Analysis of Analgesic Tablets by Analysis of Analgesic Tablets by Thin Layer Chromatography (TLC)Thin Layer Chromatography (TLC)

Organic Chemistry Lab IOrganic Chemistry Lab IFall 2008Fall 2008

Dr. MilkevitchDr. MilkevitchOct 12 & 14, 2009Oct 12 & 14, 2009

Separating MoleculesSeparating Molecules

TodayToday: Learn a separation technique: Learn a separation technique

Molecules can be separated by a number of Molecules can be separated by a number of methods:methods:– ExtractionExtraction– PrecipitationPrecipitation– ChromatographyChromatography

Chromatography = “to write in color”Chromatography = “to write in color”– Originally developed to separate components of inksOriginally developed to separate components of inks

Thin Layer ChromatographyThin Layer Chromatography

Thin Layer ChromatographyThin Layer Chromatography:: “The separation of “The separation of moderately volatile or nonvolatile substances based upon moderately volatile or nonvolatile substances based upon differential adsorption on an inert solid (differential adsorption on an inert solid (stationary phasestationary phase) ) immersed in a organic solvent or solvent mixture (immersed in a organic solvent or solvent mixture (mobile mobile phasephase)”)”

Makes use of:Makes use of:– A stationary phaseA stationary phase: solid support: solid support– Mobile phase:Mobile phase: a fluid a fluid

Basic principle of separation:Basic principle of separation: Differential interactions of molecules with these 2 phasesDifferential interactions of molecules with these 2 phases

How the separation worksHow the separation works

Molecules can move with the mobile phaseMolecules can move with the mobile phase

Or, they can remain fixed on the stationary Or, they can remain fixed on the stationary phasephase

Called Called partitioningpartitioning

How molecules partition between the 2 How molecules partition between the 2 phases depends on:phases depends on:– Properties of the phasesProperties of the phases– Properties of the molecules being separatedProperties of the molecules being separated

How this works, continued How this works, continued Molecules that partition into Molecules that partition into the mobile the mobile phase:phase:– Will move more rapidly than molecules that partition into Will move more rapidly than molecules that partition into

the stationary phasethe stationary phase– Therefore, they will Therefore, they will separateseparate from those that are partitioned from those that are partitioned

into the stationary phaseinto the stationary phase

Two component mixtureapplied to TLC plate

Slower moving componentpartitioned more into stationary phase

Faster moving componentpartitioned more into mobile phase

Solvent movement

““Nuts and Bolts” of the MethodNuts and Bolts” of the Method

You must carefully select mobile and stationary You must carefully select mobile and stationary phases to achieve full separation of mixtures of phases to achieve full separation of mixtures of moleculemolecule– Trial and errorTrial and error

Stationary phase:Stationary phase: thin layer of silica spread on a thin layer of silica spread on a surfacesurfaceMobile phase:Mobile phase: solvent system that migrates through solvent system that migrates through the silicathe silica

Samples are spotted on the plateSamples are spotted on the plate

Moved along by the solvent(s) migrating through the Moved along by the solvent(s) migrating through the silicasilica

RRff Values ValuesMolecules that are separated will migrate as “spots” and their Molecules that are separated will migrate as “spots” and their migration can be measuredmigration can be measured

Migration usually reported as an “RMigration usually reported as an “Rff” value” value

Calculation of an RCalculation of an Rff value: value:– Ratio of sample migration (how far spot moved) to solvent Ratio of sample migration (how far spot moved) to solvent

migration (how far the solvent moved)migration (how far the solvent moved)

Can be used to identify components in a mixture Can be used to identify components in a mixture – Compared to standardsCompared to standards

Today: look at the components of analgesic tablets by TLCToday: look at the components of analgesic tablets by TLC– Look for the “actives”Look for the “actives”

Calculating an RCalculating an Rff Value Value

Origin

Solvent Front

Spot 1 Spot 2

Solvent Front = 5 cm

Spot 1 moved 1 cm Rf = 1/5 = 0. 2 cm

Spot 2 moved 4 cm Rf = 4/5 = 0. 8 cm

Analgesic MedicationsAnalgesic Medications

““Analgesia”:Analgesia”: Greek Greek for the deadening or absence of pain for the deadening or absence of pain without loss of consciousnesswithout loss of consciousness– Analgesics: Analgesics: compounds that relieve paincompounds that relieve pain

Range from aspirin to morphine, and other related Range from aspirin to morphine, and other related narcoticsnarcotics

Many OTC (over the counter) analgesics availableMany OTC (over the counter) analgesics available– TylenolTylenol– AdvilAdvil– ExcedrinExcedrin– Aspirin (various preparations)Aspirin (various preparations)

ActivesActives

OTC analgesics contain OTC analgesics contain many compoundsmany compounds

The “active” is what The “active” is what relieves painrelieves pain

Other compounds are Other compounds are binders, flavorings, binders, flavorings, colorants, stabilizers, colorants, stabilizers, solubility enhancerssolubility enhancers

OH

N

O

Paracetamol (acetaminophen)

OH

O

Ibuprofen

N

NN

N

O

O

Caffeine

ProcedureProcedureObtain a silica gel plate from meObtain a silica gel plate from me– This plate will have a UV indicator in itThis plate will have a UV indicator in it

Draw a straight line (lightly, in pencil) approximately 2 cm from the bottom Draw a straight line (lightly, in pencil) approximately 2 cm from the bottom edge of your TLC plate. Use a ruler to do this. edge of your TLC plate. Use a ruler to do this. This is called the origin. This is called the origin.

Draw a straight line (lightly, in pencil) about 1 cm from the top edge of your Draw a straight line (lightly, in pencil) about 1 cm from the top edge of your TLC plate. Again, use a ruler to do this. TLC plate. Again, use a ruler to do this. This is called the solvent front. This is called the solvent front.

Mark 5 spots (in pencil) along the origin. Distribute the spots evenly along Mark 5 spots (in pencil) along the origin. Distribute the spots evenly along the origin.the origin.

Obtain the TLC standards. These are solutions of known components of Obtain the TLC standards. These are solutions of known components of the analgesic tablets. They include:the analgesic tablets. They include:

CaffeineCaffeineAcetylsalicylic Acid (aspirin)Acetylsalicylic Acid (aspirin)IbuprofenIbuprofenAcetaminophenAcetaminophen

Procedure, con’tProcedure, con’tSpot the TLC plates with the TLC standards and 1 unknown. Use a Spot the TLC plates with the TLC standards and 1 unknown. Use a

spotting capillary or the small diameter plastic dropper. Make the spots spotting capillary or the small diameter plastic dropper. Make the spots small (approx 1 mm in diameter).small (approx 1 mm in diameter).

•Before you do this, ask me or Scott/Nicole to demonstrate on your Before you do this, ask me or Scott/Nicole to demonstrate on your plateplate

Preheat the TLC plate in the 50 C oven for 5 min. This will dry the spots. Preheat the TLC plate in the 50 C oven for 5 min. This will dry the spots. Sometimes you will need to dry the plates for a longer period of time.Sometimes you will need to dry the plates for a longer period of time.

What your TLC Plate will Look What your TLC Plate will Look LikeLike

origin

Solvent front (drawn lightly in pencil)

Procedure (con’t)Procedure (con’t)

Make a TLC tank (called a developing tank). Make a TLC tank (called a developing tank). Use a 250 ml beaker. Add a few ml of the Use a 250 ml beaker. Add a few ml of the mobile phase to the beakermobile phase to the beaker

Mobile phase: ethyl acetate Mobile phase: ethyl acetate

Place a piece of filter paper in the beakerPlace a piece of filter paper in the beaker– Will allow the beaker to fill with solvent vaporWill allow the beaker to fill with solvent vapor

Cover TLC tank with a watch glass/parafilmCover TLC tank with a watch glass/parafilm

TLC TankTLC Tank

Mobile phase

Filter paper

Watch glass

DevelopingDevelopingPlace TLC plate (spotted) in the tank. Solvent level must be Place TLC plate (spotted) in the tank. Solvent level must be belowbelow the origin, otherwise you’ll lose your spots the origin, otherwise you’ll lose your spotsTilt plate over a bit onto the side of the beakerTilt plate over a bit onto the side of the beaker– Shouldn’t touch the filter paperShouldn’t touch the filter paper

Watch the migration of the solventWatch the migration of the solventRemove the plate from the TLC tank when the solvent is exactly Remove the plate from the TLC tank when the solvent is exactly at the solvent front lineat the solvent front lineLet the plate dryLet the plate dryVisualize the plate using the UV boxVisualize the plate using the UV box– See me to demonstrate use of the UV boxSee me to demonstrate use of the UV box

After this, further develop the plate in the iodine tankAfter this, further develop the plate in the iodine tank– Iodine will react with the spotsIodine will react with the spots– Makes the spots yellowMakes the spots yellow– When fully developed, remove and circle the spots with When fully developed, remove and circle the spots with

pencilpencil

ResultsResults

Measure the distance the solvent Measure the distance the solvent (mobile phase) traveled (mobile phase) traveled – Bottom to the solvent front lineBottom to the solvent front line

Measure the distance from the center of Measure the distance from the center of each spot to the origineach spot to the origin

Calculate RCalculate Rff values for each spot values for each spot

Distance spot traveled

Distance solvent traveledRf =

Results IIResults II

Attempt to identify what the spots are in your unknownAttempt to identify what the spots are in your unknown– Compare the RCompare the Rff values of the standards versus the R values of the standards versus the Rff values values

of the spots in your unknownof the spots in your unknown– If they match closely, then you have that component in your If they match closely, then you have that component in your

unknownunknown– Can you identify what unknown you have?Can you identify what unknown you have?

PossibilitiesPossibilities: Advil, Tylenol, Excedrin: Advil, Tylenol, Excedrin

Be sure to draw a figure of your TLC plateBe sure to draw a figure of your TLC plate– Identify all standards, and all spotsIdentify all standards, and all spots– Show calculations of your RShow calculations of your Rff values values

Conclusions: Things to Think AboutConclusions: Things to Think About

What can we conclude?What can we conclude?– Did your TLC plate work?Did your TLC plate work?

Were you able to spot your plate successfully, and develop the Were you able to spot your plate successfully, and develop the plate?plate?Did you see the spots for the standards?Did you see the spots for the standards?For your unknown, did you see a spot or spots?For your unknown, did you see a spot or spots?Were you able to resolve the different spots?Were you able to resolve the different spots?

– Did the spots separate from each other?Did the spots separate from each other?– This is what we are trying to do!!!!This is what we are trying to do!!!!

Could you identify your unknown?Could you identify your unknown?– See me for the identity of your unknownSee me for the identity of your unknown– If you did, then you have successfully utilized TLC to If you did, then you have successfully utilized TLC to

identify your unknown compoundidentify your unknown compound