Analysis of a Compound Mixture Using Thin Layer Chromatography

Abstract

Isolation of organic compounds via Thin Layer Chromatography (TLC) technique alongside the identification of an unknown compound from a sample was achieved in the experiment. The most appropriate solvent for the technique was also established. It was identified by the use of lowly polar solvents at the start of the experiment, then highly polar solvents. Hexane and ethyl acetate was low and medium polar solvents respectively, while acetone and methylene chloride were highly polar. Results indicated the unknown compound as fluorenol, while methylene chloride was the best solvent.

Introduction

Thin-layer chromatography is an analytical technique used to identify compounds in a mixture and their identities. It also helps establish the polarities of solvents (Nichols, 2020). Column chromatography and ion-exchange chromatography are widely used techniques to purify biomolecules. The sample’s stationary phase is applied before the mobile phase. The Ion-exchange chromatography involves electrostatic interactions between charged groups and a matrix. Other types of chromatography include gel chromatography, gas chromatography, and gas chromatography (Smith, 2013).

A thin layer chromatography is fast, sensitive, inexpensive, and simple in separating a mixture. The technique entails spotting the analyte at the solvent front. An adsorbent helps the solvent rise the plate by capillary action. Separation ensures according to the mixture components and their adsorption. Note stronger components adsorb on the stationary phase, and take less time in the mobile phase before migrating up the plate (Smith, 2013).

TLC aids purify a sample by comparing its absorption values and that of the standard. Extra spots on the chromatogram indicate impurities. Natural products such as waxes, steroids, and essential oils can be identified via TLC. In the foods and cosmetics industry, the technique helps separate and identify preservatives, colors, and sweetening agents. Cations and anions within a compound can also be identified via the TLC method (Lederer & Lederer, 1954).

Figure 1: Schematic of TLC. (a) The plate is put into the development solvent, allowing the solvent to travel up the plate. After a while (b) less polar materials travel the longest distance, unlike those more polar. The red and blue spots denote more polar and less polar substances, respectively. 

Reagents/apparatus

Table 1: Properties of reagents in TLC

Chemical	Formula weight (g/mol)	Boiling point (℃)	Melting point (℃)	Density (g/cm3)
Fluorine	18.998	-188.0	-219.0	0.00155
Fluorenone	180.192	83.5	342.0	1.130
Fluorenol	182.220	367.5	153.0	1.300
Benzoin	212.240	344.0	137.0	1.310
Hexane	86.180	68.5	-96.0	0.661
Benzyl	210.32	346.0 to 348.0	94.0 to 96.0	1.230
Acetone	58.0800	56.5	-96.0	0.785
Methylene chloride	84.930	39.6	-96.7	1.330

 

Table 2: Apparatus used during TLC.

1	A soft pencil
2	TCL plate
3	Beaker
4	Filter paper
5	A working bench
6	A paper towel

Procedure

1. Preparation of TLC plates

Fluorine, fluorenone, and unknown sample were provided. A beaker and a watch glass were used to make a TCL chamber. A 5 x 5 cm TLC plate was used so that it fits inside the chamber and becomes saturated with vapors, preventing eluent evaporation. The baseline was marked at 1 cm from the bottom of the plate. The analyte was dissolved in methylene chloride, ensuring the solution was 1%. Spots were positioned, and appropriate solutions marked, before allowing the solvent to evaporate in every spot. 

2. Developing a TLC plate

A small volume of methylene chloride was added into a beaker and cut filter paper leaned inside the beaker. The TLC plate was placed into the chamber, which was covered with aluminum foil. After 5 minutes the plate was retracted from the chamber and the spots moved were marked. The above procedure was repeated twice. Note the adsorption points on the TLC plate were visualized under UV light and the distances traveled marked. The retention factor values were calculated and the entire procedure was repeated using methylene: hexane = 4:1 and methylene chloride: hexane = 1:1 

3. Solvent optimization

Two assigned compounds were obtained, and the TLC plates were prepared. A pencil was used to draw a baseline of 1 cm from the bottom of the plate. Three marks were made at a 0.7 cm interval on the baseline, and the samples were placed on each spot. A chamber was developed using the solvent system given, and the process in part 2 was repeated to develop a TLC plate.

Observation and Data

Table 3: TLC data for methylene chloride

Sample name	(A) Distance traveled by the sample (cm)	(B) Distance between baseline and solvent front (cm)	Retention factor (A/B)
Fluorenone	4.4	6.1	0.72
Fluorenol	1.5	6.1	0.25
Fluorene	5.6	6.1	0.92
Unknown	1.5	6.1	0.25

 

The unknown was fluorenol and methylene chloride was the best solvent.

Table 4: TLC data for methylene: hexane = 4:1

Sample name	Distance traveled by the sample (cm)	Distance from baseline to solvent front (cm)	Retention factor
Fluorenone	2.6	6.1	0.42
Fluorenol	2.9	6.1	0.48
Fluorene	4.1	6.1	0.68
Unknown	2.9	6.1	0.48

 

Methylene chloride is a polar solvent, with higher concentrations than hexane. The unknown sample was fluorenol. 

 

Table 5: TLC data for methylene chloride: hexane = 1:1

Sample	Distance traveled  by sample (cm)	Distance from baseline to solvent front (cm)	Retention factor
Fluorenone	4.4	6.1	0.72
Fluorenol	1.5	6.1	0.25
Fluorene	5.6	6.1	0.92
Unknown	1.5	6.1	0.25

 

Hexane is nonpolar and has no effect on  polar compounds. The unknown sample was fluorenol.

Table 6: Retention factor for benzoin and benzene under varying solvents

Solvent	Acetone	Methylene chloride	Hexane
Benzoin	0.37	0.73	0
Benzil	0.64	0.73	0
Benzoin/benzene mixture	0.37, 0.64	073	0

The results show that separation did not take place when hexane was used since it is nonpolar.

Table 7: Retention factor for vanillin and vanillyl alcohol under varying solvents

Sample	Acetone	Toluene-ethylacetate (1:1)	Hexane
Vanillin	0.48	0.51	0
Vanillyl alcohol	0.53	0.58	0.35

 

Table 8: Retention factor for diphenylmethanol and benzophenone under different solvents.

Sample	Acetone	Acetone-hexane (3:7)	Hexane
diphenyl methanol	0.48	0.81	0.99
benzophenone	0.53	0.73	0

Discussion

Part A involved the isolation of organic compounds and determining the identity of the unknown.  The best solvent for TLC was also determined in this step. The analyzed samples exhibited similar characteristics in terms of intermolecular forces of attraction and polarity. The results from table 1 indicate that fluorenol was the most polar solvent and traveled the least distance on the plate. Fluorine was the least polar solvent and traveled the longest distance, while fluorenone was in the middle. The unknown compound was fluorenol, and methylene chloride was the best solvent. Hexane is nonpolar and cannot dissolve polar compounds. Theoretically polar dissolves polar and non-polar dissolves non-polar.

Part C analyzed benzoin, benzyl, vanillin, vanillyl alcohol, diphenyl methanol, and benzophenone. Benzoin and benzyl were more polar in methylene chloride than in acetone, while no effect was observed in hexane. Hence the conclusion that acetone and methylene chloride were the best solvents for TLC. Diphenylmethanol possesses a carbonyl group and an alcohol group that makes it partially dissolve in hexane. However, the results present that polar solvents were most suitable for TLC.

Conclusion

The TLC technique was used to separate organic compounds and to identify the unknown sample. The best solvent for the separation was also established. Results of the unknown sample tallied with that of fluorenol because they traveled the same distance on the TLC plate. Among the solvents used in the experiment, methylene chloride was the best choice due to its higher polarity than that of other solvents. The experimental objective was achieved.

References

 

1. 1. Benchflydotcom. (2009, August 6). How to pull capillary tubes. YouTube. https://www.youtube.com/watch?v=2yKHvKCatmM
   2. Khan Academy. (2013, September 17). Calculating retention factors for TLC. YouTube. https://www.youtube.com/watch?amp;v=_DEScXFyI8s
   3. Nichols, L. (2020, August 14). 2.3: Thin layer chromatography (TLC). Chemistry LibreTexts. https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Book%3A_Organic_Chemistry_Lab_Techniques_(Nichols)/02%3A_Chromatography/2.03%3A_Thin_Layer_Chromatography_(TLC)

• Lederer, E., & Lederer, M. (1954). Chromatography. A review of principles and applications. Chromatography. A review of principles and applications.

1. Smith, I. (Ed.). (2013). Chromatography. Elsevier.