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Sample Considerations for Amino Acid Analysis

Adequate sample purification is essential for accurate compositions and quantitative data. Unfortunately, even the purification protocols used can contribute both to sample contamination and loss. There are numerous factors to consider when trying to prevent sample contamination. Reagent or solvent solutions, glassware, pipetting devices, dust, fingers or anything else that comes in contact with the sample may introduce background free amino acids or proteins. The entire sample-handling environment must be scrupulously clean for high sensitivity analysis.

Tips to help reduce Amino Acid Sample Contamination

  • Use dedicated or disposable sample handling devices (glassware, pipetting devices, etc.).
  • Store everything that contacts the sample covered or in a sealed container to prevent the inclusion of dust in the sample.
  • Wear gloves (powder free) when dealing with small amounts of samples.
  • Use high purity reagents.
  • Maintain dedicated or clean equipment (centrifuges, lyophilizers, etc.).
  • Maintain a clean dust free sample handing environment.
  • Carefully choose the buffers and detergents used in the isolation and purification procedures to minimize the effect on the derivatization chemistry.
  • Open and close all sample vials carefully.
  • Do not leave the sample vial open for longer than necessary for aliquoting.
  • Avoid unnecessary opening of the sample vial and remove aliquots for multiple procedures at one time.

Samples Submitted for Amino Acid Analysis

Ideally, samples submitted for amino acid analysis should be free of salts, buffers, amino-containing substances, trace metals, and detergents for accurate results. Realistically, it is often impractical or impossible to completely desalt small amounts of sample without risking significant sample loss. The analyzer can produce reproducible, accurate analyses on a wide variety of samples when compatible buffers, salts or detergents are used. Below is a list or common buffers, detergents and salts along with the effect each has on the analysis:

Effects of Common Buffers, Salts and Detergents

Additive

Effects and %Recovery

Ammonium acetate No negative effect
Sodium acetate His, Cys - 50%
Ile, Leu, Phe, Lys - 80%
Triethylammonium acetate His, Thr, Phe - 90%
Ammonium bicarbonate Thr - 90%
Sodium bicarbonate Met - 40%
His, Tyr - 60%
Ile, Leu, Phe, Lys - 80%
Sodium borate No negative effect on result
Sodium chloride No negative effect on result
Sodium phosphate Very low and variable yields of most amino acids
Triethylammonium phosphate No negative effect on result
CAPS Very large late eluting peak obscures Phe, Lys
HEPES Gives artifact peak which co-elutes with Met
TRIS His - 60% artifact peak co-elutes with Tyr
SDS His, Thr - 90%
Cys, Lys - 110%
Triton X-100 His, Thr - 90%
Cys, Lys - 110%


Taken From: Applied Biosystems 420/H Users Manual, Table 2-2

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