Learn how to accurately determine peptide concentration using ultraviolet-visible light absorption
Imagine you've just synthesized a new peptide in the lab. Before you can run biological assays, test its activity, or publish your results, you need to answer one fundamental question: How much peptide do I actually have?
Accurate concentration determination is essential for:
By the end of this guide, you will be able to:
At the heart of UV-Vis spectroscopy is the Beer-Lambert Law, which describes the relationship between light absorption and concentration. When light passes through a solution, some wavelengths are absorbed by the molecules in solution. The amount of light absorbed is directly proportional to the concentration and the distance the light travels through the sample.
Since we measure absorbance (A) and know the path length (l = 1 cm for standard cuvettes), we can rearrange the equation to solve for concentration:
For best results, aim for absorbance values between 0.1 and 1.0. If your absorbance is too high (>1.0), dilute your sample. If too low (<0.1), use a longer path length cuvette or concentrate your sample. This ensures you're working in the linear range of the spectrophotometer.
Remember that ε is in M⁻¹cm⁻¹, not L/(mol·cm). These are equivalent but M⁻¹cm⁻¹ is the standard notation. When you calculate c, you get molar concentration (M), which you can convert to mg/mL by multiplying by your peptide's molecular weight in g/mol and dividing by 1000.
The 280nm method is the most commonly used approach for peptides containing aromatic amino acids. At this wavelength, tryptophan (Trp) and tyrosine (Tyr) strongly absorb UV light due to their aromatic ring structures, while other amino acids contribute minimally.
The choice of 280nm isn't arbitrary - it's the wavelength where aromatic amino acids show peak absorption while minimizing interference from peptide bonds and other chromophores. This makes it ideal for specific quantification of peptides containing Trp and/or Tyr.
The beauty of the 280nm method is that you can predict ε₂₈₀ directly from your peptide sequence using the following formula:
| Amino Acid | Three-Letter | One-Letter | ε₂₈₀ (M⁻¹cm⁻¹) | Notes |
|---|---|---|---|---|
| Tryptophan | Trp | W | 5,500 | Strongest absorber at 280nm |
| Tyrosine | Tyr | Y | 1,490 | Moderate absorption |
| Cystine | Cys-Cys | C-C | 125 | Only when forming disulfide bonds |
| Phenylalanine | Phe | F | ~0 | Absorbs at 257nm, not at 280nm |
Although phenylalanine has an aromatic ring with absorption maximum at 257nm (ε ≈ 197 M⁻¹cm⁻¹), it absorbs essentially zero at 280nm. This is why only Trp (5,500 M⁻¹cm⁻¹) and Tyr (1,490 M⁻¹cm⁻¹) are counted in the 280nm method. For peptides lacking Trp/Tyr, use the 214nm method instead where Phe contributes strongly (5,200 M⁻¹cm⁻¹).
Cysteine (Cys, C): The free amino acid with a thiol (-SH) group. Does not contribute
significantly to 280nm absorption.
Cystine: Formed when two cysteine residues create a disulfide bond (Cys-S-S-Cys).
Each disulfide bond contributes 125 M⁻¹cm⁻¹. If your peptide has 4 cysteines forming 2 disulfide
bonds, add 250 M⁻¹cm⁻¹ to ε₂₈₀.
Let's calculate ε₂₈₀ for melittin, the principal active component of bee venom and a classic example in peptide chemistry:
Best for: Peptides with at least one Trp or Tyr
Advantages: Fast, non-destructive, highly specific
Limitations: Cannot be used for peptides lacking aromatic amino acids
At 214nm, we're detecting the π→π* transition of the peptide bond itself. This makes the 214nm method universal - it works for ALL peptides, regardless of their amino acid composition. However, it's less specific than the 280nm method and requires more careful technique.
Every peptide bond absorbs light at 214nm due to the electronic transition in the C=O group. This means a 10-residue peptide (with 9 peptide bonds) will have a baseline absorption from these bonds alone, plus additional contributions from certain amino acid side chains.
While all peptides absorb at 214nm due to peptide bonds, different amino acids make additional contributions based on their side chain chromophores. The table below shows the complete breakdown:
| Amino Acid | Code | ε₂₁₄ (M⁻¹cm⁻¹) | Relative to Peptide Bond |
|---|---|---|---|
| Peptide Bond Baseline | - | 923 | 1× |
| Tryptophan | W | 29,050 | 31.5× (strongest) |
| Tyrosine | Y | 5,375 | 5.8× |
| Phenylalanine | F | 5,200 | 5.6× |
| Histidine | H | 5,125 | 5.6× |
| Proline (non-N-terminal) | P | 2,675 | 2.9× (see warning) |
| Proline (N-terminal) | P | 30 | 0.03× (special case!) |
| Methionine | M | 980 | 1.1× |
| Cysteine | C | 225 | 0.24× |
| Glutamine | Q | 142 | 0.15× |
| Asparagine | N | 136 | 0.15× |
| Arginine | R | 102 | 0.11× |
| Glutamic Acid | E | 78 | 0.08× |
| Aspartic Acid | D | 58 | 0.06× |
| Isoleucine | I | 45 | 0.05× |
| Leucine | L | 45 | 0.05× |
| Valine | V | 43 | 0.05× |
| Lysine | K | 41 | 0.04× |
| Threonine | T | 41 | 0.04× |
| Serine | S | 34 | 0.04× |
| Alanine | A | 32 | 0.03× |
| Glycine | G | 21 | 0.02× |
Proline at position 1 (N-terminus): ε = 30 M⁻¹cm⁻¹
Proline at any other position: ε = 2,675 M⁻¹cm⁻¹
This is an ~89-fold difference! When calculating ε₂₁₄ for Pro-rich peptides, you MUST account for
whether proline is at the N-terminus. For example:
• PAPAPA: First P uses 30, others use 2,675 each
• APAPAP: All P's use 2,675 (none at N-terminus)
Ignoring this can cause 25%+ errors in concentration calculations for Pro-rich sequences!
Kuipers, B.J.H. & Gruppen, H. (2007). "Prediction of molar extinction coefficients of proteins and peptides using UV absorption of the constituent amino acids at 214 nm to enable quantitative reverse phase high-performance liquid chromatography–mass spectrometry analysis." Journal of Agricultural and Food Chemistry, 55(14), 5445-5451.
Best for: Peptides lacking Trp/Tyr, HPLC monitoring, peptide libraries
Advantages: Universal (works for all peptides), good sensitivity
Limitations: Less specific, buffer interference, ±5-10% typical accuracy
Enter your peptide sequence to automatically calculate ε₂₈₀:
Convert measured absorbance to concentration:
Cause: Concentration is too high for accurate measurement
Solution: Dilute your sample 2-10 fold and remeasure. Multiply your final
concentration by the dilution factor. Example: If you dilute 1:10 and measure 0.50, your
original sample was 10× more concentrated.
Cause: Concentration is too low, or peptide lacks chromophores at this wavelength
Solution:
• Concentrate your sample if possible
• Use a longer path length cuvette (5 cm or 10 cm)
• For 280nm: Switch to 214nm method if no aromatic amino acids
• For 214nm: Check for buffer interference
Cause: Improper blanking, dirty cuvettes, or air bubbles
Solution:
• Blank with your exact buffer (same pH, salt concentration)
• Clean cuvettes thoroughly with ethanol and lint-free wipes
• Check for air bubbles - tap cuvette gently to release them
• Ensure sample and blank temperatures match
Cause: Temperature fluctuations, peptide aggregation, or instrument drift
Solution:
• Allow samples to equilibrate to room temperature
• Add 0.1% Tween-20 or similar detergent to prevent aggregation
• Run a standard protein (BSA) to verify instrument performance
• Ensure cuvette positioning is consistent
• At 280nm: Most common buffers (PBS, Tris, HEPES) are fine
• At 214nm: Avoid buffers with strong UV absorption (acetate, imidazole)
• Always blank with the same buffer used to dissolve your peptide
• pH can affect Tyr absorption at 280nm (pKa ≈ 10.1)
• Centrifuge samples to remove particulates (13,000 rpm, 5 min)
• Use fresh buffer - old buffers can develop contaminants
• Store peptide solutions at -20°C if not using immediately
• Avoid multiple freeze-thaw cycles when possible
• Measure in triplicate and report average ± standard deviation
• Run a wavelength scan (250-300nm) to check for impurities
• Verify peptide identity with mass spectrometry when possible
• Document all conditions (pH, buffer, temperature)
START HERE: Does your peptide have Trp or Tyr?
↓ YES → Use 280nm method (specific, accurate, easy)
↓ NO → Use 214nm method (universal, works for all peptides)
Special applications:
• HPLC monitoring → Always use 214nm (universal detection)
• Peptide libraries → Use 214nm (works for all sequences)
• Aromatic-rich peptides → 280nm is best (minimal interference)
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