User:Douglas M. Fox/Notebook/AU CHEM-571 F2011 Lab Support/2014/09/02: Difference between revisions
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==Stock Solution Preparations== | ==Stock Solution Preparations== | ||
Before we begin, we must ask ourselves several questions | 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? | |||
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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. | 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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I used the maximum and minimum molar mass of amino acids to get a range for the solid sample and the single 200 μM solution for the solution sample. If you use a solid sample, use the higher concentration of NaOH, because you can always dilute but will have a harder time concentrating the solution if needed. If you use the solution, prepare a 200 μM NaOH solution. | I used the maximum and minimum molar mass of amino acids to get a range for the solid sample and the single 200 μM solution for the solution sample. If you use a solid sample, use the higher concentration of NaOH, because you can always dilute but will have a harder time concentrating the solution if needed. If you use the solution, prepare a 200 μM NaOH solution. | ||
<br> | <br><br> | ||
Now what about quantities needed? That will depend on which method you use to titrate. | Now what about quantities needed? That will depend on which method you use to titrate. If you titrate solids, you only need about 25 mL of solution for the UV and fluorescence studies. Preparing 25 mL of 200 μM would require 0.375 mg - 1.02 mg, which would be difficult to prepare accurately. So, I would recommend 100 mL. Amino acids are non-toxic, and the amounts used are still low, so there will be little waste. If instead, you are titrating the prepared solutions, then you will need at least 100 mL, preferably 250 mL. Finally, the titrant will be used for standardization and 5 amino acid titrations. You want enough to perform three trials of each, even if you do not have time to do it today. So you will need: | ||
<br> 6*3*(20 mL) = | |||
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Revision as of 14:55, 1 September 2014
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OverviewToday, 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
Stock Solution PreparationsBefore we begin, we must ask ourselves several questions
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