what is a lambert beer law and Calibration of UV

 

Monochromatic light(I) pass through sample in  transparent Vessel  they absorb some light,  some loss  of light intensity due to scatter by particle in solution and other light(IO) came out to detector

Hear I is less than (IO)

Relationship between I and  IO Depend of pathlength of absorbing medium, and concentration of absorbing solution its call lambert beer law

  






Lambert Law :-

Its Intensity Decrease Exponentially as length of absorbing medium increase

Beer Law :-

Its Intensity Decrease Exponentially as Concentration  of absorbing medium increase

Use of Uv Spectroscopy  :-  detection of functional group

What is call chromofrm :-Colour compound   

Calibration of Uv Spectroscopy :

Which Lamp Use :- Sodium and mercury Lamp   

Frequency Of Calibration :Monthly Calibration done in pharma company 

UV/VIS Spectrometer

• Various type of software are use the calibration of uv spectrometer e.g Lamda 35  
1. Control of wavelength
2. Control of Absorbance
3. Limit of Stray Light
4. Resolution
5. Resolution Power
• uv spectrometer are work as visible wave length

1. Control of wavelength

• 1.4M perchloric acid
• 4 % Holomium perchlorate solution

Wave length

• 241.15
• 287.15
• 361.50
• 536.30

Acceptance Criteria  + or – 2

2. Control of Absorbance

• 0.005 M Sulphuric Acid
• potassium Dichromate Solution preparation
• previously Dry 130 C
• 57.0 mg to 62.0 mg
• take in 1 Liter 0.005 M sulfuric acid solution
• Wave are use 235nm,257nm,313nm,350 nm
• 430 nm are also same but make in 100ml 0.005 M sulfuric acid solution

Calculation

Absorbance (A%) = Observation absorbance X 10000

Weight of potassium Dichromate(mg)

 

Absorbance (A%) = Observation absorbance X 1000

Weight of Pottassim Dichromate(mg)

Result :

1% = 1cm

Tolerance are depend instrument

3. Limit of stray Light

1.2 % W/V Kcl are make in 100 ml water

Acceptance Criteria :absorbance > 2.0

Wavelength 200 nm

4. Resloution

0.02 % V/V Toluene diluted 0.02ml Toluene in 100 ml hexane.

Wavelength are 269nm and 266 nm

Acceptance Criteria : Ratio Should be >1.5

5. Resloution power

 Same as above parameter are use but solution are use instated of toluene use methanol