Experiment 1 Amino Acids as Ampholytes

Biochemistry 14.1 HEJ Espanol. Kaitgbak. Pabilane.

Introduction

Weak Acids
HA A- + H+ Ka = [A-][H+] [HA] Henderson-Hasselbach equation
pH = pka + log [A-]_ [HA] Half-equivalence point: pH = pka, if [A-] = [HA]

Monoprotic or polyprotic AMINO ACIDS!!

Introduction

2-aminocarboxylic acids Functions:

Amino Acids
(Williams Group, n.d.)

Component of peptides and proteins

20 proteinogenic, classified according to R group
Nonproteinogenic: ornithine, citrulline, Dopa, Selenocysteine Neurotransmitters, Precursors of metabolites, Transport molecule for NH2 groups

Chirality: -C, enantiomers (L and D form), *Not Glycine* Dominant form in solution is pH dependent Isoelectric point: pI = (pKa1 + pKa2)/2
Zwitterion form neutral net charge Amphoteric with acidic and basic properties. AMPHOLYTE!

Buffered
(Koolman and Rohm , 2005)

Introduction

Buffers
Solutions which resist drastic changes in pH when a small amount of acid or base is added Made up of weak acids or bases and their salts In biological systems:
Weak acid and conjugate base Amino acid and protein

Introduction

The Experiment
To understand the acid/base properties of amino acids and buffers in general
To prepare buffers of certain pH and concentration To construct and interpret the titration curve of amino acids To describe the buffering action of amino acids

Methodology

Preparing a buffer
Select pH
Mix in a container Adjust pH
pH of buffer ~ pka of acid 0.1M buffer, compute for the value of needed reagents

Acid to water

pH meter

Dilute
Store

Volumetric flask Distilled water Vtotal = 250mL

Acid: colored bottles, e.g. Amber Refrigerated

Methodology

Constructing a titration curve

Prepared 20mL H3PO4 solution and titration set up. Plot pH vs volume of NaOH added. Interpret.

Titrate and record pH and VNaOH added.

Results

Preparing a buffer
250 mL 0.1 M Phosphate buffer Group No. 1 and 2 3 and 4 pH 6.0 7.4 Starting Material Salts Salts Amount needed 2.73g NaH2PO4 and 0.3227g Na2HPO4 0.072g NaH2PO4 and 0.028g Na2HPO4

5 and 6
7 and 8 9 and 10

7.4
8.0 10.0

6M H3PO4 and 6M NaOH

Salts Salts

4.17mL H3PO4 and 7.15 mL NaOH

0.273g NaH2PO4 and 3.23g Na2HPO4 g NaH2PO4 and g Na2HPO4

Results

Preparing a buffer from salts (Sample Computations)

Given: 250mL 0.1 M phosphate buffer, pH=6.0, pKa2=7.0 Equations: 1. 6.0 = 7.0 + log {[HPO42-]/[H2PO4-]} 2. 0.1 = [HPO42-] + [H2PO4-] Using system of equations: [HPO42-] = [Na2HPO4] = 9.09 x 10-3 M [H2PO4-] = [NaH2PO4] = 0.09091 M NaH2PO4 : 0.9091 mol/L = X(1mol/120g)(1/0.250L) X = 2.7273 g NaH2PO4 Na2HPO4: 9.09 x 10-3 mol/L = Y(1mol/142g)(1/0.250L) Y = 0.3227 g NaHPO4

Results

Preparing a buffer from stock solutions

Given: 250mL 0.1 M phosphate buffer, pH=7.4, pKa2=7.0 Equations: 1. 7.4 = 7.0 + log {[HPO42-]/[H2PO4-]} 2. 0.1 = [HPO42-] + [H2PO4-] Using system of equations: [HPO42-] = 0.07151 M [H2PO4-] = 0.02849 M ICE table X mL NaOH = (0.0250 mol + 0.017875 mol)/(6 mol/1 L) X = 7.15 mL NaOH Y mL H3PO4 = (0.0250 mol)/(6.0 mol/1 L) Y = 4.17 mL H3PO4

Results

The titration curve (data)

Volume of NaOH pH of Solution 0 1.61 1 1.69 2 1.75 3 1.81 4 1.89 5 1.95 6 2.06 7 2.15 8 2.25 9 2.35 10 2.47 11 2.61 12 2.77 13 3.03 13.5 3.27 14 3.59 14.5 4.67 14.7 5.12 15 5.46 16 5.98 17 6.23 18 6.41 19 6.55 20 6.77 21 22 23 24 25 26 27 27.2 27.4 27.6 28.6 29.6 30.6 31 31.4 31.8 32.2 32.6 33 33.4 33.8 34.2 34.6 35 6.89 7.02 7.18 7.36 7.61 7.98 8.99 9.69 10.01 10.25 10.68 11.08 11.25 11.3 11.34 11.39 11.42 11.43 11.45 11.49 11.54 11.56 11.57 11.58 35.4 35.8 36.2 36.6 37 37.4 37.8 38.2 38.6 39 39.4 39.8 40.2 40.6 41 41.4 41.8 42.2 42.6 43 43.4 43.8 44.2 44.6 45 11.6 11.62 11.64 11.66 11.69 11.69 11.71 11.74 11.76 11.78 11.79 11.79 11.81 11.83 11.85 11.86 11.88 11.89 11.91 11.92 11.93 11.95 11.96 11.98 11.99

Results

The titration curve

pH vs VNaOH (mL)
14 12 10 8 6 4 2 0 0 3 6 9 12 14 15 18 21 24 27 27.6 30.6 31.8 33 34.2 35.4 36.6 37.8 39 40.2 41.4 42.6 43.8 45 pH

Experimental titration curve of H3PO4 Theoretical Curves courtesy of [6] and [7] JDR says: disregard the pH=average of pKas

Discussion

Amino acids and buffers

Amino acids are: ampholytes dominant form is pH dependent: buffered In preparing buffer solutions, consider the desired pH for the buffer solution the pKa of the weak acid (near the pH) the concentrations of the acid and its salt
Individual concentration range of weak acid and salt: 0.05 0.5 M (ionic interaction)

The amount of the stock salt and/or weak acid needed to prepare a buffer can be calculated using the Henderson Hasselbach equation.

Discussion

Amino acids and buffers

Phosphoric acid buffer

H3PO4 + H2O H3O+ + H2PO4-

H2PO4- + H2O H3O+ + HPO4-2

HPO4-2 + H2O H3O+ + PO4-3
H3PO4 and PO4-3 cannot function as a buffer system. Neutralization would occur.

Discussion

The titration curves

Relationship of pH and added Levelling out buffering range pH = pKa if [HA] = [A-] pI = (pKa1 + pKa2)/2

Theoretical Curves courtesy of [6] and [7] JDR says: disregard the pH=average of pKa

Conclusion and Recommendation

Conclusion
Amino acids have acidic and basic properties: ampholytes. Buffered to stabilize pH Buffer preparation and action Le Chateliers Principle Concentration, pH, volume, temperature, nature of components Titration curve construction and interpretation Titration curve of polyprotic acids and amino acids are similar. In amino acids: pI [isoelectric point], zwitterionic form

Conclusion and Recommendation

Recommendation
Mind your lab techniques. Mind the TEMPERATURE! Mind your mind. Be smart!

Answers to Guide Questions

Guide Question 1
How would you prepare a 0.1 M buffer using 0.5 M stock solutions of the acid and its salt?
1.Choose total volume, pH and buffer concentration. 2.Compute for the volume of stock solution needed to satisfy the given concentration using HHE. 3. Dissolve in distilled water in flask following proper lab techniques. 4.Adjust pH accordingly using pH meter. 5.Dilute to desired volume using a volumetric flask. 6.Store in a glass bottle kept in a refrigerator.

Answers to Guide Questions

Guide Question 2
What is the biological significance of pH? How does it affect biological activities and functions?

1. pH measures the amount of protons in a biological system; physiological pH is from 6.8-7.4. 2. pH affects chemical reactions in biological systems in that it limits the existence and spontaneity of reactions. Outside the pH range:
1.membranes disrupted 2.Low enzyme activity 3.Inefficacy and denaturation of proteins 4.Precipitation of other nutrients

3. A solution: buffer systems 4. E.g. Blood. [Recall Maam Lirazans lec]

Answers to Guide Questions

Guide Question 3
Why is the observed pH different from the actual pH?
1. The temperature factor
1. increase in temp, decrease in pKa and pH

2. pKa not so near pH of buffer 3. Random and systematic error

Answers to Guide Questions

Guide Question 4
What is the significance of the levelling shown in your diagram in terms of the buffering action of your amino acid?
1.Levelling: shows range of the buffering capacity of an amino acid. 2.At levelling point: [species 1] = [deprotonated species 1], pH=pKa

Answers to Guide Questions

Guide Question 5
What is the significance of the titration curve of your acid?
1.Shows complex relationship between pH and amount of added base 2.Levelling: buffering capacity of acid, pH=pKa 3.Steep curve: Equivalence point 4.Determine the species present in solution.

Answers to Guide Questions

Guide Question 6
How does it [titration curve of acid] compare with amino acids [titration curve]? 1. Similar. Very similar.
1. As more base is added, pH increases 2. Area of maximum buffering capacity, pH = pKa +- 1

2. Difference: Isoelectric point. Amino acid exists as zwitterions [net neutral charge].

References
1. 2.

3.
4. 5. 6. 7.

8.

Committee on Biochemistry. (n.d.). Laboratory Manual in Biochemistry. Philippines: Department of Physical Sciences and Mathematics, College of Arts and Sciences, University of the Philippines Manila. Nicholas, M. (2002). Modules in Biochemistry Part I: Structure and Function. Philippines: University of the Philippines Manila. Koolman and Roehm. (2005). Color Atlas of Biochemistry, 2nd ed. Germany: George Thieme Verlag Rudigerstrasse. Williams Group. (n.d.). Biological Mass Spectrometry & Biophysical Chemistry. Retrieved August 10, 2011 from http://www.cchem.berkeley.edu/erwgrp/science_old.html _____. (2005). Titration Set-up. The Florida State University. Retrieved August 10, 2011 from http://www.chem.fsu.edu/chemlab/chm1046lmanual/titration/background.html. Bialkowski. (2004). Triprotic Acid Titration with Strong Base. www.chem.usu.edu. Retrieved August 10, 2011 from http://www.chem.usu.edu/~sbialkow/Classes/3600/Overheads/H3A/H3A.html. Department of Biochemistry and Molecular Biophysics. (2003). Biochemistry 462a: Aminio acids and Peptides. www.biochem.arizona.edu. Retrieved August 10, 2011 from http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/Amino_Acids/amino_acids.htm. Ballad and Dones. ()n.d.) Amino acids as ampholytes. www.docstoc.com. Retrieved August 11, 2011 from http://www.docstoc.com/docs/22135989/Amino-Acids-as-Ampholytes.