Amino acid separation and identification-bidirectional analysis
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Last Update: 2020-11-03
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Source: Internet
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Author: User
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Objective It is(1) to learn the principle of two-way paper analysis.
(2) master the paper layering method to separate the
amino acids
the operation method.
the principles ofPaper lamination is
filter paper
as a support, with a certain solvent system to expand, so that the mixed samples reach the separation analysis of the layering method. Its general operation is to dissolve the sample in the appropriate solvent, dot sample at one end of the filter paper, and then select the appropriate solvent system, from one end of the sample through the capillary phenomenon to the other end, unfolded, take out the filter paper to dry or dry, and then with the appropriate color agent or ultraviolet lamp, fluorescent lamp under the observation of its map. After the sample is expanded, the position of a substance on the paper chromatography spectrum is often expressed
the
of the Rf.
Rf
paper layering can be regarded as a continuous withdrawal of the solute (sample) between the fixed phase and the flow phase, and separation is achieved due to the different distribution coefficients of the solute between the two phases. A certain substance has a fixed distribution coefficient between the two phases, so under constant conditions (liquid, PH, temperature), each substance has a fixed
Rf
value, according to which the purpose of analysis and identification can be achieved.
Because the filter paper fiber can absorb 20 to 25% of the water, and 6 to 7% of the hydrogen bond form and cellulose hydroxyl binding, under general conditions difficult to get rid of;
paper analysis operation according to the direction of solvent development can be divided into up, down and radial three. Amino acid separation is generally used up-line. The up-and-down method is divided into one-way (samples with simpler components) and two-way (one-way speckle overlap is inseparable, so in its vertical direction with another solvent system layer). Bi-phase chromatography can distinguish more than a dozen samples.
analysis solvent requirements:
(1) the separated substance in the solvent system
Rf
between 0.05 and 0.8, each component
Rf
value difference is preferably greater than 0.05, so as not to overlap spots.
chemical reaction between any component in the solvent system and the separated substance.
(3) the distribution of the separated substance in the solvent system is more constant, does not change with the temperature, and easily achieves equilibrium quickly, so that the resulting spots are more rounded.
this experiment used eight mixed amino acids as samples, two-way analysis with acidic and alkaline solvents, withtritones as a color agent, to obtain a clear separation of the layering map, as shown in Figure 3.2.
Figure 3.2 Bidirectional layering map of eight mixed amino acids
reagents
and equipment 1, reagents 0.1% (W/V)tritone acetone liquid.
solvent system: first phase: positive butanol: 88% methyl acid: water: 15:3:2
(V/V);
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