Chromatography

 

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

purpose

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

materials

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
1
water
2
70 % ROH
3
15 % ROH
4
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)