Introduction to the HPLC ChemStation and Acquisition

2 In This Section, We Will Discuss: How to work in the Microsoft Windows Environment The structure of the ChemStation Software. How to set up an acquisition method. How to run a single sample.

3 l Delete temporary files on a regular basis. Use Clean Disk for Windows 2000 and XP. l Use Checkdisk to find and correct errors on the disk. l Defragment the hard drive. l Use Virus detection software. l Create an Emergency Repair disk. Maintaining the Computer System Accessories Right-click drive letter

4 The HPLC ChemStation Software Add Instruments Schedule ChemStation Tasks Access Instrument and Software

5 Method and Run Control View Sampling Diagram System Diagram Online Plot Navigation Pane ChemStation Explorer Navigation Buttons

6 ChemStation Explorer Views

7 ChemStation Views Views  Method and Run Control  Data Analysis  Report Layout  Verification (OQ/PV)  Diagnosis Full menu or Short menu Change Views

8 View Preferences Allows you to configure the contents of the ChemStation Explorer Specifies naming convention for sequence data containers Specifies how signals are loaded

9 Parts of a Method l Method information l Instrument control parameters l Data analysis parameters l Run Time Checklist What is a Method? A method comprises all the parameters necessary to perform data acquisition and data analysis, including integration and calibration parameters, for one sample. Pre- and post-run tasks may be specified by a command or macro in the run-time checklist. The method is identified by a file name with a.m extension. Master methods are stored in Chem32\#\Methods.

10 Instrument Parameters and Control: System Diagram or Menus Click on GUI for parameters. Instrument control via menus or GUI Click here for instrument control.

11 Create a Method In the Method and Run Control view, Select New Method, or double-click on DEF_LC.M. DEF_LC.M is loaded. This method is a template file that cannot be overwritten.

12 You may use Edit Entire Method to sequentially move through instrument parameters required to acquire data for one analysis or access the parameter windows by selection. Note: Edit Entire Method does not access all instrument parameters such as More Pump > Auxiliary, etc. Editing a Method Using “Edit Entire Method”

13 Select Portions of Method to Edit Information about the method Instrument parameters found in Method and Run Control view Parameters for post-acquisition processing found in Data Analysis Parts of the method to run

14 Method Information Fill in any information you want stored with the method.

15 Time programmable composition, flow, and pressure. Pump Parameters

16 Injector Parameters - Agilent 1100/1200 Standard Sample capacity l 100 x 2 ml vials in 1 tray l 40 x 2 ml vials in 1/2 tray l 15 x 6 ml vials in 1/2 tray l Microvials with sleeves Injection volume l  l standard l Up to 1500  l with multi-draw kit. l Up to 900  l in a single draw using expanded injection upgrade kit.

17 l NEEDLE WASH to reduce carry-over to the absolute minimum l MULTI DRAW MODE for injection volumes greater than 100 ul l SWITCH VALVE TO BYPASS to decrease standard loop delay volume (300ul) to a minimum (bypass) delay volume of 6.2 ul l INJECTOR PROGRAM for programming custom injection steps Widest dynamic injection range: 0.1 µl-1.5 ml From pump To column Metering device To waste 4-port rotor seal Agilent 1100/1200 Injector Special Functions

18 Agilent 1200 High Throughput Samplers Sample Capacity 2 well plates (96 and 384) plus 10 additional 2-mL vials. 108 x 2-mL vials in 2 x 54 vial plate plus 10 additional 2-mL vials. 30 x 6-mL vials in 2 x 15 vial plate plus 10 additional 2-mL vials. 54 Eppendorf tubes (0.5/1.5/2.0mL) in 2 x 27 Eppendorf tube plate. Also compatible with the Agilent 1200 Series sample capacity extension for further expansion of the sample capacity.

19 Agilent 1100/1200 DAD Parameters  5 signals (standard DAD).  8 signals (SL).  Sample signal nm.  Slit programmable; 1, 2, 4, 8 and 16 nm settings.  Time programmable.  80 Hz data rate DAD (SL) for rapid resolution columns. DAD – SL Window shown

20 VWD Parameters Time Programmable Set peak width to narrowest chromatographic Peak width. VWD – G1314C

21 Column Thermostat Parameters l 10 degrees below ambient to 80 degrees C (Standard). l 10 degrees below ambient to 100 degrees C (SL). l Two separate heated zones for two columns. l Optional valve for column switching applications. l Compartment holds up to 30 cm column. l Column identification module with injection record for GLP. (TCC –SL shown)

22 Run Time Checklist Select items to execute during the method. Send your report to Excel using a custom macro.

23 Saving the Method Save a method by selecting Save Method or Save Method As from the Method menu, or select the Save Method Tool.

24 Prepare the Instrument – UV Lamp On Turn on a UV lamp at least 20 minutes prior to your first analysis for warm-up by clicking the control button. Balancing Ready

25 Prepare the Instrument – Prime the Pump Purge Valve 1.Make certain the vacuum degasser is on (if applicable). 2.Open the purge valve. 3.Pump 5 mL/min of 100 % A until all air bubbles have cleared.

26 Prepare the Instrument – Prime the Pump 4.Pump 5 mL/min at 100%B until all air bubbles have cleared. 5.Pump 5 mL/min at 100 % for each remaining channel. 6.Change the composition to that of your next run and continue. 7.Change the flow rate to that of your next run. 8.Close the purge valve.

27 Prepare the Instrument –Instrument Actuals Allows you to review your instrument parameters, check pump pressure and module status at a glance.

28 Prepare the Instrument - Instrument Actuals

29 Edit Signal Plot Click Change

30 Run One Single Injection To inject an individual sample, select Sample Info..., then Run Method.

31 Start Method

32 Follow Acquisition

33 Logbook Entries Check how the run proceeded in the Logbook.

34 Directory Structure for Data Files Instrument # UV Spectra UV Signal Chromatograms Logbook for Run

35 Turn Off System Remember to flush buffers from the system. Do not leave 100% Acetonitrile in the system. Do not leave the TCC at high temperatures without column flow for extended periods.