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column and thin layer chromatography
Topics: Chromatography, Thin layer chromatography / Pages: 10 (2274 words) / Published: Nov 12th, 2013

Column and Thin Layer Chromatography

Beverly

Abstract: Plant pigments were separated and concentrated from a crude spinach extract through the use of column chromatography and an eluatropic series of hexanes, hexane/acetone, and methanol. The pigments were analyzed using thin layer chromatography with a 30% ethyl acetate/hexane developing solvent.

Introduction:

Chromatography is a technique used to separate a mixture of two or more components based on differences in their physical properties. It can be used as a method of purification (and can be used on a small scale) along with analyzation of mixtures. Chromatography exploits differences in the physical properties, such as boiling point and/or polarity, of components of a mixture in order to separate them between the mobile and stationary phases. A 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. Components of a mixture partition between the two phases nu explanation of the partition coefficient relating to chromatography, where the coefficient of component x is equal to the ratio of the concentration of x in the stationary phase over the concentration of x in the mobile phase: KP (x) = [x]stationary / [x]mobile . This partition coefficient can be analyzed qualitatively; if it is larger than one, then x spent a longer period of time in the stationary phase as it was washed with the mobile phase, indicating that x has a higher affinity for the stationary phase as opposed to the mobile phase. If the coefficient is less than one, the reverse is true, and x spend more time in the mobile phase, indicating it has higher affinity for the mobile rather than stationary phase. There are many types of chromatography and all use the partition coefficient as a means of describing the separation of a mixture between stationary and mobile phases, but they can be differentiated by the types of phases, and the techniques of separation involved. In Gas Chromatography, the stationary phase is a high boiling point liquid, while the mobile phase is a gas. The mixture is vaporized into the gas phase and interacts with the gaseous mobile phase, that pushes it through the liquid stationary phase, which is always a high boiling point liquid (so it does not vaporize itself) that is usually polyethylene glycol (which will separate the components based on boiling point or polarity), or methyl siloxane (which will separate components based on boiling point alone). Separation of the components of a mixture being analyzed by gas chromatography partition based on their volatility and the differing polar interactions with the stationary phase (Padias, 180). In Column Chromatography, the mobile phase is a liquid, while the stationary phase is usually a polar solid that does not dissolve in the mobile phase. There are two typical stationary solids, silica gel (SiO2, which is slightly acidic) and alumina (Al2O3, which is slightly basic) both in their finely powered forms. The stationary phases are very polar, therefore components of a mixture that are polar will adhere to the stationary phase, and components which are non-polar will easily elute through it with a non-polar mobile phase. Consequently, the mobile phase, or eluent, is also chosen based on its polarity. A very non-polar eluent will elute all non-polar components of a mixture, while an eluent that is more polar than the stationary phase will elute polar components of a mixture. A gradient of eluents with increasing polarities can be used to separate a mixtures into its components in a stepwise function; this is called using an eluatropic series to create a step-gradient elution. This incremental increase of eluent polarity manipulates the KP value of the components (whether they adhere more to the mobile of stationary phase), so that the most non-polar components will elute first with the most non-polar eluent, and as the eluent is changed and becomes more polar, the more polar components of the mixture will elute with each incremental increase in the polarity of eluent. The most common eluents used in an eluatropic series, ranging from non-polar to polar, are alkane mixtures (Pet. Ether, a mixture of hydrocarbons; ligroin, C7 - C11 saturated hydrocarbons; or Hexanes, the most common, a mixture of isomers of C6H14), acetone (C3H6O), methanol (CH3OH), and acetic acid (CH3COOH). Column Chromatography is mainly used as a separation mechanism, whereas Thin Layer Chromatography is mainly used as a means of analysis; it can be used to monitor the process of a reaction, test for an impurity within a substance, or identify components of a mixture. However, it can also be used for separation of small amounts of material (Padias, 165). Like column chromatography, the stationary phase is a solid and the mobile phase is a liquid. However there are large differences in the way these two types of chromatography are run. In thin layer chromatography, the stationary phase is a coating of silica gel supported by metal, creating a plate called the TLC plate. The plate is “spotted” with the mixture that is desired to be separated or analyzed, and is placed vertically in a small amount of the mobile phase, or developing solvent. The developing solvent, selected based on it’s polarity, ascends the plate, bringing with it components of the spotted mixture, which will ascend to different heights on the plate depending on their polarity. The ascent of the developing solvent is due to capillary action, the combined effects of surface tension forces (molecules of a mixture adhere to each other) and adhesive forces (molecules of a mixture adhere to the walls of their container). These forces operate in a way that makes a mixture travel up a tube of a small diameter, or, in this case, the porous stationary phase. The partition coefficient of the mixture between the mobile phase, called the developing solvent, and the stationary phase determines how fast different spots of the mixture will move up the plate. The distance a spot moves up the TLC plate can be explained using its retardation factor, or Rf value, which is calculated by measuring the distance a spot as traveled and dividing by the total distance the solvent traveled, both from a standard bottom-line drawn directly below the spots.
Rf = A (bottom standard to spot) / B (bottom line to solvent line)
In many cases, a non-polar developing solvent is used as the mobile phase, in particular 30% ethyl acetate in hexanes (30% EtOAc 70% isomeric C6H14). In the case of using a non-polar developing solvent, a less polar component of the mixture will have a larger Rf value because it will have a large A; the non-polar component of the mixture will travel farther up the plate with the non-polar developing solvent. A less polar component will have a lower Rf value because it will have a smaller A; it will not travel very far with the non-polar developing solvent. Comparing the Rf between spots, or against a standard spot on the same TLC plate, can decipher the identity of the components (as having the same Rf as a spot on the standard), identify two components as the same (if they have the same Rf values), or identify two components as different (if there is a large difference in their Rf values) (Padias, 170).

Reagent Table:
Reagent
Chemical Formula
Molecular Weight (g/mol)
Amount (mol)
Volume (mL) density or conc.
Spinach Extract
Polymer, C34H29CuN4Na3O6
722.13

2.0
Hexanes
Isomers of C6H14
86.18

73.5
Acetone
C3H6O
58.08

30.0
Methanol
CH3OH
32.04

50.0
Ethyl Acetate
CH3COOC2H5
88.11

1.5 (Shaanxi Sciphar Hi-Tech Industry Co. Ltd., 2013; Sigma-Aldrich, 2013)

Experiment:

Instruments and Apparatuses:

Column (Packed with cotton, sand, and Alumina Powder)
Thin Layer Chromatography plate and chamber

Column Chromatography was used to separate 2mL of concentrated spinach extract into three polar classes that were then concentrated. The three concentrated separations of the extract were then analyzed against a standard of original spinach extract using Thin Layer Chromatography and 30% ethyl acetate in Hexanes developing solvent.

Results:

The original spinach extract was a deep green color. During column chromatography, a the first layer of extract eluted (using 9:1 Hexanes/acetone) was a solution that was very light yellow, which after concentration appeared light yellow film and boiling stones were dyed yellow. The second eluted layer (using 1:1 Hexanes/acetone) was a very light green that reduced to a light green concentrate where the boiling stones were dyed light green. The final eluted layer (using methanol as eluent) yielded a light green solution reduced to a green concentrate. Upon spotting the TLC plate, the original spinach extract spot was a very dark green, the layer 1 spot was invisible, the layer 2 spot was a light green, and the layer 3 spot was green.

The Rf values for the standard were calculated for four separate spots:
RfS1 = AS1 / B = 1.4 cm / 5.1 cm = 0.27
RfS2 = AS2 / B = 1.6 cm / 5.1 cm = 0.31
RfS3 = AS3 / B = 2.3 cm / 5.1 cm = 0.45
RfS4 = AS4 / B = 2.7cm / 5.1cm = 0.53
No Rf value could be calculated for layer one, since no spot was visible on the TLC plate. There were two spots for layer two, however, whose Rf values were calculated:
Rf1 = A1 / B = 2.4 cm / 5.1 cm = 0.47
Rf2 = A2 / B = 2.7 cm / 5.1 cm = 0.53
The Rf1 value closely corresponds with the RfS3 value from the sample spot, indicating that these two may be the same compound. The Rf2 value is the same as the RfS4 indicating it is likely these two samples are the same compound.
The third sample had one spot whose Rf value was calculated:
Rf3 = A3 / B = 2.3 cm / 5.1 cm = 0.45.
Rf3 is the same as the RfS3 value, indicating it is likely these two samples are the same compound, which may be the same compound as in spot 2.

Discussion:

The spinach extract was separated into its pigmented components based on their polarity, with the least polar pigment eluting off first with the 9:1 Hexanes/acetone eluent. In spinach, carotenoids are the least polar pigment, since it is composed of pure hydrocarbon chains, and it therefore a non-polar compound (Koster, 2008). Carotenoids are a yellow/orange color, which was reflected in the pigment of our sample 1 concentrate (C1), however concentrate 1 did not create a visible spot on the TLC plate, so it is unclear whether or not carotenoids were present in concentrate 1. However, since concentrate 2 (C2) yielded 2 spots on the TLC plate, it is possible one of these spots was a carotenoid. In particular, spot 2 has the largest Rf value, indicating it was the least polar compound because it had the largest A value, traveling the farthest up the TLC plate with the non-polar developing solvent. It can be hypothesized, therefore, that spot 2 is a carotenoid pigment, lining up exactly with the S4 sample spot that had the largest Rf value. The pigment chlorophyll is also present in spinach extract, and chlorophyll is polar molecule that contains carbon-oxygen, carbon-nitrogen, and nitrogen-magnesium bonds. It is a darker green color, corresponding to the third concentrate (C3) collected from the column after full elution with polar methanol. The carbon-oxygen and carbon-nitrogen bonds of chlorophyll are polar, and the nitrogen-magnesium bond is an extremely polar, nearly ionic bond, making chlorophyll much more polar than carotenoid pigments. This would mean its Rf value is the smallest, since it will travel for the least amount of time with the non-polar developing solvent used on the TLC plates. The smallest Rf value corresponded to spot 3 on our TLC plate from concentrate 3, although this spot was not very different in Rf value from spots 1, and especially close to the value of spot 2, indicating it may not be chlorophyll, since the actual polarities of the molecules would probably make their Rf values much more different. Spinach extract also contains the pigment pheophytin, which is a polar compound consisting of carbon-nitrogen and carbon-oxygen bonds (Pearson, 2013). The pigment presents as a light green color, which was present in the concentrated second sample (C2) that was eluted through the column using 1:1 hexanes and acetone. Concentrate 2 yielded two spots (1 and 2) on the TLC plate. Since it was hypothesized that spot 1 was a carotenoid, it can be hypothesized that spot 2, with the second highest Rf value, contains pheophytins, seeing as it is more polar than carotenoids, but less polar than chlorophyll. It is unclear whether spots 2 and 3 are the same compound, since they are very close in their Rf values, but not identical, and concentrate 3 did present much darker than concentrate 2 (which would indicate some chlorophyll was present in concentrate 3). It could be that the column did not adequately separate the pigments, which would explain why two pigments were present in concentrate 2, none presented in concentrate 1, and the separation of the Rf values was unsatisfactory at best. It must also be noted that the 2mL of spinach extract that was separated using column chromatography was a different portion of the extract than the sample used to spot the TLC plate. This is why the sample has spots with smaller Rf values that probably correspond to chlorophyll, which might not have been present in large quantities in our concentrates.

References:

"Acetone, CHROMASOLV® Plus, for HPLC, ≥99.9% | CH3COCH3 | Sigma-Aldrich." Sigma-Aldrich: Analytical, Biology, Chemistry & Materials Science products and services. | Sigma-Aldrich. N.p., n.d. Web. 13 Oct. 2013. .

"Ethyl acetate, CHROMASOLV® Plus, for HPLC, 99.9% | CH3COOC2H5 | Sigma-Aldrich." Sigma-Aldrich: Analytical, Biology, Chemistry & Materials Science products and services. | Sigma-Aldrich. N.p., n.d. Web. 13 Oct. 2013. .

"Hexane, mixture of isomers, anhydrous, ≥99% | C6H14 | Sigma-Aldrich." Sigma-Aldrich: Analytical, Biology, Chemistry & Materials Science products and services. | Sigma-Aldrich. N.p., n.d. Web. 13 Oct. 2013. .

Koster, Ph.D. , Sandra K. . "Isolation of Chlorophyll and Carotenoid Pigments from Spinach." La Crosse . University of Wisconsin, n.d. Web. 13 Oct. 2013.

References: "Acetone, CHROMASOLV® Plus, for HPLC, ≥99.9% | CH3COCH3 | Sigma-Aldrich." Sigma-Aldrich: Analytical, Biology, Chemistry & Materials Science products and services. | Sigma-Aldrich. N.p., n.d. Web. 13 Oct. 2013. . "Ethyl acetate, CHROMASOLV® Plus, for HPLC, 99.9% | CH3COOC2H5 | Sigma-Aldrich." Sigma-Aldrich: Analytical, Biology, Chemistry & Materials Science products and services. | Sigma-Aldrich. N.p., n.d. Web. 13 Oct. 2013. . "Hexane, mixture of isomers, anhydrous, ≥99% | C6H14 | Sigma-Aldrich." Sigma-Aldrich: Analytical, Biology, Chemistry & Materials Science products and services. | Sigma-Aldrich. N.p., n.d. Web. 13 Oct. 2013. . Koster, Ph.D. , Sandra K. . "Isolation of Chlorophyll and Carotenoid Pigments from Spinach." La Crosse . University of Wisconsin, n.d. Web. 13 Oct. 2013.

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