Waters HPLC Instructions

Waters Ion Chromatography Instructions

Susan J. Kohler

June 1, 1998

Introduction: These instructions are meant to be a guide to the routine use of the Waters LCI instrument in its ion chromatography mode of operation. It does NOT cover the use of this instrument when it is configured for HPLC; 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: Ion chromatography 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 ion chromatography configuration consists of a Pump, Ion Chromatography Column, Pulse Dampener, UV/Vis Detector, Conductivity 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).

When the system is configured for ion chromatography and the pulse dampener is in place, the ranges of acceptable flow rates and pressures are reduced. 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. If the power has been turned off, turn on the power switch on the left side panel at the lower front corner of the instrument. Also turn on the power for the separate conductivity detector. 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. If the Conductivity Detector has been turned off, turn it back on. It takes a long time to warm up, and should normally be left on.

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%, setting the flow rate to a reasonably high value (depending on the column limitations) and using a syringe to withdraw and then inject the eluent into the flowing system, as follows:

Set Flow Rate: If necessary, press the Direct function button to get to the Direct Control screen. Set the flow rate to an appropriate rate for the column you are using. Do not exceed 1.0 mL/min for the IC-Pak A HR column which is normally in use.

Set Eluent Composition: Use the arrow keys to move to the percent composition area on the Direct Screen. 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."

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. 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).
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 Ion Chromatography.

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

Main Unit: 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 (if using UV detector), 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!

Conductivity Detector: (See the Conductivity Detector Operator’s Guide for more information). If the green light over the Remote button is lit, press the button to return to local control. Then press the Auto Base and Auto Zero buttons to set the base sensitivity range and zero the baseline. The BASE reading should be somewhat higher than the background conductivity displayed as Conduct. Since the output of this detector is from the Integrate terminals, the sensitivity range is set as Integral Sensitivity by pressing the Shift key, then the Sens. Range key. This is normally set to the maximum sensitivity of 10 uS/1V and normally will not need to be changed.

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: Simply turn the flow rate down to 0.01 mL/min, and make sure there is an adequate supply of eluent in the reservoir. You may also turn down the sparge rate to conserve helium. Do not turn off the power to the system.

Long Term:

Flush the column with an appropriate solvent for long-term storage. In the case of the IC Pak A HR column, this is the borate/gluconate buffer.
Remove the column, replace it with a through-connector, and cap the ends of the column for storage.
Flush the system with HPLC-quality solvents. Do not flush with solvents which are not miscible with the eluents used in the experiment. The final flush before storage should be with methanol. CAUTION: Make sure the column has been removed before the methanol flush!
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.
Turn off the main valve on the helium gas tank.

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% borate/gluconate 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).