6. Perform the extraction three more times to maximize the level
of crude compounds.
7. Dry the extract in a sterile LAF and store at À20
C until
further use.
5.2 Preparation of
Stock Solution
The lyophilized ethyl acetate extract is used to prepare stock using
distilled water (1mg/ml) and filtered through 0.2 m filter to avoid
contamination.
5.3 Purification and
Structural
Identification of the
Compound
5.3.1 Column
Chromatography
1. Subject crude extract of Xenorhabdus and Photorhabdus to silica
gel column chromatography to isolate into its respective component fractions.
2. Use silica gel as stationary phase and use different ratios of
solvent (n-hexane, chloroform, methanol, and water) in
increasing polarities as mobile phase.
3. Take a glass column (80 cm in length and 5 cm in diameter),
rinse with solvent and dry it absolutely before use.
4. Cover the column using a cotton plug.
5. Pack the column with activated silica gel (70–230 mesh size)
slurry with continuous tapping.
6. Dissolve the crude extract (10 g) in little amount of solvent and
mix with silica gel (1:2 w/w), and load onto the column.
7. Elute the extract with increasing polarity of solvents. Elute and
collect 10 ml fractions.
8. Subject the fractions to TLC. Spot the aliquots of fractions on
baseline of precoated silica gel TLC sheets and allow for few
minutes until dry.
9. Place the TLC plates in TLC chamber saturated with solvent
and then keep the plates in iodine chamber to visualize the
spots.
10. Pool the fractions with distinct spots and save Rf value of
solvent system and condense using rotary evaporator.
5.3.2 High-Performance
Liquid Chromatography
(HPLC)
1. HPLC is used to establish the purity of all the pooled fractions
with single spot in TLC analysis.
2. Inject 5 μl of sample using Hamilton syringe.
3. Acetonitrile: 0.05% phosphoric acid in water mixture is used as
mobile phase, which is filtered using 0.2 μ membrane filter with
flow rate of 1 ml/min.
5.3.3 Fourier Transform
Infrared (FT-IR)
Spectroscopy
1. The functional groups of isolated biocompounds can be analyzed using Bruker alpha FT-IR instrument.
138
Laboratory Techniques for Symbiotic Bacteria
of crude compounds.
7. Dry the extract in a sterile LAF and store at À20
C until
further use.
5.2 Preparation of
Stock Solution
The lyophilized ethyl acetate extract is used to prepare stock using
distilled water (1mg/ml) and filtered through 0.2 m filter to avoid
contamination.
5.3 Purification and
Structural
Identification of the
Compound
5.3.1 Column
Chromatography
1. Subject crude extract of Xenorhabdus and Photorhabdus to silica
gel column chromatography to isolate into its respective component fractions.
2. Use silica gel as stationary phase and use different ratios of
solvent (n-hexane, chloroform, methanol, and water) in
increasing polarities as mobile phase.
3. Take a glass column (80 cm in length and 5 cm in diameter),
rinse with solvent and dry it absolutely before use.
4. Cover the column using a cotton plug.
5. Pack the column with activated silica gel (70–230 mesh size)
slurry with continuous tapping.
6. Dissolve the crude extract (10 g) in little amount of solvent and
mix with silica gel (1:2 w/w), and load onto the column.
7. Elute the extract with increasing polarity of solvents. Elute and
collect 10 ml fractions.
8. Subject the fractions to TLC. Spot the aliquots of fractions on
baseline of precoated silica gel TLC sheets and allow for few
minutes until dry.
9. Place the TLC plates in TLC chamber saturated with solvent
and then keep the plates in iodine chamber to visualize the
spots.
10. Pool the fractions with distinct spots and save Rf value of
solvent system and condense using rotary evaporator.
5.3.2 High-Performance
Liquid Chromatography
(HPLC)
1. HPLC is used to establish the purity of all the pooled fractions
with single spot in TLC analysis.
2. Inject 5 μl of sample using Hamilton syringe.
3. Acetonitrile: 0.05% phosphoric acid in water mixture is used as
mobile phase, which is filtered using 0.2 μ membrane filter with
flow rate of 1 ml/min.
5.3.3 Fourier Transform
Infrared (FT-IR)
Spectroscopy
1. The functional groups of isolated biocompounds can be analyzed using Bruker alpha FT-IR instrument.
138
Laboratory Techniques for Symbiotic Bacteria