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By mar_quez Feb 19, 2014 993 Words

Chromatography – Analyzing Analgesics by TLC and Isolation of β-Carotene by Column Chromatography

Flavonoids are an important group of additives that can be defined as pure substances either natural, extracted from raw materials or synthetic. Chromatography is the separation of two or more compounds or ions caused by their molecular interactions with two phases – one moving and one stationary (Weldegirma 2012). Three types of chromatography are used in organic chemistry: Thin Layer Chromatography (TLC), Gas Chromatography (GC), and Column Chromatography (CC). TLC stationary phase is a solid while the mobile phase is liquid. GC stationary phase is liquid and its mobile phase is liquid. CC is one of the most useful methods for the separation and purification of both solids and liquids when carrying out microscale experiments (Weldegirma 2012). CC stationary phase is liquid and mobile phase is gas, the difference between CC and TLC is that CC uses glass while TLC uses silica gel. TLC provides a simultaneous analysis of dozens of samples in a relative short period of time. The major advantage is that its low cost, low solvent consumption and only small amounts of analytes reacquired (Vlajovic 2013). The thin layer stationary phase in TLC requires small sample size and shorter development distances to reveal their separation potential and provide a faster separation, better resolution and the spots are more compact and are favorable for in situ detection (Ligor 2007). In TLC, Rf is defines as the ratio of the distance traveled by the compound to the distance traveled by the solvent: Rf = Distance traveled by the compound / Distance traveled by the solvent (Weldegirma 2012)

The objective of the first part of the experiment is to analyze several analgesics by Thin Layer Chromatography (TLC) and the second part is to apply the technique of Column Chromatography (CC) to separate of β-Carotene from a mixture of many plant pigments found in spinach. Experimental:

Part I: Analyzing Analgesics by TLC

+Apply sample using capillary tubes
+Insert TLC plate into solvent jar (95% ethyl acetate: 5% acetic acid
+Withdraw TLC plate when the solvent reaches the solvent front.
+See plate under UV light
+ Calculate Rf for each compound

Part II: Isolation of β-carotene by Column Chromatography
Stage 1: Prepare the microcolumn

+ Half fill it with silica gel
+ Add sand (0.5 cm)

Stage 2: prepare Sample

+15 ml ethyl acetate
+Shake vigorously
+mash it with glass rod (10-15 min)
+Decant the colored solution

+ Remove solvent by evaporation

+1/2 drops of ethyl acetate

Stage 3: Isolation of β-carotene

+wet microcolumn by petroleum ether
+transfer sample
+fill the column with pet. Ether
+flash chromatography
+collect β-carotene as yellow/green fraction
+evaporate petr. Ether
+ cret β-carotene as yellow/green solid substance

Stage 4: TLC comparison
Compare isolated β-carotene gathered to standard β-carotene Table of Chemicals:
AcetaminophenAspirin CaffeineIbuprofen


Molecular Formula
Melting point
169 – 170.5°C
Molecular weight
5.5 – 6.5
Varies with concentration

Part 1: Analyzing Analgesics by TLC
Calculating Rf value= Distance travelled by the compound / Distance travelled by the solvent

Distance travelled of compound from starting line (cm)
Length travelled of solvent from starting line to solvent front (cm) 3.5
Rf value

Unknown 3
Unknown 4
Distance travelled of compound from starting line (cm)

Length travelled of solvent from starting line to solvent front (cm) 3.7
Rf value
It was determined that unknown compound 3 was caffeine and unknown compound 4 was Ibuprofen.
Part 2: Isolation of β-carotene by Column Chromatography

Isolated β-carotene
Standard β-carotene
Distance travelled of compound from starting line (cm)
Length travelled of solvent from starting line to solvent front (cm) 3.4
Rf value

Weight obtained from isolated β-carotene: 100.6 g
The experimental results that were performed in this lab were concrete and supported the obtained results. During the first part of the experiment, four analgesics were given and they we analyzed by TLC and Rf values were calculated based on the TLC method. Two unknowns were given and the TLC method was used in order to compare the unknowns with the four given analgesics. The Rf values of the unknowns were compared with the Rf of the analgesics. The closes value to unknown 3 was caffeine with a difference of 0.108, while the closes value to unknown 4 was Ibuprofen with a difference of 0.054. During the second part of the experiment, β-carotene was isolated from spinach, in order to determine if the isolation was prepared correctly, the obtained isolated β-carotene Rf value was compared with the Rf value of a standard β-carotene sample. The results demonstrated that there was a significant difference of 0.235 between the two values and the isolated β-carotene traveled 0.3 cm on the silica plate while the standard β-carotene traveled 1.1 cm. The results that were gathered demonstrated that isolating β-carotene was not as successful as thought. Conclusion:

The purpose of the experiment was to analyze analgesics by TLC and isolating β-carotene by CC. The theory of TLC was that it was a sensitive, fast, simple, and inexpensive technique to analyze compounds. The information revealed that TLC is a simple technique to analyze compounds and it’s a method to determine the compounds. The TLC is a worldwide technique, it’s used to determine food colorants in various matrices and compare synthetic additives to the natural compounds and determine the compensation of the synthetic additives to the natural compounds (Vlajkovic 2013). TLC remains popular for isolation and determination of components from natural products, cosmetics, pharmaceutical products, therapeutic monitoring of drugs in biological fluid, and among other applications (Ligor 2007).

Ligor, Magdalena, and Boguslaw Buszewski. "Thin Layer Chromatographic Techniques (TLC, OP TLC) For Determination Of Biological Activated Compounds From Herb Extracts." Journal Of Liquid Chromatography & Related Technologies 30.17 (2007): 2617-2628. Academic Search Premier. Web. 5 Feb. 2014. Vlajković, Jelena, et al. "Development And Validation Of A Tlc Method For The Analysis Of Synthetic Food-Stuff Dyes." Journal Of Liquid Chromatography & Related Technologies 36.17 (2013): 2476-2488. Academic Search Premier. Web. 5 Feb. 2014. Weldegirma, Solomon. Experimental Organic Chemistry. N.p.: Cengage Learning, 2012. Print.

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