SPINACH CHROMATOGRAPHY QUESTIONS

1. why is it necessary to evaporate the acetone used to extract the pigments from spinach leaves and then redissolve the mixture in hexane before loading the column?

2. What would happen if you mistakenly began eluting the column with a mixture of 90% acetone 10% hexanes rather than 90% hexanes and 10% acetone?

3. How could you modify this experiment to achieve better separation of chlorophyll a and chlorophyll b?

4.If you were to use a mixture of methylene chlorine and ethyl acetate to elute the column (rather than a mixture of hexanes and acetone) would you begin with a mixture comprised on mostly methylene chloride (CH₂Cl₂) or ethyl acetate (CH₃COOCH₂CH₃). Explain your choice.

lab information:

The objective of the lab was to determine the separation of dyes and spinach pigments through two different chromatographic methods. In this lab, both column chromatography and thin layer chromatography were explored. To conduct column chromatography, a solid adsorbent is first packed into a column. Then, the mixture to be separated is added to the column followed by an eluting solvent (eluent) The time it takes for the components of the mixture to go through the mobile phase is called the retention time. Retention time is explained by polarity. A highly polar compound would be more bound to a polar stationary phase while a highly nonpolar compound would elute down the column in the mobile phase. In order to move a highly polar compound through the stationary phase, a highly polar solvent would be required.

The objective of the lab was to determine the separation of dyes and spinach pigments
through two different chromatographic methods. Chromatography is the process of
separating a chemical mixture into two immiscible phases: a mobile phase and a
stationary phase (Gilbert 175). This allows for the extraction of a pure substance. In this
lab, both column chromatography and thin layer chromatography were explored. The
purpose was to ultimately figure out the relationship between solvent polarity and eluting
power. The first method that was performed in the lab was column chromatography,
which was used for the separation of two dyes. The goal for using column
chromatography was to determine which solvent caused maximum separation of the dye
mixture. The second method that was performed was thin layer chromatography (TLC),
which was used to separate pigments in spinach leaves. The goal for using TLC was to
determine which ratio of acetone and hexane would create the mixture that separated the
spinach pigments the best.

Column chromatography is a type of solid-liquid absorption chromatography, which
means that there is a solid stationary phase and liquid mobile phase (Gilbert 184). To
conduct column chromatography, a solid adsorbent is first packed into a column. Then,
the mixture to be separated is added to the column followed by an eluting solvent (eluent)
(Gilbert 185). When the added solvent/eluent runs through the column, a part of the
mixture is bound to the stationary phase while the other is bound to the mobile phase,
causing a separation in the mixture. The separation causes the part bound to the mobile
phase to elute down the column. The time it takes for the components of the mixture to
go through the mobile phase is called the retention time. Retention time is explained by
polarity. A highly polar compound would be more bound to a polar stationary phase
while a highly nonpolar compound would elute down the column in the mobile phase. In
order to move a highly polar compound through the stationary phase, a highly polar
solvent would be required. Therefore, when a polar solvent is added to the mixture, a less
polar compound would travel down the column quicker. A challenge in column
chromatography is that if the solvent is too polar for the adsorbent and the mixture that
needs to be separated, the mixture could elute too quickly and the separation would be
difficult to measure. In this lab, the adsorbent used was silica gel, but other compounds
such as alumina would be appropriate choices as well. The elutropic series of eluting
power and polarity of solvents are shown below:
The stationary phase serves as an adsorbent through which the mobile phase is passed.
Many compounds with varying functional groups may be used as the stationary phase and several types of interactions can aid in developing the desired separation (i.e., hydrogen bonding, dipole dipole, interactions, electrostatic interactions, Van der Waals forces, size exclusion, affinity, etc.). The following sequence illustrates the general affinity of the functional groups towards a polar stationary phase like silica:

ionic > acids/bases > amides > alcohols > ketones > aldehydes > esters > ethers > halides > unsaturated hydrocarbons > saturated hydrocarbons

Spinach leaves, contain chlorophyll a, chlorophyll b and -carotene as major pigments as well as smaller amounts of other pigments such as xanthophylls. In this experiment you will isolate and separate the spinach pigments using differences in polarity to affect the separation. Since the different components are colored differently, the separation is easily followed visually.

Note that the -carotene, which is a hydrocarbon, is nonpolar. By contrast, both chlorophylls contain several polar C-O and C-N bonds and also a magnesium ion chelated to the nitrogen atoms. The distinctions between the chlorophylls, which are more polar than -carotene is slight: chlorophyll a has a methyl group (Y=CH3) in a position where chlorophyll b has an aldehyde (Y=CHO). This makes chlorophyll b slightly more polar than chlorophyll a. Pheophytins are chlorophyll molecules without the Mg2+ -ion and two nitrogen atoms protonated instead. Xanthophylls contain hydroxyl groups rendering them even more polar than chlorophylls. You will take advantage this polarity difference to separate the mixture using column chromatography.