College Chemistry : Chromatography
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Example Questions
Example Question #1 : Chromatography
A scientist wants to separate and purify a small, positively charged protein. Which of the following techniques might be employed to accomplish this task?
None of these
Reverse phase chromatography
Thin layer chromatography
Normal phase chromatography
Size exclusion chromatography and cation exchange chromatography
Size exclusion chromatography and cation exchange chromatography
Size exclusion chromatography is a good technique for separating proteins based on size due to tiny pores in the beads in the column (the smaller proteins will elute out last). Cation exchange chromatography is a good technique for separating proteins based on charge due to negatively charged beads in the column (the positively charged molecules will elute out last). Normal and reverse phase chromatography are methods of separation based on a molecule's polarity; in normal phase chromatography, a nonpolar mobile phase is employed so that polar, hydrophilic molecules elute last. The opposite is true for reverse phase chromatography - a polar, aqueous mobile phase is used and any nonpolar molecules tend to adsorb to the hydrophobic beads, causing them to elute out last. Thin layer chromatography is not a good method of purifying substances, rather, it is a better indicator of reaction progress via elucidation of the retardation factor, 
Example Question #1 : Laboratory Techniques And Analysis
In reverse phase chromatography, the more __________ molecules will be eluted __________.
hydrophobic . . . first
None of these answers
hydrophobic . . . last
More than one of these
hydrophilic . . . last
hydrophobic . . . last
In reverse phase chromatography, the more hydrophobic molecules will be eluted last (the more hydrophilic molecules will be eluted first). This is due to the mobile phase being polar, and thus the nonpolar/hydrophobic molecules adsorbing to the stationary phase. The opposite is true in normal phase chromatography - this technique employs a nonpolar mobile phase and a polar stationary phase. The polar stationary phase will attract polar molecules, and thus the nonpolar molecules will be eluted first along with the nonpolar/hydrophobic mobile phase.
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