PKKK/300/UP/016 HPLC Flexar Perkin Elmer

Pengenalan alat

This instrument consists of the following components:

FLEXAR LC Pumps

FLEXAR PDA Plus LC Detector

FLEXAR LC Column Ovens

FLEXAR Autosamplers

Computer

Printer

Operasi alat

Switch ON the battery

Double click <TC Navigator> to enter Total Chrom

At this moment, “Hands-on” button grey-out

Starting the Total Chrom Navigator

Seizing – establishing software & HPLC communication

Seizing – Communicating (L) and Communication established. Hands on available (R)

Preparation

To Start Pump – Click instrument setup

Browse & Choose a sequence to trigger pump

Browse sequence

A sequence displayed 🡪 Action 🡪 Setup

Status: Setting up

Status: HPLC Ready / Pump Off 🡪 Click Hands On

Hands-On 🡪 Pump (or Purge)

Pump – Adjust the composition 100% of mobile phase.

Key in flow rate 3-5mL/min

Repeat 1 – 6 for other line if necessary

Tighten purge valve and remove

Syringe after completed.

Connect the column to the inlet and outlet tubing (could be not connected)

The flow rate and % composition of mobile phases could be change

Equilibrate the column from by slowly increasing flow rate to 1mL/min or as required by method

Benchmark of column C18 (150mm x 4.6mm x 5µm)

At 1mL of 75% MeOH 25% UPW, pressure redback is 2250psi

Hands On 🡪 Autosampler 🡪 Flush Autosampler (perform a few times to get rid of air bubbles in syringe)

Double click Oven.exe

Key-in temperature 🡪 Enter

End of preparation: Run 🡪 Clear Setup

End of preparation: Run 🡪 Clear Setup 🡪 Status

Status: No Method – ready for use

Sequence

Running sample – Click “Sequence”

Browse or select sequence / Create New

Specify the Sample Type, Vial Number, Instrument Method

Double click the Data column to browse the directory (folder to save raw file) and name the raw file.

Example of directory and raw files (xxx.raw)

Highlight rows to run, or just click Actions 🡪 Set Up

HPLC Status: Setting Up – Method parameters being send to HPLC (wait a while)

Status of pre-injection & injection

Ready – Equilibrium (green) (L) and Pump start / equilibrating flow rates (R)

Status – Flushing & Running

Flushing loop, syringe, injecting & running (blue)

Details – view current run sample

Method

Overview of Method Editor (Only run instrument)

Overview of Method Editor (with calibration & processing Method)

Autosampler – Set Injection volume & temperature

Pump configuration

Pump Program (Isocratic)

Pump Program (Gradient)

Summary of Gradient Mode

Instrument Timed Events – Leave blank 🡪 OK. Save the Method Editor

Processing Method Integration & Method

External Calibration

Chromatogram of Data001 will be displayed. Notice that this data file is linked to this method (eg. Test1)

To optimize the processing method, click <Process><Noise/Area Threshold>

You will notice that most of the noises will not be integrated

Go to <Edit Components…> to name the components.

Move the box to the first peak of your interest and type the name of the compound in <Name> field. Click Next. Continue naming the peaks in similar manner.

To open the method file, click <Other><Method>

The method file is opened. Now you can see your component name and retention time appeared in the component list.

Go to <Component><Edit Component>

Click OK

Go to <Component><Calibrate…>

Highlight the data file and select the appropriate level. Click Add.

Click OK

You can see other’s calibration by highlighting the next component.

Save the method by clicking the save icon

Totalchrom – Format Report Editor

A report format is opened with same name as method file name (.rpt)

Go to Report and select whichever column you like to include as well as the column number

Example here showed how to insert column 2 with Component Name

You must insert Raw Amount column if you have done your calibration. This column is indicating the amount / concentration directly calculated from the calibration curve.

Go to Option

To insert the chromatography plot between header and report body, tick <Print replot with reports>

Build a sequence to reprocess stored data

To build a sequence using a template

Method Editor 🡪 Other 🡪 Sequence Editor

Sequence Editor 🡪 File 🡪 New

Sequence Editor 🡪 File 🡪 New 🡪 Global Parameters

A sequence displayed 🡪 Double click “Data” to input .raw files for each row 🡪 Browse & load the correct raw files for each calibration level & samples.

Sequence Editor 🡪 Action 🡪 Batch

Ensure starting & ending row no. set correctly 🡪 Set the rest as following

Batch reprocessing 🡪 Automatically proceed or click the button as showed in the picture

Browse the result file one by one (.rst file)

File 🡪 Print Preview Report

Print Preview Report

System Suitability

TotalChrom Navigator 🡪 Apps 🡪 Suitability

Option 🡪 Suitability Option

Signal to Noise (Peak Height vs. baseline)

Choose the SST criteria 🡪 Input the limits 🡪 OK

File 🡪 Save As 🡪 OK 🡪 Enter File Name (*.sui)

Results Files Selection 🡪 Choose a Result File (.rst file)

The report displayed the criteria that user has chosen

Print report as usual

View / Print Chromatogram

View chromatograms 🡪 Click Overlay icon

Overlay Chromatograms 🡪 2D

Options 🡪 3-D Preview

Adjust X and Y axis 🡪 OK

3-D Chromatogram 🡪 File 🡪 Print 🡪 Check criteria to print

Summary: Repeatability of multiple injections

Double click on the summary button

File List 🡪 Select one or more result files (.rst) 🡪 Add

Files to summarize – displayed the selected .rsst files 🡪 OK

Load from Result File (or Method)

File 🡪 Open Format 🡪 Select a Summary template (.sum file)

Summary interface – grey turned to white

IRIS – Processing software

Browse the first .rst file for viewing spectra

Main View : Chromatogram, contour, spectra & 3d view

Use Compare Tab

Browse and open the 2nd .rst file for comparison

Compare 2 Chromatogram side by side

Overlay chromatogram

Right click on one of the chromatogram, select ”Overlay Chromatogram”

Stack View of Chromatogram & Overlay Spectras

Wavelength Maximum

Move the cursor on spectra to read and label the optimum wavelengths.

Print chromatogram & overlay spectras

File 🡪 Print 🡪 Choose printer (or PDF) 🡪 Choose All Panels in view

File 🡪 Save Results

File 🡪 Print 🡪 All Pane 🡪 OK