Aim: Analysis of pharmacopoeial compound and their formlations by UV Visible spectrophotometer
References:
1. SkoogD. A, Holler J. G, Gouch S. R, “ Instrumental Analysis” Published by Ceugage learning, 3rdedition, 2009, Pg. No- 392-397.
2. Chatwal G. R. Anand S. K. “Instrumental Methodes Of Chemical Analysis”, Himalaya Publishing House. Pg. No- 2.116 – 2.122.
3. Beckett A. H, Stenlake T. B, “Practícale Pharmaceutical Chemistry”, 4th edition, part-2, CBS Publication, Pg. No- 264-274.
UV-VISIBLE SPECTROPHOTOMETER.
COMPONENTS OF SPECTROPHOTOMETER.
1. Radiation source
2. Wavelength selector: -
Both filter and monochromator allow the light of the required wavelength.
A light filter is a device that allows light of the required λ wholly or partially. Thus a suitable filter can select a desired λ band.
i. Absorption filters- it works by selective absorption of unwanted λ. An absorption filter is a solid sheet of glass that has been colored by a pigment, which is dissolved or dispersed in the glass. Dyed gelatin or similar materials can also be used as absorption filters. There are classified as either cut off or band pass filters. They cover the range from 390-700nm.
ii. Interference filters-
Narrow bandwidth is obtained with these filters. These functions on interference phenomenon at desired λ, thus permitting rejection of unwanted radiation by selective reflection. A semi-transparent metal film is deposited on a plate of glass, then it is coated with thin layer of some dielectric material {MgF2} followed by another plate of glass is kept over the films for the mechanical protection. Several outgoing rays undergo constructive interference for those wavelengths which are exactly even multiplies of the distance separating the two for other λ, the beam undergoes destructive interference band pass of 100-150A°& peak transmittance of 40-60%.
It successfully isolates band of λ usually much more than a narrow filter. It has an entrance slit, a dispersing element {a prism or grating} and an exit slit. Function of a prism or grating is to disperse the heterochromic radiation into its component wavelengths. Materials used are glass for visible, quartz for UV. Replica gratings are cheaper than prisms but the defect of gratings is that they produce more than one order of diffraction. Interference can be removed by employing filters in front of the entrance slit to absorb interfering radiations. Grating consists of large no. Of parallel lines {grooves} ruled on a highly polished surface such as alumina 15,000-30,000 lines per square inch are drawn for UV & visible region. These grooves act as scattering centres with different angles & directions, reinforcement or constructive interference may take place. Replica grating are produced after coating with epoxy resin and then aluminizing it for reflection. Slit width used as controlling device.
3. Detectors: -
The light or intensity of transmitted radiation by a sample is collected on a detector device. This is to measure the amount of transmitted radiation. Most modern detectors generate an electric current after receiving the radiation, which is then amplified and passed on to a meter or recorder.
Ideal characteristics of the detectors-
1. It should give quantitative response.
2. It should have high sensitivity & low noise level.
3. It should have a short response time.
4. It should provide signal or response quantitative in wide spectrum of radiation.
5. It should generate sufficient current or signal to be recorded.
a. Photomultiplier tube:
It consists of electrode covered with a photo emissive material. This tube also contains a large no. of plates, known as dynodes. Each dynode is covered with a material, which emits several electrons for each electron striking on its surface. So electrons striking keep on multiplied as per the potential of dynode with a fixed proportion. The process is repeated by applying no. dynodes to get the required signal. Electron falling on collector measures the intensity of light incident on cathode surface. The dynodes are maintained at 75-100v more positive than preceding dynode amplification is 106. Response time is 10-9s. It should be carefully shielded from stray light.
b. Phototubes or photo emissive cells:
It consists of evacuated glass bulb. Inside the bulb there is light sensitive cathode in the form of a half cylinder of metal. The inner surface of cathode is coated with a light sensitive layer such as caesium or potassium oxide and silver oxide. A metal ring inserted nears the centre of the bulb act as anode. When radiation incident on cathode photoelectrons are emitted. These are attached and collected on an anode. These are then returned via external circuit. Due to the flow there occurs internal drop across the resistor R that is proportional to the current. This current may be amplified by electronic means & is takes on a measure of the amount of light striking the photo emissive surface.
The Absorption Law:
Lambert’s law: - The intensity of a beam of parallel monochromatic radiation decreases exponentially as it passes through a medium of homogenous thickness. More simply it is stated that the absorbance is proportional to thickness (Path length of the solution). The expression can be given as
-dI/db I Where b = path length
-dI/db = k’ I k’ = proportionality constant
Applying log on both sides
Log Io/It = k’ b
Log Io/It = k’/ 2.303
The quantity log Io/It is % absorbance A is equal to reciprocal of common logarithmic of transmittance i.e.
A= log10 (Io/It)
i. e. Alog10 (1/T)= -log T
i. e. A=2-log (%T)
Beer’s law: - the intensity of beam of parallel monochromatic radiation decreases exponentially with the no. of absorbing molecules more simply it is stated that the absorbance is proportional to the concentration,
-dI/dc I Where c = concentration
-dI/dc = k’ I k’ = proportionality constant
Applying log on both sides
Log Io/It = k’ c
Lamberts – Beer’s Law
Log Io/It = acb
A = acb
Molar absorptivity: - The name& volume of ‘a’ depends on limit of concentration where ‘c’ is in mole/lit. The constant is called molar absorptivity. It also has a symbol ’ε’ and equation is as follows,
a =ε. b. c
Specific absorbance: - Another form of Beer-Lambert’s law proportionally constant is the specific absorbance, which is the absorbent of a specific concentration in a cell of specific path length. The most common in pharmaceutical analysis is the A (1%, 1cm), which is the substance of 1gm/100ml. i.e. (1% w/v solution) in a 1cm cell.
A = A (1%, 1cm). b. c
Where c = 1gm/100ml
b = cm
A = simple derived equation allows interconversion of ε & A (1%, 1cm).
ε = A (1%, 1cm) X molecular weight.
When does the law not obey
What is single beam and double beam
the diagram showing double beam
RESULT:
Remark:
Practical Performance
(2)
Conduct in Lab
(2)
Journal
(2)
Observations and Results
(2)
Viva-Voce
(2)
Total
(10)
Signature of Faculty In charge
EXPERIMENT NO:6
