Operation and Calibration of UV-Visible Spectrophotometer
Operation
·
Ensure that the instrument is clean & calibrated.
·
Turn On the main paper of instrument &
computer connected with the instrument.
·
After Turning ON the power UV
Spectrophotometer will starts the initialization to checks the function of all parameter,
initialization will complete about after 5 minutes.
·
Wait for 15 minutes for the proper
stabilization and illumination of UV lamp.
·
Lab Solution icon will have displayed on your desktop screen. Double
click on that icon. Software becomes open.
·
Enter User name & password and then click
on ok.
·
Now go to instruments on the left hand side & select project as per
required or current, then double click on the icon UV.
·
Now Window display will show.
·
Click on connect in the bottom of the screen if any test (Spectrum &
Photometric) is open.
·
When all the parameters are checked (in green Status), means your
instrument is initialized. Click on ok button.
·
After this you have to select which test you are going to do (either
spectrum or photometric).
· Now the following window will have opened:
Spectrum test: Operation and Calibration of UV-Visible
Spectrophotometer
·
While doing spectrum test just click on the spectrum test first as shown
in the
figure:
·
Before doing anything you have to make a method first for this just
click on the
Edit and click on method button as shown in the figure:
·
For make method, you have to specify the range at which you have to do
the
test.
·
After this make the test speed according to you as fast, medium, slow,
very slow.
·
Also you can specify the scan mode as single, auto or repeat. In scan mode
single, in the end of the scan, a window will pop up, there you have to specify
the name of the sample by clicking file name and edit file name .in the auto
scan mode you have to specify the storage location by clicking on filename
(three dots). In the repeat scan mode, you can scan the same sample more than
once. For this you have to specify the no. of scans & time interval between
each scan.
· Specify your method of making the sample or standard as its volume, dilution, path length (fix path length at 10 mm as it is the path length of the cuvette).
· Click on Instrument parameters and click on measurement mode as
absorbance or transmittance as follows:
· After this press ok.
·
Now keep your base/ blank on both sides & then press base line
button.
·
while doing baseline a window will pop up as:
· This is the window you have mentioned the wavelength range as in the
method above. Just click on ok button. While doing this all the buttons are
getting invisible as
·
When baseline is over then all the buttons are highlighted as earlier.
·
Now put your standard/ sample on front side (towards the user) &
press start button to start the test.
·
At the end when your test is done it will ask to give the path where you
have to save the file. Also specify the name or batch number. (only in the
single scan mode).
·
Press the save icon at the end. Your test results are saved at your
desired location.
·
Now the system is ready to scan other sample. If your blank is remaining
same, you can directly scan your next sample without doing baseline.
Photometric Test: Operation and Calibration of UV-Visible
Spectrophotometer
·
While doing Photometric test just click on the Photometric test first as
shown in the figure: -
·
Before doing anything, you have to make a method first for this just
click on the method button as shown in the figure: -
·
In making method you have to specify the wavelength or the wavelengths
at
which,
you have to take the absorbance. Every time you have to add the
wavelength
otherwise you can’t go further.
·
If you don’t click on add button, then a window will pop up as:
·
After adding the wavelength or wavelengths, click next, another window
will
pop
up as:
·
Select type Raw data, single data & Multi point.
Operation and Calibration of UV-Visible
Spectrophotometer
·
After this you have to select the type as Multi point, Single point, K
Factor, or raw
data.
(In multi-point you may use different standards, in single point you can use
only
one standard, in raw data no standards are used).
·
Select the wavelength WL1 & units used for your sample, then click
on next
(if doing multi point then have to specify
absorbance or concentration vise, in single point specify the standard
concentration and in raw data just click on next).
·
Specify the path where you have to save the results data as: -
·
After clicking finish button, another window will pop up to select
absorbance or
transmittance as.
·
Now go to instrument parameter to select one of the above (Absorbance or
Transmittance as in the figure. Now click on close button.
·
Then do the test as there are two sides, reference side and sample side,
keep
your
base/ blank in both sides. Then press auto zero if only one wavelength is
used.
But if multi wavelengths are used then you have to click base line.
·
If you are just checking your sample without any standard, then only one
window will pop up for sample only. But if you
are also using standards then there are two windows, one for standard &
another for sample as shown in the figure:
·
After this keep sample/standard on front side (towards the user).
·
While doing sampling, if standards are used the “Read Std” (means read
standard)
button will not be highlighted for this just follow the next point.
·
Write your batch no. or name of the sample/standard in Sample ID. Then
click
on
type your icon in the bottom is highlighted. (If Single point, then
concentration will be automatically highlighted, if multi point, then you have
to enter the concentration of the standard. Then only Read Std. will be
highlighted).
·
Click on read std. or read unknown (in the sample case), results are
shown in the table.
·
Press save icon. results are saved.
To open the saved data from
computer:
·
Go to file & click on open. Give the Name of the Project where file
is already. Open that, spectra or photometric data will be opened on the
screen.
Printing the spectrum or
photometric data:
·
Take out the printout after saving data click on window and click on
report
generator.
·
For taking printouts, select the report just after opening the data as
follows:
·
Just select your templet for the printout as spectrum peak pick,
spectrum point
pick,
spectrum peak area, photometric report or quantitation report
·
See the print preview then take the print out of desired samples.
Calibration:(Frequency: Half
yearly or after major maintenance)
·
Connect the instrument with the main power supply, and switch ON
the
instrument.
·
Calibration will be carried in verification and diagnosis mode.
·
The Spectrophotometer shall be calibrated for following parameters.
· Control of absorbance
· Resolution Power
· Limit of stray light
· Control of wavelength
· Linearity
· Quartz cell pair verification
· Resolution
· System Precision
·
Preparation of 0.005M Sulphuric
acid Solution: Take 0.27 ml of concentrate
Sulphuric acid in one-liter volumetric
flask and make volume up to mark with
purified water & shake.
Potassium dichromate Solution:
Solution (i): Dry potassium dichromate at 130°C up to constant
weight. Weigh about 0.057 g to 0.063 g
of potassium dichromate and dissolve in sufficient 0.005M sulphuric acid
solution to produce 1000 ml.
Solution (ii): Dry potassium dichromate at 130° C up to constant
weight. Weigh about 0.057 g to 0.063 g
of potassium dichromate and dissolve in sufficient 0.005M sulphuric acid
solution to produce 100 ml.
Procedure: Fill the potassium dichromate
solution (i) into the sample cell of spectrophotometer and place in the sample
compartment. Take absorbance at nm using 0.005M sulphuric solution as blank.
Further change the wavelength to 257, 313 and
350 nm, and repeat the steps using 0.005M sulphuric acid solution as blank.
Fill the potassium dichromate solution (ii) into the sample cell of
spectrophotometer and place in the
sample compartment. Take absorbance at using 0.005M sulphuric solution as
blank.
Calculate the E 1% value of 1-cm cell for each wavelength.
Sample absorbance x 1000
Specific
absorbance = ---------------------------------------------------- =
Sample wt. (g) x 100
·
The tolerance limits of the E1% (1cm) for each wavelength are given in
table:
|
Wavelength (nm) |
Specific absorbance |
Maximum tolerance |
|
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 |
|
430 |
15.9 |
15.7
to 16.1 |
· Record the document activity
in Calibration Record of UV-VIS Spectrophotometer Format (At the End of
article).
Resolution Power
·
Preparation of solution: Take 1 ml of toluene in 50 ml
of hexane, further dilute 1 ml of above
solution into 100 ml with hexane. (0.02%v/v solution).
·
Fill this solution in the sample cell and read the absorbance at 269 and
266 nm by using hexane as blank solution.
·
The ratio of absorbance at maximum at about 269 nm to that at minimum at
about 266 nm should not less than 1.5. Record the
document activity in Calibration Record of UV-VIS Spectrophotometer Format (At
the End of article)
Limits
of Stray Light
Preparation
of solution: Take 1.2 g of potassium chloride in 100 ml of purified water (1.2 %
w/v).
·
Fill the above potassium chloride solution in the sample cell of path
length of 1 cm and observe
the absorbance at about 200 nm
and it should be greater than 2.0 when compared with purified water as
reference liquid.
·
Record the document activity in Calibration Record of UV-VIS
Spectrophotometer
·
Preparation of 1.4 M Perchloric acid solution:
Take
50 ml water in 100 ml volumetric flask add Perchloric acid 70 % about 11.50 ml
(11.48 ml) diluted with water.
·
Preparation of holmium perchlorate solution (4.0 % w/v):
Take 400 mg of holmium oxide in 10 ml
volumetric flask and add 5 ml 1.4 M
perchloric acid shake & dissolve dilute
with 1.4 M perchloric acid.
·
The permitted tolerance is ±1 nm for the range of 200 nm to 400 nm and
±3 for the range of 400
to 600 nm.
·
Control of wavelength shall be checked with Holmium Perchlorate solution
or alternatively,
Holmium
filter shall be used.
·
Enter the following parameter in the instrument:
Wavelength Max. 600nm
Wavelength Min.
200nm
Speed Slow
·
Set the zero absorbance and place the 1.4 M Per chloric acid solution in
the
sample compartment of spectrophotometer.
·
Start the scanning, and take the printout.
·
Check the maxima. It should appear at 241.15 (±1nm), 287.15 (±1nm),
361.5
(±1nm) and 536.3nm (±3nm).
·
Record the document activity in Calibration Record of UV-VIS
Spectrophotometer (At the End of
article)
·
Fill the Calibration Tag and affix it on the
instrument
Format No.
(At the End of article)
·
Report any discrepancy observed during calibration of instrument to
Quality Control In-Charge
or his designee for corrective and preventive action.
·
Quality Control In-Charge or his designee informs the defect to
Maintenance Department service
engineer to rectify the defect. Affix ‘Under Maintenance’ label on the
instrument.
·
Linearity:
Stock solution preparation: Weigh accurately 100 mg of
potassium dichromate AR grade
previously dried at 1300C up to constant weight and transfer to 100 ml volumetric flask
and dilute up to volume with 0.005M H2SO4.
·
Prepare the solution for linearity test by diluting stock solution.
1
ml stock solution – 100 ml with 0.005M H2SO4 10
ppm
2
ml stock solution – 100 ml with 0.005M H2SO4 20
ppm
2
ml stock solution – 50 ml with 0.005M H2SO4 40 ppm
3 ml stock solution – 50 ml with 0.005M H2SO4 60 ppm
2
ml stock solution – 25 ml with 0.005M H2SO4 80
ppm
10
ml stock solution – 100 ml with 0.005M H2SO4 100
ppm
·
Measure the absorbance of each solution in duplicate after auto zero
with 0.005M H2SO4 at
each selected wavelength of 235 nm, 257 nm, 313 nm, 350 nm and 430 nm.
Acceptance criteria:
Correlation coefficient should not less than 0.99 for each wavelength
Quartz cell pair verification: Operation
and Calibration of UV-Visible Spectrophotometer
·
Clean the quartz cells with soap solution and rinse thoroughly with
purified, dry the cell.
·
Auto zero the system without cells, use air as a blank.
·
Take 1st cell, rinse and fill it with purified water. Measure
the transmittance against air at 220 nm and 240 nm.
·
Take 2nd cell, rinse and fill it with purified water. Measure
the transmittance against air at 220 nm and 240 nm.
·
Transmittance limit at 220 nm -
NLT 75%
Transmittance limit at 240 nm
– NLT 75%
·
Resolution (2nd
derivative)
·
0.02% v/v solution of toluene
in methanol: - Transfer 1 ml of toluene in 50 ml VF and dilute up to volume with
methanol, further dilute 1 ml of this solution to 100 ml with methanol.
·
Fill the quartz cells with methanol and start base line correction from
290 nm to 230 nm.
·
Fill the quartz cells with solution and Scan the sample in the range of
290 to 230 nm, the second order derivative spectrum by click on operation
followed by manipulate then select transform, derivative(2ndorder)
&delta (1). Finally click on calculate & take graph print on peak pick
format.
·
The spectrum shows a small negative extreme (or trough) located between
Two large negative extreme at about 261 nm and 268 nm should be clearly visible.
·
Measure the absorbance at 261 nm, 263 nm and 265 nm.
·
The ratio of difference between absorbance at 263 nm and 265 nm to the difference
in the absorbance at 263 nm and 261 nm should not less than 0.2.
·
Precision:
·
Use the potassium dichromate solution (i) as prepared for Control of absorbance.
·
Fill the potassium dichromate solution (i) into the sample cell of spectrophotometer and place in the
sample compartment.
·
Take 10 replicate absorbance of the same solution at 235 nm using 0.005M sulphuric solution as blank.
Acceptance criteria for
precision: Relative standard deviation not
more than 2.0%
·
Cleaning & Precautions:
·
Dip the cell in Dilute Nitric Acid for 10
minutes and remove. Wash with purified water to remove acid.
Protect
the cell from scratches.
·
Always wipe the optical surface of the cell
using a clean tissue paper to keep it dry and free from finger marks, just
before placing it in the cell holder.
·
Take care not to spill liquids on the
spectrophotometer and if there is any spillage, then immediately clean all
spilled materials from the affected area and wipe it with tissue paper or lint
free cloth.
·
Do not leave samples, particularly which
produces fumes or evaporates on standing, in the sample compartment.
·
Clean the cell with purified water before use.
·
Clean the cell after use with purified water
and then with Isopropyl Alcohol.
Dry it with tissue
paper and keep in respective place.
Format: Operation
and Calibration of UV-Visible Spectrophotometer
CALIBRATION RECORD OF UV-VISIBLE
SPECTROPHOTOMETER
Frequency of calibration : Half yearly
Date of calibration :
Next due date for calibration:
Control
of Absorbance
Preparation of Potassium Dichromate Solution:
Solution-(i): Take ………..g (0.057g to 0.063g) of K2Cr2O7
à 1000ml with 0.005M sulphuric acid.
Solution-(ii): Take ………..g (0.057g to 0.063g) of K2Cr2O7
à 100 ml with 0.005M sulphuric acid.
Make of
K2Cr2O7…………………....
Lot No. / Batch
no. of K2Cr2O7………………………… (Previously dried at 130°C)
Balance ID
No………………………………
Oven ID No.
……………………………….
|
Wavelength |
Observed absorbance |
E1% (1 cm ) |
Acceptance criteria E1% |
|
235 nm |
|
|
122.9 – 126.2 |
|
257 nm |
|
|
142.8 – 146.2 |
|
313 nm |
|
|
47.0 – 50.3 |
|
350 nm |
|
|
105.6 – 109.0 |
|
430 nm |
|
|
15.7-16.1 |
Calculation for
E1% (1 cm) =
Sample absorbance x 1000
(1) For 235 nm =
-------------------------------------------------
=------------------------------------
Sample weight (g) x 100
Sample
absorbance x 1000
(2) For 257 nm =
--------------------------------------------------
=------------------------------------
Sample weight (g) x 100
Sample
absorbance x 1000
(3) For 313 nm = -----------------------------------------------
=------------------------------------
Sample weight (g) x 100
Sample absorbance x 1000
(4) For 350 nm =
-------------------------------------------------
=------------------------------------
Sample weight (g) x 100
Sample absorbance x
100
(5) For 430 nm = ------------------------------------------------
=------------------------------------
Sample weight (g) x 100
Resolution Power
Preparation of toluene solution (0.02%v/v): Take …….. ml (1ml) Toluene in…….. ml (50 ml)
of Hexane, further dilute 1 ml of above
solution in to 100 ml with Hexane.
Make : Toluene …………………… Hexane ……………………….
Lot No/ Batch No: Toluene…………………….
Hexane……………………….
|
Observed absorbance at |
Ratio of Absorbance (absorbance at about 269
nm/ absorbance at about 266 nm) |
Acceptance criteria |
|
|
About 269 nm ( ) |
About 266 nm ( ) |
||
|
|
|
|
NLT 1.50 |
Limit
of Stray Light
Preparation of
potassium chloride solution (1.2 %w/v): Take …….. g (1.2 g) in …….. ml (100 ml) of purified
water.
Make/supplier
……………..
Lot No/ Batch no.
of KCl…………………………
Balance ID
No………………………………
|
Wavelength |
Observed absorbance |
Acceptance criteria |
|
198 - 202 nm (200 nm) |
|
NLT 2.0 |
Control
of Wavelength:
Preparation of
1.4 M Perchloric acid solution:
Take 50 ml water in 100 ml volumetric flask add Perchloric acid 70 %
about
11.50 ml (.......... ml) diluted with water.
Preparation of
holmium perchlorate solution (4.0 % w/v):
Take ................ mg (about 400 mg) of holmium oxide in 10 ml
volumetric flask and add 5
ml 1.4 M Perchloric acid shake & dissolve dilute with 1.4 M
Perchloric acid.
Make/supplier
……………..................................
Lot No/ Batch no.
of Ho2O3………………………….
Balance ID
No………………………………...............
|
Observed Wavelength for absorption Maxima |
Wavelengths as per Manufacturer’s COA |
Tolerance |
|
|
241.15 nm |
± 1nm |
|
|
287.15 nm |
|
|
|
361.5 nm |
|
|
|
536.3 nm |
± 3nm |
Linearity:
Potassium dichromate B.No.
……………………….. Make ………………………
Stock solution preparation: Weigh accurately ………(100 mg) of potassium
dichromate AR grade and transfer to 100 ml volumetric flask and dilute up to
volume with 0.005M H2SO4.
1
ml stock solution – 100 ml with 0.005M H2SO4 10
ppm
2 ml stock solution – 100 ml with 0.005M H2SO4 20
ppm
2 ml stock solution – 50 ml with 0.005M H2SO4 40 ppm
3 ml stock solution – 50 ml with 0.005M H2SO4 60 ppm
2 ml stock solution – 25 ml with 0.005M H2SO4 80
ppm
10 ml stock solution – 100 ml with 0.005M H2SO4 100
ppm
Take absorbance
in duplicate at each selected wavelength of 235 nm, 257 nm, 313 nm, 350 nm and
430nm. of all solutions (10ppm to 100ppm). Use average of absorbance for
correlation coefficient.
|
1st Stage |
|
Correlation coefficient for 235 nm …………………. (NLT 0.99) |
|
Correlation coefficient for 257 nm ………………… (NLT 0.99) |
|
Correlation coefficient for 313 nm ………………… (NLT 0.99) |
|
Correlation coefficient for 350 nm ………………… (NLT 0.99) |
|
Correlation coefficient for 430 nm ……………….. (NLT 0.99) |
Quartz cell pair verification
a. Take 1st cell, rinse and fill it
with purified water. Measure the transmittance against air at 220
nm ………………… (NLT
75%)at 240 nm …………………
(NLT 75%)
b. Take 2nd cell, rinse and fill it
with purified water. Measure the transmittance against air at 220 nm ………………… (NLT 75%)at
240 nm …………………
(NLT 75%)
Resolution (2nd
derivative)0.02% v/v solution of toluene in methanol:- Transfer 1 ml of toluene in 50 ml VF and
dilute up to volume with methanol, further dilute 1 ml of this solution to 100
ml with methanol.
Toluene B. No. ………………….. Make ……………………..
Methanol B.No………………….. Make ……………………
|
Sr. No. |
Wavelength |
Absorbance |
Difference
in absorbance 263 nm and 265 nm
|
|
1. |
261 nm |
|
absorbance at 263 – absorbance
at 265 = ………………………… = ……………… |
|
2. |
263 nm |
|
|
|
3. |
265 nm |
|
Difference
in absorbance 263 nm and 261 nm
|
|
absorbance at 263 – ( absorbance at 261 = ………………………… = ……………… |
Difference in absorbance at 263 nm and 265 nm
= …………………………………………………………………………………………………………………
Difference in
absorbance at 263 nm and 261 nm
=
………………………………………..… = …………………………………………... (Acceptance criteria: NLT 0.2)
Precision
Use the potassium
dichromate solution (i) as prepared for Control of absorbance
Absorbance at 235 nm
|
Sr. No. |
Absorbance |
|
1. |
|
|
2. |
|
|
3. |
|
RSD …………………. (NMT 2.0%
Remark: Instrument meets / does not meet with acceptance criteria. The calibration is OK /
