°C for 1 hour in a desktop constant temperature shaking box. After standing still, take the supernatant and measure the concentration of chlorogenic acid by UV-vis at 327nm. Measure three times in parallel, take the average value, calculate the concentration of chlorogenic acid according to the standard curve, and then Get the amount of adsorption.

${\mathrm{<span\; class="notranslate">\; Q</span>}}_{\mathrm{<span\; class="notranslate">\; e</span>}}<span\; class="notranslate">\; =</span>\frac{<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; 0</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; e</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; V</span>}}{\mathrm{<span\; class="notranslate">\; m</span>}}$

Among them, V is the volume of the solution (mL), m is the mass of the imprinted membrane (mg), C ₀ , C _e (mmol·L ^-1 ) are the initial concentration of the solution and the concentration after adsorption equilibrium, and Q _e is the equilibrium Adsorption capacity (μmol·g ^-1 ).

The static adsorption curve further drawn from the experimental data is shown in the upper right panel of Fig. 7. The experimental results showed that with the increase of chlorogenic acid concentration, the adsorption capacity of chitosan blank membrane and chlorogenic acid molecularly imprinted chitosan membrane increased, but the adsorption rate decreased. In addition, at the same concentration, the adsorption capacity of chlorogenic acid molecularly imprinted chitosan membrane to chlorogenic acid was always significantly greater than that of chitosan blank membrane, which indicated that the adsorption capacity of chlorogenic acid molecularly imprinted chitosan membrane on chlorogenic acid The adsorption is mainly specific adsorption, in other words, the "imprinted holes" left by the imprinted molecules in the chlorogenic acid molecularly imprinted chitosan membrane and the active sites on the holes determine the effect of the chlorogenic acid molecularly imprinted chitosan membrane on the green. Affinity and specific recognition of ortho acid. For the chitosan blank membrane, since it does not contain "imprinted holes", although there are functional groups on it that can bind to the substrate, the arrangement of the functional groups is random, so there is no molecular recognition for the molecule. performance.

The data were analyzed using the Scatchard model to further evaluate the binding properties of the chitosan membrane.

The Scatchard equation is as follows:

$\frac{{\mathrm{<span\; class="notranslate">\; Q</span>}}_{\mathrm{<span\; class="notranslate">\; e</span>}}}{{\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; e</span>}}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; Q</span>}}_{\mathrm{<span\; class="notranslate">\; m</span>}\mathrm{<span\; class="notranslate">\; a</span>}\mathrm{<span\; class="notranslate">\; x</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; Q</span>}}_{\mathrm{<span\; class="notranslate">\; e</span>}}}{{\mathrm{<span\; class="notranslate">\; K</span>}}_{\mathrm{<span\; class="notranslate">\; d</span>}}}$

Among them, Q _e and Q _max are the equilibrium adsorption capacity and maximum adsorption capacity of the adsorption site (μmol·g ^-1 ), respectively; C _e is the equilibrium adsorption concentration (mmol·L ^-1 ), and K _d is the equilibrium of the adsorption site Dissociation constant (mmol·L ^-1 ).

The big picture in Figure 7 is the Scatchard curve. For the chlorogenic acid molecularly imprinted chitosan membrane, it can be found that Q _e /C _e has a nonlinear relationship with Q _e , but two parts can show a good linear relationship, indicating that in the chlorogenic acid molecularly imprinted chitosan membrane There are two types of adsorption sites with different properties in the membrane, and two straight lines are obtained by linear fitting of them respectively, and the dissociation constant and maximum apparent adsorption capacity of the two types of affinity sites are obtained from the intercept and slope of the straight lines . For high affinity site: K _d1 ＝0.17mmol·L ^-1 , Q _max ＝139.57μmol·g ^-1 ; for low affinity site: K _d2 ＝1.27mmol·L ^-1 , Q _max ＝287.86 μmol·g ^-1 . For the chitosan blank film, it can be found that Q _e /C _e has a linear relationship with Q _e , indicating that there are only non-specific adsorption sites in the chitosan blank film. Among them, K _d =0.76 ^{mmol·L -1} , Q _max =83.84 μmol·g ^-1 .

(4) Selective permeation performance research

Fix the membrane between the two osmotic pools, add 150mL of 650mg·L ^-1 chlorogenic acid 20% ethanol solution to the raw material pool, add 150mL 20% ethanol solution to the permeation pool, both pools are electrically stirred, and every once in a while The liquid was taken from the sampling port of the permeation cell, and the absorbance was measured by UV-vis, so as to obtain the concentration of the permeate and residual liquid, and determine the permeability in the single-component solution permeation experiment.

The permeation experiment of the mixed solution is similar to the above experiment, except that 150mL of 650mg·L- ¹ chlorogenic acid and 650mg·L ^-1 caffeic acid and 20% ethanol mixed solution are added to the side of the raw material pool, and 150mL of 20% ethanol is added to the side of the permeation pool. For the ethanol solution, the two cells were electrically stirred. After permeating for 24 hours, the liquid was taken from the sampling port of the permeation cell, and the peak area was measured by high performance liquid chromatography (HPLC), so as to obtain the concentration of the permeate and determine the permeability.

The permeability is calculated as follows: before doing HPLC, draw the standard curve with the standard solution solubility as the abscissa and the peak area as the ordinate to obtain the curve equation, and then calculate the concentration of the permeate according to the peak area of the permeate detection HPLC, and use the permeate The concentration of the solution is divided by the concentration of the raw material solution (650mg·L ^-1) to obtain the permeability.

The permeation curve of the single component solution drawn from the experimental data is shown in Fig. 8. The experimental results show that with the extension of permeation time, the higher the permeate concentration, the more molecules permeate the membrane. Since the chitosan blank membrane only has non-specific adsorption holes, it can only adsorb a small amount of chlorogenic acid to the surface of the membrane on the side of the raw material pool and is difficult to transfer to the side of the permeation pool, so the permeability is extremely low. And because the chlorogenic acid molecularly imprinted chitosan membrane also has specific adsorption holes, according to the literature, this hole will form a channel inside the membrane, so the chlorogenic acid can be transferred from one side of the imprinted membrane to the side of the membrane through the channel. On the other hand, so the content of chlorogenic acid permeating the imprinted membrane increases with time and the content of chlorogenic acid in the residual liquid decreases with time, but the permeation rate decreases with time, This is because in the initial stage, chlorogenic acid mainly seeps through the concentration gradient force, and the adsorption is fast; as the adsorption process progresses, the concentration of chlorogenic acid in the solution gradually decreases, and at the same time, the adsorbate diffuses inward along the micropores. Increasing resistance leads to a slower adsorption rate. Throughout the whole picture, the concentration of chlorogenic acid in the permeate of chlorogenic acid molecularly imprinted chitosan membrane is always higher than that of chitosan blank membrane permeate, and the concentration of chlorogenic acid molecularly imprinted chitosan membrane The concentration of chlorogenic acid in the residual liquid of the chitosan blank membrane was always lower than that of the chlorogenic acid in the residual liquid of the chitosan blank membrane; after permeation for 24 hours, the permeability of chlorogenic acid in the molecularly imprinted chitosan membrane of chlorogenic acid was calculated as The permeability of chlorogenic acid in chitosan blank membrane was 22.43%, and the permeability of chlorogenic acid in chitosan blank membrane was 20.26%, so the permeability of chlorogenic acid molecularly imprinted chitosan membrane was slightly better than that of chitosan blank membrane.

The HPLC analysis of the mixed solution permeation experiment is shown in Figure 9. Because caffeic acid and chlorogenic acid are similar in structure, caffeic acid was chosen as the analogue, and the transmittance through chlorogenic acid molecularly imprinted chitosan membrane and chitosan blank membrane were measured when the two coexisted. According to calculation, the permeation rates of chlorogenic acid and caffeic acid in chlorogenic acid molecularly imprinted chitosan membrane were 33.54% and 31.99% respectively; in chitosan blank membrane, the permeation rates of chlorogenic acid and caffeic acid were 22.12% , 23.15%. It can be seen that the permeability of chlorogenic acid molecularly imprinted chitosan membrane is slightly better than that of chitosan blank membrane, and the selectivity is also slightly better. The reason for the insufficient selectivity may be that the chemical structure of caffeic acid is the same as that of chlorogenic acid. Part of caffeic acid also contains -COOH and -OH, so it can also bind to the imprinted site. Comparing the permeation experiment of single component solution and the permeation experiment of mixed solution, it can be seen that when chlorogenic acid and caffeic acid coexist, the permeability of chlorogenic acid in chitosan membrane imprinted with chlorogenic acid molecule and chitosan blank membrane are the same. Increased, and chlorogenic acid molecularly imprinted chitosan film is more significant, this may be because the mixed solution contains chlorogenic acid and caffeic acid, so the concentration of the solution is higher than that of the single component solution, and the concentration gradient force is greater.

Claims (7)

1. a preparation method for chlorogenic acid molecular engram chitosan film, is characterized in that described method comprises the steps:

(1) casting solution is prepared: shitosan and chlorogenic acid are dissolved in the acetum of volume fraction 2%, filtered through gauze after fully stirring, filtrate deaeration, obtained casting solution; The mass ratio of described shitosan and chlorogenic acid is 10:1, and the volumetric usage of described acetum counts 40mL/g with the quality of shitosan;

(2) be poured on the cleaned glass plate of horizontal positioned by casting solution obtained in step (1), use scraper knifing, after dry, obtained thickness is the hyaline membrane of 60 μm;

(3) hyaline membrane obtained in step (2) is placed in 0.5molL ^-1sulfuric acid solution in soak 24h and carry out cross-linking reaction, by the film eluent after crosslinked, described eluant, eluent is the mixed solution of ethanol, acetic acid, water volume ratio 2:3:7, the acetic acid of film excess surface is washed away again with volume fraction 20% ethanolic solution, drying, namely obtains chlorogenic acid molecular engram chitosan film.

2. the method for claim 1, is characterized in that in described step (1), described in be stirred in 60 DEG C of lower magnetic forces and stir 6h.

3. the method for claim 1, is characterized in that in described step (1), and described filtrate deaeration adopts vacuum defoamation.

4. the method for claim 1, it is characterized in that, in described step (2), baking temperature is 60 DEG C, drying time is 12h.

5. the chlorogenic acid molecular engram chitosan film that the method as described in one of Claims 1 to 4 prepares.

6. the application of chlorogenic acid molecular engram chitosan film as claimed in claim 5 in absorption chlorogenic acid.

7. the application of chlorogenic acid molecular engram chitosan film in adsorbing separation chlorogenic acid and caffeic acid as claimed in claim 5.