Chromatography

 

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

This laboratory activity is based on investigation 5 in your AP chemistry lab manual; thus, it provides some relevant background for the following prelab questions and this laboratory activity. While Investigation 5 refers to paper chromatography, this lab will use column chromatography; the basis / rationale of both chromatography techniques is based on an understanding of intermolecular forces.

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

materials

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

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

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

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

methods

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 (or food coloring dye mixture) solution onto cartridge

to remove the dyes from the chromatography colume, use a 10 mL syringe containing the eluent at a rate of ~ 1 drop / second, collect ~5 drop effluent into the microwell plate, and record your observations (e.g. use colored pencils) until no changes are seen in the cartridge or effluent.

at the end of each experiment, use a 10 mL syringe to rinse the cartridge with ~5 mL 70 % ROH, followed by ~5 mL water, then ~ 10 mL air; reload cartridge with Kool-Aid, then elute with the next experimental condition.

composition of the isocratic & psudeo gradient eluation solutions - YOU have to "discover" it - using your "critical thinking skills" & knowledge from the prelab assignment.

Content of lab report   [40 points]

methods & results  [10 points]

describe the experimental conditions (i.e. alcohol concentration) for both experimental conditions (i.e. isocratic- & psuedo-gradient eluation), that successfully separated the blue and red dyes in Grape Kool Aid

describe your observations, include both colors of the effluent 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):

C!8 cartridge

(source:  stationary phase's surface)

isoproply alcohol

(source:  isopropyl alcohol - mobile phase)

 

blue dye

(source:  blue dye)

red dye

(source:  red dye)

this question satisfies one of the NGSS science & engineering practices.

supplemental questions: based on questions on pp. 46 - 47 in the AP chemistry lab manual in regards to Rf  [20 points]

1. what is Rf ?

2. what would be the relative value of Rf for the red and blue dye (above) using the same material in the stationary phase and the same composition of isoproply alcohol in the isocratic eluation in this lab in a paper chromatography lab ? justify / rationalize your response.

3. what would be the relative value of Rf for the red and blue dye (above) using the same material in the stationary phase and the same composition of isoproply alcohol in the first phase of the psudeo-gradient eluation in this lab ? also, compare these Rf values to the situation in question 2. justify / rationalize your response.

4. what would be the relative value of Rf for the red and blue dye (above) using the same material in the stationary phase and the same composition of isoproply alcohol in the second phase of the psudeo-gradient eluation in this lab ? also, compare these Rf values to the situation in question 2. justify / rationalize your response.


additional background information

@ ucdavis chem wiki on chromatography, e.g. I & II

composition of the mobile phase, e.g.  I & II

chromatography simulation   [link for guidance in the use of the simulation]

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

TLC (thin layer chromatography) video; similar to paper- & column- chromatography

isocratic versus gradient eluation