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[MUSIC PLAYING]

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PROFESSOR: Thin Layer
Chromatography, or TLC,

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is a common analytical technique
used in organic laboratories.

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TLC can be helpful
when you are trying

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to identify the
components of a mixture

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or assess the
purity of a sample.

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It is also used to monitor both
reactions and purifications.

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It can take some time
to become a pro at TLC,

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but this video should
help to familiarize you

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with the basic technique.

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TLC plates are composed
of a thin layer

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of a polar adsorbent,
either silica or alumina,

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which has been bound to a plate
of solid support, generally

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plastic or glass.

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In most cases, a
fluorescent powder

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is also mixed in
with the adsorbent

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to aid with
visualization, as you

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will see later in this video.

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A sample is applied
to a TLC plate

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using a thin glass spotter.

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Micropipets, such as the
one shown here, can be used

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and are conveniently
commercially available.

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However, in many
laboratories, TLC spotters

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are prepared by heating
and pulling capillary tubes

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or disposable Pasteur pipets.

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One benefit of making
your own spotters

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is that these
homemade spotters are

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usually thinner
than commercially

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available micropipets.

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You can watch the advanced
thin layer chromatography

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video for a demonstration
of this procedure.

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Once a TLC plate has been
spotted with a sample,

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it is developed in a developing
chamber that can easily

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be assembled from a
glass jar with a lid,

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a piece of filter paper,
and 5 to 10 milliliters

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of an appropriate
developing solvent.

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Before you get started,
make sure you also

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have a pair of tweezers,
a pencil, and a ruler

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close at hand.

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Assembly in the
developing chamber

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is very straightforward.

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First, slide the filter
paper into the jar

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so that it is flat
against the wall.

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Next, pour in approximately
8 millimeter layer

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of the appropriate developing
solvent into the jar.

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Tilt the jar to moisten
the filter paper,

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and close the lid to
prevent evaporation.

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You should end up with
a layer of solvent

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no more than 5 to
8 millimeters deep.

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The moist filter paper ensures
that the air in the chamber

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is saturated with solvent vapor.

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It prevents evaporation of
solvent from the TLC plate

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during development.

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Once you have put together
your developing chamber,

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it is time to prepare
the TLC plate.

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In this video, plastic-backed
alumina plates will be used.

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For more information on
preparing glass plates,

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you can watch the
advanced TLC video.

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Before you start, there are
a few things to remember.

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Always use a pencil and never
a pen to mark your TLC plates.

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Ink is soluble in
organic solvents

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and will be developed
along with your sample.

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Make sure that you
always mark and spot

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your sample on the dull and
not the shiny side of the TLC

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plate.

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Remember, the dull side is
coated with the adsorbent.

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Even though you should
be wearing gloves

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while handling TLC
plates, it is important

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that you do not touch the face
of the plate with your fingers.

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Oils from your skin
or other contaminants

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can adsorb to the plate
and affect your results.

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Before you can apply your
sample to a TLC plate,

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it is important
to mark the plate

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so that you can keep track of
where the sample is applied.

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One way to do this is to draw
a straight line approximately 1

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centimeter from the
bottom of the plate.

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Draw small ticks through
the line at each point

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where you will apply
a spot of sample.

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It is important that
the spots are not

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too close to the
edge of the plate,

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or evaporation from
the sides of the plate

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will result in
inconsistent results.

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It is also important that
the spots are not too close

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together, or you will end
up with overlapping spots

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after you develop the plate.

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Applying your
sample to the plate

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is probably the
trickiest part of TLC,

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and it may take some practice
before you can consistently

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apply the right amount of
sample in a small enough spot.

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Your TLC sample should
be fairly dilute,

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containing approximately 1%
to 2% of the desired compound.

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If the sample is too
dilute, you will not

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be able to visualize the spots.

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If the sample is
too concentrated,

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you will observe large streaky
spots on your final plate.

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To spot the plate, dip the
glass spotter into the sample

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and touch it lightly and
quickly to the plate.

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Wait briefly for the solvent to
dissolve before spotting again.

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Generally, 1 to 3
spots will suffice.

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It is crucial that you
do not leave the spotter

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on the plate for too
long, or you will end up

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with large diffuse spots.

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It's difficult to separate
mixtures on a TLC plate

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when the spots are too big.

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Try to keep the spots 1 to
2 millimeters in diameter.

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The smaller, the better.

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Once you have
applied your sample

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and the spots have dried, it's
time to develop the plate.

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Using your tweezers, pick
up the plate and place

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it in the developing chamber.

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Make sure that the
solvent level is

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below the spots on
the plate, otherwise

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you will end up with your sample
dissolved in the developing

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solvent.

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Replace the cap to prevent
evaporation of solvent off

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of the plate, and try not to
let the edges of the plate

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touch the filter paper.

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This will disturb the
capillary motion of the solvent

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on the plate.

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Keep a close eye on
the solvent front.

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Do not let the solvent get
closer than 5 to 10 millimeters

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from the top of the plate.

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When the solvent front
gets too close to the top,

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evaporation from the top of
the plate becomes a problem.

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The spots keep
moving up the plate,

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but the solvent front
appears to stop.

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This leads to
incorrect Rf values.

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[MUSIC PLAYING]

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When the solvent front has
reached an appropriate height,

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remove the plate
and immediately draw

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a line at the solvent front.

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This line will be necessary for
the calculation of Rf values.

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When the compound of
interest is brightly colored,

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no extra steps need to be
taken to visualize the spots.

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However, most organic compounds
are colorless and cannot be

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seen on a TLC plate
with the naked eye.

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Fortunately, most TLC
plates contain an additive

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that causes the
plates to fluoresce

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under ultraviolet light.

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Certain UV active compounds
are capable of quenching

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this fluorescence.

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As a result, they appear as dark
spots on the glowing TLC plate.

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UV lamps such as
this one are commonly

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found in laboratories
for just this purpose.

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Just place your
plate under the lamp,

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turn it on, and mark
the spot with a pencil.

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A number of TLC stains have
also been developed to aid

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in the visualization of spots
that cannot be seen under a UV

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light.

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You can watch the advanced
thin layer chromatography

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video for a demonstration
of a few of these staining

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procedures.

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TLC data is described
in terms of Rf values.

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Under a specific
set of conditions,

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a particular compound should
always exhibit the same Rf.

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Let's quickly run
through the procedure

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for calculating an Rf value.

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First, measure the
distance from where

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the spot started on the
plate to where it ended up.

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Always measure from the
center of the final spot.

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We'll call this distance A.
Next, measure the distance

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from where the spot started to
where the solvent front ended

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up.

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We'll call this distance B.

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Do not measure from the
bottom of the plate.

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This is a common
mistake that will

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lead to incorrect Rf values.

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The Rf value is
defined as the ratio

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between the distance
the spot moved,

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A, and the distance
the solvent moved,

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B. This value is dependent on
the polarity of the compound

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and the polarity of
the developing solvent.

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Polar compounds will
have lower Rf values

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than nonpolar compounds under
the same developing conditions.

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This series of plates
illustrates the effect

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of solvent polarity on Rf.

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As the polarity of
the developing solvent

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is increased from left
to right, the spot

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moves further up the plate.

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The value of A gets larger
while the solvent front, B,

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stays the same.

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Increasing the polarity
of the developing solvent

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generally increases the Rf
values of all of the spots.

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It is a good idea
to use a developing

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solvent that gives you Rf
values between 0.2 and 0.8.

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This will generally give you
the most effective separation

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when you have more than one
compound in your sample.

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For example, a mixture
of two compounds

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was spotted and developed in
hexanes, a nonpolar solvent.

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The final plate shows only
one spot with a very low Rf.

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The same mixture was
developed in ethyl acetate,

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a polar solvent, and once
again, the final plate

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shows only one spot, this
time with a very high Rf.

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When a mixture of
hexane and ethyl acetate

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was used as a
developing solvent,

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the two spots were resolved.

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Notice that both spots are
near the center of the plate.

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TLC is frequently
used to compare

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the identity of two compounds.

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In a perfect world, TLC data
would be consistent from plate

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to plate and chamber to chamber.

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Unfortunately, small deviations
in development conditions

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affect the observed Rf values.

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As a result, it is most
convincing to compare

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Rf values of different
samples on the same TLC plate.

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For example, you know that
the unknown sample marked

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with a question mark consists of
either compound A, compound B,

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or a mixture of the two.

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You can use TLC to determine
the identity of the unknown.

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You will need two TLC plates.

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Make three marks on the first
TLC plate, one for the unknown,

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one for A, and one in the
center for the co-spot.

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Do the same with
the second plate,

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except substitute B for
A. On the first plate,

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spot compound A on the center
mark and one of the side marks,

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and allow both
spots to dry well.

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Drain the excess liquid from
the spotter onto a paper towel.

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And rinse your spotter in
clean solvent before switching

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to a different sample.

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Now, spot the unknown
mixture on the center mark

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and the other side mark
of the first plate.

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Repeat this procedure with
compound B on the second plate,

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and develop both plates.

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Now, compare the two plates.

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You can see that
the co-spot with A

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shows two distinct spots
while the co-spot with B

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shows only one.

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These results indicate that the
unknown sample contains only

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compound B.

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In this video, we have
surveyed the basic techniques

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that you need to know
to use thin layer

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chromatography in the lab.

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You have learned how to set
up a developing chamber, how

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to mark and apply your
sample to a TLC plate,

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how to develop the plate
in your developing chamber,

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how to visualize the spots
on your developed plate

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using a UV lamp, and how to
correctly calculate Rf values.

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You have also
learned how to choose

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an appropriate developing
solvent for your system

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and how to compare the
identity of two compounds.

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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.

249
00:15:07,240 --> 00:15:08,500
Now it's your turn.

250
00:15:08,500 --> 00:15:11,470
Good luck.

251
00:15:11,470 --> 00:15:13,620
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