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[Music]
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thin layer chromatography or TLC is a
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common analytical technique used in
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organic Laboratories TLC can be helpful
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when you are trying to identify the
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components of a mixture or assess the
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purity of a sample it is also used to
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monitor both reactions and
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purifications it can take some time to
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become a pro at TLC but this video
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should help to familiarize you with the
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basic technique
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TLC plates are composed of a thin layer
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of a polar absorbent either silica or
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alumina which has been bound to a plate
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of solid support generally plastic or
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glass in most cases a fluorescent powder
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is also mixed in with the absorbent to
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Aid with visualization as you will see
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later in this
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video a sample is applied to a TLC plate
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using a thin glass spotter microp pipets
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such as the one shown here can be used
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and are conveniently commercially
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available however in many Laboratories
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TLC spotters are prepared by Heating and
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pulling capillary tubes or disposable
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pasture pip pets one benefit of making
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your own spotters is that these homemade
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spotters are usually thinner than
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commercially available
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micropipets you can watch the advanced
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thin layer chromatography video for a
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demonstration of this procedure
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once a TLC plate has been spotted with a
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sample it is developed in a developing
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chamber that can easily be assembled
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from a glass jar with a lid a piece of
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filter paper and 5 to 10 mL of an
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appropriate developing
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solvent before you get started make sure
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you also have a pair of tweezers a
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pencil and a ruler close at hand
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assembling the developing chamber is
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very
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straightforward first Slide the filter
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paper into the jar so that it is flat
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against the
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wall next pour an approximately 8 mm
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layer of the appropriate developing
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solvent into the
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jar tilt the jar to moisten the filter
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paper and close the lid to prevent
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evaporation you should end up with a
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layer of Sol vent no more than 5 to 8 mm
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deep the moist filter paper ensures that
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the air in the chamber is saturated with
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solvent Vapor it prevents evaporation of
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solvent from the TLC plate during
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development once you have put together
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your developing chamber it is time to
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prepare the TLC
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plate in this video plastic backed
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alumin plates will be used for more
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information on preparing glass plates
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you can watch the advanced TLC
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video before you start there are a few
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things to
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remember always use a pencil and never a
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pen to mark your TLC plates ink is
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soluble in organic solvents and will be
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developed along with your
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sample make sure that you always Mark
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and spot your sample on the dull and not
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the shiny side of the TLC plate remember
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the dull side is coated with the
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absorbit
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even though you should be wearing gloves
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while handling TLC plates it is
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important that you do not touch the face
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of the plate with your fingers oils from
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your skin or other contaminants can
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absorb to the plate and affect your
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results before you can apply your sample
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to a TLC plate it is important to Mark
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the plate so that you can keep track of
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where the sample is
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applied one way to do this is to draw a
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straight line approximately 1 cm from
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the bottom of the plate draw small ticks
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through the line at each point where you
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will apply a spot of
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sample it is important that the spots
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are not too close to the edge of the
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plate or evaporation from the sides of
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the plate will result in inconsistent
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result it is also important that the
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spots are not too close together or you
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will end up with overlapping spots after
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you develop the
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plate a applying your sample to the
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plate is probably the trickiest part of
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TLC and it may take some practice before
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you can consistently apply the right
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amount of sample in a small enough
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spot your TLC sample should be fairly
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dilute containing approximately 1 to 2%
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of the desired compound if the sample is
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too dilute you will not be able to
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visualize the spots if the sample is too
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concentrated you will observe large
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streaky spots on your final plate
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to spot the plate dip the glass spotter
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into the sample and touch it lightly and
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quickly to the
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plate wait briefly for the solvent to
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dissolve before spotting
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again generally one to three spots will
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suffice it is crucial that you do not
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leave the spotter on the plate for too
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long or you will will end up with large
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diffus spots it's difficult to separate
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mixtures on a TLC plate when the spots
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are too big try to keep the spots 1 to 2
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mm in diameter the smaller the
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better once you have applied your sample
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and the spots have dried it's time to
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develop the
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plate using your tweezers pick up the
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plate and place it in the developing
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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
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dissolved in the developing
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solvent replace the cap to prevent
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evaporation of solvent off of the plate
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and try not to let the edges of the
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plate touch the filter paper this will
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disturb the capillary motion of the
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solvent on the plate keep a close eye on
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the solvent front do not let the solvent
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get closer than 5 to 10 mm from the top
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of the plate when the solvent front gets
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too close to the top evaporation from
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the top of the plate becomes a problem
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the spots keep moving up the plate but
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the solvent front appears to stop this
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leads to incorrect RF values
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[Music]
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when the solvent front has reached an
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appropriate height remove the plate and
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immediately draw a line at the solvent
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front this line will be necessary for
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the calculation of RF
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values when the compound of interest is
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brightly colored no extra steps need to
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be taken to visualize the spots however
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most organic compounds are colorless and
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not be seen on a TLC plate with the
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naked
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eye fortunately most TLC plates contain
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an additive that causes the plates to
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fluoresce under ultraviolet
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light certain UV active compounds are
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capable of quenching this fluorescence
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as a result they appear as dark spots on
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the glowing TLC
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plate UV lamps such as this one are
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commonly found in Laboratories for just
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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
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pencil a number of TLC Stains have also
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been developed to Aid in the
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visualization of spots that cannot be
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seen under a UV light you can watch the
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advanced thin layer chromatography video
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for a demonstration of a few of these
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staining
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procedures TLC data is described in
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terms of of RF values under a specific
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set of conditions a particular compound
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should always exhibit the same RF let's
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quickly run through the procedure for
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calculating an RF
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Value First measure the distance from
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where the spot started on the plate to
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where it ended up always measure from
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the center of the final spot we'll call
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this distance
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a next measure the distance from where
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the spot started to where the solvent
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front end it up we'll call this distance
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B do not measure from the bottom of the
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plate this is a common mistake that will
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lead to incorrect RF
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values the RF value is defined as the
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ratio between the distance the spot
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moved a and the distance the solvent
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moved B this value is dependent on the
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polarity of the compound and the
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polarity of the developing solvent polar
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compounds will have lower RF values than
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nonpolar compounds under the same
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developing
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conditions this series of plates
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illustrates the effect of solvent
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polarity on
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RF as the polarity of the developing
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solvent is increased from left to right
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the spot moves further up the plate the
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value of a gets larger while the solvent
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front B stays the same increasing the
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polarity of the developing solvent
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generally increases the RF values of all
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of the SP
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spots it is a good idea to use a
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developing solvent that gives you RF
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values between 0.2 and
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0.8 this will generally give you the
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most effective separation when you have
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more than one compound in your
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sample for example a mixture of two
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compounds was spotted and developed in
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hexanes a non-polar solvent the final
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plate shows only one spot with a very
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low
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RF the same mixture was developed in
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ethyl acetate a polar solvent and once
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again the final plate shows only one
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spot this time with a very high
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RF when a mixture of hexane and
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ethylacetate was used as a developing
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solvent the two spots were resolved
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notice that both spots are near the
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center of the
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plate TLC is frequently used to compare
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the identity of two compounds in a
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perfect world TLC data would be consider
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consistent from plate to plate and
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chamber to chamber unfortunately small
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deviations in development conditions
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affect the observed RF values as a
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result it is most convincing to compare
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RF values of different samples on the
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same TLC
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plate for example you know that the
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unknown sample marked with a question
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mark consists of either compound a
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compound b or a mixture of the two you
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can use TLC to determine the identity of
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the
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unknown you will need two TLC plates
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make three marks on the first TLC plate
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one for the unknown one for a and one in
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the center for the c-spot do the same
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with the second plate except substitute
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B for
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a on the first plate spot compound a on
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the center Mark and one of the side
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marks and allow both spots to dry
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well drain the excess liquid from the
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spotter onto a paper
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towel and rinse your spotter in clean
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solvent before switching to a different
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sample
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now spot the unknown mixture on the
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center Mark and the other side Mark of
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the first
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plate repeat this procedure with
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compound B on the second plate and
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develop both
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plates now compare the two plates you
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can see that the co-s spot with a shows
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two dis distinct spots while the co-s
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spot with B shows only one these results
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indicate that the unknown sample
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contains only compound
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B in this video we have surveyed the
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basic techniques that you need to know
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to use thin layer chromatography in the
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lab you have learned how to set up a
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developing chamber
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how to Mark and apply your sample to a
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TLC
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plate how to develop the plate in your
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developing
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chamber how to visualize the spots on
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your developed plate using a UV
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lamp and how to correctly calculate RF
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values
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you have also learned how to choose an
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appropriate developing solvent for your
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system and how to compare the identity
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of two
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compounds remember this video is
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intended to help you prepare for lab by
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providing a demonstration of the proper
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experimental technique it is not
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intended as a replacement for reading
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your lab manual or the supplementary
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material in order to become a great
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experimentalist it is important that you
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understand both Theory and technique now
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it's your turn good luck
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[Music]
