1. CHMI 2227 - E.R. Gauthier, Ph.D. 1 CHMI 2227E Biochemistry I Refresher: - acid-base chemistry - spectrophotometry

2. CHMI 2227 - E.R. Gauthier, Ph.D.2 Acid base chemistry - strong acids HCl  H + + Cl - pH = - log [H + ] Where [H + ] is in molar (M) concentration units.

3. CHMI 2227 - E.R. Gauthier, Ph.D.3 Acid base chemistry - weak acids Ka = [H + ] x [A - ] [HA] pKa = -log Ka Ka = 1 10 pKa HA H + + A -

4. CHMI 2227 - E.R. Gauthier, Ph.D.4 Acid base chemistry - weak acids pH = pKa + log [A - ] [HA] Ka = [H + ] x [A - ] [HA] pH = - log [H + ]

5. CHMI 2227 - E.R. Gauthier, Ph.D.5 Acid base chemistry - weak acids: titration pH NaOH Mid-equivalence point: - 50% HA - 50% A - 0.5 pH at mid-equivalence point = pKa pKa HA H + + A - 100% HA 100% A -

6. CHMI 2227 - E.R. Gauthier, Ph.D.6 Acid base chemistry - weak acids Example 1: What is the pH of a 0.1 M solution of acetic acid (Ka = 1.76 x 10 -5 M).

7. CHMI 2227 - E.R. Gauthier, Ph.D.7 Acid base chemistry - weak acids Example 2: What will be the pH of a solution made of 0.3 M acetic acid and 0.1 M acetate (pKa = 4.8).

8. CHMI 2227 - E.R. Gauthier, Ph.D.8 Spectrophotometry Incident light Transmitted light Incident light Transmitted light Detector Cuvette Light source

9. CHMI 2227 - E.R. Gauthier, Ph.D.9 Spectrophotometry

10. CHMI 2227 - E.R. Gauthier, Ph.D.10 Spectrophotometry Absorbance intensity Wavelength (nm) 400450500550600

11. CHMI 2227 - E.R. Gauthier, Ph.D.11 Spectrophotometry

12. CHMI 2227 - E.R. Gauthier, Ph.D.12 Spectrophotometry - Beer-Lambert equation -logT = log (1/T) =  cl =A Where: T = transmittance A = absorbance l = light path (cuvette size)(cm) c= concentration of analyte (M)  = absorption coefficient (M -1 cm -1 )

13. CHMI 2227 - E.R. Gauthier, Ph.D.13 Standard curves Absorbance intensity Concentration(units) 36912 Absorbance of the unknown Concentration of the unknown Non-linear part of the curve. BEWARE!!!

14. CHMI 2227 - E.R. Gauthier, Ph.D.14 Spectrophotometry