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II, Materials and "
1, Acid-to-expander: positive butanol: 88% foryic acid: water: 15:3:2 , take 150 ml of orthodol, 88% foric acid 30 ml, distilled water 20 ml, placed in the fractionfunnel full oscillation. This fluid needs to be newly preparationd.
2, 12% ammonia: take 60 ml of ammonia, add 70 ml of distilled water dilution.
3, alkali-to-expander: positive butanol: 12% ammonia water : 13:3, the amount of 130 ml of positive butanol, 30 ml of 12% ammonia, placed in the liquid funnel fully oscillating.
4, 0.2%tritone coloring solution: 0.2g3 ketones, dissolved in 100ml of acetone.
5, 0.5% standard amino acid solution: said to take a variety of amino acids each 0.5g, respectively, placed in 100 mlcapacity bottle, with 10% isopropylone solution dissolved, fixed to the scale.
6, microsyrectifiers, laminate cylinders, Xinhuafilter paper, sprayers, amino acid mixtures (optional 2-3)
Table 2-4 Standard Rf values for a variety of amino acids
amino acids | standard rf value | amino acids | standard Rf value. | |
first phase (acid orientation) | second phase (alkali phase) | < td style: "width: 94.0px;" > first phase (acid orientation)
glutamate
lysine
lysine
hithionine
glycine
hydroxybutyrine Acid
r-amino butyric acid
alanine
tyrosine
0.35.
0.12
0.15
0.11
0.30
0.37
0.46
0.47
0.25
0.01
0.03
0.05
0.10
0.06
00 .10
0.08
0.14
0.23
isoestionine
phenylalanine
cysteine
serine
proline
. 0.67
0.64
0.77
0.79
0.73
0.08
0.28
0.48
0.32
0.48
0 .52
0.57
0.58
0.02
0.06
0.16
1, layering filter paper preparation: take two 19cm×23cm Xinhua filter paper number one, in the lower right corner from the bottom of the second edge of each 2cm intersection with a pencil to draw a origin, while drawing the original line on both sides.
2, dot: the 10ul sample liquid point on a filter paper origin, with a trace syringe points many times. After one point, blow dry a second time. The diameter of the point should be less than 0.5cm.
3, expand: the two filter paper after the sample in the same operating conditions with the up-line method for two-way development.
(1) first-way (acid phase) unfolded: to filter paper 19cm long side for high, with the stitch into a cylinder shape (note that the two sides of the paper do not meet), placed in a layer tank containing acid phase expander saturation 1h. Then place the dot sample in the expander and remove when the front of the solution rises to about 1cm from the top, blowing dry until there is no butanol odor.
(2) second-way (alkali phase) unfolded: the filter paper turn 900, to 23cm long side for high, with the wire stitched into a cylinder shape (note that the two sides of the paper do not meet), placed in a layer tank containing alkali phase expander saturation 1h. Then put the dot sample end into the expander, to the front of the solution rose to about 1cm away from the top when removed, blow-dried. Then, under the same conditions, expand again in the alkali phase expander and remove the blow-dry.
4, color: after the end of the expand, the filter paper with 0.2% tritone solution spray color, 65 degrees C drying.
, qualitative identification: the type of amino acids in the sample is determined according to the size of the RF value, compared with the stratographic map of the sample and the stratographic map of the standard amino acid map.