4 Instrument Instructions for CE

Transcription

1 4 Instrument Instructions for CE Overview: the vial trays There are three instrumental steps in the experiment: a) Condition the capillary; b) Set up and, if necessary, optimize your method for a single sample; c) Set up and run a sequence, including all your samples and standards. You will need vials of liquid for each step. Although the steps are independent, you should prepare all the vials in advance and place them in the holders before starting the conditioning. The paragraphs that follow discuss each step, but before that we will have a quick look at the vial holders. The CE contains three vial trays. At the front left is the inlet vial tray; to the right is the outlet vial tray; and at the back left is the sample inlet tray. The front trays, which are identical, look like this: A B C D E F Inlet vial tray A B C D E F Outlet vial tray The sample inlet tray, at the rear, is similar, but has eight rows instead of six. Your samples, standards, spiked solutions and QC all go in the rear tray; remaining solutions go in the front trays. The inlet vial tray holds liquid used to condition or wash the column, and also buffer used during the separation. The outlet vial tray holds empty vials, into which waste is delivered, and more vials of buffer. So how do all these vials get used? When you condition the column, or do a rinse, liquid is extracted from a vial in the inlet vial tray, passed through the capillary, and dumped into an empty vial in the outlet vial tray. During a separation, all three trays are used. First the capillary is washed, again by extracting liquid from the inlet tray and expelling it to a vial in the outlet tray. Then a sample is selected from the rear tray and injected into the capillary. Finally, the left end of the capillary is placed in buffer in the left front tray, while the other end goes into buffer in the right front tray; the voltage is applied and the separation begins. The rinse solvents are gradually consumed and the separation buffer gets contaminated, so fresh solution is required periodically. However, you do not need to refill the vials. There is enough room on the trays for several vials of each solvent and of buffer, so instead of using one vial of each liquid you will use several. How many? Well, you will need fresh solvents and buffers for every four samples. Therefore, if you have, say, 17 samples (including standards, QC, ) you will need 5 sets of solvents and buffer. You will also need a set of the preconditioning solvents; see step 4 below.

2 So, let's get started. 1. Power on: Turn on the instrument by pressing the power button on the front. The CE takes several minutes to boot up, so start it 10 minutes before you need to use it. 2. CARE!: You can cause serious damage by opening the plastic cover whilst the instrument is moving the vial trays. Do not open the cover while the instrument is going through the startup. 3. Prepare the sample vials: Work carefully so that you do not muddle up your vials; your experiment will fail if the wrong vial is used for a separation. For each of your samples, standards etc. label a vial, fill it just to the shoulder and cap the vial. Count the total number (N) of standards, samples, spiked samples and QC sample vials you have. 4. Prepare the solvent and buffer vials: For every four of your N samples, you need (i) 3 vials of buffer, (ii) 1 vial of 0.1M NaOH, (iii) 1 vial of DI water, and (iv) 1 empty vial. In addition, for the preliminary conditioning you will need a further 3 vials of buffer, 1 vial of HPLC grade methanol, 1 vial of 0.1M NaOH, 1 vial of DI water, and an empty vial. Prepare all these vials and label clearly. 5. Start the software: Once the CE has completed its start up, turn on the computer and ask your TA to log on for you. Start the 32Karat 8.0 application on the desktop, then open the MDQ 2 module. When this new window opens, an Instrument Wizard window will open automatically. You don't need this yet, so click OK to close this wizard. 6. Initial control: MDQ2 establishes communication between the computer and the instrument. Under Control, select Direct Control, then View. This will create a window you can use for several instrument commands. Click the Load button. You will hear noise as the instrument brings the buffer trays forward for loading. Once the noise stops, open the cover. 7. Insert the vials for the conditioning: Lift the vial trays carefully out of the instrument. Place two buffer vials in positions A1 and A2 of the inlet tray; place the MeOH, NaOH and water vials in positions A3-A5 of the same tray. Place a second buffer vial in position A1 of the outlet vial tray and an empty vial (with lid) in position B1 of the outlet tray. Record the position of each vial. 8. Insert the remaining inlet solvent and buffer vials: Recall that for each four samples, you need a fresh set of wash solutions and buffers. From position A6 onwards on the inlet tray continue to add vials: two buffer solutions, an NaOH vial and a water vial for each set of four samples. The order of these vials does not matter, but you must make a note of where each vial is, as you must specify those positions later. You will find that a (neat and clear!) diagram will be very useful. 9. Insert the remaining outlet vials: Insert the remaining buffer and empty vials in the outlet tray. Do not use columns D, E or F on the outlet tray, or the trays may clash during operation. Again, record the position of each vial. 10. Insert the sample vials: Finally, place your standards, samples, QC, etc. in the sample inlet vial tray. As before, note the location of each vial. Return the trays to the instrument, making sure they are correctly seated, and close the cover. The vial trays will move back to the Home position. 11. Condition the Capillary: You will now prepare a fresh surface on the inside of the capillary, using the Direct Control window. First, flush the capillary with water. To do this, move your mouse over the Pressure reading in the Direct Control window. The cursor will change to a small dial. Click on this box, to open a dialog box. Enter a

3 pressure of 25 psi and a duration time of 2 minutes, then click on the Trays button to select the vial positions. On the inlet side, select the position in which you placed the vial of water. On the outlet side, select vial B1, where you placed the empty vial. This will serve as your waste vial for the conditioning. Check that forward direction and pressure type are selected, then click OK to close the Trays window. In the Rinse Window the tray positions are specified, with the BI: and BO: before the letter and numbers of the vial positions standing for the buffer inlet tray and buffer outlet tray, respectively. Click OK again to start the rinse. The vial trays will move as the instrument begins this operation. 12. Rinse in progress: You will see a little cloud in the Direct Control window, on the lefthand side by the thermometer, as pressure is applied and the capillary is rinsed. If you use the small LED torch that sits on top of the AA PC and shine it through the transparent window on the CE, you may be able to see drips coming out of the outlet end of the capillary as the rinse proceeds. 13. Continuing the conditioning: Check the countdown clock in the Direct Control Window. Wait for the rinse to complete (pressure will fall to zero) before starting the next rinse. Run the following additional rinses, in order, in the same manner. For the buffer rinse, choose the second buffer vial on the inlet tray that you put in place for this purpose (i.e. a rinse buffer vial). This should not be the vial in position A1. Solution Pressure (psi) Time (min) Methanol 25 1 Water 25 2 NaOH 20 3 Water 20 2 Buffer 20 2 Now the capillary should be like new and you can build a method. 14. Build a Method: A method combines several steps, including (each time) preconditioning of the column, sample injection, and applying voltage for the separation. To build a method, you need an offline instrument window. Leave the online window open, but go back to the 32 Karat main screen. Right click on the MDQ 2 module and select Open Offline to create an offline instrument window. Click OK to close the instrument wizard window that appears. Select File/Method/New from the menu bar, and then Method/Instrument Setup. A window with three tabs will open. Set the detection wavelength to 214 nm using the UV Detector Initial Conditions tab and the Wavelength option. Under the Electropherogram channel heading, select Acquisition enabled in order to collect and save data. The rest of the settings can be left as they are. 15. Using the Time tab: Select the Time Program tab. The Time Program window is arranged like a spreadsheet with one event per line, executed in order from top to bottom. To program a line, click the Event box. Click on the down arrow to open a menu of events. Select an event to open a dialog box for that event. In your first line, select Rinse pressure as the event. The Rinse dialog box will appear. The first rinse will be a pressure rinse in the forward direction, for two minutes, at 20 psi, with 0.1 M NaOH. The outlet for all rinses in this conditioning step will be the empty vial in the outlet tray (position B1). You can select the position of the trays graphically, by clicking on the Trays button. Don't worry about all the coloured vials that may be displayed; just click on the two positions required for this rinse and they will be highlighted in red. Select OK once

4 you have entered all this information. For the next 2 lines, select the following rinses: Solution Pressure (psi) Duration (min) Water 20 2 Buffer 20 3 After these rinses, the fourth line of your method will be for the injection of the sample. Click on the Event box and select Inject. In the Inject dialog box, enter a time of 5 seconds at a pressure of 0.5 psi. The pressure and time are small to ensure a small injection, which minimizes band broadening. Select the sample inlet tray position for the sample you want to inject. The outlet position should be the buffer vial you will use for the subsequent separation. You will be creating a sequence later; for now, select the option to Allow Override under Sequence Table. 16. Program the separation: On the next line, click on the Event box and select Separate from the drop-down menu. The Separate dialog controls the conditions for the separation process. You will be separating by voltage, so enter a voltage of 20 kv, and a duration time of 14 minutes. The tray positions must be specified so that both the inlet and the outlet of the capillary are in buffer. Remember, use fresh buffer every four runs. Enter a time value of 0 in the time column next to the Separate action. 17. Autozero: The next and last line of your method will be to autozero the detector. Under the Event drop-down menu, select Autozero. Enter a time of 0.5 minutes, and then click OK. 18. Save the method: Your method is now complete. Save it by selecting File/Method/Save As in the C:\CE data\chem361 directory. Create a folder in this directory (click on the icon of a file with a sparkle in the top of the window) with your own name for saving your methods, sequence, and data files. 19. Prepare to run the first method: Do not close the offline module you used to build the method, but return to the active MDQ 2 module. This is the initial window you opened when you were conditioning the capillary. Load the new method by clicking File/Method/Open. Select your method to load it. To make sure you are viewing it properly, select Method/Instrument Setup. Check that the uv light is on by looking to see that the relevant light is lit on the front of the instrument. To perform a single run, select Control/Single Run from the menu bar. Edit the data path so that your results are sent to the correct directory. You must provide a Sample ID and Data File. For the Data File, you may name your file after the Sample ID, Date, and/or several other options. You can view these by clicking on the arrow to the right of the Data File box. The remaining settings can be left unchanged. 20. Run the method: Click Start to begin the run. Don't open the cover after Start has been clicked! To begin with, nothing much will appear to happen, apart from a message "Running sample " below the display. However, once the washing steps have been completed, the electropherogram should start to appear in the window; if it does not, click on Window/UV to reveal the display. 21. View the results: After the sample has run, you can view it in the offline window. Go back to this window and select File/Data/Open. Find your data file and open it. You can switch to the UV (detector) window by choosing

5 Window/UV 214nm. 22. Optimize the method: If the separation is inadequate, improve your method by changing the separation voltage (see step 17), and do another run to see the effect. Continue trying new voltages until you are happy with the result. In a good separation the peaks are well resolved, and the run time minimized. It may help in your choice of a voltage to know that the time at which a peak appears is roughly inversely related to the separation voltage. 23. Build the rest of the methods: Once you have a method you are happy with, you will need to build several methods to run all your samples. Each method will be slightly different since, for every four runs, you will be using different buffer, rinse buffer, NaOH rinse, water and empty waste vials. Be sure to record the information for each method so you can identify them later. Once you have prepared these methods, you can build a sequence. 24. Building a sequence: A sequence is a list of methods and data files that will be used to run a batch of samples. To create a new sequence, select File/Sequence/New from the Instrument window. Scroll over to the Method column. Click on the grey button in the box and select the method you want to use. This will automatically fill in most of the other boxes in the row. Because you allowed overrides when you built the method, you can change the Sample Inject Inlet and Outlet locations. Leave the run type as unknown for all runs. Fill in the Sample ID column with something to identify your sample. For example, standard 1 could be entered for standard #1. You can change the number of replicates for a sample by changing the number in the Reps column. Remember to use your different methods as you built them for the different buffer locations. 25. Save the sequence: Once you have finished the list for your injections, go to the Filename column. Double click in this box (NOT on the little green square in the bottom right of the square. If you click this it will open a window. Cancel to get out of this window). Enter a file name that includes your initials at the beginning and something to identify the sample. At the end of the name enter <D> to add the date and time to the filename. After you have entered all your runs, in the last line of the sequence, select the lamp off.met method from the C:\CE Data\Chem361 directory; this will turn off the uv lamp when all runs are completed. When you have finished editing the table, select File/Sequence/Save As from the menu bar. Give the sequence a suitable name and save it in the same location as your method and data files. 26. Check that the detector lamp is on: If you run your sequence with the uv lamp off, every method will fail in turn because of the lack of a detector signal, so check the indicator lamp at the front of the instrument to be sure the lamp is on. If it is not, go to the Instrument status window under Control and turn it on. 27. Start the sequence run: Before you leave the laboratory, start the sequence by clicking the green arrow icon, or selecting File/Sequence/Start. Bring up the uv signal window and check that all is well before you leave; the sequence will run overnight. A TA or SLI will print out the data the following day and leave it in the folder outside the laboratory for you to collect, BUT see point 28 below 28. Finish: You MUST remove your vials from the buffer trays the morning after your analysis. To do this, go back into the online instrument window (which should still be open from your runs yesterday). Go to Window/Direct Control. Click on the Load button to bring the vial trays forward. Do not open the cover until the trays have stopped

6 moving! Once movement has stopped, open the cover and remove your vials. Do not throw out these vials, but clean them, and once clean, return them to your TA. Problems and solutions Steps 3/4: The software doesn't communicate with the instrument: Call for help from a TA. The driver card in the PC is probably at fault. Go to the Windows control panel and click on Add new hardware; the PC should find a NI GPIB card, search for a suitable driver, download and install it. Communication should then work. Step 13: I get an autozero error: This usually indicates that the lamp is off: check the UV lamp indicator on the instrument. If it is off, click on the lamp icon in the Direct Control window if it is displayed, or use the Instrument status window, which you ll find under the Control menu, to turn it on. Steps 17/18: My results are poor, or totally missing: Check the current window and make sure it looks ok: there should be little noise, with a fairly steady current of around ma. If it is noisy, there is no buffer present for the injection, which suggests that the vial locations you have specified are wrong. If current is OK, then the injection has failed check the method to ensure that the sample vial has been correctly specified. I don't see three peaks on the electropherogram: First check that you are looking at the detector output (UV), not the display of the capillary current. If the detector output shows fewer than three peaks, try running the QC, which certainly should give three peaks. Still don't see three? You probably have chosen too short a separation time, or too low a voltage, so try increasing the separation time and/or increasing the separation voltage. If you see more than three peaks, the smaller ones are probably contamination.