Thin Layer Chromatography

7/17/2019 Thin Layer Chromatography

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Thin Layer Chromatography (TLC)

TLC is a simple, quick, and inexpensive procedure that gives the chemist a quick answer as

to how many components are in a mixture. TLC is also used to support the identity of a

compound in a mixture when the R f of a compound is compared with the R f of a knowncompound (preferaly oth run on the same TLC plate!.

" TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid

adsorent (usually silica or alumina!. " small amount of the mixture to e analy#ed is spotted

near the ottom of this plate. The TLC plate is then placed in a shallow pool of a solvent in a

developing chamer so that only the very ottom of the plate is in the liquid. This liquid, or

the eluent, is the moile phase, and it slowly rises up the TLC plate y capillary action.

"s the solvent moves past the spot that was applied, an equilirium is estalished for each

component of the mixture etween the molecules of that component which are adsored on

the solid and the molecules which are in solution. $n principle, the components will differ insoluility and in the strength of their adsorption to the adsorent and some components will

e carried farther up the plate than others. %hen the solvent has reached the top of the plate,

the plate is removed from the developing chamer, dried, and the separated components of

the mixture are visuali#ed. $f the compounds are colored, visuali#ation is straightforward.

&sually the compounds are not colored, so a &' lamp is used to visuali#e the plates. (The

plate itself contains a fluorescent dye which glows everywhere except where an organic

compound is on the plate.!

How To Run a TLC Plate

Step 1: Prepare the developing

container

The developing container for TLC can e a

specially designed chamer, a ar with a lid, or

a eaker with a watch glass on the top (the

latter is used in the undergrad las at C&!.

)our solvent into the chamer to a depth of

ust less than *.+ cm. To aid in the saturation

of the TLC chamer with solvent vapors, you

can line part of the inside of the eaker withfilter paper. Cover the eaker with a watch

glass, swirl it gently, and allow it to stand

while you prepare your TLC plate.



Step 2: Prepare the TLC plate

TLC plates used in the organic chem teaching

las are purchased as + cm x * cm sheets.

-ach large sheet is cut hori#ontally into plates

which are + cm tall y various widths themore samples you plan to run on a plate, the

wider it needs to e. /hown in the photo to the

left is a ox of TLC plates, a large un0cut TLC

sheet, and a small TLC plate which has een

cut to a convenient si#e. 1andle the plates

carefully so that you do not distur the

coating of adsorent or get them dirty.

2easure *.+ cm from the ottom of the plate.

&sing a pencil, draw a line across the plate at

the *.+ cm mark. This is the origin3 the line on

which you will spot the plate. Take care not to press so hard with the pencil that you distur

the adsorent. &nder the line, mark lightly the

name of the samples you will spot on the

plate, or mark numers for time points. Leave

enough space etween the samples so that

they do not run together aout 4 samples on a

+ cm wide plate is advised.

Step : Spot the TLC plate

$f the sample is not already in solution,

dissolve aout 5 mg in 5 mL of a volatile

solvent such as hexanes, ethyl acetate, or

methylene chloride. "s a rule of thum, a

concentration of 56 usually works well for

TLC analysis. $f the sample is too

concentrated, it will run as a smear or streak

(see trouleshooting section elow! if it is not

concentrated enough, you will see nothing on

the plate. /ometimes you will need to use trial

and error to get well0si#ed, easy to read spots.

7tain a a microcapillary. $n the organicteaching las, we use 5*8L microcaps 0 they

are easier to handle than the smaller ones used

in research las. 9ip the microcap into the

solution and then gently touch the end of it

onto the proper location on the TLC plate.

9on:t allow the spot to ecome too large 0 if

necessary, you can touch it to the plate, lift it

off and low on the spot. $f you repeat these

steps, the wet area on the plate will stay small.



This example plate has een spotted with

three different quantities of the same solution

and is ready to develop. $f you are unsure of

how much sample to spot, you can always

spot multiple quantities and see which looks

est.

Step !: "evelop the plate

)lace the prepared TLC plate in the

developing eaker, cover the eaker with thewatch glass, and leave it undistured on your

ench top. The solvent will rise up the TLC

plate y capillary action. 2ake sure the

solvent does not cover the spot.

"llow the plate to develop until the solvent is

aout half a centimeter elow the top of the

plate. Remove the plate from the eaker and

immediately mark the solvent front with a

pencil. "llow the plate to dry.

Step #: $i%uali&e the %pot%

$f there are any colored spots, circle them

lightly with a pencil. 2ost samples are not

colored and need to e visuali#ed with a &'lamp. 1old a &' lamp over the plate and

circle any spots you see. ;eware< &' light is

damaging oth to your eyes and to your skin<

2ake sure you are wearing your goggles and

do not look directly into the lamp. )rotect

your skin y wearing gloves.



$f the TLC plate runs samples which are too

concentrated, the spots will e streaked and=or

run together. $f this happens, you will have tostart over with a more dilute sample to spot

and run on a TLC plate.

1ere:s what overloaded plates look like

compared to well0spotted plates. The plate on

the left has a large yellow smear this smearcontains the same two compounds which are

nicely resolved on the plate next to it.

TLC Solvent% Choice

%hen you need to determine the est solvent or mixture of solvents (a >solvent system>! to

develop a TLC plate or chromatography column loaded with an unknown mixture, vary the polarity of the solvent in several trial runs3 a process of trial and error. Carefully oserve and

record the results of the chromatography in each solvent system. ?ou will find that as you

increase the polarity of the solvent system, all the components of the mixture move faster

(and vice versa with lowering the polarity!. The ideal solvent system is simply the system that

gives the est separation.

TLC elution patterns usually carry over to column chromatography elution patterns. /ince

TLC is a much faster procedure than column chromatography, TLC is often used to determine

the est solvent system for column chromatography. @or instance, in determining the solvent

system for a flash chromatography procedure, the ideal system is the one that moves the

desired component of the mixture to a TLC R f of *.+0*.A+ and will separate this componentfrom its nearest neighor y difference in TLC R f values of at least *.*. Therefore a mixture

is analy#ed y TLC to determine the ideal solvent(s! for a flash chromatography procedure.

;eginners often do not know where to start3 %hat solvents should they pull off the shelf to

use to elute a TLC plateB ;ecause of toxicity, cost, and flammaility concerns, the common

solvents are hexanes (or petroleum ethers=ligroin! and ethyl acetate (an ester!. 9iethyl ether

can e used, ut it is very flammale and volatile. "lcohols (methanol, ethanol! can e used.

"cetic acid (a caroxylic acid! can e used, usually as a small percentage component of the

system, since it is corrosive, non0volatile, very polar, and has irritating vapors. "cetone (a

ketone! can e used. 2ethylene chloride or and chloroform (halogenated hydrocarons! are

good solvents, ut are toxic and should e avoided whenever possile. $f two solvents areequal in performance and toxicity, the more volatile solvent is preferred in chromatography

http://orgchem.colorado.edu/Technique/Procedures/Columnchrom/Columnchrom.html

http://orgchem.colorado.edu/Technique/Procedures/Columnchrom/Columnchrom.html



ecause it will e easier to remove from the desired compound after isolation from a column

chromatography procedure.

"sk the la instructor what solvents are availale and advisale. Then, mix a non0polar

solvent (hexanes, a mixture of 0caron alkanes! with a polar solvent (ethyl acetate or

acetone! in varying percent cominations to make solvent systems of greater and lesser polarity. The charts elow should help you in your solvent selection. ?ou can also download

this pdf chart of elution order .

'nteraction% etween the Compound and the d%or*ent

The strength with which an organic compound inds to an adsorent depends on the strength

of the following types of interactions3 ion0dipole, dipole0dipole, hydrogen onding, dipole

induced dipole, and van der %aals forces. %ith silica gel, the dominant interactive forces

etween the adsorent and the materials to e separated are of the dipole0dipole type. 1ighly

polar molecules interact fairly strongly with the polar /i71 groups at the surface of these

adsorents, and will tend to stick or adsor onto the fine particles of the adsorent while

weakly polar molecules are held less tightly. %eakly polar molecules generally tend to movethrough the adsorent more rapidly than the polar species. Roughly, the compounds follow

the elution order given aove.

The R + value

The retention factor, or R f , is defined as the distance traveled y the compound divided y the

distance traveled y the solvent.

@or example, if a compound travels .5 cm and the solvent front travels .D cm, the R f is *.E+3

http://orgchem.colorado.edu/Technique/Procedures/TLC/Elution.pdf

http://orgchem.colorado.edu/Technique/Procedures/TLC/Elution.pdf



The R f for a compound is a constant from one experiment to the next only if the

chromatography conditions elow are also constant3

• solvent system

• adsorent

• thickness of the adsorent

• amount of material spotted

• temperature

/ince these factors are difficult to keep constant from experiment to experiment, relative R f

values are generally considered. >Relative R f > means that the values are reported relative to a

standard, or it means that you compare the R f values of compounds run on the same plate at

the same time.

The larger an R f of a compound, the larger the distance it travels on the TLC plate. %hen

comparing two different compounds run under identical chromatography conditions, thecompound with the larger R f is less polar ecause it interacts less strongly with the polar

adsorent on the TLC plate. Conversely, if you know the structures of the compounds in a

mixture, you can predict that a compound of low polarity will have a larger R f value than a

polar compound run on the same plate.

The R f can provide corroorative evidence as to the identity of a compound. $f the identity of

a compound is suspected ut not yet proven, an authentic sample of the compound, or

standard, is spotted and run on a TLC plate side y side (or on top of each other! with the

compound in question. $f two sustances have the same R f value, they are likely (ut not

necessarily! the same compound. $f they have different R f values, they are definitely different

compounds. Fote that this identity check must e performed on a single plate, ecause it is

difficult to duplicate all the factors which influence R f exactly from experiment to

experiment.

Trou*le%hooting TLC

"ll of the aove (including the procedure page! might sound like TLC is quite an easy

procedure. ;ut what aout the first time you run a TLC, and see spots everywhere and

lurred, streaked spotsB "s with any technique, with practice you get etter. -xamples of

common prolems encountered in TLC3



• The compound run% a% a %trea, rather than a %pot: The sample was overloaded.

Run the TLC again after diluting your sample. 7r, your sample might ust contain

many components, creating many spots which run together and appear as a streak.

)erhaps, the experiment did not go as well as expected.

• The %ample run% a% a %mear or a upward cre%cent: Compounds which possess

strongly acidic or asic groups (amines or caroxylic acids! sometimes show up on aTLC plate with this ehavior. "dd a few drops of ammonium hydroxide (amines! or

acetic acid (caroxylic acids! to the eluting solvent to otain clearer plates.

• The %ample run% a% a downward cre%cent: Likely, the adsorent was distured

during the spotting, causing the crescent shape.

• The plate %olvent +ront run% croo,edly: -ither the adsorent has flaked off the sides

of the plate or the sides of the plate are touching the sides of the container (or the

paper used to saturate the container! as the plate develops. Crooked plates make it

harder to measure R f values accurately.

• -any random %pot% are %een on the plate: 2ake sure that you do not accidentally

drop any organic compound on the plate. $f get a TLC plate and leave it laying on

your workench as you do the experiment, you might drop or splash an organic

compound on the plate.

• .ou %ee a *lur o+ *lue %pot% on the plate a% it develop%: )erhaps you used an ink

pen instead of a pencil to mark the originB

• /o %pot% are %een on the plate: ?ou might not have spotted enough compound,

perhaps ecause the solution of the compound is too dilute. Try concentrating the

solution, or spot it several times in one place, allowing the solvent to dry etween

applications. /ome compounds do not show up under &' light try another method of visuali#ing the plate (such as staining or exposing to iodine vapor!. 7r, perhaps you

do not have any compound ecause your experiment did not go as well as planned. $f

the solvent level in the developing ar is deeper than the origin (spotting line! of the

TLC plate, the solvent will dissolve the compounds into the solvent reservoir instead

of allowing them to move up the plate y capillary action. Thus, you will not see spots

after the plate is developed. These photos show how the yellow compound is running

into the solvent when lifted from the developing ar.

TLC Techni0ue ui&



/ee how well you understand TLC y taking the online TLC Technique Gui#<

7riginal content H &niversity of Colorado at ;oulder, 9epartment of Chemistry and

;iochemistry. The information on these pages is availale for academic use without

restriction. This page was last updated on "ugust A5, *5+.

http://orgchem.colorado.edu/Technique/Procedures/TLC/Quiz.html

http://orgchem.colorado.edu/Technique/Procedures/TLC/Quiz.html

Thin Layer Chromatography

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