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 cock 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 cock.
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 cock, 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 cock, 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 cock 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 cock 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.