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UV VIS instrumentation parts:
- Light source
- Entrance Slit
- Collimating mirror
- Diffraction grating ( Wavelength selector)
- Focusing Mirror
- Exit Slit
- Sample cuvette
- Detector (PMT or Computer Chip)
The intensity of light depends on
- Intensity vary with wavelength
How the instrument compensate for the reduction of the lamp intensity?
Instruments are designed so that their slit opening increases slightly as wavelength increase, so the energy reaching the detecotr is kept realtively constant
Narrow Range of wavelength is called__________ and it is selected by ___________
- Band pass or band width
- Monochromator ( Prism or diffraction Grating)
- A = ε b c + intercept(it is 0)
- b= path length
Why Cuvettes has to be machined to a high accuracy
Because absorbance depends on pathlength ; Cuvettes must be machined to high accuracy with parallel sides and known pathlength (1cm)
requirements of a detector
- Production of electrical output which is proportional to the intensity of the radiation hitting it
- needs to be able to produce signal from reasonably small number of photon
How does the electrons generates in the PMT detector?
The Cathode has _____ charge, while Dynode has a ________ charge.
- When a photon of light hits the Cathode which is coated with photoemassive material, an electron is being emitted and repelled by the negative charge.
- Negative, Positive
UV/VIS affects ________ electrons.
External ( Valence Electron)
Electronic Transition (4 types) is due to ___________
Excitation of bonding electrons
Lower energy to break Pi bond indicates:
Higher energy to break sigma bond indicates:
- Not much over lapping, absorption is in visible region
- More over lapping, Absorption is in UV region
Can a molecule be absorbed in a different wavelength? why?
Yes, sigma and Pi bonds are absorbed in different wavelength
Absorption of UV/VIS is restricted by a functional group called
Chromophores ( gives information about Functional Group of a molecule)
Sigma to Sigma* has a __________ energy and absorbs________ region. Pi to Pi* has a __________ energy and is absorbed in___________ region of light.
- Greater, UV
- Lower, Visible
As the degree of unsaturation increases, a shift to a _____ wavelegth will be observed.
- Unsaturaion is due to the multiple bonds and resonance structure. This unsaturation is need for Pi* orbitals.
- A group which extends the conjugation of a chromophore by sharing of NON-bonding electrons
- Shifts the wavelength where most absorption occurs
What type of shifting are caused by Auxochromes?
- 1-Bathochromic Shift: Shift of absorption to alonger wavelength ( red shift)
- Analogy: Most people like to take Long Bath, and If they stay in the bath for long time, their skin turns RED :)
- Hypsochromic Shift: shift of absorption to a shorter wavelength ( Blue Shift)
Why the measurement should be taken at Wavelength the max?
- to minimize errors
- achieve max sensitivity
Position of wavelegth max is influenced by________
Nature of solvent
How would you solve a problem of a molecule which does not absorb in UV/VIS region?
- Add Colour reagent.
- Colour reagents are organo metalics and are known as charge transfer. This addition causes a formation of complex which in return form resonance. These species tend to show very large abosebtivities.
How would you solve a problem of many sepcies absorb at the same wavelength? (Over lapping)
- Use masking Agent.
- Masking agent covers one analyte, measures the total A, then measures the A of the covered analyte
- total A - UnMasked analyte A = A of Masked species
- Ex: Cl, ammonia, cyanide
What is the purpose of Dual beam? Indicate their loctions.
- Double beam is used to measure the difference in A of two samples.
- One is placed in the sample beam and the other in the reference beam
How would you find the end point of a titration in which both titrant and titrand absorb radiation?
Use photometric Titration
What is the cause of Deviation in Beer's Law? Give two types of deviation
- Deviation caused by instrumental and chemical interferences.
- Two types of deviations are:
- Positive deviation: greater absorbance
- Negative deviation: decreased absorbance
What is the affect of power fluctuation? How can it be fixed?
- Power fluctuation changes source intensity and detector response
- To fix the problem use:
- UPS: uninterrupted power supplies ( levels off the fluctuation and incase of turning of the main power source, it is a good back up source)
Types of Instrument Noise that are related to the detector
Johnson's noise and Signal shot noise