Experiment 1
Experiment 1
Experiment 1
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]
Introduction
Amino Acids
(Williams Group, n.d.)
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
Methodology
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
Results
Results
Results
Results
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
The amount of the stock salt and/or weak acid needed to prepare a buffer can be calculated using the Henderson Hasselbach equation.
Discussion
Discussion
Theoretical Curves courtesy of [6] and [7] JDR says: disregard the pH=average of pKa
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
Recommendation
Mind your lab techniques. Mind the TEMPERATURE! Mind your mind. Be smart!
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.
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
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
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
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.
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
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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.