Preparation of heparin by ion exchange method
Preparation of heparin by ion exchange method
Heparin (Heparin) is a class of acidic mucopolysaccharides that are stable to alkali and heat, and exists in organisms as complexes bound to proteins, which are commonly separated by salting out and enzymolysis, and then removed by heating and filtration. Since heparin molecules are strongly negatively charged under alkaline conditions, they can be adsorbed by anion-exchange resins at a certain pH and ionic strength, and the crude product can be obtained by enrichment, elution and precipitation. (Source: Laboratory instruction of biochemistry and molecular biology, Shao Xueling et al, Wuhan University Press, 2003)
Operation method
ion exchange method
Principle
The main methods for the determination of heparin are bioassay, UV spectrophotometry, and chemical methods. The heparin molecule is strongly negatively charged and can combine with positively charged molecules to form complexes. Azure A is a basic dye, and the complex with heparin exhibits "photoisochromism", i.e., it changes the original absorption spectrum of the dye. Controlling the concentration of the dye, the relationship between the light absorption at 505 nm and the concentration of heparin in a medium with a low concentration of heparin at pH 8.6 is in accordance with Lang-Beer's law.
Materials and Instruments
Intestinal mucosa or pig liver Move Solution Preparation: For more product details, please visit Aladdin Scientific website.
Barbiturate buffer Gum arabic Azure A Heparin
Beakers Test tubes Test tube holders Glass rods Magnetic stirrer Electric stove Filter suction flasks Brinell's funnel Centrifuge 722 Spectrophotometer Volumetric flasks Vacuum dryer Vacuum pump Water bath Filter paper Cloth cloth. Precision pH test paper
1. Barbiturate buffer (pH 8.6, 0.05 mol/L): weigh 1 g of sodium hydroxide dissolved in 50 ml of boiling water, then accurately weigh 5052 g of dihexylbarbituric acid dissolved in the above solution, cooled and diluted to 500 ml, and the pH meter was corrected to pH 8.6.
2. 0.1% gum arabic solution: weigh 0.5 g, first dispersed with a small amount of water, then diluted to 500 ml, filtered and spare.
3. 0.1% azurite A solution (weigh 0.5 g, first dissolved with a small amount of water in a mortar and pestle grinding, and then diluted to 500 ml, filtered, refrigerator reserve. Dilute 5 times more when used.
4. Heparin standard solution: accurately weigh a certain amount of heparin standard, use sterilized water to form a standard solution of 100 U/ ml, refrigerate for temporary storage and dilute 100 times with water when used.
Procedures:
1. Extraction (salting-out method)
Fresh pig intestinal mucosa 2 kg (or pig liver crushed), add sodium chloride 80 g (4% concentration), stir to dissolve with 40% sodium hydroxide solution to adjust the p H to 9.0 (with a wide range of pH paper), water bath heating to 50 ~ 55 ℃ stirring extraction for 2 h, and then warmed to 90 ℃ to maintain 10 min, cooled to 60 ℃ filtered with a cloth bag, and the filtrate to be taken to the ion exchange.
2. Ion exchange
Dilute the filtrate 3 times with water, add 120 g of D254 resin (chlorine type) (6% according to the amount of feed, W/W), stir slowly for 6 h (can be sampled every 1 h to determine the content of the remaining heparin in the extract), filter and take the resin.
3. Wash, elution
Take the resin after ion exchange and wash it with tap water first, and then with distilled water. Take the washed resin and add an equal volume of 1.4 mol/L NaCl solution and stir slowly for 1 h. Filter the resin. Continue to use 0.5 times the volume of 5 mol/L sodium chloride solution stirring slowly for 2 h, filtration to collect the filtrate (take a sample to measure the content). Add 0.5 times the volume of 3 mol/L NaCl solution to the resin, stir slowly for 1 h, filter and collect the filtrate (sample for content). Resin then 0.5 times the volume of 3 mol/L sodium chloride solution stirred slowly for 2 h, filtration, filtrate collection (sampling for content), combined three times the elution filtrate.
4. Crude heparin
Take the combined filtrate and add 1.5~2 times the volume of 95% ethanol wash once, acetone wash twice, vacuum drying that is crude heparin.
5. Refined heparin
Weigh the crude heparin powder with 1% sodium chloride solution (pre-cooled but) appropriate amount of dissolution, adjust pH1.8 or so, centrifugation for 15 min, the supernatant through the sand funnel to remove the fat on the surface of the centrifugal solution. The suspension was heated to 80～90 ℃ on a water bath, 0.0003 mol/L potassium permanganate solution was added dropwise (according to 0.1～0.2 mol/billion units), and the end point was detected by the filter paper method, and the filtrate was added with 30% hydrogen peroxide, adjusted the p H to 11, and placed in a refrigerator for 36 h (in a refrigerator). Extract filtration, the filtrate adjusted pH to 6.4, add 1 times the volume of 95% ethanol, centrifugation, the precipitate was washed twice with 95% ethanol, acetone washed twice, vacuum drying that is, the refined heparin.
6. Azure method for the determination of potency
(1) the production of standard curve: take a larger test tube (easy to mix) 6, to 0 ~ 5 number, according to the following table. After adding gum arabic must be shaken well before adding the dye, after mixing with a 722-type spectrophotometer to measure the absorption value at 505 nm, with the absorption value as the vertical coordinate, the number of units as the horizontal coordinate to draw the standard curve.
(2) Determination of sample: weigh the sample accurately about 10 mg, prepare 1 mg/ml solution, then dilute it into 1 mg/100 ml solution and 2 mg/100 ml measurement solution in two 100 ml volumetric flasks, operate according to the following table and record the absorption value, check the corresponding number of units on the standard curve, calculate the potency of the sample according to the following formula, and then take the average value. 
The unit is U/mg, where Pi is the number of units found on the standard curve from the A-value; V is the total number of milliliters of the sample; Vi is the number of milliliters of the sample used in the determination; Sw is the number of milligrams of the sample weighed.