Liquid Chromatography

2 September, 2013
Introduction
In this lab, the goal is to separate mixtures by Liquid Chromatography. Liquid Chromatography is using the properties of a stationary phase and the moving phase to separate the mixtures. The component will pass through a system, and the more attracted for the mobile phase will make it move faster through the system. On the other hand, if it is more attracted to the stationary phase, it will move at a slower speed. Stationary phase is usually a solid, and the mobile phase is usually a gas or a liquid. The different speed will allow the components to separate into bands, and exiting the system as different division. This lab will be conducted by injecting Kool-Aid into a cartridge. Then liquid chromatography will be used to attempt to separate Kool-Aid into two components.

Formula
Wave = bandwidth (end of band - start of band)
VM = 0.49 mL
Center of Band
VRave = Vstart + ½ Wave
Capacity Factor k1 = (VRave - VM) / VM
Separation Factor α = k1Blue / k1Red
Resolution
R = (VRave(Blue) - VRave(Red)) / [ 0.5 (WBlue + WRed) ]
Data/Results
Part 1. Isocratic Separation Red Dye Blue Dye | Run #1 | Run #2 | Run #3 | Run #1 | Run #2 | Run #3 | Start of Band (mL) | 1.0 | 1.0 | 1.0 | 1.9 | 1.5 | 1.4 | End of Band (mL) | 1.9 | 1.5 | 1.4 | 4 | 3.7 | 3.5 | W (mL) | .9 | .5 | .4 | 1.6 | 1.7 | 1.6 | VRave (mL) | 1.45 | 1.25 | 1.2 | 2.37 | 2.35 | 2.2 | k1 | 1.96 | 2.27 | 1.45 | 4.51 | 3.8 | 3.49 |

Calculated Values | Run #1 | Run #2 | Run #3 | α | 2.55 | 2.55 | 2.04 | R | 3.57 | 1.78 | 1.66 |

Part 2. Step Gradient Separation Beaker | Eluent | Observations - Eluted Fractions | 1 | H2O | clear color liquid | 2 | 5% isopropyl alcohol | reddish color liquid | 3 | 28% isopropyl alcohol | blueish color liquid | 4 | 70% isopropyl alcohol | clear color liquid | * It is hard to distinguishing between the color of the blue dye and the red dye, so you must record as the experiment is taking place. * 1 mL of Kool-Aid is not enough, so you can add 2 mL of Kool-Aid instead of 1 mL. * Remember to slowly inject the components into the cartridge, it might mess up the solution if you inject it too soon. * After the end of the blue dye bandwidth, everything else is clear.
Inquiry
Material
Red Leaf Extract, 3 mL 5% isopropyl alcohol, 15 mL
70% isopropyl alcohol, 30 mL 28% isopropyl alcohol, 15 mL distilled water, 50 mL
50 mL Beakers, 4 Syringe 3 mL with male Luer tip
10 mL Graduated cylinder Syringe 10 mL with male Luer tip
Sep-Pak C18 cartridge
Procedures
Pretreat cartridge
1. Fill 10 mL syringe with 10 mL 70% isopropyl alcohol
2. Inject it through the cartridge over the duration of 1 minute, using the the 10 mL graduated cylinder to keep track of your pace.
3. Repeat steps 1-2 again using distilled water
Into the Lab
1. Fill the 3 mL syringe with 2 mL of red leaf dye
2. Inject it into the cartridge slowly
3. Using the 10 mL syringe, pump 5 mL of distilled water into the cartridge and collect the effluent in Beaker #1
4. Inject 10 mL of 5% isopropyl alcohol into the cartridge and collect it in Beaker #2
5. Use 10 mL of 28% isopropyl alcohol to the elute and collect in Beaker #3
6. Use 10 mL of 70% isopropyl alcohol to elute, and collect in Beaker #4
7. Record your observations of the colors using a Data Table

Beaker | Eluent | Observations - Eluted Fractions | 1 | 5 mL of H2O | reddish brown color liquid | 2 | 10mL of 5% isopropyl alcohol | yellow color liquid | 3 | 10mL of 28% isopropyl alcohol | orangish brown color liquid | 4 | 10mL of 70% isopropyl alcohol | brown color liquid |

* When injecting the last 2 mL of water, the effluent was clear. * There is murky remains inside the cartridge when injecting the isopropyl alcohols * The effluent of the injection of the red leaf dye have a color. * After injecting all of the solution into the cartridge, you have to wait awhile,