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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#