Question

Describe two sources of error in the determination of the molar extinction coefficient of methylene blue. What is(are) the largest contributor(s)?

Answer 1

Ans. Beer-Lambert’s Law, A = e C L - equation 1,

where,

A = Absorbance

e = molar extinction coefficient at specified wavelength (M-1cm-1)

L = path length (in cm)

C = Molar concentration of the solute

The molar extinction coefficient of methylene blue (or any other dye) can experimentally be determined by preparing aliquots of gradual dilutions. The absorbance of aliquots on Y-axis is plotted against respective concentrations on X-axis. The slope of the graph gives extinction coefficient of the dye.

# Possible sources of errors:

I. Concertation of stock dye and standard aliquots: Any error in the concentration of the standard aliquots, whether during making dilutions or incorrect calculations, will give erroneous graphs and slope. Thus, resultant value of molar extinction coefficient will be inaccurate, too.

II. Path length: The path length for most spectrophotometer is generally 1.0 cm. However, it may vary depending on the make of instruments, too. If path length is not 1.0 cm, it must be accounted accordingly during calculations.

III. Choice of wavelength- Instruments: Some spectrophotometer are sophisticated enough to record absorbance exactly at the specified wavelength. Some spectrophotometer, cheaper in cost, may allow setting the wavelength in 10 or multiple of 10, or so on – in such cases absorbance of sample is taken as the nearest wavelength filter available in the instrument.

Greater is the deviation from exact wavelength, greater would be the error in calculated molar extinction coefficient of the dye.

IV. Several other factors like finger prints on cuvette, contamination of stock and standard aliquots, and homogeneity of the aliquots prepared, etc. may adversely affect the calculated values of molar extinction coefficient of the dye.

# Largest contributor: The factor that yields largest error in the experimental value of molar extinction coefficient would depend on the extent of deviation from the particular step of the experiment- it can be anything and at any step of the experiment.

For example, if dilution of standard aliquots is NOT prepared accurately, large deviations may creep. If calculations are not accurately, you may get unacceptable values, too.