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
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Use dedicated or disposable sample handling devices (glassware, pipeting devices, etc.)
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Store everything that contacts the sample covered or in a sealed container to prevent the inclusion of dust in the sample.
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Wear gloves (powder free) when dealing with small amounts of samples.
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Use high purity reagents.
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Maintain dedicated or clean equipment (centrifuges, lyophilizers, etc. ).
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Maintain a clean dust free sample handing environment.
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Carefully choose the buffers and detergents used in the isolation and purification procedures to minimize the effect on the derivatization chemistry.
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Open and close all sample vials carefully.
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Do not leave the sample vial open for longer than necessary for aliquoting
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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
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Additive
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Effects and % Recovery
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Ammonium acetate
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No negative effect
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Sodium acetate
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His and Cys~50%; Ile, Leu, Phe and Lys ~80%
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Triethylammonium acetate
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His, Thr and Phe ~90%
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Ammonium bicarbonate
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Thr ~90%
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Sodium bicarbonate
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Met ~40%; His, Tyr ~60%; Ile, Leu, Phe and Lys ~80%
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Sodium borate
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No negative effect on result
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Sodium chloride
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No negative effect on result
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Sodium phosphate
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Very low and variable yields of most amino acids
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Triethylammonium phosphate
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No negative effect on result
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CAPS
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Very large late eluting peak obscures Phe, Lys
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HEPES
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Gives artifact peak which co-elutes with Met
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TRIS
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His ~60%, artifact peak co-elutes with Tyr
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SDS
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His and Thr ~90%, Cys and Lys ~110%
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Triton X-100
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His and Thr ~90%, Cys and Lys ~110%
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