the idea of gas chromatography is to separate materials based upon their behavior as a gas
but what about this guy this is glucose
what about this guy
this is arachidonic acid I believe it's the C20 or C22 carboxylic acid
it is an Omega 6
what about this guy is a very common material in biological systems
how about these guys ..these are ....androgens is the category
steroids, estrogen, progesterone, testosterone all kinds of steroids there's ba--zillions (lots) of them
how about this guy... this is tetrahydrocannabinol the Delta nine metabolite
very common analyte in chemistry laboratories
so we say
"well , we will just heat them up and get them hot enough and they will turn into a gas"
ehhh... not really...heat these guys up and heat them up and they will decompose
before they ever turn into a gas
they just don't do gas..
they are commonly analyzed by gas chromatography
organic chemists, chromatography people,, look at the molecules and say
"how are we going to make this fly"
like, What are we going to have to do to make this...go into a gas
these molecules tend to stick together and the way to make them go into a gas
is to tie up the groups that make them stick together
which are particularly -OH groups.... hydroxyl groups... carboxylic acid groups.... the -COOH group
and the way to do it is to derivatize them so that these groups are gone
and then they become different molecules but you can still quantitate them
the rule in analytical chemistry is you are less concerned about the specific details
as much as that they are reproducible
so you run your standards and your controls and your blank
and your samples all through the same procedure
and you're analyzing for something that is really not the same as what you started out with but you can still
identify and quantity it
let's put together a very quick example
We're going to run two samples
we need a blank.... a standard
remember this is a one point...this is a two point calibration.
but one of them is " 0,0 " so we only need one more standard
so blank ..standard ...we need a control which is an independent standard ...essentially
and then let's say we have two samples... so we have five tubes that we're going to run
get five test tubes ....label the test tubes... put them in a rack
put 1 mL exactly of the sample in the bottom of each one
1 mL exactly ...
This is an internal standard quantitation
so I say "1 mL exactly"
the operative word is "exactly" (exactly the same)
this is an internal standard ... add 250 microliters, exactly
of the internal standard ...okay ....so add the internal standard
it is not very much
just put in a little bit of internal standard
a real common stage in extractions is to acidify the samples a little bit
so put in 50 ÁL of acid
and then add a milliliter of .....and lets pick ethyl ether as a solvent
so add a ml of ether... to each one
so, you've got the blank would be a ml of water, the standard would be a ml of the standard
a milliliter of the control ..a milliliter sample one
and a milliliter of sample two
so let's look very quickly at the chemistry of it
we have the sample which is hydrophilic
it is a water-based material
the sample is typically urine or maybe blood or a biological laboratory specimen of some sort
but is hydrophilic
the internal standard could be added dissolved in isopropanol or something like
that it really doesn't matter
the ether is, of course, hydrophobic.... the ether will be on top
and then vortex the whole thing
vortex it using a little tabletop vortexer.. just do them one at a time
it will just take you a minute maybe about 10 seconds apiece or fifteen seconds apiece
vortex them really good
and put them back in the rack
they will perhaps quickly start to separate the ether on top and hydrophilic layer on the bottom
remember that ..an ordinary solvent, if it's not chlorinated will go to the top
so the ether will be on top
of common procedures at this point is to put the tubes in a centrifuge
and centrifuge them, to help this separation process
centrifuge them.... it will just take a few minutes..
put them back on the rack. Now ...the ether is nicely separated on top
hydrophilic layer, which is now junk, is now on the bottom.
So the big picture is...
we want the hydrophilic layer, which is now junk, gone
we want the ether...
we want the ether gone too and we want the extracted material left in one of these little guys
...in a little autosampler vial
a technique would be to take the tube.. take the vial...
put a half a ml from the tube ..off the top.. a half a ml into here and blow it down
put another half a ml into here and blow down
put another half a ml mail into here and blow it down and
the ether will be gone.... the hydrophilic layer will be left behind
and all the analytes will be left the in the autosampler vial
so basically just manually ...it's not real simple really
just manually.... a little bit at a time .... transfer the ether layer to autosampler vial
and blow it down
transfer a little more and blow it down
transfer a little more and blow down
transfer a little more and blow it down
in actual fact the way this would work would be you would have the five tubes
five disposable pipettes like Eppendorf pipettes
and five of these guys ...labeled... and you would be constantly
cycling them into ...like you some in the first one
some of the second one
some in the third one
some in the fourth one
some in the fifth one
wait a few minutes...do a little more
wait a few minutes... do a little bit more
wait a few minutes
you'll have a block that will help you blow it down
you want to warm it up just a little bit
the ether is really volatile and it does not take as long as you might think to transfer
the ether into one of these vials
they use "reactivials" for this
it's a little autosampler vial but it has a conical bottom
I don't think of them as being disposable
so you're going to want to probably put them in a furnace
or something before you use them
and then... so now you have the....... ether is gone
the analytes are stuck along the side of the reactivial
you can't see them
take these vials ...you have five of them
put the 50 ÁL of your derivatizing agent in them
vortex them... follow the directions for the particular derivatizing agent
you're using
but you're probably going to want to heat them up for a while
like maybe 20 minutes , or something like that
vortex them put them in a heating block
maybe vortex them once in a while ,while they are in the heating block
let the reaction take place and then without doing anything else
take these vials and put them in the autosampler
and set up a program and see what you get
this analysis involves an extraction and a transfer step
the extraction has a percent efficiency
the transfer step is a quantitation
.....there's a quantitation error introduced with the transfer step
it's very important to not transferred any of the hydrophilic layer
it's more important to not get the hydrophilic layer
than it is to get all of the ether layer
so you're going to have some ether left behind
it's not going to be the same amount every time
you're using an internal standard so you don't really need to worry too much about it
the nature of the mass spec is such that the sensitivity is not going to be much of a problem
so don't worry too much about getting every tiny little bit of it
be very careful not to get any of the hydrophilic layer
and the quantitations will come out fine
if it is set up right ,initially
And run it through the mass spec
the GC mass spec will give retention times according to what you're looking at
it will give ions according to what you are looking at
which aren't really the same as what you started out with
but it doesn't matter
follow it through the same , with the standards and the samples
and the standard will tell you where your retention time is
you'll be looking it up in a reference obviously
for what you're ions will be
your standard will confirm that
and your standard will confirm the retention time and
you can do very good quantitative work with this
the...so you say "well.. but... but,..."
derivatized gas chromatography...... so I want to make a guess that 30% (probably much higher than 30%)
of all gas chromatography is with a derivatized analyte
this is not an obscure branch of gas chromatography