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SOP for Operation of Agilent 1260 HPLC

To lay down the procedure for operation of HPLC (Agilent 1260) when using either the Diode Array (DAD), Fluorescence (FLD), and Refractive Index (RID) detectors.

This SOP is applicable for Quality control / Stability department. This procedure applies to established procedures of analysis using the standard configuration for the 1260 HPLC.

Assistant – QC / Stability to operate the HPLC.

Manager - Quality control / Stability To ensure the compliance for established procedure

Equipment (Standard Configuration)
  • Agilent 1260 Infinity Quaternary HPLC
  1. Mobile Phase: Methanol, Water, Acetonitrile
  2. Detector: DAD, FLD, RID
  3. HPLC Column: Agilent Poroshell 120 EC-C18 HPLC Column

Initial Setup
  • Turn on the seven LC modules (Regardless of requirement)
  • Ensure all the solvents have a minimum of 500 mL
  1. Anticipate the amount of solvent you for the completion of experiment. Speak to the TRACES Lab Manager to confirm the amounts if you are unsure.
  2. Establish the identity and channel of all the solvents needed
  • Ensure the HPLC waste bottle is empty (or at least ¾ empty)
  • Login to the computer with the appropriate password and username
  1. Click on Instrument 1 online
  • Confirm that all the modules you require are not greyed out.
  1. Power down all greyed-out modules.

HPLC Setup Section
Purge Solvents
  • Open the Purge Valve by turning it counterclockwise
  • RCM (right click mouse) in the Quat Pump module
  1. Click on Method
         👉   Increase flow to 5 mL/min
         👉   Purge each channel for 5-10min
         👉   Click Okay
  • RCM in the Quat Pump module
  1. Click Control
         👉   Turn pump to ‘On’

Load a Method
  • Click on Method and Load a method from the list provided
  1. If you wish to use this loaded method.
  2. To create a new method, use the loaded method as a template
  3. Save the Method under a different name.

Create a Method
  • Click on the Instrument Module windows to change a parameter
  • Quat Pump
  1. RCM in the Column Comp. module
  2. Click on Method
         👉   Set eluent composition, gradient and flow rate

Column Heating
  • RCM in the Temperature Column Compartment (TCC) module
  • Click on Method
  1. ID the HPLC Column and the parameters
  2. Set the Temperature Control for the HPLC column loaded
  3. Ramping temperature is possible in the Timetable area

  • RCM on the Sampler module
  1. Click on Method
         👉   Set the Injection Volume


A.   DAD
  1. RCM in the DAD module
     a. Click Method
  • Set the characteristic wavelength(s) and reference wavelength (if necessary)
  • Set the peak width: default values 5-20 Hz
  • Spectrum stored: All in Peak
  • Zero Offset: 5% (default)
  • Slit: 2nm (default)
  • Auto balance during the Pre-run
  • UV Lamp on between 190-400nm
  • Vis lamp on between 400-950nm

     b. Timetable
  • Change signal channels or wavelengths during a run

     c. Lamps ON
  • RCM in the DAD module
  • Click Control
  • Lamps
                        i. UV –On (if necessary)
                       ii. Vis –On (if necessary)
                      iii. At Power Off (default): Do not use

B.   FLD
  1. RCM in the FLD module
     a. Click Method
  • Signal to be set:
          👉   Excitation:200-1200nm
          👉   Emission: 200-1200nm
  • Set the peak width: default values 2-5 Hz

     b. Click Control
  • On
  • Multiplier Factor: 1.00

C.   RID
CAN NOT be in-line with the other detectors OR with the Fractionator
  1. RCM in the RID module
     a. Click Method
  • Optical Unit Temperature set
  • Set the peak width: default values 2-20Hz
  • Polarity (+) is default value
  • Auto zero Before Analysis: On
  • Automatic Recycling After Analysis: Off

     b. Click Control 
  • On

Save Method
  • Use a different name from any of the loaded methods

     a. Sequence Parameter
  • Set a new subdirectory for EVERY new sequence
  1. The data may be overwritten
  2. Change to prefix/counter and set a base name to id samples
  3. Sequence output: As specified by method
     b. Sequence Table
  • Transcribe for each sample:
  1. Vial number
  2. Sample name
  3. Method
  4. Injections/Vial
  5. Sample Type: Set to sample
  • Fill down Wizard can be used
  • Press Okay

Method > Save
  • Use an exclusive method name

HPLC Analysis Run Section
  • Analysis Run
     a. Single Run
  1. Select ‘Run Control’
  2. Sample Info…
  3. Populate Required Area
          👉   Run Method

     b. Multiple Run
  1. Run Sequence
          👉   Select ‘Run Control’  ----->  Run ‘Sequence’

Print Report
  • Press Okay to print to the default printer
  • If the need is to auto print the Report go to #16(c)(i)
  • If the Report is unsatisfactory go to #16(b) and alter accordingly

Data Evaluation Section
  • Click on Instrument Offline
  • Click Data Analysis tab
  • Click on File ------> Load Signal
  1. Load the data file of interest

  • Print (default parameters)
  • Specify Report (specify parameters to print)
     1.  Determine Qualitative Results Info
          👉   Calculate: Percent
          👉   Based On: Area
          👉   Sorted by: Signal

     2.  Report Style: Short
          👉   Add Chromatogram Output box checked

     3.  Report Layout for Uncalibrated Peaks
          👉   With Calibrated Peaks box checked
  • Destination
     1.  Printer box checked
          👉   Reports are auto printed at the end of a run
                  a. Ensure a default printer is available
     2.  Screen box checked
        👉   Reports are shown on the screen at the end of run where they can be printed
          👉  The subsequent run report will remove the preceding run report from the screen

     3.  File box checked
          👉   File Setting
                  a. Check the Unique pdf file box
                  b. Check the desired file format
                       i.  Files are saved in the xxxx.D file format

  • Using the CS Integrator
  1. Integrate Manually
          👉   Using Integration Events
  • Auto Integrate

  • Calibration Table
  1. Set the Analyte Name, Level and Concentration
  2. Calibrate

Method > Save
  • This will lock the Report/Integration/Calibration Results, to the specific method for successive analysis. All default values will be saved for future analysis.

On instrument control diagram: hit on to start the ovens, pumps and lamps. Wait until all sections are ready (diagram color from yellow to green). Observe the baseline in Online Plot (View-> Online Signals-> Signal Window 2); it should be stabilized before you start running samples.



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