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Chromatography of Analgesics

 

NAME:_________________________________________ PERIOD:__________

 

Prelab

 

1. Determine the Rf values of each of the components (A and B) on the thin layer chromatograph below. Show your data and calculations.

 

Solvent line

 

Spotting line

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



Chromatography of Analgesics

 

I. Theory:

 

            Different compounds contain molecules that exert different forces of attraction on each other resulting in different solubilities in a given solvent. These differences in solubilites make it possible to separate the component substances in a mixture and form the bases of a wide range of chromatographic techniques.

            Chromatography usually involves two components, a stationary phase and a mobile phase. The separation is achieved by a difference in the degree of attraction to the stationary and mobile phases by the compounds in the mixture. In thin-layer chromatography, the stationary phase is coated as a thin layer of the solid adsorbant on a sturdy support material such as a glass plate or a sheet of plastic. The stationary phase may be very polar (i.e. alumina Al2O3), intermediately polar (i.e. silica gel), or weakly polar (i.e. cellulose, starch, sugar). The advantages of thin-layer chromatography are the wide range of polarities of the stationary phase which allows a wide range of compounds to be separated, the small sample size needed, the relatively short separation time, and the ease of handling the plates. The plates can be purchased already coated and can contain a fluorescent compound, which helps to locate colorless compounds when the plate is exposed to ultraviolet light.

            The sample is applied near the bottom of the plate. The mobile phase (developing solvent) is allowed to flow up the plate by capillary action. The components in the mixture dissolve to varying extents in the mobile phase and are carried up the plate. Polar compounds tend to be strongly attracted to the coating on the plate and do not travel up the plate as rapidly as less polar molecules. If a more polar mobile phase is used, polar compounds will dissolve well in it and will travel more rapidly up the paper. If too polar a solvent is used, all the compounds will travel up the plate at the same rate and no separation will be achieved. By careful choice of the solvent or by use of a mixture of solvents, the compounds in the mixture can be separated and will be distributed along the length of the plate. The compounds will appear as somewhat diffuse spots or zones on the plate. The identification of the compound is based on the Rf value of the compound, which is the ratio of the distance traveled by the compound to the distance traveled by the solvent. The Rf value is characteristic of the compound for a particular coating, developing solvent, and temperature system.

 

 

            Thin-layer can be run in ascending (solvent moving up the plate) or descending (sample spotted at the top of the plate and the solvent moving downwards) manner. The Rf values for a given compound will not be the same for the different thin-layer materials. Ascending and descending chromatography will also give different Rf values for the same compound. Compounds identification is determined by measuring the Rf value for a compound with a particular stationary phase and solvent system and comparing it to the known values for compounds using the same system. While thin-layer chromatography is primarily used for identification purposes, it can be used to separate larger amounts of the compounds.

            In this experiment, thin-layer chromatography will be used to separate the compounds in commercial pain relievers. The structures for the common pain relievers are shown below.

 

                                   

                                                       

                       

            Aspirin (Acetylsalicylic acid)                             Acetaminophen (4-acetamidophenol)

           

                                

 

                        Caffeine                        Ibuprofen  ((+) 4-isobutyl-a-methylphenylacetic acid)

 

                     

                        Naproxen                                             Naproxen sodium

( (+) 6-methoxy-a-methyl-2-naphthalene acetic acid)                    (naproxen, sodium salt)

 

                       

                        Salicylamide

 

Experimental Procedure:

 

1. Place 25 ml of ethyl acetate in tank. Cover the tank.

 

2. Allow the tank to stand for several minutes before putting in the plate so the atmosphere inside the tank can become saturated with the solvent vapor. Keep the tank tightly covered during the experiment.

 

3. Obtain two 5 cm x 10 cm silica gel thin layer plate with fluorescent coating.

 

4. Draw a light pencil line 1.0 cm. and 9.0 cm. from the bottom (see Figure 1 and 2). Be careful not to scrap off the coating.

 

5.The first line is where the samples will be spotted and the second line will be the line to which the solvent front will rise. It is important to use pencil for the lines since the developing solvent will dissolve ink. The spotting line should not be below the solvent level in the beaker because the sample will dissolve off the plate and it should not be too high because it will take a long time for the solvent to reach the sample and begin the separation. The ending line for the solvent rise is used in calculating the Rf values and is arbitrarily chosen. The greater the distance the solvent is allowed to rise, the better is the separation but a longer time will be required.

 

6. Write only above the solvent line. Writing below the solvent line or spotting line can sometimes interfere with the solvent flow. 

 

7. Write a K in the top left corner of one plate, which will be for the known compounds (Fig.1).

 

 

8. Write a UK in the top left corner of the other plate which will be the for the unknown samples (Fig. 2).

 

 

 

 

 

 

 

 

 

 


           

 

 

 

 


                    Figure 1                                                               Figure 2

 

9. Label the knowns as AC (acetaminophen), ASP (aspirin, acetylsalicylic acid), C (caffeine),

I (ibuprofen), N (naproxen), S (salicylamide). Write abbreviations for the names of the samples about the solvent line. See Figure 3.

 

10. Label the unknowns as AD (AdvilTM), T (TylenolTM), BC (BC powderTM), EX (Excedrin Extra StrengthTM), G (Goody’s powderTM). Write abbreviations for the names of the samples about the solvent line. See Figure 4.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


                      Figure 3                                                          Figure 4

 

11. The samples are dissolved in 1:1 ratio of 95% ethanol and methylene chloride, which evaporates easily, and are applied to the plate with a fine glass capillary tube. The capillary tube is dipped in the sample solution and is briefly touched to the plate to form a spot that is about the diameter of the capillary tube. Use a different capillary tube for each sample. Too large a spot will result in spots that tend to trail too much and may cause overlap in the sample areas. The spot only needs to be large enough to be clearly visible under the UV light. Do not spot the samples too close to the edge of the plate or too close to each other.

 

12. Practice spotting a sample using some colored water on a piece of paper towel or filter paper.

 

13. Spot each sample on the spotting line directly below the label.

 

14. Place the Knowns plate in the solvent in the tank labeled Known .

 

15. Place the Unknowns plate in the solvent in the tank labeled Unknown .

 

16. Handle the plates only by the top edge.

 

17. Lean the plate against the side of the tank and keep the plate as vertical as possible.

 

18. Cover the tank.

 

19. Allow the solvent to rise up the plate until it reaches the solvent line.

 

20. When the solvent has reached the line, remove the plate from the solvent.

 

21. Dry the plate in the hood (or near a window or outside).

 

22. To view the position of the compounds, place the plate under a UV light in a darkened room.

 

23. Mark the top of each spot with a light pencil line.

 

24. For each spot, measure and record the distance from the top (leading edge farthest from the spotting line) of the spot to the point where the sample was applied (the original spotting line). See Figure 5.

 

25. For the solvent distance, measure and record the distance from the solvent front to the point where the sample was applied (the original spotting line). See Figure 5.

 

26. Calculate (as a decimal) and record the Rf value for each known and for each compound in the unknown.

 

27. Identify the components in each unknown mixture.

 

 

 

 

 

 

 


           

 

 

 

 

 

 

 

                                                                  Figure 5

 

While the intensity of the spot can give you a relative comparison of the amount of the compounds present, it is difficult to make thin-layer chromatography quantitative.

 

            This experiment is adapted from: “Thin-Layer Chromatography of Analgesics- An Update”, John W. Elder, Journal of Chemical Education, Vol. 72, No. 11, 1995.


 

Chromatography of Analgesics

 

NAME:_____________________________________  PERIOD:_____________

 

LAB PARTNER:_______________________________ COURSE:____________

 

Data Table

 

 Thin Layer Chromatography

 

Pure Compound Data:

 

Compound

Solvent Distance cm.

Sample Distance cm.

Rf

Acetaminophen

 

 

 

Acetylsalicylic acid

 

 

 

Caffeine

 

 

 

Ibuprofen

 

 

 

Naproxen

 

 

 

Salicylamide

 

 

 

 

Unknown Data:

 

Sample

Solvent Distance cm.

Sample Distance cm.

Rf

Compound

AdvilTM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TylenolTM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

BC PowderTM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ExcedrinTM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Goody’s PowderTM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

How did the Rf values of the compounds in the mixture compare to the Rf values of the pure compounds?

 


 

Chromatography of Analgesics

 

Thin-Layer Chromatography

Setup

 

48        5cm x 10cm silica gel coated plates cut from commercially prepared plastic backed plate

            with fluorescent dye.

Thin glass capillary tubes for spotting

Developing tanks

UV Lamp

 

 1 set of sample vials of knowns: AC (acetaminophen), ASP (aspirin, acetylsalicylic acid), C (caffeine), I (ibuprofen), N (naproxen), S (salicylamide) in 1:1 95%ethanol: methylene chloride.

 

 1 set of sample vials of unknowns: AD (AdvilTM), T (TylenolTM), BC (BC powderTM), EX (Excedrin Extra StrengthTM), G (Goody’s powderTM) in 1:1 95%ethanol: methylene chloride.

 

Ethyl acetate for the developing tanks.


 

Chromatography of Analgesics

 

Instructor’s Notes

 

1. Use a prepared silica gel plate with a plastic backing cut to about 5cm x 10 cm. Commercial plates are 20cm x 20cm. Turn the plate face down on a clean piece of paper so that it doesn’t pick up anything from the bench top. Use a thin permanent maker to draw lines in the plastic backing. Draw a line at 10cm across the plate in one direction. Draw lines 5cm apart in the other direction. Use sharp scissors to cut the plates to minimize chipping off the coating.

 

2. The amount of developing solvent used depends on the tank used. A 1 pint (16oz) canning jar with a screw cap lid can be used. Use a different jar for the known and unknown plate. The solvent can be reused but should be poured into a storage bottle when the experiment is finished and the jars allowed to dry.

 

3.

Pure Compound Data:

 

Compound

Solvent Distance cm.

Sample Distance cm.

Rf

Acetaminophen

 

 

 

Acetylsalicylic acid

 

 

 

Caffeine

 

 

 

Ibuprofen

 

 

 

Naproxen

 

 

 

Salicylamide

 

 

 

 

 

Unknown Data:

 

Sample

Solvent Distance cm.

Sample Distance cm.

Rf

Compound

AdvilTM

 

 

 

I

 

 

 

 

 

 

 

 

 

 

TylenolTM

 

 

 

AC

 

 

 

 

 

 

 

 

 

 

BC PowderTM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ExcedrinTM

 

 

 

AC

 

 

 

 

C

 

 

 

 

ASP

Goody’s PowderTM

 

 

 

AC

 

 

 

 

C

 

 

 

 

ASP

 

 

4. The knowns and unknowns should be in small screw top vials in separate wooden blocks with small test tubes behind each vial to hold the capillary tubes. Place the vials in the same order in which they will be spotted.

 

5. A smaller set of known and unknowns can be used to run both on the same plate. Aspirin, acetaminophen, caffeine, and salicylamide separate well. BC and Extra Strength Excedrin would be good unknowns.

 

6. Other samples might be AnacinTM (aspirin, caffeine), TylenolTM (acetaminophen), and various generic tablets.

 

7. Other samples can be used. Goody’s powders have the same compounds as Extra Strength Excedrin. The ketoprofen has a Rf similar to salicylamide so samples containing it weren’t used. The salicylamide fluorescences blue, which could be used to distinguish it.

 

8. The mixtures were prepared by grinding two tablets or one powder with 6 ml of a 1:1 mixture of 95% ethanol and methylene chloride. The mixture was suctioned filter using a small Hirsch funnel and 50 ml suction flask to remove the insoluble starch, sugars, and binders. Gravity filtration can be used but use a small size filter paper and funnel to prevent having a large amount of the sample being absorbed by the paper.

 

9. The knowns were made to be 2% solutions using 0.100g of the compound dissolved in 5 ml of 1:1 mixture of 95% ethanol and methylene chloride.

 

10. Infrared spectra were run on the known compounds and peak assignments made based on the structure of the compound.

 

11. The structure of ketoprofen is:

                       

 

12. The Apple II program Separations can be run a prelab activity or a co-lab activity where half the students are running the program and the other half is running the experiment.