Contents
Why Measure Peptide Concentration?
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:
- Apply the Beer-Lambert Law to convert absorbance measurements into concentrations with proper units
- Determine when to use the 280 nm method versus the 214 nm method
- Explain how to calculate extinction coefficients from peptide sequences
The Beer-Lambert Law
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.
Beer-Lambert Law Formula:
Figure 1: The Beer-Lambert Law in practice. Light at 280nm passes through a 1 cm cuvette containing 100 µM peptide solution with one Trp residue. The sample absorbs 72% of incident light (A = 0.55), producing the simulated characteristic UV absorbance spectrum shown on the right.
Understanding Each Variable
Rearranging for Concentration
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:
With l = 1 cm:
c (M) = A / ε
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.
280 nm Method: Aromatic Amino Acids
The 280 nm 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.
Why 280 nm?
The choice of 280 nm 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.
Figure 2: Aromatic amino acids and their UV absorption maxima. Only Trp and Tyr absorb significantly at 280 nm due to their extended aromatic ring systems (highlighted in blue/purple).
Calculating ε₂₈₀ from Sequence
The beauty of the 280 nm method is that you can predict ε₂₈₀ directly from your peptide sequence using the following formula:
This calculation method was established by Gill & von Hippel (1989),1 with improved extinction coefficient values determined by Pace et al. (1995)2 for native proteins in water. The values used here (Trp: 5,500 M⁻¹cm⁻¹, Tyr: 1,490 M⁻¹cm⁻¹, Cystine: 125 M⁻¹cm⁻¹) represent the most accurate estimates for peptides and proteins under native conditions.
| Amino Acid | Three-Letter | One-Letter | ε₂₈₀ (M⁻¹cm⁻¹) |
|---|---|---|---|
| Tryptophan | Trp | W | 5,500 |
| Tyrosine | Tyr | Y | 1,490 |
| Cystine | Cys-Cys | C-C | 125 |
| Phenylalanine | Phe | F | ~0 |
Although phenylalanine has an aromatic ring with absorption maximum at 257 nm (ε ≈ 197 M⁻¹cm⁻¹), it absorbs essentially zero at 280 nm. This is why only Trp (5,500 M⁻¹cm⁻¹) and Tyr (1,490 M⁻¹cm⁻¹) are counted in the 280 nm method. For peptides lacking Trp/Tyr, use the 214 nm 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 280 nm 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.
Practical Example: Melittin
Let's calculate ε₂₈₀ for melittin, the principal active component of bee venom and a classic example in peptide chemistry:
Melittin: GIGAVLKVLTTGLPALISWIKRKRQQ
• Tryptophan (W): 1
• Tyrosine (Y): 0
• Cysteine (C): 0 (no disulfides)
ε₂₈₀ = (1 × 5,500) + (0 × 1,490) + (0 × 125)
ε₂₈₀ = 5,500 M⁻¹cm⁻¹
If measured absorbance A₂₈₀ = 0.55:
c = 0.55 / 5,500 = 0.0001 M = 100 μM
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
214 nm Method: Peptide Bonds
At 214 nm, we're detecting the π→π* transition of the peptide bond itself. This makes the 214 nm method universal - it works for ALL peptides, regardless of their amino acid composition. However, it's less specific than the 280 nm method and requires more careful technique.
The Peptide Bond Chromophore
Every peptide bond absorbs light at 214 nm 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.
Amino Acid Contributions at 214 nm
While all peptides absorb at 214 nm 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!
Complete Formula for ε₂₁₄
Where n = number of residues
(n-1) = number of peptide bonds
Example for GIGAVLKVLTTGLPALISWIKRKRQQ (26 residues):
= 25×923 + 1×29,050 + other amino acids
≈ 64,321 M⁻¹cm⁻¹
Academic Reference
The amino acid-specific extinction coefficients at 214nm used in this guide are from Kuipers & Gruppen (2007),4 who systematically measured the molar extinction coefficients of all 20 standard amino acids and the peptide bond at 214nm in the presence of acetonitrile and formic acid (typical RP-HPLC conditions).
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
Interactive Calculators
🧮 Calculator 1: Extinction Coefficient from Sequence
Enter your peptide sequence to automatically calculate ε₂₈₀:
🧮 Calculator 2: Concentration from Absorbance
Convert measured absorbance to concentration:
Worked Examples
Example 1: Melittin (Simple Case)
• Sequence: GIGAVLKVLTTGLPALISWIKRKRQQ (26 residues)
• Molecular Weight: 2,845.7 g/mol
• Measured A₂₈₀: 0.55
• Path length: 1 cm (standard cuvette)
Count aromatic residues: 1 Trp, 0 Tyr, 0 disulfides
ε₂₈₀ = (1 × 5,500) + (0 × 1,490) + (0 × 125) = 5,500 M⁻¹cm⁻¹
c = A / ε = 0.55 / 5,500 = 0.0001 M = 100 μM
c (mg/mL) = c (M) × MW (g/mol) × 1000 (mg/g) / 1000 (mL/L)
c (mg/mL) = 0.0001 × 2,845.7 = 0.285 mg/mL
Example 2: Aromatic-Rich Peptide
• Sequence: WYWYKY (6 residues)
• Measured A₂₈₀: 1.20
• Molecular Weight: 1007.5 g/mol
Count: 2 Trp, 2 Tyr, 0 disulfides
ε₂₈₀ = (2 × 5,500) + (2 × 1,490) = 11,000 + 2,980 = 13,980 M⁻¹cm⁻¹
c = 1.20 / 13,980 = 0.0000858 M = 85.8 μM
c = 0.0000858 × 1007.5 = 0.0864 mg/mL
Example 3: Peptide with Disulfide Bonds
• Sequence: CYIQWCY (7 residues)
• Contains 1 disulfide bond (Cys1-Cys7)
• Measured A₂₈₀: 0.68
Count: 1 Trp, 1 Tyr, 1 disulfide bond
ε₂₈₀ = (1 × 5,500) + (1 × 1,490) + (1 × 125) = 7,115 M⁻¹cm⁻¹
c = 0.68 / 7,115 = 0.0000956 M = 96 μM
Example 4: When to Use 214 nm Instead
• Sequence: AAAKAAAK (8 residues - no Trp or Tyr)
• Attempted measurement at A₂₈₀: 0.02 (very low signal)
ε₂₈₀ = 0 M⁻¹cm⁻¹ (no Trp or Tyr)
Cannot use 280 nm method!
Switch to 214 nm method where peptide bonds provide strong signal.
Approximate ε₂₁₄ for this sequence ≈ 8,600 M⁻¹cm⁻¹
(7 peptide bonds × 923 + amino acid side chain contributions)
Example 5: Using 214 nm Method
• Sequence: AAPAAKAPA (9 residues with proline)
• Measured A₂₁₄: 0.750
• Molecular Weight: 766.4 g/mol
• Peptide bonds: 8 × 923 = 7,384
• Proline at position 3 (not N-terminal): 1 × 2,675 = 2,675
• Proline at position 8 (not N-terminal): 1 × 2,675 = 2,675
• Alanine: 5 × 32 = 160
• Lysine: 2 × 41 = 82
Total ε₂₁₄ ≈ 12,976 M⁻¹cm⁻¹
c = 0.750 / 12,976 = 0.0000578 M = 57.8 μM
c = 0.0000578 × 766.4 = 0.0443 mg/mL
Troubleshooting & Tips
Common Problems and Solutions
❌ Problem: Absorbance Too High (>1.0)
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.
❌ Problem: Absorbance Too Low (<0.1)
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 280 nm: Switch to 214 nm method if no aromatic amino acids
• For 214 nm: Check for buffer interference
❌ Problem: Negative or Strange Absorbance Values
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
❌ Problem: Inconsistent Measurements
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
Best Practices
• At 280 nm: Most common buffers (PBS, Tris, HEPES) are fine
• At 214 nm: 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 280 nm (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 (250-300 nm) to check for impurities
• Verify peptide identity with mass spectrometry when possible
• Document all conditions (pH, buffer, temperature)
Decision Tree: Which Method to Use?
🌳 Quick Decision Guide
START HERE: Does your peptide have Trp or Tyr?
↓ YES → Use 280 nm method (specific, accurate, easy)
↓ NO → Use 214 nm method (universal, works for all peptides)
Special applications:
• HPLC monitoring → Always use 214 nm (universal detection)
• Peptide libraries → Use 214 nm (works for all sequences)
• Aromatic-rich peptides → 280 nm is best (minimal interference)
🚀 Coming Soon: PepDraw Pro Features
Upgrade to PepDraw Pro for advanced UV-Vis spectroscopy tools
Sequence-specific extinction coefficient prediction with all 22 amino acids
Automatic handling of special cases like N-terminal Pro
Detailed contribution analysis showing which residues dominate absorption
Database of buffer interference at 214 nm with recommendations
Upload CSV of sequences and get concentration data for entire libraries