Thursday, 14 August 2014

The Biochem Life: Experiment 1

As I mentioned in my previous post, I've quite enjoyed Biochemisty this year and have even gone so far as to take a practical Biochemistry subject called Techniques in Molecular Science. Here's a run down of what we've done so far!

Experiment 1

Our first experiment focused on two things: pipetting and spectrophotometry.

For the most part, the pipetting stuff was pretty straight forward. We played around with the pipettes and used scales to work out how accurate our pipetting technique was. Mine wasn't too bad, although I buggered up my work with the smallest pipette (A20)—an error of 11%! The thing I was most pleased about with the pipettes was the fact that the set I have to work with this semester is set 19, which is my birthday!

My guns

After we were done with the pipetting, we moved onto the spectrophotometry. If you're a VCE Chemistry kid, you've probably heard this called spectroscopy or, more specifically, UV-vis spectroscopy. This is a pretty nifty technique based on some basic science. Light is essentially a wave of particles, with different colours of light having different length waves. All molecules absorb light at specific wavelengths better than others. A red car, for example, is really bad at absorbing red light, instead reflecting it. This is why we see the car as red, because the red light is returned to our retinae.

Armed with this knowledge and a spectrophotometer, we can do some pretty nifty science. The first thing that we did was try to work out how the compound potassium chromate (K2CrO4) absorbs light. We managed to do this by whacking some potassium chromate into a spectrophotometer and generating something called an absorbance spectra. The spectrophotometer shines beams of light at different wavelengths through the sample of potassium chromate and measures how much light passes through. The more light that passes through, the less light that was absorbed. For the mathematically inclined, the absorbance is the log of the the 1/T, where T is transmittance. Transmittance is the fraction of light that makes it through the sample, and is given by the received light divided by the incident light.


This sounds like a really complicated process, but for the most part it's all about just pressing a couple of buttons and letting the machine do the work. The technology is relatively old as well, with the machine looking as though it came from the 1920s!

The Beast
When we ran the experiment, we actually generated two spectra for potassium chromate. The spectra we generated corresponded to different dilutions of the potassium chromate solution. Apparently the results start to become a bit wonky if the absorbance gets too high, because the fraction of light that makes it through becomes smaller and smaller and thus more prone to external interference or error in measurement. To allow for this, we compared a 1/10 dilution and a 1/30 dilution, and thankfully got pretty similar results.

Absorbance spectra of potassium chromate (λmax=372nm)
Lower curve is 1/30 dilution
Higher curve is 1/10 dilution

After we had our fun with the potassium chromate, it was time to get to apply some spectrophotometry to a problem. As the potassium chromate curves show, the absorbance is dependent on the concentration of the solution (note that the 1/30 dilution absorbs a lot less than the 1/10 dilution). The spectral properties (that is what light it absorbs best) are the same for both mixtures—the shapes of the graph don't change; it's merely elongated in the 1/10 dilution.

Using this information, we can construct a relationship between absorbance and concentration. This relationship is called the Beer-Lambert equation and also contains parameters called the molar extinction coefficient and path length. For those interested, the molar extinction coefficient is a constant that tells us how that solution behaves when absorbing light and path length is just the length of solution that the light has to flow through, which is obviously a variable.

A= e x c x l

Where, A is absorbance, e is the molar extinction coefficient, c is the concentration and l is the path length. 

So, with all that science dealt with, what did we do? We were given solutions of DNA and told to calculate the concentration of DNA in that solution. Kindly, we were given the molar extinction coefficient for DNA. The path length of the sample is 1cm, so that's all dealt with. This meant that we had to use the spectrophotometer on our sample of DNA to work out the concentration of DNA in that sample.

Absorbance spectra for DNA
The maximum absorption for DNA in our sample is 259nm, so this is the peak that we'll use. By taking the value from the absorption here and plugging it into the Beer-Lambert equation, we can use some pretty simple mathematics to work out the concentration of the DNA in the sample. And that, is precisely what we did. I won't bore you with the details of that!

All in all, it wasn't a particularly bad prac. It didn't take up the whole three hours (early home time yay!) and was all fairly straight forward. I was particularly surprised by how inaccurate micropipettes could be at times and a little bit disconcerted about the potential for error when using them. They are vastly better than the "first year pipettes" though. Using the spectrophotometer was quite fun as well. When we learned about advance analytical techniques in Chemistry in year 12, everything seemed so mystical. I, perhaps naïvely, always expected that these machines were only available to men in white coats, hidden in bunkers in the middle of the desert. Part of me wishes there were more bells and lights on the spectrophotometer; more fun!

That's experiment 1! I will do my best to keep you updated about coming experiments, though I won't be able to update you about ones for which we write proper reports. Stay mass spec-tacular!


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