Thin_Layer_Chromatography

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THIN LAYER CHROMATOGRAPHY

Reference:

1. Schoffstall, Gaddis, Druelinger, Microscale and Miniscale Organic

Chemistry Laboratory Experiments, 2nd edition, McGraw Hill

2. Dickson, Kittredge, Sarquis, J.Chem.Ed, Vol81, No 7, PP 1023 1025,

July 2004

Theory

Chromatographic techniques are used extensively in organic chemistry

laboratories for routine analysis. Thin layer chromatography (TLC) can be used

to determine the purity of a compound, to analyze the composition of a mixture or

to follow the progress of a reaction. The components of a mixture are

differentiated by exposing to two competing phases, the stationary and the

mobile phases. In TLC, the stationary phase is a polar adsorbent such as silica

gel or alumina, which has been coated on a plastic plate. The mobile phase is an

organic solvent. The solvent moves up the plate by capillary action.

A coated and dried glass or plastic plate is called a thin-layer plate. The mixtureto be analyzed is dissolved in a suitable solvent and applied as spots near the

base of the plate. This process is known as spotting. Spotting is achieved by

repeated applications of the mixture from a capillary pipet. When the filled pipet

touches the plate, the liquid is delivered on to the plate by capillary action. The

plate is immersed in a development chamber (usually a beaker) that contains a

solvent or a mixture of solvents. The solvent in the development chamber is also

referred to as the eluent or eluting solvent. As the solvent rises up the plate by

capillary action, it carries the mixture with it. The components of the mixture are

separated on the stationary phase depending on their polarity. This process is

known as developing or running the TLC plate. Polar compounds are attracted to

the silica gel and are held more tightly compared to nonpolar compounds. A

separation results due to the difference in rates at which individual components

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move on the plate. In general, nonpolar compounds move faster than polar

compounds on a TLC plate coated with silica gel.

When the plate has been developed, it is removed and allowed to dry. There will

a series of vertical spots on the plate. Each spot corresponds to a separate

component of the original mixture. If the components are colorless, the spots are

invisible. To see the spots, a visualization method is used. Ultraviolet lamp is a

common visualization method.

The distance traveled by each component is measured and this value is called

the retardation factor, designated as Rfvalue.

Rfvalue for a component is calculated using the following expression.

Rf = Distance traveled by the component

Distance traveled by the solvent

There is a Rfvalue associated with each developed spot on the TLC plate.

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Figure 1: TLC

plate before

development in a

beaker

Figure 2: TLC

plate in a

beaker


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d solventd sample

Rf = dsampledsolvent

origin

solvent front

Procedure

In this guided inquiry lab, you will investigate properties such as hydrogen

bonding and polarity of various samples and solvents in context to TLC. You will

also apply your knowledge to a common industrial application such as separationand identification of components in an analgesic medication.

Part 1: Effect of the length of TLC plate on Rf value

Obtain three TLC plates of varying lengths, a development chamber (400 mL

beaker and watch glass), a pencil and a ruler. Follow these instructions for all

three TLC plates. With a lead pencil (do not use a pen), draw a faint line 1 cm

from the bottom of a TLC plate. Mark a dot on this line. This dot will be the origin

for the substance (benzyl alcohol) that you will spot. With a Microcap capillary

tube, apply a tiny drop of benzyl alcohol (diluted by ethyl acetate in the ratio of

1:5) at the origin and air dry. Do not blow air. Place the TLC plate in a

developing chamber. Trickle down 60% ethyl acetate-40% hexanes (using a

pipet) along the sides of the beaker, making sure that the level of the solvent

is below the spotting line. The volume of the solvent used in this experiment

will vary with the size of the beaker, the distance of the spotting line from the

bottom plate and other dimensions of the TLC plate. Place a watch glass on the

beaker. You may also insert a piece of filter paper (as shown in Figure 2). When

the level of the solvent reaches ~ 0.5 cm from the top of the TLC plate, remove

the plate, quickly mark the solvent front with a pencil and let it air dry. Do not

shake the plate to dry it. Visualize the plate under a short wavelength UV lamp,

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Figure 3: TLC

plate after

development


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in a darkened hood. Lightly outline the spot with a pencil. Calculate Rfvalues for

benzyl alcohol on each TLC plate.

You may also choose to run all three TLC plates simultaneously (in separate

beakers). Make a sketch of the plates in your lab report.

Part 2: Effect of hydrogen bonding on Rf value

Obtain a TLC plate and spot the following compounds on the same plate. Make

sure the TLC plate is large enough to place all samples with good separation on

the spotting line. Use a new micropipette for each compound.

The compounds that you will use in this experiment are anisole, benzyl alcohol,

acetophenone, benzoic acid and phenyl acetate (dissolved in ethyl acetate).

Develop the TLC plate in a beaker with 60% ethyl acetate-40% hexanes. Circle

the spots on the TLC plate under UV light. Calculate the R fvalue for each

sample.

Part 3: Effect of polarity of the solvent on Rf

valueObtain three TLC plates and spot benzyl alcohol on all the plates. Develop the

plates in three beakers labeled A, B and C. The beakers contain the solvent

mixtures listed below:

Beaker Solvent MixturesA 5% ethyl acetate 95% hexanesB 10% ethyl acetate 90% hexanesC 20% ethyl acetate 80% hexanes

Visualize the spots under a UV lamp. Calculate the R fvalue for benzyl alcohol on

each TLC plate.

Part 4: Analysis of analgesic tablets

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Over the counter pain medications, such as Excedrin, Tylenol, and Advil,

commonly contains aspirin, ibuprofen, acetaminophen, and/or caffeine. TLC can

be used to determine whether an analgesic tablet contains one or more active

ingredients.

Your instructor will assign an analgesic tablet to your team. With a mortar and

pestle, pulverize the tablet. Add 25 mg of the powdered tablet to a vial and

dissolve in 0.5 mL methylene chloride (may also be labeled as dichloromethane).

Add 2 drops of ethanol (may also be labeled as ethyl alcohol) to help in the

dissolution of the tablet. Not all of the tablet will dissolve, because the analgesics

usually contain an insoluble binder. In addition, they may also contain buffering

agents or coatings that are insoluble in methylene chloride and ethanol. Similarly,

prepare solutions of aspirin, ibuprofen, acetaminophen, and caffeine in four

separate vials. Instead of 25 mg, use 1-2 mg for these compounds. These

solutions are the standards. Remember to label all vials clearly using a red

marker. Obtain a silica gel TLC plate (a sample plate is available to estimate the

dimensions), a development chamber (400 mL beaker and watch glass), a pencil

and a ruler.

With a lead pencil (do not use a pen), draw a faint line 1 cm from the bottom ofthe TLC plate. Mark five dots on this line. These will be the origins for the

substances that you will spot. You will spot solutions of the analgesic tablet and

the standards on these dots. With a Microcap capillary tube, apply a tiny drop of

each solution at the origin and air dry. Do not blow air. Place the TLC plate in

the developing chamber. Trickle down ethyl acetate (using a pipet) along the

sides of the beaker, making sure that the level of the solvent is below the spotting

line. The volume of the solvent used will vary with the size of the beaker, the

distance of the spotting line from the bottom plate and other dimensions of the

TLC plate. Place the watch glass on the beaker. When the level of the solvent

reaches ~ 0.5 cm from the top of the plate, remove the plate, quickly mark the

solvent front with a pencil and let it air dry. Do not shake the plate to dry it.

Visualize the plate under a short wavelength UV lamp, in a darkened hood.

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Lightly outline all the spots with a pencil. Calculate Rfvalues of the assigned

analgesic, and of caffeine, ibuprofen, aspirin, and acetaminophen. List the active

ingredients in the assigned analgesic tablet. Make a sketch of the plate in your

lab report. Make conclusions about the contents of each tablet.

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THIN LAYER CHROMATOGRAPHY

YOUR NAME __________________

Questions

1. List any 2 factors that affect Rf values in TLC.

2. Explain how to prepare sample solutions.

3. Describe the principle that governs the TLC process.

4. When should a mixed solvent system be used for TLC?

5. What tool would you use to analyze the separated components in a

TLC process?

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6. What would happen if

a) You had too much solvent in the developing chamber that it

covered the application spot.

b) You spotted a very concentrated solution of the analgesic.

c) You spotted a very dilute solution of the analgesic.

d) You placed the TLC plate slanted in the developing chamber.

e) You used a pen instead of a pencil to mark the origin spots.

7. What is meant by the term green chromatography? What advantages

does this green method have over traditional types of

chromatography?

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8. Excipients were traditionally assumed to be inert. However, new

results show that they interact with the drug, affecting its dissolution,

absorption and bioavailability. Explain the role of excipients in drug

action.

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Results and Conclusion

Part 1: Effect of the length of TLC plate on Rf value

Make a sketch of the three TLC plates. Label the plates. DO NOT TURN IN THE

ORIGINAL PLATE. DISCARD THEM AS INSTRUCTED.

Make conclusions about the effect of the length of TLC plates on R fvalues.

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Part 2: Effect of hydrogen bonding on Rf value

Calculate the Rf value for each sample.

Name of the

compound

RfValue

anisolebenzyl alcoholacetophenonebenzoic acidphenyl acetate

Based on the Rfvalues, classify the compounds in groups (if possible). Identify

the least and the most polar compound.

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Part 3: Effect of polarity of the solvent on R fvalue

Calculate the Rf value for benzyl alcohol on each TLC plate.

Beaker Solvent Mixtures Rf

A 5% ethyl acetate 95% hexanesB 10% ethyl acetate 90% hexanesC 20% ethyl acetate 80% hexanes

From your data, arrange the solvent mixtures in the order of increasing polarity

(least to most).

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Part 4: Analysis of analgesic tablets

Sketch the developed TLC plate to the right.

Name of the Analgesic

RfValue of the Analgesic

Rf Values of Active Ingredients Aspirin

Ibuprofen

Acetaminophe

n

Caffeine




Ibuprofen

Acetaminophe

n

Caffeine

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Ibuprofen

Acetaminophe

n

Caffeine




Ibuprofen

Acetaminophe

n

caffeine

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