1.

PURPOSE AND SCOPE

This Standard Operating Procedure describes the procedure for use, maintenance and performance
check of the UV-VIS Spectrophotometer of Model No. _______
PRECAUTION/SAFETY
 Always ensure that laboratory personnel should use proper personal protective equipments like
safety apron, safety goggles (If require) and gloves before using this instrument.
 Do not open the sample compartment during initialisation.
 Do not open sample compartment during baseline correction, auto zero and sampling measuring
mode.
 Before placing the cuvettes in the sample holder clean the outer surface of cuvettes with tissue
paper.
 Do not block any ventilation grills present on the computer.
 Any spills in the sample compartment should be wiped up immediately.
 Properly wash the cuvettes after using them.
 Don’t rub the transparent outer wall of cuvettes with coarse material.
 Do not hold the cuvettes from its transparent optical surface.
 This instrument contains an intense light source (Xenon). Direct viewing of the light source will
damage eye. Operators must never remove the main cover.
 Take any reading after closing the cell compartment.
 Clean the cuvettes after every analysis, wash the cuvettes several time with purified water
and then rinse with methanol or acetone and wipe with tissue paper, dry and store in the
box.
2. PROCEDURE

3.2 Use

 Ensure that there should not be cell inside the sample compartment and make sure that beam
paths are free.
 Switch on the main power and then computer.
 Switch on the power supply to the instrument by pressing power button which is on the right
corner.
 The instrument will internally calibrate for approximately 2 – 3 minutes. Do not open the sample
compartment during this calibration/initialization.
 After finishing the calibration/initialization, green colour appears on power button which means
the instrument is now ready to use. Then open the ‘______________ icon in desktop by double
clicking on it.
 Several icons of modes of measurements will appear on the screen e.g.,
(i) Advance reads: Used for measuring the absorbance of one or more solutions at particular
wavelength or multiple wavelengths without generation of calibration curve.
(ii) Scan: Used for measuring the absorbance of solution from a certain range of wave lengths.
(iii) Concentration: Used for measuring the concentration of solution at particular wavelength
with auto-generation of calibration curve.
(iv) Kinetics: Used for measuring the absorbance of solution with respect to time.
(v) Simple read: Used for measuring the absorbance of one solution at multiple wavelengths
without generation of calibration curve.
(vi) Validate: Used for performance check.
 Warm up time of the instrument is not required after initialization.
 After that select the appropriate mode by double clicking on that icon and set up the required
parameters to run that mode.

3.2.1 Method creating and quantification of compound using standard Curve at particular
wavelength

I. Click on ‘Concentration’ icon, after opening the window go to ‘Set up’ in left corner.
II. Under ‘Cary’ put the wavelength as required, under ‘Standard’ put number of points of
standards, Concentration of standards, unit of standards, under ‘Sample’ put no of
sample, sample name, under ‘Report’ put operator name, comment (if any) and then
click on ‘OK’.
III. Then click on ‘Zero’ in left corner. A window will show ‘Load blank’. Press OK to
read.
IV. Place solvent blank in the cell and put the cell in microcell holder. Click on ‘OK’.
V. Then go to ‘Start’ on the top of the window. Keep standards serially and then samples
one by one in the cell and click on ‘Start’.
VI. After completing the analysis a curve will appear on screen. Save the method in
respective folder and take print out of the curve if necessary.
VII. Carefully wash the cell with distilled water and appropriate solvents and keep it in cell
box.

3.2.3 Method creating for analysis of Sample at particular wavelength without taking standard
curve

I. Click on ‘Advanced reads’, after opening the window go to ‘Set up’ in left corner.
II. Under ‘Cary’ , put the wavelength as required, under ‘Sample’ put no of sample,
sample name, under ‘Report’ put operator name, comment (if any) and then click on
‘OK’.
 III. Then click on ‘Zero’ in left corner. A window will show ‘Load blank. Press OK to
read.
IV. Place solvent blank in the cell and put the cell in microcell holder. Click on ‘OK’.
V. Then go ‘Start’ on the top of the window. Keep samples one by one in the cell and
click on ‘Start’.
VI. After completing the analysis save the method in respective folder and take the print
out if necessary.
VII. Carefully wash the cell with distilled water or appropriate solvent and keep it in cell
box.
Edit a existing method: Open file menu by clicking > Open Method menu by clicking > select the
desire method and open the clicking the method > then click on “ Save as” option from file menu>
give name of the new method > edit the method > save it as new method.
Print out of method or data: Go to print menu > then click on it.

3.3.3 Shutdown procedure

I. After use, save the data, close that icon, then Close the ‘Cary WinUV’ software.
Then switch off the power button of spectrophotometer.
II. Switch off the main power supply from plug and then shutdown the computer.
Use/operational activities shall be documented in the log book of instrument Register ID:
CISCO/RG/15(15).

4 MAINTENANCE

 Preventive maintenance (PM) shall be carried out as per protocol mentioned in AMC (annual
maintenance contract) at least once in a year by external service engineer/ provider.
 In-House/ internal maintenance will be done at least one in three months with the following
points
(i) The exterior surface/ housing and cell compartment of the Spectrophotometer should be kept
clean always. Cleaning should be done with either tissue paper or soft cloth moist with water
or mild detergent. Do not use any organic solvent in cleaning purpose. Power plug shall be
checked visually.
 If any major part is changed or repaired or returned to service or brought back then In-house
performance check is performed to check the performance of instrument as per below section.
 If any malfunction is observed and continued, service engineer shall be called for rectification,
and the same shall be recorded in the instrument maintenance record, Format no. CISCO/EQP &
MNT RCRD/FM/08.
5 PERFORMENCE CHECK

In-House Performance check: Instrument in-built Performance check procedure will be used to
check the performance of instrument by internally / in-house with the following procedure at least
once in every 6 months interval.
Purity checking of Hexane for Resolution power
Before starting the checking purity of hexane, go for “Scan” to check the purity of hexane used in
performance check parameter with respect to blank as MilliQ water.
 Open the ‘Cary WinUV’ icon, click on ‘Scan’.
 Go to ‘Set up’. Under Cary put the following details:
X Mode = Wavelength range (nm), Range Start 420 nm to Stop 260 nm
Y Mode = % T (Transmittance)
 Under Report put operator name and comment (if any). Click on ‘OK’.
 After completion of the test save the method & clean the cuvette with distilled water.
% Transmittance (T) accepted limit of Purity of Hexane = >97% (if below then take another one)
5.1 Three Performance checks Procedure

(i) Resolution Power check (ii) Limit of Stray light test check (iii) Control of Absorbance
accuracy Performance at different wavelengths with K 2Cr2 O7 at UV and VIS regions.
(a) Preparation solutions for each check
For Resolution Power test: 0.020 % (v/v) Toluene in Hexane .i.e. 0.02 ml Toluene in 100 ml
Hexane solution.
For Limit of Stray light: 1.2% (w/v) KCl in MilliQ water i.e. 1.2 g KCl in 100mL solution
For Control of Absorbance accuracy Performance at different wavelengths with
K2Cr2O7 at UV and VIS regions: (i) .005M Sulfuric acid solution 3000 ml or according to
requirement of volume.
(ii) for UV region = 60 ±2 mg reference standard of K2Cr2O7 in 0.005 M Sulfuric acid
(iii) For VIS region = 600 ± 3 mg /lit.in 0.005M Sulfuric acid
(b) Procedure of each cheek
 Open the ‘Cary WinUV’ icon. Click on ‘Validate’.
 Go to ‘Test’. Under ‘Test’ go to ‘EP/BP Test’.
 Under ‘EP/BP Test’ select “Toluene/ Hexane, Stray light and Control of Absorbance
K2Cr2O7”.
 Then go to ‘Control of Absorbance K 2Cr2O7’ and put weight of K2Cr2O7 in mg for UV and
VIS regions.
 Click on ‘OK’
 Then go to ‘Set up’
  Under report window put operator name. Click on ‘OK’.
 Click on ‘Zero’, and then click on ‘Start’.
 A window will show as below where sequential instruction will come to perform the check

Resolution Power Check:

Toluene/Hexane test about to occur

Please place the Hexane blank solution in the sample compartment for
zeroing. Press OK to continue or cancel to abort.
 Put Hexane in cuvette and close the door of the instrument. Click on ‘OK’.
 After completion of the test, below window will come
Toluene/Hexane test about to occur
Please replace the Hexane blank with 0.020 % v/v Toluene in Hexane
sample and close the sample compartment lid.
 Put 0.020 % v/v Toluene in Hexane solution in cuvette and close the door of the instrument.
Click on ‘OK’.
 After completion of the test below window will occur and clean the cuvette

Stray Light Check:

Stray light test about to occur

100 % transmission reading about to occur. Please insert water blank
solution in the front beam. Press OK to continue or cancel to abort.
 Put distilled water in cuvette & close the door of the instrument. Click on ‘OK’.
 After completion of the test, below window will come
Stray light test about to occur
Please insert KCl (198 nm) sample. Press OK to continue or cancel to abort.
 Put KCl solution in cuvette & close the door of the instrument. Click on ‘OK’.
 After completion of the test, below window will come and clean the cuvette
Control of Absorbance accuracy Performance at different wavelengths with K 2Cr2O7 at UV
and VIS regions:

K2Cr2O7 Control of Absorbance test about to occur

Please insert a blank cell containing only solvent in the sample position
for zeroing. Press OK to continue or cancel to abort.
 Put 0.005 (M) H2SO4 solution in cuvette & close the door of the instrument. Click on ‘OK’.
 After completion of the test, below window will come.
K2Cr2O7 Control of Absorbance test about to occur
Please insert the 60 mg/L K 2Cr2O7 solution for the 235 nm, 257 nm, 313
 nm & 350 nm test in the sample position. Press OK to continue or cancel
to abort.
 Put the solution in cuvette & close the door of the instrument. Click on ‘OK’.
 After completion of the test, below window will come.
K2Cr2O7 Control of Absorbance test about to occur
Please insert the 600 mg/L K2Cr2O7 solution for the 430 nm test in the sample
position. Press OK to continue or cancel to abort.
 Put the solution in cuvette & close the door of the instrument. Click on ‘OK’.
 After completion of the test save it in respective folder & take the print out.
 Carefully wash the cuvette with distilled water and keep it in box.

Accepted limits of Criteria of above three Performance Checks:

Parameters Accepted Limit

Resolution Power Ratio= ( Abs at 269 nm) / (Abs at 266 nm)= Limit <1.5
Limit of Stray Light Abs. at 198, 199.0, 200.0, 201.0 and 202.0 nm =Limit > 2.0000
Wave length Set value of Limit of Criteria
(nm) Specific Abs. of Specific Abs.
(A1%1cm)
235.0 124.5
at UV
Control of region 257.0 144.5
± 1.7
Absorbance 313.0 48.6
350.0 107.3
at
VIS 430 15.9 ± 0.2
region

Calculation of Specific Abs. ((A1%1cm) = (Absorbance × 10000)/ Wt. of K2Cr2O7 in mg.

The observed performance check data shall be documented in the Performance Check Record Format
No. CISCO/EQP & MNT RCRD/FM/05, entitled “Instrument Performance Check Record for UV-
VIS Spectra”.

6 DOCUMENTS RETENTION

Old certificates and reports shall be kept in Cup board for three years while current Intermediate
performance check record, Preventive Maintenance report, any Service/break down report (if any)
shall be available in instrument file for the reference as photocopy.
Log Book of Instrument and Log Book of Maintenance shall be kept in retained area after completion
of respective log book, while the current log book (with Register number) shall be with the Instrument
File for recoding the activities.
7 REFERENCES

i) User’s Guide Agilent Cary 60 Spectrophotometer (Agilent Technologies).