1.0 Objective:
To provide written procedure for Operation
and Calibration of Photofluorometer.
2.0 Scope:
This
SOP covers Operation and Calibration of Photofluorometer.
3.0 Responsibility:
Jr. Research Officer, Research
Officer: Responsible for operation, calibration and maintenance of the
instrument as per procedure.
Head of Department: Responsible
for calibration and maintenance, timely as per schedule.
QA Officer/QA Manager: Review the records and governing the document.
4.0 Procedure:
4.1 Ensure that the instrument is
clean and free from dust.
4.2 Connect the mains plug of instrument
to the 230 V A.C. mains.
4.3 Set the sensitivity control in LOW
position, STANDARD and BLANK (coarse & fine) controls to minimum i.e.
counter clockwise.
4.4 Insert ‘Primary’ and ‘Secondary’
filters in the appropriate slots marked.
Primary filters marked 1) 366
nm
2) 5113
nm
Secondary filters 1) 4308 nm
2) 3486 nm
4.5 Switch
on the instrument and ensure that the neon lamp on the front panel is glowing.
4.6 Allow the instrument to warm up for a
period of 30 minutes prior to use.
4.7 MEASURING PROCEDURE: [Direct method with computed blank
correction]
4.7.1 Set sensitivity switch control in HIGH
position.
4.7.2
Do not press the SHUTTER,
adjust display to zero by BLANK controls with Coarse and
fine knob.
4.7.3
Insert the test tube containing
the ‘standard solution’ of known concentration into the
test tube holder and align the index marking
on the test tube with the marking on the
holder.
4.7.4 Depress the ‘Shutter and adjust the ‘STANDARD’
controls ‘Coarse’ and ‘Fine’ to set the display reads standard reading or 100
counts to full scale exactly. Always adjust 100 full scale at the lowest
possible sensitivity. In case 100 full scale is not possible at LOW
sensitivity, try at MEDIUM, and if still not possible at MEDIUM try at HIGH.
Call this reading ‘S’. DO NOT DISTURB BLANK CONTROLS.
4.7.5 Replace the standard solution with blank
solution. Depress the shutter and note the display reading ‘B’.
4.7.6
Replace the bank solution with unknown
solution. Depress the shutter and note the reading ‘ U ’.
4.7.7 The blank reading ‘ B ’ is part of the
reading ‘ S ’ and ‘ U ’ and must be
subtracted before ‘ S
’ and ‘ U ’are compared. After subtraction the remainders are proportional
to the concentration of the
considered fluorophor in the ‘Standard’ and the ‘Unknown’.
U
- B = Concentration of Unknown
S - B Concentration
of Standard
OR Concentration
of Unknown = U - B X Concentration of Standard
S
- B
4.7.8
Record the details in usage logbook
(Annexure - 1).
5.0 Calibration procedure:
5.1 SENSITIVITY:
Check the sensitivity of the instrument with the help of 0.2 ppm
solution of Riboflavin in 0.2 N HCl (use solution c prepared for linearity)
secondary wave length 3486 nm and primary wave length 5113 nm as per the
measuring procedure. The solution should show response.
5.2 LINEARITY:
5.2.1 Glacial Acetic Acid : Reagent Grade.
5.2.2 Standard Riboflavin solution :
5.2.2.1 Weigh accurately about 50.0 mg of Riboflavin
working standard in a 500 ml volumetric flask;
add 5 ml glacial acetic acid and about 300 ml of water.
5.2.2.2 Heat
on steam bath, until solution is complete clear, cool and make to mark with
water.
5.2.2.3 Pipette
5 ml of std. solution in 50 ml volumetric flask & dilute to mark with 0.2 N HCl
mark solution (A).
Section (A)
1 ml -----à200
m ( 0.05
mcg / ml ) [ solution a]
|
1 ml----à100
ml ( 0.10 mcg / ml ) [ solution b]
|
2 ml----à100
ml ( 0.20 mcg / ml ) [ solution c]
|
3 ml---à100
ml ( 0.30 mcg / ml ) [ solution d]
|
7 ml---à100
ml--à25
ml--à50
ml ( 0.35 mcg / ml ) [ solution e]
|
all volumetric flask dilute to
mark with 0.2 N HCl.
5.2.3 Measure the readings with the above
solutions.
5.2.4 Record the observations in the calibration
record format (Annexure - 2).
5.2.5 Plot
the graph concentration verses reading; it should show a linear graph.
5.2.6 Frequency: Once in a month and after each maintenance job.
6.0 Precautions:
6.1 Do not spill the sample liquid in the
sample compartment.
6.2 Ensure that the instrument is kept
away from any vibration forming devices.
6.3 Ensure proper handling and
storage of the sample tubes and filters.
7.0 Routine maintenance:
7.1 Clean the instrument with dry
cloth.
7.2 Clean the pre filter and
secondary filter with tissue paper.
8.0 Documentation:
8.1 Annexure – 1 - Photoflurometer usage log book. XXX/FRM/000
8.2 Annexure – 2 - Photoflurometer
calibration record format. XXX/FRM/000
9.0 History of Revision:
Revision No.
|
Effective
Date
|
Revision
details
|
Reason for
revision
|
Annexure – 1
Date
|
Name of Sample
|
A. R. No.
|
B. No.
|
Used by
|
Remark
|
SOP No. : XXX/SOP/000-00 Format No. : XXX/FRM/000-00
|
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Annexure – 2
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CALIBRATION DATA OF
PHOTOFLUOROMETER
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Calibration
Date : Next
Due date for Calibration :
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1.0 Sensitivity :
Working standard no. :
Weight of Riboflavin taken : in a 500 ml volumetric flask, add
5 ml glacial acetic acid and about 300 ml of water. Heat on steam bath, until solution is complete
clear, cool and make to mark with water. Pipette 5 ml of std. solution in 50 ml volumetric flask
& dilute to mark with 0.2 N HCl
mark solution (A).Pipette 2 ml solution (A) in 100 ml volumetric flask
& dilute to mark with 0.2 N HCl.
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2.0
Linearity : SECTION A
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Calibration
status : Satisfactory / Not
satisfactory.
Calibrated
by : Checked by :
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SOP No. : XXX/SOP/000-00 Format No. : XXX/FRM/000-00
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