Background: Most biologically important molecules, large and small, are water-soluble. Since few moderately sized to large neutral molecules are soluble in water, even if polar (WHY?), most macromolecules contain ionizable groups and are charged at physiological conditions. Their ionic character derives from weakly acidic and/or basic groups in their structure. These ionizing groups impart water solubility, are crucial for the formation of native three-dimensional conformations and often are essential for biological function (as in the catalytic ability of enzymes). The ionization state of such groups depends on the pH of their solution and on the pKa value of the protonated structure.
In this experiment, we will determine the effect of pH on the ionization state of an amino acid in aqueous solution. Amino acids contain at least one ionizable acidic group, the α-carboxyl group, and one basic group, the α-amino group. Some amino acids contain an additional acidic or basic group in the amino acid side chain. Each has its own characteristic pKa value.
This experiment will study the reaction of typical amino acids with H+ and OHˉ ions. As acid or base is added to a solution of an amino acid, we will monitor the pH with a pH meter. The resulting data can be plotted to generate the titration curve for that particular amino acid.
Like other weak acids and bases, amino acids show buffering regions. They have 2 or 3 buffering regions, usually well separated, that represent the transition between protonated and deprotonated forms of one of the ionizable groups. Amino acids are also characterized by an isoelectric point, pI, which is the pH at which the amino acid has no net electrical charge. For a simple amino acid with only an α-carboxyl and α-amino group, pI = (pKa1 + pKa2)/2. However, for an amino acid with an ionizable side chain, pI is the average of only two of the three pKa values, the two that involve a neutral species in the ionization equation.
After this experiment, you will be able to
- Write the structural formula of an amino acid in the correct ionization state at various pHs.
- Construct a graph that represents the titration curve of an amino acid.
- Identify the pKa of each ionizable group in a particular amino acid from its titration curve.
- Calculate the pI of any amino acid, given the pKa values of its ionizable groups.
- Solid amino acid sample, glycine, alanine, serine, valine, or histidine
- Standard buffer solutions for pH meter calibration (pH 4.00, 7.00 and 9.00 or 10.00)
- pH meter or Venier Labquest & pH probe
- 1 M (or higher) HCl and NaOH solutions
- KHP and oxalic acid + phenolphthalein indicator
- TRIS (free amine) and bromocresol green indicators
- Magnetic stirrer and stir bar
Standardize NaOH: Prepare approximately 250 mL of a 0.2 M NaOH solution from the stock solution. Standardize the solution with solid KHP (KHC8H4O4) or oxalic acid (H2C2H2O4) and determine the precise molarity of the base solution. Three good trials will work. Use about ~0.5 g of the solid if you are using KHP, and ~0.15 g if you are using oxalic acid. Record the precise mass before you start the titration.
If you need to review standardization with KHP, please see part A of LACC’s Standardization of NaOH experiment (from Intro Chem). If you want to use oxalic acid, please see LACC’s Standardization of NaOH experiment (from Gen Chem).
Set up and calibrate the pH meter. Rinse the electrodes with deionized water after calibration.
Weigh out about 400 mg of your assigned amino acid and record the precise mass. Dissolve the sample in 20 mL of deionized water in a 50-mL beaker. If the sample does not dissolve readily, gently heat the solution to about 50 to 60 ˚C and allow it to dissolve then cool the solution to room temperature before the titration.
Place the amino acid solution on the magnetic stirrer, add a stir bar and spin the stir bar slowly. Introduce the pH electrode so that it is immersed about ¼ inch and so that it is not being struck by the stir bar. Mount the base-filled buret so its tip is inside the beaker just above the solution.
Record the initial buret volume and initial solution pH. Then add a small portion of base, stir it in and record the buret volume and pH. Continue making small additions of acid and record the data until the pH reaches 12.00.
Similarly, titrate a 20-mL water blank (contains no solute) with the base, recording the buret readings and pH initially and after each addition. Continue until the solution reaches pH 12.0.
Prepare approximately 250 mL of a 0.2 M HCl solution from the stock solution. Standardize the solution with solid TRIS and determine the precise molarity of the acid solution. Prior to use, dry TRIS to a constant mass (~2 h) at 104 °C and cool off in a desiccator. Use about ~0.4 g of TRIS for each titration. You’ll need bromocresol green as the indicator. Bromocresol green changes from blue, through green, to yellow. The proper stopping point is at the intermediate green color, this corresponds to a pH of 4.7. Alternatively, the stockroom may have standardized HCl solutions ready for use.
Again, prepare 400 mg of your assigned amino acid as in part A and dissolve it in water.
Place the amino acid solution on the magnetic stirrer, add a stir bar and spin the stir bar slowly. Introduce the pH electrode so that it is immersed, and mount the acid-filled buret.
Record the initial buret volume and initial solution pH. Then add a small portion of acid, stir it in and again record the buret volume and pH. Continue making small additions of acid and recording data until the pH reaches 1.0.
Similarly, titrate a 20-mL sample of deionized water (a water blank containing no solute) with acid, recording the buret readings and pH initially and after each addition. Add the acid one drop at a time initially; you may add the acid more generously after about 10 drops have been added. Continue until the solution reaches pH 1.0.
To determine the true titration curve of the amino acid, you must determine how much titrant is consumed in titrating the solvent (the water blank) to each pH and then you must subtract this from the amount of titrant added to the amino acid solution at the same pH.
First, plot the uncorrected titration curves of water and of the amino acid, showing pH as a function of mL of acid solution added from the buret. Reading from the graphs, prepare a table listing pH values every 0.5 unit, the volume of acid (mL) required to titrate the amino acid sample to that pH and the volume of acid needed to titrate the water to that same pH. Then, take the difference between the two volume values and add this corrected volume data to your table.
Calculate the number of millimoles of acid represented by each corrected volume of titrant. Then, plot the pH versus mmol of acid, with the mmol axis increasing toward the LEFT from the zero point.
Use the same method to correct the data for the titration with NaOH. To the acid titration curve, add data for the mmol of NaOH consumed in the titration toward the RIGHT side of the zero point then plot the corresponding pH values. Finally, draw smooth curves connecting the data points to generate the full titration curve of the amino acid from pH 1.0 to 12.0.
Calculations and Questions
The following should be addressed in the Results and Discussion section of your report.
- For both the amino acid sample and the water blank and on the same axes, plot the volume of acid added in mL versus the pH of the solution. Clearly label each curve, label the axes and give the graph a title.
- Similarly prepare the uncorrected base titration curves of water and the amino acid on a single graph.
- Prepare and complete the data table that relates pH to corrected titrant volume for both the acid and base titration. Calculate the number of millimoles of titrant consumed for each pH.
- Construct a single titration curve for your amino acid from pH 1.0 to 12.0. Note the buffer regions (the almost flat regions with sharply rising or falling pH at each end of the plateau) on your curve. Each represents the ionization of one acidic or basic group in your amino acid. The midpoint of the plateau is the pKa of that group. Estimate the pKa of each ionizable group of your amino acid and compare it to the literature values. If there are differences, what are some of the sources or error?
- Draw the dominant structure of your amino acid at a) pH = 2.0; b) pH = 7.5 and c) pH = 11.0.
- Calculate the pI of the amino acid from YOUR data.
Follow up Questions
These questions can be answered in a separate section following the “Results & Discussion” portion of your paper.
- Calculate the theoretical pI of glutamic acid.
- How would the corrected titration curve of aspartic acid differ from that of glycine?
- How would the corrected titration curve of lysine differ from that of glycine?
- Suppose that a student performed this experiment with samples of two different masses of amino acid. For example, s/he weighed out 403 mg for the acid titration but only 385 mg for the base titration. How would this affect the titration curve and the determination of the pKa values for the ionizable groups in this student’s experiment?
- Could you use a similar procedure to determine the pI of a dipeptide glycylvaline? Explain. What is the theoretical value of the pI of the dipeptide glycylvaline? Draw the Lewis structure of this dipeptide at pI.