Formula Used
Corrected A280 = Sample A280 − Blank A280
Corrected A260 = Sample A260 − Blank A260
Purity Ratio = Corrected A280 ÷ Corrected A260
Protein Concentration = Corrected A280 × Dilution Factor ÷ Extinction Coefficient ÷ Path Length
Purity Score = A280/A260 Ratio ÷ Target Ratio × 100
How to Use This Calculator
Enter the absorbance reading at 280 nm and 260 nm.
Add blank values from your buffer or reagent control.
Enter the dilution factor used before measurement.
Use 1 cm for a standard cuvette path length.
Enter your extinction coefficient when known.
Set a target ratio and warning limit for your method.
Press calculate to view purity, concentration, and status.
Use CSV or PDF export to save the result.
Protein Purity by A280/A260
Protein samples are often checked with ultraviolet absorbance readings. The 280 nm reading mostly reflects aromatic amino acids. The 260 nm reading reflects nucleic acid signal more strongly. A comparison between both readings can help estimate sample cleanliness. This calculator uses that comparison in a practical way.
Why the Ratio Matters
A protein preparation may contain DNA, RNA, salts, buffer compounds, or reagent traces. These materials can affect downstream tests. Enzyme assays may shift. Binding tests may weaken. Quantification may also become less reliable. The A280/A260 ratio gives a fast screening clue before deeper analysis.
Blank Correction
Blank correction is important. The buffer may absorb light. Plasticware, salts, or reducing agents may also contribute. Subtracting blank readings gives a cleaner estimate. This calculator corrects A280 and A260 separately before calculating the ratio.
Dilution and Path Length
Dilution changes concentration results. Path length also changes absorbance response. A standard cuvette often uses a 1 cm path. Microvolume instruments may use shorter paths. Enter the correct value for better concentration estimates.
Extinction Coefficient
The extinction coefficient links absorbance to concentration. Some proteins absorb strongly at 280 nm. Others absorb weakly. Use a known coefficient when available. A default value can still help with relative checks, but it may not give exact concentration.
Purity Status
The calculator compares your ratio with a target value. It also checks a warning limit. A low ratio suggests possible nucleic acid contamination. A moderate ratio means the sample may need review. A high ratio usually supports better purity, when readings are stable.
Best Practice
Use clean blanks. Mix samples gently. Avoid bubbles. Measure replicates when possible. Review raw readings before trusting the final score. Very low absorbance values can produce unstable ratios. Export results for traceability. Keep notes about buffer, instrument, dilution, and preparation date.
FAQs
What does A280 measure?
A280 measures absorbance near 280 nm. It is commonly linked with aromatic amino acids in proteins. It helps estimate protein amount when the extinction coefficient is known.
What does A260 show?
A260 absorbance is often linked with nucleic acids. A high A260 reading in a protein sample may suggest DNA or RNA contamination.
Why use the A280/A260 ratio?
The ratio compares protein signal against nucleic acid related signal. It gives a quick purity check before advanced testing or downstream experiments.
Can this calculator confirm exact purity?
No. It gives an absorbance based estimate. Confirm critical samples with SDS-PAGE, chromatography, mass analysis, or another validated laboratory method.
Why is blank correction needed?
Buffers and reagents can absorb ultraviolet light. Blank correction removes that background from sample readings and improves the ratio calculation.
What is the extinction coefficient?
It is a factor that connects absorbance to concentration. It depends on the protein sequence and measurement conditions.
What if corrected A260 is zero?
The ratio cannot be calculated reliably. Check the blank, repeat the measurement, and confirm that the instrument reading is valid.
Can I export my calculation?
Yes. Use the CSV button for spreadsheet records. Use the PDF button for a simple printable laboratory report.