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Ionic Properties of Amino Acids

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

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Ionic Properties of Amino Acids

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  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 Ka • At pKa ratio of charged to uncharged is 1 • Two pH units away from pKa 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 pKa functional group is in its base form (Deprotonated) • pH is less than pKa functional group is in its acid form (Protonated) pKa

  4. Alanine: An Ampholyte zwitterion Different pKa

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

  6. Henderson-Hasselbach Equation • pH=pKa + 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 BufferPage 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 B A Exists in 3 forms

  9. [3 forms of Glycine] at pH red black blue

  10. H-H equation and acid-base equilibrium • @ pKa, half of the group is protonated, half deprotonated • @ pKa + 1 (ph Units), 90% deprotonated (ionized) • @ pKa + 2, 99% deprotonated • @ pKa – 1, 10% deprotonated • @ pKa – 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 CO2 • 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

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