Calibration of a UV instrument is essential to ensure accurate measurements and performance. Here's a general guide to UV calibration, depending on the application. UV Spectrophotometer Calibration Parameters as per IP are:
1. Control of Absorbance
2. Calibration of Wavelength
A. Holmium Perchlorate Solution
B. Discharge Lamps
C. Holmium Filter
3. Linearity
4. Limit of Stray Light
5. Resolution
1. Control of Absorbance:
- Materials Required are Potassium dichromate, Sulphuric Acid, Weighing balance, 1000 ml and 100 ml of volumetric flask, Butter paper, Beaker. Laboratory oven.
- First of all dry the potassium dichromate in oven at 130 degree celcius.
- Prepare 0.005 M Sulphuric Acid.
Calculation Procedure for 0.005 M sulphuric acid.
Mostly Sulphuric acid which is available in the market is about 98% Purity. So it's properties are as
Density of Sulphuric Acid: 1.84
Molecular Weight of Sulphuric acid: 98 g/mol
So first of all we have calculate the molarity of 98% Sulphuric acid by using given formula:
M= %age of H2SO4 x Density of H2SO4 x10 divided by molecular weight of H2SO4.
M=(98 x 1.84 x 10)/ 98
M=18.4
How much sulphuric acid is required to prepare 0.005 M sulphuric acid of 1000 ml.
Here, M1=Molarity of 97% H2SO4=18.4 M,
V1= Volume of 97% H2SO4 required=?
M2=Molarity of final solution=0.005 M,
V2=Final volume of solution= 1000 ml
By using Formula M1V1=M2V2
V1= M2V2/M1
V1=(0.005 x 1000)/18.4
V1=0.2717 ml
- Take 1000 ml volumetric flask containing 500 ml water. Add 0.2717 ml of Conc. Sulphuric acid into the volumetric flask and make up volume up to mark with water.
- After that prepare solution A by dissolving 60 mg of potassium dichromate in 1000 ml of 0.005 M sulphuric acid.
- Also prepare Solution B by dissolving 60 mgof potassium dichromate in 100 ml of 0.005 M sulphuric acid.
- In UV set the method as control of absorbance.
- Auto zero using 0.005 M Sulphuric acid
- Check the absorbance at different wavelengths as described in table:
|
Solution |
Wavelength (nm) |
Standard Absorbance (1%,1cm) |
Observed Absorbance |
Calculate Absorbance (1%,cm) |
Tolerance Value (Limit) |
|
Solution
A |
285
nm |
124.5 |
|
CA=(Absorbance
x 1000)/ wt.
in mg |
122.9-126.2 |
|
Solution
A |
257
nm |
144.5 |
|
142.8-146.2 |
|
|
Solution
A |
313
nm |
48.6 |
|
47.0-50.3 |
|
|
Solution
A |
350
nm |
107.3 |
|
105.6-109.0 |
|
|
Solution
B |
430
nm |
15.9 |
|
CA=(Absorbance
x 100)/ (wt. in gm x 100) |
15.7-16.1 |
2. Calibration of Wavelength:
A. Holmium Perchlorate Solutions:
- Prepare a 1.4 M perchloric acid solution:
Calculation and procedure to prepare 1.4 M perchloric acid
Mostly Perchloric acid which is available in the market is about 72% Purity. So it's properties are as
Density of Perchloric Acid: 1.67 g/ml
Molecular Weight of Perchloric acid: 100.46 g/mol
So first of all we have calculate the molarity of 98% Perchloric acid by using given formula:
M= %age of HClO4 x Density of HClO4 x10 divided by molecular weight of HClO4.
M=(72 x 1.67 x 10)/ 100.46
M=11.754 M
How much Perchloric acid is required to prepare 1.4 M Perchloric acid of 100 ml.
Here, M1=Molarity of 72% HClO4=11.754 M,
V1= Volume of 72% HClO4 required=?
M2=Molarity of final solution=1.4 M,
V2=Final volume of solution= 100 ml
By using Formula M1V1=M2V2
V1= M2V2/M1
V1=(1.4 x 100)/11.754
V1=11.91 ml
- Take 100 ml volumetric flask containing 75 ml water. Add 11.91 ml of Conc. Perchloric acid into the volumetric flask and make up volume up to mark with water.
- Prepare Holmium (III) perchlorate solution. Dissolve 0.5 gm holmium oxide in 2.4 ml of 1.4 M perchloric acid (72% AR grade) by gently heating and shaking and dilute to 10 ml in volumetric flask with water.
- Select method in UV as control of wavelength. Set the absorption spectrum from 500 nm to 230 nm.
- Auto zero with 1.4 M perchloric acid and record the absorbance.
Acceptance criteria:
The absorbance maxima should be observed at 241.5 nm, 279.4 nm, 287.5 nm, 361.5 nm, 452.4 nm.
B. Discharge Lamp:
- Prepare 1000 ml of 0.05 N Potassium Hydroxide solution.
Potassium Hydroxide has 1 replaceable hydroxyl ion (OH) so acidity of KOH is 1
Normality= Molarity x Acidity= 0.05 x 1= 0.05N
So, we can say that 0.05N is eqaul to 0.05M
Formula for calulation: Mass (g)= Moarity (M) x Molar mass (g/mol) x Volume (L)
Mass= 0.05 x 56.11 x 1 =2.8055 gm
- Take 1000 ml volumetric flask containing 750 ml distilled water and add 2.8055 gm potassium hydroxide. Dissolve and make up volume upto mark.
- Prepare Standard solution: Dissolve 100 mg of potassium dichromate in 500 ml of volumetric flask containing 20 ml of 0.05 N KOH and make up volume up to mark.
- Set the absorption spectrum wavelength from 340 nmto 400 nm.
- Auto zero using 0.05 N KOH solution. Record the absorbance.
Acceptance criteria: Maxima wavelength observed at 370 nm.
C. Holmium filter:
- Here we can take any sample solution for this test. We can take previous potassium dichromate solution.
- Set the absorption spectrum from 500 nm to 280 nm.
- Also use slowest scan speed and narrowest slit setting.
- Auto zero and record the absorbanc.
Acceptance criteria:
Three fused bands cetered at 452.2 nm.
Single band observed at 360.9 nm
Other maxima may be observed at 452.2 nm, 360.9 nm, 287.5 nm, 279.4 nm, 241.5 nm
3. Linearity:
- Prepare 0.05 N KOH solution as described in discharge lamp of wavelength calibration.
- Prepare 4,8,16,24,32 µg/ml solution of KOH.
- Prepare Stock solution by dissolving 40 mg of potassium dichromate in 100 ml volumetric flask with 0.05 N KOH.
For 4 µg/ml (0.004 mg/ml): Dilute 1 ml stock solution of potassium dichromate in 100 ml V.F. with 0.05 N KOH
For 8 µg/ml (0.008 mg/ml): Dilute 1 ml stock soluion of potassium dichromate in 50 ml V.F. with 0.05 N KOH
For 16 µg/ml (0.016 mg/ml): Dilute 1 ml stock soluion of potassium dichromate in 25 ml V.F. with 0.05 N KOH
For 24 µg/ml (0.024 mg/ml): Dilute 3 ml stock soluion of potassium dichromate in 50 ml V.F. with 0.05 N KOH
For 32 µg/ml (0.032 mg/ml): Dilute 2 ml stock soluion of potassium dichromate in 25 ml V.F. with 0.05 N KOH
- Set absorbance wavelength at 370 nm. Auto zero with 0.05 N KOH.
- Record absorbance of each standard soution. Plot the linearity graph.
Acceptance criteria: Value of R square should be more than 0.999.
4. Limit of Stray Light:
- First of all heat the potassium chloride in oven at 130 degree celcius.
- Weigh accurately 120 gm of potassium chloride in 100 ml volumetric flask having 50 ml water. Disssolve and meake up volume up to mark with water.
- Select the method file limit of stray light and set wavelength at 200 nm.
- Baseline correction or auto zero with water. Record the absorbance.
Acceptance criteria: Absorption should be more than 2.0
5. Resolution:
- Prepare 0.02% v/v solution of toulene in hexane. Dilute 0.02 ml of toulene in 100 ml volumetric flask with hexane.
- Select the method of file resolution power in UV setting.
- Auto zero with hexane. Record the absorbance.
Acceptance criteria:
Solution absortion maxima should be 269.
Solution absorption minima should be 266.
Ratio of Maxima/Minima should be equal to or more than 1.5
Post a Comment