Chromatography
background [prelab assignment (15 points); due
at the beginning of the lab]
basis of adsorption chromatography; include / define the terms: (i) reverse phase chromatography, (ii) stationary phase, and (iii) mobile phase in your response
describe isocratic versus gradient eluation
MSDS data on isopropyl alcohol
purpose
to separate the blue and red dyes in Cool-Aid Grape drink mix by developing 2 experimental protocols
- isocratic eluation: use a single concentration of alcohol (i.e. use a single diluted alcohol solution to elute both dyes separately)
- psudeo-gradient eluation: use 2 different concentration of alcohol, i.e. use a low concentration of alcohol to elute a single dye followed by a higher concentration of alcohol to elute the other dye
materials
Sep-pak C-18 cartridge ("cartridge")
syringe (1 & 10 mL); microplate (to collect eluant (sample coming out of the cartridge)); pipets
Cool-Aid Grape drink mix
isopropyl alcohol ("ROH"); water; red, blue, & purple pencils (supply your own colored pencils)
methods
rinse cartridge with ~5 mL 70 % ROH, followed by ~5 mL water & ~ 5 mL air
load ~1 mL Cool-Aid Grape solution onto cartridge
elute (i.e. apply ~ 10 mL ROH (or water) using 10 mL syringe) onto cartridge at a rate
of ~ 1 drop / second), collect ~5 drop eluants and record your observations (e.g. use colored pencils) until no changes are seen in the cartridge or eluant.
at the end of each experiment, rinse cartridge with ~5 mL 70 % ROH, followed
by ~5 mL water & ~5 mL air; reload cartridge with Kool-Aid, then eluate with the next experimental condition.
specific composition of eluation solution - YOU have to "discover" it - using your "critical thinking skills" & knowledge from the prelab assignment, i.e. you'll be "lost" without adequate prior knowledge
Content of lab report [20 points; no lab report revision]
methods & results [10 points]
describe the experimental conditions (i.e. alcohol concentration) for both experimental conditions, to separate the blue and red dyes in Grape Kool Aid
describe your observations, include both colors of the eluant and cartridge as a function of fraction #.
discussion [10 points]
based on concepts involving IMF, rationalize the basis of your successful separation of the 2 dyes using both methods. hints: hydrophobic interaction, reverse phase chromatography (read section on polarity), & chemical structures of various chemicals in this lab (shown below):
