Thin-Layer Chromatography:

Applying TLC as a Method to Monitor the Synthesis of Aspirin

Barbara Jennings

Chem 203.1

Desk 109.D

TA: Tian Chen

1
Introduction:

Chromatography has been an effective means of separating a mixture of chemical

compounds for over a hundred years. Originally chromatography had been preformed by

separating components in a column of calcium carbonate. In 1938, Nikolai Izmailov and his

postgraduate student Maria Schreiber developed a newer form of chromatograph known today as

Thin-layer chromatography (TLC). TLC is a solid-liquid form of chromatography that consists

of a thin plastic backing coated with a polar solid absorbant, usually silica gel, and a mobile

phase consisting of organic solvents with varying polarities. TLC separates a mixture of two or

more compounds based on polarity. As the mobile phase solvent carries the compounds up the

plate through capillary action, the more polar compound will interact with the stationary phase

separating itself from the less polar compound that will stay in the mobile phase since it is less

attracted to the stationary phase. The mobile phase consists of a mixture of solvents that are just

polar enough to allow the polar compound to elute slightly, but just nonpolar enough to achieve a

greater separation by partition from the nonpolar compound (see figure 1, appendix A). This fast,

effective, and inexpensive means of chromatography has played a major role in food science,

medicine, and industry. One major application the pharmaceutical industries use TLC analysis

for is to monitor the synthesis of a drug or to identify the active ingredients of medicine.

Monitoring the reaction of a drug by TLC is very significant because it allows pharmaceutical

companies to know when the reaction is complete and also how pure their product is or if further

purification is needed due to the presence of impurities.

2
 TLC has been used to monitor the synthesis on many medicines, but one particular over

the counter drug has been aspirin. Aspirin, also known as acetylsalicylic acid, was invented in

1893 by Felix Hoffman originally as a treatment for arthritis pain and inflammation. Aspirin is

now commonly used as an anti-inflammatory, analgesic, and antipyretic. Acetylsalicylic acid is

synthesized by acetylating the phenolic functional group of salicylic acid with acetic anhydride.

This reaction is a similar mechanism to Fisher esterification or otherwise known generally as a

nucleophilic acyl substitution mechanism. The final product is formed protonating the acetic

anhydride, with the catalyis phosphoric acid, forming an electrophile that can then bond to the

nucleophilic phenolic substituent of salicylic acid. The attached acetic anhydride is then broken

apart leaving the acetyl group attached to the subsituent thus forming the final product

acetylsalicylic acid and byproduct acetic acid (see mechanism Figure 2, appendix B).

Purification of the final product can be achieved by recrystallization in a slightly polar solvent,

such as ethyl acetate. To determine if the synthesis has gone to completeness, the reaction

mixture is spotted on a TLC plate every 15 minutes and developed in a TLC chamber with a

mixture of ethyl acetate and hexane as the mobile phase until only one spot is observed in the

reaction mixture lane. Further analysis, by nuclear magnetic resonance (1H-NMR) spectroscopy,

is used to determine the purity of the final product.

The purpose of this experiment was to determine what mixture of ethyl acetate and

hexane will achieve the greatest separation of salicylic acid and acetylsalicylic aid. Then use this

solvent system to monitor the synthesis reaction of acetylsalicylic acid from the reactants

salicylic acid and acetic anhydride. Finally the purity of the final product, acetylsalicylic acid,

was determined by melting point analysis and by.

3
Experimental:

TLC Mobile Phase Determination

An appropriate solvent system for TLC mobile phase to achieve a large separation of

salicylic acid and acetylsalicylic acid was determined by trial and error. Eight trials of hexane

and ethyl acetate were mixed in various percents (see Table 1). Each mixture was used as a

mobile phase to develop a TLC plate spotted with standards salicylic acid (1%) and

acetylsalicylic acid (1%) in a saturated TLC chamber. The mobile phase was allowed to develop

up the plate until the solvent line reached approximately one centimeter from the top of the TLC

plate. The plate was allowed to dry and then examined under ultraviolet (UV) light to visualize

the spots. The retardation factor (Rf) value was determined for each spot (see sample calculation

Figure 3, appendix A). The goal was to determine which solvent system created the best

separation of the standards. It was determined that trail four, 50% ethyl acetate and 50% hexane,

achieved the greatest separation of salicylic acid (Rf 0.945) and acetylsalicylic acid (Rf 0.291)

because it had the largest difference in Rf values (0.654).

4
 Mobile Phase Rf Rf
Mixture % Ethyl % Hexane Salicylic Acetylsalicylic
Composition Acetate Acid Acid
Number
1 100 0 0.530 0.545
2 90 10 0.620 0.600
3 70 20 0.255 0.291
4 50 30 0.945 0.291
5 30 50 0.360 0.280
6 20 70 0.270 0.250
7 10 90 0.182 0.164
8 0 100 0 0
Table 1: Mobile Phase Mixture combinations for the separation of Salicylic Acid (1%) and
Acetylsalicylic acid (1%)

Synthesis of Aspirin

Synthesis of aspirin took place by placing salicylic acid (100mg, 0.724 mmol) in a 13 x

100 mm test tube with acetic anhydride (0.25 mL, 2.25mmol) and one pipet drop of phosphoric

acid (85%). The tube was corked, gently mixed and placed in a warm beaker of hot water just

below boiling point. After every 15 minutes the reaction was checked by TLC using a mobile

phase 50/50 mixture of ethyl acetate/hexane and a TLC plate spotted with three lanes: pure

salicylic acid (1%), pure acetylsalicylic acid (1%), and the reaction mixture (R). Rf values were

calculated for each spot that formed. The reaction is said to have synthesized to completeness

when there is only one spot left, the acetylsalicylic acid spot, meaning there is no more salicylic

acid present.

5
 Figure 4: TLC plates used to monitor the synthesis if acetylsalicylic
acid at 15, 30, and 45 minutes. Lane S: Salicylic Acid (1%)
Standard. Lane R: Reaction Mixture. Lane AS: Acetylsalicylic Acid
(1%) Standard
Time (minutes) Salicylic Acetylsalicylic Reaction Reaction
acid Rf acid Rf Mixture Rf Mixture Rf
Lower Spot Upper Spot
15 0.385 0.346 0.385 0.481
30 0.320 0.340 0.360 0.500
45 0.264 0.321 0.321 0.491
Table 2: Retardation factor (Rf) values for TLC of salicylic acid, acetylsalicylic acid, and
reaction mixture in a 50/50 ethyl acetate/hexane solvent system over time.
After 45 minutes the tube was removed for the warm water bath and distilled water (0.7

mL) was added to quench the reaction. Slow cooling allowed crystals to form and vacuum

filtration removed the reaming solvent. The crystals were allowed to dry for one week and

melting point was then taken three times to determine purity of crystals. Further purification was

not need since. Acetylsalicylic acid was obtained as flaky white crystals (97mg, 75% yield); mp

135-136° C (lit. acetylsalicylic acid 134-136° C, lit. salicylic acid 158-161° C) and a 1H-NMR

spectrum was obtained ( see Figure 5 (Appendix C) and Table 3) .

6
 Shift (ppm) Splitting Observed Integral Observed Type of hydrogen
11.649 ppm Singlet 1.00 -COOH
7.074-8.206 ppm Multiplet 4.35 Ar-H
2.346 ppm Singlet 3.00 H3-C-COOR
Table 3: 1H-NMR data, obtained for Reaction sample (35mg) with deuterated chloroform solvent on a
60 MHz 1H-NMR.

Sample calculation for percent yield of acetylsalicylic acid:

%yield = (actual yield / theoretical yield) x 100

75% = (97mg / 130mg) x 100

7
Results, Discussion, Conclusion:

When determining the best mobile phase to monitor the synthesis of aspirin, it was

important to find the correct combination of ethyl acetate and hexane that would produce the

highest separation of salicylic acid and acetylsalicylic acid. However, as seen in Table 1, many

different results occur depending on the ration of ethyl acetate to hexane, only the right

combination can achieve significant separation between the reactants and products for the

synthesis of aspirin. For example, when using pure hexane the entire solvent has a nonpolar

composition. Therefore acetylsalicylic acid, the more nonpolar compound, will be more attracted

to the mobile phase and move with the hexane solvent while salicylic acid, the more polar

compound, will be extremely attracted to the polar stationary phase and will not move at all

(Table 1). When using a solvent system that is %100 ethyl acetate both the acetylsalicylic acid

compounds and salicylic acid elute too fast since the nonpolar compounds are not attracted to

either the stationary phase or mobile phase, while salicylic acid will elute just as fast since the

compounds are more attracted to the mobile phase than the stationary phase. From the results, a

50/50 mixture of ethyl acetate and hexane achieve the greatest theoretical separation of

acetylsalicylic acid and salicylic acid because there is a greater distance between the developed

spots and has the biggest difference in Rf values (0.654).

Using solvent system should theoretically produce the greatest separation of the products

and reactants when monitoring the synthesis of aspirin. However, this mobile system did not

produce ideal separation of products and reactants. The salicylic acid spot of the reaction mixture

was expected to have a much lower Rf value than the acetylsalicylic acid, since the salicylic acid

appears to have a much higher polarity for the stationary phase due to the phenolic substituent.

8
However, salicylic acid and acetylsalicylic acid were only separated slightly from one another,

having an Rf difference of 0.096, much lower than the theoretical separation of the standards (Rf:

0.654) (Table 2). One possible explanation for this occurrence may be due to internal physical

interactions of the salicylic molecules with each other in the reaction mixture. This may be

because the hydrogen on the phenolic substituent of salicylic acid interacts with the double

bonded oxygen of the other substituent, carboxylic acid, through hydrogen bonding thus

decreasing its polarity significantly. Due to higher interaction among molecules, salicylic acid is

less attracted to the stationary phase and will move further up the plate thus having a higher Rf

value than the standard salicylic acid and a closer Rf value to the acetylsalicylic acid in the

reaction mixture. Another complication that occurred was that the reaction mixture had an upper

spot that consistently higher than both the standards it was develop with (Figure 5). This may be

due to the fact that acetylsalicylic acid in the reaction mixture was had many other molecules

interacting and blocking the acetylsalicylic acid. Thus it’s polar carboxylic acid substituent had

less chance to interact with the stationary phase than the pure standard acetylsalicylic acid.

As time progressed the TLC plates showed no sign that the reaction had run to

completeness. There were still two separate spots that appeared on the TLC plate after 45

minutes. This may be due to the fact that even though the reaction may have been completed, the

acetylsalicylic acid compounds were actually more polar than expected, due to the carboxylic

acid substituent, causing a greater attraction to the polar stationary phase and streaking in two

close spots. Since the Rf value of the lower spot (0.321) had the exact same Rf value of the

standard acetylsalicylic acid (0.321), this confirmed that the final TLC plate had only

acetylsalicylic acid was left in the reaction mixture, thus the reaction was complete. Since the

melting point (134-135° C) was very close to the actual melting point of acetylsalicylic acid

9
(134-136° C) there was no further purification needed. Isolating the final product, acetylsalicylic

acid, was a success. Further 1H-NMR spectrometry analysis was preformed to confirm the

identity of the product.

The 1H-NMR spectrum data matched perfectly to the theoretical peaks that would

be seen if pure acetylsalicylic acid 1H-NMR data were obtained (Figure 6, Appendix D). There

was a small peak at 11.649 ppm with an integral value of 1.00 due to the carboxylic acid

hydrogen, multiple peaks ranging form 7.074-8.206 ppm with an integral value of 4.35

indicating the four hydrogens on the aromatic ring, and a significant single peak at 2.346 ppm

with an integral value of three indicating the hydrogens on the ester (-COOCH3) substituent. The

peak at 0.00 ppm is a TMS peak, typical when using the solvent deuterated chloroform. There

were no other significant peaks presented in the spectrum, indicating the isolated product was

pure. The obtained spectrum is a near perfect match with the theoretical spectrum of standard

acetylsalicylic acid. For these reasons it is reasonable to confirm that an esterification reaction

that took place between salicylic acid and acetic anhydride formed pure acetylsalicylic acid. The

percent yield supports that fact that the synthesis of aspirin was successful because the product

obtained had a high yield of 97 mg (75% yield) from a theoretical yield of 130mg. Some residual

crystals may have been left on glassware, accounting for the decrease in yield.

All in all the 50/50 mixture of ethyl acetate and hexane did not prove to be helpful in

monitoring the progression of the synthesis of aspirin. Since the spots were visualized as streaky

and close together is was difficult to tell whether the reaction was complete or there were still

reactants present. For future experimentation, more trial and error solvent systems should be

tested to determine the best solvent system that will stop internal physical interactions while

provide clear separation of components on the TLC plate. Melting point and 1H-NMR analysis

10
were very helpful when determining the true identity of the product. The synthesis of aspirin

proved to be successful at achieving a high yield final acetylsalicylic acid product. In

conclusion, the techniques, knowledge, and skills gain from this experience have not only proven

to be helpful for this experiment, but also for future scientific work.

11
References:

1. Bortiatynski, J.; Masters, K.; Minard, B.; Halmi, T.O.; Williamson, K.L. Lab Guide for
Chemistry 203 & 213B, 2009-2010, pp. 165-188.

2. Kapp, Khail. Chapter 7, Thin Layer Chromatography, March 2, 2010.

3. Kaine, Mary Ann. Chapter 7, Thin Layer Chromatography, March 2, 2010.

4. McMurray, J.; Simanek, E. Fundamentals of Organic Chemistry, 6th ed.; Thomson

Brooks/Cole: Belmont, CA, 2007, pp. 307-308.

5. Sigma-Aldrich. Acetylsalicylic Acid. http://www.sigmaaldrich.com/united-states.html.

(accessed March 28, 2009).

6. Young, Wei. Chapter 7, Thin Layer Chromatography, March 2, 2010.

12
Appendix A:

Figure 1: Sample thin-layer chromatography (TLC) plate,

sample C shows the compound with the least polarity;
sample A shows the compounds with the most polarity

Appendix B

13
 Figure 2: Nucleophilic acyl substitution mechanism for the synthesis of
acetylsalicylic acid from salicylic acid and acetic anhydride

Retardation Factor (Rf) = distance spot travels / distance solvent travels

Example: Table 1, Mobile Phase Mixture Composition Number 1, salicylic acid

Rf = 2.9cm/5.5cm

= 0.53
Figure 3: Retardation Factor (Rf) theoretical and sample calculation.

14
Appendix C

TMS

Figure 5: Annotated 1H-NMR spectral graph for the product of synthesized of acetylsalicylic
acid

15
Appendix D

Figure 6: Predicted chemical shifts, splitting patterns, and integral values of pure salicylic acid
and acetylsalicylic acid.

16