茚 the amino acid content by tritone chromosomes.

. ObjectiveLearning Methods for determining the content of
amino acids
by
three ketone coloringAmmonia reactsand reductiveto produce purple
compounds
.the color of the compound is directly related to the amino acid content, the amino acid content can be determined by measuring the light density at 570nm.
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. Third,
reagents
materials(1) standard amino acid solution: with 0.3mmol/L solution.
(2) pH5.4, 2mol/L acetic acid buffer: take 86mL 2mol/L sodium acetate solution, add 14mL 2mol/L acetic acid mixed. Check the correction with pH.
(3)tritone coloring solution: said to take 85 mg throme and 15 mg reduction tritone, dissolved with 10mL glycol methylene ether.
if the three ketones become slightly red, you need to press the method to recrystase: 5gtritones dissolved in 15 to 25mL hot distilled water, add 0.25g activated carbon, gently stirred.
heating
30min and then
hot
, the filter is placed in the fridge overnight. The next day out of yellow and white crystallization, filtration, with 1mL cold water washing crystallization,
drying
drying, loaded into a brown glass bottle for preservation.
also prototypetritones press the legal preparation: said to take 5gtritones, with 125mL boiling distilled water dissolved, get yellow solution.
5g vitamin C is dissolved with 250mL of warm distilled water, stirring while the vitamin C solution droplets are added to thetritone solution, and precipitation continues to occur. After titration, continue to stir 15min, and then cooled in the refrigerator to 4 degrees C, filtration, precipitation with cold water washing 3 times, in the phosphorus phosphate vacuum dryer dryer drying, spare. If the glycol methylene ether is placed for too long, the following method should be used to remove the oxide: in 500mL glycol methylene ether added 5g of iron sulfate, oscillating 1 to 2h, filtered to remove iron sulfate, and then distilled, collected boiling point of 121 to 125 degrees C of distillation, for colorless transparent ethyl glycol methylene.
(4) 60% ethanol.
(5) sample fluid: 0.5 to 50 μg amino acid per milliliter.
(6)
a 100-dlight
.
(7) water bath pot.
, the operation steps . 1. The standard curve is
to take 0.3mmol/L of standard amino acid solution 0,0.2, 0.4, 0.6, 0.8, 1.0mL in
test tube
, water to 1mL. Each add 1mL pH5.4, 2mol/L acetate buffer, then add 1mL tritone color solution, mix well, cover the test tube mouth, heat 15min in a 100C water bath, cool with tap water. After placing 5min, add 3mL60% ethanol dilution, shake well, and measure OD570nm with a terrolight meter. (Proline and hydroxy proline react yellow and OD440nm should be determined.)
is based on OD570nm as the ordinate and the amino acid content as the horizontal coordinate, and the standard curve is drawn.
2. The determination of amino acid samples
take sample fluid 1mL, add pH5.4, 2mol/L acetate buffer 1mL and tritone coloring solution 1mL, mix and heat 15min in a boiling water bath at 100 O, tap water cooling. After placing 5min, add 3mL 60% ethanol dilution and shake to determine OD570nm (the resulting color is stable within 60min).
the OD570nm measured in the sample with the standard curve to determine the amino acid content in the sample. .