Absorption by molecules in solution produces changes in electronic transitions as well as vibrational and rotational changes. For example the carbonyl group bonds contain sigma and pi electrons. These electrons may transition from bonding to antibonding levels.
Figure II-8 Sigma and Pi Bond Levels
Each of these transitions would result in a single peak in the absorbance / wavelength spectrum except for the broadening effect of the rotational and vibrational transisitions.
Figure II-9 Plot of Absorbance versus Wavelength
As light pases through a compound in solution the intensity is reduced.
Figure II-10 Light Absorbed through a Solution
The longer the pathlength the more light is absorbed. Also, the higher the concentration of compound in solution the more light is absorbed. Absorbance is proportional to pathlength and the concentration (Beer-Lambert's law)
Thus, A = a ¥ b ¥ c where a = absorptivity (epsilon - molar absorptivity includes pathlength and wavelength), b = pathlength (commonly 1 cm), and c = concentration (molar if molar absorptivity)
If b is 1 cm and c is in g/100ml the absorptivity is given as A1%1 cm at wavelength (lambda).
Figure II-11 Turner Model 330 Spectrophotometer
The Turner model 330 single beam spectrophotometer has a cell holder for the sample and dials for zero adjustment, 100% transmission and wavelength. Absorbance is read from the upper scale on the meter.
Calibration of the Spectrophotometer
The spectrophotometer should be turned on at least 30 minutes before calibration. Adjust the wavelength to the required setting. With the 'Press for Zero Set' button depressed adjust the 'Zero Adjustment' dial until the meter needle is aligned with 0 % transmission (lower scale). Then place an appropriate blank in a cell in the cell holder and adjust the '100 % Transmission' dial until the meter needle is aligned with 100 % transmission (lower scale). The blank can be replaced with the sample(s) of interest and the absorbance read from the upper scale. This procedure should be made for each wavelength of interest. The spectrophotometer should remain on until all the required readings are made.
Figure II-12 Schematic of a Double Beam Spectrophotometer
Analysis of Drugs by Visible Spectroscopy
Figure II-13 Absorbance versus Concentration
Extraction of Salicylic Acid
One Compartment - IV Bolus
One Compartment - IV Infusion
One Compartment - Oral
One Compartment Model - IV Bolus - Multiple Dose
Copyright 2001 David W.A. Bourne