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. Objectivechromosome analysis method has been widely used in the separation and analysis of
amino acids
,
nucleic acids
,
hormones
,
vitamins
, sugars, etc. The advantages are: the ability to separate and analyze in the composition, structure and properties of very similar substances;
aim of this experiment is to master the general principles and operating techniques of paper layering. , the principlepaper layering method is based on
filter paper
as a support of the distribution of the layer analysis method. Distribution layering method is the use of substances in two different mixed solvents in the distribution coefficient is different, and to achieve the purpose of separation. The allocation factor is usually represented by an assignment of . Under certain conditions, the distribution coefficient of a substance in a solvent system is a constant. α - The concentration of the solute in the stationary phase (Cs)/the concentration of the solute in the flow phase (CL)
This experimental laminate solvent is composed of
organic
solvent and water. Because the filter paper fiber and water have a strong affinity (there are many - OH-based and water connected by hydrogen bonds), adsorption of a lot of water, generally up to about 22% of the filter paper weight (of which about 6% of the water and cellulose combined into a complex), so that this part of the water diffusion effect reduced to form a static phase.
, the affinity of the filter paper is very weak, it can flow freely in the capillary tube of the filter paper, and the flow phase is formed. For layering, immerse one end of the filter paper in the layering solution. The organic solvent continuously passes through the origin of the sample at the point, so that the solute in it is distributed between the two phases according to its own distribution coefficient.
distribution process: a portion of the solute moves away from the origin of the organic phase and enters the insoluble region and is redistributed, i.e. a portion of the solute enters the water phase from the organic phase.
as the organic phase moves forward, the solute continuously distributes reversiblely between the two phases and moves forward continuously. Because of its different distribution coefficients, the distribution coefficients between the two phases are different, and the solutes with small distribution coefficients are distributed in the flow phase in large quantities and move slowly. Therefore, various solutes can be separated from each other during the analysis process due to the different speed of movement.
speed is generally expressed as a move rate RF value: . The distance from the origin to the center of the analysis point /the distance from the origin to the front of the solvent
a variety of
compounds
under constant conditions, after the analysis has its own certain RF value, that is, there is a certain position in the stratography spectrum. In this way, the purpose of qualitative, separation and identification can be achieved.
rf value is determined by many factors, the most important of which is the distribution coefficient of the separated substance. The distribution coefficient of matter is determined by the following factors:
(1) the polarity of matter. The polarity of water is very strong, the general polarity of the material is easy to enter the water phase, non-polar material is easy to enter the organic solvent. For example, amino acids with -OH and -NH2 and -COOH in the side chain are easily distributed in the water phase, the Rf value is smaller, and the polarity of the molecules of substances containing more non-polar basis (e.g. -CH3) decreases and the Rf value increases.
(2) the texture of the filter paper and the degree of moisture saturation. The texture of filter paper must be equal, pure, thick and appropriate, with a certain mechanical strength, before layering should be water and organic solvent steam saturated.
(3) solvent purity, PH value and water content. Changes in PH values and moisture content can change the polarity of amino acids and laminate solvents, as well as changes in Rf values.
(4) the temperature and time of the layering. The temperature change changes the organic phase moisture content in the solvent and the Rf value changes. When all conditions are the same, the layering time is short, and the RF value is small.
, during the analysis process, the above factors affecting rf values must be strictly controlled.
.