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Amino Acid Analysis requires adequate purified sample amounts for accurate composition and quantitative data analysis. 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, pipeting 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 and Cys~50%; Ile, Leu, Phe and Lys ~80%
Triethylammonium acetate
His, Thr and Phe ~90%
Ammonium bicarbonate
Thr ~90%
Sodium bicarbonate
Met ~40%; His, Tyr ~60%; Ile, Leu, Phe and Lys ~80%
Sodium borate
No negative effect on result
Sodium chloride
Sodium phosphate
Very low and variable yields of most amino acids
Triethylammonium phosphate
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 and Thr ~90%, Cys and Lys ~110%
Triton X-100
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