Calibration of HPLC

TITLE: CALIBRATION of HPLC

Calibration of HPLC
SOP NO.:	QC00X-01	EffectiveDate:	DD/MM/YYYY	Company Logo
Supersedes :	NA	Page No.:	01 of end
Department:	Quality Control	Review Date:	DD/MM/YYYY

I           OBJECTIVE 

• To provide a procedure for calibration of the HPLC (High-Performance Liquid Chromatography).          

II         SCOPE

•  This procedure is applicable for the HPLC series, used in the Quality Control department of          ABC Ltd, Location. 

III        RESPONSIBILITY 

•  It is the responsibility of all the QC personnel involved in the analysis using the system.    

IV        DOCUMENT REFERENCE(s)

            SOPs               :  Policy on calibration (Current version of QA 0XX)

            Forms              :

S.No	Details	Format No. (Current version)
1	Calibration of the HPLC system	QC0XX/F0X-XX

 

 

  

V         SCHEDULE:

•   Calibration shall be carried out once every 6 months.

VI        PROCEDURE  

1.0       Calibration Procedure

1.1       To Perform the Calibration of HPLC as follows.

1.2       Calibration shall be carried out with the below parameters.

1.2.1    Pump flow calibration

1.2.2    Wavelength accuracy

1.2.3    System precision

1.2.4    Carryover

1.2.5    Detector linearity

1.2.6    Injector linearity

1.2.7    Temperature verification for oven and sample cooler.

1.2.8    Gradient Performance Check

1.2.9        Signal Noise and Drift

1.3             Enter the calibration details in the tag and appear the same to the respective instrument

1.4              Calibration procedure shall be carried out as follows

            Conditions:

   Column  : Zorbax SB-C8 150mm x 4.6mm, 3.5u or equivalent

            Wavelength                 : 273 nm

            Flow Rate                   : 1.0ml/minutes

            Injection Volume        : 20 μl

            Runtime                      : 10 min

            Temperature                : Ambient temperature

1.5       Mobile Phase Preparation

A filtered and degassed mixture of Methanol and Water in the ratio of 60:40V/V.

1.6       Preparation of Caffeine stock solutions

Weigh accurately 100 mg of Caffeine into 100 volumetric flasks and make up to the mark with the mobile phase.

1.7       Sample Solutions Preparation:

1.7.1   Sample solution-1 (0.02mg/ml concentration) preparation: Take 2.0 ml of Caffeine stock solution in a 100 ml volumetric flask and make it to the mark with the mobile phase.

1.7.2   Sample solution-2 (0.04mg/ml concentration) preparation: Take 4.0 ml of Caffeine stock solution in 100ml volumetric flask and make it to the mark with the mobile phase

1.7.3   Sample solution-3 (0.06mg/ml concentration) preparation: Take 6.0ml of Caffeine stock solution in a 100 ml volumetric flask and make it to the mark with the mobile phase.

1.7.4     Sample solution-4 (0.08mg/ml concentration) preparation: Take 8.0ml of Caffeine stock solution in a 100 ml volumetric flask and make it up to the mark with the mobile phase.

1.7.5     Sample solution-5 (0.1mg/ml concentration) preparation: Take 10.0ml of Caffeine stock solution in a 100 ml volumetric flask and make it to the mark with the mobile phase.

1.8       Pump flow rate calibration

1.8.1    Measure the flow rate ua sing 10 and ml, 25 ml volumetric flask, and calibrate the ted stop clock. Measure the flow rates of 1.0ml/min, 1.5 ml/min, 2.0 ml/min, and 2.5 m/min.               

                                           Volume in ml  X 60

         Flow Rate     =  ___________________________________                                                   

                                 Actual time taken to collect the volume in seconds

1.8.2    Acceptance Criteria    :  ± 2.0%.

1.9       Wavelength Accuracy

1.9.1    Chromatographic conditions  

            Column                   :    Zorbax SB-C8 150mm x 4.6mm, 3.5 micron or equivalent

            Flow rate                 :   1.0 ml/min.

            Injection volume     :   20 ml

            Run time                 :   10 min

1.9.2  Preparation of Mobile phase: Prepare a filtered and degassed mixture of methanol and water in the ratio of 60:40 v/v.

1.9.3 Prepare 0.1 mg/ml caffeine solution in the Mobile phase. Inject caffeine solution at each length rise from 200nm – 210nm, from 239nm – 249nm, and from 268nm-278nm (increment of   1nm), record the details in HPLC calibration format record as per format No: QC0XX/F0X-0X

1.9.4   Acceptance Criteria

•    Maximum peak response must be at wavelength: 205 ± 2 nm for200 nm-210 nm 
•   Maximum peak response must be at wavelength: 273 ± 2   nm for 268 nm-278 nm
•   Minimum peak response must be at wavelength: 245 ± 2 nm for 239nm-249nm               

1.10   Detector Linearity

1.10.1  Inject Sample solution-1 to Sample solution-5 into the chromatographic system.

1.10.2  Record the area of Caffeine in each sample solution and calculate the Correlation coefficient. Draw the linearity graph with area vs. concentration.

1.10.3    Acceptance Criteria: The Correlation coefficient should be not less than 0.99.

1.11     System Precision:

1.11.1  Inject 5 times sample solution-5 into the Chromatographic system and calculate the           %RSD for Retention time and %RSD for Areas of the sample solution in 5 injections.

1.11.2  Acceptance Criteria: %RSD for Area and Retentions times should be less than 2.0.

1.12     Carryover:

1.12.1  Applies only if carry-over is run immediately after precision.

1.12.2  Inject diluent as a blank. And record the chromatogram.

1.12.3  Calculate the carryover percent using the fifth injection area of precision.

                                                                Blank area/height

                            Carryover (%) =      ------------------------    x 100

                                                               Sample area/height

1.12.4   Acceptance Criteria: NMT 0.2 % for the area and NMT 0.4 % for height. 

1.13     Temperature verification for Oven and Sample cooler:

1.13.1  Check the oven temperature at different set temperatures at 30°C, 40°C, 50°C, 60°C using calibrated Digital thermometer.

1.13.2 Check the Sample cooler temperature at 5°C and 10°C

1.13.3 Acceptance Criteria: ±1°C

1.14     Injector Linearity:    

1.14.1  Inject Sample solution-5. 10µl, 20µl, 30µl, 40µl, 50µl into    Chromatographic system.

1.14.2  Record areas of Caffeine in each injection and draw the linearity graph with area vs injection volume.

1.14.3  Calculate correlation coefficient.

1.14.4  Acceptance Criteria: The correlation Coefficient should not be less than 0.99. 

1.15    Gradient performance check:( GPV Test)

1.15.1 Preparing the Instrument:

1.15.2 Connect with union 

1.15.3 Filter and degas methanol, 0.5% acetone in methanol

1.15.4 Method Parameters:

1.15.5 Detector wavelength of 254 nm

1.15.6 Binary gradient with a total flow of 2.0 ml/min

1.15.7 Gradient table as per below table for pumps A and B.    

1.15.8 Running the Gradient proportionate Valve test:                                                                                                

Time	A                        Methanol	B  0.5% Acetone in Methanol
0.01	100	0
4.01	90	10
8.01	100	0
12.01	75	25
16.01	100	0
20.01	50	50
24.01	100	0
28.01	25	75
32.01	100	0
36.01	0	100
40.01	100	0
44.0	100	0

.          

 

 

 

 

 

 

 

1.15.9    Purge all the pumps with a flow rate of 2 to 5 ml/min for about 2 to 5 minutes.

1.15.10  Set the total flow rate at 2.0ml/min and wait until the baseline is stable.

1.15.11  Set the gradient profile for Pumps A and B.

1.15.12  Record the height of peaks.

1.15.13 Consider height at B conc. At 100% 100 calculate the other peaks' heights.

1.15.14 For Quaternary systems, repeat the study for other channels of C and D.

1.15.15  Consider height at D conc. At 100% 100 calculate the peak heights.

    Calculation:

                                          Height of B/D peak at different concentrations

 Height % of Peak B/D = ----------------------------------------------------------  X100

                                         Height of full-scale peak (100% B/D peak height)

 

1.15.16 Acceptance criteria: The difference in the proportion of B or D concentration should not vary by more than ± 1.5% for the set concentration.

1.15.17 then select the Setup Detector signal option enter the required values and click OK.

1.15.18 to her that if already method already was saved we have to open it, and if it any necessary to modify the method we can change it.

1.15.19 now run the sample, the sampler will pick the sample after the sample is picked the needle is washed from the additional vial kept for purposes
5.20 Check the flow on running and we can get the result.

1.15.21 here select printer and press OK. Again select the Report/print option. The report is printed and filed.

1.16.0   Signal noise and Drift:

1.16.1   Fix the union as a column.

             Mobile phase: Water.

             Flow rate        : 1.0 ml/min

             Wavelength    : 254 nm

             Run Time       : 24 min

             Start Time      : 03 min, End Time: 20 min

1.16.2    Run the chromatogram and record the data.

1.16.3    Evaluation of the noise and drift between 3 min to 20 min.

1.16.4    Determine the noise and drift using software.

1.16.5    Acceptance criteria: Should be less than 40µV for noise and Should be less than 500µV/Hz 8.3µV/min.                     

2.0     CARE AND MAINTENANCE:

2.1 Ensure the details are documented in the column usage log after the analysis & washing activity are completed

2.2   Use 85:15 Acetonitrile and water for the plunger wash.

2.3   Use mobile phase/diluent vial for syringe needle wash.

2.4 Verify the performance of the system as per the schedule/after a major breakdown of the instrument.

2.5    Replace the inlet filters based on their performance.

2.6    Enter the breakdown details & spares utilized in the Instrument malfunction record. 

* All mobile phase solutions shall be prepared as per the analysis requirement. However mobile phase solutions stored beyond 48 hrs shall not be used. And document the details in the mobile phase and mobile phase log

REFERENCE:

*  Instrument Manual of  HPLC

REVISION HISTORY

Version	Effective	Summary of Revision
0.0	DD/MM/YYYY	New SOP

 

END O F THE DOCUMENT

                                     

                                     Prepared By           Checked By             Approved By

        Sign & Date

        Name & Dept    QA Executive         QC Manage               QA Manager.   

#HPLC #Calibration, #GPV #Performance 

Operation of Agilent HPLC

 

Company Logo

TITLE: OPERATION OF AGILENT HPLC
SOP NO.:	QC00X-01	Effective Date:	DD/MM/YY
Supersedes :	QC00X-00	Page No.:	1 of end
Department:	Quality Control	Review Date:	DD/MM/YY

I    OBJECTIVE

To provide a procedure for the operation of Agilent HPLC (High-Performance Liquid Chromatography).

II SCOPE

 This procedure is applicable for the HPLC series, used in the quality control department of ABC Ltd.

III  RESPONSIBILITY 

 It is the responsibility of all the QC personnel involved in the analysis using the system.

IV  DOCUMENT REFERENCE(s)

  SOPs     :  Operation and calibration of Analytical balance (Current version of QC00X)

 Forms               :

S.No	Details	Format No. (Current version)
1	HPLC Instrument Log	QC00X/F01
2	HPLC Column usage Log	QC00X/F02
3	Mobile Phase log	QC00X/F03
4	Mobile Phase	QC00X/F04

 

 

 

V         PROCEDURE  

1.0       For Agilent Model                                     

            Power on all the individual modules of HPLC sequentially i.e.-degassing module, Pump,  

            Auto-injector, Column thermostat and detector.

            Switch on the computer.

            After switching on the instrument of all modules, the VWD initialization process takes Place.

 New project creation:

Then go to the computer, click Start, and then EZ Chrome Elite shall display from this 

Only Administrator can create new project and assign a user to access that project with different rights in below screen, all other options are disabled, after the administrator login, it will be enabled. To access the menu, click on Tools. Enterprises login

 Enter the administrator username and password

Click on the Tool  System administration wizard….

Select project. Click on Next

Select create a new project. Click on Next

Enter the name of the project. Click on Next.

If you want to enable Audit trail settings then select automatically enable audit trail. Also, you can give a list of reasons here. Click on Next

In the below screen you can add E-Signature roles. Click on Next

In below screen, list of all users will be displayed in Available users. You have to select the users from that list and move it right side, so only those users will access the project which you are creating now. Click on next

Here you can give different rights to access the project to different users. For that you have to select the user first from list of users and then select privileges from unassigned privileges and move it to right side. Screen after giving privileges to the user.  Click on Next 
Click on Finish

Same way you can modify the project setting, assigned users to projects etc. With admin login. After completion of this admin activity, you need to admin logout. For that click on To  Enterprise logout.

Login in to EZChrom elite:

Double click on EZChrom elite icon on the desktop OR

 You can click on start           programs         chromatography           EZChrom Elite

EZchrom Elite window will appear from there you can open it online and offline.

If you want to open an online session then double click on the HPLC-01(LC or GC name) icon.

One login window will appear, in which you have to enter your user name, password and you have to select your working project from scroll down menu. Then click on login tab.

If you want to open your offline session then click on HPLC-01 (LC or GC name) then clicks on open offline. Then same login window will appear. click on OK

In this window you will find navigation pane left side. From that directly access the method, sequence, control, report menus.

 New method creation:

Click on file >method > New

The instrument set up window will appear aslow or you can go to this instrument set-window by clicking MeaInstrument setup or from the navigation pane.

Instrument setup window you will find all the modules which are configured with software. Here you can see VWD (UV detector), TCC (column oven), Quat pump, ALS (auto sampler with sample cooler if installed)

In the VWD tab: Enter the wavelength of analysis and select stop time AS pump/injector. To further optimize the method change the peak width, otherwise no need to change another parameter in the same window if clicked on. Advanced then you will find some other parameters related to VWD. Refer above screenshot.

In TCC tab: Enter the temperature of the column, if you can give temp for left side, right side or both side here. If you select Not controlled then TCC will work at ambient temperature. If you select combined then TCC left and right side temp will be same as given in left side. Select stop time as pump/injector. No need to change other parameter. To access other parameter related to TCC you can click on Advanced.

In Quat pump tab:  Enter Flow in ml/min, solvent concentration by selecting A, B, C, D lines pressure limits (Max). No need to change Min Pressure limit (always set zero). Here you can type you mobile phase detail in blank space. Select stop time i.e. analysis run time in minutes. From right side you can create gradient program by add, remove etc. button. No need to change other parameter. Also you can access other parameter related to pump by selecting Advanced.

            In the ALS Tab:  Enter Injection volume in uL. 

            Select either Standard injection or injection with needle wash.

In standard injection, the Needle will not be cleaned during injections.

In injection with needle wash mode when you select this mode location option wil be enabled. Here you have to enter the wash vial number, Needle outer surface will be cleaned after sample sucked from the sample vial.

Enter stop time in minutes same as given in Quat pump parameter.

If ALS thermostat (sample cooler) is installed then enter temperature (sample).

No need to change other parameter. For further method optimization you can have some other Advanced parameter related to Auto sampler in same window.

In Aux Traces tab: You can also able to acquire the below signals during the analysis like pressure, graph, column oven temp graph etc. select which ever you want to see along with detector signal.

In last tab trigger:  Normally where auto sample is installed, it is External. No need to change. Detail also mentioned in same window for different option. 

            After editing all the method parameters, you need to save all the method by clicking 

Save method screen will appear. Give method filename in blank space (For example: Demonstration)

After saving method file, download it into instrument by clicking control         Download method.

After downloading the method you can see the online signal before injection i.e. to check baseline gets stabilized or not. Click on view online signal.

How to do single run Acquisition:

Click on control single Run. One window will appear in that need to enter sample ID, vial number and injection volume.

How to create new sequence:

 Click on the file         Sequence            Sequence wizard

Sequence wizard window will appear as below. In this window, Select method using browse button, select data file type for acquisition click on Next.

Enter sample ID, Data path (browse your path if required), Data file name. In sample ID and Data file name by clicking triangle symbol ►you will be able to give different of naming sample ID and data file here let select increment number. Give number of unknown runs in sequence (i.e. numbers of lines want to create in sequence, for exp.give:10). Also you can give repetitions per run. After filling detail in below window, click on A newt.

New window will appear as below. 

In Unknown vials of sequence: Enter First vial and incrthe the ement by and give injection volume in μl by giving increment By: 1, 10 lines will be generated in sequence with file names automatic using viaautomaticallyally. Click on  new 

New window will appear as below. No need to change in this window. Click on nexa a t

Now window will appear as below. No need to change in this window. Click on fiThe sequencequence will be generated as below.

You can choothe the se number of columns the the in sequence table clickingick on sequence table

      PropertiThe belowlow screen will appear. Selethe ct required columns from this list.

Edit sequence table, i.e. give correct vial no, repetitions per runs, inrunion volume, method nand and ame if required. Verify the method path, data patand and h by clicking sequence         es.

To save this sequence click on File      

Give sequence file name in Blank space

How to start sequence run

 Click on control sequence Run OR Right click on sequence tabl Run sequence.

One Run sequence window will appear, here you can select all the lines or some selected lines or range of lines from the sequence table. DefaultThe defaultion will be all. Click on start. Your sequence will be startou can see the status in sequence the table when analysis is going on.

How to access acquired data, integrated data, Assign peak name, process sequence

From main EZ Chrom Elite window right click on HPLC-01(LC or GC name)         Open offline.

One login window will appear, in which you have to enter your user and name, password and you have to select your working projectthe the  from scroll down menu. Then clithe the ck on login tab. click on file DataOpen

Select data file and from option select Original/Acquisition

That will open data file with original method with which data acquired

Data file will open with related method.

To integrate chromatograms, integration events are available

By clicking on the chromatogram  Graphical programming list will be displayed, you can use different integration events like integration off, valley to valley etc.

OR you can integrate chromatogram using graphical tools i.e. in same window.

All the integration entries you have applied that you can see from method      Integration Events.

Below screen will appear. From this table also integration can be set. You can add integration in event column using scroll down menu and manually entering start/ end time, value.

Now at this time chromatogram window will disappear behind the table. To see the chromatogram click on view        Tile data.

To check the effect of integration you have applied on chromatogram, click on A  Analyze

For Assigning Peak Name:

Right click on chromatogram        Graphical programming       Define peaks

Enter the range of peaks you want to define (i.e. starting point and end point). After this click OK.

Click on method       Peaks/ Groups.

The peaks/ groups window will appear as below.

Give correct peak names from their RT in this table in the name column. You can change also the RT window from this table.To see the namess of peaks on the chromatogram, click on Analysis    Analyze. Click view       Tile data.

Then right-click on chromatogram      Annotations. The below window will appear

Select Name, Area, RT, or whatever you want to see on the chromatogram. Select it and move to the right side.  Then click on OK.

After proper integration and assigning peak names, you have to save this integration into the method so click on File, Method, and Save. It will save the method which you opened last, you can see on top method name.

To apply the same integration set-in method to all data files in sequence

Open the sequence by clicking File Sequence. Select the sequence you want to process. Here take care that your method in the sequence must be set with proper integration events before.

Click on sequence process OR click on Sequence         Process sequence.

Process sequence window will appear as below. You can select All/Made selection/Give range. Click on Start.All data files in sequence table will be processed using integration events saved in respective method

Print out of chromatograms:

Open the data file by clicking File  Data open. Then Select your interested data file. Open the respective method for that data.   

Click on Reports   View       Area% (you can select any report template listed)

Below is area % report generated.

To give print of this report simply right-click on report Print.

You can edit OR create new report template by clicking Reports         Report Template Editor

After above action, you can edit the report template by right click on report        List will be displayed. Then you can import reports all ready existing and edit it or create new report template.

After editing report template save the report template by clicking File      Report template      Save As    

 How to find system suitability of repeat runsOpen the sequence you have already run before. Set proper integration for method used in sequence For example: open sequence injection precision seq.In this sequence table demonstration met method is used. This method is already set with proper integration events. Also it is compulsory to assign peak names in chromatograms and save it in method. Open that method.After proper integration and peak assigning chromatogram s.To take print out of this curve and calculation. Right click on curve .Print all peaks/Groups

 CARE AND MAINTENANCE: 

Ensure the details are documented in the column usage log after the analysis & washing activity is completed.

Use 85:15 Acetonitrile and water for the plunger wash.

Use mobile phase / diluent vial for syringe needle wash

Verify the performance of the system as per the schedule / after a major breakdown of the instrument.

Replace the inlet filters based on their performance

 Enter the breakdown details & spares utilized in the Instrument malfunction record.

REFERENCE:

* Instrument Manual  of Agilent HPLC System 

REVISION HISTORY

Version no.	Effective date	Summary of revision
0.0	DD/MM/YYYY	New SOP

END O F THE DOCUMENT

                                                Prepared By               Checked By                         Approved By

Sign & Date

Name & Department               QA Executive               QC Manager                                QA Manager