I;m tired with the TLC and column chromatography and updating this blog. So today i'll be writing in notes...so sorry! =P

The test tubes were covered with aluminium foil before i left yesterday, but when I arrived today, the test tubes filled with eluent had now contained only half of the initial amount. (pet.ether was the solvent). So anyway, I continued from where I left off, and about 10 test tubes later, my advosor told me to fill in the column with new ratio of elunet which is PE 95%, EA5%. The new mixture definitely caused the eluent to drip even faster.

Now I'm at my 46th vial, still counting. Hopefully can finish up till 60.

Note to self: Should have filled up my test tubes with eluent by 80% instead of 60 or 50%. I'm using up test tubes fast, and there's still more sample eluents to be collected!

Wednesday

It's finally the day where my synthesis can finally be completed!

With 4 hours still remaining, I did a TLC test to see what was the progress if the reaction.

After spotting the silica gel paper with the 2 reactants and the product, and placed in jar with 4:1 Pet.ether : Ethyl acetate, dried and placed in UV light, the product line still had reactants' spots and also extra 6 more colourful spots.

This is a problem because i'm suppose to be obtaining one product only, hence there should be extra one spot only.

Also, something i forgot to mentioned. yesterday I performed a TLC around 12pm. The Product showed 3 spots that does not corrolate with the spots in reactants 1 and 2. My advosor told me it was probably because the product is still its formation process.

After my break, finally I was given the green light by my advisor to stop the stirring process and continue with stopping the reaction by acidifying the mixture with 10% HCl (acidity tested with PH paper-orange version), then washed with EtOAc (ethyl acetate), poured in seperating funnel, organic layer separated, aqueous layer washed with EtOac, EtOAc separated from aqueous layer and mixed into organic layer. Organic layer washed with brine, seperated again, MgSO4 added to organic layer, organic layer filtered.

The product was evaporated under pressure, and I noticed that right after the broen syrup cools to room temperature, the syrup crystallizes to yellow/orange cloudy crstals. Heating up the crystal with some EtOAc will dissolve the crystals back to the brown syrup. (did this twice!)

Then I had to prepare for column chromatography. A column (slightly bigger than what i used in uni, but still smaller compared to the humongous column another friend of mine was using). Cotton wool was added, to block the stationary phase from escaping. Poured some PET ether (4-6). Then added about 50g of silica (had to wear face mask all the time, these silicas fly everywhere!!) with Pet.ether slurry.

(accidentally spilt some of the slurry, so had to wait until the pet.ether evaporates and had to use a feather duster to dust away the silica that was on my desk, my books, my other apparatuses, my arms, everywhere!)

Conditioning the stationary phase: Let 200mL of Pet.ether run down the column until the eluent's surface levels above the silica surface.

Then it was time to add the sample. As I said, the sample had turned to cystals. And so my advisor, did the same thing as I did (adding solvent and doing rotatory evaporation). But this time, the sample stayed as syrup for a longer while, but we could see that it was hardening to cystals, so she sucked the syrup with a long pasteuer pippete and added the sample into teh column, but some portion of the syrup tuned into crystals and some of the sample left in the container had also turned to crystal I.E Percentage yield will be low!

100mL of pet.ether was added to the column, and eluent was allowed to run for sometime until yellowish colour eleunt reached the middle of the stationary phase.

That's where I collected a portion of the eluent as it drips through the column with a capillary tube and spotted on a silica gel paper and the spot was observed under the UV light. This was done every 5-10 minutes. If a spot is visible under the UV light, means there's presence of compound, meaning then I could start collecting the eluent with the 80 test tubes that I had already washed, dried and prepared!

And by the end of the day, I had collected 3 test tubes of eluent with very very very diluted sample, so diluted, it would be very hard to see under the UV light.

Tuesday

Still had to wait for the 48 hours of synthesis to be complete.

Thankfully i brought my laptop and hence spent most of my time updating this blog and studying TLC and column chromatography methods.

Helped some of my friends with their things.

And prepared my apparatus.

Monday

Actually the TLC was conducted today, which showed that there was no reaction occuring at all.

So my advisor gave me a new project. Also product synthesis. But this time, there will be two synthesis of products.

First is the Prenylation of aromatic aldehyde.

Second is the synthesis of chalcone from the prenylation product and aromatic ketone via aldol condensation.

Which means I have to synthesize one of the reagent for the second synthesis. I'm thrilled, because I like synthesis, because chemical reactions are at play here, but the first reaction takes 48 hours to yield some amount of products, so, I'm not really happy having to waste time waiting.

Anyway, to the first synthesis:

The first synthesis required 3-hydroxy-4-methoxybenzaldehyde (1g) which i dissolved in 12ml of 10% KOH solution (self-prepared), which turned the mixture transparent yellow.

Weighing 1-bromo-2-methyl-but-2-ene (Reactant 2, simplified: PrBr) was a [roblem. PrBr exists in liquid, and is both photosensitive and sensitive to air. And also flammable and corrosive.

Since I definitely could not use a transparent container to place in PrBr, so I took a glass vial with a stopper, wrapped it with aluminium foil to use as a container.

As I transfered the amount I needed, my eyes gradually became incredibly painful, (which was the exact same pain I remembered as I prepared wasabi paste from wasabi powder in my japanese restaurant working days) and I tried my best not to inhale in the halogen-ish smell.

So yes, I considered weighing in a fume hood, but there were no weighing scales in the fume chambers. So I just had to bear it through.

Then the PrBr was added drop by drop into the round bottom flask containing the yellow solution of R1 and 10% KOH. Adding PrBr drop by drop made the mixture turn gradually more cloudy and the colour form yellow to reddish brown.

then the reaction mixture was stirred with magnetic stirrer.

Guess how long will I have to wait? 48 hours! Again!!

This time the yield had better be satisfactory!

But the advisor did tell us that failed synthesis is normal so I dont have to worry.

4th day (Friday)

waited until 3pm (reaction started on 12 pm on wednesday) because 12 pm is lunch time until 2.40pm.

(helped the others with their stuff while preparing my apparatus too)

Liquid was an amber brown colour with trace amount of white solids

Liquid was washed with squirts of cold water from the dropper and acid HCL (10%) (a few squirts too, more white solids were formed) was added to stop the reaction (by reacting with KOH).

Chloroform was added to dissolve the organic product, and separate the organic product from the aqueous solution.

Mixture was poured into separating funnel, aqueous layer was above and and organic layer was below (because chloroform is more denser than water). Organic layer was separated from aqueous layer into a conical flask. Aqueous layer was washed with chloroform (because some products could be trapped in the aqueous layer) and the chloroform was added into the organic layer. Organic layer was cloudy, meaning there's still water in the organic mixture. Brine (supersaturated NaCl solution) was added into the organic layer to extract water, and water was eliminated by adding MgSO4.

Mixture was filtered to remove MgSO4, and then the next process was to conduct a simple TLC.

Conducting TLC.

Purpose of conducting TLC is to determine if any reaction occured to your reactants and to see if there are any products.

3 things were prepared:

a little bit of solid reactant 1 was dissolved in chloroform in a small vial.

Same thing for solid reactant 2.

A glass jar was added 4ml petroleumn ether and 1 ml ethyl acetate lefta while fior the mixture vapour to fill the jar.

4 spots were spotted on a piece of silica paper (4cmx5cm), R1, R2, P1 and P2 (2 spots of product, just to be safe) and placed vertically into jar.

Waited for solvent to soak the paper approx 90%, , paper taken out, dried, put under shortwave light.

Spots were circled, and analyzed.

The 2 spots on both P1 and P2 corrolated with the spots on R1 and R2, meaning there was NO REACTION TO GIVE PRODUCT.

T_T

My two and half days wasted, thank you very much claisen-schmidt condensation!

3rd Day

This will be very short because I posted a long essay initially but apparently I can't use both blogger (signed in with one google account) with a google email account (another google account) simultaneously. Google had a seizure when I tried to post it. Damn you, Google.

Switched on stirrer first thing in the morning. Colour had turned from yellow yesterday to dark orange today. Obeserved white solids in the transparent liquid, which is worrying, because my partner's mixture was yellow transparent.

Helped friend with filtration of organic solvent from shredded plants after soaking them in the solvents for overnight. Then transfered some amounts of liquids to test tubes to test for compounds tannin and another compound (forgot)

Will tell you the methods that I used later.

Also did TLC for another friend while she collected elutes from her column chromatography.

Helped another friend with rotatory evaporation (by playing 'eliminate-the-bubbles')

2nd Day

I finnaly met my supervisor, she is one of the research officers in the Natural Products division where she and another officer are in charge of Product synthesis. She gave me a brief overview of what Natural products division do:

The herbatologists collect and identify the species of plants, and then chemists determine if there are any useful compounds in the plants, then the compounds are converted into other types of compounds to enhance the benefits of the original compound.

She gave me my assignment, which is synthesis and identification of chalcone via Claisen - Schmidt condensation.

First I had to calculate the masses of the two reactants needed for the product synthesis by determining the RMM and moles of the compounds. Then after weighing the two compounds, I combined both the solids in a round bottom flask with EtOH, and then added KOH solution of determined concentration that I prepared by dilution. and then swirled the contents until the solids were dissolved.

The round bottom flask was placed on an electric heating plate with a magnetic stirrer. The plate was heated to about 38 degrees and the contents were left to stir for 48 hours.

Since the addition of reactants up until the stirring was done at approx 12.00pm, I had to wait for 48 hours before continuing with the next procedures. Hence with nothing to do, I went to my friend's place and observed her doing her column chromatography. Then I also observed how another friend of mine was extracting chemical compounds in plants parts that were soaked in organic solvent. And then helped another friend of mine with his evaporation of organic compounds under pressure.

Overall, a rather dull day.

I will post up my assignment details later, which I am grateful I get to do some product synthesis while others are doing qualitative analysis.

About my supervisor.

I shall not indulge in the details of her name, appearance or anything personal. To surmise what type of supervisor she is...she is a very...elaborative person. She did not leave out any details in her explanations, and explained even the most basic procedures to me. She insisted that we ask her any questions, and made sure that we could understand what procedures we were doing, and how to do it, even simple steps like dilution of acid. I'm really grateful to have her as I feel secure that she will guide me well.

Claisen-Schmidt Condensation (aldol condensation)

(from wikipiedia)

An aldol condensation is an organic reaction in which an enolor an enolate ion reacts with a carbonyl compound to form a β-hydroxyaldehyde or β-hydroxyketone, followed by a dehydrationto give a conjugated enone.

In its mechanism, it involves the nucleophilic addition of a ketone enolate to analdehyde to form a β-hydroxy ketone, or "aldol" (aldehyde + alcohol), a structural unit found in many naturally occurring molecules and pharmaceuticals.

An aldol condensation differs from an aldol reaction in that a H20 molecule is lost to form a C=C double bond.

While an aldol reaction involves a nucleophilic addiction of a ketone enolate (nucleophile) to an aldehyde, where the H of a C=C bond attaches to the O=C of the aldehyde to form OH and the C= of nucleophile bonds with C of aldehyde to form a C-C single bond.

(Diagrams always speaks clearer than words =))

Literature Review

Looking through the journals that my supervisor has co-authored, looks like I'll probably be dealing with synthesis of flavanoids and its collection, reaction monitoring, identification and maybe some microbiological screening. ^^

...I need to refresh back on my Spectroscopy for organic chemistry...

I am thankful, of my 2 years of laboratory experience in university. Because the laboratory methods I'm learning in my internship now, is an advancement or continuation of what I have learnt in my chemistry courses.

Which reminds me of what my lecturer once told me (by memory, modified): "The courses that you have learnt, is sufficiently enough to be applied in any outside laboratories" when I voiced out my concern that I am not capable enough intellectually and experience wise to work in the outside world.

This makes me even more prouder of my Chemistry Department of University of Malaya!

Which means...in future, I would have to take on all courses , both compulsory and electives, with requisite laboratory work to improve myself!

Go Chemist-Joanne!

First Day

(I am nearly always accompanied with my fellow 5 interns, but here, I will use "I" )

My first stop at FRIM on a dull quiet morning was at the HR department, where I and my fellow interns were given the relevant documents to fill up and hand in. Then I headed to the Natural Products division laboratory, where I was supposed to meet my supervisor.

Unfortunately, she was on leave, but she gave some directions to me, which was to read some of the journals (co-authored by her) and manual on column chromatography and TLC methods.

Since reading can be done at a later time, I chose to observe my fellow interns as they did their work while being instructed by their supervisors. There were two groups I obseved:

1st:

Isolation of organic compounds by soaking raw materials in organic solvent first and then do claisen-schmidt condensation with rotary evaporator, and then compound isolation and identification with TLC

2nd:

Weighing of 32 different plant raw materials, before soaking overnight in solvent before compound isolation

In short, mostly learnt about isolation of compound and TLC method. Also introduced to HPLC method and column chromatography.

Given a trip around the laboratories and introduced to many analytical machines such as HPLC, FTIR, etc

Homework: read journals and get to know different stationary eluents for column chromatography, and know the principles behind HPLC.

Night before First Day

I the night before the first day of my internship, I was busy with monsoon cleaning (instead of spring cleaning, because it was raining) my rented condominium with my fellow five FRIM intern crewmates. And it was around 2am before I finally settled down with everything ready for the next morning. However a mixture of excitement, nervousness, uncertainty and unfamiliarity was inside my guts, hence the only way to calm me down? Internet surfing until 2.30am, and my head finally touched the pillow with comforter wrapped around me at 2.40am.