background [prelab assignment (15 points); due at the beginning of the lab]

brief description on the applications of chromatography

using the concept of IMF, describe the basis of adsorption chromatography; include / define the terms "stationary-" & "mobile-" phase in your response. hint: while the animation describes a normal phase (i.e. polar) stationary phase, where this lab uses a reverse phase stationary phase, the concepts are similar in both types of stationary phases, i.e. both involves IMF

MSDS data on isopropyl alcohol


analzye (i.e. separate) the dyes in Cool-Aid Grape drink mix


Sep-pak C-18 cartridge, a reverse phase chromatography column material

syringe (1 & 10 mL); microplate (to collect eluant (solution coming out of the cartridge)); pipets

Cool-Aid Grape drink mix; water (or 6 drops red food dye + 8 drops blue food dye in 100 mL water)

70% isopropyl alcohol ("ROH");  red, blue, & purple pencils  (supply your own pencils)

methods [delegate work]

use a 10 mL syringe to rinse cartridge with ~5 mL 70 % ROH, followed by ~5 mL water then ~ 10 mL air

use a 1 mL syringe to load ~1 mL Cool-Aid Grape solution onto chromatogrpahy column

to remove the dyes on the chromatography column, using a 10 mL syringe at a rate of ~ 1 drop / second (see below table for composition of the eluent ), collect ~5 drop of the effluent into the microwell plates, and record your observations (use colored pencils) until no changes are seen in the chromatography column or effluent.

experiment # eluent with ~10 mL
70 % ROH
15 % ROH
5% ROH, then 70% ROH

at the end of each experiment, use 10 mL syringe to rinse cartridge with ~5 mL 70 % ROH, followed by ~5 mL water then ~ 10 mL air

repeat with the next experimental condition.

for experiment 3, prepare 15 % ROH by mixing 3.0 mL 70 % ROH + 11 mL water

for experiment 4, prepare 5% ROH by mixing 1.0 mL 70% ROH + 13 mL water


Content of lab report [30 points; no lab report revision]

results   [10 points]

draw / describe the color of the eluant  AND  cartridge in each experimental condition as a function of the fraction number

discussion   [20 points]

rationalize your experimental observations based on the concept of IMF (explicitly identify molecules involved in IMF interaction); organization of your response:

experiment 1: effect of water

  • rationale for none of the dyes to be eluated

experiment 2: effect of 70% ROH

  • rationale for both dyes to be eluated simultaneously

experiment 3: effect of 15% ROH

  • rationale for both dyes to be eluated separately
  • rationale for sequence / order of dye eluation

experiment 4: effect of 5% ROH, then 70% ROH

  • 5% ROH: rationale for only a single dye eluated; rationale for this specific dye eluated
  • 70% ROH: rationale second dye eluated by 70% ROH, but not 5% ROH
hints:  hydrophobic attraction & reverse phase chromatography (read section on polarity) & chemical structures of various chemicals in this lab (shown below):

C!8 cartridge

(source:  stationary phase's surface)

isoproply alcohol

(source:  isopropyl alcohol - mobile phase)


blue dye

(source:  blue dye)

red dye

(& red dye)