Functional Genomics Research Stream. Lecture: February 24, 2009 Buffer & Reagent Production, ph

Transcription

1 Functional Genomics Research Stream Lecture: February 24, 2009 Buffer & Reagent Production, ph

2 Agenda State of the Union Acid / Base Theory Buffers in Physiology Buffers in the Laboratory Assignment Six

3 End of Phase I Phase I: Molecular Lab Skills & Communication Phase II: Cell Growth & Maintenance Phase III: Molecular Techniques & Diagnostics Phase IV: Biological Assays & Interpretation

4 Acids & Bases

5 Acid / Base Theory (aka Brønsted Lowry Theory) Acid: any chemical species (molecule or ion) that is able to lose, or donate a hydrogen ion (proton). Base: any chemical species with the ability to gain or accept a hydrogen ion (proton). Wikipedia, General Chemistry: An Integrated Approach: Hill, Petrucci, 4th Edition, Mark P. Heitz

6 Strong Acids & Bases Strong acids and bases completely dissociate in aqueous solution. Mix strong acid + strong base: 1) Mix. 2) Ionic dissociation. 3) Salt & Water. Example: 1) HCl + NaOH 2) H + Cl - Na + OH - 3) NaCl + H2O

7 Weak Acids Dissociates incompletely. Does not release all hydrogens in solution. Does not donate all of its hydrogens.

8 Weak Base Dissociates incompletely. Does not accept all hydrogens in solution. Some times called incomplete protonation.

9 Weak Acids & Bases Incomplete dissociation occurs. Wikipedia, General Chemistry: An Integrated Approach: Hill, Petrucci, 4th Edition, Mark P. Heitz

10 Conjugate Acids & Bases The conjugate acid of a base is the base plus the attached proton and the conjugate base of an acid is the acid minus the proton. Wikipedia, General Chemistry: An Integrated Approach: Hill, Petrucci, 4th Edition, Mark P. Heitz

11 Conjugate Acids & Bases HCl Hydrochloric acid Acids Cl Chloride ion Bases H2SO4 Sulfuric acid HSO4 Hydrogen sulfate ion HNO3 Nitric acid NO3 Nitrate ion H3O + Hydronium ion H2O Water HSO4 Hydrogen sulfate ion SO4 2 Sulfate ion strength H3PO4 Phosphoric acid H2PO4 Dihydrogen phosphate ion strength HC2H3O2 Acetic acid C2H3O2 Acetate ion H2CO3 Carbonic acid HCO3 Hydrogen carbonate ion H2S Hydrosulfuric acid HS Hydrogen sulfide ion H2PO4 Dihydrogen phosphate ion HPO4 2 Hydrogen phosphate ion NH4 + Ammonium ion NH3 Ammonia HCO3 Hydrogencarbonate (bicarbonate) ion CO3 2 Carbonate ion HPO4 2 Hydrogen phosphate ion PO4 3 Phosphate ion H2O Water (neutral, ph 7) OH Hydroxide ion HFSbF5 Fluoroantimonic acid SbF6 Hexafluoroantimonate ion Wikipedia, Conjugate Acids & Bases

12 Measuring Strength

13 Dissociation Constant Ka acid dissociation constant acidity constant acid-ionization constant Quantitative measure of the strength of an acid. Higher Ka means stronger acid. Wikipedia, General Chemistry: An Integrated Approach: Hill, Petrucci, 4th Edition, Mark P. Heitz

14 Log Transformation Common in science, bioinformatics. Order of magnitude changes. pka = -log10 Ka Weak acids have higher pka compared to strong acids, which release all of their hydrogens when dissolved in water.

15 ph Measure of acidity or basicity of a solution. Minus log of free hydrogen ions activity. Order of magnitude changes (7, 8, 9, etc). Wikipedia, General Chemistry: An Integrated Approach: Hill, Petrucci, 4th Edition, Mark P. Heitz

16 Controlling ph (using conjugate acids & bases to do our evil bidding)

17 Biological Buffers: Purpose Biological systems tend to dislike significant shifts in ph. Our environment is full of weak acids and bases that can easily shift biological systems from a ph. Buffer systems prevent these shifts.

18 Biological Example: Blood Ideal blood ph ~ 7.4 As low as 6.8, death. As high as 7.8, death. CO2 and H + produced during processing of glucose.

19 Biological Example: Blood When blood ph rises, carbonic acid dissociates to form bicarbonate and H +. H2C03 HC H + When blood ph drops, bicarbonate binds H + to form carbonic acid. HC H + H2C03 General: HC H + H2C03 H2O + CO2

20 Similar Laboratory Concerns (not death, but rather ph shifts during experiments)

21 Buffer Solutions Solutions used in a laboratory setting to control the ph of experimental settings. Goal = ph is held nearly constant. The ph is easily shifted by addition of: other reagents temperature evaporation

22 Buffer Solutions: Examples Common Name pka at 25 C Buffer Range Mol. Weight Full Compound Name TAPS {[tris(hydroxymethyl)methyl]amino}propanesulfonic acid Bicine N,N-bis(2-hydroxyethyl)glycine Tris tris(hydroxymethyl)methylamine Tricine N-tris(hydroxymethyl)methylglycine HEPES hydroxyethyl-1-piperazineethanesulfonic acid TES {[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid MOPS (N-morpholino)propanesulfonic acid PIPES piperazine-n,n -bis(2-ethanesulfonic acid) Cacodylate dimethylarsinic acid MES (N-morpholino)ethanesulfonic acid Wikipedia, Buffer Solutions

23 Work This Week

24 Assignment Six Purpose: Phase I Review (no exams in this course) Learning ph Meter Use & Calibration Buffer Production (ph) Buffer Titrations, Excel Data Analysis Laboratory Notebooks (always) Due: Tuesday, March 3

25 Section A Quick review of concepts to date. In lieu of examinations. End of Phase I.

26 Section B: ph Meters Calibration & Use

27 Section C Buffer / Reagent Production With ph Adjustment Multi Component Solution Preparation

28 Section D Buffer titrations. Characterize two buffers, two titration methods. Attempt titration, reverse titration.

29 Titration Curves 14 equivalence pt equivalence point ph ½ equivalence pt ½ equivalence point 1 ml ½ equivalence pt ml equivalence pt 1 addition of x ml base (NaOH) ~30

30 Section E Microsoft Excel data analysis. Data input. Chart production.

31 Questions?