ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A.

LEGASPI, 2020

ACTIVITY #1: PREPARATION OF BUFFERS

A buffer is a mixture of a weak acid or a weak base and its conjugate base or its conjugate
acid respectively. Since it has both an acidic and a basic component, it is capable of resisting
drastic changes in pH. The Henderson-Hasselbalch equation is used in calculating the pH of
buffers:

Biomolecules are typically pH sensitive, hence, appropriate buffer is needed to preserve their
native conformation. The following sample exercises will guide you how to calculate the pH of a
buffer and how to prepare a particular buffer.

Sample Exercise 1- pH of a Buffer: Acetic acid (50.0ml, 0.100 M) and sodium acetate (20.0ml,
0.200 M) were mixed to form a buffer. Calculate the pH of the solution given that the Ka of
acetic acid is 1.75 x 10-5.

Solution: pH = -log (1.75 x 10-5) + log (0.200 M x 0.0200 L)

(0.100 M x 0.0500 L)
pH = 4.757 + (-0.0969) = 4.66

Sample Exercise 2- Addition of Acid and Base to a Buffer: What will be the pH of the above
buffer system after addition of (a) 30.0 ml of 0.010M HCl or (b) 15.00 ml of 0.025M NaOH?

Solutions:

(a) HCl will deplete the base and will increase the amount of acid

pH = -log (1.75 x 10-5) + log (0.20M x 0.020 L) – (0.010M x 0.030 L)

(0.10M x 0.050 L) + (0.010M x 0.030 L)
pH = 4.757 + (-0.156) = 4.60

(b) NaOH will deplete the acid and will increase the amount of base

pH = -log (1.75 x 10-5) + log (0.20M x 0.020 L) + (0.025M x 0.015 L)

(0.10M x 0.050 L) - (0.025M x 0.015 L)
pH = 4.757 + (-0.0241) = 4.73

The above calculations show that the pH was changed minimally although a relatively large
amount of acid or base was added.

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

Sample Exercise 3 - Preparation of Buffer: How will you prepare a 2.00L of 0.500 M acetic
acid-sodium acetate buffer with pH of 5.00 from 6.00 M acetic acid solution and solid sodium
acetate (CH3COONa, MW=82.0 g/mol). The Ka of acetic acid is 1.75 x 10-5.

Solution:
Step 1 - Determine the ratio of the conjugate base and weak acid:

5.00 = -log (1.75 x 10-5) + log [ A-] 5.00 = 4.757 + log [ A-]
[HA] [HA]

antilog(5.00 – 4.757) = antilog ( log [ A-] ) 1.75 = [ A-] 1.75 [HA] = [A-]
[HA] [HA]

Step 2 - Calculate the molarity of the weak acid and conjugate base needed by considering
the final molarity of the buffer:

Substitute 1.75 [HA] = [A-] to [A-] = 0.500 M – [HA]

1.75 [HA] = 0.500 M - [HA] 1.75 [HA] + [HA] = 0.500 M

2.75 [HA] = 0.500 M [HA] = 0.500 M / 2.75 = 0.182 M

Therefore, [A-] = 0.500 M – 0.182 = 0.318 M

Step 3: Calculate the amount of acid and its conjugate base that should be taken from the
stock solution or solid reagent stock.

HA (acetic acid) will be taken from a stock solution:

M1V1 = M2V2 V1 = M2V2 / M1 = (0.182 M) (2.00 L)/(6.00 M) = 0.0607 L or 60.7ml

A- (sodium acetate) will be taken from solid reagent stock:

M = g / MW VL g = M MW VL = (0.318 M) (82.0 g/mol) (2.00 L) = 52.152 g

Step 4: Weigh 52.152 g of sodium acetate using analytical balance then transfer it
quantitatively to a 2.00 L volumetric flask. Add 60.7 ml of 6.00 M acetic acid to the flask then
half-fill with distilled water and swirl to dissolve the solids. Fill to the 2.00 L mark, and then
shake thoroughly by inverting the flask several times. Make sure that all the sodium acetate
is dissolved completely. Check the pH using a pH meter and adjust the pH by adding HCl or
NaOH if necessary.

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

Activity # 1: Preparation of Buffers

Name:________________________________________________ SCORE: __________

Group No._____ Section:________ Lab Instructor:__________________________________

Date Performed:_____________________ Date Submitted:_________________________

(1) What will be the pH when 50.0 ml of 2.00M carbonic acid is mixed 20.0 ml of 3.00 M
sodium bicarbonate? The Ka of carbonic is 4.30 x 10-7. (5 pts)

(2) For the above buffer system, what will be the pH when (a) 15.0 ml of 0.500 M HCl and (b)
when 15.0 ml of 0.750 M NaOH is added? (10 pts)

(3) How will you prepare 500 ml of 0.750 M citrate buffer with pH of 4.25 from solid citric acid
(H3C6H5O7) and solid sodium citrate (NaH2C6H5O7).The Ka of citric acid is 7.40 x 10-4. (15 pts)

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

EXPERIMENT #1: BIOLOGICAL BUFFER SYSTEMS

Introduction

Buffers are very important in biological systems as biomolecules & biochemical reactions
are very sensitive to pH conditions. The physiologic pH is narrowly maintained around 7.40 by
biological buffer systems. Great deviations from this pH lead to acid-base disorders which may
result to serious deleterious effects including possible death if left uncorrected.

Experiments in Biochemistry typically require the use of appropriate buffer system

especially if proteins and nucleic acids are involved. The most common buffer system used is
the phosphate buffer. Phosphoric acid is a weak triprotic acid that dissociates as illustrated below.

- + -3
H3PO4 + H2O H2PO4 + H 3O Ka1 = 7.5 x 10
- -2 +
H2PO4 + H2O HPO4 + H3O Ka2 = 6.2 x 10-8
-2 -3 + -13
HPO4 + H 2O PO4 + H3O Ka3 = 4.2 x 10

Phosphate buffer can be prepared using either H3PO4-H2PO4- or H2PO4—HPO4-2 system.

The choice of which system to use depends on the desired pH of the buffer. If an acidic pH is
required, then the H3PO4-H2PO4- buffer system should be used. For buffer with pH value near or
above the physiologic pH, the H2PO4—HPO4-2 system should be utilized.

Objectives

The experiment will help you learn how to systematically prepare a buffer system. It will
also help you see the capacity of a buffer in resisting drastic changes in pH.

Reagent & Materials

K2HPO4 Distilled water 25 ml volumetric pipette

KH2PO4 250-ml volumetric flask 5 ml volumetric pipette
0.100 M HCl 125-ml Erlenmeyer flask Clean plastic bottle with cap (250 ml)
0.100 M NaOH pH meter Weighing or filter paper

Procedures

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

1. Calculate the needed reagents in preparing 250 ml of 0.200 M H2PO4—HPO4-2 buffer

with pH of 7.40. Show the results of your calculation to your teacher to check whether
your calculations are accurate.
2. Weigh the exact amount of reagents in separate weighing or filter papers using an
analytical balance.
3. Transfer the reagents qualitatively into 250-ml volumetric flask.
4. Half-fill the flask with distilled water. Stopper then swirl until the solid dissolves. Add
a little more water if some reagent remained undissolved then swirl again.
5. Fill-to-mark when the reagents are completely dissolved. Stopper and invert the flask
repeatedly for at least twenty times to ensure complete mixing.
6. Transfer 25.0 ml of the buffer using a volumetric pipette to two separate 100-ml
Erlenmeyer flasks. Measure the pH using a pH meter. Determine the percent error in
the pH of your buffer.
7. To one flask add 10.0 ml of 0.100 M HCl and 10.0 ml of 0.100 M NaOH to the other
flask. Swirl the contents then measure the pH of each sample.
8. Repeat procedure in #7 using distilled water instead of the buffer.
9. Place the remaining buffer in a clean plastic bottle. Label appropriately and store it in
your locker as it will be used in subsequent experiments.
10. Combine all the used samples for neutralization and dispose them into the acid waste
jar.
11. Complete your data table and needed calculations. Research for your answers on the
follow-up questions & applications.

Experiment # 1: Biological Buffer Systems

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

Name:________________________________________________ SCORE: __________

SCHEMATIC DIAGRAM

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

Experiment # 1: Biological Buffer Systems

Name:________________________________________________ SCORE: __________

Group No._____ Section:________ Lab Instructor:__________________________________

Date Performed:_____________________ Date Submitted:_________________________

A. Buffer Preparation Calculations (10 pts)

B. Addition of Acid or Base (20 pts)

pH
Phosphate Buffer Distilled Water
Theoretical Experimental % Error
Initial
+ 10.0 ml 0.100M HCl
+ 10.00 ml 0.100 M NaOH

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

C. Follow-up Questions and Applications (20 pts)

1. Are there significant differences in the theoretical & experimental pH of your buffer? If
yes, then explain the possible sources of errors. (2 pts)

2. What makes a buffer capable of resisting drastic changes in pH as compared to

distilled water? (2 pts)

3. Give two examples of synthetic organic buffers that are use instead of inorganic buffer
like phosphate buffer and what are the advantages of using them? Draw the chemical
structures of your examples. (6 pts)

4. What is the buffer system in our blood plasma & extracellular fluid (ECF) and how does
our body regulate the physiologic pH through this system? (5 pts)

5. What are the types, causes, and effects of acidosis & alkalosis? (5 pts)

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

ACTIVITY #2: AMINO ACIDS AND PEPTIDES

A. Standard Amino Acids

Amino acids are the building blocks of proteins. There are three structural features in
amino acids: the α-amino group, the α-carboxylic acid, and the variable functional group (R) as
illustrated in Figure 1 below.

H H
+ -
H2N C COOH H3N C COO
R R
formal structure zwitterionic structure

Figure 1. General Structure of an Amino acid

As shown above, amino acid can exist in its zwitterionic form in solution as the H+ of the carboxylic
acid is donated to the amine. The zwitterion has equal number of positive & negative charges
so its net charge is zero. The α-amino group is located to the left side, hence, the complete
designation for amino acids is L-α-amino acid. All standard amino acids are therefore said to be
left-handed. The designation also implies the chiral nature of amino acids which make them
capable of rotating plane-polarized light in solution.

The structure of the R group determines the identity of an amino acid. There are twenty
standard amino acids as listed in the Table 1. Their names are abbreviated either by a one letter
symbol or a three-letter contraction. They can be classified according to the structure & nature of
their respective R groups.

B. Peptides

The α-carboxylic acid group of one amino acid can react with the α-amino group of another
amino acid to form a special amide bond called a peptide bond. This process is illustrated in
Figure 2 below:

H O H H O H
H2N C C OH H N C COOH H2N C C N C COOH
CH2 H CH2 CH2 CH2
+
COOH H2O COOH

Aspartic acid Phenylalanine Aspartylphenylalanine dipeptide

Figure 2. Peptide Formation

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

1. Glycine (Gly or G) 8. Tyrosine (Tyr or Y) 15. Arginine (Arg or R)

H H
+ - + -
H H3N C COO H3N C COO
+ - CH2 CH2
H3N C COO CH2
H CH2
NH
+
C NH2
OH NH2

2. Alanine (Ala or A) 9. Tryptophan (Trp or W) 16. Histidine (His or H)

H H
H + - + -
+ - H3N C COO H3N C COO
H3N C COO
CH2 CH2
CH3
+
HN NH
N
H
3. Valine (Val or V) 10. Serine (Ser or S) 17. Aspartic acid (Asp or D)
H H H
+ - + - + -
H3N C COO H3N C COO H3N C COO
CH CH2 CH2
H3C CH3 OH -
COO

4. Leucine (Leu or L) 11. Threonine (Thr or T) 18. Glutamic acid (Glu or E)

H H H
+ - + - + -
H3N C COO H3N C COO H3N C COO
CH2 HC OH CH2
CH
CH3 CH2
H3C CH3
-
COO
5. Isoleucine (Ile or I) 12. Cysteine (Cys or C) 19. Asparagine (Asn or N)
H H H
+ -
+ - + - H3N C COO
H3N C COO H3N C COO
CH2
HC CH3 CH2 C
CH2 O NH2
SH
CH3
6. Proline (Pro or P) 13. Methionine (Met or M) 20. Glutamine (Gln or Q)
H H H
+ - + -
+ - H3N C COO H3N C COO
HN C COO
CH2 CH2
CH2 CH2
S C
O NH2
CH3

7. Phenylalanine (Phe or F) 14. Lysine (Lys or K)

H H
+ -
+ - H3N C COO
H3N C COO
CH2
CH2
CH2
CH2
CH2
NH3+

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

Notice that aspartic acid has the free α-amino group while phenylalanine has the free α-
carboxylic acid. They are referred to as the N-terminal & C-terminal amino acids respectively.
Peptides are always named from N- to C-terminal and the suffix –yl is added with the deletion of
–ine/-ic acid except for the C-terminal amino acid, and the number of amino acids is specified
afterwards.

B. Acid-Base Behavior of Amino Acids and Peptides

Amino acids are amphoteric as they can behave both as an acid and as a base. A
prototropic or ionizable group can accept or donate H+ at a particular pH. Table 2 lists the pKa
values of the various prototropic groups of amino acids.

Table 2. pKa Values of Protropic Groups in Amino Acids

Amino pKa1 pKa2 Amino pKa1 pKa2 pKa3

Acid α-COOH α-NH2 Acid α-COOH α-NH2 R group
Gly 2.34 9.60 Met 2.28 9.21 ---
Ala 2.34 9.69 Asn 2.02 8.80 ---
Val 2.32 9.62 Gln 2.36 9.13 ---
Leu 2.36 9.60 Tyr 2.20 9.11 10.07
Ile 2.36 9.60 Cys 1.71 10.78 8.33
Pro 1.99 10.6 Asp 2.09 9.82 3.86
Phe 1.83 9.13 Glu 2.19 9.67 4.25
Trp 2.83 9.39 Lys 2.18 8.95 10.79
Ser 2.21 9.15 Arg 2.17 9.04 12.48
Thr 2.63 10.43 His 1.82 9.17 6.00

Titration is a useful way of investigating the acid-base behavior of amino acids and
peptides. In this process, we start with the most acidic or fully protonated form of the amino acid
by adding HCl then the solution is titrated by adding NaOH incrementally. For example, the
titrimetric profile for histidine is illustrated in Figure 3 below.

Figure 3. Titrimetric Profile of Histidine

During titration, the prototropic groups lose H+ successively as the pH reach their respective pKa
values. The net charge of histidine at the start of titration is +2 and as each group loses H + the
net charge decreases by one unit. The titrimetric profile clearly shows which form of the amino
acid predominates at a particular pH. It also reveals the isoelectric pH (pI) which is the pH where

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

the amino acid is zwitterionic. The value of pI is taken as the average of the pKa values that
border the zwitterion. For histidine, its pI is the average of 6.00 and 9.17 that is 7.59. Figure 4
shows the titration curve for histidine which emphasizes the pKa values and pI of the amino acid.
Notice that the pKa values of the prototropic groups can be determined from the titration curve.
They are taken as the pH value at the various inflection points of the curve. Meanwhile the
buffering regions of the amino acid are the medial zones between two pKa values where pH
change is slowest. The pI is actually within the buffering zone of an amino acid and for histidine
the pI of 7.59 makes it an effective buffer at physiologic pH. The above analysis can be extended
to peptides.

Figure 4. Titration Curve of Histidine

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

Activity # 2: Amino Acids & Peptides

Name:________________________________________________ SCORE: __________

Group No._____ Section:________ Lab Instructor:__________________________________

Date Performed:_____________________ Date Submitted:_________________________

1. Glutathione is a naturally-occurring tripeptide that has potent antioxidant property. It has

become a popular ingredient in various whitening pills and cosmetic products. Its systematic
name is γ-glutamylcysteinylglycine tripeptide. Draw the structure of glutathione and label the
N-terminal and C-terminal amino acids. [Note: the term gamma (γ) signifies that the peptide
bond is formed by the γ-carboxylic acid of the side chain of glutamic acid) (10 pts)

2. Show the titrimetric profile of glutamic acid and calculate its pI? (10 pts)

3. Name the peptide below, show its titrimetric profile, and determine its pI. (10 pts)

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

EXPERIMENT #2: TITRATION OF AMINO ACIDS & PEPTIDES

Introduction

Titration is a useful way of analyzing the acid-base properties of amino acids and peptides.
The titrimetric profile reveals which species predominates at a particular pH. It is also useful in
determining the isoelectric pH of the sample. The titration curve meanwhile, reveals the pKa of
the various prototropic groups in amino acids and peptides, their buffering zones, and of course
their respective pI values. Amino acids and peptides are important buffers in biological systems.
They can resist drastic changes in pH due to the presence of both acidic and basic components.
Titration is also a useful way of identifying an unknown amino acid based on its pKa values and
pI.

Objectives

The experiment will help you understand the acid-base behavior of amino acids and
peptides. You will also be able to identify an unknown amino acid base on the analysis of its
titration curve. Lastly, you will be able to deduce the implication of the acid-base behavior of
amino acids and peptides as biological buffers.

Reagent & Materials

Unknown amino acids powder Distilled water Volumetric pipette

Aspartame sweetener 250-ml Erlenmeyer flask Filter paper
0.200 M HCl Burette 1ml syringe
0.200 M NaOH pH meter

Procedures
1. Weigh exactly 0.250 g sample of the unknown amino acid powder assigned to your on
a watch glass or weighing paper group using an analytical balance.
2. Transfer the sample quantitatively to separate 250-ml Erlenmeyer flask. Add 15.0 ml
of distilled water to each flask using volumetric pipette and dissolve the sample by
swirling.
3. Add small increments of 0.200 M HCl to the dissolved sample using a burette. Swirl
the flask then measure the pH. Repeat the procedure until pH of 1.50 is reached.
4. Titrate each acidified solution separately with 0.200M NaOH by adding 0.20 ml
increments of the base. Swirl the flask then measure the pH. Record the pH value at
each amount of base added. Repeat the procedure until at least pH 11.0 is reached.
5. Repeat procedures numbers 1-4 using commercial aspartame as your sample.
6. Construct the titration curves of your sample and that of aspartame in MS Excel®
program using the average of your data on two trials. Determine the pKa values and
the isoelectric pH from the graph.
7. Identify your amino acid sample based on its pKa values and pI. Check on your
teacher if your identification is correct. If yes, then draw the titrimetric profile of your
sample and that of aspartame. Calculate the percent error of your pKa values and pI.

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

Experiment # 2: Titration of Amino Acids & Peptides

Name:________________________________________________ SCORE: __________

SCHEMATIC DIAGRAM

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

Experiment # 2: Titration of Amino Acids and Peptides

Name:________________________________________________ SCORE: __________

Group No._____ Section:________ Lab Instructor:__________________________________

Date Performed:_____________________ Date Submitted:_________________________

A. Identification of Unknown Amino Acid by Titration(30 pts)

1. Titration of the Unknown Amino Acid (10 pts)

Vol. NaOH, pH Vol. NaOH, pH Vol. pH Vol. pH

ml ml NaOH, ml NaOH, ml

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

2. Titration Curve of the Unknown Amino Acid (10 pts)

(Paste your Excel® graph here)

3. Identity of the Unknown Amino Acid and its Titrimetric Profile (10 pts)

Experimental Theoretical % Error

pKa1
pKa1
pKa1
pI
Identity of Unknown Amino Acid:

Titrimetric Profile of the Amino Acid

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

B. Titration of Aspartame (30 pts)

1. Titration of Aspartame (10 pts)

Vol. pH Vol. pH Vol. pH Vol. pH

NaOH, ml NaOH, ml NaOH, ml NaOH, ml

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2. Titration Curve of Aspartame (10 pts)

(Paste your Excel® graph here)

3. pKa Values, pI, and Titrimetric Profile of Aspartame (10 pts)

Experimental Theoretical % Error

pKa1
pKa1
pKa1
pI
Identity of Unknown Amino Acid:

Titrimetric Profile of Aspartame

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ESSENTIALS OF BIOCHEMISTRY LABORATORY © GIDEON A. LEGASPI, 2020

C. Follow-up Questions and Applications (15 pts)

1. Are there significant differences in the theoretical & experimental pKa values and pI of
your amino and aspartame? If yes, then explain the possible sources of errors. (3 pts)

2. What are the buffering zones of your amino acid and that of aspartame? (3 pts)

3. Base on the pI of your amino acid, is it suitable as buffer on acidic, physiologic, or

basic pH? Why? (3 pts)

4. Aspartame is now widely used as artificial sweeteners in many food products. What
are the benefits and health risks of aspartame consumption? (6 pts)

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