Thin layer analysis of amino acid cellulose

related topicsThin layer analysis (TLC) technology 1, purposeto learn cellulose
thin layer analysis
operation methods, master the principle of distribution of layering.II, principleto cellulose as a support, it is evenly coated on the glass plate into a thin layer, and then on this thin layer of layer analysis is cellulose thin layer analysis. Cellulose is an inert support, it has a strong affinity with water and
organic
solvent affinity is weak. The water absorbed on cellulose during layering is fixed phase, while the spread solvent is the flow phase. When the distribution coefficients of the substances to be separated in the fixed and flow phases are different, they can be separated.III, experimental materials, instruments and
reagents 1. Experimental materials mung bean sprouts or sprouted wheat seeds 2.instrumentBerrible
50mL×1; glass plate 5 cm×20cm×1; layering cylinder; capillary tube; sprayer; research.3. ReagentsStandard
Amino Acids
Solutions: Serine, Tryptophan, Liline, 0.01mol/L hydrochloric acid into a solution of 4mg/mL.cellulose powder (for layering) or microcrystalline cellulose (for layering); Stratified solvent system: positive butanol (analytical pure): ice acetic acid (analytical pure): water: 4:1:1 (V/V). Coloring agent: 0.1%tritone-acetone solution.4, operation step 1. Extraction of amino acids take the sprouted wheat seed 2g (or green bean sprouts under the embryo shaft 2g), put into the research, add 95% ethanol 4mL and a small amount of quartz Sand, after the study of homogenization, poured into the
centrifugal tube
centrifuge 3,000r/min, 15min, the upper liquid is amino acid extract, with a dropper carefully inhaled sample bottle spare.2. Platetake a small amount of sodium methyl cellulose (about 12 mg), fully ground in the research, then called cellulose powder 3g in the research grinding, and then add 14mL water grinding slurry, fiber Pour the slurry on the washed and dried glass plate, gently vibrate, so that cellulose is evenly distributed on the glass plate, horizontally placed air-dried, before use into 100 to 110 degrees C
bast
to live 30min. Here, sodium methyl cellulose is the adhesive, it enables cellulose powder to adhere more firmly to the glass plate, adding too much will destroy the thin layer of cellulose capillary action and make the lamination speed delay, the addition of too little is not firm adhesion, so attention needs to be paid to the increase control.3. Dot samplethe left and right sides of the thin sheet with a blade to scrape off 0.5cm to prevent "edge effect". Gently mark with a pencil at 15mm from one end on a cellulose sheet. The distance between the sample points is 1.3cm. Samples are taken with capillary tubes and sampled at the mark, with the sample speckled diameter controlled at about 2mm.4. The layerthe end of the sheet with the sample is immersed in the laminate tank where the spread solvent is stored, and the surface of the layer solvent cannot be higher than the sample line. When the spread layer solvent walks to the top of the sheet 0.5 to 1cm, remove the sheet (about 1 to 2h), use a pencil at the front to make a mark and blow dry by the wind.5. Coloringspraytritone colorant on the board, with a hot blow for a few minutes, (or dry in a 70 to 80 degrees C oven) can be observed fuchsia amino acid spots, proline exception, yellow spots. The amino acid spots are circled with a pencil, the distance between the front of the solvent and the distance between the center and the starting point of each spot is measured, and the RF value of each amino acid is calculated. The Rf value is the rate of migration (rate of flow, Rf), and each amino acid has its own RF value under constant conditions. The Rf value is expressed as:Based on known standard amino acids and RF values, compared to the RF value of amino acids in wheat (or mung bean sprouts) extracts, determine which amino acids are contained in the extract., Note 1. During operation, the hand must be washed and can only touch the corners of the sheet layer;2. When the preparation of the exhibition layer agent, to use pure solvents, should be ready to use, so as not to leave for too long its composition changes (esterification)., thinking question 1. What is the distribution coefficient?2. Which is faster than the polar amino acids and non-polar amino acids in thin layer analysis?3. What is the "edge effect" and how can it be mitigated or eliminated?the answer 1. The distribution coefficient refers to the concentration ratio of a solute in two phase solvents when the solute dissolves to balance in two non-soluble solvents. The allocation factor is a constant, expressed in K, after the layering conditions are determined.The distribution coefficient is related to the properties of solvents and solutes, and is also affected by temperature and pressure. Therefore, the distribution coefficients of different substances are different. Under
constant temperature
constant pressure, the distribution coefficient of a substance in the defined layering system is a constant.2. The separation of layering is based on the nonpolar nature of amino acids. The smaller the polarity of the substance (i.e., the greater the non-polarity), the greater the migration rate when distributed in organic solvents; Therefore, non-polar amino acids spread the layer in organic solvents faster than polar amino acids.3. When spreading with a multi-system, solvents with weak polarity and lower boiling point (e.g. chloroform in chloroform in a chloroform-methanol system) are volatile on both sides of the lynte plate, so their concentration on both sides of the thin layer is smaller than in the middle, that is, on both sides of the thin layer than in the middle contains more polar or higher boiling point solvents, so the Rf value on both sides of the thin layer is higher than the middle, that is, the so-called "edge effect". In order to reduce or eliminate the edge effect, the
filter paper
, which is soaked with the expander, can be attached to the inner wall of the analyzer, increasing the saturation of solvent vapor in the analyzer.