Practical Advanced Pharmaceutical Analysis University of Mosul
UV-Vis. Spectroscopy--- Lab-1- College of Pharmacy
Measuring the absorption spectrum and
determining λ max
Raghad Riyadh
B.Sc., M.Sc. Pharm
ELECTROMAGNETIC RADIATION:
Electromagnetic radiation can be considered a combination of alternating electric and
magnetic fields that travel through space with a wave motion.
Ultraviolet (UV) and visible radiation comprise only a small part of the electromagnetic
spectrum, which includes such other forms of radiation as radio, infrared (IR), and X rays.
FUNDAMENTALS OF SPECTROPHOTOMETER
Because radiation acts as a wave, it can be classified in terms of either
wavelength or frequency, which are related by the following equations:
E=h =c
E = h c/
= distance of one wave (nm)
= frequency: waves per unit time (sec-1, Hz)
c = speed of light (3.0 x 108 m • sec-1)
h = Plank’s constant (6.63 x 10-34 J • sec)
PRINCIPLE OF THE UV-VIS SPECTROSCOPIC PROCESS
UV–visible spectroscopy: is a measurement technique in which the recording of the
absorption spectra of different samples using ultraviolet (UV) and visible (Vis) light is
achieved by a spectrophotometer, i.e. an instrument able to measure the spectrum of a sample
in the UV/Vis range.
When the sample is subjected to the
radiation in the UV-Vis. and if a particular
electronic transition matches the energy of
a certain band of this spectrum, it will be
absorbed. The remaining light passes
through the sample. From this residual
radiation a spectrum is obtained with
“gaps” at these discrete energies – this is
called an absorption spectrum.
Absorption spectrum Is a graph between absorbance of the analyte versus
the change in the wavelength.
from absorption spectrum we find the wavelength with the highest
absorbance, the wavelength of the absorption peak (λ max), at this wavelength
the spectrophotometric method is most sensitive for the analyte
- Effects of Conjugation
When two or more chromophores are conjugated the absorption maxima is shifted to a larger wavelength.
- Prescence of auxochrome
It is a group which itself does not act as a chromophore but when attached to a chromophore, it shifts the absorption
towards longer wavelength (why?) along with an increase in the intensity of absorption. e.g. -OH, -NH2, -OR.
Solvent
Concentration of the sample
pH of the sample
Temperature of the sample
INSTRUMENTATION
contents of the spectrophotometer
(1)Radiation source – Visible = tungsten filament lamp; UV = deuterium and hydrogen
lamps
(2)Monochromators/filters – Restricts wavelength to offer narrow band of radiation
(3)Sample containers (cuvettes or cells) – glass, plastic or quartz (1 cm)
(4)Phototubes/ photomultiplier tubes – emit photoelectrons after being irradiated,
current is then amplified and measured.
(5)Measuring system.
Types of spectrophotometer instruments
1 single beam
2 double beam
UV-VISIBLE SPECTROPHOTOMETRY APPLICATIONS IN PHARMACEUTICALS
Analyzing pharmaceutical active compounds with spectrophotometers requires a
small sample quantity to obtain accurate results. UV spectrophotometry for the
pharmaceutical industry ensures patient health and safety through applications
such as:
•Chemical quantification: Based on the Lambert-Beer Law, the concentration of a
compound in a solution can be determined quantitatively by UV-Vis. spectroscopy.
•Quality control: Spectrophotometers can obtain the highly accurate
measurements needed to ensure the purity and quality of the product. A high
degree of quality control is critical for pharmaceuticals.
•Confirmation of compound identity Although UV-Vis. spectra do not enable
absolute identification of an unknown, they frequently are used to confirm the
identity of a substance through comparison of the measured spectrum with a
reference spectrum.
Measuring the absorption spectrum and determining λ max of
potassium permanganate solution:-
Procedure:
1 - Prepare the sample solution with concentration of 100 μg/ml by dissolving
potassium permanganate in D.W.
2 - Rinse one of the cuvettes with blank solution, put the cuvette in the sample
compartment, set the wavelength , then set the absorbance (A) to zero.
3 - Rinse a second cuvette and fill with sample solution, place the cell in the
sample compartment, measure the absorbance at 450 nm and record.
4 - Repeat this procedure (steps 2 and 3 above) for the two cuvettes at
wavelengths 470, 510, 520,525, 530, 540, 570, and 600 nm, first setting A = 0 for
the cuvette with blank, then measuring A for the cuvette with sample solution,
recording the absorbance at each wavelength, record in data table.
5 - Prepare a graph of absorbance (A) vs. wavelength (λ) and determine λ max.
Wavelength (nm) Absorbance
450
470
510
520
525
530
540
570
600
