|
When measuring the absorbances of several samples that have different concentrations, it is important to identify an appropriate wavelength. This wavelength is the one at which the samples absorb the most light and is called lambda max.
Lambda max is determined by plotting absorbance versus wavelength and noting where the sample absorbs the most light. Thus, it is necessary to collect absorbance data at different wavelengths. When absorbance versus wavelength is plotted the resulting plot is referred to as an absorption spectrum.
To record an absorption spectrum, set the wavelength on the Spectronic 20™ to 400 nm and calibrate the maximum and zero absorbance using the reference solution. Insert the sample solution and record its absorbance at this wavelength.
Change the wavelength by increasing the wavelength by 20 nm (to 420 nm). Record the absorbance. Remember to recalibrate the maximum and zero absorbance each time the wavelength is changed. Continue collecting data for different wavelengths of light. Initially, change the wavelength in 20 nm increments, but when the absorbance begins to rise rapidly decrease the wavelength increment from 20 nm to 2-5 nm. This smaller increment allows a more accurate determination of the wavelength of maximum absorbance (lambda max). After the absorbance has peaked and again begins to decrease, change the wavelength increment back to 20 nm. Continue recording absorbance data until the upper wavelength is reached.
|
