HPLC Method

PROCEDURAL VIDEOS TO WATCH:

• • Correctly using a volumetric pipet

  • Correctly using a volumetric flask

CHEMICALS PROVIDED (FOUND ON THE SHELF UNLESS OTHERWISE INDICATED)

• • Phenol (C6H5OH, MW = 94.11 g/mol; MSDS)

  • Choline chloride, ChCl (MW = 139.62 g/mol; MSDS)

  • Tetrahydrofuran, THF (found in the hood, MSDS)

  • Butyl benzoate solution, 10 mg/L in methanol (MSDS)

  • Sodium chloride (MSDS)

  • Phthalate standard mixture, 20 mg/L in methanol (MSDS)

  • Acetonitrile, HPLC-grade (MSDS)

  • Juice unknown solution (get this from me); BE SURE TO RECORD THE UNKNOWN NUMBER

REQUIRED VOLUMETRIC GLASSWARE

• • 25-mL volumetric flask (4)

  • 10-mL glass pipet (6)

SPECIAL EQUIPMENT (FOUND ON THE SHELF UNLESS OTHERWISE INDICATED)

• • 1000-µL micropipette (on the bench next to the sink)

  • Filtering system (on the bench next to the sink)

  • 50-mL Erlenmeyer flasks and rubber stoppers (6)

  • 15-mL glass centrifuge tubes (6)

  • Vortex mixer (on shelf next to the fume hood)

  • Centrifuge (in ISC 331)

  • 5-mL or 10-mL disposable syringes

  • 0.45-µm syringe filters

  • Autosampler vials and caps (6)

  • 1-mL glass syringes (3, each labeled for its particular use)

PROCEDURE:

A.  Preparation of HPLC Mobile Phase. The mobile phase we will use is a mixture of ultrapure water and acetonitrile (CH3CN). Both need to be very filtered to protect the HPLC column. The acetonitrile is purchased as HPLC-grade and is pre-filtered, however we need to filter our ultrapure water as described here.

1. Pour approximately 500 mL of ultrapure water into a beaker and bring it to the filtering system (next to the door in ISC 306),

2. Identify all the various parts as shown in the image below. (There should also be a black rubber cone that is not shown.)

2.  Log onto the HPLC computer if needed. The user name is Admin and the password is 3000hanover.

3.  Open the HPLC software by double clicking the Infinity II (online) icon on the desktop.

4.  When the Loading Method box appears, select Download to Instrument.

5.  Right-click on the Binary Pump control and select Bottle Fillings.  

6.  Fill in the actual volumes of solvent in the A2 and B1 bottles, and make sure both boxes are checked under Actions. ( Make sure the A1 and B2 volumes show something greater than 0.1 L, otherwise the pump thinks we're running out of solvent and gives us an error.)

7.  We must now "prime" the pump. This will fill the pump heads with solvent and remove air bubbles. Begin by opening the doors to the pump and opening the purge valve by turning it in a counterclockwise direction. This will divert any solvent from the pump directly to waste instead of the column.

8.  Right-click on the Binary Pump control and select Method.

9.  In the Method window that appears, set the pump flow rate to 5 mL/min, and make sure that Solvent A2 is selected (water) at 100%. Then click OK.

10.  Click the Pump On button to begin pumping. You should start to see solvent movement in the pump tubes.  The pressure should never read higher than ~25 bar, so hit the Pump Off button if this occurs and contact me or a TA. You should also see steady drops of solvent coming from the waste line into the waste reservoir (VIDEO). Let the pump continue for 3-4 minutes, then hit Pump Off.

11.  Now open the pump method window again as in step 8 above, and this time set up the Method to prime the acetonitrile in solvent line B1 (see below). Then repeat step 10 above.

12.  When both lines are primed and the pump is stopped, close the purge valve that you opened in step 7 above and close the doors to the pump. 

C.  Method Set-Up and Column Equilibration

1. 1. 1. From the Method menu, select Edit Entire Method...

      2. When the Edit Method: Infinity II box appears, make sure all the boxes are check and click OK.

      3. Enter details about your experiment in the Method comments box and click OK. Something like: Separation of phthalates. Solvent A: water; Solvent B: Acetonitrile. Gradient elution. Detection at 230 nm. Column temp = 35oC

      4. Select AIs as the injection source. This is the autosampler.

      5. The Setup Method window should now appear. Configure the Binary Pump tab. The window should look like the following:

6.  Click the Sampler tab and set up the sampler to show an injection volume of 2 µL and the same Stoptime as the pump.

7.  Skip the Sampler Injector Program tab and go to the Column Oven tab. Set the oven temperature to 35oC. Again make sure the Stoptime is the same as the pump.

8.  Go to the detector (DAD) tab and select both 230 nm as the detection wavelength. 

9.  Skip the Instrument Curves tab and click OK to accept the parameters.

10.  Skip the Signal Details window by clicking OK, then again select OK in the Edit Integration Events window. Make the Specify Report window look like the following:

11.  Skip the Instrument Curves window by clicking OK, then make the Run Time Checklist look like this:

12.  Under the File menu, choose Save As..., then Method...  Give your method a unique name (e.g., MBJ HPLC Method).  (You can skip the Audit Trail and just click OK.)

13.  Now start the instrument by clicking the System on button (see image). The pump will start and the detector lamp will turn on. Keep the system running while you begin preparing samples in part D below. The pressure should eventually stabilize around 400-430 bar. If this doesn't happen, there is most likely a bubble in one or both lines and you'll need to re-prime. (See me if you think this is the case.)

D.  Preparation of the Deep Eutectic Solvent

1. Phenol should be used in the fume hood, so move the top loading balance into the fume hood.  Make sure to wear gloves as well.

2. Weigh 3.8 g of phenol into a weigh boat.

3.  Weigh 2.8 g of choline chloride into a second weigh boat.

4. Transfer both solids into a 50-mL beaker, add a stir bar, and stir for 10 min. This is your deep eutectic solvent (DES). 

E.  Sample Preparation

1. 1. 1.  The juice sample must first be filtered through a 0.45-µm syringe filter to remove any small particles that may clog the column. Pour the sample into a clean beaker and use a clean disposable 10-mL syringe to suck as much sample as possible up into the syringe. Screw a syringe filter onto the end of the syringe and push the sample through the filter back into the test tube. (VIDEO)

      2. Cover the beaker with a watch class or parafilm to prevent evaporation.

F.  Preparation of Standards

1. 1. 1. Obtain four clean 25-mL volumetric flasks and label them Std 1, Std 2, Std 3, and Std 4.

      2. Use the 1000-µL micropipette to add the following volumes of the 20.0 mg/L phthalate standard solution to each labeled flask. Dilute to the line with ultrapure water and invert the flasks 20-30 times to mix.

G.  Extraction 

1. 1. 1. Obtain six 50-mL Erlenmeyer flasks and rubber stoppers. Label the flasks Std 1, Std 2, Std 3, Std 4, Blank, and Unknown.

      2. Pour ~20 mL of ultrapure water into a clean, dry beaker. You will use this for your blank.

      3. Using six clean, dry 10-mL glass pipets, accurately deliver 10.00 mL of each standard, your ultrapure water blank, and your unknown solution into the labeled flasks. Put a rubber stopper on each flask to prevent evaporation.

      4. To each Erlenmeyer flask add 1.6 g NaCl and 800 µL of the benzyl benzoate internal standard solution. With each, replace the stopper and swirl to dissolve the salt. (The salt is added to aid in pushing the phthalates out of the aqueous phase and into the extraction solvent.)

      5. Obtain six 15-mL centrifuge tubes (and caps) and label the tubes Std 1, Std 2, Std 3, Std 4, Blank, and Unknown. Pour each solution from its Erlenmeyer flask into the similarly labeled centrifuge tube. Cap each tube. (Be careful that the cap is on perfectly straight before screwing it down - these are cheap tubes and may leak otherwise ☹️.)

(Be sure to wear gloves in steps 6-10)

1. 1. 6. Use a 1-mL glass syringe to add 800 µL (0.8 mL) of DES (extraction solvent) to each of the six centrifuge tubes. (Rinse the syringe and needle several times with RO water when finished).

      7. Vortex each capped centrifuge tube for 1 min.

      8. Working in a fume hood, use another 1-mL glass syringe to add 800 µL (0.8 mL) of THF (dispersive agent) to each of the six centrifuge tubes. 

      9. Vortex each capped centrifuge tube for 1 min.

      10. Go to the centrifuge in ISC 331 and centrifuge the tubes for 7 min at 3000 rpm. (VIDEO COMING - SEE ME ABOUT THIS UNTIL THEN!!)

H. Preparation of HPLC Samples

1. Obtain six HPLC vials and caps. Label the vials Std 1, Std 2, Std 3, Std 4, Blank, and Unknown.

2. Use a third 1-mL glass syringe to remove 300 µL (0.3 mL) of the top layer of each extraction solution and transfer into the appropriately labeled HPLC vial. Do several rinses with RO water between samples.

3. Use the 1000-µL micropipette (with a new tip) to add 130 µL of acetonitrile to each of the six vials. 

4. Place a cap on each vial and tighten so the seal is snug but not so tight that the septum in the cap starts to curl up. 

5. Gently shake each vial to mix the extract and acetonitrile.

6. Fill a seventh HPLC vial with the 20-mg/L phthalate stock solution and cap this as well. (This will be used to clearly identify the retention times for each phthalate.)

I.  Data Acquisition (Please note this not account for the reference vial. Make sure to add this as a seventh vial/run.)

1. Place all six vials in the autosampler tray, noting the location of each. Make sure the tray is pushed all the way in and then close the door.

2.  Click the Sample Entry tab. 

3.  In the Sample - Half Trays window, click on the tray you are using (P1 is left and P2 is right). The corresponding tray should then appear in the Container window in the middle of the screen. Double click each vial location you are using, and each vial should be added to the Sample List. 

4.  Add your method name to line 1 under the Method Name column heading. You can copy the method to the rest of the rows by first left-clicking the column label "Method Name", then right-clicking and selecting Fill down - Copy.

5.  Fill in a sample name for each vial. Your screen should now resemble the following:

6,  Click the Add to queue button (circled in the figure above), then choose as soon as possible from the window that appears.

7.  The instrument will now begin collecting data. Click the Instrument Control tab to view the chromatogram. (See me or a TA about adjusting the X and Y scales.) Each run will take 10 min. (You may be prompted to save the data file after a run, but for some reason this doesn't happen every time. Don't worry - your data is being saved automatically.)

8.  When all runs are complete, you need to wash the column with pure acetonitrile. As before, right click the Binary Pump control and select Method. Change the solvent to 100% B1 and click OK.  Allow the acetonitrile to wash the column for 10-15 minutes.

9.  While the column is being washed with acetonitrile, pull up your data and print the reports as pdf files. Click the Data Analysis tab in the lower left of the screen. From the File menu, choose Load Signal. Look for your data folder - it's probably labeled with the date and time the data was collected. Open this folder and you should see your data files. Choose a file to open and double click on it. Now go to Print Preview - Report. A report should appear showing your chromatograms and your peak information. Click the Save icon and save the pdf report to a flash drive. Repeat this procedure for all of your data files, then upload the files to LabArchives.

10.  When the column wash is complete, shut down the instrument by clicking they System Off button shown in step C-13 above. Exit the software and log out of the computer.

J.  Waste Disposal / Clean Up

1. 1. 1. Pour your remaining phthalate standard solutions in the waste container labeled "Aqueous Phthalate Waste"

      2. Any remaining solutions containing DES and THF should be poured into the container labeled "Phenol/Choline Cl/Water Waste"

      3. Place all used pipets in the "Pipets to Be Washed" bin. 

      4. Rinse all volumetric flasks 5-6 times with tap water and then 2-3 times with RO water, and return to the shelf. (Use acetone to wash off any markings.) Similarly rinse all glassware from your drawer and return it to your drawer.

      5. Use the sponge in your drawer to clean your bench area.