chem 30bl lecture 6a tlc
TRANSCRIPT
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Lecture 6aT hi n La yer C hr omat o g r a ph y
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Introduction I
• Chromatography was discovered by Russian botanist Mikhail Semyonovich
Tsvett, who separated plant pigments using calcium
carbonate columns (1!1"#
• Martin and Synge ( Noble Prize in Chemistry, 1952" established
many o$ the basic techni%ues in partition chromatography
i#e#, paper chromatography, gas chromatography, &'C, etc#
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)pplications I
• TC is usually per$ormed on a glass, plastic or
aluminum $oil that is covered with an adsorbent
(*stationary phase"#
• Uses
• Identi$y compounds in a mi+ture by comparison with internal standards#
• etermine the purity o$ a compound#
• Monitor the progress o$ reactions to determine its completion#
• -ptimi.ing a solvent mi+ture (mobile phase" $or column
chromatography#
• The compound in the mi+ture separate su$$iciently well on the TC plate#
• It is important to use the same stationary phase (ideally same %uality" as used
in the chromatography later on#
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)pplications II
• Separation o$ dyes in pen ink (i#e#, black
ink"/• )s can be seen, the black ink is actually a mi+ture
o$ di$$erent dyes#
•
Separation and determination o$ pigments in plants (i#e#, spinach"/
• The TC shows di$$erent compound such as
carotene, lypocene, chlorophylls, +anthophylls,
pheophytins, etc#
•
Monitor the progress o$ $ermentation inwine making (T*tartaric acid, M*malic acid,
*lactic acid"#
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Stationary 'hase
• Commonly used are silica, alumina, cellulose (i#e#, paper chromatography", etc#
• These stationary phases are considered polar due the presence o$ hydro+yl groups
on the sur$ace and their ability to $orm hydrogen bonds with polar
and protic solvents#
• Silica0coated TC plates are primarily used in organic labs because most
o$ the compounds analy.ed in the lab are (weakly" polar due to the presence
o$ carbonyl groups, hydro+yl $unctions, etc#
• The type o$ stationary phase used in a given separation problem depends
on the polarity o$ compounds and the separation mechanism#
Al
O
Al
O
Al
O
Al
O
Al
O
Al
O
Al
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Mobile 'hase
• The eluting power o$ the mobile phase depends on the polarity o$ the solvent vs# the polarity
o$ the stationary phase/
• 'olar solvents i#e#, alcohols have a high eluting power on polar stationary phases because
they interact strongly with the polar stationary phase via their hydrogen bonding donor and a hydrogen bond acceptor ability#
• Medium0polar solvents like ketones, esters and ethers possess a medium eluting power
because they only act as hydrogen bond acceptors#
• on0polar solvents i#e#, toluene, he+ane, etc# have a low eluting power on polar stationary
phases because their interaction with polar stationary phase is weak#
•
The general a$$inity o$ $unctional groups towards silica is/
WaterMethanolEthanolPropanolAcetoneEthyl acetateDiethyl etherChloroform
Methylene ChlorideTolueneHexane
if nonpolar stationary phasethen increasing eluting power
if polar stationary phasethen increasing eluting power
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2+perimental I
• TLC Plate
• The plate is coated with a very thin layer (3!#45 mm" o$
a mi+ture o$ a stationary phase and a binder i#e#, gypsum#
• The stationary phase o$ten also contains a $luorescent indicator
(.inc silicate, .inc cadmium sul$ide",
which appears bright green when e+posed to
short wavelengths (λ*456 nm"#
• Preparation of the TLC plate
• The white surface should not be touched.
• raw a very thin start line with pencil or mark the plate
on the lower end on each side (3!#5 cm $rom the bottom"#
• Do not use a pen for this step.
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2+perimental II
• Spotting
• ) capillary spotter (drawn $rom a 'asteur pipette" or a commercial spotter should be
used $or spotting (top/ melting point capillary,
bottom/ commercial spotter"#
• elting point capillaries! syringe needles! etc. "as is# are not
suitable for the spotting process because they produce a hugespot that o$erloads the plate "%tailing! see also last slide#&
• The spots have to be e%ually spread at the starting line and not be located too close to
the outer edges#
• The spots have to be small in diameter (3104 mm"#
• ) diluted solution o$ the compound in a low0boiling, low polarity solvent i#e#, diethyl
ether, he+ane, ethyl acetate, etc# has to be used(5 mg7m"#
• I$ the compound cannot be detected with the naked eye, the TC
plate has to be dried and then be inspected under the 890lamp
prior to development i#e#, ben.il, diben.yl ketone are colorless
in low concentrations#
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2+perimental III
• De$eloping the Plate
• ) :ar or a small beaker covered with a watch glass is used as development chamber, lining the walls
with wet $ilter paper is usually not necessary i$ the :ar is kept close#
• The solvent level in the :ar has to be below the starting line#
• The TC plate is placed straight in the chamber, which is le$t undisturbed#
• The compounds move up the plate at di$$erent rates (i$ the proper mobile phase is used"#
• ote that the rate o$ movement is not constant (Why?"#
• The solvent $ront is allowed to move up the plate until 31 cm $rom the top#
•The plate is then remo$ed and the sol$ent front immediately marked.
t*! min t*1 min t*5 min
;rown/ Mi+ture
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2+perimental I9
• 'isuali(ation ($or more reagents see SKR 326 "/
• )irst! the plate has to be dried thoroughly.
• UV light * It is only use$ul i$ the compounds are 89 active and the stationary phase
contains a $luorescent indicator#
• Iodine* It is used $or unsaturated and aromatic compounds (brown stain, not permanent"#
•
Permangante: It is used $or compounds that can be o+idi.ed easily (mostly yellow on purple"#
• Vanillin* It works well $or hydro+yl and carbonyl compounds (appear in di$$erent colors
depending on the compound"#
• Ceric staining C!"#* It is good general stain but particularly sensitive
$or hydro+yl, carbonyl, epo+ides (dark blue upon heating"#
•
$inhydrin* It is used $or amino acids, amines (o$ten pink or purple"#• %romocresol green/ It is mainly used to detect carbo+ylic acids#
• +fter marking the spots with pencil! the diagram is transferred to the notebook
"taking a picture with the cell phone could not hurt either#. Do not take the TLC
plate home because the silica will rub off. Silica powder can cause a lung disease
called silicosis.
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ata )nalysis
• Determination of , f -$alue*
• Measure the distance o$ the center o$ the spot $rom
the starting line (g, r "#
• Measure the distance o$ the solvent $ront $rom the
starting line (s"#
• The R $ 0value is de$ined as the ratio o$ the
travel distances#
• The R $ 0value is a ratio and thus is a unitless number#
• The R $ 0value has to be between =!> and =1>#
gr
s
?6#!sg"green(R
@5#!s
r "red(R
$
$
==
==
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Solvent 2$$ect
• Changes in the mobile phase have an impact on the movement o$ all compounds (with varying
degree though due to changes in various parameters"/
• The more eluting power the mobile phase (given as e!0values on silica above" has, the more the
compounds move because the mobile phase interacts stronger with the stationary phase# This makes
it more di$$icult $or the compounds to interact with
the stationary phase leading to higher R $ 0values and (o$ten" poorer separation#
&e+anee!*!#!
Chloro$orme!*!#4@
iethyl ether e!*!#?A
) ; ' ) ; '
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Common 'roblems
• Problem 1/ )ll spots are grouped together on the lower (upper " end o$ the plate#• Solution 1/ The eluting power o$ the solvent was too low (high" $or this separation
problem# ) more polar (less polar " solvent should be added to
the mobile phase (see previous slide"#
• Problem 2/ The spot is spread over a large part o$ the lane or does
not look round#• Solution 2/ The student spotted too much o$ the sample on the plate
that leads to tailing# ess sample should be spotted using the proper
spotter#
• Problem 3/ The spot has a crescent shape a$ter the development#
• Solution 3/ The solvent used to dissolve the sample was too polar
and was not allowed to evaporate completely#
• Problem 4: The spots seem to run into each other on the top#
• Solution 4: 2ither the spots were to close at the start line or the TC plate
was not placed straight in the :ar#