SOP For Operation and Calibration of HPLC

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 to ensure smooth operation and its use monitored to obtain consistent and reproducible results minimize downtime and meet regulatory requirements.

This procedure is to be applied at the time of Operation and Calibration of HPLC.   


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

Operation and Calibration of HPLC 


Start the EZChrom Elite Software by double-clicking the EZChrom Elite icon located on your desktop.

Make sure the sample to be run is properly loaded in the autosampler of your   instrument

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.

Create or modify a method

This button starts the Method Wizard that will enable you to step through creating or modifying a method.

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

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

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.

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.

Run sequence of samples this button opens the Run Sequence dialog where you can start data acquisition using a stored sequence.

Save the method. When you have completed setting up the method parameters, from the   File menu, follow Method and click Save As. Type a name to be used for your method and then click OK

Maintain Logbook of HPLC on XYZ/CQA/SOP-044/FR-01  Logbook for  HPLC (Appendix I)

Report any discrepancy observed during the operation and calibration of the instrument to the Section In-charge or his representative for corrective and preventive action.

Section In-charge or his representative will take the necessary action and report the same to the Manager – Quality Assurance.

5.0.14   Affix ‘Under Maintenance’ label on HPLC




For calibrating the Column oven use only a calibrated thermometer.

For calibration of pump flow rate use only a calibrated measuring cylinder and calibrated stopwatch.


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

Pump Calibration 

Flow rate Accuracy :

Use Milli Q water (Degassed & filter) as a mobile phase for calibration of a pump.

Before starting the calibration purge the system two or three times to remove air bubbles.

Before starting calibration set the pump at constant flow and discard the volume for five minutes.

Set the Flow rate to to1.0ml(through channel A) allow the system to stabilize and measure the flow using a 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

Repeat steps to for calibration of pump B (if applicable).

Record 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%

Flow rate Consistency:

From the injection under injector precision i.e.( 20µL(5 replicate injections) calculate the %RSD  of  RT

Acceptance criteria: % RSD of RT shall not be more than 1%

Record the Calibration data on XYZ/CQA/SOP-044/FR-02 Flow Rate Consistency (Appendix II).

Gradient Composition Accuracy

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:


Time in minute

% Solvent  A

%Solvent  B






























Time in minute

% Solvent  A

%Solvent  B






















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 the 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.

SOP For Operation and Calibration of HPLC

Injector Calibration 

Injector Accuracy

Fill a standard vial with HPLC grade water(MilliQ) and seal with the cap.

Weigh this vial and record the weight as( W1) at ambient temperature.

Inject 50µL from the vial and repeat it 5 more times.

Program HPLC system for a flow rate of 1.0ml/min and run for 1 min using water as per mobile phase.

After completion of 6 injections, remove the vial and weigh again(W2) at ambient temperature.

Calculate the average volume injected per injection using the formula:

W3 = [(W1 –W2)/6}*1000=mg of water withdrawn per injection= µL per injection 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)

Injector Precision and Linearity


Acetonitrile (HPLC grade)

Water (HPLC grade) /MilliQ       


Set up the chromatographic system using the following parameters:

Flow : 1.0ml/min

Column:  C18 250X4.6mm, 5µ (inertial ODS column is suitable)

Wavelength:  273nm

Runtime: 15min

Mobile phase:  Water: Acetonitrile (85:15)

Prepare a solution of 250ppm of Caffeine using mobile phase as diluent.

Inject mobile phase as blank run

Inject Caffeine with an injection volume of 5 µL. (Duplicate),10 µL.(Duplicate),20 µL. (Five replicates),  50 µL. (Duplicate) ,100 µL(Duplicate)   

From the data obtained plot a graph of Area vs. injection and calculate the value of the correlation coefficient (R2).

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%

Carryover Test 


Acetonitrile  (HPLC grade)

Water (HPLC grade)/MilliQ


Set up a chromatographic system using the following parameters.

              Flow:  1.0 ml/min

             Column:  C18 250X4.6mm, 5µ (inertial ODS column is suitable)

             Wavelength:   273nm

             Run time:  15 min

             Mobile phase: Water:  Acetonitrile  (85:15)

             Injection volume:100 µL

Prepare a solution of 250ppm of Caffeine using mobile phase as diluent.

Inject 100 µL. of the following solutions:

(a) Pre-blank- mobile phase

(b) Caffeine solution -250ppm

(c) Post blank –mobile phase

The peak area measured in post blank compared with the peak area of Caffeine solution indicates the amount of carryover.

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.

Record the Calibration data on   XYZ/CQA/SOP-044/FR-02  Carry Over Test (Appendix II)

Acceptance criteria; ≤ 0.05%

Detector Calibration

Linearity of Detector Response

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

Wavelength: 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 it and dilute to volume with the mobile phase.

(1)  Preparation solution 1(100 ppm): Dilute 10 ml of the standard solution to 100 ml with the mobile phase.

(2Preparation solution 2(50 ppm): Dilute 25 ml 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 10 ml 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.


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 the 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.

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

Wavelength accuracy  (Caffeine in  water)

Using Caffeine Solution

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

Sample preparation: Weigh accurately 25mg of caffeine, transfer it into a 100 ml volumetric flask,  dissolve, and dilute to volume with purified water.  (250 ppm Caffeine in purified water).

Switch ON the detector UV lamp for at least 1 hour.

Inject the Caffeine solution in the mobile phase at 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


273.0 nm

± 1.0nm



Record the Calibration data on XYZ/CQA/SOP-044/FR-02 as  Wavelength Accuracy (Appendix II )

Column Oven Calibration (Temperature Calibration)

Switch on the column oven and set the Temperature at 40°C

Remove the right-side plastic opening center clip and insert the standard calibrated thermometer up to the middle of the column oven.

Properly pack the side space of the thermometer with cotton for proper insulation.

Now, wait to position the lamp (up to set temperature is achieved).

Observe the oven temperature displayed reading (current) and the standard thermometer reading. (Appendix  II)

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.

Summarize the calibration data on XYZ/CQA/SOP-044/FR-03  HPLC Calibration summary sheet (Appendix III).


Generate the Instrument Calibration number on the calibration datasheet as INSCALXXYYZZZ

Where INS denotes Instrument, CAL denotes Calibration

XX denotes the year, YY denotes the Month ZZZ denotes the sequence number.


INS CAL: Instrument Calibration

Download:- Logbook & Appendix I, II, III,

>SOP For Operation and Calibration of FTIR


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