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[MUSIC PLAYING]

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PROFESSOR: TLC Advanced.

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While the basic TLC video
covers the essential steps

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for using thing
layer chromatography,

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a few advanced techniques
may also come in handy.

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For instance, learning how to
cut your own glass TLC plates

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or pull your own spotters is
essential in a research lab

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environment.

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It is also important to know
how to visualize spots using

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TLC stains as well as UV light.

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This video will cover
the advanced techniques

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involved in carrying out
thin layer chromatography.

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In the basic TLC
video, you learned

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how to set up a
developing chamber,

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mark and spot a TLC
plate, develop the plate,

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visualize the spots using a UV
lamp, and calculate Rf values.

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You also learned how to
choose a developing solvent

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and compare the identity of
two compounds using a co-spot.

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This video will demonstrate some
advanced techniques commonly

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used in research laboratories.

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You will learn how to cut glass
TLC plates from large plates,

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how to pull spotters
from capillary tubes,

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and how to stain TLC
plates to visualize spots.

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Let's start out
with the TLC plates.

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Plastic-backed TLC
plates such as those

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used in the basic video are
found in many laboratories

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because they are cheap, usually
pre-cut into a usable size,

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and if not, easy to
cut with scissors.

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In spite of these
conveniences, many chemists

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prefer glass-backed TLC plates.

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They can withstand more heat
than a plastic-backed plate,

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which is important when
you are using stains

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to visualize spots.

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They also frequently
offer better resolution.

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Unlike plastic
plates, glass plates

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are generally purchased as
large squares, approximately 20

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centimeters by 20 centimeters.

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This means that it's up to you
to cut the plate into smaller

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plates of usable size.

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With a few well-placed
cuts, you can generally

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get approximately 40 to 60
small plates from one big plate.

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The small plates are
commonly 5 centimeters long

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and about 1 to 2
centimeters wide.

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Cutting glass TLC plates
without breaking them

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is something of an art.

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It will take some practice,
but here are a few tips

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to get you started.

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First, you will
need a glass cutter

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like the one shown
here to score the glass

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and allow you to break
it in a straight line.

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Start by making three
well-placed cuts

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approximately 5
centimeters apart.

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This will give four
long strips that can

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be cut into individual plates.

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Start by placing the large
glass plate adsorbent side down

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on a clean, dry surface.

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Use a pencil and a
ruler to mark the cuts

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that you are going to make.

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Next, use the glass cutter
to score a line in the plate.

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You may be tempted to
score the plate repeatedly

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to deepen the cut in the
glass, but this will actually

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make it more difficult
to break the plate

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in a clean, straight line.

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Instead, firmly score the glass
with one sweep of the glass

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cutter, and using the edge
of your bench for support,

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break the plate in a
clean, straight line.

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Continue cutting the long
strip into individual TLC

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plates until you have
amassed a nice stockpile.

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With your plates
ready to go, now it's

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time to pull some TLC spotters.

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While commercially
available capillary spotters

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can be used to apply
samples to TLC plates,

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many people prefer to
pull their own spotters.

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This is because pulled spotters
have a smaller diameter,

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so you can apply smaller
spots to the plate.

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This means that you
get better resolution,

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and you are able to spot
more spots per plate.

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Because you can
conserve plates, this

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makes pulling your own
spotters the economical choice.

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Put the center of
a capillary tube

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into the blue flame
from a Bunsen burner,

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and hold it there until the
center starts to soften.

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Then remove the
capillary from the flame

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and pull on the
ends of the tube.

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This causes the center of the
tube to stretch and narrow.

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This will take a
little bit of practice.

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If the tube isn't hot
enough when you pull it,

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you won't be able to stretch
it, but if you leave it

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in the flame for too long,
it'll turn into a saggy mess.

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Once you have heated and
pulled the capillary tube,

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break it into two
pieces at the center.

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You should end up with two
fine-tipped TLC spotters

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from every capillary tube.

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With a bit more experience
and an acetylene torch,

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you can also pull TLC
spotters from Pasteur pipets.

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On a good day, you can
get dozens of TLC spotters

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from one pipet.

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But for safety
reasons, this technique

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is best learned from an
experienced lab mate.

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With your plates and
spotters in hand,

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you're ready to learn about
using some TLC stains.

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A standard TLC station
in an organic laboratory

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consists of a UV lamp,
which you learned

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about in the basic video,
a variety of TLC stains,

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some paper towels,
and a heat source,

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either a heat gun
or a hot plate.

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Before you do any staining, spot
and develop your plate as usual

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and allow all of the solvent
to evaporate from the surface.

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Then dip your plate
into the stain.

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Allow excess stain to
drain back into the jar,

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and dry the back of the
plate on a paper towel.

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Don't be discouraged if
you don't see any spots

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on your plate just yet.

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You have to heat the plate
to activate the stain.

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Turn on the heat gun,
and hold the plate just

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above the barrel.

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Watch closely, and you
should see your spots appear.

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Don't heat for too
long or you'll end up

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with a blackened TLC plate.

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While numerous TLC stations
have been developed,

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some good common ones are
Phosphomolybdic Acid, PMA,

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Ceric Ammonium Molybdate, CAM,
and potassium permanganate.

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Let's finish up with
a few general tips

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for using TLC stains.

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Remember that staining
is a destructive form

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of visualization.

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The stain will
react irreversibly

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with the compounds
on your plate.

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This means that
you should always

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view your plate under the
UV lamp prior to staining.

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Not all compounds will
show up in all stains,

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so you may have to screen
a variety of stains

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to find one that works for you.

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Remember, this video
is intended to help

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you prepare for lab by
providing a demonstration

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of the proper
experimental technique.

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It is not intended
as a replacement

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for reading your lab manual
or the supplementary material.

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In order to become a
great experimentalist,

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it is important that you
understand both theory

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and technique.

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Now it's your turn.

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Good luck.

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[MUSIC PLAYING]