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

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 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

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

Alanine: An Ampholyte

zwitterion

Different pKa

Effects of Ph on Amino Acid R-group Charge

pK=pI

Henderson-Hasselbach Equation

• pH=pKa + log [base]/[acid]

• Description on page 49

Buffering Capacity

• Molecules which resist changes in pH

• Ability to resist change in pH is referred to as strength of buffer or buffering capacity

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

[3 forms of Glycine] at pH

red black blue

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

Glutamic Acid

4 forms

Histidine

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

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

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

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

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??

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

0

1

2

3

4

5

6

1000 2000 3000 4000 5000 60000

VHCl (l)

pH

Acid titration

ProtonatesCOO COOH

5

6

7

8

9

10

11

2000 4000 6000 80000

VNaOH (l)

pH

12

Base titration

Deprotonates

NH3 NH2

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

5

6

7

8

9

10

11

2000 4000 6000 80000

VNaOH (l)

pH

12

Report

• See page 58 “report requirements”

Look at thought questions

• Page 59

• Some of these may show up on exams

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.

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

Clean Up

All materials down sink with running water

Value of this lab

• Number of labs = 1

• Lab Report = 10 Points

• Pre lab questions = 3 Points