biochem - c3 lab con 02 - titration of amino acids
DESCRIPTION
Biochem expt. for UST. For 1st yr med students.TRANSCRIPT
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TITRATIONOF
AMINOACIDS
Biochem C3
I-CMED Class 2016
Mendez | Mendiola | Mendoza | Mendoza | Mendoza
Mercado | Mercado | Miguel | Milante | Miranda
Mirano | Mojica | Momani | Montalbo | MontalboMontenegro | Mopia | Morales | Moya
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INTRODUCTION
I
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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
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reflects acid and base
X : volume of the titrant
Y : pH of titrant-analyte
TITRATIONCURVE
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DEFINITIONOFTERMS 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 exactlyequivalent to amount of analyte
End point
point in titration when physical change associated with
condition of chemical equivalence occurs
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APPLICATIONOFTITRATION 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
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TYPESOFTITRATION Acid-Base
neutralization
Complexometric / Chelatometric
volumetric analysis
colored complex as endpoint
Oxidation-reduction
redox reactions
Precipitation
ionic compounds of limited solubility
silver nitrate
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neutralization reaction
acid/base of
concentration (titrant)
reacts with acid/base ofunknown concentration
(analyte)
ACIDBASETITRATION
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amphoteric electrolytes
(ampholytes)
ionizable groups as weak
acids/bases
ionizable R groups influences behavior
during titration
Amino
Carboxyl
Guanidinium
P-hydroxyphenyl
Imidazole
AMINO ACIDS
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AMINO ACIDS
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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
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IONIC PROPERTIES OF AMINO ACIDS
Henderson-Hasselbalch equation:
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unprotonated = protonated
(concentrations)
ratio equals 1
log 1=0 pKapH
at which concentrations of
protonated and
unprotonated forms of anionizable species are equal
at which the ionizable
group is at its best
buffering capacity
IONIC PROPERTIES OF AMINO ACIDS
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Isoelectric Point (pI)
pH at which net charge on
a molecule is zero
(zwitterions)
If pH pI, net charge is
negative
average of two pK values
IONIC PROPERTIES OF AMINO ACIDS
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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
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MATERIALS REAGENTS
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glassware
burettes
beakers
pipettes
pH meter
stir bar
MATERIALS
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Burettes volumetric graduation
stopcock
Miscellaneous
beakers
pipettes
GLASSWARE
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Probe
thin-walled glass bulb at
tip
activity of hydrogen
cations
Glass Electrode Principle
electric potential from
electrode in solution
sensitive to changes in ioncontent (H+ in titration)
PH METER
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magnetic stir bar or flea
rotating magnet at
platform
teflon or glass coated
STIRBAR
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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
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highly caustic metallic
base
very soluble in water
prototypical base
SODIUMHYDROXIDE
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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
HYDROCHLORICACID
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side chain: H
NEUTRAL
non-polar
non-essential
pk1(-COOH) = 2.35
pK2(-NH3) = 9.78
G
LYCINE
(G
LY
, G)
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side hain: (CH2)4NH2
BASIC
polar
essential
pk1(-COOH) = 2.16
pK2(-NH2) = 9.06
pKR(R-group) = 10.54
L
YSINE
(L
YS
, K)
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side chain: CH2(COOH)
ACIDIC
polar
non-essential
pk1(-COOH) = 1.99
pK2(-NH2) = 9.90
pKR(R-group) = 3.90
A
SPARTIC
A
CID
(A
SP
, D)
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METHANAL
rarely found in original
state
used in preservation
water-soluble
aldehyde
donates H
+
to amino acid lowering pH
F
ORMALDEHYDE
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METHODOLOGY
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1
Prepare two pipettes and fill the first with0.1 N HCL and the second with 0.1 NaOH
2 Prepare two beakers for each amino acidand 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 ata time (2ml, 2ml, 1ml, 1ml, 2ml, 2ml, 2ml,
2ml, 1ml ,1ml, 2ml, 2ml)
5 Determine pH after each addition
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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 ofneutralized formaldehyde solution in
each amino acid
10
Titrate again each solution getting pHafter each addition
Pl t H ( di t )
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11
Plot pH (ordinate)vs equivalentacid/base (abcissa)
12
Solve for the pIand pK values ofyour amino acids
13 Construct the
titration curves
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pH at the midpoint ofbuffering region pH changes only slightly
after addition of acid/base
inflection point in thetitration curve
apparent pK values for 2dissociation stepsextrapolated from themidpoints
PKVALUE
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pH at which amino acid
has net zero charge
for simple diprotic amino
acid:
pIfalls halfway betweenthe 2 pK values
for acidic amino acids
pI = (pK1+ pK2)
for basic amino acids
pI= (pK2+ pK3)
ISOELECTRICPOINT(PI)
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RESULTS
ASPARTIC ACID
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ASPARTIC ACID
Acidic: ASPARTIC ACID in 0.1 N HCl0.1 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 4.465 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
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ASPARTIC ACID
Acidic: ASPARTIC ACID in 0.1 N NaOH0.1 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 9.835 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
A A (A )
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ASPARTICACID(ACID)
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pKa39.3
pKa37.8
pKa12.7
pKa12.6
pKa23.7
pKa23.9
Protonated
Deprotonated
Zwitterion
GLYCINE
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GLYCINE
Neutral: GLYCINE in 0.1 N HCl0.1 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 2.315 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
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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 9.756 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 11.4815 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
G (N )
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GLYCINE(NEUTRAL)
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pKa29.2
pKa25.8
pKa12.0
pKa12.0
Protonated
Deprotonated
Zwitterion
L (B )
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LYSINE(BASE)
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pKa29.5
pKa26.5pKa12.0
pKa12.2
pKa311.7
pKa310.9
Protonated
Deprotonated
Zwitterion
T O K V S
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TABLEOFPKAVALUESAmino Acid
w/o HCHOObtained pKa1
Theoretical
pKa1Obtained pKa2
Theoretical
pKa2
Obtained
pKa3
Theoretical
pKa3
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
pKa1
Theoretical
pKa1 Obtained pKa2Theoretical
pKa2
Obtained
pKa3
Theoretical
pKa3
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 Acidw/o HCHO
ObtainedpI
TheoreticalpI
Aspartic
Acid3.2 2.77
Glycine 5.6 5.97
Lysine 10.6 9.62
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DISCUSSION
S ' D
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Soren Peter Sorensen
end point not reached
without neutralized
formaldehyde
SORENSEN'SDISCOVERY
SORENSENS DISCOVERY
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presence of NH2 aminogroup balances CO2 (acidic)
original amino acid thus
initially neutral takes up H+
H+ from ionization of -COOH
forms -NH3
not possible to titrate andestimate total acidity
SORENSENSDISCOVERY
A F
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destroys basic nature of
NH2
acidicCOOH free for
titration readily combines with
free unprotonated amino
groups
dimethylol derivatives
proton to be titrated
directly
ADDITIONOFFORMALDEHYDE
A F
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ADDITIONOFFORMALDEHYDE
FORMALDEHYDE
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HCHO reacts with -NH2
of amino acid
forms methyloyl
derivative H+made available for
reaction
without = pH
with = pH
FORMALDEHYDE
MAXIMUM BUFFERING CAPACITY
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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
MAXIMUMBUFFERINGCAPACITY
GLYCINE
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GLYCINE
pKa29.2
pKa25.8
pKa12.0
pKa12.0
Protonated
Deprotonated
Zwitterion
LYSINE
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LYSINE
pKa29.5
pKa26.5pKa12.0
pKa12.2
pKa311.7
pKa310.9
Protonated
Deprotonated
Zwitterion
ASPARTIC ACID
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ASPARTICACID
pKa39.3
pKa37.8
pKa12.7
pKa12.6
pKa23.7
pKa23.9
Protonated
Deprotonated
Zwitterion
R GROUP FROM TITRATION CURVE
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determination of pKa
values for each
dissociable group
extrapolating themidpoint of buffering
region or plateau within
curve
R-GROUPFROMTITRATIONCURVE
R GROUP FROM TITRATION CURVE
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neutral: two pKs
acidic: three pKs, which
two are acidic
basic: three pKs, whichtwo are basic
R-GROUPFROMTITRATIONCURVE
LYSINE
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LYSINE
pKa29.5
pKa26.5pKa12.0
pKa12.2
pKa311.7
pKa310.9
Protonated
Deprotonated
Zwitterion
ASPARTIC ACID
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ASPARTICACID
pKa39.3
pKa37.8
pKa12.7
pKa12.6
pKa23.7
pKa23.9
Protonated
Deprotonated
Zwitterion
GLYCINE
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GLYCINE
pKa29.2
pKa25.8
pKa12.0
pKa12.0
Protonated
Deprotonated
Zwitterion
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CONCLUSION
CONCLUSION
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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
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CONCLUSION Formaldehyde
combines withNH2
destroys basic nature (H+ removal fromCOO)
H+ free for titration
lowers pH
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RECOMMENDATIONS
AVOIDING ERRORS
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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
AVOIDINGERRORS
THE END
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THEEND