biochem - c3 lab con 02 - titration of amino acids


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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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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



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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



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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

biochem - c3 lab con 02 - titration of amino acids

Documents

analyte end point point

ph of titrant

isoelectric point pi

end point dye

titrant y

aqueous solution

base x

net charge isnegative