Oh and here is the extra question from last week High-performance Liquid Chromat
ID: 1029743 • Letter: O
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Oh and here is the extra question from last week High-performance Liquid Chromatography (HPLC) is a common technique used to separate chemicals in labs these days. In HPLC, like in paper chromatography, there is a mobile phase and a stationary phase. The constituents of the chemicals either stay behind on the stationary phase or move with the mobile phase and elute (dissolve in the solvent and move with it) out. The chemicals are identified by their elution times. So a chemical which moves with the mobile phase has a lesser elution time than the one that stays behind in the mobile phase. Let us say that you are given a solution of spirulina/spinach (whichever you did) to separate using an HPLC column with silica as the mobile phase and tetrahydrofuran (THF) as the stationary phase. What would the order of your elution times be (fastest to slowest)? Why do you observe what you observe?Explanation / Answer
Spinach mixture would be used to separate a mixture of pigments such as chlorophyll (chlorophyll a and b) and carotenoids majorly -carotene (plant derivatives known as terpens). The solid phase used in HPLC is silica and liquid phase is tetrahydrofuran (THF). Components of mixtures are separated in chromatography on the bases of their interactions with stationary or mobile phase. All the pigments contain polar groups but both chlorophyll a and b are much more polar then -carotene.
Chlorophyll a and b have very slight differences as chlorophyll a contains methyl group (-CH3) at a position whereas chlorophyll b contains an aldehyde (-CHO) group. That’s why chlorophyll a is slightly less polar than chlorophyll b.
Differences in polarity will be useful in separation of these pigments as polarity ranges from high to low (chlorophyll b > chlorophyll a > -carotene). Separation of different pigments is based on their interactions with stationary phase (silica; hydrophilic) and mobile phase (tetrahydrofuran; hydrophobic). Hydrophilic molecules shows more interaction with stationary phase again order of interaction strength will be based on their more of less hydrophilic nature. More hydrophilic means more interaction and vice versa.
The molecule with less hydrophilic nature will move faster along with mobile phase as it has less interaction with stationary phase and vice versa. Therefore as mentioned earlier chlorophyll b with more hydrophilic nature will be showing more interaction with silica and will be separated faster followed by separation of chlorophyll a and -carotene.
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