CN115537036B - Process for purifying safflower red pigment by macroporous resin - Google Patents
Process for purifying safflower red pigment by macroporous resin Download PDFInfo
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- CN115537036B CN115537036B CN202211329258.7A CN202211329258A CN115537036B CN 115537036 B CN115537036 B CN 115537036B CN 202211329258 A CN202211329258 A CN 202211329258A CN 115537036 B CN115537036 B CN 115537036B
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- 239000011347 resin Substances 0.000 title claims abstract description 80
- 229920005989 resin Polymers 0.000 title claims abstract description 80
- 239000001054 red pigment Substances 0.000 title claims abstract description 63
- 244000020518 Carthamus tinctorius Species 0.000 title claims abstract description 48
- 235000003255 Carthamus tinctorius Nutrition 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 38
- 238000011084 recovery Methods 0.000 claims abstract description 55
- WLYGSPLCNKYESI-RSUQVHIMSA-N Carthamin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1[C@@]1(O)C(O)=C(C(=O)\C=C\C=2C=CC(O)=CC=2)C(=O)C(\C=C\2C([C@](O)([C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)C(O)=C(C(=O)\C=C\C=3C=CC(O)=CC=3)C/2=O)=O)=C1O WLYGSPLCNKYESI-RSUQVHIMSA-N 0.000 claims abstract description 43
- DYQVDISPPLTLLR-HJQYTNQXSA-N Carthamin Natural products CC[C@H]1O[C@H]([C@H](O)[C@@H](O)[C@@H]1O)[C@]2(O)C(=C(C=C/3C(=O)C(=C(O)[C@](O)([C@@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4O)C3=O)C(=O)C=Cc5ccc(O)cc5)C(=O)C(=C2O)C(=O)C=Cc6ccc(O)cc6)O DYQVDISPPLTLLR-HJQYTNQXSA-N 0.000 claims abstract description 43
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 84
- 238000011068 loading method Methods 0.000 claims description 44
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 28
- 239000007788 liquid Substances 0.000 claims description 18
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 238000001179 sorption measurement Methods 0.000 claims description 15
- 230000003068 static effect Effects 0.000 claims description 14
- 239000012071 phase Substances 0.000 claims description 13
- 238000005303 weighing Methods 0.000 claims description 13
- 238000003795 desorption Methods 0.000 claims description 12
- 238000001914 filtration Methods 0.000 claims description 9
- 239000000463 material Substances 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 239000012043 crude product Substances 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 5
- 241000628997 Flos Species 0.000 claims description 4
- 238000002791 soaking Methods 0.000 claims description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 239000012074 organic phase Substances 0.000 claims description 3
- 238000010298 pulverizing process Methods 0.000 claims description 3
- 238000009210 therapy by ultrasound Methods 0.000 claims description 3
- 239000001052 yellow pigment Substances 0.000 claims description 3
- 238000001704 evaporation Methods 0.000 claims description 2
- 238000000746 purification Methods 0.000 abstract description 29
- 238000012216 screening Methods 0.000 abstract description 6
- 238000001953 recrystallisation Methods 0.000 abstract description 4
- 230000009897 systematic effect Effects 0.000 abstract description 3
- 238000010801 machine learning Methods 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 36
- 238000012360 testing method Methods 0.000 description 29
- 239000000523 sample Substances 0.000 description 27
- 238000011835 investigation Methods 0.000 description 17
- 238000013461 design Methods 0.000 description 16
- 230000000694 effects Effects 0.000 description 14
- 239000003480 eluent Substances 0.000 description 14
- 238000010828 elution Methods 0.000 description 14
- 239000012488 sample solution Substances 0.000 description 14
- 238000003062 neural network model Methods 0.000 description 13
- 230000002068 genetic effect Effects 0.000 description 12
- 238000011160 research Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- 230000004044 response Effects 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 239000000049 pigment Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000013558 reference substance Substances 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 3
- 238000000540 analysis of variance Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 238000004040 coloring Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 239000011435 rock Substances 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 241000208838 Asteraceae Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000013528 artificial neural network Methods 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- KINGXFAMZNIVNL-SXQDSXCISA-N safflor yellow A Natural products OC[C@@H]1O[C@H]2[C@H](OC3=C2C(=O)C(=C(O)C=Cc4ccc(O)cc4)C(=O)[C@]3(O)[C@@H]5O[C@H](CO)[C@@H](O)[C@H](O)[C@H]5O)[C@@H](O)[C@H]1O KINGXFAMZNIVNL-SXQDSXCISA-N 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000009495 sugar coating Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B67/00—Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
- C09B67/0096—Purification; Precipitation; Filtration
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Saccharide Compounds (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The application relates to a process for purifying safflower red pigment by macroporous resin. The application provides a high-efficiency, simple and stable method for obtaining high-purity safflower red pigment and industrialized mass production of the red pigment through systematic screening of macroporous resin types and optimizing purification process conditions by machine learning. Under the determined macroporous resin purification process conditions of HPD-400, the recovery rate of the carthamin red pigment can reach 96.68 percent, which is 3 to 4 times that of the prior reported purification method. The mass fraction of the carthamin is 91.76% after macroporous resin purification, which is about 3 times that of the carthamin before purification. After recrystallization operation, the mass fraction of the carthamin red pigment can reach 98.74 percent.
Description
Technical Field
The invention relates to a process for purifying safflower red pigment by macroporous resin.
Background
Safflower red (carthamin) is a natural pigment extracted from dried tubular flower of safflower Carthamus tinctorius L, which is a plant of the genus safflower of the family Compositae, and is widely used for coloring food such as bread, candy, beverage, etc. in various countries in east Asia, and can be processed to prepare liposoluble colorant for coloring high-grade cosmetics such as lipstick, rouge, etc. In addition, the safflower red pigment is also an active ingredient in safflower, and can also be used for medicine sugar-coating dyeing.
As the safflower is rich in safflower yellow and other components besides red pigment, the crude safflower red pigment obtained by extraction often contains a large amount of impurities, which affects the quality, so the crude safflower red pigment needs to be refined. However, the stability of the carthamus tinctorius red pigment is poor, the photo-thermal and pH-sensitive performance is high, the color of the carthamus tinctorius red pigment is yellow due to the high temperature and alkaline environment, the degradation rate is high, and the selection of a proper purification method is important in the study of the carthamus tinctorius red pigment.
The macroporous resin adsorption separation has the characteristics of simple operation, good selectivity, low cost, repeated use and the like, and has been widely used for the separation and purification of various natural compounds. The existing carthamus tinctorius red pigment research does not carry out systematic screening on the type of macroporous resin, and adopts an ultraviolet-visible spectrophotometry to calculate the color value as a reference index, so that the purification effect cannot be quantitatively explained; the optimal condition is compounded by adopting a single-factor highest point, and the reliability is poor. Therefore, the research firstly screens the macroporous resin model through a static adsorption-elution test, then adopts a single factor, a partial factor design, a response surface test and a genetic neural network model to examine the influence of different process conditions on the recovery rate of the carthamin, and seeks the optimal process for purifying the carthamin by using the macroporous resin.
Disclosure of Invention
In order to solve the problems, the invention provides a process for purifying the safflower red pigment by using macroporous resin, which is efficient, simple, convenient and stable.
A method for purifying safflower red pigment by macroporous resin comprises the following steps:
(1) Dissolving the crude red pigment into red pigment solution by pure water;
(2) Soaking macroporous resin in 95% ethanol for full swelling, eluting with ethanol until the eluate is not turbid, washing with a large amount of water until no ethanol smell exists, and suction filtering; the macroporous resin is HPD400 type macroporous resin or X-5 macroporous resin;
(3) Weighing the pretreated wet resin, placing the wet resin into a conical bottle with a plug, respectively adding a carthamin solution, oscillating at room temperature, centrifuging to obtain supernatant, performing HPLC (high performance liquid chromatography) after constant volume, calculating the concentration according to the peak area, and calculating the static adsorption rate; then adding ethanol solution to desorb the resin, oscillating at room temperature, centrifuging to obtain supernatant, determining the volume, calculating the concentration according to the peak area, and calculating the static desorption rate;
(4) Weighing pretreated and activated resin, loading the resin on a column by a wet method, adding a carthamus tinctorius red pigment solution, eluting with ethanol, collecting an eluent, rotationally evaporating, transferring to a measuring flask, fixing the volume with ethanol, measuring by an HPLC (high performance liquid chromatography), and calculating the recovery rate Q according to the chromatographic peak area, wherein Q=S/S 0, S is the peak area of carthamus tinctorius red pigment after macroporous resin treatment, and S 0 is the peak area of carthamus tinctorius red pigment solution before untreated.
Preferably, the macroporous resin is HPD400 type macroporous resin.
Preferably, the pH of the sample solution in the step (4) is 6-7.
Preferably, in step (4): the mass concentration of the sample loading liquid is 0.05-0.125 g.mL -1.
Preferably, in step (4): the volume fraction of the eluting ethanol is 58% -70%.
Preferably, in step (4): the volume fraction of the eluting ethanol is 58%, the mass concentration of the loading liquid is 0.125 g.mL -1, and the pH of the loading liquid is 6.0.
Preferably, in step (4): the eluent dosage is 4BV; the volume flow of elution is 0.5-1.5 mL min -1; the ratio of the sample loading amount to the resin is 0.3-0.4; the sample loading volume flow is 0.5-1.0 mL.min -1.
The method specifically comprises the following steps:
(1) Dissolving the crude product of the haematochrome into haematochrome solution with the concentration of 0.10 g.mL -1 calculated by the raw medicinal materials by using pure water;
(2) Soaking HPD400 macroporous resin in 95% ethanol for 24 hr to swell, eluting with ethanol until the eluate is not turbid, washing with water until no ethanol smell is present, and suction filtering;
(3) Weighing 2g of the pretreated wet resin, placing the wet resin into a conical flask with a plug, respectively adding 25mL of 0.10g.mL -1 of carthamin solution, oscillating for 2 hours at room temperature, centrifuging to obtain supernatant, fixing the volume to 25mL, performing HPLC (high performance liquid chromatography), calculating the concentration according to the peak area, and calculating the static adsorption rate; then 25mL of 60% ethanol solution is added to desorb the resin, the resin is oscillated for 2 hours at room temperature, the supernatant is centrifugally taken, the volume is fixed to 25mL, the concentration is calculated according to the peak area, and the static desorption rate is calculated;
(4) 3g of pretreated and activated resin is weighed, loaded on a column by a wet method, 10mL of 0.10 g.mL -1 of carthamin solution is added, the mass concentration of the loading liquid is 0.125 g.mL -1, the pH of the loading liquid is 6.0, the loading volume flow is controlled to be 0.5 mL.min -1, 4BV of ethanol is used for eluting at the volume flow of 1.0 mL.min -1, the volume fraction of the eluted ethanol is 58%, the eluent is collected, rotationally evaporated and transferred to a 10mL measuring flask, the volume of the 60% ethanol is fixed, the volume is fixed, the HPLC is used for measuring, the recovery rate Q is calculated according to the chromatographic peak area, Q=S/S 0, S is the peak area of carthamin after macroporous resin treatment, and S 0 is the peak area of carthamin solution before treatment.
Wherein, the preparation of the crude carthamin red pigment comprises the following steps: washing Carthami flos with water to remove most of yellow pigment, drying at 25deg.C, and pulverizing into powder; precisely weighing 10g of safflower powder in a conical flask with a plug, adding 58% acetone solution according to a liquid-to-material ratio of 23:1, adjusting the temperature to 40 ℃, carrying out ultrasonic treatment for 41min, filtering, placing the filtrate in a separating funnel, adding a proper amount of ammonium sulfate, shaking uniformly, standing to separate layers, taking the upper phase as an organic phase and the lower phase as a water phase, collecting the upper phase, recovering acetone, drying, and freeze-drying to obtain a crude product of the haematochrome.
According to the invention, response surface design and test are carried out on main influencing factors screened out by partial factor design, and the genetic neural network model is fitted and predicted to optimal parameters.
The application provides a high-efficiency, simple and stable method for obtaining high-purity safflower red pigment and industrialized mass production of the red pigment through systematic screening of macroporous resin types and optimizing purification process conditions by machine learning. Under the determined macroporous resin purification process conditions of HPD-400, the recovery rate of the carthamin red pigment can reach 96.68 percent, which is 3 to 4 times that of the prior reported purification method. The mass fraction of the carthamin is 91.76% after macroporous resin purification, which is about 3 times that of the carthamin before purification. After recrystallization operation, the mass fraction of the carthamin red pigment can reach 98.74 percent.
Drawings
FIG. 1 elution profile of red pigment through HPD400 macroporous resin.
FIG. 2 is a graph showing the effect of various factors on recovery.
Wherein,
Figure 2a effect of eluted ethanol volume fraction on recovery (n=3);
Fig. 2b effect of eluent amount on recovery (n=3);
fig. 2c effect of elution volume flow on recovery (n=3);
Fig. 2d effect of loading on resin recovery (n=3);
fig. 2e effect of sample volume flow on recovery (n=3);
fig. 2f effect of sample mass concentration on recovery (n=3);
Figure 2g effect of loading pH on recovery (n=3).
Pareto plot of the normalization effect of fig. 3 (response Q, α=0.05).
FIG. 4 error analysis results of experimental data and model predictive data.
Detailed Description
2 Materials and methods
2.1 Instruments and materials
Agilent1260 type high performance liquid chromatograph, TG16-WS bench top high speed centrifuge, FE28 type pH meter, swing bed, etc. AB-8, ADS-8, CAD-40, D-101, DM130, HPD-300, HPD-400, NKA-9, S-8, X-5 type macroporous resin, carthami flos, acetone, ethanol, potassium carbonate, and citric acid.
2.2 Preparation of crude carthamin
Washing Carthami flos with water to remove most of yellow pigment, drying at 25deg.C, and pulverizing into powder. Precisely weighing 10g of safflower powder in a conical flask with a plug, adding 58% acetone solution according to a liquid-to-material ratio of 23:1, adjusting the temperature to 40 ℃, carrying out ultrasonic treatment for 41min, filtering, placing the filtrate in a separating funnel, adding a proper amount of ammonium sulfate, shaking uniformly, standing to separate layers, taking the upper phase as an organic phase and the lower phase as a water phase, collecting the upper phase, recovering acetone, drying, and freeze-drying to obtain a crude product of the haematochrome.
2.3 High Performance liquid chromatography method for preparing safflower haematochrome
2.3.1 Preparation of control
Since no carthamus tinctorius red pigment standard product is sold in the market, the red pigment standard product is obtained by extracting the carthamus tinctorius red pigment standard product by an alkali extraction and acid precipitation method according to a method established in the past of a subject group, separating the extracting solution by a preparation type high performance liquid chromatograph, and freeze-drying the extracting solution to obtain a red pigment reference product (purity is more than 98%).
2.3.2 HPLC determination of the red pigment
Preparation of a control solution: precisely weighing a certain amount of red pigment reference substance, dissolving with 50% acetone, and fixing volume to obtain red pigment reference substance solution with concentration of 0.3 mg.mL -1.
Sample solution preparation: precisely weighing a certain amount of crude safflower red pigment, dissolving with 50% acetone, and filtering with 0.22 μm microporous membrane.
The chromatographic conditions were ALLTECH ALLTIMA C 18 chromatographic columns (4.6 mm. Times.250 mm,5 μm) and ALLTECH ALLTIMA C 18 guard columns (4.6 mm. Times.12.5 mm,5 μm); the mobile phase is 0.3% phosphoric acid water-methanol-acetonitrile (57:5:38), the flow rate is 1.0 mL-min -1, the column temperature is 35 ℃, the sample injection amount is 20 mu L, and the detection wavelength is 520nm.
2.3.3 Methodology investigation of HPLC quantification
Precision test: taking carthamin reference substance solution, continuously sampling for 6 times within 1 day and continuously sampling for 3 days respectively for measurement, wherein the RSD of carthamin peak area is 0.41% (within day) and 1.43% (within day), which shows that the instrument precision is good.
Stability test: precisely weighing the same batch of crude red pigment, dissolving with 50% acetone, passing through 0.22 μm microporous filter membrane, preparing into test solution, storing at 4deg.C in dark place, and sampling and measuring at 0, 2,4, 6, 8, 10, and 12 hr respectively. The RSD of the red pigment peak area of the safflower in the test solution within 12 hours is 2.16 percent, which shows that the test solution is stable within 12 hours under 4 ℃ light-proof environment.
Repeatability test: precisely weighing 6 parts of the crude red pigment in the same batch, respectively dissolving with 50% acetone, filtering with 0.22 μm filter membrane, preparing into sample solution, and sampling for determination. The RSD of the peak area of the red pigment in 6 parts of the solution was 1.60%.
Sample addition recovery test: precisely weighing the same batch of crude red pigment, dissolving with 50% acetone solution, adding red pigment reference substances according to the ratio of 0.8:1, 1:1 and 1.2:1 to prepare sample solutions, measuring 3 parts of each group by HPLC (high performance liquid chromatography) sample injection, and calculating the sample injection recovery rate, wherein the average sample injection recovery rate is 99.31% (RSD=1.20%).
The established method meets various requirements of content measurement through methodology investigation.
2.4 Static adsorption and static resolution of macroporous resins
Soaking 10 types of macroporous resin with 95% ethanol for 24h to fully swell, eluting the macroporous resin with ethanol until the eluate is not turbid, washing with a large amount of water until no ethanol smell exists, and suction filtering for later use.
2G of each of the 10 wet resins after pretreatment is weighed and placed in a conical flask with a plug, 25mL of 0.10 g.mL -1 carthamin red pigment solution (the crude red pigment prepared by 2.2 is dissolved into red pigment solution with the concentration of 0.10 g.mL -1 calculated according to the raw medicinal material by pure water), the mixture is oscillated for 2 hours at room temperature, the supernatant is centrifugally taken, the volume is fixed to 25mL, HPLC is carried out, the concentration is calculated according to the peak area, and the static adsorption rate is calculated. Then 25mL of 60% ethanol solution is added to desorb the resin, the resin is oscillated for 2 hours at room temperature, the supernatant is centrifugally taken, the volume is fixed to 25mL, the concentration is calculated according to the peak area, and the static desorption rate is calculated. By comparing the static adsorption rate, desorption rate and recovery rate of 10 resins, an appropriate resin type was selected.
The static adsorption rate, desorption rate and recovery rate of the macroporous resin are calculated according to the following formula:
Adsorption rate= (C 1-C2)/C1 ×100%
Desorption rate=c 3/(C1-C2) ×100%
Recovery = C 3/C1 x 100%
Wherein: c 1 is the concentration of the carthamin solution before resin adsorption; c 2 is the concentration of the carthamus tinctorius red pigment solution after resin adsorption; c 3 is the concentration of the carthamin solution in the resin desorption solution.
2.5 Single factor test
2.5.1 Investigation of the volume fraction of eluent
3G of pretreated and activated resin is weighed, loaded into a column by a wet method, 10mL of 0.10 g.mL -1 of carthamin solution is added, the loading volume flow is controlled to be 1.0 mL.min -1, 5BV of ethanol is used for eluting at the volume flow of 1.0 mL.min -1, the volume fractions of the eluting ethanol are respectively adjusted to be 50%, 60%, 70%, 80% and 90%, the eluent is collected, the rotary evaporation is carried out, the eluent is transferred to a 10mL measuring flask, the volume is fixed by 60% of ethanol, the volume is measured by an HPLC instrument, the recovery rate Q is calculated according to the chromatographic peak area, Q=S/S 0, S is the peak area of the carthamin solution after macroporous resin treatment, and S 0 is the peak area of the carthamin solution before treatment.
2.5.2 Investigation of the eluent usage
The volumes of the eluting ethanol are respectively adjusted to be 2 BV, 3 BV, 4 BV, 5 BV and 6BV, the sample is loaded and eluted, and the other steps are operated under the condition of 2.5.1, so that the recovery rate Q of the carthamus tinctorius is calculated.
2.5.3 Investigation of elution volume flow
The elution volume flows are respectively adjusted to be 0.25, 0.5, 1.0, 1.5 and 2.0 mL.min -1, loading elution is carried out, and the other operations are carried out according to the item of 2.5.1, so that the recovery rate Q of the carthamus tinctorius is calculated.
2.5.4 Investigation of the sample loading and resin ratio
And (3) respectively adjusting the loading amount and the resin ratio to be 0.1, 0.2, 0.3, 0.4 and 0.5, loading and eluting, and otherwise operating according to the item of 2.5.1, so as to calculate the recovery rate Q of the carthamus tinctorius.
2.5.5 Investigation of the sample volume flow
And (3) respectively adjusting the loading volume flow to 0.25, 0.5, 1.0, 1.5 and 2.0 mL.min -1, loading and eluting, otherwise operating according to the item of 2.5.1, and calculating the recovery rate Q of the carthamin.
2.5.6 Investigation of the sample solution Mass concentration
The sample loading liquid mass concentrations are respectively adjusted to be 0.025, 0.05, 0.10, 0.15 and 0.20 g.mL -1 for loading elution, and the other steps are operated according to the item of 2.5.1, so that the recovery rate Q of the carthamus tinctorius is calculated.
Investigation of the pH of 2.5.7 sample solutions
The pH of the sample solution was adjusted to 6, 7, 8, 9 and 10, and the sample was eluted, and the recovery rate Q of the safflower red pigment was calculated by operating under the condition of "2.5.1".
2.6 Partial factor design test screening of Primary influencing factors
And selecting a part of factor design tests to screen the significant factors influencing the recovery rate of the carthamus tinctorius. 7 experimental factors were selected: the volume fraction of the eluting ethanol (A), the dosage of the eluting agent (B), the eluting volume flow rate (C), the loading volume (D), the loading volume flow rate (E), the loading mass concentration (F) and the loading liquid pH (G). The partial factor test factors and horizontal designs are shown in Table 2, and include 16 test points and 4 center repeat points, for a total of 20 sets of tests, each set of tests repeated 3 times. And determining the high and low 2 levels of each factor by combining the results obtained by the single factor experiment with the conditions among the factors, and calculating the recovery rate Q of the carthamin. And performing analysis of variance on the result by using Minitab software.
2.7BBD test
The 3 main influencing factors screened out through the partial factor design test are independent variables, and the BBD test optimizes the macroporous resin purification process of the safflower red pigment. Fitting analysis is carried out on experimental data by adopting Design expert 8.0 software, the low, medium and high levels of each factor are respectively encoded by-1, 0 and 1, 17 groups of experiments are carried out, and each group of experiments is repeated 3 times.
2.8 Establishment and prediction of genetic neural network model
The method takes 3 main influencing factors screened by a partial factor design test as independent variables as input variables of a genetic neural network, and takes the recovery rate Q value of carthamin as output variables. According to the leave-one-out method, 15 groups of data in BBD design are used as training sets of the neural network model, and 2 groups of data are used as test sets.
2.9 Verification test of optimal Process conditions
And (3) operating under the optimal purification process condition predicted by the genetic neural network model, and measuring the recovery rate of the carthamus tinctorius pigment for 3 times.
3 Results of experiments
3.1 Screening of resin model
TABLE 1 influence of resin model on the recovery of Red flower pigment
In the experiment, 10 resins with different properties are selected, and the purification effect of the resins with different types on the carthamus tinctorius red pigment is examined in terms of adsorption rate and desorption rate. As can be seen from Table 1, the adsorption rate, desorption rate and recovery rate of the HPD400 on the carthamin are all high, so that the subsequent experiments adopt HPD400 macroporous resin to carry out optimization investigation on the purification process of the carthamin. The elution curve of the carthamin through HPD400 macroporous resin is shown in FIG. 1.
3.2 Single factor experiment
3.2.1 Investigation of the ethanol volume fraction of the eluent
The volume fraction of ethanol eluted was 50%, 60%, 70%, 80%, 90%, with the corresponding recovery rates of 82.50%, 93.98%, 91.39%, 85.09%, 83.21%, and the volume fraction of ethanol eluted was 60% with the maximum recovery rate, so the volume fraction of ethanol eluted was determined to be 60%. As shown in fig. 2 a.
3.2.2 Investigation of the eluent usage
The recovery rates for the eluent amounts of 2, 3, 4, 5 and 6BV were 61.28%, 67.77%, 74.27%, 68.48% and 64.24%, as shown in FIG. 2 b.
The corresponding eluent dosage is 4BV when the recovery rate is maximum, so the eluent dosage is determined to be 4BV.
3.2.3 Investigation of elution volume flow
The recovery rates for elution volume flows of 0.25, 0.5, 1.0, 1.5, 2.0mL min -1 were 67.46%, 89.93%, 93.48%, 91.96%, 66.66% as shown in FIG. 2 c.
The corresponding elution volume flow rate at the maximum recovery rate was 1.0 mL/min -1, so that the elution volume flow rate was determined to be 1.0 mL/min -1.
3.2.4 Investigation of the sample loading and resin ratio
The recovery rates corresponding to the loading and resin ratios of 0.1, 0.2, 0.3, 0.4, and 0.5 were 55.69%, 57.35%, 64.93%, 62.57%, and 60.92%, as shown in fig. 2 d.
The corresponding loading to resin ratio at the maximum recovery rate was 0.3, so the loading to resin ratio was determined to be 0.3.
3.2.5 Investigation of the sample volume flow
The recovery rates corresponding to the sample volume flows of 0.25, 0.5, 1.0, 1.5 and 2.0mL min -1 are 89.33%, 94.41%, 91.97%, 87.50% and 82.77%, as shown in FIG. 2 e.
The corresponding sample volume flow rate at the maximum recovery rate was 0.5 mL/min -1, so that the sample volume flow rate was determined to be 0.5 mL/min -1.
3.2.6 Investigation of the sample Mass concentration
The mass concentrations of the sample solutions were 0.025, 0.05, 0.10, 0.15 and 0.20 g.mL -1, and the recovery rates were 86.47%, 92.89%, 78.90%, 64.65% and 56.18%, as shown in FIG. 2 f.
The corresponding sample solution mass concentration at the maximum recovery rate was 0.05 g.mL -1, so that the sample solution mass concentration was determined to be 0.05 g.mL -1.
3.2.7 Investigation of the pH of the sample solution
The recovery rates corresponding to the pH values of the sample loading solutions of 6, 7, 8, 9 and 10 are 74.64%, 80.90%, 44.87%, 18.68% and 16.67%, as shown in FIG. 2 g.
The pH of the sample solution was 7 when the recovery rate was maximum, and therefore, the pH of the sample solution was determined to be 7.
3.3 Partial factor design test screening of major influencing factors
TABLE 2 partial factor design test and results
TABLE 3 analysis of variance for partial factor design test
The significance analysis was performed on each factor using Minitab software, and the resulting regression equation Q＝0.43761+0.02618A+0.02516B+0.02096C-0.01610D+0.00771E+0.03266F-0.16450G-0.04620A*B-0.00495A*C-0.0 2406A*D-0.01766A*E-0.02680A*F-0.01113A*G+0.06109B*D-0.00941A*B*D+0.2788Ct Pt. table 3 results showed that model p=0.001, reached the level of extreme significance, and determined coefficient R 2 = 0.9988, indicating that the variability of the experimental data of 99.88% could be explained by this regression model. The significance order of the effect of each factor on Q value was G > F > A > B > C > D > E, with G, F, A having a very significant difference in the effect on recovery. See Pareto plot of normalized effect of fig. 3 (response Q, α=0.05)
3.4 BBD test design and results
Table 4 BBD test design and results (n=3)
TABLE 5 response surface quadratic regression equation analysis of variance
In the dynamic purification process, the nonlinear equation model P=4.0x10 -4 obtained by BBD shows that the model is very remarkable, the correction decision coefficient R 2 = 0.9608 shows that 96.08% of variability of experimental data can be explained by the regression model, and the reliability is high. The mismatch test P <0.0001 has significant difference, which indicates that the predicted value of the regression model has a certain gap from the actual situation. The influence of each factor on the regression rate is C > A > B. Subsequent attempts have employed genetic neural network models, since the response surface model does not fit well.
3.5 Establishment and prediction of genetic neural network model
15 Sets of data in the BBD design are used as training sets of the neural network model, and 2 sets of data are used as test sets. The relevant weights for this genetic neural network model are shown in table 6. Fig. 4 is an error analysis of experimental data and model predictive data, r train＝0.9846,rtest =0.9957, showing that the genetic neural network model containing 4 hidden layer neurons is substantially identical to the experimental data.
And finally predicting and obtaining the optimal purification process parameters of 58.10% of the volume fraction of the eluting ethanol by utilizing MATLAB software, wherein the mass concentration of the loading liquid is 0.1265 g.mL -1, the pH of the loading liquid is 6.00, and the recovery rate of the carthamin red pigment is 98.92%.
TABLE 6 weights of 4 hidden layer neurons for predicting carthamin recovery
See FIG. 4 for experimental data and model predictive data error analysis results
3.6 Validation test of optimal purification Process conditions
The optimal purification process conditions predicted by the genetic neural network model are as follows: the volume fraction of the eluting ethanol was 58.10%, the mass concentration of the loading solution was 0.1265 g.mL -1, the pH of the loading solution was 6.00, the volume fraction of the eluting ethanol was 58% in consideration of the practical feasibility, the mass concentration of the loading solution was 0.125 g.mL -1, the pH of the loading solution was 6.0, the operation was carried out under this condition, the recovery rate of the carthamus tinctorius was measured, and the results were repeated 3 times, and are shown in Table 6. The optimal purification process predicted by the genetic neural network model obtains the carthamin red pigment recovery rate average value of 96.68%, the mass fraction of 91.76% and the RSD of 0.18%.
Table 7 verification test of genetic neural network model to predict optimal purification Process
3.7 Comparison with X-5 macroporous resin purification methods
The method for purifying the safflower red pigment by using the X-5 type macroporous resin is compared with that reported by Yao Xiuling et al (Yao Xiuling, lv Xiaoling, periclan, zhang Jinfeng. Research on the separation of the safflower red pigment by adsorption with macroporous resin [ J ]. University of Tianjin technology, 2009,24 (03): 39-42), periclan (periclan. Extraction and purification of the safflower red pigment and research on blood lipid reduction [ D ]. University of Tianjin technology, 2009).
Yao Xiuling and the like, the process conditions for purifying the safflower red pigment by adopting the X-5 macroporous resin are obtained only according to static adsorption and desorption: adsorbing at room temperature of 25 deg.c with pigment liquid absorbance of 0.5-1.5 and pH 7.0-9.0; the desorption adopts 60% ethanol solution, and the pH value is 7.0-9.0. The research is operated under the condition, and the recovery rate of the obtained red pigment is 19.83-35.01% and the mass fraction is 64.12-83.70%.
The method for purifying the safflower red pigment by adopting X-5 type macroporous resin for surrounding rocks comprises the following steps: the absorbance of the sample loading liquid is 0.597, the dilution is 10 times, the sample loading flow rate is 4.5BV/h, the elution speed is 2.0BV/h, and the recovery rate of the red pigment is 20.67% and the mass fraction is 58.69% under the condition.
In addition, the two methods have the problem of extremely low mass concentration of the sample solution, under the condition of the same sample loading amount, the sample loading volume of the method such as Yao Xiuling is 40 times (the absorbance value of the pigment solution is 1.0) of the research, and the sample loading volume of the method of surrounding rock is nearly 700 times of the research.
3.8 Recrystallization
Taking eluent obtained under the optimal purification process condition of 3.6 HPD-400, and determining the carthamin content by an HPLC method, wherein the mass fraction of the carthamin after HPD-400 macroporous resin purification is 91.76%, which is about 3 times that before purification.
Volatilizing part of ethanol at room temperature, freeze-drying to obtain crude red pigment, dissolving with acetone to supersaturate, adding pure water at a ratio of pure water to acetone=4:1, standing at 4deg.C, centrifuging after precipitation, and lyophilizing the precipitate to obtain red pigment with mass fraction of 98.74%.
The HPD-400 macroporous resin purification process established by the research is simple and convenient to operate, and the recovery rate of the target compound is high. After recrystallization operation, the mass fraction of the carthamin red pigment can reach 98.74 percent.
Claims (2)
1. The method for purifying the safflower red pigment by using the macroporous resin specifically comprises the following steps:
(1) Dissolving the crude product of the haematochrome into haematochrome solution with the concentration of 0.10 g.mL -1 calculated by the raw medicinal materials by using pure water;
(2) Soaking HPD400 macroporous resin in 95% ethanol for 24 hr to swell, eluting with ethanol until the eluate is not turbid, washing with water until no ethanol smell is present, and suction filtering;
(3) Weighing pretreated wet resin 2 g, placing in a conical flask with a plug, respectively adding 25 mL of 0.10 g.mL -1 carthamin solution, oscillating 2 h at room temperature, centrifuging to obtain supernatant, fixing the volume to 25 mL, performing HPLC, calculating concentration according to peak area, and calculating static adsorption rate; then adding 25 mL of 60% ethanol solution to desorb the resin, oscillating at room temperature for 2 h ℃, centrifuging to obtain supernatant, fixing the volume to 25 mL, calculating the concentration according to the peak area, and calculating the static desorption rate;
(4) Weighing pretreated activated resin 3g, loading the resin on a column by a wet method, adding 10mL of 0.10 g.mL -1 carthamin solution, controlling the mass concentration of the loading liquid to be 0.125 g.mL -1, controlling the pH of the loading liquid to be 6.0, controlling the volume flow of the loading liquid to be 0.5 mL.min -1, eluting with 4BV ethanol at the volume flow of 1.0 mL.min -1, collecting the eluting ethanol with the volume fraction of 58%, rotary evaporating the eluting liquid, transferring the eluting liquid to a 10mL measuring flask, fixing the volume by 60% ethanol, measuring by an HPLC instrument, and calculating the recovery rate Q according to the chromatographic peak area, wherein Q=S/S 0, S is the peak area of the carthamin after macroporous resin treatment, and S 0 is the peak area of the carthamin solution before the treatment.
2. The method according to claim 1, characterized in that: the preparation of the carthamin crude product comprises the following steps: washing Carthami flos with water to remove most of yellow pigment, drying at 25deg.C, and pulverizing into powder; precisely weighing 10 g safflower powder in a conical flask with a plug, adding 58% acetone solution according to a liquid-to-material ratio of 23:1, adjusting the temperature to 40 ℃, carrying out ultrasonic treatment at 41: 41 min, filtering, placing the filtrate in a separating funnel, adding a proper amount of ammonium sulfate, shaking uniformly, standing to separate layers, taking the upper phase as an organic phase and the lower phase as a water phase, collecting the upper phase, recovering acetone, drying, and freeze-drying to obtain a crude product of the haematochrome.
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