Introduction
to Column Chromatography
Objective: To separate the pigmented compounds contained within a beet plant using column chromatography.
Credits: Shoestring Biotechnology Project from NABT and occc program students, class of 2005.
Chromatography is a process by which individual components of a mixture can be separated. Chromatography has a mobile phase (liquid) and stationary phase (solid). The mixture components interact differentially with the mobile and stationary phases to effect the separation of components.
Materials:
Mobile phases:
· Wash Buffer #3 No Salt (deionized water only)
Procedure:
1. Weigh 1g of beet. Be sure to get some stem (red) and some leaf (green) for a variety of colors (half and half).
2. Place weighed sample into the mortar and add enough equilibration buffer to cover the beet. Grind with Pestle as much as possible getting the smallest pieces of plant you can.
3. Place ground liquefied plant material (beet extract) in a 2 mL centrifuge tube.
4. Centrifuge at 500-1000 rpm for 5 minutes to form pellet of plant solids plus liquid on top (supernatant).
5. Transfer the supernatant (the top liquid, solid plant material should be stuck at bottom) into a fresh 2 mL tube. Discard tube with pellet.
6. Set up a series of clean empty microcentrifuge tubes in a rack and number them in the order they will be used.
7. Position the column on a stand above the test tube rack.
8. Close the column stopcock, add 4ml of equilibrium buffer. Open the stopcock as you add ~ 2 mL of your Phenyl Sepharose or Sand slurry, depending on your assignment. The bed height should be about 3-4 cm high. DO NOT let your slurry/sand run dry. If you run out of Equilibration Buffer, just add more to keep slurry/sand wet.
9. Continue to run equilibration buffer through the column until the solid material is completely level and settled down into compact bed. Discard outflow.
10. Once the meniscus of the equilibration buffer has reached the top of the bed, close the stopcock and add 500 mL of beet extract. Drizzle this extract gently and slowly down the side of the column so that you do not disturb the bed.
11. Open stopcock and allow the sample to flow into the bed. When the top meniscus of the sample touches the top of the bed, close the stopcock. Discard outflow. The beet pigmented compounds bind to column in presence of equilibrium buffer.
12. Gently Pipette 3 mL
of Wash Buffer #1 into column (BE SURE NOT TO DISTURB THE BED!) and drain until
the meniscus is just above the column bed.
The high salt concentration of
wash buffer #1 causes pigments with lots of exposed hydrophilic groups to come
off column in this wash. Observe the
column for protein (color) elution or movement down the column. When the first
color begins to leave the column, begin the collection procedure: collect 10-drop samples in 1.5 mL plastic microcentrifuge tubes. When meniscus for wash buffer #1 reaches top
of column bed, close stopcock.
13. Gently pipette 3 mL of Wash Buffer #2 onto the column; again, take care not to disturb the bed. The lower salt concentration of washing buffer #2 will allow pigments with fewer hydrophobic groups to come off column during this wash.
14. Be sure to note which collections are taken from which buffers.
15. When Wash Buffer #2 has drained to the top of
the bed, stop flow. Then pipette 5 mL of Wash Buffer #3, restart flow and continue collecting
samples.
Clean up. If you have sand slurry just dump the sand from the column into trash. If using Phenyl Sepharose, which is very expensive, “wash” the column with 5mL of 8mM NaOH and then collect and save the phenyl sepharose slurry.
Observations and Results:
The column bed was composed of:
Observations from eluted liquid from wash buffer #1:
Observations from eluted liquid from wash buffer #2:
Observations from eluted liquid from wash buffer #3:
Conclusions: (relate the colored materials that came off with each wash to the relative hydrophobicity or hydrophilicity of the material compared to wash buffers)
Check your understanding:
4. Define hydrophilic:
6. Define extract: