The laboratory is an area of endless research, incredible technical skill and a large amount of mishaps! As an biochemistry undergraduate, I spend a considerable amount of time in the lab with the sole purpose of trying to learn the technical methods that enhances scientific understanding and inevitably proceeds research. On my Instagram stories, I asked you guys what posts you would like to see on my blog and overwhelmingly, you guys were interested in my labs. This semester I have labs every other week and a few labs in the middle of the semester, so I will be doing a series here talking about each of these labs. These will include the background behind the lab, the procedures that I learned during the actual lab and post-lab work. I hope this gives you guys a good insight into how labs might be like at university, and hopefully you find it enjoyable learning about all that can go wrong (only occasionally does it go right!) in the lab.
In this specific blogpost, I’ll be talking about a lab I completed during my second week of semester B. This was for my Metabolic Pathways module. I’ll start off by talking about the background information about measuring absorbance and transmittance, why these measurements are useful, the actual lab procedure itself, and my analysis afterwards.
Background
So this lab was concerned about learning about the importance measuring absorbance using the spectrometer and using the appropriate cuvette for the appropriate wavelength. We also did a little bit of Thin Layer Chromatography (TLC), but the majority of the lab was focused on using the spectrometer and the mathematics surrounding absorbance and transmittance. The appropriate equation to use is the Beer-Lambert Law. Take a look below to see how it is.
*image of equation*
Essentially, what this law tells us is that the loss of light intensity when transmitted within a medium is directly proportional to the intensity and the path length. It also describes that “the absorbance of light is proportional to the concentration of light-absorbing molecules”. From this equation we can deduce concentrations of solutions, and if we know the concentration – we can work out the absorbance, transmittance and associated rates and patterns.
This lab was split into three practical experiments, the latter two were concerned with the ideas of absorbance, transmittance and Beer-Lambart laws whilst the first experiment utilised TLC to distinguish between compounds.
The Practical
Experiment One
In this experiment, we were given three different urine samples (A, B and C) and one sample of pure progesterone. We were tasked with determining which urine sample was from which patient. The three patients were as followed:
- A 30-year-old female at the beginning of her oestrous cycle
- A pregnant woman
- A 20-year-old male
The experiment was fairly simple. On TLC strips, we pencilled a line 1.5cm from the bottom of the strip and put 4 crosses along this line. We pipetted each solution to its respective cross, and this TLC plate was placed in a chromatography tank so it can develop in a solvent mixture. This was a mixture of chloroform and ethanol at a 9:1 ratio. After about an hour, we got our TLC plate back and examined our results. What we saw was that the progesterone travelled the entire distance (and thus, is the solvent front), A split into two compounds at different distances – 3.6cm and 4cm. B travelled the same distance as A, but the second separation was much thicker and C travelled the same distance but only produced one spot. From these results we can conclude that sample A was the menstruating woman, B was the pregnant woman and C was the young man. This conclusion arose from analysing the interactions between the compounds (and their relative concentration) in each urine samples and the solvent. For example, it is likely that the pregnant woman had more progesterone in her urine, and thus produced a larger spot on the TLC plate than the menstruating woman.
Experiment Two
This experiment was concerned with teaching the importance of utilising the correct type of cuvette in the spectrometer. Essentially, we had three different cuvettes and we measured the transmittance value of these after blanking with air (so there was nothing in the cuvette holder, and air was used as the baseline to compare absorbance). We also switched modes so we could see the % transmittance too. From this, we saw that the highest absorbance was varied amongst cuvettes depending on what wavelength was used. We saw that cuvettes A and C read an absorbance reading of ‘>2.5’ in some cases, meaning the spectrometer could not accurately determine the absorbance at a certain wavelength, rendering them incompatible for our next experiment.
Experiment Three
This last practical was concerned with determining the absorbance spectrum of riboflavin – a B vitamin that is required for normal cell growth and function. This was done by setting the spectrometer to wavelengths from 220nm-500nm. Cuvette B was used, and a blank was prepared by pipetting 2ml of distilled water into one cuvette. Another cuvette was filled with 0.5ml of riboflavin and 1.5ml of distilled water, and the absorbance spectrum was determined by simply blanking the instrument and then placing the riboflavin-containing cuvette in the instrument. A graph was produced, and important data was noted.
After obtaining this spectrum, one grain of sodium dithionite was added to the cuvette and a new spectrum was found.
Data Analysis
The data analysis for this lab was somewhat simple. Calculations were focused on determining correct dilution factors and as mentioned previously, manipulating the Beer-Lambert law to gain appropriate information from our data. For example, for experiment three we utilised our data to calculate the molar absorptivity of riboflavin at its peak wavelength. We also had a variety of post-lab questions and exercises which were centred around how to choose the correct dilution of samples and calculating % coefficients – concepts I was not familiar with before.
Conclusions
In hindsight, this was a simple lab to get us used to the spectrometer and comfortable with the necessary calculations for both lab preparation and data analysis. Throughout the semester, our labs built on these concepts and new enzymatic reactions were used to see how absorption differs throughout a reaction, and what this means for the data and the actual enzyme.
I hope you guys enjoyed this slightly different style of blogpost – let me know if you liked this focus on labs, and I’ll try to deliver more!
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(Photo Credit: CDC)
The Anonymous Biochemist 