The principle and operation method of paper layering of amino acids.

. First, the purpose and requirements1. Learn the principles and methods of distribution analysis.
2. Master
techniques of
on-paper on-paper analysis (including dot, balance, layering, coloring, identification). , the principle
the method of layer analysis, also known as the color layer separation method,
chromatography
method. It is a very effective physical and physical chemical analysis method for separating and analyzing multi-composition mixed substances.
chromatography is one of the most important methods in various chemical separation methods. Because of its high separation efficiency, high separation speed, low sample dosage and so on, has become a widely used separation technology in all fields related to chemistry
paper layering is
filter paper
as the carrier (inert support) distribution layering. What is an assignment? The dissolution process of substances between two insoluble solvents is generally referred to as distribution. When equilibrium is reached, the ratio of the concentration of dissolved solutes in the two solvents is called the distribution coefficient, also known as the equilibrium constant.is the distribution coefficient of a substance in a solvent system and is a constant at a certain temperature.
filter paper fiber and water have a strong affinity, filter paper fiber can absorb 25-29% of the water, of which 6-7% of the water with hydrogen bonds and cellulose hydroxyl binding, under normal conditions more stable, not easy to remove. This water group becomes a fixed phase. The
solvent
the filter paper is very weak, so the organic solvent is the flow phase (flowing in the gap between the fibers), it moves along the filter paper. The solvent moves from the bottom up, called the up-line method, and the top-down movement, called the down-line method.
the sample is layered on filter paper (this point is called the origin), and the various solutes in the sample (e.g. various amino acids) are continuously distributed in two-phase solvents. Because of their different distribution coefficients, the rate at which different solutes move with the flow phase varies, so they are separated to form a layer of analysis that is not equal to the origin.the rate at which
solutes move on the filter paper is expressed in RF values
the distance from the origin to the center of the analysis point
Rf-————————————
the distance from the origin to the front of the solvent
as long as the conditions (e.g. temperature, composition of the spread solvent, quality of the filter paper, etc.) remain constant. Therefore, qualitative analysis can be made according to rf value.,
reagents
and equipment(i) reagents
1, acidic expanders: 4 water-saturated orthotinol and 1 acetic acid mixture. Put 20 ml of orthotinol and 5 ml of acetic acid into a liquid
lepper
, mix with 15 ml of water, fully shake, layer after rest, release the lower layer of water. Pour the extender into
medium
dish for spare.
2, color agent: 0.1%tritone coloring solution: 0.1g3 ketones, dissolved in 100ml of butanol.
3, unknown amino acid solution
4, standard amino acid solution.
(ii) equipment
1, capillary tubes.
2, specimen cylinder.
3, Xinhua filter paper No. 1. (If the filter paper is not clean, 0.4mol/LHCL can be soaked for 20 hours or overnight, washed to neutral with distilled water, then rinsed with 95% ethanol and dried for use.) (
4, sprayer.
5, Petri dishes.
6, split funnel.
7, hair dryer.4, operation 1, the preparation of the layering filter paper
in 15cm×20cm filter paper on one end of the edge 3cm with a pencil gently draw a straight line, in this line each interval of 3cm to make a mark.
2, dot: use capillary tube to point each amino acid sample in these four positions; The diameter of the point should be less than 0.5cm, three to four times per point.
3, balance and unfold
the filter paper sewn into a cylinder shape (note that the two sides of the paper do not meet), hanging on the hook inside the head of the analysis cylinder, cover the lid saturation 20 to 30min. Then put the filter paper cartridge into a Petri dish filled with an expander (dot-like end down, the liquid surface of the expander needs to be 1cm below the dosage line), until the solvent front rises to 1cm from the top, blow dry until there is no butanol odor, draw the solvent frontier line with a pencil.
4, color: with 0.1%tri ketone solution uniform spray color, 65 degrees C drying or blow-drying can show a variety of amino acids of the layering spots. 5, qualitative identification
to calculate the RF value of various amino acids, with a variety of amino acids standard RF value (25±1 degrees C) see the main reference book 1 p49 Table 2-4) control identification is what amino acids.
, note
1, filter paper to keep clean, do not touch by hand, carry
gloves
or only take corners.
spots should be as small as possible, the maximum does not exceed 0.5 cm diameter, after each sample with a cold wind dry and then point a second time.
2, expand the point do not immerse in organic solvents, at least balance half an hour or 1 to 2 hours.
3, color when the spray should be evenly small, uneven will be speckled (solute) washed down.
4, solvent must be freshly preparation and shake to use. .