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TLC and Bioautography. Determining which compound in a plant extract is bioactive. Bioactivity detected– now what?. Microbial Assay has shown bioactivity Next Step? Separate the compounds in the extract. Determine which compound in the extract is the bioactive one.

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tlc and bioautography

TLC and Bioautography

Determining which compound in a plant extract is bioactive

bioactivity detected now what
Bioactivity detected– now what?
  • Microbial Assay has shown bioactivity
  • Next Step?
    • Separate the compounds in the extract.
    • Determine which compound in the extract is the bioactive one.
tlc separation of compounds
TLC: Separation of Compounds
  • TLC = Thin Layer Chromatography
  • Consists of 2 phases:
    • Stationary Phase (remains immobile)
    • Mobile Phase (flows across the stationary phase)
how tlc works
How TLC works
  • 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”
concentrations of extracts
Concentrations of Extracts
  • 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
Separation
  • 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)
separation cont
Separation (cont.)
  • 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
viewing the separated compounds
Viewing the Separated Compounds
  • Some of the compounds will be visible already
  • To see other compounds you will use a UV light  mark where the compounds are
bioautography
Bioautography
  • 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.
bioautography1
Bioautography
  • 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 negatives
False Negatives
  • 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