Sample preparation of amino acid estimation

For estimating the amino acid composition of foodstuff, feed or any protein, it has to be first hydrolyzed. The free amino acids may be extracted from the tissues or foods in ethanol. The amino acids may be determined by chromatography techniques or in an amino acids analyzer. If the extract sample contains higher amounts of salts and/or sugars they have to be removed before estimation of amino acids.

Content
A. Free Amino Acids
B. Acid Hydrolysis of Protein
C. Deproteinization
D. Desalting
E. Hydrolysis of Large Samples

   A. Free Amino Acids

Materials

» Ethanol
» 0.01N HCl

Procedure

1. Weigh accurately sufficient quantity of the sample which should have 2 to 6m moles of each amino acids.
2. Extract the warm (60°C) 70% ethanol or 0.01M phosphate buffer pH 7.0, three to six times. Use extractant five times the weight of the sample for each extraction.
3. Pool the extract after filtration or centrifugation and evaporate in a rotary vacuum evaporator to dryness.
4. Take the residue in 1-10mL of 0.01N HCl or in a suitable sample diluting buffer.

  B. Acid Hydrolysis of Protein
Material
» 6N HCl
» 0.01N HCl

Procedure

1. Weigh accurately sufficient quantity of the sample containing above 10mg protein in a thick-walled heavy glass tube having a constriction at 7.0cm from top.
2. Add 5mL of 6N HCl and place the tube in a liquid nitrogen.
3. Evacuate the frozen sample to 0.01mm Hg.
4. Thaw the sample by shaking the slight warning to allow any dissolved air bubble out.
5. Freeze the contents and evacuate to 0.005mm Hg again.
6. Seal the tube at the constriction using a flame till the tube is under evacuation.
7. Place the tube in a hot air oven at 110 ±1°C for 22h to hydrolyze protein
8. Break open the seal and evaporate the contents in a rotary evaporator to remove hydrochloric acid.
9. Add water and repeat evaporation two times.
10. Take the residue in 1-10mL 0.01N HCl or 50mm citrate buffer (pH 2.2)

Note
Some amino acids may be degraded partly or completely during acid hydrolysis. Glutamine and asparagines are converted to their respective acids. Cysteine may be converted to cysteic acid. Serine and threonine are very rapidly hydrolyzed from the protein and are lost to some extent. Tryptophan is also partially destroyed during acid hydrolysis.



  C. Deproteinization

Materials

» 1% Picric acid
» Dowex 1 x 8 Cl^- Resin
» 0.02N HCl
» 0.01N HCl

Procedure

1. Add 50mL of 1% picric acid to 10mL of the sample.
2. Centrifuge at 3,000rpm for 10min and reserve the supernatant.
3. Place the supernatant in a glass column (2 x 10cm) containing Dowex 1 x 8 Cl^- resin to a height of 3cm.
4. Immediately after the sample sinks into the resin wash the sides of the column with 3mL of 0.02N HCl.
5. Repeat washing five times.
6. Collect all the eluate and evaporate it to dryness.
7. Take the residue in 0.01N HCl.

Notes

Centrifugation of the sample for 30min at 2,000rpm is a better method for deproteinization. The ultra filtrate may be used for analysis. Picric acid does remove some amino acids.

  D. Desalting

Materials

» Dower 5 x 8 (H⁺) or amberlite CG-120 (Na⁺)
» 2N NH4OH
» 0.01N HCl

Procedure

1. Load 10-20mL of sample over the column of resin (2 x 10cm) and allow it to be absorbed by the resin.
2. Elute the amino acids with ammonium hydroxide.
3. Collect the eluate (about 100mL) and evaporate to remove ammonia.
4. Wash the residue twice with water and repeat evaporation.
5. Take the amino acid residue in 0.01N HCl.

Note
This procedure removes sugar impurities.

E. Hydrolysis of Large Samples
Feeds and foods being highly hetrogenous, it is advisable to take larger samples to get better results.

Procedure

1. Weigh 2g of sample into a two liter round bottom flask.
2. Add 900mL of 6N HCl.
3. Pass nitrogen gas (oxygen-free) for 30min.
4. Reflux for 20h in an oil bath at 137°C with continuous passage of nitrogen.
5. Filter through suction and dilute to one liter with water.
6. Evaporate an aliquot of it (containing about 25mg protein) in a rotary evaporator.
7. Wash with water twice and evaporate to dryness.
8. Take the residue in 0.01N HCl for analysis.