I.

PURPOSE :
To provide a procedure to operate and Calibrate the spectrophotometer, Model
Shimadzu, UV1700 UV-VIS
II. SCOPE:
The procedure is applicable to the UV/Visible Spectrophotometer being used for
analytical purpose at the Quality Control laboratory,

III. RESPONSIBILITY:
It is the responsibility of the Quality Control personnel involved in the
UV/Visible Spectrophotometer Calibration, to follow the procedure and schedule as mentioned
in this Standard Operating Procedure.
IV. DOCUMENT REFERENCE:
SOPs : Core procedure, Calibration of Equipment
Operation of UV Visible Spectrophotometer
Forms : UV Visible Spectrophotometer Calibration Record
V. OPERATION:
1. 0 PRELIMINARY CHECK:

1.1 Check and ensure that the instrument is clean and suitable for starting the
operation. If it is not, clean and ensure the suitability.
2.0 BASIC OPERATION:
2.1 Put Power button `ON’ provided on left hand side Instrument.
2.2 Keep LCD unit in vertical position.

2.3 As soon as, power is kept ON, LCD will show, initialisation checking of
different parts will start. Each test `PASS’ will be indicated by .

2.4 After every check is over & passed it will display mode.

2.5 When the power is turned `ON’ the spectrophotometer is checked and initialised.
As each item enters it’s initialisation operation it is highlighted. However, if any
abnormality is detected, the initialisation process is interrupted at that item,
without it’s star being highlighted.
2.6 Wait for 15 minutes after automatic initialisation for optimum illumination of UV
lamp.
 3.0 BASIC MODE MENU:

3.1 Photometric :- Used to measure the ABSORBANCE or TRANSMITTANCE at

desired wavelength.
3.2 Spectrum :- Used to scan the wavelength range to measure the absorbance or
transmittance of range selected.

3.3 Quantitation :- Creates a calibration curve from a standard sample and quantitates an
unknown sample.
3.4 Kinetics : - Used to measure the activity from the time dependent changes in absorbance
& % transmittance
3.5 Time scan: - Used to measure the Change in absorbance, transmittances or energy with
time in the fixed wavelength.
3.6 Multi-Component : - Used for multi - component determination.
3.7 Photometric (multi ) : To measured the absorbances or transmittances up to eight
wavelength.
3.8 Optional program pack: Used for optional program pack.
3.9 Utilities: Used for setting and changing the basic operating parameters of the instrument.
D2 lamp off time should be .
On routine basis following modes are used with the given operations.

For measurement of Absorbance / Transmittance :

3.10 After initialisation of UV the basic mode menu will be displayed on the monitor. Press
‘1’ to go in the photometric mode.
3.11 Then press GOTO WL and give the required wavelength e.g. 277 nm & press ‘ENTER’
to get that wavelength.
3.12 Press F1 key to select Absorbance / Transmittance mode.
3.13 Fill the cuvettes with blank solution and place it in both the cell holders & press. Auto
Zero the current wavelength will automatically be set to `0’ Abs. (100% T)
3.14 Then remove front side cuvette, discard the blank solution. Rinse the cuvette with sample
solution. Then fill the cuvette with sample solution & replace in the cell holder. The
monitor will display the Absorbance of the sample solution at the wavelength displayed.
3.15 Then press ‘MODE’ to come back to the basic mode menu.
 To get the spectrum in the desired range of wavelengths:

3.16 When in basic mode menu press ‘2’ to go in the spectrum mode. Then ‘6’ different
parameters will be displayed.
3.17 Press `1’ to select Absorbance / Transmittance / Energy mode.
3.18 Press ‘2’ & then give the higher wavelength of the desired range and press ‘ ENTER’.
Then give the lower wavelength & press ‘ENTER’.
3.19 Then press, ‘3’ and give higher value of Absorbance which is greater than the expected
Absorbance value & then press ‘ENTER’. Lower value should be kept zero only, so
press ‘ENTER’.
3.20 The speed of the scanning can also be changed. Press ‘4’. Then select ‘1’, ‘2’,`3’`4’
and `5’ for scanning with `Very Fast’, `Fast’, ‘Medium’, ‘Slow’ and `Very slow’
speed respectively.
3.21 Press `6’ to select display mode i.e. sequential or overlay.
3.22 Place the cells filled with blank solutions in both the cuvette holders and press ‘F1’ so
that baseline correction will be done.
3.23 Rinse the cuvette with sample solution. Place the sample in the front side cuvette holder.
Then press ‘START’ key.
3.24 The scanning in the given wavelength range will be done.

3.25 To know the Absorbance value at a particular wavelength in the range given, make use of
‘<-----’ and ‘------> ‘ keys.

3.26 Once scanning is over. Press F2 and select 3 for peak detection and F4 for valley. Press
graph to obtain scan with peak detection. Then press print key.

3.27 Press print key in each step to get required copy.

3.28 Then press ‘Return’ & ‘Mode’ to come back to the basic mode menu.

4.0 MAINTENANCE :

4.1 Do not keep cells above the outside body of the spectrophotometer. Clean the cells
perfectly and wipe out sidewalls with the tissue paper in one direction only. Do not hold
the cells with their optical surfaces between the fingers.
4.2 Press the keys and handle the instrument very carefully.
4.3 The extinction of the cells intended to contain the solution to be examined and the
reference liquid when filled with the same solvent, should be identical. If this is not the
case, an appropriate correction must be applied. The tolerance on the path length of the
cells used is +/- 0.005 cm. Cells should be cleaned and handled with great care.

4.4 When not in use, put off the power switch of the instrument and then the mains.

4.5 Check condition of Silica gel for effectiveness and replace once in a week (or) earlier if
required.

4.6 Ensure that during spectrophotometer initialisation process spectrophotometer makes

various checks with preset initialised parameters such as Detection of wavelength,
lighting of WI lamp, optical alignment of D 2 lamp, Energy check of D2 lamp, optical
alignment of WI lamp, Energy check of WI lamp, Baseline correction etc. which were
appropriately evaluated by the instrument with no error message. In case any
abnormalities observed in the above parameters, contact service engineer.
VI. CALIBRATION PROTOCOL: Calibration frequency: Once in three
months
1.0 Control of wavelengths

1.1 Preparation of 1.4 M Perchloric acid solution :

Take 11.5 ml of Perchloric acid (about 60%w/w strength) into a clean 100 ml
volumetric flask.
Dissolve and dilute to the volume with water
Note : Dilute 8.5 ml to 100 ml with water in case of 70% Perchloric acid.

1.2 Preparation of Holmium perchlorate :

01. Weigh accurately about 400 mg Holmium oxide.
2. Transfer into a 10 ml volumetric flask containing 5 ml of 1.4 M Perchloric acid

solution.
03. Shake the solution well to dissolve Holmium oxide.
04. Make up the volume to the mark with 1.4 M Perchloric acid solution.
Procedure:
01. Take a pair of matched quartz cells having a path length of 1 cm.
 02. Perform the base line correction with 1.4 M Perchloric acid solution from 200 to
600 nm.
03. Remove the cell from sample compartment.
04. Rinse the sample cell with Holmium perchlorate solution, fill the cell with the
same, clean the smooth surfaces of the cell with tissue paper and place it in the sample
compartment

Acceptance criteria:

Wavelength Maximum Tolerance

241.15 nm 240.15 to 242 .15 nm
287.15 nm 286.15 to 288.15 nm
361.50 nm 360.50 to 362 .50 nm
536. 30 nm 533.30 to 539.30 nm

2.0 Control of absorbances:

2.1 Preparation of 0.005 M Sulphuric acid :
01. Take a clean 100 volumetric flask and fill to the mark with water.
02. Cautiously add 2.8 ml Conc. Sulphuric acid to the above flask.
03. Dissolve and dilute to the volume with water.
04. Take 5 ml of the above solution into a 500 ml volumetric flask, dissolve and
dilute to the volume with water.
Preparation of K2Cr2O7 UV solution :
01. Take about 200 mg of Analytical Reagent grade K 2Cr2O7 into a weighing bottle
and heat at 130oC in a laboratory oven for 2 hours.
2. Cool it in a desiccator and weigh about 120 mg in a 100 ml volumetric flask
containing 50 ml of 0.005 M Sulfuric acid.
03. Shake the flask well to dissolve Potassium dichromate completely. Make up the
volume with 0.005 M Sulfuric acid.
04. Dilute 5ml of the above solution to 100 ml with 0.005M Sulfuric acid.
2.2 Procedure :
01. Take a pair of matched quartz cells having a path length of 1 cm.
02. Perform base line correction with 0.005 M Sulphuric acid from 200 to 400 nm.
03. Remove the cell from sample compartment.
 04. Rinse the sample cell with potassium dichromate UV solution, fill the cell with
the same, clean the smooth surfaces with tissue paper .
05. Place the cell in sample compartment, scan from 200 to 400 nm and measure the
absorbances at 235 nm, 257 nm, 313 nm and 350 nm. Calculate the specific
absorbance using the following equation.
Specific absorbance at x nm = Absorbance at x nm
Concentration of Potassium dichromate
06. The specific absorbances against each wavelength should be as follows :

Wavelength (nm) Specific absorbance Maximum tolerance

(1%, 1 cm)
235 124.5 122.9 to 126.2
257 144.5 142.8 to 146.2
313 48.6 47.0 to 50.3
350 107.3 105.6 to 109.0

3.0 Photometric accuracy :

3.1 Preparation of Potassium nitrate stock solution(14.20 % w/v)

Weigh accurately about 14.2 g of Potassium nitrate in a 100 ml volumetric flask, dissolve
and dilute to volume with water.

3.2 Preparation of solution A (1.065% solution):

Pipette out 7.5 ml of the stock solution into a 100 ml volumetric flask, dissolve and
dilute to the volume with water.

3.3 Preparation of solution B (0.710% solution):

Pipette out 5.0 ml of the stock solution into a 100 ml volumetric flask, dissolve and
dilute to the volume with water.

3.4 Preparation of solution C (1.065% solution):

Pipette out 2.5 ml of the stock solution into a 100 ml volumetric flask, dissolve and
dilute to the volume with water.

3.5 Procedure:
Measure the absorbances of the above solutions at about 302 nm against water blank.
Check the absorbance of the above solutions at 302 nm using milli Q water as blank.
 Record the absorbance at each wavelength in the Calibration Data Sheet.
Check the Absorbance for each solution against the acceptance criteria given below :
Calculate correlation coefficient using mean of the absorbance of respective mean
concentration.
3.6 Acceptance Criteria:

01. Solutions Absorbance Acceptance Criteria

% w/v at 302 nm ± 0.010

1.065 0.751 0.741 to 0.761

0.710 0.500 0.490 to 0.510
0.355 0.250 0.240 to 0.260

02. Correlation coefficient: NLT 0.999.

4.0 LIMIT OF STRAY LIGHT:

4.1 Preparation of 1.2% W/V Potassium Chloride Solution:

01. Take about 3 gm of Potassium chloride dry at 250OC for about 1 hours.
02. Weigh accurately about 1.2 g of Potassium chloride.
03. Transfer into a 100 ml volumetric flask containing 75 ml water.
04. Shake the flask well to dissolve completely and make up to the volume with water.
4.2 Procedure :
01. Take a pair of matched quartz cells having a path length of 1 cm.
02. Perform the base line correction with water.
03. Remove the cell from sample compartment.
04. Rinse the cell in the potassium chloride solution, fill the cell with the same, clean the

smooth surfaces with tissue paper and place it in sample compartment.

05.. Measure the absorbance at 200 nm.
4.3 Acceptance Criteria: The absorbance should be more than 2.

5.0 RESOLUTION POWER :

5.1 Preparation of 0.020 % v/v of toluene in Hexane solution: Dilute 2 ml of Toluene to

200 ml with Hexane. Further dilute 2 ml of above solution to 100 ml with Hexane.
(Note : Use only spectroscopy grade Toluene and Hexane)
5.2 Record the spectrum in the range 260 to 275 nm of 0.020 % v/v toluene in Hexane
using Hexane in the reference cell.
5.3 Calculate the ratio of the absorbance at the maximum at about 269 nm to that at the
minimum at about 266 nm.
Acceptance Criteria : Ratio is not less than 1.5.

6.0. SPECIFICATION OF CELLS:

In `Photometry mode ’ Select transmittance mode. Set the wavelength at 200 nm. Then
press `autozero’ to get 100 % transmittance with air blank in both the cell holders. Then
place the cell in the sample compartment filled with milli Q water using air blank and
read the transmittance. Similarly repeat the operation for 220 nm and 240 nm. Repeat
the test for second cell. The transmittance values should meet the requirement as given
in the following table.
Acceptance Criteria :

Wavelength % Transmittance
200 nm 73 to 77 %
220 nm 83 to 87 %
240 nm 85 to 89 %
7.0- IO - LINE FLATNESS :

a. With air blank in the sample and reference compartment, perform the test.
b. Scan in the wavelength range 220 to 700 nm.
c. Obtain the spectrum in the % transmittance mode.
Acceptance Criteria : The Io line obtained should not deviate from a horizontal
line at any point by more than 99.54 - 100.46 % transmittance.