1. Ionic Properties of Amino Acids Lab B.1, pages 48 to 64 In lab manual

2. Bronsted Acids and Bases Have groups which can accept or donate hydronium ions (H + ) Have acid dissociation constants K a –At pK a ratio of charged to uncharged is 1 –Two pH units away from pK a functional groups are completely (almost) charged or discharged. –Within this range there exists a ratio between the charged and uncharged groups Amino Acids have at least 2 of these

3. Acid-Base Pairs pH greater than pK a functional group is in its base form (Deprotonated) pH is less than pK a functional group is in its acid form (Protonated) pK a

4. Alanine: An Ampholyte zwitterion Different pK a

5. Effects of Ph on Amino Acid R- group Charge pK=pI

6. Henderson-Hasselbach Equation pH=pK a + log [base]/[acid] Description on page 49

7. Buffering Capacity Molecules which resist changes in pH Ability to resist change in pH is referred to as strength of buffer or buffering capacity

8. Glycine As a Buffer Page 50&51 Ampholyte –Has both acidic and basic groups –As base is added the acidic group group looses protons first Glycine can buffer in two ph ranges –2.3 & 9.6 Exists in 3 forms A B

9. [3 forms of Glycine] at pH redblackblue

10. H-H equation and acid-base equilibrium @ pK a, half of the group is protonated, half deprotonated @ pK a + 1 (ph Units), 90% deprotonated (ionized) @ pK a + 2, 99% deprotonated @ pK a – 1, 10% deprotonated @ pK a – 2, 1% deprotonated

11. Glutamic Acid 4 forms

12. Histidine

13. Technique can be used to characterize proteins Determines Isoelectric point –Point at which 50% protonated 50% deprotinated –Protein has neutral charge and comes out of solution A mechanism for separating proteins according to their amino acid composition

14. Gameplan Will show you how to use Henderson- Hasselbach equation –To determine the buffering capacity of an unknown amino acid –Determine the molecular weight of the unknown Know mg unknown Know moles of acid or base to completely protinate or deprotonate Expect lab exercise ~ 2 hours

15. Important This lab works when you measure carefully Read pH only after allowing 30 seconds for mixing –Do not mix at speeds that make bubbles CO 2 –Be sure that magnetic stir bar does not hit electrode End point of titrating a group is when pH changes rapidly with addition of acid/base

16. Use titration procedure described on page 52 &53 Add a volume of acid or base to your unknown or to water and then determine resulting pH Plot as Figure B.1-4 page 54(see also page 64) –Spread sheet on page 62 & 63 In the NaOH titration you will need about 7 ml of base to complete titration

17. Data collection See manual page 55-56 Enter data in table format for water and water + unknown –Do for acid titration and base titration Examples of titration plots on page 64 Calculate molecular weight as on page 56 for both acid and base. –B. The base calculation is more accurate WHY??

18. Tips Calibrate pH meter for Acid OR Base –Page 51 Titrate water Weigh out unknown Titrate unknown + water (do not recalibrate Repeat for other titration

19. Acid titration Protonates COO COOH

20. Base titration Deprotonates NH3 NH2

21. Protocol Follow dilution protocols on page 62 & 63 Calculate molecular weight after subtracting water titration –Moles NaOH to titrate unknown minus moles to titrate water to same pH equals moles to titrate amino acid –Moles NaOH equals Moles amine

22. Report See page 58 “report requirements”

23. Look at thought questions Page 59 Some of these may show up on exams

24. SAFETY FIRST! Acid burns sting or burn. Base burns feel slippery; after time, skin will be raw. Splashes on skin: Wash with lots of water.

25. Lab Exit Requirements Page 57 Show TA #s 1 & 2 ( Data table and titration curves 62-63) Calculate molecular weight TA will provide name of the unknown

26. Clean Up All materials down sink with running water

27. Value of this lab Number of labs = 1 Lab Report = 10 Points Pre lab questions = 3 Points