TITRATION OF

AMINO ACIDS
Biochem C3
I-CMED Class 2016
Mendez | Mendiola | Mendoza | Mendoza | Mendoza
Mercado | Mercado | Miguel | Milante | Miranda
Mirano | Mojica | Momani | Montalbo | Montalbo
Montenegro | Mopia | Morales | Moya
I
total acid neutralizing
capacity of solution
etymology
Greek Titulus (Title)
French Titre (Rank)
determination of the
quantity of substance A
by adding measured
increments of substance B
TITRATION
reflects acid and base
X : volume of the titrant
Y : pH of titrant-analyte

TITRATION CURVE
DEFINITION OF TERMS
Titrant
standardized substance reacted with analyte to determine
analyte concentration
Analyte
substance being analyzed
Indicator
used to mark end point ; dye or pH meter
Equivalence Point
point when amount of added standard reagent is exactly
equivalent to amount of analyte
End point
point in titration when physical change associated with
condition of chemical equivalence occurs


APPLICATION OF TITRATION
Medical
drug concentrations
IV drip
CBG
pregnancy test
urinalysis
Food Industry
fatty acid chain length
Biodiesel Production
acidity of waste vegetable oil
Aquarium Water Testing

TYPES OF TITRATION
Acid-Base
neutralization
Complexometric / Chelatometric
volumetric analysis
colored complex as endpoint
Oxidation-reduction
redox reactions
Precipitation
ionic compounds of limited solubility
silver nitrate

neutralization reaction
acid/base of
concentration (titrant)
reacts with acid/base of
unknown concentration
(analyte)

ACID BASE TITRATION
amphoteric electrolytes
(ampholytes)
ionizable groups as weak
acids/bases
ionizable R groups
influences behavior
during titration
Amino
Carboxyl
Guanidinium
P-hydroxyphenyl
Imidazole

AMINO ACIDS
AMINO ACIDS

contains
acidic (amine) group
basic (carboxyl) group
in aqueous solution
side chain which ionize
depending on the pH
can behave as acid &
base


IONIC PROPERTIES OF AMINO ACIDS


IONIC PROPERTIES OF AMINO ACIDS

Henderson-Hasselbalch equation:

unprotonated = protonated
(concentrations)
ratio equals 1
log 1=0
pKa pH
at which concentrations of
protonated and
unprotonated forms of an
ionizable species are equal
at which the ionizable
group is at its best
buffering capacity


IONIC PROPERTIES OF AMINO ACIDS

Isoelectric Point (pI)
pH at which net charge on
a molecule is zero
(zwitterions)
If pH<pI, net charge is
positive
If pH> pI, net charge is
negative
average of two pK values

IONIC PROPERTIES OF AMINO ACIDS

OBJECTIVES
to determine the acid-base behavior of three
amino acids upon titration with a strong acid
and a strong base
to determine concepts in the reactions as
represented in various points on the titration
curve
to observe & characterize the effect of
formaldehyde on the titration curve of amino
acids
glassware
burettes
beakers
pipettes
pH meter
stir bar

MATERIALS
Burettes
volumetric graduation
stopcock
Miscellaneous
beakers
pipettes




GLASSWARE
Probe
thin-walled glass bulb at
tip
activity of hydrogen
cations
Glass Electrode Principle
electric potential from
electrode in solution
sensitive to changes in ion
content (H+ in titration)


PH METER

magnetic stir bar or flea
rotating magnet at
platform
teflon or glass coated
STIR BAR
0.1 N NaOH
0.1 N HCl
0.1 M glycine solution
0.1 M lysine solution
0.1 M aspartic acid solution
neutralized formaldehyde


REAGENTS
highly caustic metallic
base
very soluble in water
prototypical base
SODIUM HYDROXIDE
highly corrosive and
strong mineral acid
consists of a hydrogen ion
and the non-reactive,
non-toxic chloride ion
one of the least hazardous
strong acids to handle

HYDROCHLORIC ACID
side chain: H
NEUTRAL
non-polar
non-essential
pk
1
(-COOH) = 2.35
pK
2
(-NH
3
) = 9.78

side hain: (CH
2
)
4
NH
2
BASIC
polar
essential
pk
1
(-COOH) = 2.16
pK
2
(-NH
2
) = 9.06
pK
R
(R-group) = 10.54
side chain: CH
2
(COOH)
ACIDIC
polar
non-essential
pk
1
(-COOH) = 1.99
pK
2
(-NH
2
) = 9.90
pK
R
(R-group) = 3.90

METHANAL
rarely found in original
state
used in preservation
water-soluble
aldehyde
donates H
+
to amino acid
lowering pH



1
Prepare two pipettes and fill the first with
0.1 N HCL and the second with 0.1 NaOH
2
Prepare two beakers for each amino acid
and fill it with 10.0 ml of the amino acid
solution
3
Measure first the pH of the amino acid
solution
4
Titrate first with 0.1N HCL adding 2.0ml at
a time (2ml, 2ml, 1ml, 1ml, 2ml, 2ml, 2ml,
2ml, 1ml ,1ml, 2ml, 2ml)
5
Determine pH after each addition
6
Titrate again this time with 0.1 NaoH,
same manner of addition with the first
7
Determine again pH after each addition
8
Repeat titration for the other amino acids
9
Repeat step two but add 5.0 mL of
neutralized formaldehyde solution in
each amino acid
10
Titrate again each solution getting pH
after each addition
11
Plot pH (ordinate)
vs equivalent
acid/base (abcissa)
12
Solve for the pI
and pK values of
your amino acids
13
Construct the
titration curves
pH at the midpoint of
buffering region
pH changes only slightly
after addition of acid/base
inflection point in the
titration curve
apparent pK values for 2
dissociation steps
extrapolated from the
midpoints
PK VALUE
pH at which amino acid
has net zero charge
for simple diprotic amino
acid:
pI falls halfway between
the 2 pK values
for acidic amino acids
pI = (pK
1
+ pK
2
)
for basic amino acids
pI = (pK
2
+ pK
3
)

ISOELECTRIC POINT (PI)
ASPARTIC ACID
Acidic: ASPARTIC ACID in 0.1 N HCl
0.1 N HCl
(mL)
mEq pH (w/
HCHO)
pH (w/o
HCHO)
0 0 6.18 6.26
2 0.2 4.91 4.88
4 0.4 4.44 4.46
5 0.5 4.26 4.28
6 0.6 4.12 4.13
8 0.8 3.81 3.86
10 1 3.54 3.55
12 1.2 3.26 3.25
14 1.4 3.01 3
15 1.5 2.88 2.88
16 1.6 2.8 2.77
18 1.8 2.66 2.63
20 2 2.55 2.5
ASPARTIC ACID
Acidic: ASPARTIC ACID in 0.1 N NaOH
0.1 N NaOH
(mL)
mEq pH (w/
HCHO)
pH (w/o
HCHO)
0 0 5.96 6.16
2 0.2 7.81 9.39
4 0.4 8.33 9.83
5 0.5 8.54 9.98
6 0.6 8.73 10.13
8 0.8 9.09 10.42
10 1 9.51 10.74
12 1.2 10.21 11.2
14 1.4 11.06 11.82
15 1.5 11.29 11.99
16 1.6 11.43 12.12
18 1.8 11.64 12.28
20 2 11.77 12.38
ASPARTIC ACID(ACID)

pKa
3
9.3
pKa
3
7.8
pKa
1
2.7
pKa
1
2.6

pKa
2
3.7
pKa
2
3.9
Protonated
Deprotonated
Zwitterion
GLYCINE
Neutral: GLYCINE in 0.1 N HCl
0.1 N HCl
(mL)
mEq pH (w/ HCHO) pH (w/o
HCHO)
0 0 4.75 6.08
2 0.2 3.44 3.45
4 0.4 3.07 3.08
5 0.5 2.94 2.94
6 0.6 2.84 2.85
8 0.8 2.66 2.7
10 1 2.54 2.55
12 1.2 2.41 2.4
14 1.4 2.32 2.3
15 1.5 2.26 2.25
16 1.6 2.21 2.2
18 1.8 2.15 2.14
20 2 2.1 2.06
GLYCINE
Neutral: GLYCINE in 0.1 N NaOH
0.1 N NaOH
(mL)
mEq pH (w/ HCHO) pH (w/o HCHO)
0 0 4.42 6.21
2 0.2 6.08 9.08
4 0.4 6.63 9.58
5 0.5 6.85 9.75
6 0.6 7.07 9.9
8 0.8 7.51 10.19
10 1 8.03 10.49
12 1.2 8.9 10.88
14 1.4 10.85 11.48
15 1.5 11.13 11.71
16 1.6 11.31 11.88
18 1.8 11.53 12.08
20 2 11.67 12.2
GLYCINE (NEUTRAL)

pKa
2
9.2
pKa
2
5.8
pKa
1
2.0
pKa
1
2.0
Protonated
Deprotonated
Zwitterion
LYSINE (BASE)

pKa
2
9.5
pKa
2
6.5
pKa
1
2.0
pKa
1
2.2

pKa
3
11.7
pKa
3
10.9
Protonated
Deprotonated
Zwitterion
TABLE OF PKA VALUES
Amino Acid
w/o HCHO
Obtained pKa
1
Theoretical
pKa
1
Obtained pKa
2
Theoretical
pKa
2
Obtained
pKa
3
Theoretical
pKa
3
Aspartic Acid 2.7 1.88 3.7 3.65 9.3 9.6
Glycine 2.0 2.34 9.2 9.60
Lysine 2.0 2.18 9.5 8.95 11.7 10.28
Amino Acid
w/ HCHO
Obtained
pKa
1
Theoretical
pKa
1
Obtained pKa
2
Theoretical
pKa
2
Obtained
pKa
3
Theoretical
pKa
3
Aspartic Acid 2.6 1.88 3.9 3.65 7.8 9.6
Glycine 2.0 2.34 5.8 9.60
Lysine 2.2 2.18 6.5 8.95 10.9 10.28
Amino Acid
w/o HCHO
Obtained
pI

Theoretical
pI

Aspartic
Acid
3.2 2.77
Glycine 5.6 5.97
Lysine 10.6 9.62
Soren Peter Sorensen
end point not reached
without neutralized
formaldehyde
SORENSEN'S DISCOVERY
presence of NH2 amino
group
balances CO2 (acidic)
original amino acid thus
initially neutral
takes up H+
H+ from ionization of -
COOH
forms -NH3
not possible to titrate and
estimate total acidity
SORENSENS DISCOVERY
destroys basic nature of
NH2
acidic COOH free for
titration
readily combines with
free unprotonated amino
groups
dimethylol derivatives
proton to be titrated
directly
ADDITION OF FORMALDEHYDE
ADDITION OF FORMALDEHYDE


HCHO reacts with -NH2
of amino acid
forms methyloyl
derivative
H
+
made available for
reaction
without = pH
with = pH

FORMALDEHYDE
pH = pKa
ratio of conjugate base to
conjugate acid is 1
pH at which solution has
strongest ability to resist
changes
at plateau near or on its
pKa values
nearly equal amounts of
proton donors and
acceptors

MAXIMUM BUFFERING CAPACITY
GLYCINE

pKa
2
9.2
pKa
2
5.8
pKa
1
2.0
pKa
1
2.0
Protonated
Deprotonated
Zwitterion
LYSINE

pKa
2
9.5
pKa
2
6.5
pKa
1
2.0
pKa
1
2.2

pKa
3
11.7
pKa
3
10.9
Protonated
Deprotonated
Zwitterion
ASPARTIC ACID

pKa
3
9.3
pKa
3
7.8
pKa
1
2.7
pKa
1
2.6

pKa
2
3.7
pKa
2
3.9
Protonated
Deprotonated
Zwitterion
determination of pKa
values for each
dissociable group
extrapolating the
midpoint of buffering
region or plateau within
curve


R-GROUP FROM TITRATION CURVE
neutral: two pKs
acidic: three pKs, which
two are acidic
basic: three pKs, which
two are basic


R-GROUP FROM TITRATION CURVE
LYSINE

pKa
2
9.5
pKa
2
6.5
pKa
1
2.0
pKa
1
2.2

pKa
3
11.7
pKa
3
10.9
Protonated
Deprotonated
Zwitterion
ASPARTIC ACID

pKa
3
9.3
pKa
3
7.8
pKa
1
2.7
pKa
1
2.6

pKa
2
3.7
pKa
2
3.9
Protonated
Deprotonated
Zwitterion
GLYCINE

pKa
2
9.2
pKa
2
5.8
pKa
1
2.0
pKa
1
2.0
Protonated
Deprotonated
Zwitterion
Acid-Base Titration of
Amino Acids
-COOH ; -NH2 ; ionizable R
group
neutralization
known acid/base (titrant)
reacted with unknown
acid/base (analyte)
Titration Curve
pK
pKa
pI


CONCLUSION
CONCLUSION
Formaldehyde
combines with NH2
destroys basic nature (H+ removal from COO)
H+ free for titration
lowers pH
different amino acid
strong acid- glutamic acid
strong base- arginine
weak base- histidine
weak acid- cysteine
improve accuracy
better pH meter
automatic titration setup
minimize contamination
proper cleaning of
materials after use

AVOIDING ERRORS
THE END