Chloro..who?? Part two.

So as I promised, here is the exciting conclusion to the experiment that I have been working on, so I’ll be the only one in the dark!!

I was instructed my Matt to be very careful with this next step as it involved and known noxious chemical that has been made famous by many movie villains…Chloroform, Reagent Plus >99.8%.  This stuff is nasty, you know the chemically soaked rag that the bad guy uses to knock out his victim, makes my experiment that much more interesting!!

  I of course turned the fume hood turned on high and stood with the glass partition separating my nose from the fumes, as I carefully measured out a mixture of Chloroform, Petroleum Ether and Isopropanol Alcohol and put them all into a developing chamber.  I then placed my TLC plates (two plates in one) into the developing chamber, set my timer for 45 minutes and once again, turned off the lights while the mobile phase takes place.  The purpose of this phase is for components of the mixture to move up the TLC places at different rates as it completes the development of the Chromatography or the visible bands of Chlorophyll A (bright green in color), Chlorophyll B (olive green in color) and Xanthropyle (yellow in color) that migrate across the silica gel that covers the TLC plates.  Once the mobile phase is over, the plates are removed from the development chamber and dried in order to scrape each of the bands off and examine them for the actual content or concentration of chlorophyll in each plant.

Calculating the concentration of chlorophyll is a pains taking process of finding the absorbency spectra of TLC purified pigment bands.  The colored silica gel is scraped off and mixed with alcohol in a centrifuge tube, placed on a vortex, and spun down once again.  The resulting liquid is placed in a cuvette and is examined in a Spectrophotometer which is a device to measure light intensity at different wavelengths.  It produces light with a light source, and after the light passes through a subject, the light is diffracted into a spectrum which is detected by a sensor and interpreted into results we can use. 

Next week I'll go into my results and give you an exciting update on my Mini-city microbes from the hay infusion lab.  Have a great week everyone!