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Examination of the Use of Column Chromatography for Spinach Purification

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Absorbance Spectra of Spinach Fractions Purified by Column Chromatography


Column chromatography is a particular branch of chemical purification, by which the components of a substance are separated from each other based on each component’s chemical interactions with certain filters – absorbents and solvents. The analyzed species, often a solid ground into a particulate solute, is dissolved in a solvent and decanted through a column of solid absorbent. If one compound in this species has significantly different polarity from that of another component, the attraction of each component to the added solvent and the absorbent of certain polarity will cause one component to travel down the column faster than the other. The more a component’s polarity matches that of the column, the more its attraction to the column will slow it down.

In this experiment, the two compounds of interest, extracted from spinach by thorough centrifugation, are chlorophyll-A and β-carotene. Because chlorophyll-A is comprised of a nonpolar hydrocarbon tail attached to a ring containing polar C-N and C-O bonds, it is a more polar compound than β-carotene, which is highly nonpolar as a total hydrocarbon (Waghulde, n.d.). Therefore, we hypothesize that when the nonpolar solvent, petroleum ether, is decanted into a column with spinach extract and a polar absorbent, silica gel, β-carotene will exit the column first because β-carotene remains attracted to the flowing solvent and is repelled from the polar absorbent. Chlorophyll-A will resist extraction and remain bound to the solidified silica gel, until the partially polar solvent, acetone, is added to the column; the attraction of chlorophyll-A to this descending solvent will overcome its attraction to the absorbent, and one can collect this fraction separately from that of β-carotene by timing the addition of solvents.

From purified fractions, chemists can derive information about specific compounds in the analyzed species, such as absorbance spectra. If the compounds purified by the column absorb radiation in the visible range of the electromagnetic spectrum, as chlorophyll-A and β-carotene do, these fractions will have distinct colors and their purity can be tested by a UV-vis spectrophoto-meter. This apparatus generates a curve of the fraction’s absorbance of light at each wavelength in a given range, called an absorbance spectrum, which can be compared with the compound’s literature spectrum. These tendencies of compounds to absorb different wavelengths of light to varying degrees result from conjugated π bonds (Soderberg, 2016). Alternation of double and single carbon bonds, as in β-carotene and chlorophyll-A, results in delocalized π bonds (Soderberg, 2016). Since the energy required for an electron to transition from the bonding orbital (highest occupied molecular orbital, HOMO) to the antibonding orbital (lowest unoccupied molecular orbital, LUMO) is so low in this conjugated system, the wavelengths of absorbed light for these compounds are relatively high and visible, resulting in reflection of non-absorbed visible light wavelengths (Soderberg, 2016).


In this experiment, my lab partners and I followed the procedure outlined in Section 14, pp. 165-173, of the General Chemistry Lab Manual by Dr. Meishan Zhao.


As expected, addition of petroleum ether as the first solvent caused a yellow β-carotene fraction to travel down the column first, and it was only after subsequent addition of the acetone solvent that a green chlorophyll fraction followed. The wavelengths of maximum absorbance for the chlorophyll fraction strongly matched those of a literature chlorophyll-A spectrum, as did those of the β-carotene for a literature β-carotene spectrum (Figs. 1, 2, and 3). However, the relative absorbance values of the two peaks of the chlorophyll fraction spectrum had a ratio more closely resembling that of chlorophyll-B than chlorophyll-A, while for β-carotene this ratio was comparable (Figs. 1, 2, and 3). Absolute absorbance values for the chlorophyll fraction deviated highly from those of the literature spectrum, and less severely so for the β-carotene fraction (Figs. 1, 2, and 3).

Discussion Questions

If acetone had been used as the first elution solvent, the chlorophyll-A fraction likely would descend first due to attraction to this polar solvent. This reversal of order would risk the collection of overlapping fractions, however, because the silica gel slurry was prepared with petroleum ether, and the spinach extract was made with an 80:20 ratio mixture of petroleum ether and acetone. Therefore, the β-carotene fraction could co-elute with the chlorophyll fraction due to attraction to the minor amount of petroleum ether solvent already present, and by the time the tail of the chlorophyll fraction reached the bottom of the pipette, the head of the β-carotene fraction could be mixed with it. Therefore, we would be unable to collect pure fractions of the separate compounds.

There is an upper limit to the number of solvent molecules with which β-carotene or chlorophyll-A can associate in opposition to the absorbent, meaning that excessively fast solvent flow could leave insufficient time for the fraction to equilibriate – this would result in non-distinct fractional bands, which could overlap and result in impure fractions.

In the phase of the experiment in which petroleum ether was used as a solvent, cracks in the silica gel absorbent would reduce the ability of this absorbent layer to serve as a chemical barrier to the chlorophyll fraction. The mechanical force of gravity would permit the chlorophyll fraction to descend the column faster than expected, even before addition of extra acetone (because the initial spinach extract was prepared with some acetone solvent), if there were spaces in the absorbent layer. Though the β-carotene fraction would still likely descend faster due to attraction to petroleum ether, the fractions could end up overlapping at the intersection of the top of the β-carotene fraction and bottom of the chlorophyll fraction – again, the purity of the fractions would be compromised.

Water is a polar solvent, by which chlorophyll could likely elute sooner than desired for the column separation. A predominantly nonpolar solvent, petroleum ether, is required first in this experiment, because for reasons described above, an initial polar solvent would have potential for fraction overlap as the β-carotene fraction descended through the trace petroleum ether in the initial silica gel.

Decreasing the length of the column would hinder chromatographic separation, because this would increase the risk of fractions overlapping by the time of column exit, and prevent collection of pure fractions.

Increasing column diameter would decrease the solvent pressure, in turn reducing the precise resolution of the separated fractions (which could permit overlapping of fractions as well).

For the same reason that decreasing column length would increase the chance of fraction overlap, increasing length minimizes this risk.

By decreasing the diameter of a column, sharper fractions would be result, in the inverse of the phenomenon described in (b).

The wavelength of maximum absorbance is 450 nm for β-carotene, as noted in Fig. 2. β-carotene has 22 sp2-hybridized carbon atoms, meaning there are 22 π orbitals. The initial energy level ni is the highest energy level filled given that each π orbital represents an energy level in this model, thus ni = 22, and when an electron is excited from the absorption of the wavelength noted above, nf = 23.

This value for the length of the conjugated π-bond network is very close to the known value of L = 2.6 x 10-9 m, suggesting both the accuracy of our data (and purity of the β-carotene fraction) and the applicability of the particle-in-a-box model (Zhao, 2015).


Chromatography columns and UV-vis spectrophotometry produced spectra with wavelengths of maximum absorbance largely consistent with those of spectra from the chemical literature, and with the known length of the conjugated π-bond chain of β-carotene. These columns showed the behavior of elution expected from the polarity of the fractions, solvents, and absorbent. Absolute absorbance values did not match these literature spectra, and the absorbance ratio of the two maxima of the chlorophyll fraction spectrum more closely aligned with chlorophyll-B than chlorophyll-A.

The absorbance values from our empirical spectra most likely differed from those of the literature spectra because of different concentrations of analyzed fractions – this does not reflect on the purity of the sample, as only wavelengths of maximum absorbance are characteristic of compounds. It is not completely unexpected that the fraction eluted with the polar acetone would not be pure chlorophyll-A, because chlorophyll-B (which spinach also contains) is also a largely polar compound of very similar structure to chlorophyll-A. Chlorophyll-A still most likely dominated this fraction, because of its green color (contrasted with the yellow color of chlorophyll-B) and wavelengths of maximum absorbance. The particular accuracy of the β-carotene fraction’s spectrum could have resulted from the absence of such a comparable compound in spinach.

From the highly accurate data acquired by these chromatographic methods, we can see that the relative polarities of compounds can result in stark differences in interactions with solvents and absorbents. These particular interactions have practical significance in that highly pure substances can be extracted from mixtures, the principle by which processes such as the biochemical isolation of medical compounds are possible (Zhao, 2015). The particle-in-a-box model applied to these chromatographic data also shows the versatility of quantum mechanics, as well as the molecular orbital theory.

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