Rolling – A New Application Technique For Luminescent Bacteria on High Performance Thin Layer Chromatography (HPTLC) Plates
1. Rolling – A New Application Technique For Luminescent
Bacteria on High Performance Thin Layer Chromatography
(HPTLC) Plates
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2. The Vibrio fischeri Bacterium
• The marine bacterium Vibrio fischeri is a Gram-negative, facultative anaerobic, comma-
shaped, rod shaped with flagella bacteria(figure1).
• The special characteristic of Vibrio fischeri is its ability to luminize and emit blue-green
light at approximately 480nm.(Figure 2 )
• Bioluminescence, the emission of light by a living organism
• For the generation of light, Vibrio fischeri employs a luciferin-luciferase system.
• degree of toxicity is inversely proportional to the luminescence.
Baumgartner Vera , Hohl Christopher, Journal of Chromatography A, Elsevier, 2011, 2692 2
3. Principle of HPTLC coupled with Vibrio fischeri
Detection
• Principle- Adsorption
• Precleaned HPTLC plate- methanol, dried for 30 min at 120°c
• Application is performed by Linomat syringe (100ml)
• Tracks made on the plate
• Preparation of mobile phase
• Run the solvent and dry the plate
• Roller is used previously dipped in bacteria suspension.
• BioLuminizer or UV chamber.
Baumgartner Vera , Hohl Christopher, Journal of Chromatography A, Elsevier, 2011 ,2693
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4. Application techniques for luminescent bacteria on HPTLC plates
• There are several application techniques by which luminescent bacteria onto the (HP)TLC plate is
preferably put –
Dipping
Spraying
Rolling (New technique)
• Disadvantage:-
• Homogeneous Application was quite difficult to achieve, bacteria were damaged due to evaporation
and while spraying, and precautionary measures were necessary due to aerosols.
Baumgartner Vera , Hohl Christopher, Journal of Chromatography A, Elsevier, 2013
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5. Rolling- New Application Technique
Rolling assembly and Components: -
a) The wooden rolling pin(1)
b) saran wrap to protect the wood from getting wet .
c) Then, a piece of window cloth, size approximately 25cm×15cm for 20cm×10cm HPTLC
plates, was applied as a pad onto the rolling pin (2)
d) A plastic cutting board was used as a support for the HPTLC plate(3).
e) A stop bar (4).
f) For the rolling process, the HPTLC plate (5)
g) A glass plate roughened with carbide for better friction was used as a forerun(6).
h) The tray
i) BioLuminizer
j) Between consecutive rolling, the cutting board and forerun were wiped dry and the roller
wetted again with bacteria solution.
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Baumgartner Vera , Hohl Christopher, Journal of Chromatography A, Elsevier, 2011
6. Automatic Rolling machine
Automatic rolling machine consist of :-
• adjustable pressure and speed control, wetting of the pad on the
roller must be homogeneous
Requirements for Material used:
• non-toxic for Vibrio fischeri (especially solvent tank)
• easy to clean (washable)
• components corrosion-resistant
Other Requirements:
• suitable for two plate sizes (10 x 10 cm and 10 x 20 cm)
• PTFE coating of the components
• automatic measurement of the luminescence
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Vera Baumgartner, 2013 , HPTLC-Biolum Detct. Methodological Improvements , 56-58
7. Biodetection and Evaluation :-
• The luminescence of the bacteria was checked
before use by adding 10 μL bacteria suspension to a
cuvette containing 2mL of a cooled 2 % sodium
chloride solution (15°c).
• The relative luminescence capacity (Irel mode) of
the suspension in the cuvette was measured
immediately with a specific photometer (LUMIStox,
measuring instrument for luminescent).
• The bacteria could be used if the Irel vale is greater
than 500.
• Images were taken using the BioLuminizer 1.0,
exposure time 55 s. The offset and gain corrections
were set in the automatic mode.
• The photos were evaluated quantitatively with the
help of ChromQuest HPLC software.
• The BioLuminizer software convert the raw data
from the camera into an image.
7Vera Baumgartner, HPTLC-Biolum Detct. Methodological Improvements ,2013, 40-41
8. Continue..
Comparison between images:-
• The process uses two parameters- Gain value and offset value
• Offset values remain same for all the images but gain value has a
particular range 5 -12.
Handling in Rolling technique:-
• Wiping is done with or without filter paper
• To wet the rolling device 80 ml of bacteria solution is required
Quantitative parameters are calculated such as:-
• Hight, Area, Width at 10% height, S/N Ratio, Standard deviation,
Relative standard deviation etc.
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Baumgartner Vera , Hohl Christopher, Journal of Chromatography A, Elsevier, 2011, 2696-2698
Figure 1 comparison between rolling (solid line) and dipping technique (dotted line).
9. Applications
• Determination of preservatives in the skin care creams.
• Detection of phenols in urine.
• Industrial Waste water analysis.
• Pollutants in tap water.
• Berberine Alkaloids in Plant Extracts.
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Vera Baumgartner, HPTLC-Biolum Detct. Methodological Improvements ,2013, 11-14
