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THIN LAYER CHROMATOGRAPHY ANALYSIS OF ANALGESIC TABLETS

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THIN LAYER CHROMATOGRAPHY ANALYSIS OF ANALGESIC TABLETS Powered By Docstoc
					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 mixture to 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 Rf value. Rf value for a component is calculated using the following expression. Rf = Distance traveled by the component Distance traveled by the solvent

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

Figure 1: TLC plate before development in a beaker

Figure 2: TLC plate in a beaker

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solvent front d solvent d sample origin Rf = dsample dsolvent
Figure 3: TLC plate after development

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 separation and 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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in a darkened hood. Lightly outline the spot with a pencil. Calculate Rf values 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 Rf value for each sample.

Part 3: Effect of polarity of the solvent on Rf value Obtain 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 Mixtures A B C 5% ethyl acetate – 95% hexanes 10% ethyl acetate – 90% hexanes 20% ethyl acetate – 80% hexanes

Visualize the spots under a UV lamp. Calculate the Rf value for benzyl alcohol on each TLC plate.

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Part 4: Analysis of analgesic tablets 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 of the 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.

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Visualize the plate under a short wavelength UV lamp, in a darkened hood. Lightly outline all the spots with a pencil. Calculate Rf values 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 Rf values.

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Part 2: Effect of hydrogen bonding on Rf value Calculate the Rf value for each sample. Name of the compound Rf Value anisole benzyl alcohol acetophenone benzoic acid phenyl acetate

Based on the Rf values, 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 Rf value Calculate the Rf value for benzyl alcohol on each TLC plate. Beaker Solvent Mixtures A B C 5% ethyl acetate – 95% hexanes 10% ethyl acetate – 90% hexanes 20% ethyl acetate – 80% hexanes Rf

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 Rf Value of the Analgesic Rf Values of Active Ingredients Aspirin

Ibuprofen

Acetaminophen

Caffeine

Name of the Analgesic Rf Value of the Analgesic Rf Values of Active Ingredients Aspirin

Ibuprofen

Acetaminophen

Caffeine

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Name of the Analgesic Rf Value of the Analgesic Rf Values of Active Ingredients Aspirin

Ibuprofen

Acetaminophen

Caffeine

Name of the Analgesic Rf Value of the Analgesic Rf Values of Active Ingredients Aspirin

Ibuprofen

Acetaminophen

caffeine

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