Subject Title: Lab Techniques
Hi, Everyone. I am the fourth to post here and I am posted to a research lab. In the lab, there is a lot more chemistry processes than biology.
The lab I am attached to deals with phytoplankton…..a single-cell organism that can photosynthesize.
For this first post, I will be discussing on these:
1)Preparation of culture
2)Solid Phase Extraction (SPE)
The rest of the steps shall be discussed in future posts =P
For the preparation of culture, several carboys of sterilized seawater from an institute on St. John Island were collected personally. (take a ferry there =D). Why the seawater is collected from there and not other places? My supervisor say because seawater at that place is much more clean….less pollution. The seawater has been filtered through several different size filters and UV-sterilised at the institute.
Nutrients are added according to a given secret recipe.
The medium is then filtered using a filter unit.
The type of filtration carried out is pressure filtration.
This means gas is used to force the content from a cylinder tank to pass through the filter unit. The gas will travel from the cylinder, enter the pressure cylinder tank and force the medium from the tank to pass through the filter unit.
This is then followed by the addition of the seed cells. Then, the cells are left to grow in optimal conditions-optimal light intensity and optimal temperature.
After growing, we harvest the culture by separating the medium (supernatant) and cells (pellet) by centrifugation.
Time to harvest depends on the growth rate of the cells…….there is no standard amount of time to grow the cells…….if a lot of cells die then it is time to harvest.
The supernatant is then filtered.
Then SPE (solid phase extraction) is carried out on the filtered supernatant.
In the lab I am working in, the SPE used is reversed phase SPE. Other types of SPE include normal phase and ion-exchange SPE. I am not very sure about these others types of SPE since only reversed phase is carried out in our lab. Reversed phase SPE extract compounds base on polarity.
Reversed phase SPE use cartridges that contains C18 silica-based sorbent (stationary phase) which retain compounds by hydrophobic interaction. C18 means the silica is linked by a link that consists of 18 carbon atoms.
So, these cartridges are placed on top of a tank.
Picture taken from www.biotage.com/Print.aspx?id=35833)
1)Then, methanol (organic solvent) is loaded first to remove any impurities in the cartridges. Next, DI water is passed through these cartridges to wash away the methanol otherwise the desired compounds cannot be retained on the sorbent.
2)Then, the sample is passed through the cartridges, and the compounds will be bounded onto the sorbent by hydrophobic interaction.
3)DI water is then used to wash away impurities that may be present in the medium. It must be ensured that no DI water is left in the cartridges after washing otherwise it will inhibit the elution step.
4)To get the compounds from the sorbent, methanol is used as solvent to elute the compounds. The methanol will disrupt the hydrophobic interaction resulting in the compounds to detach from the sorbent and flow out of the cartridges with the methanol.
Actions carried out on the eluent and how to get the compounds from the eluent shall be explained in future posts. =)
Since, throughout the SIP, I will be dealing with a lot of methanol, PPE such as goggle, lab coat, and gloves are necessary. Also, the experiments are carried out in fume hood whenever possible. (Methanol is toxic when inhaled/consumed and can cause blindness.)
Hope everyone is enjoying your SIP. ^_^
Lim Xin Ni (0607325H)
TG02
Group 9