Assays for free amino groups (–NH₂) are used to quantify amino acids, peptides, proteins, or other molecules containing primary or secondary amines. Here’s a post on the main methods used, grouped by type and with key details:
1. Ninhydrin Assay
Principle:
Ninhydrin reacts with free α-amino groups to produce Ruhemann’s purple, a deep blue–violet chromophore measurable at 570 nm.
Reaction:
Amino acid + Ninhydrin → Ruhemann’s purple + CO₂ + Aldehyde + NH₃
Applications:
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Detection and quantification of free amino acids or peptides. It will also cover proline too which many other assays fail to measure.
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Used in chromatography (e.g., amino acid analyzers).
Notes:
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Proline and hydroxyproline give a yellow product instead of purple.
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Sensitive down to low microgram levels.
2. Trinitrobenzene Sulfonic Acid (TNBS) Assay
Principle:
TNBS reacts with primary amines to form trinitrophenyl (TNP) derivatives, giving a yellow colour measurable at 335–345 nm.
Reaction:
R–NH₂ + TNBS → R–NH–TNP
Applications:
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Quantifies free amino groups in peptides and proteins.
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Commonly used to measure degree of modification or cross-linking (e.g., in gelatin or chitosan).
Notes:
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Works well in aqueous solutions at mild alkaline pH (≈8.5).
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Interference possible from other nucleophiles.
3. Fluorescamine Assay
Principle:
Fluorescamine reacts rapidly with primary amines to form highly fluorescent products (excitation ≈ 390 nm, emission ≈ 475 nm).
Applications:
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Very sensitive detection of low levels of amines (nanomolar range).
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Used for protein and peptide labeling and quantification.
Notes:
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Reaction is almost instantaneous at room temperature.
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Reagent itself is nonfluorescent (advantageous for sensitivity).
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Requires organic solvents (e.g., acetone or acetonitrile).
4. O-Phthaldialdehyde (OPA) Assay
Principle:
OPA reacts with primary amines in the presence of a thiol (e.g., β-mercaptoethanol or N-acetylcysteine) to form a fluorescent isoindole derivative (excitation ≈ 340 nm, emission ≈ 455 nm).
Applications:
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Quantitative detection of amino acids and peptides.
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Used in amino acid analyzers and HPLC derivatization.
Notes:
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Secondary amines (e.g., proline) do not react.
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Rapid and sensitive.
5. Dansyl Chloride (DNS-Cl) Assay
Principle:
Dansyl chloride reacts with amino groups to produce fluorescent dansyl derivatives (excitation ≈ 340 nm, emission ≈ 520 nm).
Applications:
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Useful for derivatization prior to chromatographic analysis.
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Enables quantification and identification of amino compounds.
Notes:
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Requires anhydrous or mildly basic conditions.
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Reaction mixtures often purified before measurement.
6. Alternative or Specialized Methods
| Method | Principle / Detection | Notes |
|---|---|---|
| FDNB (Sanger’s reagent) | Formation of dinitrophenyl (DNP) derivatives; UV absorbance ~360 nm | Classic method for identifying N-terminal amino acids |
| Isothiocyanate reagents (e.g., PITC) | Derivatization to phenylthiocarbamyl derivatives for HPLC | Used in Edman degradation |
| TNBSA (2,4,6-Trinitrobenzene Sulfonic Acid) | Similar to TNBS but more water-soluble | Used for proteins and biopolymers |
| Sulfosuccinimidyl esters (NHS esters) | React with primary amines; fluorescence or absorbance tagging | Useful for labeling lysine residues |
Summary Table
| Assay | Detection Mode | Sensitivity | Primary Use |
|---|---|---|---|
| Ninhydrin | colourimetric (570 nm) | µg range | Free amino acids |
| TNBS/TNBSA | colourimetric (335–345 nm) | µg range | Free amino groups in proteins/polymers |
| Fluorescamine | Fluorescence | ng range | Very low amine levels |
| OPA | Fluorescence | ng range | Amino acids, peptides |
| Dansyl chloride | Fluorescence | ng range | Derivatization for HPLC |
| FDNB/PITC | colourimetric or HPLC | µg–ng range | N-terminal analysis |
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