Question 2: Compare the separation of spinach pigments on your \'\'best\'\' and

Question

Question 2: Compare the separation of spinach pigments on your ''best'' and ''worst'' plates. To do so, refer to the composition of the solvent systems used in each to explain the difference in the number of bands (and thus individual pigments)detected or the distance specific bands moved. Hint: The issue is how the polarity of specific functional groups in the separated pigments determine their tendency to move in the two solvents (3.0 pts) Question 3: A team of four students isolated the components of a mixture and then verified the purity and identity of each component by comparing its movement on a TLC plate with that of reference samples. Each student developed the plate with a solvent he chose and located the sample spots with a UV light to determine the Rf value of each. Student A found one spot in each lane, each with Rf value of ~ 0. Student B also found one spot in each lane with an Rf value of 0.55. Both students Concluded that their isolated component was identical to the reference tested. Is that a valid conclusion? To answer the question, address the following: Is there evidence that the partitioning between mobile and stationary phases necessary to separate species on TLC took place in each case? Describe what you know about the samples and the solvent used by student A? Which student has better reason to think the conclusion about his analysis is valid? Why? (2.0 pts)

Explanation / Answer

Question #2 is the laboratory experiment. Spinach extract can be spotted on TLC plates and plates can be developed in different solvent systems (different composition of constituent solvents). Upon observing all TLC plates, the one which contains well separated band is best plate i.e. solvent system composition is good. The one which does not show well separated band is not good. Such observations are due to difference in polarity of the solvent system.

Spinach extract spotted on TLC plate gets partitioned between stationary phase (TLC surface) and mobile phase (solvent system). Different constituents of extract have different partition coefficient (different ability to get partitioned between stationary phase and mobile phase), hence in the appropriate solvent system, the constituents of extract are observed as different bands after developing TLC plate. The difference in partition coefficient of constituents of mixture is mainly due to difference in the functional groups present in their molecules.

Question #3

Answer: Student A could locate the sample and reference spot at Rf = 0, i.e. in the solvent system chosen by student A, the sample and reference compound did not partitioned in to the mobile phase. Hence, both the spots did not move up. In order to check purity with TLC it is important to select a solvent system such that given sample gets partitioned into solvent system and mobile phase and Rf of spots should be in between 0 to 1. Thus, conclusion of student A is not valid.

In case of student B, the sample and reference spots were at Rf=0.55. This shows that both sample and reference are same as they have same ability to get partitioned between mobile and stationary phase. The solvent system chosen by student B is of proper polarity.

Thus, conclusion of student B is valid.

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