Day 5 - In Pictures

Today we complete the dialysis! Then move on to freeze-drying!

The Lab: 

 Step 1: DaWei pours out the 5% acetic acid and removed urea 

Step 2: He then replaces it with a fresh solution of 5% acetic acid free of urea! 

Step3: Steps 1-2 are repeated once more at 2-3 hours each time.

Step 4: Placing it in rack and puncturing a hole. This is to ensure that the frozen water and acetic acid can be sublimed when in the freeze dryer, leaving only the protein

It it then placed in a free dryer for 1 h and 30 mins to ensure that everything is frozen.

The -80 Freezer!

Step 5: DaWei quickly places the rack containing the vial in a special glass which can withstand pressures close to vacuum. This has to be done quickly to ensure that the solution does not melt! 

   Done!

This is left overnight to ensure that all water and the 5% acetic is removed, leaving only the protein.

The freeze-dryer machine!

Our attempts successful!:

Day 5 - Completing Dialysis, onto obtaining pure denatured protein

We are halfway through our shadowing programme. Today's procedure is similar to yesterday's.

The dialysis bag containing urea, denatured protein and 5% acetic acid was further processed so that all the urea can removed from the bag. This step was repeated twice, each time roughly 2-3 hours. The result was a bag containing water, 5% acetic acid and the protein, meaning that urea has been successfully removed.

In order to obtain the pure protein powder, the bag was placed in a freezer at -80 degrees Celsius. The bag then turned solid hard. Afterwards, Dawei placed the bag in a freeze dryer. Ice and acetic acid which were initially present soon sublimed. Liquid water and aqueous acetic acid were not  formed as both substances were sublimed. 

The final product contains the protein only. 

Day 4 - Protein Extraction

Pre - Preparation: Protein (Polymer)

DaWei needs to obtain the Unfolded Protein Strand.. First he adds urea to the protein. This urea denaturises the protein into a polymer. After doing so he needs to remove the denatured protein. He then makes adds acetic acid at 5%.

Dialysis:

Step 1: Taking out the right vial containing his prepared solution.

Step 2: The bag to contain the solution is soaked in deionised water!

Step 3: DaWei proceeds to homogenise the 5% acetic acid in deionised (total 3 Litres!) water using a Homogeniser. The white motion blur you see is a magnetic mixer which is spun by a electromagnet below the machine.

Step 4: DaWei Proceeds to carefully add the protein into the bag.

Step 5: Securing the bag with clips!

Step 6: The bag is placed into a container containing roughly 800 mililitres of 5% acetic acid. This first round will be put overnight. Subsequent ones are done for 2-3 hours to remove any excess urea. Stay tuned!

How this last step works: 

The 5% acetic acid is maintained to make sure to make sure the protein stays soluble in the solution. The urea molecules are small enough to pass through the semi-permeable membrane of the bag. This is done repeatedly, removing any remaining urea.

Points to note: Dialysis although share similar steps to osmosis, they are different, to understand do check out a website by clicking here.

Day 4 - More application

Unfortunately my partner has fallen sick, so I will be putting my own thoughts here.

Today's laboratory work is very similar to what I have learnt. They make use of the process of osmosis and in this case, it is termed dialysis. More advanced equipment are used to make the process more effient and make it a much quicker process. Careful measures are also taken to ensure the wanted proteins are not contaminated. Once again, this really lets me appreciate how the practices we do at our level are applied at this higher level. Stay tuned, for further information on the completed work!

Day 3 - FTIR (Spectroscopy) and Hydraulic Press

Hydraulic Press (Preparatory Steps)

Step1 : Cleaning all Equipment with Ethanol! So that samples made will not be contaminated.

Step 2:  Placing the samples into a manual crusher! 

Grinding it... HARD!

Step 4: Placing sample powders and making sure it is level!

Step 5: After encasing it, the equipment holding the sample is screw tightly into place.

Step 6: Making use of the (hidden) hydraulic system, up to 10 tonnes of Pressure is applied!

Removing equipment holding sample....Carefully!

The crushed powder under pressure forms into a clear circular sheet, allowing it to be placed into a FTIR sample holder.

Front view of the Hydraulic Press

FTIR (Fourier Transform Infrared Spectroscopy) Machine

 The sample is placed into the FTIR machine, where the sample will be beamed with a wide spectrum of light. These different lights will be blocked(absorbed) depending on the types of bonds the sample has. The remaining light then passes through the sample and is picked by the detector.
The (lowest) peak are recorded and the graph is printed for analysis.

Post 3 - Engineering for thought

Benedict:
All of the experiments today, most were new and unusual to me.
Polymerisation on one hand was something that I have learnt a bit in 'O' level chemistry. This was the first time I am able to appreciate the physical process of polymerisation that I has only read in textbooks on. FIrst, the monomer was collected then extracted, then converted to polymer using chemical processes.
The precipitation was slightly more complex than what I had did in 'A' level experiments.
FTIR and hydraulic press were new processes to me. However I was unable to see the process of FTIR as it happened.

Day 3 - Precipitation and Polymerisation

In the afternoon, we went to meet LiHong in the Biomaterial Lab located at the basement of MSE building. She is working on a project to use amino acids to synthesis materials. Her work is on the polymerisation of Serine and Tyrorine with varying composition.

The process of polymerisation of proteins is similar to our A level syllabus where we learnt how amino acids chain up together using a peptide bond between the C terminal and the N terminal.

Firstly, a solution was placed within the Rotary Evaporator. Pressure was decreased so that water can be boiled off and removed at roughly 50 degrees Celsius. The solvent (in this case, water) is being removed so that residue can be obtained. The end product of this procedure is a concentrated glue like substance. 

Rotary Evaporator

Next, she continued to precipitate out the amino acids from the solution using Hexine. In the picture below, Hexene is placed in a glass bottle with the drying agent.

Hexene with Drying Agent to Remove Water

However, the yield of precipitate using Hexine to precipitate out the amino acid is very low. She then tried using deionised water. Fortunately a clear precipitate was obtained using deionised water. She proceeded to use the whole beaker of the concentrated solution and placed it in a freezer at 4 degrees Celsius. 

Precipitation in Progress

After the amino acids were precipitated, LiHong removed the excess water in the beaker. She placed the beaker containing the viscous amino acid into a vacuum oven to obtain the final reactant for polymerisation,

Her previous work in progress: