Overview
Today, we will learn about the properties of amino acids. Each group will choose a different set of amino acids with the 5 different R groups (hydrophilic, aromatic, aliphatic, acidic, and basic). You will examine the acid/base behavior, UV/VIS absorbance, and fluorescence. As you conduct the experiments, note how the absorbance spectra and fluorescence spectra match. The characterizations will introduce you to the techniques of solution preparation, titration, UV/VIS spectrometry, and Fluorescence spectrometry.
Tasklist
- Prepare Stock Solutions
- Standardize titrant
- Titrate Amino Acids
- UV/VIS Spectra
- Fluorescence Spectra
- Collect data/sample from DSC
Stock Solution Preparations
Before we begin, we must ask ourselves several questions
- How do we know what concentrations to make?
- In which experiments will these solutions be used?
- How accurate must the concentrations be?
- How much will we need?
For this experiment, we only want to examine some of the properties of amino acids so we can understand what causes the behaviors we observe with our proteins. So, the amino acid solutions will only be used for this experiment. The concentration is rather arbitrary, but we want to make sure that the UV/VIS absorbance peaks are no higher than 1 (absorbances higher than 1 no longer obey the Beer-Lambert Law) and that the fluorescence does not saturate the detector. For the former, we can estimate maximum concentrations using literature values for the extinction coefficient (molar absorptivity). For the latter, we would need the quantum yield, the laser power of the fluorimeter, and a method to correlate these to fluorescence counts. It would be faster to simply use the UV/VIS as the maximum concentration and dilute if needed or use a solution that is 10 - 20% of the UV/VIS concentration. (Note: Lower concentrations are used because fluorescence is a more sensitive instrumental technique with a lower detection limit and lower maximum concentration. As you design your own experiments next semester, you may want to consider this if you have extremely low concentrations of molecules that can fluoresce.) If you go lower, you'll get fluorescence spectra, but the UV/VIS spectra will no longer have discernible peaks.
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