This document details the procedure for the Operation and Calibration of HPLC(AGILENT 1120 COMPACT LC). or Agilent HPLC Calibration SOP, HPLC calibration by caffeine
It is the policy of Xyz pharmaceuticals Limited that the written procedure shall be followed for the operation and calibration of HPLC in order to ensure smooth operation and its use monitored to obtain consistent and reproducible results and minimize downtime and meet regulatory requirements.
This procedure is to be applied at the time of Operation and Calibration of HPLC.
RESPONSIBILITY & ACCOUNTABILITY
Persons along with their responsibilities and accountability are
given below:
Executive - Corporate Quality Assurance:- To prepare SOP.
Trainee Analyst/ Jr. Analyst/ Analyst/ Sr. Analyst:- To follow the SOP accordingly
Manager - Corporate Quality Assurance/Quality control:- To ensure implementation of SOP
PROCEDURE (OPERATION)
5.0.1 Start the EZChrom Elite Software by double-clicking the EZChrom Elite icon located on your desktop.
5.0.2 Make sure the sample to be run is properly loaded in the autosampler of your instrument
5.0.3 Each time you start an instrument application (by double-clicking the instrument icon from the Main window), an Instrument Wizard will appear. This wizard is designed to direct you to the basic functions of the instrument window.
5.0.4 Create or modify a method
This button starts the Method Wizard that will enable you to step through creating or modifying a method.
5.0.5 When you start the Method Wizard, click to create a new method. The wizard will guide you through the method sections. For each section, enter the run parameters required for the sample you will be injecting
5.0.6 Data acquisition and instrument control parameters –
(a) Instrument Setup. Select each tab and enter the correct parameters for running this sample
(b) Integration
events . Additional integration events are not
necessary for a test sample.
(c). Calibration of Peaks/Groups.
Calibration parameters are not necessary for a test sample.
(d). Advanced method options. Advanced method options are not necessary for a test sample.
(e). Reports. From the File menu, select Report Template and then click Open. From the list of templates, select Area% .rep to generate an Area %report when running the sample
5.0.7 Create a sequence This button starts the Sequence Wizard that steps you through the creation of an acquisition reprocessing sequence. Or for creating a sequence enter a file menu that displays Sequence, from Sequence enter a new method.
5.0.8 Run one sample this button opens a dialog where you can use a stored method to run a single sample.
In the Single Run Acquisition dialog box, enter a number to be used as a Sample ID. Click the method open button ▶ and then select the method file that was provided or the method you saved in the previous step.
Enter a Data file name you wish to use or click the adjacent button to select a parameter that will be used to create the data file name for you.
Enter the Vial number and Injection Volume to be used.
5.0.9 Run sequence of samples this button opens the Run Sequence dialog where you can start data acquisition using a stored sequence.
5.0.10 Save the method. When you have completed setting up the method parameters, from the File menu, followed by Method and click Save As. Type a name to be used for your method and then click OK
5.0.11 Maintain Logbook of HPLC on XYZ/CQA/SOP-044/FR-01 Logbook for HPLC (Appendix I)
5.0.12 Report any
discrepancy observed during operation and calibration of instrument to Section
In-charge or his representative for corrective and preventive action.
5.0.13 Section In-charge or his representative will take the necessary action and report the same to Manager – Quality Assurance.
5.0.14 Affix ‘Under Maintenance’ label on HPLC
5.1 HPLC CALIBRATION PROCEDURE
5.1.1 CALIBRATION FREQUENCY: QUARTERLY
5.1.1.1 Precautions
5.1.1.2 For calibrating the Column oven use an only
calibrated thermometer.
5.1.1.3 For calibration of pump flow rate use only calibrated
measuring cylinder and calibrated stopwatch.
5.1.2 CALIBRATION PARAMETERS
The following components
of the system shall be calibrated.
A)Pump Calibration i) Flow rate
accuracy
ii) Flow rate consistency
iii) Gradient composition accuracy
B)injector calibration: i) Injector accuracy
ii) Injector Precision & Linearity
iii) Carryover test
C)Detector Calibration i)
Linearity of Detector Response
ii) Wavelength accuracy
D)Column Oven Temperature i)
Temperature Calibration
5.1.3 Pump Calibration
5.1.3.1 Flow rate
Accuracy :
5.1.3.2 Use Milli Q water
(Degassed & filter) as a mobile phase for calibration of a pump.
5.1.3.3 Before starting
the calibration purge the system two or three times to remove air bubbles.
5.1.3.4 Before starting
calibration set the pump at constant flow and discard the volume for five minutes.
5.1.3.5 Set the Flow rate to1.0ml(through channel A) allow the system to stabilize and measure the flow using calibrated measuring cylinder and with the help of calibrated stopwatch for the delivered volume10ml at the flow rate of 1.0ml/min and 2.0ml/min Calibrate Flow rate by measuring the time required for 10ml vol. delivered(with the help of calibrated stopwatch)Repeat the same operation another 4 times for both 1.0ml/min and 2.0ml/min flow rate respectively and determine % RSD for observed flow rate (five replicates). Also, determine the observed flow rate and flow accuracy using the formula.
Observed Flow rate = Volume collected / time
Flow accuracy = Observed
flow rate X 100
Set
flow rate
5.1.3.6Repeat step
5.4.1.1 to 5.4.1.1.4 for calibration of
pump B (if applicable).
5.1.3.7Record Calibration
data on XYZ/CQA/SOP-044/FR-02 Calibration of Pumps (Appendix II).
Acceptance
criteria
Flow accuracy: Between 95% and105% of the set flow rate
Flow precision: %RSD of five
measurements at each flow, should not be more than 2.0%
5.1.3.8 Flow rate
Consistency:
5.1.3.9 From the injection under injector precision i.e.(5.4.2.2) 20µL(5 replicate injections) calculate the %RSD of RT
Acceptance criteria: % RSD
of RT shall not be more than 1%
5.1.3.10Record the Calibration data on XYZ/CQA/SOP-044/FR-02 Flow Rate Consistency (Appendix II).
5.1.3.2 Gradient Composition Accuracy
5.1.3.2.1 Set the
following chromatographic conditions
Column : Restriction capillary (~0.12mm ID)
Solvent for Pump A:
Filtered and degassed purified water
Solvent for Pump B: 0.3%
Acetone in purified water
Flow rate : 1.0 ml/min
Wavelength:
254 nm
Injection volume:
0.00 µl
Run time: 50 min
Gradient programming:
|
Step |
Time in minute |
% Solvent A |
%Solvent B |
|
1 |
0.01 |
100.0 |
0 |
|
2 |
1.0 |
100.0 |
0.0 |
|
3 |
2.0 |
90.0 |
10.0 |
|
4 |
10.0 |
90.0 |
10.0 |
|
5 |
10.01 |
50.0 |
50.0 |
|
6 |
20.0 |
50.0 |
50.0 |
|
7 |
20.01 |
10.0 |
90.0 |
|
Step |
Time in minute |
% Solvent A |
%Solvent B |
|
8 |
30.0 |
10 |
90 |
|
9 |
30.01 |
0 |
100 |
|
10 |
40.0 |
10 |
100 |
|
11 |
40.01 |
100 |
0 |
|
12 |
50.0 |
100 |
0 |
5.1.3.2.2 Procedure:
Set the flow rate at 1.0ml per minute. Keep the system in this flow rate for some time to equilibrate. Start the gradient programming. After completion of Gradient programming calculate actual concentration by using the formula below
Actual conc of X % = absorbance level of
set conc(X%) – absorbance level of set conc(0%)
absorbance level of set conc100%- absorbance level of set conc 0%
Where X =10,50,90
Acceptance criteria: At each absorbance level ±1 of set concentration.
5.1.3.3 Injector Calibration :
5.1.3.3.1 Injector Accuracy
5.1.3.3.2 Fill a standard
vial with HPLC grade water(MilliQ) and seal with the cap.
5.1.3.3.3 Weigh this vial
and record the weight as( W1) at ambient temperature.
5.1.3.3.4 Inject 50µL from the vial and repeat it 5 more times.
5.1.3.3.5 Program HPLC system for a flow rate of 1.0ml/min and run for 1 min using water as per mobile phase.
5.1.3.3.6 After completion of 6 injections, remove vial and weigh again(W2) at ambient temperature.
5.1.3.3.7 Calculate
average volume injected per injection using the formula:
W3 = [(W1 –W2)/6}*1000=mg of water withdrawn per injection= µL per injection
5.1.3.3.8 Record the calibration data on XYZ/CQA/SOP-044/FR-02 Injector Calibration (Appendix II)
Acceptance criteria: Average volume of injection shall be 50±1.0 µL.(49 µL to 51 µL)
5.1.3.4
Injector Precision and Linearity:
Requirements:
5.1.3.4.1 Acetonitrile
(HPLC grade)
5.1.3.4.2 Water (HPLC
grade) /MilliQ
5.1.3.4.3 Procedure :
5.1.3.4.4 Set up
chromatographic system using following parameters:
Flow : 1.0ml/min
Column: C18
250X4.6mm, 5µ (inertsil ODS column is suitable)
Wavelength:
273nm
Runtime: 15min
Mobile phase: Water: Acetonitrile (85:15)
5..1.3.4.5 Prepare a
solution of 250ppm of Caffeine using mobile phase as diluent.
5.1.3.4.5 Inject mobile
phase as blank run
5.1.3.4.6. Inject Caffeine with injection volume 5 µL.(Duplicate),10 µL.(Duplicate),20 µL. (Five replicates), 50 µL. (Duplicate) ,100 µL(Duplicate)
5.1.3.4.7 From the data
obtained plot a graph of Area vs. injection and calculate the value of the correlation coefficient (R2).
5.1.3.4.8 Record the calibration data on XYZ/CQA/SOP-044/FR-02 as Injection Precision and Linearity (Appendix II)
Acceptance criteria: Correlation
coefficient (R2).should not be less than 0.999
RSD of an area of
replicate injection should not be more than 1%
5.1.3.5 Carryover Test :
Requirements :
5.1.3.5.1 Acetonitrile (HPLC grade)
5.1.3.5.2 Water (HPLC
grade)/MilliQ
5.1.3.5.3 Procedure:
5.1.3.5.4 Set up
chromatographic system using the following parameters.
Flow :
1.0 ml/min
Column : C18 250X4.6mm , 5µ (inertsil ODS column is
suitable)
Wavelength : 273nm
Run time
: 15 min
Mobile phase : Water : Acetonitrile
(85 :15)
Injection volume :100 µL
5 1.3.5.5 Prepare a
solution of 250ppm of Caffeine using mobile phase as diluent.
5.1.3.5.6 Inject 100 µL.
of following solutions:
(a) Pre blank- mobile phase
(b) Caffeine solution -250ppm
(c) Post blank –mobile
phase
5.1.3.5.7 The peak area measured in post blank compared with the peak area of Caffeine solution indicates the amount of carryover.
5.1.3.5.8 If there is any
peak area measured in the pre-blank at the same retention time the same shall
be subtracted from the peak area of the post-blank before it is compared with the Caffeine
solution.
5.1.3.5.9 Record the
Calibration data on XYZ/CQA/SOP-044/FR-02 Carry Over Test (Appendix II)
Acceptance
criteria; ≤ 0.05%
5.1.3.6 Detector
Calibration
5.1.3.6.1 Linearity
of Detector response
5.1.3.6.2 Chromatographic
conditions:
Column : C18 250 x
4.6mm, 5µ (Inertsil ODS column is suitable)
Mobile phase : Acetonitrile : water (15 : 85 )
Flow rate :
1.0ml/min
Wave length :
273nm
Run Time : 15min
Column
oven temperature : Ambient
Injection volume : 20µl
(A) Preparation
of standard solution (1000ppm): Weigh accurately about 0.1g of Caffeine in 100ml volumetric flask dissolve and dilute to
volume with the mobile phase.
(1) Preparation solution 1(100 ppm):
Dilute 10ml of standard solution to 100 ml with the mobile phase.
(2) Preparation solution 2(50 ppm):
Dilute 25ml of solution 1 to 50 ml with the mobile phase.
(3) Preparation solution 3(25 ppm):
Dilute 25ml of solution 2 to 50 ml with the mobile phase.
(4) Preparation solution 4(10 ppm):
Dilute 10ml of solution 1 to 100 ml with the mobile phase.
(5) Preparation solution 5(5ppm):
Dilute 25ml of solution 4 to 50 ml with the mobile phase.
5.1.3.6.3 Procedure:
After stabilization of the system, Inject the blank (mobile phase) and then inject each of solution 5, solution 4 (in duplicate), solution 3 (five times) solution 2, and solution 1 (in duplicate) injecting blank at each step and record the chromatograms. Calculate the average peak area of caffeine from the duplicate injections at each level. Plot a linearity graph between the Concentration of caffeine (ppm) on the x-axis and the average area of Caffeine at each level on the y-axis.
5.1.3.6.4 Record
the Calibration data on XYZ/CQA/SOP-044/FR-02 Linearity of Detector Response (Appendix II)
Acceptance criteria: The linearity coefficient ‘r’ obtained from the linearity graph of caffeine for different levels should not be less than 0.999
5.1.3.7 Wavelength accuracy (Caffeine in water)
5.1.3.7.1 Using
Caffeine Solution
5.1.3.7.2 Set the following chromatographic
conditions.
Mobile phase :
Water: Acetonitrile (85:15)
Flow rate :
1.0 ml/minute
Column : C18 250 x 4.6mm, 5µ (Inertsil
ODS column is suitable)
Run time : 15 min
Injection Volume : 20µl
5.1.3.7.3 Sample preparation: Weigh accurately 25mg of caffeine, transfer into a 100 ml volumetric flask, dissolve and dilute to volume with purified water. (250 ppm Caffeine in purified water).
5.1.3.7.4 Switch ON the detector UV lamp for at
least 1 hour.
5.1.3.7.5 Inject the Caffeine solution in the mobile
phase at the wavelengths 271, 272, 273, 274 & 275, record
the results, and check the maxima by
plotting a graph w.r.t area v/s wavelength.
Acceptance
Criteria:
|
Standard wavelength maxima |
Limit |
|
273.0
nm |
± 1.0nm |
5.1.3.7.6 Record the Calibration data on XYZ/CQA/SOP-044/FR-02 as Wavelength Accuracy (Appendix II )
5.1.3.8 Column Oven Calibration (Temperature
Calibration)
5.1.3.8.1 Switch on the
column oven and set Temperature at 40°C
5.1.3.8.2 Remove the right side plastic opening center clip and insert the standard calibrated thermometer up to the middle of the column oven.
5.1.3.8.3 Properly pack the side space of the thermometer with cotton for proper insulation.
5.1.3.8.4 Now, wait up to
ready position lamp (up to set temperature is achieved).
5.1.3.8.9 Observe the oven temperature displayed reading (current) and the standard thermometer reading. (Appendix II)
5.1.3.8.10 Similarly
repeat the calibration for 50°C.
Acceptance Criteria :
The observed reading (current) and standard thermometer reading do not differ by more than ± 2°C from the set temperature.
5.1.3.8.11 Summarize the
calibration data on XYZ/CQA/SOP-044/FR-03 HPLC Calibration summary sheet (Appendix III).
5.2 GENERATION OF INSTRUMENT CALIBRATION NUMBER
Generate Instrument Calibration
number on the calibration datasheet as INSCALXXYYZZZ
Where INS denotes Instrument, CAL denotes
Calibration
XX denotes the year, YY denotes Month ZZZ denotes sequence number.
ABBREVIATIONS
INS CAL: Instrument Calibration
Download:- Logbook & Appendix I, II, III,
>SOP For Operation and Calibration of FTIR
