Introduction:

The MS in MCAL uses electro-spray ionization (ESI) and Ion Trap technology to detect sample ions. The analyte ions are nebulized and dried (and become charged ions).  The ions can be scanned in full scale mode, MS-MS mode or can be further fragmented and the product ions scanned. The MS is connected to a binary LC system.  

Laboratory Week 2: HPLC-MS

Prior to using the LC-MS, there are a few important points to check:

·         Ensure the Solvent bottle has significant levels: 30% Acetonitrile.  Both pump lines should be in this bottle.   

·         Ensure the source on the Mass spec is connected to the LC via the red tubing  rather than the syringe pump

·         Equilibrate the column for at least 5 min

·         Always slowly increase flow rate (ramping over 1-2 min) as to avoid sudden pressure surges to the system

·         Make sure you wash the autosampler before injecting your first sample (ask the instructor to show you how, it just needs to be done before you start your samples). 

Part A: Start up LC-MS

1.      At the top of the system control software; select window in the tool bar and select 212 LC to open the pump control window.  There will be a graph display plotting each pump’s pressure as well as solvent. 

2.      Click the manual control button and a new window will pop up.  Enter 0% under pump b and a flow rate of 250 uL. Ramp time 1 min.  Click start and the pumps should start and begin recording on the graph.

3.      Monitor the pressure.  It should be fairly stable. Slight fluctuations are normal; pay attention to the scale.  If it is fluctuating more than 30 p.s.i. there may be a problem such as an air bubble.  Consult the instructor if this is the case.

Part B: Prepare Solutions

Stock standards (dissolved in M-Q water):  

Caffeine (1 mg/ml)

Theophylline (1mg/ml).  

30% Acetonitrile + 0.1% Formic Acid

Standards for caffeine determination with theophylline as internal standard.

The easiest way to prepare standards is serial dilution, but remember you need 1.0mL of each standard to put in the HPLC vial for analysis. Also, to do serial dilution you will need to use Theophylline as the diluent in order to keep the concentration constant.   

1. Make a 10 mL solution of 80 ug/mL  Theophylline and 2 mL solution of of 160 ug/ml both using  the stock and 30% Acetonitrile/formic Acid. 

2. Prepare the following Standards by serial dilution ( using Theophyline as diluent)

a.   80 ug/mL Caffeine.

Theophylline 40 ug/ml

b.  Caffeine: 10 ug/ml

     Theophylline 40 ug/ml

c.  Caffeine: 20 ug/ml

     Theophylline 40 ug/ml

d.  Caffeine: 40 ug/ml

     Theophylline 40 ug/ml

e.  Caffeine: 80 ug/ml

     Theophylline 40 ug/ml

Caffeine Unknowns:

·         Cola, Diet Cola, diluted 1 to 5 and 1 in 10

·         Energy drinks 1 in 10 and 1 in 20

All your unknowns should be diluted in 30% acetonitrile, 0.1% formic acid.  Add      theophylline to each of the caffeine unknowns so that the final concentration is 40 ug/ml.  This is your internal standard.

Part C: Set up Method and Run Standards and samples

Set up an LC-MS method:

1.   Open the method builder located on the tool bar at the top right of the screen, it is the second from the top.  Click File then New Method and save it in the 3590 2016 folder. Click next three times and then finish and you will be able to save your method.

2.   On the left is a navigation pane of all components of the method.  Click on pump program and set the following pump program in the window on the right:

            0-12min: 100% A

            The flow rate should be set to 250 uL/min .

3.      Double Click the Mass Spec Aqcuisiton in the navigation window:

a.       Make sure Scan Type is full

b.      Under Active Segment name the segment and set the segment end time.

c.       Under Electrospray Ionization Parameters, set the nebulizing pressure to 50 and the drying gas pressure to 30.  Drying gas temp should remain at 350. 

d.      Under the Full Scan parameters,  set the Capillary voltage to 70, the start and end mass to 100 and 400 respectively, and the RF loading to 85%.

4.   Save your method and upload. To upload, go to the System Control window and there is a button at the top displaying the active method.  Left click and select Activate Method. Find yours and open it and it will activate.

5.   Under the 500 MS window; select File then New Sample list and save it in the 3590 2016 folder. 

a.       Enter  names of standards and your unknowns; the correct vial # position and an injection volume of 10 uL.

b.      Click Data Files in the bottom right hand corner and make sure the data is allocated to your folder.

c.       Click Begin.  Observe the tool bar.  After a minute or so; you should see the NOT READY change to WAITING and you will hear the autosampler start up.

Part D: Data Analysis

1.      Open the MS data review software (found on the same toolbar as the method editor)

2.      Locate your chromatogram from your folder on the left hand navigation window and click on it.

3.      Use the curser and click on the peak of interest in the TIC; this will open the RIC in a window below and display the masses associated with that chromatographic peak. 

4.      Integrate the area of  each of your peaks in your standard and unknowns to create a curve in excel and determine the concentration of your unknown:

a.       Extract the molecular ion by typing in the window at the bottow left of the TIC.  This will filter out only the molecular ion from the rest of the ions through this peak.

b.      Locate the set single click action button on the tool bar above the TIC and set it to integrate.

c.       Click the mouse cursor where you want to start the integration and drag it to where you want to end.  The peak will fill in color and provide an area and RT at the top.  If you can’t see it; you will need to zoom in or out by resetting the single click action back to zoom. 

d.      Record the area

e.       Repeat for all peaks in all samples.

Refer to original lab report for Data Analysis procedures – Internal and External Standard.

Once samples are finished; move the inspirator for pump B into 100% Acetonitrile and change the  to 80 % in the pump manual control window.  Run for 10 minutes to clean out the column.  

Once finished; you need to turn off the pumps from the 212 pump window by clicking Stop Pumps.  Also turn of the trap clicking on it in the schematic; then turn the ONLY  the ion source back on.