Question

A. A beginning scientist measures an absorbance of 3.0 at 260 nm for a DNA sample. Is this a meaningful number and why or why not ?(Consider the amount of light passing through the sample, ie. the % transmittance). What range should values of absorbance fall in?

B. A 6.0 mg/L solution of adenine in water has an absorbance of 0.59 at 261 nm in a 1 cm pathlength cell. Adenine has a molecular weight of 135.1 g/mol.What is the extinction coefficient of adenine at 261 nm (ε₂₆₁) in units of M^-1cm^-1?

Answer 1

A.

Absorbance (A) of a sample is related to % transmittance of light through the sample as follows

Hence, if a sample has an absorbance of 3.0, the % transmittance can be calculated as

It means only 0.1 % of the incident light has passed through the sample and 99.9% of the light is absorbed. This can only happen if the concentration of the DNA sample is too high. Hence, if we are to measure the concentration of the sample using the Beer-Lamberts law with absorbance of 3.0, the relationship between the concentration and absorbance is not linear anymore as predicted by the equation

Where c is the concentration

l is the path length

is the extinction coefficient.

Note that all the high concentrations with absorbance > 3.0 will result in an absorbance of close to 99.9% incident light and it will be practically impossible to relate the concentrations with absorbance as they all give greater than 99.9% absorbance. In other words, high absorbance values like 3.0 leads to non-linearity in the absorbance vs concentration relationship and makes it impossible to predict the correct concentration from absorbance data.

Hence, 3.0 is not a meaningful number for absorbance.

Instead, we dilute our solution enough to get a transmittance of at least 10% where the absorbance value is 1.

Hence, a reading of absorbance between 0 to 1 is preferred where the absorbance and concentration is linear and easily predicted by the Beer Lambert law. Hence, instead of taking a highly concentrated solution which gives a high absorbance reading, we should dilute it to bring the absorbance to 0-1 range.

B.

Concentration of the adenine solution is 6.0 mg/L.

Hence, there are 6.0 mg of adenine in 1 L of the solution.

Molar mass of adenine is 135.1 g/mol

Hence, number of moles of adenine in 1 L is

Hence, of adenine is present in 1 L of solution. Hence, the molar concentration of adenine solution is

Now, it gives an absorbance reading of 0.59 in a 1 cm path length cell with a light of 261 nm wavelength.

Hence,

Now using the Beer-Lamberts law,

Hence, the extinction coefficient of adenine at 261 nm is about (rounded to two significant figures)