Calibration of UV- Visible spectrophotometer
What is Calibration?
Calibration is the act of ensuring that a method or
instrument used in measurement will produce accurate results.
Calibration is performed using primary reference standard
Need of Calibration
When a specific time period is elapsed.
When a specific operating hours has elapsed.
With a new instrument.
When observations appear questionable.
Procedure of Calibration
For ultraviolet region
For visible region
Limit of stray light
Control of Wavelength
CHEMICALS: 1.4M perchloric acid, holmium perchlorate
STEP 1: Dissolve 1 gram of holmium oxide in 1.4M
perchloric acid with the aid of heating on water bath, cool and dilute to 25ml with
STEP 2: Record the spectrum holmium perchlorate
solution from 200nm to 600nm using 1.4M perchloric acid as reference solution
Note down the maxima observed at wavelength against the acceptance
criteria given below:
Web length Accuracy
Control of Absorbance
STEP 1: Weigh 57 to 63mg of potassium dichromate primary
standard and transfer to 1000ml volumetric flask. Dissolve in 0.005m sulphuric acid
and make up to the mark with the same acid.
Step 2: Measure the absorbance at 235 nm, 257nm, 313nm
and 350nm using 0.005M sulphuric acid as reference
FOR VISIBLE REGION: Whole procedure is same as UV
region but at the end measure the absorbance at 430nm.
CALCULATION: Value of A(1%,1cm)
A(1%,1cm)= absorbance x 100
weight in gm x 100
Limit of Stray Light
STEP 1: Prepare the solution of 1.2%v/v potassium chloride and
dissolve with 50ml distilled water.
STEP 2: Determine the absorbance using path length of 1cm at
200nm against purified water as blank
Absorbance at 200nm should be Greater then 2.0
STEP 1: Prepare a solution 0.02%v/v toluene in hexane
STEP 2: Record the spectrum of 0.02%v/v toluene in hexane from
250nm – 300nm using hexane as reference
STEP 3: record the absorbance at 269nm (max) and 266nm (min)n
STEP 4: Calculate the ration of absorbance by dividing the absorbance
at maxima and minima
Absorbance at 269nm to 266nm should be not less then 1.5
When the instrument not produce calibrated results it should
be labeled “FAUL TY” and should be repaired by service engineer of instrument.
The instrument should be always free from dust
Place the instrument as far as possible from any strong magnetic
or electric field or any electric
apparatus generating a high frequency.
Avoid places where vibrations exist or direct sunlight.
To extent the life of source lamp have them powered only when
The temp. Of all solution used in the test should not differ
by more then 0.5ºC.