Paper layering of amino acids.

, the purpose of the experiment1. To understand and master
principles and methods of
amino acids and paper layering.
2. Learn the operating techniques of paper layering and analyze the composition of amino acids in unknown samples.
, the principle of experiment . The method of layering with filter paper as support is called paper layering, which is a kind of distribution layering method. Most of the spread solvents used in paper
are
solvents. Filter paper fiber - OH-based hydrophobic group, can adsorption of water in organic solvents as a fixed phase, organic solvents as a flow phase, it moves along the filter paper from the bottom up, called upward layering;
the sample point on the filter paper for layering, the sample of various amino acids in the two phases of continuous distribution, because of their respective distribution coefficients are different, so that the rate of movement in the flow phase is not the same, so that different amino acids are separated and purified.
paper layering method is mainly based on the difference of mixed parts to the two-phase distribution coefficient, but there is also a certain degree of adsorption and ion exchange phenomenon. Amino acids are layered to form a layering point on the filter paper that is not from the origin, and the rate at which the amino acids move on the filter paper is expressed in RF.
Rf - The distance from the origin to the center of the stratography speckle / the distance from the origin to the front of the solvent
as long as the experimental conditions (e.g. temperature, components of the spread solvent, pH, quality of the filter paper, etc.) remain unchanged, the Rf value is constant and can therefore be used for qualitative analysis reference.
If there are more amino acids in the solution or some of these parts of the RF value is the same or similar, with one-way layering should not separate them, for this reason can be two-way analysis, after the first solvent expanded the filter paper turn 90 degrees, to the first spread of the layer obtained by the analysis point as the origin, and then use another solvent layer, can achieve the purpose of separation. Because amino acids are colorless, theof amino acids can be used to color the amino acids, thus qualitatively and quantitatively.
III, instruments and
reagents instruments
thography filter paper,
beech
, scissors,
culture
dishes, monkey head sprayer, microscope or capillary tube, hair dryer one, ruler, pencil, etc.
reagents
. 1. Mixed amino acid solution (hydrolysed amino acid dry powder)
glycine solution: 50 mg glycine dissolved in 5mL water.
solution: 25mg methionine dissolved in 5mL water.
solution: 25 mg of limeine dissolved in 5mL water.
amino acid mixture: glycine 50mg, lilacine 25mg, methionine 25mg dissolved in 5mL water.
2. Spread layer solvent
alkali phase solvent: positive butanol: 12% ammonia: 95% ethanol: 13:13:13 (V/V)
acid solvent: positive butanol: 80% methicillin: water: 15:3:2 (V/V), shake and place for more than half a day, take the liquid backup.
3. Color-showing storage fluid: 0.4mol/Ltritone-isopropanol: foric acid: water: 20:1:5.
4.0.1% copper sulfate: 75% ethanol x 2:38, prorated before use. 4, experimental step (1) standard amino acid single upward line layering method
1. draw a baseline
put on a finger sleeve or rubber
gloves
, in the length of about 20cm, width of about 17cm filter paper, 2.5cm from the short side, draw a line with a pencil, that is, the baseline.
2. Dot-like
on the original line, starting from 4cm from the long side of the paper, every 3cm with a trace syringe or capillary tube in turn dot glycine, methionine, lilacine and mixed amino acid solution. After the sample point is dry, the point can be repeated 1 to 2 times. The diameter of each point does not exceed 2mm, and the sample amount is suitable for each amino acid containing 5 to 20ug.
3. Spread layer
will point a good kind of filter paper rolled into a barrel shape, filter paper on both sides do not touch each other, with the line fixed, the lower end of the original line immersed in a petri dish filled with solvents, without the need for balance can be immediately spread layer.
exhibition layer agent is an acidic solvent system, in the spread layer solvent to add color-showing storage liquid (0.1 to 0.5mL per 10mL spread layering agent) for the spread layer, the baseline must be kept above the liquid surface, so as to avoid direct contact between amino acids and solvents. Cover the analysis cylinder and remove the filter paper when the solvent front is 2cm from the end of the paper (approximately 3h).
4. After the color
filter paper is removed, blow-dry or bake 3 to 5min in a
oven
around 80 degrees C, i.e. a fuchsia amino acid layering spot appears. With a pencil to cut down the layering spots, can be qualitative, quantitative determination.
(2) mixed amino acids two-way up paper
. 1. Filter paper preparation
the filter paper cut into about 28cm2 square, from the filter paper adjacent to each side of the intersection of 2cm, with a pencil to draw a point, as the origin.
2. Dot
mixed amino acid solution (5mg/mL) 10 to 15 μL, respectively, at the origin.
3. The display layer and color
roll the dosing filter paper into a half-barrel shape, standing in a petri dish, the origin should be at the lower end. Take a small amount of 12% ammonia with a small beech, cover the analyzer and balance overnight. The next day, take out ammonia water, add a moderate amount of alkali phase solvent (first-) and Petri dish, cover the layering cylinder, up the spread layer, when the solvent cutting edge filter paper top 1 to 2cm, take out the filter paper, cold wind dry.
turn the filter paper to 90o, and then roll into a half-barrel shape, erected in a clean petri dish, and in a small beech at least a amount of acid phase solvent, cover the layering cylinder, balance overnight, the next day will be added to the color agent acid solvent (every 10mL layering agent plus 0.1 to 0.5mL of color storage fluid) poured into the petri dish, for the second spread layer. Spread the layer, take out the filter paper, dry with a hot wind, blue-purple spots will appear.
, calculate the . The RF value of one-way stratography is calculated by measuring the distance from the rf- origin to the center of the layered speckle/the distance from the origin to the front of the solvent.
bidirectional stratification RF value, consisting of two values, measured once in the first direction and measured once in the second direction, respectively, compared with the RF value of the known amino acid in the acid-base system, can initially determine what kind of amino acid spot it is, cut it Under, cut a piece of the same size blank paper on the same sheet of paper as a control, with copper sulfate-ethanol solution washed away, with 722 type
-photometric
to determine its absorbance, in the standard curve to find out the amino acid content..