1.1. Concentrated hydrochloric acid (GR)
1.2. 6 mol/L Hydrochloric acid: Concentrated hydrochloric acid mixed with water in the volume ratio 1:1.
1.3.Phenol: need to be re-distilled.
1.4.(0.0025 mol/L) mixed Amino Acid standard solution (Sold by the equipment manufacturing company)
1.5.Buffer
1.5.1. Sodium citrate buffer with pH2.2: Weigh 19.6g sodium citrate(Na3C6H5O7•2H2O) and 16.5mL concentrated Hydrochloric acid, dilute with water to 1000mL, adjust pH to 2.2 using concentrated Hydrochloric acid, or 500g / L of Sodium Hydroxide solution.
1.5.2. Sodium citrate buffer with pH3.3: Weigh 19.6g Sodium Citrate and 12mL concentrated Hydrochloric acid, dilute with water to 1000mL, adjust Ph to 3.3 by using concentrated Hydrochloric acid or 500g/L Sodium Hydroxide solution.
1.5.3.Sodium citrate buffer with pH4.0: Weigh 19.6g sodium citrate and 9mL concentrated Hydrochloric acid, dilute with water to 1000mL, adjust ph to 4.0 by using concentrated Hydrochloric acid or 500g/L Sodium Hydroxide solution.
1.5.4.Sodium citrate buffer with pH3.3: Weigh 19.6g sodium citrate and 46.8g NaCl (GR), dilute with water to 1000ml, adjust ph to 6.4 by using concentrated Hydrochloric acid or 500g/L Sodium Hydroxide solution.
1.6. Ninhydrin solution
1.6.1.Solution of Lithium acetate dihydrate with pH5.2: Weigh 168g Lithium hydroxide(LiOH•H2O), then adding 279ml glacial acetic acid (GR), dilute with water to 1000mL, adjust ph to 5.2 by concentrated Hydrochloric Acid or 500g/L Sodium Hydroxide solution.
1.6.2. Ninhydrin solution: take 150mL Dimethyl sulfoxide (C2H6OS) and 50mL Lithium acetate dihydrate solution, add 4g (C9H4O3•H2O) and 0.12g Restore Ninhydrin(C18H10O6•2H2O), stir it until completely dissolved.
1.7.High-purity nitrogen: Purity 99.99%
1.8.Refrigerant: mix the salt sold in market with ice in mass ratio 1:3.
2. Instruments and equipment
2.1 Vacuum Pump
2.2 Ovens with constant temperature.
2.3 Hydrolysis tube: pressure resistant glass tube with screw cap or hard glass tube, volume 20ml – 30ml, rinse and dry by Deionized water.
2.4.Vacuum Dryer(Temperature can be adjusted)
2.5 Amino Acid Analyzer
3. Sample handling
Homogenized the samples by homogenizer (or try to crush samples completely), store in the low-temperature refrigerator, unfreeze samples when analyzing.
4. Analysis Steps
4.1 Weigh Sample
Take a certain amount of uniform sample, such as milk, etc., accurate to 0.0001g(the sample protein content within 10mg ~ 20mg; sample in poor uniformity, such as meat, etc. in order to reduce the error, increase the sample weight properly, dilute again before measurement. Put the sample into Hydrolisis tube.
4.2 Hydrolysis
4.2.1. Take 6mol / L hydrochloric acid 10ml ~ 15ml (depending on protein content in the
sample) into the hydrolysis tube, add equal volume of concentrated Hydrochloric Acid into the sample of high water content (such as milk), add newly distilled Phenol of 3 – 4 drops, put the Hydrolysis tube in refrigerant, freeze for 3 – 5 min, connect to the exhaust pipe of vacuum pump, vacuum ( close to 0 Pa), inflate high purity nitrogen; vacuum and inflate nitrogen, repeat 3 times, seal the screw cap close when inflating nitrogen, put the Hydrolysis tube in the costant temperature oven at 110 ℃ ± 1 ℃, hydrolyze for 22 hours, draw out and cooling.
Open the Hydrolysis tube, filter the hydrolysis solution, rinse the hydrolysis tube by deionized water. Remove the hydrolysis solution into 50ml volumetric flask, dilute with deionized water to 50ml. Draw filtrate of 1ml into 5ml volumetric flask, dry at 40℃~ 50℃ by vacuum dryer,dissolve the residues in water of 1-2ml, and dry, repeat twice, then dry completely, dissolve with 1ml pH 2.2 buffer, keep and for use of instrumental determination
5. Determination
draw exactly 0.200ml standard mixed Amino Acid, dilute with pH2.2 buffer to 5ml, the concentration of this standard dilution solution is 5.00 nmol/50μL, use it as the criterion of amino acid determination when on the instruments. Determine the amino acid content of sample solution using the External Standard Method by Amino Acid analyzer.
6. Calculation
Calculate as the expression as below:
Thereinto:
X ---- Amino acid content in sample, unit as g/100g;
C ---- Amino acid content in sample solution, unit as nmol/50μL;
F ----- Dilute multiple of sample;
V ----- Dilution volume after hydrolysis, unit as ml;
M ---- Molecular weight of Amino acid;
m ---- Mass of sample, unit as g;
1/50 ---- Amino acid content of per milliliter sample, unite asμmol/L;
109 ------ Coefficient of convertion of sample content from ng to g.
The molecular weight of the 16 amino acids: Aspartate 133.1, Threonine 119.1, Serine 105.1, Glutamate 147.1, Proline 115.1, Glycine 75.1, Gly Ala 89.1, Valine 117.2, Methionine 149.2, Isoleucine 131.2, Leucine 131.2, Tyrosine 181.1, Phenylalanine 165.2, Histidine 155.2, Lysine 146.2, Arginine 174.2.