TLC and Bioautography - PowerPoint PPT Presentation

TLC and Bioautography

Determining which compound in a plant extract is bioactive

• Microbial Assay has shown bioactivity
• Next Step?
• Separate the compounds in the extract.
• Determine which compound in the extract is the bioactive one.

• TLC = Thin Layer Chromatography
• Consists of 2 phases:
• Stationary Phase (remains immobile)
• Mobile Phase (flows across the stationary phase)

• Compounds in the extract have different degrees of attraction

to the 2 phases

• Compounds are initially adsorbed on to the stationary phase
• Adsorbed = “stuck to”

• Because the extract disc contained a large amount extract, you don’t know what concentration of extract will be bioactive
• You will test 3 different concentrations of your extract– determined by how many dots of extract you put on

• Separation is achieved based on the relative affinity (how attracted it is) a compound has for the mobile phase as it flows across the stationary phase
• Stationary phase = silica gel (SiOH groups – polar)

• Highly polar molecules adsorb more to the SiOH groups  move SLOWER
• Less polar molecules adsorb less to the silica gel  move FASTER up TLC plate

• Some of the compounds will be visible already
• To see other compounds you will use a UV light  mark where the compounds are

• Bioautography allows you to test the separated compounds on E. coli and yeast
• Compounds on the TLC plates are transferred to the agar plates to see if they can inhibit growth.

• If a particular compound is bioactive then after 24 hrs of growth there won’t be growth on the agar where it was transferred to

• False negative is possible
• False negative = seems like a negative result, but it may actually be bioactive
• Why might you see a false negative in bioautography?
• Lower concentrations might not be powerful enough to show bioactivity
• It might be a combination of compounds that contributed to the bioactivity