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)
pKa
• pH is less than pKa functional group is in
its acid form (Protonated)
Alanine: An Ampholyte
zwitterion
Different pKa
Effects of Ph on Amino Acid Rgroup 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
B
A
Exists in 3 forms
[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 HendersonHasselbach 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
Acid titration
6
Protonates
COO COOH
5
4
pH 3
2
1
0
0
1000
2000
3000
VHCl (l)
4000
5000
6000
Base titration
12
11
Deprotonates
NH3
NH2
pH
10
9
8
7
6
5
0
2000
4000
6000
VNaOH (l)
8000
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
12
11
10
pH
9
8
7
6
5
0
2000
4000
6000
VNaOH (l)
8000
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