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Hi, I'm Daniel Sharp and I am a graduate research assistant in Doctor Shellhammer's lab at Oregon
State University, and today I'm going to be showing you a new proposed method for measuring
bitterness in beer using solid phase extraction. For this method you'll need HPLC-grade methanol,
concentrated phosphoric acid, one octanol, a wash solution which is 60 percent methenol
and 40 percent deionized or highly purified water, and this is acidified with 200 ml.
of phosphoric acid per 100 ml of solution; elution solvent which is 100 ml of methanol
acidified with 100 micro leaders of phosphoric acid. You'll need some milacue or deionized
water. You'll need a supell clean LCEASPE column. You also either a positive pressure
or a negative pressure column to push or pull your sample through the column. That can be
done using a vacuum manifold as we have here for multiple samples. For single samples,
it's much cheaper to use a side-arm flask hooked up to a water aspirator, or if you
don't have a vacuum source or are having difficulty getting a constant flow rate, you can use
a supelo single vile syringe that allows you more control. Also, you'll need two large
beakers for de-gassing your sample. A 250 meter graduated cylinder. Some pipettes, 1-5
mil. adjustable auto pipette and a 200 micrometer to 1000 micrometer adjustable pipette, some
syringe filter to filter your sample afterwards. You'll need to measure out about 100 mls.
of your sample. It's also important to allow your sample to come to room temperature, and
just pour it back and forth 20 times. And then we add a drop of octonol to help knock
down the foam, and try and use as little as possible and then pour back and forth an additional
six times, and it's important to collect the foam here as you do this. Now measure out
100 mls. of your sample using a graduated cylinder. And then transfer to a flask, add
200 micrometers of concentrated phosphoric acid. After we've prepared our sample, we're
going to start the extraction process. Before we do that we need to condition our SPE column,
and today we're going to be using a sidearm flask for single sample measurement. Insert
your SPE column into the stop *** and also the teflon needle that goes into the sidearm
flask. Turn on the water aspirator. This is what you'll use to control your flow rate
and not the stop ***. First place 2 mls. of ethanol into the column. With all of these
steps, it's very important not to exceed a flow rate of 4.5 milliliters per minute. We
generally shoot for 2-3 milliliters per minute. Otherwise, you'll wash all of your compounds
of interest out of the packing material. Add four milliliters of water to the column. We'll
add two mls. of our sample. During this step the compounds of interest will be retained
in the packing material here, and everything that drops into the sidearm flask can be discarded
later. For the washing step, we're going to ad 5 ml of wash solution. Once it's turned
dry, stop the stop ***, turn off your aspirator or the vacuum source, and we're going to dispose
of all the waste in the side arm flask. In order to collect your sample, we've found
that it's easy to use a 10-mil volume metric flask with a rubber stopper taped to the bottom
that we insert through the top. Use tweezers if you need to to reposition it. Next, replace
your SPE column inserting the teflon needle into volume metric flask inside the sidearm
flask and make sure you have a good seal with your rubber stopper. Start your vacuum source
again, and we'll begin sample collection using the elusion solvent. We'll be adding 4 ml
of the elusion solvent to the SPE column. Gently open the stop ***, and adjust your
vacuum source to get the desired flow rate. We'll be adding another 4 ml of elusion solvent
to the column. Once it's run dry you can turn off your vacuum source, close the stop ***
and carefully remove the column. Remove the flask with some tweezers. And then make up
the volume with more of the elusion solvent. Mix the sample well by inverting it a few
times, and then we'll filter it using .45 micrometer PDEF syringe filters, and if we're
only doing a single sample, you can filter directly into a quartz UV spec cuvette, otherwise,
if you're doing multiple samples into a glass test tube or something like that. Once you've
filtered your sample into the quartz cuvette, you can take a measurement using the UV spec.
It's important to use the elusion solvent as a blank in the spectrophetomicer.
And that's the SBU Method. To use a syringe, back up the bottom disc here, it's on a screw.
Pull the plunger up to the top, apply your sample, wash, water, whatever step you're
using to the column. There's no need to use a stop *** because it won't run throughout
the bottom of the packing material. Put the single vile syringe into your packed column.
Make sure there's a good seal, and slowly turn the top disc approximately a quarter
turn per 10 sections will give you the desired flow rate of about 2-3 mils per minute.