Sunday, 8 November 2015

Calibration Procedure for UV - VIS Spectrophotometer


UV-Vis SpectroThese instructions apply to the Varian CARY 1 UV - Vis Spectrophotometer. The calibration is carried out as per AS 3753 - 1990 “Recommended practice for chemical analysis by ultraviolet/visible spectrophotometry”.



AS 3753 - 1990 Section (c) and UV-Vis Cary 1E & 3E Operation manual - Section 7.7. ;

1. Using a purchased Holmium perchlorate standard (5 % w/v solution holmium oxide (HO2O3) in 1.4 N perchloric acid).

2. The wavelengths to be monitored are:

241.0, 250.0, 278.5, 333.0, 345.0, 361.5, 385.5, 416.5, 451.0, 486.0, 536.6, 536.6, 641.0 nm.

3. From the Index page, press the P key to take you to the Parameters page.

4. Press the “Edit” (F2) function key and set the following parameters:

Abscissa nm
Ordinate (Y) min/max 0.5/4.0
Abscissa (X) min/max 236.0/246.0 )*
SBW (nm) 0.2
Signal averaging time (sec) 0.067
Data interval 0.02
Scan rate 18
Lamps on UV-Vis
Base line correct off
Auto scale Yes
Auto store data No
Auto store report No
)* Set the min/max wavelength at least 5 nm away from the wavelength being monitored.

5. Press the Esc key to confirm your selections and return to the command line.

6. Press the A key to go to the Advanced Parameters page.

7. Press the “Edit” (F2) function key and set the following parameters.

Press change (nm) 700
Beam mode Single
Gain 170
Beam interchange Normal
Cycle count/Replicate 1
Collection mode Default

The other parameters will not affect your measurement.

Press the Esc key to confirm your selections and return to the command line.

8. Fill a quartz cell with holmium perchlorate solution and place in the cell holder, close the sample compartment lid and press the “Start” (F8) function key.

9. When the scan is complete, press the M key and then the C key to activate the cursor.

10. Press the Enter key to select ‘Current” and then the X key to select the Peak cursor.

11. The cursor will move to the top of the peak. The wavelength and the absorbance will show in the command line.

12. Press Clt key and Print Screen key to print out the peak.

13. Record the wavelength at which the absorbance is at a maximum on ENFM (73).

14. Repeat steps 3 to 13 for each of the selected wavelengths in turn.

15. The recorded wavelength should be within  1 nm of the nominal wavelength , at all selected wavelengths. Notify the Laboratory Manager if the values are out of specification.


AS 3753 - 1990 Section 3.1.4.

1. Dry a quantity of analytical reagent grade potassium dichromate by heating to constant mass at 130 ± 5 degC.

2. Weigh 0.600 ± 0.003 g of potassium dichromate, and dissolve in sufficient 0.05 mol/L sulphuric acid to produce 1 L of solution.

3. Pipette 10.0 mL of this solution into a 100 mL volumetric flask, and dilute to volume with distilled water.

4. Measure the absorbance of the solution, using distilled water as the reference solution, at 235 nm, 257 nm, 313 nm and 350 nm . The absorbance of the solution at each of these wavelengths should be within ± 0.01 absorbance units of the value specified in Table 1.

Table 1. Absorbance Values for Potassium Dichromate Solution

Wavelength (nm) Absorbance
235 0.747
257 0.864
313 0.292
350 0.640


Use calibration data from routine laboratory work.


AS 3753 - 1990 Section 3.1.3.

1. Fill a 10 mm cell with distilled water and place in the reference cell position.

2. Fill another 10 mm cell with the required test liquid, and place in sample position.

3. Set the wavelength at a reading 100 nm higher than the test wavelength, and set 100 % transmission.

4. Measure the transmission at the test wavelength.

5. Repeat for each test wavelength.

NOTE: Any transmission recorded is due to stray light. Stray light within the instrument should be less than 1 % transmission.

Table 2. Liquids and Instrument Conditions for Determining Stray Radiation.

Spectral range (nm) Test Wavelength (nm) Liquid
210 - 259 220 1% (m/V) Aqueous Nal
250 - 320 285 Aceton
300 - 385 350 5% (m/V) Aqueous NaNO2

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