User:Douglas M. Fox/Notebook/AU CHEM-571 F2011 Lab Support/2014/09/02

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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

  1. Prepare Stock Solutions
  2. Standardize titrant
  3. Titrate Amino Acids
  4. UV/VIS Spectra
  5. Fluorescence Spectra
  6. 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.

      Amino Acid      λmax (nm) ε (M-1cm-1) Max conc (M)
      Tryptophan (Trp,W)          280       5050     200 μM
      Tyrosine (Tyr,Y)          274       1440     700 μM
      Phenylalanine (Phe,F)          257         220     4.5 mM


If you want to make all solutions of the same concentration, then you can probably get away with making 200 μM solutions.
So what about the NaOH solution concentration? For any titrant, you want to have a concentration that will use approximately 20 mL of titrant to reach equilibrium. This will provide better precision (higher volume) without producing too much waste. If you use solid amino acid, you want to use 0.1 - 0.25 g (same reason of precision without waste). If you use a solution, you want to use 10 - 20 mL using a volumetric pipet. Both of these quantities will provide you with 3 - 4 significant figures. Let's calculate what would be needed for these:

       (0.2 g glycine)/(75 g/mol glycine)*(1 mol NaOH/mol glycine)/(0.0200 L NaOH) = 0.133 M NaOH
       (0.2 g Trp)/(204 g/mol Trp)*(1 mol NaOH/mol Trp)/(0.020 L NaOH) = 0.0490 M NaOH

       (0.0200 L Trp)*(0.000200 mol/L Trp)*(1 mol NaOH/mol Trp)/(0.0200 L NaOH) = 0.000200 M NaOH

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, you would prepare a 200 μM NaOH solution. In this case, we will be adding some HCl initially to ensure a low initial pH, so I would stick with the solid samples.

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:
       6*3*(20 mL) = 360 mL + 40 mL for buret rinsing
Note that you do not need to prepare the solution using a volumetric flask, since you will be standardizing it.

Titrant Standardization

NaOH is hydroscopic and cannot be measured accurately enough to use as a primary standard. The primary standard most often used for NaOH is potassium hydrogen phthalate (KHPhth). Standardization is performed by simple titration

  • Obtain a clean buret
  • Rinse the buret with about 10 mL of your NaOH solution. Be sure to drain some through the tip. You may use a funnel to fill.
  • Empty completely and repeat 1 - 2 times.
  • Fill completely with NaOH and drain some through the tip. Tap to remove all air bubbles. Remove the funnel.
  • Record the initial volume. It does not have to equal one, but you need at least 25 mL of titrant. Remember, top line reads 0 mL.
  • Transfer about 0.5 g of KHPhth to an Erlenmeyer flask and add 15 - 30 mL of water. (How did I get these values?)
  • Or, transfer 20 mL of 0.2 mM KHPhth that you have prepared (or 1 mL of 4 mM if you needed higher mass for accuracy)
  • Add a few drops of methyl red phenolphthalein solution
  • Titrate until the solution turns orange pink. If it is yellow dark red, you overshot. You can periodically rinse sides with wash bottle.
  • Repeat at least twice

Amino Acid Titration

  • Transfer 0.2 g of solid amino acid to a clean and dry 100 mL beaker (Why does it need to be dry?)
  • Add 10 mL of 20 mM HCl and 10 mL water
  • insert your pH probe and record the initial value
  • Add titrant in 2 - 4 mL intervals. Swirl gently, being careful not to splash. Record the pH.
  • Continue until the pH reaches at least 10 or 11.
  • For the first trial, you will likely skip past the inflection points. On 2nd & 3rd trials, slow addition to 0.2 or 0.1 mL near inflections.
  • Repeat for all 5 amino acids

Spectra

Be sure to record the spectra of your solutions

  • Examine your AA solutions, NaOH stock, protein stock, and gold stock solutions
  • You may need to dilute to obtain absorbance below 1
  • Note how absorbance wavelength and shape compares to the fluorescence
  • Try fluorescence with both 230 nm and 280 nm excitation wavelengths
  • If you have time, try an excitation spectra using your maximum emission wavelength from your 280 nm excitation curve
  • Remember that you can't have an emission at wavelengths below the excitation wavelength