Beer-Lambert Law Calculator and A260 DNA Concentration

Two panels share this page. The first solves the Beer-Lambert law for any one of its four terms. The second applies the same law to nucleic acids, turning a 260 nm reading into ng/µL and checking purity from the 260/280 and 260/230 ratios.

Panel 1: the Beer-Lambert law

How to use it

Fill in three of the four main boxes and leave the fourth empty. The defaults solve for concentration:

• Absorbance A: 0.5
• or % transmittance: empty. If you type a value here while the absorbance box is empty, it fills A for you.
• Molar absorptivity ε: 6220 L/(mol·cm), the value for NADH at 340 nm
• Path length l: 1 cm
• Concentration c: empty, with M, mM or µM for entering a value

A solved concentration is shown in whichever unit gives a readable number. If the absorbance is above 2, whether you typed it, it came from % transmittance or the tool solved for it, you get a warning to dilute and measure again.

The formula

A = ε × l × c

A is absorbance (no unit). ε is the molar absorptivity (also called the molar extinction coefficient) in L mol⁻¹ cm⁻¹, which belongs to one compound at one wavelength. l is the path length of the light through the sample in cm, 1 cm for a standard cuvette. c is the concentration in mol/L.

Absorbance comes from the light that gets through: A = log10(I0 ÷ I) = -log10(T), with T as a fraction. 10 % transmittance is A = 1, and 1 % is A = 2. Above about A = 2 so little light reaches the detector that stray light dominates, which is why the tool warns there. Readings between about 0.1 and 1 are the safest on most instruments.

Worked example with the default values

c = A ÷ (ε × l) = 0.5 ÷ (6220 × 1) = 8.039 × 10⁻⁵ M

The tool shows "Concentration c: 80.39 µM". This is the calculation behind enzyme assays that follow NADH at 340 nm.

When you do not know ε: the standard curve

Many assays (protein dye assays, colorimetric kits) have no reliable ε, so you build a standard curve instead. Make a stock with the molarity calculator, dilute it into five to eight standards with the serial dilution calculator, and measure each against a blank of the same buffer. Plot A against concentration. If the points fall on a straight line, its slope is ε × l, and unknowns are read off the line. Only trust readings inside the range of your standards.

Panel 2: DNA and RNA concentration from A260

How to use it

Pick the sample type (dsDNA, RNA or ssDNA), then type the readings. The defaults are A260 0.75, A280 0.41, A230 0.36 (optional) and a dilution factor of 1. Use readings for a 1 cm path. Microvolume instruments convert to 1 cm for you; with a cuvette, check its path length and divide the reading by it if it is not 1 cm. Below an A260 of about 0.1 the tool warns that the result is rough.

The formula

concentration (ng/µL) = A260 × factor × dilution factor

The standard conversion factors are 50 for double-stranded DNA, 40 for RNA and 33 for single-stranded DNA: one A260 unit equals about 50, 40 or 33 µg/mL. A µg/mL is the same as a ng/µL. If you diluted the sample before reading it, enter that dilution so the result refers to the undiluted stock.

Worked example with the default values

• Concentration: 0.75 × 50 × 1 = 37.5 ng/µL
• 260/280: 0.75 ÷ 0.41 = 1.83, in the usual range
• 260/230: 0.75 ÷ 0.36 = 2.08, in the usual range

Switch the same readings to RNA and the concentration becomes 30 ng/µL.

Purity rules of thumb

A 260/280 well above 1.8 in a DNA prep often means RNA is present. These are rules of thumb, not pass marks: the 260/280 ratio also shifts with the pH and salt of the blank, and slightly acidic water can pull it down. Blank with the same buffer the sample is in.

Common mistakes

• Using ε for the wrong wavelength, or entering it per mM while the box expects per M.
• Forgetting the dilution factor or the path length of a short cuvette.
• Trusting readings below about 0.1, where noise dominates, or far above 1.
• Treating A260 as specific. Everything that absorbs at 260 nm counts: RNA in a DNA prep, free nucleotides, leftover primers. For low concentrations or sequencing libraries, fluorescent dye assays are more specific.

FAQ

What is the Beer-Lambert law?

It states that absorbance is proportional to concentration and path length: A = εlc. With ε known for your compound and wavelength, one absorbance reading gives the concentration. It holds best for dilute solutions read within the instrument's linear range.

What is a good A260/A280 ratio?

About 1.8 for DNA and about 2.0 for RNA. Clearly lower values point to protein or phenol left over from the extraction. Treat the ratio as one clue among several, because pH and the blank also move it.

What does a low A260/A230 ratio mean?

Something that absorbs near 230 nm came through the purification, most often guanidine salts from a column kit, carbohydrate, EDTA or phenol. Values of about 2.0 to 2.2 are typical for clean samples, and the tool flags anything below 1.8.

How much DNA should I add to a PCR?

Polymerase suppliers' guidelines commonly suggest roughly 1 ng to 1 µg of genomic DNA per 50 µL reaction, and far less, picograms to about 10 ng, for plasmid DNA. Use the range in your enzyme's protocol. At the default 37.5 ng/µL, 1 µL adds 37.5 ng.

Can I enter percent transmittance instead of absorbance?

Yes. Leave the absorbance box empty and type the % transmittance; the tool converts it with A = -log10(T). For example, 50 % transmittance becomes an absorbance of about 0.301.