Convert percent transmittance to absorbance or reverse the calculation, then review transmitted and nontransmitted light and optional Beer–Lambert concentration. Instrument and method limits must be validated separately.
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Percent Transmittance to Absorbance Formula
The following formula converts percent transmittance to absorbance:
- A is the absorbance (unitless)
- T is the percent transmittance (%)
Dividing %T by 100 converts to fractional transmittance. The negative base-10 logarithm yields absorbance. The inverse formula recovers %T: T% = 100 x 10^(-A).
| Percent Transmittance (%) | Absorbance (A) |
|---|---|
| 0.1 | 3.000 |
| 0.2 | 2.699 |
| 0.5 | 2.301 |
| 1 | 2.000 |
| 2 | 1.699 |
| 3 | 1.523 |
| 5 | 1.301 |
| 10 | 1.000 |
| 20 | 0.699 |
| 30 | 0.523 |
| 40 | 0.398 |
| 50 | 0.301 |
| 60 | 0.222 |
| 70 | 0.155 |
| 80 | 0.097 |
| 90 | 0.046 |
| 95 | 0.022 |
| 99 | 0.004 |
| Formula: A = -log10(T/100). Absorbance is unitless. | |
Why Absorbance Over Percent Transmittance
Under suitable Beer–Lambert conditions, absorbance is proportional to concentration: A = εlc, where ε is molar absorptivity in L/(mol·cm), l is optical path length in cm and c is concentration in mol/L. Use an absorptivity valid for the analyte, wavelength and measurement conditions. Each additional absorbance unit represents ten times less transmitted light: 1.0 A transmits 10%, 2.0 A transmits 1% and 3.0 A transmits 0.1%.
| Absorbance (A) | %T | Light Not Transmitted | Measurement Note |
|---|---|---|---|
| 0.05 | 89.1% | 10.9% | Use validated method range |
| 0.1 | 79.4% | 20.6% | Use validated method range |
| 0.2 | 63.1% | 36.9% | Use validated method range |
| 0.3 | 50.1% | 49.9% | Use validated method range |
| 0.5 | 31.6% | 68.4% | Use validated method range |
| 1.0 | 10.0% | 90.0% | Use validated method range |
| 1.5 | 3.16% | 96.8% | Use validated method range |
| 2.0 | 1.00% | 99.0% | Use validated method range |
| 3.0 | 0.10% | 99.9% | Use validated method range |
| These are mathematical conversions, not universal quality bands. Nontransmitted light may include reflection and scattering as well as absorption. | |||
Instrument Limits and Stray Light
The usable absorbance range depends on the instrument, wavelength, measurement settings, noise and stray light. Stray light can cause measured absorbance to underestimate true absorbance and distort Beer–Lambert linearity, particularly when sample transmission is very small. A displayed value alone does not establish measurement accuracy.
| Stray Light (%T) | −log10(stray-light fraction) | Interpretation |
|---|---|---|
| 1.0 %T | ~2.0 A | Not an accuracy specification |
| 0.1 %T | ~3.0 A | Not an accuracy specification |
| 0.01 %T | ~4.0 A | Not an accuracy specification |
| 0.001 %T | ~5.0 A | Not an accuracy specification |
| The logarithms illustrate a limiting transmission-floor concept in a simplified model. They are not maximum reliable absorbances or instrument-class specifications. Follow the instrument and assay validation, including any required dilution. | ||
Common Analyte Wavelengths
Measurement wavelength and usable range depend on the analyte, assay, pH, optical path and instrument. The following are common wavelength examples, not universal protocols or purity criteria. Follow the validated method for your sample.
| Analyte | Wavelength (nm) | Validated A Range | Notes |
|---|---|---|---|
| dsDNA | 260 | Follow validated method | A260/A280 is often near 1.8 for DNA; assess spectrum, conditions and downstream performance |
| Protein (aromatic residues) | 280 | Follow validated method | Tryptophan and tyrosine absorbance |
| NADH / NADPH | 340 | Follow validated method | Use the coefficient for the specified wavelength and conditions |
| Hemoglobin (oxyhemoglobin) | 415, 541, 577 | Follow validated method | Soret band at 415 nm has highest absorptivity |
| p-Nitrophenol | 405 | Follow validated method | Common enzyme assay reporter at pH greater than 8 |
| Bradford assay (protein) | 595 | Follow validated method | Coomassie brilliant blue G-250 dye complex |
| BCA assay (protein) | 562 | Follow validated method | Preferred for detergent-containing samples |
| Molar absorptivity (epsilon) varies by analyte and wavelength. Use Beer-Lambert law (A = epsilon x l x c) to convert absorbance to concentration. | |||
What is Absorbance?
Absorbance is dimensionless and defined by A = −log10(I/I0), where I0 is the reference incident intensity and I is transmitted intensity. Under appropriate Beer–Lambert conditions it is proportional to concentration and optical path length. Absorbance is commonly reported in UV-Vis and absorbance-mode microplate measurements. Instrument response, sample chemistry, scattering and reflection can affect measured transmission; there is no universal absorbance threshold at which every method becomes nonlinear.
How to Calculate Absorbance from Percent Transmittance
- Measure or obtain the percent transmittance (T) from the spectrophotometer readout.
- Apply the formula: A = -log10(T / 100).
- Compare the reading with the instrument and method’s validated range. Follow the assay’s dilution and blank/reference instructions where required; the conversion alone does not establish measurement reliability.
Example Problem:
Percent Transmittance (T) = 35%
A = -log10(35 / 100) = -log10(0.35) = 0.4559
The conversion gives approximately 0.456 absorbance. Whether the measurement is reliable must be assessed using the instrument and method validation; this number alone does not establish accuracy.
