Amino Acid Analysis

Shane M. Rutherfurd1, G. Sarwar Gilani2

1 Riddet Institute, Massey University, Palmerston North, New Zealand, 2 Health Canada, Nutrition Research Division, Food Directorate, Health Products and Food Branch, Ottawa, Ontario, Canada
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 11.9
DOI:  10.1002/0471140864.ps1109s58
Online Posting Date:  November, 2009
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Amino acid analysis is used to determine the amino acid content of amino acid–, peptide‐ and protein‐containing samples. With minor exceptions, proteins are long linear polymers of amino acids connected to each other via peptide bonds. The first step of amino acid analysis involves hydrolyzing these peptide bonds. The liberated amino acids are then separated, detected, and quantified. The method was first developed by Moore, Stein and coworkers in the 1950s using HCl acid hydrolysis, and, despite considerable effort by many workers, the basic methodology remains relatively unchanged. This unit provides an overview and strategic planning for amino acid analysis, discussing a range of methodologies and issues. In addition, several common methods used for analysis of L‐amino acids are described in detail, including: HCl acid hydrolysis, performic acid oxidation for methionine and cysteine analysis, base hydrolysis for tryptophan analysis, analysis of free amino acids, and analysis of reactive lysine. Curr. Protoc. Protein Sci. 58:11.9.1‐11.9.37. © 2009 by John Wiley & Sons, Inc.

Keywords: amino acids; hydrolysis; derivatization; chromatography

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Acid Hydrolysis of Proteins and Peptides for Amino Acid Analysis
  • Basic Protocol 2: Performic Acid Oxidation of Proteins for Cysteine and Methionine Analysis
  • Basic Protocol 3: Base (LiOH) Hydrolysis of Proteins for Tryptophan Analysis
  • Basic Protocol 4: Analysis of Free Amino Acids by HPLC
  • Basic Protocol 5: Analysis of Reactive Lysine Content in Food Samples
  • Support Protocol 1: Preparation of Samples to be Subjected to Amino Acid Analysis
  • Reagents and Solutions
  • Literature Cited
  • Figures
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Basic Protocol 1: Acid Hydrolysis of Proteins and Peptides for Amino Acid Analysis

  • Sample (see protocol 6)
  • Constant boiling 6 M HCl containing 0.1% phenol (see recipe)
  • Internal standard (norleucine) solution (see recipe)
  • Buffer suitable for downstream applications
  • Hydrolysis tubes: 100 × 12–mm Schott Duran rimless test tubes (
  • Glass blowing torch (Wale Apparatus;; if the laboratory has a methane or natural gas tap, then use this and bottled oxygen to provide the fuel mix)
  • Tweezers
  • Chemically resistant, oil‐free PTFE diaphragm pump with a vacuum controller and vacuum gauge (e.g., Buchi V‐700 pump with V‐850 controller)
  • Rubber vacuum hose to attach tube to vacuum pump
  • 110°C forced air oven
  • Low‐speed centrifuge (capable of 2600 × g) with a rotor adapter that will accommodate 100 mm × 12 mm glass tubes
  • Glass‐etching pen
  • Glass rod
  • Savant Speedvac (centrifugal concentrator) or equivalent that will accommodate 100 mm × 12 mm glass tubes, resistant to acid
  • Sonication bath
  • 0.22‐µm syringe filters (13‐mm diameter)
  • 1‐ml luer‐lock syringes
  • HPLC vials (2 ml or 4 ml depending on the HPLC autosampler)
  • Vortex mixer
  • Additional reagents and equipment to prepare samples for amino acid analysis (see protocol 6)

Basic Protocol 2: Performic Acid Oxidation of Proteins for Cysteine and Methionine Analysis

  • Sample (see protocol 6)
  • Performic acid (see recipe), freshly prepared and ice cold
  • 48% hydrobromic acid, ice‐cold
  • Savant Speedvac (centrifugal concentrator) or equivalent that will accommodate 100‐mm × 12‐mm glass tubes, resistant to formic acid
  • Additional reagents and equipment to prepare samples for amino acid analysis ( protocol 6) and hydrolysis of proteins/peptides for amino acid analysis ( protocol 1)

Basic Protocol 3: Base (LiOH) Hydrolysis of Proteins for Tryptophan Analysis

  • Sample (see protocol 6)
  • Lysozyme from chicken egg white (Sigma, cat. no. L 7651)
  • 4.3 M lithium hydroxide (see recipe)
  • Internal standard (5‐methyltryptophan) solution (see recipe)
  • Constant boiling 6 M HCl containing 0.1% phenol (see recipe)
  • HPLC buffer for LiOH hydrolysis (see recipe)
  • 30‐ml screw‐cap Teflon containers (Nalge, cat. no. DS1630‐0001), thoroughly cleaned and rinsed with 18 MΩ deionized water.
  • Balance (accurate to 5 decimal places)
  • Nitrogen cylinder equipped with a flexible tube to which a Pasteur pipet can be attached
  • 110°C forced‐air oven
  • 25‐ml volumetric flask
  • 0.22‐µm syringe filters (13‐mm diameter)
  • 1‐ml luer‐lock syringes
  • HPLC vials (2‐ml or 4‐ml depending on the HPLC autosampler)
  • HPLC system with column heater
  • C 8 column (4.6 × 150 mm)
  • Peak integration software for HPLC

Basic Protocol 4: Analysis of Free Amino Acids by HPLC

  • Sample (see protocol 6)
  • Internal standard (norleucine) solution (see recipe)
  • 2× HPLC loading buffer (see recipe)
  • Refrigerated microcentrifuge
  • Microcon tube (MWCO, 3000 Da; Millipore) or similar centrifugal ultrafiltration device
  • HPLC vials (2 ml or 4 ml depending on the HPLC autosampler)
  • Additional reagents and equipment for pre‐column derivitization (see )

Basic Protocol 5: Analysis of Reactive Lysine Content in Food Samples

  • Sample (see protocol 6)
  • 0.6 M OMIU (see recipe)
  • 20°C shaking water bath
  • Savant Speedvac (centrifugal concentrator) or equivalent that will accommodate 100‐mm × 12‐mm glass tubes
  • Additional reagents and equipment to prepare samples for amino acid analysis ( protocol 6) and hydrolysis of proteins/peptides for amino acid analysis ( protocol 1)

Support Protocol 1: Preparation of Samples to be Subjected to Amino Acid Analysis

  • Sample for analysis
  • Hydrolysis tubes: 100 × 12–mm Schott Duran rimless test tubes (
  • Muffle furnace (capable of attaining 500°C)
  • Freeze dryer
  • Grinder with mesh pore size ≤1 mm
  • Balance (accurate to 5 decimal places)
  • Savant Speedvac (centrifugal concentrator) or equivalent that will accommodate 100 × 12–mm glass tubes
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  •   FigureFigure 11.9.1 Derivatization reactions for amino acid analysis.
  •   FigureFigure 11.9.2 Stretching the neck of the hydrolysis tube prior to degassing and sealing.
  •   FigureFigure 11.9.3 Degassing the hydrolysis tube using a vacuum pump.
  •   FigureFigure 11.9.4 Apparatus for the preparation of constant‐boiling HCl.


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