The Titration of Amino Acids

Introduction: Alpha amino acids are the building blocks of proteins.

Almost all proteins consist of various combinations of the same 20
amino acids. Amino acids are compounds containing an amine group,
-NH
2
, and a carboxlic acid group, -!""H. #n addition there is an $%$
group that di&ers for each amino acid. 'he smbol $%$ is used here to
represent a generali(ed abbreviation for an organic group.
C COOH H
2
N
H
R
#n phsiological sstems )here the pH is near neutralit, the amino
group of an amino acid )ill be protonated and the carboxlic acid
group )ill be deprotonated. 'his is called the ()itterion form.
C COO H
3
N
H
R
#n strongl acidic solutions the carboxlic acid group )ill also be
protonated, )hile in strongl basic solutions both the carboxlic acid
group and the amino group )ill both be unprotonated.
C COOH H
3
N
H
R
C COO H
2
N
H
R
Acidic Basic
'he acid-base behavior of amino acids is best described b the
*r+nsted-,o)r theor of acids and bases. A simple amino acid -that
does not have an acid or base group in the $%$ group. is a diprotic acid
in its full protonated form/ it can donate t)o protons during its
complete titration )ith a base. 'he titration )ith Na"H is a t)o-stage
titration represented b the reactions belo).
0
NH
1
!H-%.!""H 0 "H
-

0
NH
1
!H-%.!""
-
0 H
2
"
0
NH
1
!H-%.!""
-
0 "H
-
NH
2
!H-%.!""
-
0 H
2
"
'he hdrochloride salt of a simple amino acid contains one mole of H!l
for each mole of amino acid such that the amino acid is full
protonated.
!l
-

0
NH
1
!H-%.!""H
'he titration curve )ill be biphasic -see diagram belo).. 'here )ill be
t)o separate 2at portions -called legs. on the titration curve. 'he
midpoint of the 3rst leg -*. is )here the amino acid is half in the acidic
form and half in the ()itterion form. 'he point of in2ection -!. occurs
)hen all of the original amino acid is in the ()itterions form -assuming
the $%$ group has no charge.. 'he actual pH at )hich this occurs is
called the isoelectric pH -or isoelectric point., and is given the smbol
p#. 4uring the pH titration of an amino acid )ith a non-ioni(able $%$
group, the e5uivalence point occurs at the p# of the amino acid. At the
midpoint of the second leg -4., half the amino acid is in the ()itterion
form and half is in the basic form. 'he apparent p6 values for the t)o
dissociation steps ma be extrapolated from the midpoints of each
step. 'his can be sho)n b the Henderson-Hasselbach e5uation:
pH 7 p6
a
0 log-8*ase9:8Acid9.
'he p6
acid
-p6
a
for the carboxlic acid group. is point -*. )here half the
acid group has been titrated. 'herefore the e5uation becomes:
pH 7 p6
a
#n the same )a, point -4. gives us the p6
amine
. #n this experiment ou
)ill titrate an unkno)n amino acid, determine its p#, p6
acid
and p6
amine
,
and compare our values to literature values.
Procedures: ;ou )ill titrate one of the amino acid solutions A or *
three times.
Titration of the amino acid solution
<. =oak the pH electrode in distilled )ater )hile preparing the amino
acid solution and setting up the buret. Also soak the electrode in
distilled )ater bet)een titrations. 4o not hit the calibrate button on
the pH meter/ the meters are alread calibrated for ou.
2. 'horoughl rinse the buret )ith distilled )ater. 'he distilled )ater
rinse should drain evenl from the inside surfaces of the buret and
leave no droplets of )ater behind. %epeat the rinsing procedure until
the buret drains cleanl.
1. "btain approximatel <00 m, of Na"H solution. %ecord the
concentration. %inse and then 3ll the buret -to the top of the
graduated markings. )ith Na"H solution. %emove an air bubbles
-especiall from the tip of the buret. and note the starting volume
-)hich ma or ma not be >0.0 m,?..
@. "btain approximatel 1A m, of one of the amino acid hdrochloride
salt solutions/ record the letter of the solution. %inse the <0 m, pipet
)ith the deioni(ed )ater and then rinse )ith t)o small portions of our
chosen amino acid solution. Bipet <0.00 m, of our chosen amino acid
solution into a clean <00 m, beaker. Add 2A m, of deioni(ed )ater -for
a total volume in the beaker of 1A m,..
A. Blace the pH electrode assembl and a magnetic stirring bar into
the beaker. !lamp the electrode so that the stirring bar )ill not hit it
as stirring occurs. #f the electrode is not properl immersed, the pH
reading )ill be erratic.
C. 'itrate the amino acid solution )ith the Na"H from the buret. 'he
3rst run -called the >dr run?. is done b adding the Na"H at D< m,
intervals -note the exact amount dispensed each time. until ou are
Eust past the 3rst endpoint/ in other )ords, keep adding < m,
increments until the pH rises abruptl -the >!? part of the ideal graph
on the previous page. %ecord all of this information in the >dr run?
data table. Note that ou can calculate the second endpointFs
theoretical volume of Na"H easil b doubling the volume dispensed in
getting to the 3rst endpoint.
G. %e3ll the buret )ith Na"H solution. Het another clean dr <A0 m,
beaker and pipet <0.00 m, of our chosen amino acid solution into it/
add 2A m, of distilled )ater. 'itrate the amino acid solution again
->'itration <? data table./ this time, use D0.A m, intervals until Eust
before each endpoint and then drop)ise until Eust after each endpoint.
'his )ill be the >real? run that ou )ill graph. !ontinue until 2.A
e5uivalents of Na"H have been added or the pH reaches about <2.A.
I. %e3ll the buret and repeat the titration process ->'itration 2? data
table. for a fresh batch of the amino acid hdrochloride salt solution.
Waste disposal: =olutions must be bet)een A and <2 pH units
before being poured do)n the drain. ;ou can lo)er the pH b adding
hdrochloric acid, or raise it b adding solid sodium bicarbonate/ if
either of these neutrali(ing chemicals are not available, please place
all )aste in a container in the hood.
Data:
#n our lab notebook record the standard lab notebook information plus
the follo)ing:
!oncentration of Na"H solution
Amino acid -A or *.
4r run titration
o Jake a table of m, Na"H added and pH
o Kirst endpoint -m, Na"H added.
o =econd endpoint -m, Na"H added/ this ma need to be
calculated, rather than measured.
'itration <
o Jake a table of m, Na"H added and pH
'itration 2
o Jake a table of m, Na"H added and pH
Analysis:
<. Lrite a brief obEective for this experiment in our o)n )ords.
2. 'he amino acids A and * used in this lab are from the follo)ing list:
glcine, aspartic acid, glutamic acid and phenlalanine. ,ook up the
structure of each of these on page CAA of the text. #dentif the % group
-the variable part. of each and dra) the structural formulae -acidic,
basic and ()itterionic. of each as the titration proceeds. Note that
some of the amino acids )ill have three forms and others )ill have
four.
1. #s the completel uncharged form of an amino acid ever seen/ in
other )ords, can the uncharged amine group and the uncharged
carboxlic acid group ever exist at the same pHM Nxplain our ans)er.
@. Brepare a graph of our results, plotting m, of Na"H versus pH.
Blot each of the t)o titrations -not the dr run. using Nxcel. %emember
to label the graph axes and provide enough tick marks along each axis
to be useful. #t is eas to forget these steps )hen using soft)are/ )rite
the information in b hand, if necessar.
A. Krom a titration curve it is eas to discern the in2ection point -p#..
'his is the point )hen < e5uivalent of base has been added. #n this
experiment ou use <0 m, of 0.< J amino acid or < millimole of amino
acid -full protonated.. As stated in the introduction, the completel
protonated amino acid can donate t)o protons during the titration. #t
)ill take one e5uivalent -< millimole. of base to titrate the 3rst proton
-on the acid group. and another e5uivalent to titrate the second proton
-on the amine group.. Kind the p# and label it on each graph.
C. =ince the p6
acid
is the midpoint on the 3rst leg, ou can 3nd the
p6
acid
at 0.A e5uivalents of base. ,ike)ise, the p6
amine
can be found at
<.A e5uivalents of base. Kind and label the p6
acid
and p6
amine
for each
graph -if possible..
G.
a. Average the p6
acid
values from the last t)o graphs.
b. Average the p6
amine
values from the last t)o graphs -if possible..
c. Average the p# values estimated from the last t)o graphs.
d. !alculate the p# b 3nding the average bet)een the average p6
acid
value and the average p6
amine
value. 'his should be a better method of
determining the p# of the amino acid. !omment on ho) close ou )ere
b eeballing the p# value o& of the graphs -in other )ords, compare
the value from part c )ith )hat ou calculated in part d..
I. #dentif our amino acid from the follo)ing list: glcine, aspartic
acid, glutamic acid or phenlalanine. ,ook up the >true? p# of the
amino acid from a reliable source -list our source., and calculate a
percent error bet)een our p# value and the >true? p# value.
O. !omment about ho) e&ective this method is at identifing amino
acids/ speci3call, if ou did not have a choice of three candidate
amino acids, )ould ou have been able to pick ours out from among
the t)ent possible amino acidsM #f our p6
amine
)as impossible to
determine, suggest a reason )h the >Eump? did not sho) up.
<0. Brepare a tped report including all of the information relating to
this analsis section: our obEective statement, data:plots, calculated
results, ans)ers to the 5uestions, etc. -'his is not a formal report, but
ou are asked to tpe up the re5uested information..