Column and Thin Layer Chromatography: the Separation of Spinach Pigment

Column and Thin Layer Chromatography: The Separation of Spinach Pigment

Abstract
Spinach extract was separated into fractions containing compounds of similar polarity by column chromatography. Based on solid-liquid phase partitioning, this separation technique exploited the different polarity of the compounds in the spinach extract. Three fractions with different colors were obtained. The extract and its fractions were analyzed using thin layer chromatography (TLC). The TLC results showed that there was one compound (Rf=0.979) in the first fraction; there were three compounds (Rf1=0.839, Rf2= 0.691, Rf3=0.149) in the second fraction; there was one compound (Rf=0.017) in the third fraction. The separations of compounds which went to the first and third fraction were relatively satisfying, while the second fraction had several kinds of compounds.
Introduction
Chromatography is the separation of compounds or ions by distribution between two phases—a mobile phase and a stationary phase. The technique is based on the differential absorptivities of the constituents between these two phases, due to different properties of the compounds to be separated and the nature of the two phases involved. If one constituent adheres more to the stationary phase than the mobile one, separation will be achieved. There are several different types of chromatography, such as thin layer chromatography (TLC), gas chromatography (GC) and column chromatography. All the chromatographic methods are based on partitioning of molecules between a stationary phase and a mobile phase. In order to measure the amount of partitioning between the two phases, each compound has an unique Partition Coefficient (Kp), which is defined as the ratio of concentrations of the compound between the stationary phase and the mobile phase: Kp=[x]sp/[x]mp. Therefore, if Kp is greater than 1, the substance adheres more to the stationary phase; if Kp is smaller than 1, the substance adheres more to the mobile phase; if the Kp is equal to 1, the probabilities of the substance to adhere to the two phases are roughly the same. The Kp value is depended on a multitude of factors: “polarity, solubility in the solvent, hydrogen bonding, volatility in the case of gas chromatography.” After the injection, the sample molecules will either stay in the mobile phase or adhere to the stationary phase. When the mobile phase is pushed through the stationary phase by the eluent, the mobile phase, the molecules will move through the column at a rate that depends on their different Kp value. For example, if one constituent is more polar than other and adheres more to the stationary phase when the stationary phase is more polar than the mobile phase, this constituent will lag behind; while the constituent that is less polar and adheres less to the stationary phase will move ahead. In this way, the sample can be separated into fractions containing compounds of similar polarity. The differences among the various types of chromatography are mostly depended on the nature of the two phases involved. Column and thin layer chromatography use solid stationary phase and liquid mobile phase; while GC uses a gas as mobile phase and a liquid as stationary phase.
Compared with TLC and column chromatography, GC is a more sophisticated method.
Reagent Table Compound | MW | MP | BP | Density | Safety Consideration | Hexane | 86.18 g/mol | -96--94°C | 68-69°C | 0.6548 g/mL | Flammable | Acetone | 50.08 g/mol | -95--93°C | 56-67°C | 0.791 g/mL | Flammable | Methanol | 32.04 g/mol | -98--97°C | 64.7°C | 0.7918 g/mL | Flammable | Chlorophyll a | 893.49 g/mol | 117-120 °C | NA | NA | | Chlorophyll b | 907.47 g/ mol | NA | NA | NA | | β- Carotene | 536.87 g/ mol | 180-182 °C | 633-677 °C | 0.94(6) g /cm3 | | Alumina | 101.96 g/ mol | 2072 °C | 2977 °C | 3.95–4.1 g/cm3 | Not health y to inhale | Ethyl acetate | 88.11 g /mol | −83.6 °C | 77.1 °C | 8.3 g/100 mL (20 °C) | Flammable |

Experimental

Results
Observations:
TLC plate:

Data
Solvent front: 5.72cm Smaple | Distances the spots traveled/cm | Rf | First fraction | 5.6 | 0.979 | Second fraction | 4.8; 3.95; 0.85 | 0.839; 0.691; 0.149 | Third fraction | 0.1 | 0.017 | Extract | 1.2; 4.0; 4.9; 5.6 | 0.210; 0.699; 0.857; 0.979 |

Discussion
The intent of this experiment was to successfully separate the constituents of spinach extract using column chromatography. The results of the separation were analyzed by TLC to reveal in which fraction the compounds of the mixture were. Since both of the samples of the first fraction and the third had only one developed spot on the TLC plate, the separation of the compounds in the first and third fractions were relatively satisfying. However, there were 3 different compounds reveled for the second fraction. Since the color of the first fraction was yellow and the color of the second and third fractions were green, the major compound in the first fraction should be Carotenes, the yellow-orange pigment; while the major compounds in the second fraction and the third fraction should be Chlorophyll.
During the process of column chromatography, the first band gained in the column was very clear and the color was yellow. The second band gained in the column was green and the thickness of the second band was greater than the first. Also, the green color of the second band varied, and colorless area was observed in the second band. Compared with the first band, the second band showed an unsatisfying separation result. Since there was colorless area and various green colors in the second band, the second fraction must contained several constituents, which was examined by the TLC results—there were 3 developed spots of the second fraction sample on the TLC plate. As for the third fraction, there was no clear band in the column while adding the last eluent. The concentration of the compound in third fraction was relatively small, resulting in a very small Rf value, 0.017. Compared with the spots of the second fraction sample, which had a much clearer spot with similar Rf value, 0.839, the compound in the third fraction must existed in a large amount in the second fraction. In addition, these 2 spots had the same light green color, which can also examine this conclusion. The lower concentration can also due to the unsatisfying spotting. The diameter of the undeveloped spot in the third fraction was larger than the first and second one, because too much solvent was used. Also, multiple times of spotting was needed.
Observing the spots color, the clearest spot in the second fraction had a more intense green color, which showed that the compound in this fraction should be Chlorophyll a. While the major compound in the third fraction should be Chlorophyll a, with a lighter color and lower Rf value.
The ideal solvent system should result in Rf values ranging from 0.24 to 0.54. However, in this experiment, none of the Rf value was in this range. Therefore, the polarity gradient of the eluents should be narrowed, which can result in more spots in the ideal range. Since there were 3 spots in the second fraction sample, over 4 eluents are needed to give a better separation, assuming the spot with lowest Rf value in the second fraction had the same compound as the spot in the third fraction sample.