Solid Phase Extraction (SPE) Lab
SCHA 319
(Draft)
Lab Objective: Using reverse phase SPE cartridges develop use a method that will allow
for the isolation and assay of caffeine from foods.
Data Sheet 205000U is provide for your information. (On Line)
http://www.alltechweb.com/productinfo/technical/datasheets/205000u.pdf
In a nutshell, you may consider this a method of selective filtration. By controlling the
polarity of your solvents you can hold back your analyte until you are ready to elute it
off. This allows you to use this method for two major applications 1) as a clean-up
method to get rid of unwanted components in your matrix or 2) as a pre-concentration
procedure.
You will be using a Fisher PrepSep 12 Port Vacuum Manifold.
Connect the vacuum hose to the water aspirator in the lab. The turn knob on the inlet will
allow for the formation of a vacuum in the chamber. The gauge will let you know when
a vacuum is present. Turning the knob counter-clockwise will release the partial vacuum
so you can open the compartment. Also each of the SPE cartridge ports turns to shut off
flow. This allows you to shut off the positions you are not going to use. This control of
vacuum gives you the ability to control the flow of the solution through the SPE
cartridge. (Your lab instructor will show you basic block operation).
Use a 1 mL pipetman to add sample / conditioning / wash and elute portions.
You will find 10.0 mL volumetric flasks very handy.
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General SepPack Procedures
1)
Condition with reagent grade methanol. ( 1 to 2 mL) This solvates the solid to
that your binding mechanism will be effective). Follow this with 1 to 2 mL of your polar
solvent.
(I would suggest that you use 1% acetic acid in HPLC water as this is part
of your HPLC mobile phase)
2)
Samples are in aqueous solution and must be added in aqueous solution!
3)
Wash with distilled water. (1 to 2 mL) (This will wash off polar matrix
compounds that might interfere with later analysis.)
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4)
Elute with methanol. (Experiment with this parameter)
You have a solution that is 1.00 mg/mL caffeine in the lab to assist your method
development.
Design an experiment to see what recovery you can get of the caffeine sample. Check
you design idea with your lab instructor before you proceed. Since you are using a
method that measures concentration then how are you going to know how much has
come off column.
Word of advice. How could you know that you have recovered any sample without
looking at a sample that has not been run through a cartridge.
You will be using HPLC to assay the amount of caffeine present in your solutions. We
will have you piggy back on the HPLC Lab to get your results.
Reporting:
(This should be done as a group report (two parts) - all will share the
glory or disgrace)
If you are not the last group to finish the quarter then you will prepare a brief memo to
pass to the group that will do this lab after you (you will also pass a copy to me). The
following week’s group will take the information that is in the report and carry on
method development and/or product assay. This group reports to the next group and to
the group(s) that went before them. The last group will send their memo back to the three
groups that went before. All groups will then prepare a summary for me (Due check out
week). You should compare how much you found in each product and compare this to
expected values. You should also let me know your final assay method. All groups in
each series will share the grade. Since you will need to get the info to the next group so
they can plan their work you should pass along the report three days after you are in the
lab (Monday’s lab by Thursday, Tuesday’s lab by Friday and Thursday’s lab by
Tuesday)
Group Sequences:
5 → 7 → 1 → 2
6→ 8 → 3 → 4
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