Formula Used
Corrected absorbance: A280 corrected = Sample A280 - Blank A280 - Baseline correction.
Molar coefficient method: Concentration mg/mL = A280 corrected × Dilution × Molecular weight g/mol ÷ Extinction coefficient ÷ Path length cm.
Mass coefficient method: Concentration mg/mL = A280 corrected × Dilution ÷ Mass coefficient ÷ Path length cm.
E1 percent method: Concentration mg/mL = A280 corrected × Dilution × 10 ÷ E1 percent coefficient ÷ Path length cm.
Unit conversions: 1 mg/mL equals 1 ug/uL. ng/uL = mg/mL × 1000. uM = mg/mL × 1000 ÷ molecular weight kDa.
How to Use This Calculator
Enter the Nanodrop absorbance values for A280, A260, and A230. Add matching blank values when available. Enter a baseline correction if you use A320 or A340 correction. Choose the coefficient method that matches your protein record. Then enter dilution, path length, molecular weight, and volume. Press calculate to view concentration, purity ratios, and yield.
Overview
A Nanodrop reading can be useful and fast. It still needs clear inputs. Protein absorbance depends on aromatic residues. It also depends on the chosen coefficient. This calculator keeps those choices visible. It corrects the sample reading first. It subtracts the blank and the baseline value. Then it adjusts for path length and dilution. The final value is shown in mg/mL, ug/uL, ng/uL, and micromolar units.
Why the Extinction Choice Matters
Many purified proteins use a molar extinction coefficient. This value normally comes from a sequence tool or a certificate. It is used with molecular weight. Some assays use a mass extinction coefficient instead. That method needs absorbance per mg/mL. A third option is the E1% coefficient. It is common in older protein references. The calculator supports all three methods, so records stay traceable.
Purity Checks
A280 is the main signal for many proteins. A260 helps flag nucleic acid carryover. A230 can show salts, buffers, phenol, or other background. These ratios do not prove purity alone. They are warning signs. A clean spectrum and a matched blank are still important. Very low absorbance can make ratios unstable. In that case, repeat the reading or concentrate the sample.
Practical Lab Use
Use the same buffer as the blank. Wipe both pedestals before each reading. Mix the sample gently. Avoid bubbles and visible debris. Enter the path length used by the instrument. Many instruments normalize readings, but manual checks help audits. Enter dilution when the sample was diluted before loading. Add sample volume when total yield is needed.
Interpreting Results
The result is only as good as the coefficient. A wrong coefficient can shift the answer greatly. Baseline correction helps when scatter is present. A320 or A340 is often used for that purpose. Negative corrected absorbance suggests a blank problem. It can also mean the sample is below range. Save the CSV for spreadsheets. Use the PDF for notebook attachments. Record the method beside each value.
Quality Notes
Run replicate readings when samples are precious. Compare the spread before reporting. Clean the pedestal after concentrated samples. Use fresh blank drops. Keep notes about buffer additives. Detergents and reducing agents may affect background. Recheck unusual ratios with a full spectrum scan quickly.
FAQs
What does A280 measure?
A280 measures absorbance near 280 nm. Many proteins absorb there because of tryptophan, tyrosine, and disulfide bonds. The value is useful when the correct extinction coefficient is known.
Why is blank correction important?
The blank removes background from buffer, salts, and the pedestal surface. Use the same buffer as the sample. A mismatched blank can create false high or low concentration values.
Which coefficient method should I choose?
Use the molar coefficient when you know molecular weight and epsilon. Use the mass coefficient when your reference gives absorbance per mg/mL. Use E1 percent when that value is supplied.
What does A260/A280 show?
A260/A280 helps flag nucleic acid contamination. Protein samples often have lower ratios than DNA or RNA. A high value does not prove contamination, but it should prompt spectrum review.
What does A260/A230 show?
A260/A230 can warn about salts, phenol, guanidine, carbohydrates, or buffer background. It is a screening ratio. Always compare it with the full spectrum and sample preparation details.
Why enter path length?
Beer Lambert calculations depend on optical path length. Some instruments normalize automatically. This field lets you document the effective path and check readings from different settings.
Why is my corrected A280 negative?
A negative corrected A280 usually means blank, baseline, or sample input is mismatched. It can also mean the protein is below the reliable reading range. Repeat the measurement.
Can this replace a colorimetric assay?
No single method fits every sample. Nanodrop A280 is fast and non-destructive. Bradford, BCA, or Lowry assays may be better for mixtures or proteins with weak aromatic absorbance.