Thin Layer Chromatography of Analgesics

Thin Layer Chromatography of Analgesics

Introduction:

Over-the-counter analgesics typically contain one or more of the following active ingredients: acetaminophen, aspirin, caffeine, ibuprofen, sodium naproxen, phenacetin, or salicylamide. Thin layer chromatography (TLC) offers a simple method of analysis for these products.

Chromatography is a term that is widely used to describe a family of closely related separation methods. There are many separation methods, but the feature that distinguishes chromatography from other physical and chemical methods of separation is that both a stationary and mobile phase, which are incapable of being mixed, come into contact and the sample to be separated interacts numerous times with these phases. The sample is carried through the system via the mobile phase, and the interactions that occur are due to the differences in the physical and chemical properties that govern the rate at which the individual components of the sample pass over the stationary phase under the influence of the mobile phase.

Thin layer chromatography (TLC) is one type of chromatography where the stationary phase is a thin layer of adsorbent particles attached to the solid plate. A small amount of sample is applied (spotted) near the bottom of the plate, and the plate is placed in the mobile phase. This solvent is drawn up by capillary action to a predetermined height. Separation occurs because each component interacts differently with the mobile phase. Each component, being different in chemical and physical composition, will interact with the mobile phase to different degrees thereby creating the individual bands on the plate. The retention time or retention factor (Rf) is used to characterize and compare components of various samples.

Rf = ____distance from origin to spot_____

distance from origin to solvent front

Purpose:

The purpose of this experiment is to determine the content of over-the-counter analgesics.

Equipment/Materials:

analgesic tabletsIbuprofencaffeine

test tubesExcedrinacetaminophen

metric ruleracetylsalicylic acid(aspirin)Anacin

capillary tubesNaproxinUV light

developing chambers w/lids1:1ethyl alcohol/methylene chloride solution

calibratedtransfer pipettes5% acetic acid in ethyl acetate solution

micro spatula (developing solution)

Safety:

• Always wear an apron and goggles in the lab.
• Use caution in handling the 1:1ethyl alcohol/methylene chloride and

5% acetic acid in ethyl acetate solutions. Avoid contact with skin and eyes. Do not ingest.

• Review SDS’ for more detailed safety and disposition instructions.
• Dispose of all chemicals according to teacher’s instructions.

• Exposure to UV light can be damaging to skin and eyes. Do not look directly at the UV light and do not shine the light in anyone else’s eyes.
• Use caution when handling capillary tubes. They are glass and can break easily causing cuts.

Procedure:

Preparation of sample

1. Using a micro spatula, transfer a small portion of the assigned analgesic to a 3-inchtest tube
2. Using a calibrated transfer pipette, add approximately 1 mL of 1:1ethylalcohol/methylene chloride solution to the analgesic. Cover with parafilm and gently shake sideways to dissolve the analgesic. The binder portion will not dissolve and will settle.
3. With masking tape, label the vial with the name of the analgesic being tested.
4. Repeat the procedure for each of the assigned analgesics.

Spotting of the sample

1. With a pencil, draw a line approximately 1 cm from the short edge of the TLC

plate. Be careful not to scrape the coating of the plate. Mark the line with two tic

marks approximately 1/3 from either side. See diagram.

2. Immerse a capillary tube into the upper liquid portion of the sample test tube

untilsome of the sample is drawn into the tube.

3. Very gently press the capillary tube onto one of the tic marks on the plate.

Keep the spots small and concentrated by applying the sample 5 times and

allowing the spot to dry between applications. Additional spots may be required

for the ibuprofen standard in order to see its spot clearly.

4. Repeat this procedure for each of the standards available.

5. Two or more samples may be applied to each plate if they are kept one cm apart.

Mark the position of the spots lightly in PENCIL and be sure to keep a record of

which spot represents each product.

Development of the TLC plates

1. Prepare a developing chamber by adding some 5% acetic acid/ethyl acetate solvent to barely cover the bottom of the bottle. The solvent should not come in direct contact with the spots. The solvent will rise via capillary action and then come in contact with the spots.

2.Place the TLC plates in the chamber so that they do not touch and cover with the

lid.Allow the solvent to rise to within one cm of the top of the plates.

3. Remove the plates, mark the solvent front using a pencil, and allow them to dry.

4. Visualize the spots by illumination under a UV lamp.

5.Trace around each spot with a pencil and then measure the distance traveled by

each component.

6.Calculate the Rf by dividing the distance of the solvent front by the distance of the

spot. The Rf value is characteristic of each substance and may be used for

identification of the substance.

Name ______Date ______Period _____

Thin Layer Chromatography of Analgesics

Data:

Draw the observed chromatographs

Record the Rf value for each of the standards and identify the components of the unknown(s)

Component / Distance spot / Distance solvent / Rf
acetaminophen
Anacin
Aspirin
Caffeine
Excedrin
Ibuprofen
Naproxen

Questions:

1. What is the stationary phase and what is the mobile phase in this experiment?
2. Why is a pencil (not a pen) used to mark the position of the spots?
3. Why is an Rf value rather than the distance the spot moved used to help identify a substance by TLC?
4. What is co-spotting?
5. Why is co-spotting a good technique for determining the identity of a component by TLC?

Thin Layer Chromatography of Analgesics

Teacher Notes

Lab Time: about 90 minutes

Preparations:

Time: less than 15 minutes

T:Make one set of equipment and materials available to each lab group.

T:Each student should bring a sample of analgesic to the lab. (At discretion of teacher – review school policy for guidance.)

T:The instructor should set up a demonstration on spotting the TLC plate.

T:All used solutions should be placed in the “Used Solution” container and returned with laboratory.

V:The standards, pipettes, solvents, UV light, and capillary tubes

will be brought by the van. Any other materials must be requested.

Answers to Questions:

1. What is the stationary phase and what is the mobile phase in this experiment?

The stationary phase is the TLC plate. The mobile phase is the developing solution composed of toluene, ethyl formate and formic acid.

1. Why is a pencil (not a pen) used to mark the position of the spots?

The inks from the pen would separate during chromatography.

1. Why is an Rf value rather than the distance the spot moved used to help identify a substance by TLC?

The distance the spot moves depends on time. It is also influenced by rate of evaporation. By using an Rf value, the movement of the solvent becomes a common factor, thus resulting in a more accurate comparison.

1. What is co-spotting?

Spotting an unknown substance and the substance it is believed to be on top of each other.

1. Why is co-spotting a good technique for determining the identity of a component by TLC?

The two substances are spotted together thus eliminating any factors due to a slight difference in spotting or any measurement uncertainty. If two spots result the substances are different.

Consideration:

This lab really allows the students to get a good understanding of TLC. The practice of making microcapillary tubes and the preparation and interpretation of the plates will give them a strong background in this type of chromatography.

Updated Feb 2017