The Separation of Spinach Pigments via Column and Thin-Layer Chromatography 5/29/14
Spinach extract was separated into fractions containing compounds of similar polarity via column chromatography. Thin layer chromatography was then used to analyze the extract and its separated components. Experimental results showed that the retention factor for carotene (0. 0.86) was the largest, followed by xanthophyll (0.38) and then chlorophyll (0.14) being the smallest.
Chromatography exploits differences in the physical properties, such as boiling point and/or polarity, of components in a mixture and separates the different compounds or ions by distribution between two phases, a stationary and a mobile phase (Padias 162). The methodology behind chromatography is based on the variant absorbability 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. The stationary phase is constantly washed with a mobile phase, and over time the added mixture will separate into its substituent components based on their physical properties, or whether a particular component adheres better to the mobile phase or the stationary phase.
By the concept of “like attracts like”, a polar stationary phase will adsorb a polar components more strongly than a non-polar component, which also indicates that a non-polar components will be removed along the solvent more easily and faster than the polar compounds. Similarly, when a polar solvent is used, the polar component will be moved along the solvent more quickly and hence, leave behind the non-polar component. The polarity of the molecules in a compound determines the forces of attraction between the molecules in the liquid state that in turn affect the boiling point of the liquid, which is the temperature at which the vapor pressure of the liquid and atmospheric pressure are equal. Higher boiling point or greater polarity leads to larger forces of attraction. Generally, nonpolar compounds have a lower boiling point than polar compound.
In chromatography, a sample is loaded onto the beginning of the stationary phase and based upon differences in the physical properties, the components start to equilibrate between the mobile and stationary phase. Non-polar components (low boiling point) are most likely to be present in the mobile phase while polar components (high boiling point) are most likely to be present in the stationary phase. The most volatile components are most likely to spend more time in the mobile phase than the less volatile components. Therefore the separation of each component is based on the components affinity for either the stationary or mobile phase, which is determined by the partition coefficient (Kp), a ratio of the concentration of each component in the stationary and mobile phase (Equation 1). The larger the Kp, the higher the affinity for the stationary phase, and therefore the component spends less time in the stationary phase. The smaller the Kp the lower the affinity for the stationary phase and so the component moves faster through the stationary phase thereby spending more time in the mobile phase. Equation 1: Kp (A) = [A] Stationary phase / [A] Mobile phase
There are many types of chromatography such as gas chromatography (GC), column chromatography and thin layer chromatography (TLC). GC is used to analyze thermally stable volatile mixtures, check sample purity, and identifies unknown components by comparing peaks of the unknown chromatograph to a standard. Column Chromatography involves running an analyte through a column and separation of the components is based on the polarity difference of the solid-liquid phase. TLC is used mostly for a fast qualitative analysis of a mixture or for very rapid separation of small amounts of material. All of the different chromatographic techniques focus on the mobile and stationary phases even though the nature of the two phases (e.g. solid-liquid, liquid- gas) determines the likelihood of using a particular technique. The method of separation in all the techniques depends on the differential absorptivity of the substances to be separated between the two phases, which is achieved if one component of a mixture adheres more to the stationary phase than another.
Although the different methods of chromatography are similar, there are certain unique characteristics of each technique that qualifies it as a suitable separation technique for specific mixtures. GC is used to separate volatile liquid mixtures. The mobile phase is a non-reactive inert gas and the stationary phase is a viscous high boiling point liquid. The analyte mixture separates base on solution-dissolution equilibrium as the analyte first interacts with the high boiling point liquid and gets pushed with the mobile phase. Therefore the main physical property exploited in separation is volatility.
Column chromatography is a heat free process with a liquid mobile phase and a polar non-reactive solid stationary phase with alumina or silica gel often used as the adsorbents. The components to be separated emerge from the stationary phase at different times under the influence of gravity. In order to achieve the best possible separation of components, the stationary and mobile phases are chosen based on the nature of the sample mixture. Since silica and alumina are highly polar, polar substances will adsorb more strongly to the stationary phase and elute late from the column because they have a large Kp, while less polar (non-polar) substances will adsorb poorly to the stationary phase and elude early because they have a small Kp. The starting mobile phase should be non-polar to insure that it does not compete with the stationary phase which is usually very polar for convenience, and each mobile phase afterwards should follow a “step-wise” increase in polarity. This means the first eluent should be nonpolar and changes to more and more polar to produce step gradient elution. Therefore the main physical property exploited in column chromatography is polarity. TLC like column chromatography is used for separating mixtures based on polarity, but is very efficient when working with small samples. In TLC the mobile phase is a solvent and the stationary phase is a thin layer of silica gel or alumina.
A good solid support is also required in TLC. The support can be a plastic or metal plate. TLC uses capillary action, the tendency for the liquid to rise due to adhesive and cohesive properties to move the solvent through the stationary phase into the mobile phase. When conducting TLC it is important to make sure that the atmosphere in the beaker is saturated with solvent vapor by lining the beaker with some filter paper and making sure the chamber is closed at all times with something like a wash glass. This will stop the solvent from evaporating as it rises up the plate. As the solvent travels up the plate the different components of the mixture travel at different rates and the mixture is separated into different colored spots at different height distances traveled on the plate. Maximum separation of the mixture is obtained when the solvent is allowed to rise until it almost reaches the top of the plate. The distance each spot will travel compared to the distance the solvent will travel is related by a feature called Retention factor (Rf) (Equation 2).
Equation 2: Rf = Distance traveled by spot / Distance traveled by solvent. Experimental:
Spinach extract was separated into fractions via column chromatography and TLC was then used to analyze the extract and its separated components. Results:
As the first eluent moved down the column, the first color band/fraction that was collected was bright yellow. When the second eluent moved down the column, the band/fraction that was collected had a very light green color. The last fraction that exited the column with the third eluent had a darker green color. In the TLC experiment, as the solvent traveled up towards the top of the plate, it carried the spots of the three pigments and standard along with it, finally leaving them at different heights. Retention factor of Reference 1 = 5.6 cm / 6.5 cm = 0.86
Retention factor of Reference 2 = 2.4 cm / 6.5 cm = 0.38
Retention factor of Reference 3 = 0.9 cm / 6.5 cm = 0.14
Retention factor of the yellow pigment (9:1) = 5.6 cm / 6.5 cm = 0.86 Retention factor of the light green pigment (1:1) = 2.4 cm / 6.5 cm = 0.38 Retention factor of the dark green (Meth) = 0.9 cm / 6.5 cm = 0.14 TLC Spots Pattern Sketch:
Spinach leaves contain chlorophyll a and b and β-carotene as major pigments as well as smaller amounts of other pigments such as xanthophyll, which is an oxidized version of carotene and pheophytin, which looks similar to chlorophyll. Each of the components was colored differently, thus in chromatography separation of the pigments could be followed visually. Carotenes are yellow-orange, xanthophyll is yellow, a-chlorophyll is blue green and b-chlorophyll is green.
In the column chromatography experiment, the stationary phase was alumina. Alumina is a very polar basic solid compound because in is made up of aluminum atoms bonded to oxygen atoms. It is the unequal sharing of electrons between the oxygen and aluminum atoms that make the alumina solid a polar compound and a good stationary phase. Three mobile phases of varying polarity were used because the three components of the spinach extract had different polarities and so each mobile phase could transport the component with similar polarity through the column. The yellow pigment eluded first, implying that it was the least polar of the three fractions. This correlates with the fact that its retention factor of 0.86 was the largest of the three components. This means that the first 9:1 hexane/acetone eluent was the least polar of the three mobile phases. Since the yellow component eluded first, it means that it spent very little time in the polar stationary phase. The yellow pigment was identified as carotene, which is indeed a non-polar compound. Even though the Kp of the carotene was not calculated in this experiment, it is safe to predict that it would be a very small value since the carotene molecules spent most of their time in the mobile phase than in the stationary phase.
The lighter green pigment eluded second, implying that it had a middle polarity, that is, it was not as non-polar as the first eluent but also not as polar as the last eluent. This observation also suggests that the light green pigment interacted equally to some degree with both the stationary solid and the second mobile eluent, which correlates with its retention factor of 0.38 because it is smaller than the first but larger than the third pigment’s. This implies that the 1:1 hexane/acetone eluent was middle in polarity. The second pigment band was identified as xanthophyll. Xanthophyll contains an additional hydroxyl group and so it makes it more polar than carotene (hydrocarbon). Therefore it makes sense that it eluded after the carotenes, since it must have adsorbed to the polar stationary phase to some extent. This was easily observed in the experiment because it took a longer time for the second band to be eluded compared to the first.
The third pigment that eluded from the column was the most polar component because it was the last to elude from the column. This means that it adsorbed to the stationary phase more than the second and first pigment, which also correlates to its retention factor of 0.14, which is the smallest amongst the three. Therefore the third solvent that was used, methanol, was the most polar amongst the solvents. The third dark green pigment is identified as chlorophyll (either a or b). Chlorophylls are much more polar than carotene and xanthophyll. It is safe to conclude that chlorophylls have a large Kp since their concentration in the stationary phase is larger than their concentration in the mobile phase.
In column chromatography, the solid support used was sand. Separation was achievable due to the gravitational force acting downward on the liquids in the column. Gravity applies the force needed to pull the mobile phase down the column, enabling separation. As the pigments travel down the column, equilibrium is established between the components dissolving in the mobile phase and adsorbing to the stationary phase. The wet/slurry packing method was done to prevent the experimenter from possibly inhaling the alumina, which is dangerous. However, dry packing, slurry packing and any other packing method could be used interchangeably depending on the experimenter. The pigment fractions collected were evaporated on the steam bath so as to have a concentrated solution of each pigment needed for TLC. The stationary phase used in the TLC was silica gel that again is very polar, the mobile phase was 30% ethyl acetate in hexanes with a medium polarity and the solid support was aluminum. The degree to which the different pigments interact with the mobile and stationary phases determines how fast they travel on the plate. Due to silica gel’s high polarity, the less polar pigments and the solvent would travel up the plate faster than the most polar pigment. How far each pigment traveled determined its retention factor.
The results of this experiment show that carotene traveled the fastest, followed by xanthophyll, with chlorophyll having traveled the slowest. These results make sense based on the fact that the least polar (carotenes) do not adsorb to the alumina, but dissolve more in the mobile phase (ethyl acetate) of medium polarity and so travel fast. The xanthophyll absorbed to a small degree to the silica TLC plate and also dissolves in the mobile phase liquid, and so traveled less than the carotenes but more than the chlorophylls. The chlorophylls traveled the slowest since they adsorbed to the alumina plate to a great degree more than the other pigments. The distance traveled by each pigment correlate to its retention factor and so the retention factor for carotene (0.86) was the largest, followed by xanthophyll (0.38) and then chlorophyll (0.14) being the smallest. The retention factors for the three spots of the spinach extract were determined to each match the retention factor of one of the pure pigments, which was expected.
A line was drawn with a dull tipped No. 2 pencil above the solvent line to prevent direct contact of the line with the solvent. Direct contact with the line would erase the line as it will dilute in the solvent and would have hindered the TLC analysis because the origin point would have been arbitrary. For the TLC chamber, a watch glass was used to cover the beaker containing the solvent so as to prevent the solvent from evaporating into the atmosphere. Therefore for a good TLC analysis, it was important to keep the chamber saturated with the solvent, by keeping the chamber closed at all times. Also, a filter paper was placed into the chamber so that the solvent could travel up the plate faster as the filter paper continuously absorbs the solvent. This also helped to keep the chamber saturated with solvent, enabling the separation process to go on gradually as each spot is pulled up the plate by capillary action. The spots on the TLC plate were circled immediately after the plate was taken out of the chamber because they are most likely to fade away over time.
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