Experiments on the analysis of amino acid composition in plant wounding fluids
Experiments on the analysis of amino acid composition in plant wounding fluids
Principles and methods of analyzing the amino acid composition of plant root wound fluids by paper spectroscopy.
Operation method
Experiments on the analysis of amino acid composition in plant wounding fluids
Principle
Paper chromatography, also known as filter paper chromatography, is an identification or quantification technique in which a mixed solution is separated on filter paper using a chromatographic solvent. Chromatographic solvent consists of organic solvents and water, in which water molecules are adsorbed by the cellulose of the filter paper and become the stationary phase, while the organic solvent becomes the driving phase. When the driving phase moves along the filter paper, the various solutes in the sample move faster due to the different partition coefficients (i.e., the ratio of solubility in the two phases) in the two phases, while the more lipophilic solutes move slower, resulting in the separation of the mixture. Finally, the separates are identified by color development using a color developer. Different substances move at different rates, so there are different Rf values (specific shift rate), which can be used as a reference for characterization. Paper spectrum analysis has one-way, two-way points. Where the composition is simple and easy to separate the available one-way chromatography, otherwise available two-way chromatography.
Materials and Instruments
Plant Root Wound Liquid Move I. Material Instrumentation and Reagents For more product details, please visit Aladdin Scientific website.
n-Butanol Glacial acetic acid 95% ethanol Aspartic acid Lysine Alanine Leucine Phenylalanine Valine Arginine Glutamic acid Glycine Histidine Proline Tryptophan Serine Threonine
Chromatography cylinder Chromatography filter paper Micropipettes or glass capillaries Hair dryer or electric oven Oven Small sprayer
1 . Materials: plant root wounding fluid.
2. Instruments and equipment: chromatography cylinder; chromatography filter paper; micro-dropper or glass capillary tube; hairdryer or electric oven; oven; small sprayer.
3. reagents: n-butanol; glacial acetic acid; 95% ethanol.
Standard amino acid solution: aspartic acid, lysine, alanine, leucine, phenylalanine, valine, arginine, glutamic acid, glycine, histidine, proline, tryptophan, serine, threonine and other amino acids are divided into 3-4 groups dissolved in 10% isopropanol, that is, the standard amino acid solution mixture, the concentration of each amino acid is 2mg . ml-1 for each amino acid.
0.1% hydrated ninhydrin ethanol solution: weigh 0.1g hydrated ninhydrin, dissolved in 100 ml 95% ethanol.
Phenol and water mixed solvent: phenol : water (ratio 3 :1).
II. Experimental steps
1. Unidirectional chromatography
(1) spot sample: take the chromatography filter paper, about 25cm long, about 4-5cm wide (such as the number of samples should be widened). At one end of the filter paper 1.5~2cm away from the edge of a pencil gently draw a line, and then use a micro burette or capillary to suck up the wound flow of liquid in the pencil line at the appropriate location on the point, and another standard amino acid mixture in the vicinity of the point (about 5μl). The size of the point should not exceed 0.3cm, and the distance between the two samples should be 1.5~2cm, and then dry the filter paper with a hairdryer or at a certain distance on the electric stove, taking care that the temperature should not be more than 80°C. If the concentration of amino acids in the wound stream is high, the filter paper should be dried with a hairdryer or a capillary. If the concentration of amino acids in the wound fluid is low, you can focus on the original point a few times (note that each point of the sample once, blow-dry and then repeat the point of the sample), the sample point blow-dry for use.
(2) Chromatography: Take an appropriate size of the chromatography cylinder, add phenol: water mixture (ratio of 3 :1) 50 ml as the solvent for chromatography, or n-butanol, glacial acetic acid and water mixture (ratio of 40:10:50), and then point the sample of the filter paper into the cylinder under the lid, the lower end of the temporary not in contact with the solvent. Close the lid, so that the filter paper and solvent vapor equilibrium 1 h, the lower end of the filter paper immersed in the solvent, (note that the sample point can not be immersed in the solvent, so as not to leach off the sample). At this time, the solvent rises along the paper, to be the solvent front from the top of the filter paper and 2cm, the filter paper from the chromatography cylinder, with a pencil in the solvent at the front edge of the mark, and then suspended in the room air-drying or drying at 60 ~ 80 ℃.
(3) color development: filter paper dry, with a sprayer evenly sprayed with 0.1% hydrated ninhydrin ethanol solution, slightly wet can be, and then in the 60 ℃ ~ 80 ℃ oven heating for about 10min, for color development, at this time there are amino acids at the violet spot that is presented.
(4) Identification: Calculate the Rf value of each spot and compare it with the Rf value of the standard amino acid to identify the type of amino acid in the wound fluid. At the same time, according to the size and color of each spot, we can approximate the amount of various amino acid content.
2. Bidirectional chromatography
Bidirectional chromatography is suitable for the analysis of samples with complex composition or difficult separation, and only one sample can be analyzed per filter paper.
(1) point sample: take two 20cm×25cm or so chromatography filter paper, in the lower left corner from both sides are 1.5~2cm, with a pencil mark, with a micropipette or capillary point sample, a point standard amino acid mixture (about 5μl); another point of the wound flow liquid, point sample point size of no more than 0.3cm is appropriate. If the concentration of amino acids in the wounded fluid is low, you can repeat the point on the original point several times (note that each point must be blown dry after sampling). After blow-drying the sample point, the filter paper rolled into a tube, the two sides can be sewn together with a line (leaving a gap in the center), as shown in the figure.
(2) Chromatography
The first chromatography: take two chromatography cylinders, add the mixture of phenol : water (ratio of 3 :1) about 50 ml as the solvent for chromatography, and then hang the filter paper with the sample into the cylinder under the lid, and the lower end is not in contact with the solvent for the time being. Close the lid, so that the filter paper and solvent vapor equilibrium 1 h, the lower end of the filter paper immersed in the solvent, (note that the sample point can not be immersed in the solvent, so as not to leach off the sample). After a period of chromatography, that is, when the solvent front from the top of the filter paper there are 2cm, the filter paper from the chromatography cylinder, with a pencil in the solvent front of the mark, and then suspended in the room air-drying or drying at 60 ~ 80 ° C, until there is no obvious solvent odor.
The second chromatography: the first chromatography of the line of the two filter paper folded, the filter paper turned 90?, so that the original left side to become the lower edge, and then rolled into a cylinder sewing as previously described. Change the chromatography solvent to a mixture of n-butanol, glacial acetic acid and water (ratio 40:10:50), and the rest of the chromatography is the same as the first chromatography.
(3) color development: the second chromatography is completed, with a sprayer in the air-dried filter paper evenly sprayed with 0.1% hydrated ninhydrin ethanol solution (slightly wet can be). Heating in the oven at 60 ℃ ~ 80 ℃ for about 10min, color development.
(4) Identification: After the color development is completed, the chromatograms and the center point are depicted with a pencil, and then the distance from the origin to the center point of the chromatogram and the solvent front is measured, and the chromatographic Rf values of the various standard amino acids and unknown amino acids in the sample are calculated for comparison and identification. Bidirectional chromatography, its Rf value consists of two values, that is, to be calculated once in the first and second direction chromatography, according to the Rf value and the unique color of each amino acid, the color spot on the two pieces of filter paper for comparison, you can know the type and name of the free amino acids contained in the sample.