Paper chromatography experiments on amino acids
This experiment was obtained from the Department of Physiology and Biochemistry, College of Plant Science and Technology, Bayi Agricultural and Reclamation University, Heilongjiang, China.
Operation method
Paper chromatography experiments on amino acids
Principle
Paper chromatography is a partition chromatography method using filter paper as a support. Distribution chromatography utilizes the different partition coefficients of substances in two different solvent mixtures for the purpose of separation. The partition coefficient is usually expressed in terms of α. Under certain conditions, the partition coefficient of a substance in a given solvent system is a constant. Chromatography solvents are chosen to consist of organic solvents and water. As the filter paper cellulose has a strong affinity for water (paper has a lot of -OH group and water connected by hydrogen bonding), adsorption of a lot of water molecules, generally up to about 22% of the weight of the filter paper (of which about 6% of the water and cellulose combined into a complex), so that part of the diffusion of water to reduce the formation of the stationary phase; and the organic solvents and the filter paper of the affinity of the paper is very weak, you can in the filter paper of the free flow of the capillary, then the formation of the Mobile phase. Chromatography, one end of the filter paper immersed in the chromatographic solvent, organic solvent continuously through the point of origin of the sample, so that the solute according to its own distribution coefficient in the distribution between the two phases. Distribution process: a part of the solute with the organic phase moves away from the origin and into the solute-free zone, and redistribution, that is, a part of the solute from the organic phase and into the aqueous phase. As the organic phase continues to move forward, the solute continues to move forward with reversible partitioning between the two phases. Various substances due to its different distribution coefficient, the number of distribution between the two phases is different, the distribution coefficient of small solutes in the mobile phase in the distribution of the number of fast forward speed; and distribution coefficient of large solutes in the stationary phase in the distribution of the number of slow moving speed. So all kinds of solutes in the process of chromatography due to the different speed of movement can be separated from each other. The moving speed is generally expressed by the moving rate Rf: various compounds under constant conditions, after chromatography have their certain Rf value, that is to say, in the chromatographic spectrum has a certain position. The Rf value is determined by many factors, the most important of which is the partition coefficient of the separated substance. The partition coefficient of a substance is determined by the following factors: (1) the size of the polarity of the substance. The polarity of water is very strong, and generally the substances with strong polarity are easy to enter the aqueous phase, and the non-polar substances are easy to enter the organic solvent. For example, the side chain containing -OH and -NH2, -COOH more amino acid distribution in the aqueous phase, the Rf value is small, while containing non-polar (such as -CH3) more substances whose molecules reduce the polarity, the Rf value increases. (2) The texture of the filter paper and the degree of saturation with water. The texture of the filter paper must be uniform, pure, thick and thin, with a certain degree of mechanical strength, before chromatography should be saturated with water and organic solvent vapor. (3) the purity of solvent, pH value and water content. pH value and water content change can make amino acid and chromatography solvent polarity change, Rf value also change. (4) Temperature and time of chromatography. The change of temperature changes the water content of the organic phase in the solvent, and the Rf value also changes. When all the conditions are the same, the chromatography time is short and the factors for measuring the Rf value must be strictly controlled.
Materials and Instruments
Mung bean sprouts Move I. Experimental materials, apparatus and reagents Common Problems Unidirectional Chromatography: After the color development is completed, the outline and center point of each chromatogram is traced with a pencil, then the distance from the origin to the center point of the chromatogram and the solvent front is measured, and the Rf values of the various known and unknown chromatograms are calculated for comparison and identification. The order of the known amino acid chromatograms for each group was supplied by the instructor. For more product details, please visit Aladdin Scientific website.
Aspartic acid Lysine Threonine Alanine Methionine Leucine
Thermostatic bath Oven Mason jar Triangular vial Evaporation dish Dispensing funnel Measuring cylinder Micropipettes Electrothermal hair dryer Chromatography cylinder Chromatography filter paper Mini-sprayer Scissors Blade Pencil Ruler Bolus Vaseline Spotting bottle
1, experimental materials: mung bean sprouts sprouted in a 25 ℃ temperature box for 2-3 days.
2, Instruments: (1) a constant temperature water bath; (2) an oven; (3) a mortar and pestle; (4) a triangular flask; (5) an evaporating dish; (6) 50 ml dispensing funnel; (7) three measuring cylinders; (8) micropipettes (or marked with a graduated capillary tube); (9) electric hair dryer; (10) chromatography cylinder and Petri dishes; (11) chromatography filter paper; (12) a small sprayer; (13) scissors, razor blade, pencil, ruler, glass rod, petroleum jelly, and spotting bottle.
3, reagents:
(1) Amino acid standard solution: concentration of 0.01M, dissolved in 10% isopropyl alcohol.
Group I: aspartic acid, lysine, threonine, alanine, methionine, leucine.
Group II: ornithine, glycine, γ-aminobutyric acid, valine.
Group III: glutamic acid, arginine, phenylalanine, histidine, proline.
Group IV: tyrosine, cysteine, tryptophan, glutamine.
Group V: cystine, hydroxyproline, isoleucine, citrulline, asparagine.
(2) Solution system:
First direction: n-butanol: formic acid: water: (15:3:2, v/v), shake well and stand aside.
Second direction: phenol: water (80:20, wt.). Preparation method: in the dispensing funnel first metered addition of a small amount of non-ionized water, then add a certain amount of freshly distilled phenol. According to the amount of phenol to make up all the non-ionized water should be added, so as to avoid the dissolution difficulties caused by the condensation of phenol. Shake well and set aside.
(3) Nonionized water: Used for the preparation of various reagents in the experiment.
(4) 10% isopropyl alcohol: used for the preparation of amino acid standard solution.
(5) 80% ethanol: take analytical ethanol to dilute with non-ionized water.
(6) 0.25% hydrated ninhydrin solution.
(7) Activated carbon.
(8) Quartz sand.
Second, the operation steps:
1, Extraction of amino acids:
Take 2 grams of fresh mung bean sprouts hypocotyls, add 80% ethanol 10 ml, add a small amount of quartz sand, in the mortar research into a homogenate, transferred to the triangular flask, with a small amount of 80% ethanol rinse the mortar, and moved to the triangular flask, add 0.8 grams of activated charcoal, put in a boiling water bath heated to boiling for 1 minute, removed and cooled, filtered through a filter paper, rinsed with a small amount of 80% ethanol filtrate. The filtrate was collected in an evaporating dish and evaporated to dryness on a 50-60°C water bath. Dissolve with 1 ml of 10% isopropanol and collect the sample solution for spotting.
2. Preparation of filter paper:
Unidirectional chromatography filter paper: take a 28 × 28 cm chromatography filter paper, in the corresponding two sides of 2 cm from each side, with a pencil and a ruler to draw a straight line parallel to the edge of the paper, one of which is the solvent front to arrive at the mark, and the other for the spotting line. Draw a short line perpendicular to the spotting line every 2 cm on the spotting line, and mark the spotting positions of the five groups of standard amino acids and samples.
Bidirectional chromatographic filter paper: take two pieces of 28×28 cm chromatographic filter paper, draw a straight line parallel to the paper edge at a distance of 2 cm from each edge, and take the intersection point of the left end of the straight line at the lower edge of the "well" grid as the origin of the spotting sample, with the right hand side of the origin as the first direction, and the other perpendicular direction as the second direction. Use one sheet for the standard amino acid profile and the other for the sample profile.
3, point sample:
When spotting the sample, use a micropipette or a capillary tube marked with a scale (1 microliter per cell), and spot 4 microliters of sample precisely at each spotting point. Each drop of sample must be dry before the second drop. In order to accelerate the drying of the sample, it can be carried out on a heated counting table or dried with a hair dryer, the diameter of the sample point should be 2-3 mm, and the temperature should not be too high when counting, otherwise the amino acids will be broken. Will point a good sample of one-way and two-way chromatography filter paper are rolled into a cylinder, in the upper, middle and lower three places tied three lines, but the two edges of the filter paper can not touch. In order to eliminate the interference of hydrochloric acid, to avoid the phenomenon of trailing, the chromatographic filter paper can be put into the chromatographic cylinder containing concentrated ammonia fumigation for 10 minutes, remove the ammonia in the oven at 45 ℃ will be expelled, and then chromatography.
4, chromatography:
This test adopts the upper chromatography method.
(1) one-way chromatography: take the appropriate size of a chromatographic cylinder, the bottom of the cylinder put a petri dish, add the first to the petri dish solvent, the liquid layer is about 1.5 cm thick, in the petri dish on the horizontal two glass rod, take the sample has been pointing a good unidirectional chromatographic filter paper to the point of the sample end down on the bottle containing chromatographic solvent chromatographic cylinder on the glass rod, cover the cover (the filter paper do not contact with the solvent). Equilibrate for one hour, then place the filter paper in the chromatographic solvent, taking care that the sample point does not enter the solvent to avoid leaching. Seal the chromatography cylinder, this time the solvent rises along the paper, when the solvent front reaches the mark line, remove it immediately, dry it with a hair dryer or dry it at 45℃, and then develop the color.
(2) Two-way chromatography: according to the above one-way chromatography method, the two-way chromatography filter paper of the sample of the standard amino acid will be ordered to carry out upward chromatography with the first solvent respectively, and when it reaches the mark line of the front edge, take it out immediately and blow-dry the solvent, subtract the part of solvent other than the front edge, then turn the paper to an angle of 90 °, and carry out the upward chromatography in the same way with the second solvent, and take it out and blow-dry or dry it under 45 ℃ immediately when the mark of the front edge of solvent is reached, and then develop the color. drying at 45℃ for color development.
5, color development:
Use a sprayer to spray 0.25% of ninhydrin color developer evenly on the chromatography filter paper, be careful not to spray too much. After drying the solvent with a hair dryer, placed in the oven at 60-65 ℃ for 30 minutes or blowing hot air with a hair dryer, that can show the color spot of various amino acids. In order to eliminate the effect of ammonium ions, can be unfolded on the filter paper after spraying 1% of potassium hydroxide in anhydrous ethanol solution, kept at 60 ℃ for 15 minutes, in order to make all the ammonia completely volatilized off, and then color development.
Two-way chromatography: The Rf value consists of two values, i.e., it has to be calculated once in the first and once in the second direction of chromatography. Based on the Rf and with the help of the unique color of each amino acid, it can be identified by comparing it with the standard amino acid respectively.