Waters HPLC Instructions

Waters HPLC Instructions

Susan J. Kohler

May 14, 1998

Introduction: These instructions are meant to be a guide to the routine use of the Waters LCI instrument in its basic HPLC mode of operation. It does NOT cover the use of this instrument when it is configured for ion chromatography; that is covered in a separate set of instructions. More detailed information on all topics is found within the LC Module I/LC Module Iplus Installation and Maintenance Guide (IMG) and the LC Module I/LC Module Iplus User’s Guide (UG). These instructions will deal with general cautions, daily setup, routine operation, once a week maintenance, and long term shutdown procedures.

General Cautions: HPLC equipment uses extremely small bore tubing throughout. Therefore great care must be taken to use only HPLC grade solvents or filtered buffers and to avoid the possibility of either particulate contamination or miscibility problems. In order for us to properly maintain this instrumentation, it is important for you to record the appropriate information in the log book each time you use the instrumentation.

Instrument Overview: The Waters LCI instrument in its basic HPLC configuration consists of a Pump, HPLC Column, UV/Vis Detector, Autoinjector, and Sample Carousel. These components are controlled by the microprocessor-driven System Controller which is accessed via a keypad and display screen. In addition to a numeric entry section, the keypad has several permanently labeled keys (Function Keys) which call up specific screens on the display. Of particular interest are the Set Up key which accesses a series of screens for instrument set up and the Direct key which accesses the screen for direct control of an experimental run. There is also a row of blank keys (Screen Keys) which take on variable tasks as identified directly above them in the display screen. Unfortunately one of the Function Keys is labeled Set Up and one of the Screen Keys is labeled Setup. In order to help distinguish between the two types of keys, they will here be referred to as Function Buttons (the permanently labeled things) and Screen Keys (the variable things).

As mentioned earlier, it is also possible to configure this instrument for ion chromatography by installing the appropriate column, connecting the optional pulse dampner, and using the conductivity detector. Instructions for use of the instrument in the ion chromatography configuration are given elsewhere. In particular, the Autoprime function may NOT be used with the ion chromatography configuration.

Daily Set Up (see UG chapter 3): The daily set up routine involves turning the system on, removing any dissolved gas or bubbles from the fluidics system (eluent reservoirs, reservoir lines, pump, and autoinjector), and performing a compression check on the autoinjector.

Power On. Turn on the power switch on the left side panel at the lower front corner of the instrument. After initialization is complete, press the Set Up Function Button. Note that the power on automatically turns on the UV source, so it will have some time to equilibrate while the rest of the preparation is in progress.

Sparge (UG 3.2).

Turn on the helium tank. The pressure must be between 50 -150 psi.
If the Pump Setup screen is not displayed, access it by pushing the Set Up Function Button.
Check the Reservoirs to Sparge. Move the cursor with the arrow keys to position it over the Enabled/Disabled space for reservoir A, and set it appropriately. It is Enabled by pressing 1 followed by the Enter key, and Disabled by pressing 0 followed by the Enter key. Enable each reservoir you plan to use, and disable the others. This does not start the sparging process, but it prepares it.
Start sparging. Press the Direct Function Button to get to the Direct Control screen. Use the arrow keys to get to the Sparge section and set the rate for 100 ml/min. Let the helium flow at this rate for 20 minutes while you finish the setup.

Purge the Reservoir Lines (UG 3.3.2).

Overview: The line from each reservoir which will be used should be purged to remove any gas bubbles. This is done by setting the eluent composition to 100%, starting autoprime, and using a syringe to withdraw and then inject the eluent into the flowing system, as follows:

Set Eluent Composition: If necessary, press the Direct function button to get to the Direct Control screen. Use the arrow keys to move to the percent composition area. Enter 100% for the eluent line you wish to purge and 0% for all other lines. You must hit the Enter button after each entry, and go all the way through the list of four solvents. When done, the composition you just entered will register as "Current."

Start Autoprime: Press the More screen key, then press the Start Autoprime screen key to start the autoprime. (NOT for ion chromatography.)

Withdraw and reinject eluent: The syringe fits into the Luer lock on the black knob inside the front of the instrument. After attaching the syringe, turn the knob counterclockwise from Run to Draw and withdraw 10-20 mL of the eluent, then turn the knob clockwise to Inject and reinject the eluent. This should be done for each reservoir to be used. Just push the Start Prime screen key to restart the prime if you don’t finish in one prime cycle. TAKE CARE not to flow immiscible eluents one after the other!

Purge the Autoinjector and Perform Compression Check (UG 3.6).

Press the Direct button and enter vial number 1 for both the initial and final vial numbers by using the arrow keys to move the cursor to the appropriate fields and then typing 1 followed be Enter. Then press the Set Up function button and then the Next Setup screen key to get to the Autoinjector Setup screen. Press the Purge Page screen key to get to the purge. Using the arrow keys, set the time to 1 minute and press Enter. Then turn off the compression check by typing 0 followed by Enter while on the compression check line. Then hit the Start screen key. The purge will take longer than one minute, and finally will put up a screen saying Purge Complete. At this point, redo the purge (press Redo Purge screen key). After two purges have been done, press the Return screen key and change the compression check to Yes by moving to that line with the arrows and typing 1 followed by Enter. Then start the purge for the third and final time. This one takes even longer. When complete, note the test results in the log book and press the Return screen key until you get back to a setup screen.

Prepare for Experiment

Press the Direct function button, and turn the sparge rate down to a more suitable value (i.e. 30 mL/min for aqueous and methanol, 15-20 for aqueous and acetonitrile, and 10-15 for aqueous alone (UG p. 3-15)).
Continue with routine operation (see below).

Routine Operation (UG Chapter 5).

Set Up Computer for Data Acquisition Using Chrom Perfect Software.

Turn on the PC and double click on the Chrom Perfect icon.

Click on Acquire Data. Then click on the Download menu heading. There will only be one choice: Chrom1Waters IC. Choose the proper method. It is not necessary to put in complete path names; in fact it is better not to. More complete instructions for the Chrom Perfect software appear elsewhere (SJK, Chrom Perfect Instructions & the Chrom Perfect User’s Manual).

Set Up HPLC.

Unless you are changing from the default conditions, it is not necessary to access any of the Set Up screens (UG Chapter 4).

Press the Direct button to get to the Direct Control screen. Use the arrows to move the cursor to set the proper operating conditions for your experiment. Make sure that the status shown at the far left of the screen key line reads IDLE while trying to move the arrows. The fields to set will include the percent mixture of the available eluents, the sparge rate, the flow rate , the injection volume, the total experiment time, detector wavelength, and the sample vial numbers. Note that the samples are loaded into the carousel which is accessed by opening the bottom door in the front of the instrument. The carousel automatically homes itself to sample vial 1 when it is placed in the instrument. To set the eluent mixture it is necessary to enter a percentage for each of the four reservoirs, and follow each entry by pressing the Enter key. CAUTION: When changing solvent compositions, beware of miscibility problems! (UG 3.1.2). Verify the new solvent composition when you are done!

Prime the Pumps (UG 3.3.1). (NOT for Ion Chromatography).

From the Direct Control screen press the More screen key. Then press the Start Prime screen key to start the two minute autoprime. This will cause the pumps to ramp up to a flow of 15 mL/min for two minutes, using the eluent composition as defined on the Direct Control screen. The column is bypassed during this operation.

Start Experiment.

Push the Setup screen key (NOT function button) to download the parameters which have been set. If the solvent composition is new, it is a good idea to let the system equilibrate for five minutes or so to flush the old solvent out of the column before starting the sample run. Then press the Start Run screen key to begin the experiment. If you have already set up the computer, the injection process will automatically trigger data acquisition on the computer system.

Shutdown.

Overnight:

Flush the system with HPLC-quality solvents. This is most easily done by priming the pump to flush the lines. Since the prime bypasses the column, it is also necessary to run the system for 5-10 minutes to flush the column. CAUTION: Do not flush with solvents which are not miscible with the eluents used in the experiment, and do not use the prime function when doing ion chromatography! The final flush should be with methanol, assuming the column is a reverse phase column such as C-18. Follow manufacturer’s guidelines for other types of columns.
Turn off the main valve on the helium gas tank.
Turn off the power switch.

Long Term:

Follow the overnight shutdown procedure. Remove the column, replace it with a through-connector, and cap the ends of the column for storage. Then make sure that both the reservoir lines and waste lines are immersed in appropriate solvents in sealed containers to reduce the chance of the lines drying out.

Routine Maintenance (IMG 3.1).

Weekly:

Fill the Needle Wash solvent reservoir with fresh solvent (IMG 1.7.1).
Record UV detector energy when set to 254 nm and 100% methanol as both the sample and eluent. This is done by following the flow chart on p. 4-15 of IMG to get to the detector 1 diagnostics screen, then displaying the energy.
Check the flow rate under pressure. With a column in place, run the waste line into a graduated cylinder and verify the flow rate (IMG 3.1.2).

Every three months:

Autocalibrate autoinjector (IMG 3.6.3).

Power Up After Long Shutdown.

The main problem is likely to be dried out lines. The normal daily startup routines should take care of the pump and reservoir lines. The needle wash line might require forcing solvent through the line with a syringe. Connect the syringe to the needle wash intake line (green), start the needle wash (IMG 3.6.4) and expel solvent into the line. Autocalibrate autoinjector (IMG 3.6.3).