OBJECTIVE:
To lay down the procedure for Operation and Calibration of High Performance Liquid Chromatography.
SCOPE:
This procedure is applicable for the Operation and Calibration of High Performance Liquid Chromatography (Agilent 1260 series)
RESPONSIBILITY:
Quality Control Officer / Executive are responsible to follow procedure as per the procedure mentioned in this SOP.
QC Head is responsible for implementation and compliance of the procedure as per this SOP.
ABBREVIATIONS
SOP : Standard Operating Procedure
QC : Quality Control
LC : liquid Chromatography.
SOP : Standard operating procedure.
STP : Standard test procedure.
PROCEDURE:
PROCEDURE FOR OPERATION :
Start the Open Lab Control panel by Double click on the Open LAB Control Panel icon located on Computer desktop.
Make sure the sample to be run is properly loaded in the auto sampler of HPLC.
Each time when start an instrument application (by double clicking the instrument icon from the Main window), an Instrument Wizard will appear. Enter the Use credentials and click ok.
Click Launch for online and Launch offline for offline.
Close the instrument wizard window.
Click file > New > and select Blank.met in New file Template and click ok. It will load the Instrument setup window.
Select each module and enter the method parameters as per method spec.
Detector: Enter wavelength nm – Peak width 0.1 - stop time should be as Pump/ injector.
Column Comp: Enter the temperature in the Left and select combined in the Right – stop time should be as Pump/ Injector.
Quaternary Pump: Enter flow rate and select the initial solvent composition (B or C or D) in the solvents – Enter the stop time in the pump. In the right side Timetable check the function centric view box and change the Gradient compositions.
Sampler: Enter the injection volume and select the injection mode (standard injection or Injection with needle wash. If second option is selected, enter wash vial number in the Needle Wash Location). – Stop time should be as pump. **Note: Along with method injection volume should entered in sequence table also. Don’t select Use method option in the sequence
Trigger: It should be external always.
After editing all the method parameters, you need to save the method by clicking File-> Save As ->Method.
How To Create New Sequence (option 1)
Click file -> New -> select sequence and blank sequence.
Here you can enter the sequence parameters.
Reps - Enter Reps ( ex: number injections per vial)
Vial – Enter vial number
Volume – Enter injection volume (** Don’t select Use Method in volume)
Method – browse and select method
File Name – Enter the data file name.
Once finished just copy the created line and insert the same and edit the vial and data file name as required.
How to access acquired data, Integrate data ,Assign Peak name, process sequence
In the Control Panel -> Select Instrument -> select the project -> click on Launch Offline.
The below screen will appear and close the instrument Wizard.
Click File -> Open -> Result Set.
The below window will appear and select the required Result Set and click on Open
The below screen will appear once you click Open. The Result Set will be opened along the Method.
To open a data just right click on any line and click on Select Record it will open the data file OR just double click on any line it will open the data file.
To integrate chromatogram, integration events are available.
By right click on chromatogram🡪Graphical Programming 🡪list will be displayed, from that you can use different integration events like integration off, valley to valley etc.
OR you can integrate chromatogram using graphical tools i.e. available at the bottom in the same Window.
If you integrate by using graphical tool, the below screen will appear with integration parameter and in the same window you will be asked to select two options, Option 1. “Add event to Method (all data)” and Option 2. “Add event to the data file only”. Here if you select Option 1, the events will be added to method and applicable for all the data files. And if you select Option2, only the particular the data which is open will be integrated.
All the integration entries you have applied can be seen from Method🡪Integration Events.
Below screen will appear. From this table also integration can be set. You can add integration in the event column using scroll down menu and manually entering start, end time and value.
How to Find system suitability of repeat runs
Click File -> Open -> Result Set… and select the required result file which you have already run before.
Define the peaks and save the method (see Define Peaks section) close the peaks and groups table if opened. Click -> Method -> System suitability.
Below screen will appear with defined peak names in the Compound column.
Select the peak name in the Compound column and in the Parameters column select Area and Retention time.
In the %RSD column enter the Area RSD limit and Retention Time RSD limit.
Click -> File -> Save -> Method.
Select Run Type column (all 6 runs) from the table. Right click 🡪Set Run types🡪System Suitability.
After this Run Type Column status will be changed as below. Click File-> Save -> Result Set.
Select the six runs and right click and click on Process Sequence.
Click on Start.
After the process click -> Reports -> View -> Sequence Report.
Below screen will appear. Select the System Suitability and click view
System suitability report will be displayed.
To take print of this report right clicks on Report🡪Print.
How to get Calibration curve and R2 value
Open the Result Set -> Open the data by double clicking the required line in the Result Sequence.
Define the peaks ( see the Define peaks section)
In the peaks and groups table enter the 5 injections concentrations in the level columns Like: Level 1, Level 2, Level 3, Level 4, and Level 5.
Enter the concentration in respective order. For example :
1st run Concentration : 1 ppm 🡪 it will be level 1 in sequence table
2nd run Concentration: 2.5 ppm 🡪 it will be level 2 in sequence table
3rd Concentration : 4 ppm 🡪 it will be level3 in sequence table
4th run Concentration : 6 ppm 🡪 it will be level4 in sequence table
5th run Concentration : 10 ppm 🡪 it will be level5 in sequence table
After editing the above table , the table wiil appera as below
Click File -> Save -> Method
Edit the Result Set by clicking Result Set -> Edit
Edit the Result sequence table as below. Select first run in the Run Type column and click Began.
Calibration for first run and End Calibration for fifth run. Enter correct level values (EX: 1 to 5) for respective runs. Click File -> Save -> Result set.
Select the 5 required runs in the Result Sequence-> right click -> Process Sequence -> click start in the next window.
To view the calibration curve. Click on Method🡪Review calibration
Calibration curve will be generated as shown below.
Change the fit type to Linear by right clicking on curves.
To take printout of this curve and calculation. Right click on Curve🡪 Print all Peaks/groups
Maintain Log book of HPLC.
Report any discrepancy observed during operation and calibration of instrument to 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 Manager Quality Assurance.
Affix ‘Under Maintenance’ label on HPLC.
CALIBRATION:
Calibration Frequency:
Half yearly or after major maintenance job done.
If only Lamp change, perform Detector Calibration Only.
Calibration of the pump:
Calibration of the pump shall be done to check the performance of the following
Flow rate accuracy
Flow rate consistency
Compositional accuracy (gradient profile)
Delay volume of the system
Flow rate Accuracy
Check the flow rate with filtered and degassed water as follows
set the flow of the pump at 0.50 ml per minute, and equilibrate the system.
Start collecting the water from column connector for 1 minute in to a tarred flask/beaker. Note down the weight of the flask/beaker and calculate the amount of water collected in ml by dividing the observed water density or 0.997 (at 25ºC).
Then Set the flow rate respectively at 1.00 ml/min, 1.5 ml/min and 2.0 ml/min to 5 minutes.
Calculate Flow Rate ml/min by using following formula
Flow Rate in ml/min = Observed weight in g X Density of water/5
Flow rate observe in triplicate for each to check flow rate precision.
Calculate the flow accuracy using by following formula (%)
Flow accuracy =Observed flow rate (ml/min) X l00 /Set flow rate
Acceptance criteria:
Flow Rate Accuracy: Flow rate should be between ± 5.0% of Set flow rate.
For flow 0.5 ml/min: Between 0.475 ml to 0.525 ml
For flow 1.0 ml/min: Between 0.95 ml to 1.05 ml
For flow 1.5 ml/min: Between 1.425 to 1.575 ml
For flow 2.0 ml/min: Between 1.90 to 2.10 ml
Flow Rate Consistency:
Done as per Injector Linearity, Take retention time of triplicate injections for 10µl (level-2), 20 µl (Level-3), Calculate Relative Standard Deviations for 3 injections retention time.
Acceptance criteria: Relative Standard Deviation for 3 retention times should not be more than 1.0%.
Compositional Accuracy (Gradient Profile)
Replace the column with Dead Volume connector.
Compositional accuracy: (Gradient Profile): Flow rate – 2 ml/min & detector: UV at 254 nm
Time (Minute) | HPLC grade water (Mobile Phase I) (Channel A, C) | 0.5% Acetone in water (Mobile Phase II) (Channel B, D) |
0 | 100 | 0 |
4 | 100 | 0 |
6 | 80 | 20 |
10 | 80 | 20 |
12 | 60 | 40 |
16 | 60 | 40 |
18 | 40 | 60 |
22 | 40 | 60 |
24 | 20 | 80 |
28 | 20 | 80 |
30 | 0 | 100 |
34 | 0 | 100 |
45 | 100 | 0 |
Run the gradient using Channel combination A &B and C& D after stabilization.
Inject 0 µl or minimum volume of HPLC grade water and record the gradient profile.
Calculate the concentration for each conc. Label 20%, 40%, 60 %, 80% and 100 % by using
Formula –
Actual concentration = Height at set % conc. (measured from 0 % conc. height)X 100/Height at 100% conc. (measured from 0 % conc. height)
The chromatogram obtained shall be checked for gradient steps & Plot a graph between each concentration & related absorbance (height)
Print the overlay plot of gradient profile A & B, and C&D.
Acceptance criteria:
Difference for Absorbance (Y Axis) should not be more than 10 mAU.
The actual concentration should not deviate by more than ± 1 of set value
Linearity coefficient should not be less than 0.999.
Delay Volume of the System:
Review the overlay chromatogram under Section 5.5.4.9 Compositional Accuracy (Gradient Profile). Note the time in minutes, at which actual change in absorbance (Lift of the baseline) had taken.
Delay Volume = (a-b) x Flow rate
a=Calculated start of composition
b= Set start of composition
Acceptance criteria:
The delay volume of the system should be not more than 1.5 ml.
Auto Injector Calibration:
Calibration of the Auto injector shall be done to check the performance of the following:
Injection Volume Accuracy
Auto injector Linearity & Precision
Carry over
Injection Volume Accuracy:
Fill a vial with HPLC grade water and seal with a cap. Weigh this vial and record weight (W1) in g.
Program HPLC system for a flow rate of 1.0 ml/min of water and run time of 1.0 ml /min. Inject 20µl from vial and repeat it for 18 times.
After completion of 18 injections, remove the vial and weigh again (W2) in g.
Calculate the injected Volume in µl by the formula.
= (W1-W2) X 1000 / 18 X D
(D - Density of water 0.997 at 25° C)
Acceptance Criteria:
Average volume of injection (µl/injection) shall be 20µl±0.4 µl.
Auto Injector Linearity and Precision:
Flush the HPLC system with HPLC grade water for about half an hour.
Chromatographic Condition:
Column: Inertsil ODS 25cm x 4.6 mm, 5 microns or Equivalent
Mobile phase: Water: ACN (85:15)
Flow rate: 1 ml/min
Wavelength: UV at 273 nm
Column temp: 30ºC
Run Time: about 10 minutes
Standard Preparation
Accurately weigh about 100 mg of Caffeine in a 100 ml volumetric flask, dissolve and dilute to volume with mobile phase. Further dilute 1 ml of this solution to 100 ml with Mobile Phase.
Inject the 5µl (level-1), 10µl (level-2), 20 µl (level-3), 50µl (level-4) and 100 µl (level 5) of the sample preparation in triplicate and record the chromatograms and position of vials as per calibration data sheet.
Calculate the average and RSD of the peak areas of caffeine from the triplicate injection at each Level.
Plot a linearity graph of injection volume on X axis and average area of caffeine at each level on the Y axis.
Acceptance Criteria:
Tailing factor of caffeine peak in the I st injection of level 1 should not be more than 1.5.
The % RSD of retention time of caffeine from the triplicate injection at each level should be Less than 1.0%.
The correlation coefficient obtained from the linearity graph of caffeine for different level should not be less than 0.999.
Carry Over Test:
Set the following chromatographic conditions.
Column: Inertsil ODS 25 cm x 4.6 mm, 5 microns Or Equivalent
Mobile phase: Water: ACN (85:15)
Flow rate: 1.0 ml/ min
Injection volume: 100µl
Wavelength: UV at 273 nm
Column temp: 30ºC
Standard Preparation: Weigh of caffeine 0.025g of caffeine standard and dissolved and diluted to 100 ml with mobile phase. (Std. Solution A). Dilute 10 ml of std. A solution to 100 ml with phase. (Std. Solution B)
Inject standard solution and blank (Mobile phase) in sequence as: Blank-1ST, Std. Solution B (1---3), blank-2nd, Std. Solution A (1----3), Blank 3rd. Calculate any carryover of caffeine in blank (3rd).
(Note: each blank should inject from separate vial)
Carry over = A X 100 / B X 10
Where,
A = Peak area response obtained from Blank -3 (after std. solution A) at the retention time of Caffeine.
B = Average peak area response of Caffeine obtained from Caffeine Standard 25 ppm solution
Acceptance Criteria: Carry over should not be more than 0.01%
Detector Performance: Calibration of detector shall be done to check the Performance of following:
Detector Noise& Lamp intensity
Detector Linearity
Wavelength Accuracy
Detector Noise & Drift & Lamp intensity: (Replace column with Connecter/Union)
Set the instrument parameters as mentioned below in chromatographic condition and keep the flow rate 1.0 ml/min. Flush the System with IPA for 15 minutes followed by methanol for 15 minutes. Further followed by normal purified water for ½ hour.
(Note: Purge after each mobile phase changeover)
Chromatographic conditions:
Column: Connecter/Union
Column oven temperature: 30°C
Wavelength: 254 nm
Mobile phase: Filtered and degassed purified water
Flow rate: 1.0ml / minute
Injection volume: 0 µl
Run Time: 20 min
Acceptance Criteria : for noise & drift
ASTM Noise [mAU]: Not more than 0.040
Drift [mAU/h]: Not more than 0.500
Acceptance Criteria : Lamp intensity:
Highest intensity: --------------- (Not less than 320000 counts)
Lowest intensity: -------------- (Not less than 6400 counts)
Average intensity: -------------- (Not less than 160000 counts)
Detector Linearity:
Set up HPLC System using following chromatographic conditions
Column : Inertsil ODS 250 cm x 4.6 mm, 5 microns Or Equivalent
Mobile phase : Water: ACN (85:15)
Flow rate : 1.0 ml/ min
Injection volume: 20 µl
Wavelength : UV at 273 nm
Column temp : 30°C
Run Time : About 10 minutes
Standard Preparation: Weigh accurately about 100 mg of caffeine in to 100 ml volumetric flask, Dissolve and dilute to volume with mobile phase. Further dilute accordingly with mobile Phase to get solutions having concentration of about 0.001, 0.01 and 0.10 mg/ml.
Inject six blank (Mobile phase) replicate injections. Calculate noise from all injections.
Inject Standard preparation of concentration 0.001, 0.01 and 0.10 mg/ml in triplicate & calculate the RSD. Plot graph of mean area count in Y-axis versus concentration (µg/ml) in x-axis and calculate the correlation Coefficient.
Acceptance Criteria:
Correlation coefficient should not be less than 0.999.
WAVE LENGTH ACCURACY:
Set the following chromatographic conditions.
Column : Inertsil ODS 250 cm x 4.6 mm, 5 microns Or Equivalent
Mobile phase : Water: ACN (85:15)
Flow rate : 1.0 ml/ min
Injection volume: 20 µl
Wavelength : UV at 273±2 nm & 245±2nm
Column temp : 30°C
Run Time : About 10 minutes
Standard Preparation: Weigh accurately about 100 mg of caffeine in to 100 ml volumetric flask, dissolve and dilute to volume with mobile phase. Further dilute 1ml to 100ml with mobile phase. (0.01 mg/ml solution)
Inject blank (mobile phase).
Inject the standard solution at wavelength range 269 nm to 278 nm as increase of 1 nm. Plot the graph of wavelength (X axis) versus Area counts (Y axis).
Repeat same as 6.3.3.4 at wavelength range 240 nm to 249 nm as increase of 1 nm. Plot the graph of wavelength (X axis) versus Area counts (Y axis).
Acceptance Criteria
Maxima - 273 ± 2 nm
Minima - 245 ± 2nm
Column oven Thermostat Verification:
Set the column Oven temperature at 30° C after about 10 minutes record the observed temperature using a calibrated digital thermometer.
Set Column temperature at 20°C, 40°C, 60°C and 80°C and after about 10 minutes record the observed temperature using a calibrated digital thermometer.
Acceptance criteria
The observed temperature should not be more than ± 1°C of the set temperature.
Maintenance of Instrument
In case of malfunction of the instrument, inform the Section In-charge for corrective action to be initiated. Put UNDER MAINTENANCE tag on the instrument.
The section in-charge will arrange for the repair of the instrument or call service engineer.
Make entries of the breakdown and corrective action taken in the instrument
After instrument repairing do the calibration, put the calibration tag & remove the UNDER MAINTENANCE tag.
ANNEXURES/REFERENCES :
Annexure I : Calibration Record of High performance liquid chromatography.