Pharmaceutical Analysis
UV‑Vis Peak Analyzer
Checks nucleic-acid purity from the A260/A280 ratio and works out molar absorptivity (ε) from a peak's absorbance.
A260 / A280
1.67
Possible contamination
Molar absorptivity
500M⁻¹cm⁻¹
Peak data
One row per absorption maximum. Peak 1 is used for ε.
200–400 nm is plotted.
Reading at the peak maximum.
200–400 nm is plotted.
Reading at the peak maximum.
Sample
Molar concentration of the solution measured, e.g. 0.001 M = 1 mM.
Standard cuvettes are 1 cm.
Try an example spectrum
Working
Simulated spectrum
Each peak drawn as a Gaussian band; overlapping bands add.
Purity standards (A260/A280)
Bands used for the reading: below 1.5 protein contamination · 1.5–1.8 possible contamination · 1.8–2.1 pure nucleic acid · 2.1 and above phenol contamination.
Purity ratio = A260 ÷ A280
ε = A ÷ (c × b)
A = absorbance (no unit), c = molar concentration (M), b = path length (cm), ε = molar absorptivity (M⁻¹cm⁻¹). The second line is the Beer–Lambert law rearranged for ε.
For nucleic-acid concentration, labs use A260 × 50 μg/mL for double-stranded DNA and A260 × 40 μg/mL for RNA (1 cm path).
Sources: Sambrook et al., Molecular Cloning; Thermo Fisher technical guide.
Keep learning
References
- Harris DC, Lucy CA. Quantitative Chemical Analysis. 10th ed. New York: W. H. Freeman (Macmillan Learning); 2019.
Common questions
About 1.8 for DNA and about 2.0 for RNA. A lower ratio suggests protein (which absorbs at 280 nm) is still present.
The calculator looks for a peak within 5 nm of 260 nm and within 5 nm of 280 nm. A peak at 255 or 285 nm is just outside that window.
Peak 1, always. Put the peak whose molar absorptivity you want at the top of the list, and enter the concentration in mol/L.
No. It is an illustration built from your peaks as idealised Gaussian bands, to show how nearby bands overlap. Use your instrument's scan for anything you report.
Prepared by the PharmaWallah student team. Verify results with your textbook or instructor. Report an error · Editorial policy