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Written by Kelvin Soo on this day of

Sunday, the 6th of June 2010

ROTAVAPOR TECHNIQUE OF CHLOROGEL MANUFACTURE

Objective
To create Chlorogel with the addition of buffered chloroplast stock solution into an ethanol-free silica
sol-gel prepared by rotavaporisation.

Equipment
i) Buffer preparation
- Anhydrous Na2HPO4
- Anhydrous KH2PO4
- Anhydrous KCl
- Sucrose sugar
- Distilled water
- Aqueous KOH or HCl (any conc.)
- 1 L glass beaker
- Glass stirrer
- Spoon
- pH meter
- Thermometer
- Parafilm
ii) Chloroplast isolation
- Fresh spinach leafs
- Distilled water
- Blender
- Cold sucrose-phosphate buffer
- Bandages
- Centrifuge tubes 50 mL
- Centrifuge
- Micropipette
- Glass funnel
- Measuring cylinder 50 mL
- Lamp/light source
- Ice bucket w/ ice
iii) Sol-gel process
- Aqueous tetraethyl orthosilicate (TEOS)
- Aqueous 0.05 M HCl
- Distilled water
- Evaporating flask
- Beaker w/ ice
- Magnetic stirrer
- Measuring cylinder 25 mL
- Ultrasonic processor
- Retort stand + clamp
- Thermometer
- Stopwatch
iv) Rotavaporisation process
- Rotavapor unit
- Vaseline
- Stopwatch
- Glass stirrer
- Container mold
Procedure
i) Buffer preparation (Estimated completion time: 20 min)
- Add 3.97 g of Na2HPO4 in 700 mL of distilled water and stir to completion.
- Add 2.99 g of KH2PO4 into solution and stir to completion.
- Add 136.9 g of sucrose and stir to completion.
- Add 0.745 g of KCl and stir to completion.
- Add distilled water to give a volume of 950 mL.
- Adjust pH of solution with KOH or HCl to 7.3.
- Add distilled water to give a final volume of 1 L.
- Sucrose-phosphate buffer hence obtained; stored at 4°C.
ii) Chloroplast isolation (Estimated completion time: 40 min)
- Wash approximately 50 g of fresh deveined spinach leafs with distilled water in the
presence of a light source for 10 minutes.
- Homogenize spinach leafs in blender with the addition of 200mL sucrose-phosphate
buffer for 10 s.
- Filter homogenate through 3 layers of bandages.
- Equally decant filtrate into an even number of (boiling-tube size) centrifuge tubes.
- Centrifuge at 1500x for 10 min.
- Discard supernatant and resuspend the pellets in 35 mL of sucrose-phosphate buffer.
- Centrifuge at 1500x for 10 min.
- Discard supernatant and resuspend the pellets in 15 mL of sucrose-phosphate buffer.
- Stock chloroplast solution hence obtained; stored at 4°C in subdued light.
iii) Sol-gel process (Estimated completion time: 60 min)
- Prepare an evaporating flask on beaker filled with cool ice at 4°C.
- Add 11.0mL of TEOS, 4.0 mL of distilled water and 1.55 mL of 0.05M HCl.
- Stir mixture vigorously for 30 min.
- Subject vessel onto an ultrasonic processor at 20-kHz freqeuency and 2.4 kJ/cm3 ultrasonic
energy for 15 min.
- Homogenous sol hence obtained.
iv) Rotavaporisation process (Estimated completion time: 35 min)
- Fix evaporating flask into the rotavapor.
- Commence process at 42°C for 25 min of controlled vacuum at 20 bar.
- Quickly decant mixture from evaporating flask into container mold and stir with stock
chloroplast solution.
- Leave sol-gel to cure for ? hours.
- Chlorogel hence obtained.
Precaution / Remarks
i) Buffer preparation
- Care must be taken not to contaminate the anhydrous salts. Always wash spoon with
distilled water and wipe with cloth each time after use.
- Crush solute into grains to ensure accurate weighing and better solubility.
- Always keep the two electrodes of the pH meter in uncontaminated distilled water.
- Take extra care not to add too much KOH or HCl into the buffer when adjusting pH
- Once buffer is prepared, cover beaker surface with parafilm to prevent contamination.
- Must be refrigerated to 4°C or less prior to chloroplast isolation experiment.
- Sucrose is used to regulate osmotic pressure of the buffer around the chloroplasts,
whilst Na2HPO4 and KH2PO4 are a good source of Na+ and K+ ions for regulating ion
concentration. H+ and PO2-4 ions maintain the proton gradient and supply inorganic phosphate
ions for ATP synthesis in chloroplasts respectively.
ii) Chloroplast isolation
- Blender, centrifuge tubes, measuring cylinders and any other containing vessels used
to house the chloroplast mixture must be pre-cooled to 4°C or less prior to the experiment.
Sterilization is necessary to prevent microbial growth in the vessels.
- Experiment must be swiftly conducted to ensure survival of chloroplasts from
changes in the external environment.
- Fresh, healthy and locally-grown spinaches harvested after 3-4 weeks must be used.
- Washing in the presence of light necessary to maintain chloroplast activity and
remove unwanted stuffs from spinach leaves.
- Stop blending after 10 seconds has gone, or once the mixture looks homogenized.
- An even number of similarly-weighted centrifuge tubes must be oppositely placed to
each other in order to balance the centrifuge rotor.
- Parafilm to be used to seal the vessels containing stock chloroplast solution to
prevent microbial contamination.
- Stock chloroplast solution to be kept at 4°C to preserve chloroplast survivability.
- Homogenization of deveined spinach leaves and filtration separates the plant cells
from the leaves. Centrifugation is performed twice; the first to separate plant cells from the
homogenized mixture, the second to separate chloroplasts from plant cells. Resuspension in
phosphate buffer solution necessary to retain chloroplast activity. Optionally, a density
gradient centrifugation can be subsequently performed to separate the intact chloroplasts
from broken chloroplasts.
iii) Sol-gel process
- Tetraethyl orthosilicate, ethyl silicate, silicic acid, tetraethyl ester, silicon ethoxide or TEOS is
labelled as Xn, a harmful hazard. Its smell is akin to that of ester. Sniffing and consumption is
not advised. Rinse with water if in contact with body.
- Add reagents in the specified volumes, otherwise the sol-gel process will be affected.
- The whole evaporating flask must be completely immersed in ice-filled beaker and wrapped
with aluminium foil to prevent drastic temperature change and melting.
- Clamp evaporating flask to a retort stand and immerse contents in the ultrasonic processor.
The water level must exceed the sol-gel level inside the evaporating flask to ensure its
contents are subjected to ultrasonic waves.
- Stirring and exposure to ultrasonic waves assist in the hydrolysis of TEOS and the
condensation of two silanol groups 2(Si-OH) into one siloxane bridge (Si-O-Si).
iv) Rotavaporisation process
- Ensure that the evaporating flask is firmly attached to the receiver with a clamp.
- Lubricate the receiver with Vaseline to ensure smooth rotation.
- Entire evaporating flask must be immersed in bath before distillation.
- Wear gloves to prevent scalding when handling evaporating flasks.
- At the end of the distillation, immediately detach evaporating flask and transfer contents into a
container mold. Wash vessel thoroughly with distilled water to prevent formation of glass.
- Stir the contents of the container mold to uniformly distribute chloroplasts within the media.

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