Sei sulla pagina 1di 12

OPERATION AND CALIBRATION OF HPLC SYSTEM (SHIMADZU)

1.0. OBJECTIVE:
The objective of this SOP is to ensure that the instrument performs satisfactorily and
gives accurate and reproducible data.

2.0. RESPONSIBILITY:
2.1 The Chemist/ Executive - Quality Control shall be:
2.1.1. Responsible for operating the HPLC as per the SOP.
2.1.2. Responsible for timely calibration of HPLC System as per the SOP.
2.1.3 Responsible for ensuring the adherence of SOP.

3.0. ACCOUNTABILITY:
Head - Quality Control

4.0. PROCEDURE:
4.1 PROCEDURE FOR GENERAL CLEANING:
4.1.1 Ensure that the power supply to the instrument is switched off and main
cord is removed from supply.
4.1.2 Clean the instrument with a clean dry cloth every day. A wet cloth dipped in
dilute soap solution may be used occasionally.
4.1.3 Precaution must be taken to clean the instrument immediately with dry
cloth.

4.2 OPERATING INSTRUCTIONS:


4.2.1 Ensure that the instrument is properly connected to the power supply.
4.2.2 Fix the column and prepare the mobile phase.

4.3 PREPARATION OF MOBILE PHASE


4.3.1Use HPLC grade solvent / water & AR/Excel/ HPLC grade reagent only.
4.3.2 Filter the mobile phase through 0.45 membrane filter after mixing it in
required proportion and de-gas on ultrasonic bath for about 5 minutes.

4.4 PROCEDURE TO GET STARTED


4.4.1 Switch on pump, auto injector, detector and then system controller.
4.4.2 Turn the drain valve knob to 180 in anti- clockwise to open the drain valve
to run the purge system.
4.4.3 After the purge is over close the drain valve.
4.4.4 Set desired flow rate by pressing function key.
4.4.5 Press pump key. The pump will run and indicator will glow.
4.4.6 Purge the auto injector by pressing purge key on auto sampler display.
4.4.7 Enter the desired wavelength on detector using function key.

4.5 PROCEDURE TO OPERATE CLOSE UP


4.5.1 Switch on the computer monitor and printer.
4.5.2 Double click on the main Menu of SHIMADZU Icon.
4.5.3 It will show Configuration, Real Time, and Sample Schedule & Post Run
Analysis.
4.5.4 Open Real Time.
4.5.5 From the file menu create a new method or load the existing method.
4.5.6 Create a new sample schedule and feel sample name, batch number,
sample volume, method name and file name, after then save the schedule.
4.5.7 Run Sample Schedule.
4.5.8 For system suitability test, inject six continuous injections of same standard
and RSD should not be more than 2.0 %, otherwise it is specified in standard test
procedure.
4.5.9 After every ten injection of sample (5 samples in duplicate), the standard
solution shall be injected 3 times and the RSD shall be calculated and ensure that it is
within the limit.
4.6 SHUT DOWN PROCEDURE
4.6.1 Close Sample Schedule, Real Time and then CLASS LC 10 to Software.
4.6.2 From Start button select Shut down and click yes.
4.6.3 Switch-off the computer.
4.6.4 Switch-off CBM.
4.6.5 Switch-off Detector then, auto sampler.
4.6.6 Stop pump by pressing, Pump key and switch off the pump module.

4.7 CALIBRATION PROCEDURE.


4.7.1 FOR PUMP:
4.7.1.1 Disconnect the column and connect the inlet and outlet tubings
with a union.
4.7.1.2 Prime all the lines at 5 ml/min flow rate with water and ensure that
flow line is free from air bubbles.
4.7.1.3 Set the flow rate at 1ml / min and collect the mobile phase (water)
in a dry preweighed beaker and collect the mobile phase for 10 min. weigh the beaker
to get the weight of mobile phase.
4.7.1.4 Calculate the flow rate by dividing the weight obtained with weight
per ml and 10 (run time).
4.7.1.5 Calculate the corresponding flow rate. Carry out the experiment in
duplicate.
4.7.1.6 Repeat the same procedure for Pump- B
4.7.1.7 Record the observation in Annexure -1
4.7.1.8 Acceptance criteria: Flow rate should be in between 0.99 to 1.01 ml
/ min.

4.7.2 FOR GRADIENT VALVE:


4.7.2.1 Install union in place of column & flush solvent lines (A&B) at flow
rate of 2ml/min with water.
4.7.2.2 Prepare the mobile phase.
4.7.2.3 Prepare 0.3% acetone with HPLC grade water.
4.7.2.4 Fill reservoir A with 100% HPLC grade water & reservoir B with
0.3% acetone in HPLC grade water as mobile phase.

4.7.3 INSTRUMENT SET UP:


4.7.3.1 Enter the following time program:

TIME FUNCTION VALUE


0.01 B CONC 10
10.00 B CONC 10
10.01 B CONC 50
20.00 B CONC 50
20.01 B CONC 90
30.00 B CONC 90
30.01 B CONC 100
40.00 B CONC 100
40.01 B CONC 0
50.00 B CONC 0

4.7.3.2 Use detector at wavelength of 254 nm.


4.7.3.3 Record the printout of gradient valve test as per Annexure - 2.
4.7.3.4 The gradient valve test shall be accepted if actual
concentration with 1% of set concentration.

4.7.4 CALIBRATION OF INJECTOR:


CHECK FOR PRECISION: Purge the injector system with 100% water to ensure
the complete washing of injector.

4.7.5 STANDARD PREPARATION:


4.7.5.1 Transfer about 50 mg of Uracil to a 250ml volumetric flask. Add
100ml of Methanol, sonicate to dissolve and make up the volume with Methanol to
obtain a solution containing about 0.02 mg/ml of Uracil.
4.7.5.2 Filter the solution through 0.45m membrane filter.
4.7.5.3 Use HPLC grade Methanol as the mobile phase.
4.7.5.4 Instrumental Set Up
Column : Hypersil ODS or equivalent 150 x 4.6 mm, 5.0m
Flow rate : 1.0 ml/min
Injector Volume: 20m litre
Detector : 254 nm
4.7.5.5 Inject the standard preparation six times in the system. The peak
areas observed shall be consistent.
4.7.5.6 The relative standard deviation for area counts calculated shall not
be more than 1.0%.
4.7.5.7 Record the observation in the format as mentioned in Annexure 3.

4.7.6 CHECK FOR LINEARITY:


4.7.6.1 Inject 10, 20, 30, 40 & 50m litre of standard preparation in
duplicate. Calculate the average area counts corresponding to each set of injection.
4.7.6.2 Tabulate the average area against each injection. Plot a graph for
area counts vs mlitre the resulting graph shall be linear.
4.7.6.3 The correlation co- efficient calculated shall be not less than 0.99.
4.7.6.4 Record the observation as per the Annexure - 3.

4.7.7 CALIBRATION OF DETECTOR:


4.7.7.1 Standard preparation. Mobile phase, Instrument set- up same as
mentioned in calibration of Injector.
4.7.7.2 Run the chromatograph at different wavelength (252 nm 262 nm
with 2 nm increment)
4.7.7.3 The largest peak response shall be at 258 nm 2nm.
4.7.7.4 Record the results in the detection calibration record as per the
Annexure - 4.
4.7.7.5 Affix a calibration status label on the instrument containing
Calibrated On, Due On and Signature.
4.7.7.6 Report to Head QC, if any discrepancy observed during
calibration or operating the instrument and affix Under Maintenance label on the
instrument.
UNDER MAINTENANCE
EQUIPMENT :
SINCE :
SIGNATURE:

4.7.8 FREQUENCY OF CALIBRATION:

4.7.8.1 Detector: Once in three months and after each maintenance job.
4.7.8.2 Pump: Once in three months and after each maintenance job.
4.7.8.3 Injector: Once in six months and after each maintenance job.
4.7.8.4 Gradient: Once in six months and after each maintenance job in the
pump.

5.0 TRAINING:
Trainer -- Head Quality Control
Trainees -- Quality Control Chemists and Assistants
Period -- One day

6.0 DISTRIBUTION:
Certified Copy No. 1 : Head of Department Quality Control
Certified Copy No. 2 : File copy in HPLC Calibration Data
Reference Copy No. 3 : Display copy Near HPLC System
Original Copy : Head QUALITY ASSURANCE.

7.0 ANNEXURES:
Annexure 1 : Format for Calibration Record of Pump (Flow Rate)
Annexure 2 : Format for Calibration Record of Gradient Proportionate Valve
Annexure 3 : Format for Calibration Record of Injector
Annexure 4 : Format for Calibration Record of Detector
8.0 REFERENCES:
In house.

ANNEXURE 1
FORMAT FOR CALIBRATION RECORD OF HPLC
REF. SOP NO.: Revision No.: 00 Page No.: 1 of 1
Effective from:

Calibration Record : High Performance Liquid Chromatograph


Instrument No. :
Make and Model :
Calibration Date :
Calibration Due On :
Calibration of Pump (Flow Rate):

Observed weight in gm.


Mean Limit in ml
Flow rate adjusted to 1.0 ml / min. Mean wt. /
1 2 Vol. / / min.
min
min
Pump-A
Weight of beaker + water after 10
min. (W2) 0.99 to
Weight of beaker (W1) 1.01
(W2 W1) / 10
Pump-B
Weight of beaker + water after 10
min. (W2) 0.99 to
Weight of beaker (W1) 1.01
(W2 W1) / 10

Weight per ml of water = ________ gm.


Calibrated By: Checked By:
Date Date:
ANNEXURE 2
FORMAT FOR CALIBRATION RECORD OF HPLC
Revision No.: 00
REF. SOP NO.: Page No.: 1 of 1
Effective from:

Calibration Record : High Performance Liquid Chromatograph


Instrument No. :
Make and Model :
Calibration Date :
Calibration Due On :
Calibration of Gradient Proportionate Valve
Parameters:
Mobile Phase : Reservoir A 100% Water with HPLC
Reservoir B : 0.3% Acetone HPLC
Wavelength : 254 nm.
Flow rate : 2 ml / min.
Height From Base Line
Actual Value Set In
Tine Range To Value Obtained
The Program
B Concentration Peak.
0.01 To 10.0 (Lt: 9.9 To 10.1)
10.01 To (Lt: 49.5 To
20.0 50.5)
20.01 To (Lt: 89.1 To
30.0 90.9)

Value: Height of B Concentration


Height of a 100 % Concentration
Remarks: Satisfactory / Not Satisfactory
Calibrated By: Checked By:
Date Date :
ANNEXURE 3
FORMAT FOR CALIBRATION RECORD OF HPLC
Revision No.: 00
REF. SOP NO.: Page No.: 1 of 1
Effective from:

Calibration Record : High Performance Liquid Chromatograph


Instrument No. :
Make and Model :
Calibration Date :
Calibration Due On :
Calibration of Injector:

1. Check for Precision:


Standard Preparation:
Take ______ mg of Uracil (about 50 mg) 250 ml. 5ml 50ml with Methanol.
Parameters:
Mobile Phase: Methanol HPLC grade. Column: ODS (150 mm x 4.6 mm. 5 micron)
Wavelength: 254 nm. Flow rate: 1 ml / min. Injection volume: 20 ml.
Inject six replicate injection of standard solution and calculate RSD for area of main
peak.
Injection no. Area % of RSD Limit
1
2
3
Not More Than 1.0 %
4
5
6

2. Check for Linearity:


Correlation coefficient
Sr. Injection Are Mea
Observed
No. volume a n Limit
results
i) Not less than
1 10 ml.
ii) 0.99
i)
2 20 ml.
ii)
3 30 ml. i)
ii)
i)
4 40 ml.
ii)
i)
5 50 ml.
ii)

Remarks: Satisfactory / Not satisfactory.

Calibrated By: Checked By:


Date Date:

ANNEXURE 4
FORMAT FOR CALIBRATION RECORD OF HPLC
Revision No.: 00
REF. SOP NO.: Page No.: 1 of 1
Effective from:

Calibration Record : High Performance Liquid Chromatograph


Instrument Sr. No. :
Make and Model :
Calibration Date :
Calibration Due On :
Calibration of Detector
Standard Preparation:
Take _____ mg of Uracil (about 50 mg.) 250 ml. 5ml 50ml with Methanol.
Parameters:
Mobile Phase: Methanol HPLC grade Column: ODS (150 mm x 4.6 mm. 5 micron)
Wavelength: 252- 264 nm with 2 nm increment. Flow Rate: 1 ml / min. Injection
Volume: 20ml.

Sr.No. Wavelength (nm) Area Mean


1)
1 252
2)
1)
2 254
2)
1)
3 256
2)
1)
4 258
2)
1)
5 260
2)
1)
6 262
2)
1)
7 264
2)

Largest peak response is observed at __________ nm. (Limit: 258 nm 2 nm)


Remarks: Satisfactory / Not Satisfactory
Calibrated By: Checked By:
Date Date:

Potrebbero piacerti anche