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Separation of nucleotides by chromatography on ion exchange columns
In this experiment, yeast RNA was used as the material, and the RNA was hydrolyzed into mononucleotide by alkali, then separated by ion exchange column chromatography, and finally identified by ultraviolet absorption method. The purpose of this experiment is to understand and master the principle and method of alkaline hydrolysis of RNA, the principle and method of separation by ion exchange column chromatography, and the method of ultraviolet absorption analysis.
Operation method
ion exchange chromatography
Principle
In this experiment, yeast RNA was used as the material, and the RNA was hydrolyzed into mononucleotides by alkali, then separated by ion-exchange column chromatography, and finally identified by ultraviolet absorption method. Meanwhile, the base composition of yeast RNA could be calculated by measuring the content of each mononucleotide.
Materials and Instruments
Yeast RNA Move 1. Base hydrolysis of RNA For more product details, please visit Aladdin Scientific website.
Anion exchange resin Polystyrene-divinylbenzene-trimethylamine quaternary ammonium formate Sodium formate KOH Distilled water Perchloric acid NaOH HCl AgNO3
Chromatography column Gradient eluent Electromagnetic stirrer Constant flow pump Automatic partial collector Acid meter Ultraviolet spectrophotometer Vortex mixer Nucleic acid protein detector Benchtop centrifuge
Weigh 20 mg of yeast RNA, put it in a graduated centrifuge tube, add 2 ml of freshly prepared 0.3 mol/ L KOH, dissolve it by stirring with a thin glass rod, and then hydrolyze it in a 37 ℃ water bath for 20 h. Then adjust the pH of the hydrolysis solution to below 2 with 2 mol/ L HClO4 (perchloric acid) (a small amount of it should be added many times and a few drops are enough). Since nucleotides are easy to depurinate under over-acidic conditions, a vortex mixer should be used to stir the solution rapidly to prevent over-acidicity, and then centrifuge the solution at 4000 r/min for 15 min, and place it in an ice bath for 10 min to complete the precipitation. Pour the supernatant into another graduated test tube, and adjust the pH value of the supernatant to 8~9 with 2 mol/L NaOH drop by drop, and use it as the sample solution for backup. Before the sample solution was loaded onto the column, 0.1 ml of the sample solution was taken and diluted 500 times, and the light absorption value at 260 nm was measured, which was used for the final calculation of the recovery of ion exchange column chromatography.
2. Pretreatment of ion exchange resin
Take 8g of 201x8 powder type strong alkali anion exchange resin (wet), soak it in distilled water for 2 h, remove the fine particles by flotation, and remove the air bubbles in the resin by decompression, and then soak it in 4 times the amount of 0.5 mol/L NaOH solution for 1 h to remove the alkali-soluble impurities in the resin. After washing with deionized water until nearly neutral, the resin was soaked in four times the amount of 1 mol/ L HCl for half an hour to remove acid-soluble impurities in the resin. The anion exchange resin is then washed with distilled water to neutral (column wash is possible), and the anion exchange resin is now of the chlorine type.
3. Ion exchange chromatography column loading method
Ion exchange chromatography columns can be used with an inner diameter of about 1 cm, a length of 10 cm, the lower end of the column has a sintered fused filter plate, and the upper end of the column uses a rubber stopper with a small hole in the middle of the stopper. Insert a thin polyethylene tube tightly, the chromatography column clamped on the iron frame, adjusted to vertical, the lower end of the column thin hose with a spiral clamp, add distilled water to the column to the height of 2/3 of the column, and then use a dropper will be pre-treated ion-exchange resin added to the column, so that the resin is free to settle to the bottom of the column, relax the spiral clamp, so that the distilled water slowly outflow, and then continue to add the resin, so that the resin finally settles to the height of about 6 ~ 7 cm. Note that in the process of loading the column and using the column later, do not dry the column, the resin should not be layered, and the liquid surface above the resin surface should be maintained at a certain height (not too high, about 1 cm), in order to prevent air bubbles from entering the resin and affecting the separation effect.
4. Transformation treatment of resin
Transformation of the resin is to make the resin take on the ions needed for elution. In this experiment, the anion exchange resin was converted from chlorine type to formic acid type. The column was first washed with 200 ml of 1 mol/ L sodium formate, and the effluent of the column was checked with 1% AgNO3 until no white AgCl precipitate appeared. Then the column was washed with about 200 ml of 0.2 mol/ L formic acid, and the A260 of the effluent was measured to be ≤0.020. Finally, the column was washed with distilled water until the pH value of the effluent was close to neutral (or the same as that of distilled water).
5. Add the sample and rinse to remove the components that are not adsorbed by the resin.
Sample addition is the transfer of RNA alkali hydrolysis products to the ion exchange chromatography column to be adsorbed by the ion exchange resin. First, the liquid in the column is gently sucked off with a dropper, so that the liquid level drops to just near the surface of the resin. Tighten the lower screw clamp, use the dropper to accurately remove 1.0 ml of RNA alkali hydrolysis sample solution, carefully add it to the surface of the resin along the wall of the column, and then loosen the lower screw clamp, so that the sample level drops to the surface of the resin, and then add a small amount of distilled water with the dropper, and when the water level drops to the surface of the resin, then wash the column with about 200 ml of distilled water, so that impurities, such as purines, pyrimidine bases, nucleosides, etc., which are not adsorbed by the anion exchange resin, will be washed down. The impurities such as purines, pyrimidine bases and nucleosides were washed down. Check the absorbance of the effluent at 260 nm until it is below 0.020. Turn off the constant-flow pump and tighten the screw clamp at the lower end of the column.
6. Gradient elution
Add 300 ml of distilled water into the mixing bottle of the gradient eluter, and add 300 ml of 0.20 mol/ L formic acid-0.20 mol/ L sodium formate mixture into the reservoir bottle (Note: the connecting tube at the bottom of the gradient eluter should be filled with distilled water beforehand to drive out the air bubbles). The outlet of the eluent and the inlet of the constant-flow pump are connected by a thin plastic tube, open the connecting valve between the two bottles and the outlet valve, turn on the electromagnetic stirrer, loosen the screw clamp at the lower end of the column, turn on the constant-flow pump, control the flow rate of 5 ml/tube/10 minutes, turn on the part of the collector, and collect the effluent in separate tubes. The A260 value of each tube at 260 nm was measured with distilled water as the control, and each tube was numbered and the tube with the highest peak was labeled.
7. Identification of nucleotides
Determine the light absorption values of the liquid in the highest peak tube between 230 and 300 nm at 5 nm intervals. This includes the points at 250 nm, 260 nm, 280 nm, and 290 nm. (Note: The liquid should be retained and not poured out. A quartz cup was used for the measurements). Since formic acid (HCOOH) has a strong light absorption value at less than 250 nm, the reference control solution used in the measurement was approximated as follows: 0.05 mol/ L formic acid-0.05 mol/ L sodium formate for the first peak. For the second peak, 0.10 mol/ L formic acid - 0.10 mol/ L sodium formate was used. The third peak uses 0.15 mol/ L formic acid - 0.15 mol/ L sodium formate. For the fourth and fifth peaks, use 0.20 mol/ L formic acid or 0.20 mol/ L sodium formate. It is also possible to select the reference solution by calculating the concentrations of formic acid and sodium formate according to the location of the highest peak.
8. Determination of the content and total recovery of various nucleotides
Combine the eluent of each component (including the highest peak tube) in the eluent tube, and measure the total volume of the solution with a measuring cylinder, and then determine the A260 value, and the reference control solution is the same as above. Based on the A260 value of the sample solution on the chromatography column and the sum of the A260 values of each component after chromatography, the recovery of ion exchange column chromatography can be calculated (Note: the molar extinction coefficient of RNA, E260, is 7. 7x103-7. 8x103, and the value of hydrolysis is increased by 40%).
9. regeneration of resin
The used ion exchange resin can be reused after regeneration. This can be done either inside the column or by removing the resin. To remove the resin, blow into the column from the lower end of the column with a rubber ball and collect the resin in a beaker. The method of resin regeneration is the same as the pretreatment of new unused resin. Alternatively, the resin can be directly soaked or washed with 1 mol/ L NaCl solution, and finally washed with distilled water until the pH of the effluent is close to neutral.
Results processing
1. Make the elution curve of nucleotide separation by anion-exchange resin column chromatography, take the number of tubes (or volume) of chromatographic effluent as the horizontal coordinate, and take the corresponding A260 value as the vertical coordinate, and make the elution curve graph.2. Make the ultraviolet absorption spectra of each mononucleotide, according to the absorbance value of each component solution in the wavelength range of 230-300 nm, take the wavelength (nm) as the horizontal coordinate, and the absorbance value as the vertical coordinate, and make their absorption spectra. The wavelengths of the maximum absorption peaks of each single nucleotide component were determined from the graphs, and the absorbance ratios of each component at different wavelengths (250/ 260, 280/ 260, 290/ 260) were calculated, which were compared with the standardized values of each nucleotide, so as to identify what kind of nucleotide was in the respective group.3. Calculate the recovery of ion exchange column chromatography based on the A260 value of the sample solution and the sum of the A260 values of the components obtained after chromatography.