Friday, September 26, 2008
It is the 14th week!!!…..left 6 more weeks to go. Jiayou pple. As I am still in a chemistry lab for the last 14weeks and for the future 6 weeks, this week post will continue from my first post. (week 4) which is on my major project which is on phytoplankton (diatom and dinoflagellate are some e.g. of phytoplankton.)
Here is a recap:
1) Seed phytoplankton cells
2) Harvest (centrifuge) when culture is ready
(Centrifuging takes 1 whole day. Why? Cause got about 20 litre of culture to spin down and there is only 1 rotar available----1 rotar can only carry 6 bottles, each bottle has a volume of 250ml, so after calculating…figure out !?!)
3) On the supernatant, carry out SPE
a. Methanol loaded first to condition the cartridges and wash away any impurities
b. Then load DI water to remove methanol
c. Next, load the supernatant (this take many days)
d. Then, load DI water again to remove salts (cause seawater contains salts)
e. Spin down the cartridges to ensure all DI water is removed from cartridges
f. Lastly, load methanol to elute out the extract.
So, now I have the eluent which contains the solvent (methanol) with the extract I want.
To get the extract and remove the methanol, evaporation is carried out.
Technique: evaporation
Used for removing solvent
Equipment used: rotary evaporator
(Image taken from http://www.eyelausa.com/category.php?cID=1)
It is a very easy process but take very very long for large quantity of eluent.
The bottle on the right is the evaporating flask and on the left is the receiving flask.
Water aspirator is connected to the hook-like thing on the condenser. Condenser pump is placed in a beaker with water covering the pump and the remaining space in the beaker is filled with ice.
So first switch on the condenser (the tall tube with coil inside) and water bath (it take time for the water bath to reach the temp set).
What temp to set for the water bath?
Temp set is around 40Cto 50C as long as it is below methanol boiling point.
Depending on the compound want to obtain, if it is stable and does not react easily (meaning it has few hydroxyl(OH) groups), then it is alright to set at higher temperature but the temp must be below the boiling point.
Eluent is poured into the evaporating flask, filling around 1/3 of the flask. If the flask is filled too full, eluent might spill out while evaporating and the whole process need to be repeated again.
After everything is set, the water aspirator is on. Water aspirator provides the vacuum required. Next, the pressure is gradually reduced. The eluent in the evaporating flask had to be monitored constantly in case there is lots of bubbles (‘boiling”). When this occurs, the pressure is released a while then reduced again. If violent boiling occurs, the desired compound may spill over to the receiving flask.
When evaporating, methanol from the evaporator become vapours and travels up to the condenser. The vapours then condense on the cool surface of the condenser. The methanol in liquid form then dripped into the receiving flask.
This whole process continue until the eluent in the receiving flask left a very small quantity, then I top it up with more eluent. When all eluent has been poured into the evaporating flask, then I evaporate the remaining eluent till dryness.
And the residue in the evaporating flask is the extract that I want to get. The residue is of a very very small quantity. To get sufficient amount for LCMS (discuss about it in next post) …. I need to accumulate about 3 times of SPE eluent.
After accumulating, I use methanol to dissolve and suspend the accumulated dry extract. Then I transfer the extract into a small tube using a dropper. As there may be some extract then cannot be removed from the walls of the flask, sonication using a sonicator is carried out to detach the extract from the walls.
Next I dry the extract in the tube using stream of nitrogen gas. (Why nitrogen? Actually nitrogen is a common gas used for drying.)
For the cells,
Methanol is used to remove the cells from the centrifuge bottles.
The methanol containing the cells is transferred into a conical flask.
The cells is left in the flask for about 1 week. Meanwhile, the flask is sonicated.
Methanol can lyse the cells to get the extract from the cells.
Sonication helps to enhance the process of cell lysis because high frequency sound waves can shear cells.
After that, the mixture (cells in MeOH) is filtered to remove the cells.
Methanol extract is evaporated as mentioned above.
LCMS on the extract will be discussed in the next post.
6 more weeks to go.
Hope my explanation is clear.
Any doubts, questions or uncertainty, juz ASK. =D
Lim Xin Ni
TG02
0607325H
=)
2:16 PM