LC/MS

Rough Draft of LC/MSD Advanced User Operating Instructions

NORMAL RUN PROCEDURE:

1. Prepare Sample. Dilute the sample in MeOH or MeOH/Water at no more than 1 mg/ml. Use only HPLC grade solvents.

WHY: The main reason that dilute solutions are used is to prevent the column from being overloaded. Overloading causes peak tailing and impairs separation.

The mass spec is capable of handling a wide range of concentrations. It is a very sensitive instrument and 1 mg/ml is somewhat high. As experience is gained with the analysis, lower concentrations may be prepared. Sometimes the same solution used for the NMR is also used on the LC/MS. If this done however, the analytes of interest must be cleanly separated from the solvent peak.

2. Filter the solution with a 5-micron filter.

Why: Particulate matter will clog the HPLC column. In extreme cases, the entire instrument may stop working because the injector or pump components are blocked. You can tell if there is a blockage if the back pressure from the column begins to rise.

3. On the menu bar, select the Method Run and Control option under View.

Why: This screen is the primary user interface. Most of the time it will be already displayed.

4. Turn on the HPLC, MSD, and DAD by clicking on the grey icons and using the Control option.

Why: Each component in the system has a distinctive control window. This allows a user to selectively turn them on, off, or place in the standby mode.

When the components are initializing their respective icons will be yellow. To find out what is not ready, you can click on the icon, select Not Ready and check on the status.

Remember to turn both the UV and Visible lamps ON for the DAD.

5. If necessary, set the mobile phase bottle and flow rate by clicking on the pump icon and choosing Setup.

Why: The default flow rate is 0.3 mL/min. However it may have to be adjusted for certain samples.

6. Check the nitrogen flow to the mass spec

Why: Click on the mass spec icon and select Spray Chamber. Read the “Drying Gas Flow” value, it should be 10 ml/min when the mass spec is operational and 3 ml/min when it is in standby mode.

Even though the instrument operates under vacuum, dry nitrogen is used to blanket the mass spec’s inlet. As a relatively inert gas, it will not react with samples. Also the hot surfaces inside the mass spec can be damaged by contact with oxygen.

7. Click on Run Control form the menu bar and select Sample Info

Why: You must enter a new sample name and then you may enter as much information as appropriate for the sample. If no changes need to be made, you still need to enter a new sample name.

Entering complete sample information will help you track the data for your projects and classes. In most workplaces, your data will not be accepted without this documentation so form good habits now. And in the happy event that you and your faculty advisor make a useful discovery, you will need that information for the patent office. (Yes, I’m serious.)

8. When finished entering sample information, click the Run Method button, which is located at the bottom of the dialog box.

Why: This button tells the system that it should prepare for a sample injection.

9. Choose YES if asked to overwrite a file.

Why: At the present time, most instrumental records are kept in paper form.

Note that the system status icon turns to a purple color. A message appears stating that the system is running and also that it is waiting for an injection. Do not allow this seeming contradiction to confuse you.

10. Wait for the MSD Manual Injection dialog box to appear but don’t click on anything yet.

Why: You need to wait because the system will need a few minutes to prepare itself. When it senses that an injection has been made, it will start automatically.

This time of preparation is invisible to the user when an autosampler is used on a system. Yet there is almost always a short waiting period between runs.

11. Check the following parameters:

Correct bottle of mobile phase. [ ]

Why: The mobile phase bottle from which the pump is drawing will be indicated on an icon.

Is there sufficient mobile phase for your needs? [ ]

Why: At the flow rates this system uses, there is generally little danger of the reservoir running dry. If you need to refill the reservoir, use only HPLC grade solvents.

Is the pump running at the correct speed and is the backpressure at an acceptable level? [ ]

Why: The speed at which the pump operates will control the analyte elution time, separation efficiency, peak widths, and the pressure inside the column. The actual speed should be checked against what you set it for.

Knowing an acceptable level of backpressure requires some experience. Get in the habit of checking the pressure each day. Shut off the pump if the pressure is not stable, has risen dramatically in the last 24 hours, or exceeds 350 bar. Any of these symptoms means that the column is clogged.

Is the degasser working? [ ]

Why: Removing dissolved gasses and gas bubbles from the mobile phase will improve your chromatography.

Check the degasser’s front panel. The light on the left should be green and the light on the right should be off. If the degasser is working properly you will also hear a chirping sound occasionally as the pump works to maintain a partial vacuum

Are all the component icons green? [ ]

Why: If a component is not ready, its icon will turn yellow. The cause will also be displayed. Should any icons remain yellow for more than a few minutes, notify your instructor.

12. Using a clean HPLC syringe, load 10 uL of sample into the injector valve. Then turn the valve clockwise to load.

Why: As the run begins, the icon for each component turns blue. If they do not, it means that the injection was not detected and you must click on the Start Run button.

If you forget to start the run and sample is already in the system, don’t panic. Simply wait for the components to elute try again.

13. When the samples have fully eluted, press the f8 key to stop the run.

Why: You can watch the total ion chromatogram in the window on the lower left corner of the screen. When all your components have been eluted, allow the run to continue about a minute more, and then stop it.

Because each sample is different, the system is configured so each operator can allow the run to continue as long as necessary. When analyzing multiple samples, a stop time can be set as one of the method’s parameters.

14. Wait for all icons to return to green.

15. Turn the injector back to LOAD.

Why: Remember that the injector works by diverting the mobile phase flow through a sample loop and sweeping the sample onto the column. A sample can only be placed in that loop when the valve is set to Load.

DATA ANALYSIS IS DESCRIBED ON ANOTHER SET OF DIRECTIONS: SEE: LCMS STUDENT DATA

SHUTDOWN PROCEDURE:

WARNING: It is very important to shut down properly in order to conserve the nitrogen gas supply and save wear and tear on HPLC pumps.

1. Return the screen to Method Run and Control under the menu bar’s View option.

2. Click on the Pump, DAD, and MSD icons and select the Control option. Turn the DAD’s UV and Vis lamps off, set the other components to Standby.

3. Confirm that the pressure in the pump returns to zero.

Why: Excess pressure over time will damage the pump. Note what happens to the total ion chromatogram when the mobile phase stops flowing. The smooth baseline is replaced by random noise.

4. Check the nitrogen flow to the mass spec

Why: Click on the mass spec icon and select Spray Chamber. Read the “Drying Gas Flow” value, it should read 3 ml/min. If not, notify your instructor immediately. Allowing gas flow to remain high causes the nitrogen tank to run dry. The system will have to be shut down.

5. Clean up! Cap your samples and store them properly. Rinse the syringe thoroughly. Remember that only standards are allowed to be stored in the mass spec lab.

Why: There is a positive correlation between cleanliness and good grades.

Use the browser's back button to return to the home page.