CALIBRATION OF UV/ VISIBLE SPECTROPHOTMETER

Ensure that the connection of the instrument are proper.

Control of absorbance

Potassium dichromate solution:

Dry a quantity of potassium dichromate by heating to constant weight at 130°C. Weigh & transfer accurately
a quantity not less than 57.0 mg & not more than 63.0 mg to 1000ml volumetric flask. Dissolve & dilute in sufficient 0.005M H2SO4 to produce 1000ml.

Measure the absorbance of potassium dichromate solution at the wavelength at the wavelengths given below.

Calculate value of A (1% 1cm) for each wavelength.

A(1% 1cm) = Absorbance X 10000 / Weight of potassium dichromate in me.

Acceptance criteria

Wavelength (nm)	A [1% 1cm]	Limit
235.0	124.5	122.9 – 126.2
257.0	144.0	142.8 -145.7
313.0	48.6	47.0 – 50.3
350.0	106.6	105.6 – 108.2

Stringent limit are adopted from IP & BP for acceptance criteria

Resolution power

Record the spectrum of a 0.02% v/v solution of toluene in hexane in the range of 260nm to 420 nm (before use check the hexane for transmittance, using water as a blank between 260nm to 420nm & use only if transmittance is not less than 97%).

Acceptance criteria:

The ratio of the absorbance at the maximum at about 269 nm to that at the minimum at about 266nm is not less than1.5.

Limit of stray light

Prepare a 1.2 % w/v solution of potassium chloride in water.

Measure absorbance of the above solution at 198.0, 199.0, 200.0, 201.0, 202.0 nm using water as blank.

Acceptance criteria:

Absorbance is greater than 2.

Wavelength accuracy, Resolution & Baseline flatness (inbuilt test)

Attach printer directly to the instrument (instead of computer).

Go to > MODE press F3 key i.e. Maintenance.

Press  ‘1 ‘.

Press Start/ Stop Key.

After screen changes, ensure that nothing is kept in the optical path & press the Start / Stop key again.

Prints will come after all the three tests are over.

Acceptance criteria:

For wave length accuracy : at 656.1 ± 0.3 nm & at 486.0 ± 0.3 nm.

Resolution: 1.0 nm or less.

Baseline flatness: ±0.002Abs.

Frequency: Once in a month.

Maintenance / Repairs

When the instrument does not comply with the requirement / tolerance range specified above, the instrument should be labeled “OUT OF CALIBRATION” and should be repaired / serviced.

After repair / maintenance, calibration the instrument.

Reference:

Indian Pharmacopoeia

British Pharmacopoeia

Instruction manual of UV-1700.

CALIBRATION REPORT OF UV/ VISIBLE SPECTROPHOTOMETER

SOP Reference -                                                                                               Date-

Instrument No-                                                                                                                Frequency – Once in a month

Make -                                                                                                                  Model –

1. Control of Absorbance:

Potassium dichromate solution:

Gross Wt:                                                                                            Balance :

Tare Wt :

Net Wt :                                                               ________ ml with 0.005M sulphuric acid.

Dissolve and dilute 57.0 to 63.0 mg of potassium dichromate in sufficient 0.005M sulphuric acid to produce 1000.0 ml.

Wavelength	Absorbance	A (1%, 1cm)	Tolerance
235.0			122.9 to 126.2
257.0			142.8 to 145.7
313.0			47.0 to 50.3
350.0			105.6 to 108.2

Calculation:

1%                          Absorbance X 10000

A1cm     =      --------------------------------------------------------   =  -------------------  =

         Weight of potassium dichromate in mg

(Limit : Not less than 1.5)

Remark : Satisfactory /Not satisfactory.

Performed By:                                                                                  Checked By:

Date:                                                                                                     Date:

2. Resolution Power:

0.02% v/v toluene in hexane:

………………… ml   -> 100.0 ml with hexane / …………… ml  -> 100.0 ml with hexane

Absorbance at 269 nm = ……………………..

Absorbance at 266 nm = ……………………..

                Absorbance at 269 nm

Ratio = ----------------------------------- = ------------------- =

                Absorbance at 266 nm

(Limit : Not less than 1.5)

Remark: satisfactory / Not satisfactory.

Performed BY:                                                                  Checked By:

Date:                                                                                     Date:

3. Limit of stray light:

Potassium chloride solution

Gross Wt :_________________                                                                              Balance : __________

Tare Wt : __________________            

Net Wt : ___________________                           _________ml with water.

Dissolve and dilute accurately 1.2g potassium chloride in sufficient water to produce 100.0 ml.

Sr. No	Wavelength (nm)	Absorbance	Tolerance
1	198.0		Absorbance is greater than 2
2	199.0
3	200.0
4	201.0
5	202.0

Remarks : Satisfactory / Not satisfactory.

Performed By:                                                                                  Checked By:

Date:                                                                                                     Date:

4. Wavelength accuracy (In built test)

Sr No	Wavelength Standard (nm)	Wavelength Observed (nm)	Difference	Limit (nm)
1	656.1			±0.3
2	486.0			±0.3

Remark: Satisfactory / Not satisfactory.

Performed By:                                                                                  Checked By:

Date:                                                                                                     Date:

5. Resolution (In built test)

Sr No	Limit	Observation
1	1.0 nm or less	Nm

Remark: Satisfactory / Not satisfactory.

Performed By:                                                                                  Checked By:

Date:                                                                                                     Date:

6. Baseline flatness (In built test)

Sr.No	Limit	Observation
1	± 0.002 Absorbance

Remark : Satisfactory / Not satisfactory.

Performed By:                                                                                  Checked By:

Date:                                                                                                     Date:

Conclusion: Instrument working is Satisfactory / Not Satisfactory.

Next Calibration Due On:

Performed By: Date:

Checked By: Date: