rush with time

Today, run DPPH assay on one sample alone (of course alone), but this is the second time I do it, so I was hoping I am not nervous and mess around that time. This time I know I need to be very very careful. Boss also there to look around and clear some things, but I am not that nervous anymore, in fact, I am calm that time…haha ^^ Oh yeah!

Make my pace as fast as possible to dry all the glasswares needed. Wow, for just to test one compound, I need more than 10 of 10mL volumetric flask, sample tubes, sample vials, a lot of micropipette tips, a lot of beakers to stabilize the flasks n tubes, and my energy to shake vigorously to mix the compounds in the flask.

Ok, use samples prepared to make up initial concentration: 500ppm. Then the next is stock solutions preparation which are 250, 100 (x2), 50 (x2), 40, 20, 10, 5, 2.5 and 0 ppm in concentration. 0 ppm is just consists of DMSO and DPPH. Well, DMSO is the solvent used to dissolve the sample. DPPH is the free radical. Firstly, 5mg or 0.005g of sample is dissolved in DMSO and top up with DMSO to 10mL in volumetric flask. Then, start the using of sample tubes…from 250 ppm to 2.5 ppm. All has to be precise…if not I will get scolded by boss…lolz. Ok, prepare stock solution. It took me a lot of time…!!! After prepare that, it is almost 1pm. Went to have lunch, to save time, I only get 100 plus.

Next is the step of preparing DPPH solution. Got the reagent from the refrigerator, take a sample vial, a beaker and one small spatula to get DPPH measured. Dissolve the 1.97 mg of DPPH powder in methanol then transfer to 50mL volumetric flask then top up with methanol then shake vigorously to mix. Cover the whole flask with aluminium foil as stated in manual and keep in dark. In the meanwhile, prepare the samples for DPPH assay. 1mL of sample solution plus 4mL of DPPH solution in sample vials. Well, DPPH solution is added in time intervals of 3 minutes between each concentration. Aiks, why I am not thinking of this, while prepare stock solution, I can prepare the solution in sample vials to avoid from using so many sample tubes (because end up I need to wash a lot of glasswares…lol).

DPPH assay can be run when I am ready. Get a stopwatch, prepare sample tubes to allow easy filling of DPPH solution to sample vials by micropipette. Get set, ready! First 4mL of DPPH solution is added to first concentration, 2.5ppm. This is the challenging part. Within time intervals I need to fill the particular amount of DPPH solution. After I fill the last sample vial of 250ppm concentration, I need to get the UV-VIS spectrophotometer set up, initialization, the cuvettes, distilled water, tissue paper, all get ready within 15 minutes. 5 minutes before the 30th minute, I need to get ready to get all my things to downstairs to the UV-VIS spec there. When it is the 30th minute, the 3 minutes time intervals is rushing me. Get the readings in triplicate. Wash the cuvette for the next concentration. Blank for this test is pure DMSO.

When I measure the first concentration, which is 0ppm, it showed 1.254 for absorbance, I was shocked there, because so far I do the spectrophotometric assay, the absorbance seldom reach that high. But this one, is high until second last concentration. I was a bit panic that time. Thought I was wrong. Then I asked a senior, luckily she done DPPH assay before, she said my result was ok. Fuh, relieved. It would get that high absorbance, as long as not reach 2.xxx for absorbance then is ok.

Ended up the absorbance of 5 and 10ppm got problem. Oh gosh!!! I already very careful. Maybe I shall be even even more careful next time. Tomorrow will be run with a standard, which I need to do 2 times of absorbance reading, standard and sample’s readings. Will make it better and more efficient for next round! I can do it!!!