NAME: MERVE MURAT

08.03.2017
ID: 2004141
SECTION / GROUP: 3 / 1
SUBMITTED TO EMRE EVN

TITRATION OF AMINO ACIDS

PURPOSE
The purposes of this experiment are to draw titration curves for Glycine and unknown amino
acid after titration of these amino acids with KOH (and HCl), and to determine the pKa and pI
values of unknown amino acid by using this titration curve.

THEORY
Titration for amino acids is a technique that provide chemical analysis. After titration process,
titration curve is formed and this curve provides an important information about amino acids
such as its pI and pKa values. Moreover, from this titration curve, buffering range of the
amino acid can be determined. In addition, the number of dissociable protons of amino acid
can be predicted by looking at the number of buffer region on a titration curve (THE
TITRIMETRIC DETERMINATION OF THE CONCENTRATION AND ACID
DISSOCIATION CONSTANTS OF AN UNKNOWN AMINO ACID, n.d.).

Amino acids can be either accept or lose more than one proton. Because of this, they are
called polyprotic. Also, they are amphoteric owing to their ionizable -amino and -
carboxylic group; that is, they can act as acid or base depending on the pH. In addition, amino
acids have ionizable groups that can act weak acid or base. If amino acids are dissolved in
water, they can be generally observed in isoelectric form (Dobson & Winter, 2014; Titration
Curves of Aminoacids, n.d.).

Each amino acid have different pI value. This value is the pH where the net charge of amino
acid is equal to zero. In other words, amino acid is in the zwitter ion form. It can be calculated
by taking the average of pKa values for given amino acid and pKa values can be found by
taking the midpoint of buffer region. When the concentration of the unprotonated amino acid
equals that of the unprotonated form at a given pH, their ratio is one. So, this pH can be called
as pKa. In addition, it can be said that the ionizable group is at its best buffering capacity at
when reaching pKa value; that is, amino acid solution can resist the pH changes most
effectively (Titration Curves of Aminoacids, n.d.).

PROCEDURE
pH meter calibration:
1. Remove the cover of glass electrode.
2. Rinse it with distilled water and dry it with absorbent tissue.
3. Immerse electrode into pH 7 buffer and measure the pH.
4. Rinse it with distilled water and dry it with absorbent tissue.
5. Immerse electrode into pH 10 buffer and measure the pH.
6. Measure the pH of tap water, distilled water, 0.05 N HCl and 0.05 N KOH, and record
them.

Glycine titration:
1. Put 25 mL of Glycine into beaker and add 25 mL of distilled water.
2. Mix the solution by using magnetic stirrer.
3. Measure the pH of solution before titration and record it.
4. Perform the titration process by adding 1 mL of KOH to Glycine solution and mix the
solution by stirrer.
5. Measure the pH of solution and record it.
6. Repeat 5th and 6th steps until solution comes to buffer region (until solution strongly
resists to pH changes.)
7. Repeat 5th and 6th steps by adding 2 mL of KOH rather than 1 mL until solution close
to the end point of buffer region.
8. Repeat 5th and 6th steps by adding 1 mL of KOH rather than 2 mL until the end of
buffer region.

Unknown amino acid titration:

1. Put 50 mL of unknown amino acid into beaker.

2. Repeat 4-9 step of Glycine titration.

CALCULATIONS
Questions:

1. What would have been the pH upon addition of 10 mL of 0.1 M NaOH to 3.0 mL 0.1
M solution of isoelectric Glycine (pKa values for Glycine are 2.4 and 9.8) ?

n
M= milimole=MmL
L
 milimole NaOH =0.110=1

milimoleGly =0.13.0=0.3

10.3=0.7 milimole NaOH remaining

At the end of the titration, 0.7 milimole NaOH remains. In other words, there is no
contribution to pH by Glycine.

So, pOH can be calculated by the help of remaining NaOH.

0.7 mmole
pOH=loq [OH ] where [ OH ] = 13 mL =0.05 M

pOH=loq ( 0.05 )=1.27 pH=141.27=12.73

2. How many mL of 0.1 M NaOH are required to titrate 0.3 g of isoelectric Arginine
(MW of Arginine is 174) ?

m 0.3
n= = =0.0017 mole Arginine
MW 174

So, we need 0.0017 mole of NaOH for titration

n n 0.0017
M= L= = =0.017 L NaOH is required
L M 0.1

3. Describe the preparation of 10 L of 0.045 M potassium phosphate buffer pH 7.5

(MW of K2HPO4 is 174, MW of KH2PO4 is 136, pKa2 = 7.2).

[ K 2 HPO 4 ] [ K 2 HPO 4 ]
pH= pKa+ log 7.5=7.2+ log
[ KH 2 PO 4 ] [ KH 2 PO 4 ]

[ K 2 HPO 4 ] [ K 2 HPO 4 ]
log =0.3 =2
[ KH 2 PO 4 ] [ KH 2 PO 4 ]

[ K 2 HPO 4 ] =2 [ KH 2 PO 4 ]

[ KH 2 PO 4 ] + [ K 2 HPO 4 ] =0.045 M
 2 [ KH 2 PO 4 ] + [ KH 2 PO 4 ] =0.045 3 [ KH 2 PO 4 ] =0.045

[ KH 2 PO 4 ] =0.015 M

0.015 M + [ K 2 HPO 4 ] =0.045 M [ K 2 HPO 4 ] =0.03 M

n
M= n=0.01510=0.15 mole KH2PO4 is required
L

n=0.0310=0.3 mole K2HPO4 is required

m
n= m=0.3174=52.2 g of K2HPO4 is required
MW

m=0.15136=20.4 g of KH2PO4 is required

So, for preparation of 10 L of 0.045 M potassium phosphate buffer pH 7.5, 52.2 g of K 2HPO4
and 20.4g of KH2PO4 should be mixed and then final volume should be completed to 10 L by
using dH2O.

pH Measurements:

pH Tap water dH2O 0.05 N HCl 0.05 N KOH

Theoretical 7.3* 7** 1.3 12.7
Measured 7.42 8.03 1.45 12.66
Table 1. Theoretical and measured pH values of reagents used in experiment.
*(T.C. ANKARA BYKEHR BELEDYES ASK GENEL MDRL ME SUYU
KALTE PARAMETRELER KIYASLAMASI, 2017)
**(Youmans, 1972)

N = n*M 0.05 = 1*M M = 0.05 molar HCl

So, 0.05 molar H+

pH = -log[H+] pH = -log(0.05) pH = 1.3 for 0.05 N HCl

N = n*M 0.05 = 1*M M = 0.05 molar KOH

So, 0.05 molar OH-

pOH = -log[OH-] pOH = -log(0.05) pOH = 1.3

pH = 14 pOH pH = 14 1.3 pH = 12.7 for 0.05 N KOH

Titration Curves:

TItr atIon cur ve for GlycIne

Glycine

12

10

6
pH 4

Ttrant Volume(mlLITER)

Graph 1. Titration curve for Glycine (titrants are 0.5 N HCl and 0.5 N KOH).
Starting point calculated by using two group data = (5.41+5.80)/2 = 5.6 = pH

According to titration curve, pKa1 values can be found.

pKa1 can be around 2.08 marked with

pKa2 can be around 9.90 marked with

So, pI value is (2.08 + 9.9) / 2 = 5.99 for Glycine

pKa Net mL HCl or KOH used Net mM HCl or KOH used

2.08 21 1.48 * 102
9.90 15 1.15 * 102
Table 2. According to pKa values of Glycine, net mL and mM KOH used.

Normality of HCl and KOH is 0.5.

 0.5
So, N=nM M= =0.5 M HCl and KOH
1

1 M =1000mM mM =500 for HCl and KOH

M1 * V1 = M2 * V2 5 * 102 * 21 = M2 * (21+50) M2 = 1.48 * 102 mM HCl

M1 * V1 = M2 * V2 5 * 102 * 15 = M2 * (15+50) M2 = 1.15 * 102 mM KOH

TItratIon c urve for unknown

Unknown

12

10

6
pH 4

Ttrant Volume(mlLITER)

Graph 2. Titration curve for unknown amino acid (titrants are 0.5 N HCl and 0.5 N KOH).
Starting point for both group (pH = 7.56)

According to titration curve, pKa values can be found.

pKa1 can be around 1.53 marked with

pKa2 can be around 6.30 marked with

pKa3 can be around 9.27 marked with

So, this amino acid can be Histidine

So, pI value is (6.3 + 9.27) / 2 = 7.79 for Histidine

pKa Net mL HCl or KOH used Net mM HCl or KOH used

 1.53 54 2.60 * 102
6.30 100 3.33 * 102
9.27 13 1.03 * 102
Table 3. According to pKa values of Histidine, net mL and mM KOH used.

Normality of HCl and KOH is 0.5.

0.5
So, N=nM M= =0.5 M HCl and KOH
1

1 M =1000mM mM =500 for HCl and KOH

M1 * V1 = M2 * V2 5 * 102 * 54 = M2 * (54+50) M2 = 2.60 * 102 mM HCl

M1 * V1 = M2 * V2 5 * 102 * 100 = M2 * (100+50) M2 = 3.33 * 102 mM HCl

M1 * V1 = M2 * V2 5 * 102 * 13 = M2 * (13+50) M2 = 1.03 * 102 mM KOH

DISCUSSION
In this experiment, Glycine and unknown amino acid titrated with KOH and titration curves
of these amino acids were drawn. For these curves, the data set resulted from titration with
HCl are taken from Group 2.

Before titration procedure, pH meter calibration was done with pH 7 and 10 buffer rather than
4 and then some pH measurements were performed by using tap water, distilled water, 0.05 N
HCl and 0.05 H KOH. For calibration, the reason of using pH 7 and 10 buffer is that titrant
used during experiment is KOH. So, calibration was done with basic buffer because KOH is a
basic reagent (pH Calibration Procedure for Optimal Measurement Precision, 2009, Two-
Point Calibration, n.d.). According to pH measurements of tap water, distilled water, 0.05 N
HCl and 0.05 N KOH, almost all measurement overlap with theoretical values. There is a
deviation only in distilled water measurement (measured value is 8.03 whereas theoretical
value is 7). The possible reason of this deviation is that beaker might not be washed properly
with distilled water and contamination (change in ion concentration) might be occur because
of tap water, or distilled water stock might not be prepared properly. In addition, these
measurements was done for control of calibration.

In the first titration process, 50 mL Glycine and dH 2O mixture titrated with 30 mL KOH. To
reach the second pKa value, 15 mL of KOH was used and 9.9 was obtained as pKa value.
This value overlap with theoretical value that is 9.6 (Amino Acids Reference Chart, n.d.).
The titration process with HCl performed by Group 2 by using 50 mL Glycine and dH 2O
mixture. This mixture titrated with 39 mL of HCl. To reach the first pKa value, 21 mL of HCl
was used and 2.08 was obtained as pKa value. This value overlap with theoretical value that is
2.34 (Amino Acids Reference Chart, n.d.). Also, its pI value was found as 5.99 and this
value overlap with theoretical pI value which is 5.97 (Amino Acids Reference Chart, n.d.).

In the second titration process, 50 mL unknown amino acid titrated with 29 mL of KOH. To
reach the third pKa value, 13 mL of KOH was used and 9.27 was obtained as pKa value. The
titration process with HCl performed by Group 2 by using 50 mL unknown amino acid. This
unknown titrated with 115 mL of HCl. To reach the first and the R group pKa values, 54 mL
and 100 mL HCl were used and 1.53 and 6.30 were obtained as pKa value respectively. By
looking at these values, it is predicted that this unknown amino acid can be Histidine because
its theoretical pKa1 is 1.82, pKaR is 6.0 and pKa2 is 9.17 (Amino Acids Reference Chart,
n.d.). Also, its pI value was found as 7.79 and this value overlap with theoretical pI value
which is 7.59 (Amino Acids Reference Chart, n.d.).

Isoelectric point (pI) applications:

- Protein purification and separation using isoelectric focusing and 2D electrophoresis,

- Study of protein heterogeneity (protein truncations, isoforms, PTMs, etc),
- Study of protein-protein interactions,
- Analysis of protein folding status (Isoelectric Point, n.d.).

REFERENCES
Amino Acids Reference Chart. (n.d.). Retrieved March 12, 2017, from
http://www.sigmaaldrich.com/life-science/metabolomics/learning-center/amino-acid-
reference-chart.html

Dobson, C. M., & Winter, N. S. (2014). The Identification of Amino Acids by Interpretation
of Titration Curves: An Undergraduate Experiment for Biochemistry. World Journal of
Chemical Education, 2(4), 5961. https://doi.org/10.12691/WJCE-2-4-3

Isoelectric Point. (n.d.). Retrieved March 12, 2017, from http://www.creative-

proteomics.com/services/isoelectric-point.htm

pH Calibration Procedure for Optimal Measurement Precision. (2009). Retrieved March 11,
2017, from http://tools.thermofisher.com/content/sfs/brochures/TN-ph-calibration-
procedure-for-optimal-measurement-precision-T-PHCAL-EN.pdf
T.C. ANKARA BYKEHR BELEDYES ASK GENEL MDRL ME SUYU
KALTE PARAMETRELER KIYASLAMASI. (2017). Ankara. Retrieved from
http://www.aski.gov.tr/yukle/dosya/pdf/icme_suyu_raporlari/2017-Subat.pdf

THE TITRIMETRIC DETERMINATION OF THE CONCENTRATION AND ACID

DISSOCIATION CONSTANTS OF AN UNKNOWN AMINO ACID. (n.d.). Retrieved
March 13, 2017, from
http://www.chem.ucla.edu/~bacher/CHEM14CL/Handouts/Titrimetric Determination of
the Concentration and Acid Dissociation Constants of an Amino Acid_Sp16.pdf

Titration Curves of Aminoacids (Theory): Biochemistry Virtual Lab I: Biotechnology and

Biomedical Engineering: Amrita Vishwa Vidyapeetham Virtual Lab. (n.d.). Retrieved
March 14, 2017, from http://vlab.amrita.edu/?sub=3&brch=63&sim=1336&cnt=1

Two-Point Calibration. (n.d.). Retrieved March 11, 2017, from

http://faculty.sdmiramar.edu/fgarces/LabMatters/ChemTech/modules/phmeter/pH2ptcal.
htm

Youmans, H. L. (1972). Measurement of pH of distilled water. Journal of Chemical

Education, 49(6), 429. https://doi.org/10.1021/ed049p429