BCH02L Analytical Methods for

Biology

Prepared by Roberth Riggs Rondilla

2021
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I. Overview

This module will be all about pH measurements. Several definitions of acids and
bases will be given first. We will also tackle the principle behind a pH meter.
Calcutions in pH will also be shown. A simulation experiment and a recorded
experiment will be shown to reinforce the topics

Throughout the discussion, exercises are given for you to practice yourself. Be sure
to answer them before looking at the answer to exercises which is provided at the
last part of the module. If you feel you didn’t grasp a certain topic, go back to that
part and read it again. If you still can’t get it, feel free to call my attention and ask
me for further clarification.

II. Objectives

At the end of this module, you should be able to

1. Identify and differentiate definition and properties of acids and bases
2. Compute for the pH and pOH of a solution
3. Know the definition and uses of buffer
4. Familiarize the utilization of pH meter and pH pen

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III. Table of Contents
I. Overview……………………………………………………………….…………….…1
II. Objective……………………………………………………………….…………….…1
III. Table of Contents…………………………………………………….…………….…2
IV. Discussion
1. Acids and Bases……………..……………………………….……………..……3
2. Neutralization Reactions……….………..…………………….……………..12
3. Titration in Analytical Chemistry……………………………...……….……13
V. Activity Proper….…………...……………………………………………………….19
VI. Laboratory Report Sheet 7………………………………………………….……..21
VII. Post-Laboratory Question 7……………………………………………….……...22
VIII. Answer to Check yourself exercises………………………...……………….….23
IX. Take Home Quiz 7……………………..………………………..……………….….24

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IV. DISCUSSION

1. Acids and Bases

An acid of which it is a substance that yields hydrogen

ions (H+) when dissolved in water. Furthermore, base is a

substance that yields hydroxide ions (OH-) when dissolved in

water. These definitions were formulated by the Swedish

chemist Svante Arrhenius to classify substances whose

properties in aqueous solutions were well known. Substances

that are neither acid nor base can be said a neutral substance. Svante Arrhenius

Acids and bases are as familiar as aspirin and milk of magnesia although

many people do not know their chemical names—acetylsalicylic acid (aspirin) and

magnesium hydroxide (milk of magnesia). In addition to being the basis of many

medicinal and household products, acid-base chemistry is important in industrial

processes and essential in sustaining biological systems.

General Properties of Acids and Bases

Acids and bases are distinct classes of compounds because of the properties

of their aqueous solutions. Those properties are outlined below:

Acids

• Acids have a sour taste; for example, vinegar owes its sourness to acetic

acid, and lemons and other citrus fruits contain citric acid.

• Acids cause color changes in plant dyes; for example, they change the color

of litmus paper from blue to red (Figure 7.1)

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 • Aqueous acid solutions conduct electricity. Some acids are strong

electrolytes because they ionize completely in water, yielding a great many

ions. Other acids are weak electrolytes that exist primarily in a non-

ionized form when dissolved in water.

• Acids react with certain metals, such as zinc, magnesium, and iron, to

produce hydrogen gas. A typical reaction is that between hydrochloric acid

and magnesium:

2HCl(aq) + Mg(s) → MgCl2(aq) + H2(g)

• Acids react with carbonates and bicarbonates, such as Na2CO3, CaCO3,

and NaHCO3, to produce carbon dioxide gas:

2HCl(aq) + CaCO3(s) → CaCl2(aq) + H2O(l) + CO2(g)

Bases

• Bases have a bitter taste, generally.

• Bases feel slippery; for example, soaps, which contain bases, exhibit this

property.

• Bases cause color changes in plant dyes; for example, they change the color

of litmus from red to blue (Figure 7.1)

• Aqueous base solutions conduct electricity. Bases can be either strong or

weak, just as acids can.

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Ionization of Acids

As mentioned above, acids and bases are also electrolytes. Some acids,

including hydrochloric acid (HCl) and nitric acid (HNO3), are strong electrolytes.

These acids are assumed to ionize completely in water; for example, when hydrogen

chloride gas dissolves in water, it forms hydrated H+ and Cl- ions:

HCl(aq) → H+(aq) + Cl- (aq)

In other words, all the dissolved HCl molecules separate into hydrated H+ and

Cl- ions. Other example of strong acids include sulfuric acid and nitric acid. Thus,

these acids are also strong electrolytes. On the other hand, certain acids, such as

acetic acid (CH3COOH), which gives vinegar its sour flavor, do not ionize completely

and are weak electrolytes. We represent the ionization of acetic acid as:

CH3COOH(aq) ⇋ CH3COO-(aq) + H+(aq)

,where CH3COO- is the acetate ion. We use the term ionization to describe the

separation of acids and bases into ions.

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 As you would notice, the ionization of acetic acid is written with a double

arrow instead if single-headed arrow to show that it is a reversible reaction; that is,

the reaction can occur in both directions. Initially, a number of CH3COOH molecules

break up into CH3COO- and H+ ions. As time goes on, some of the CH3COO- and H+

ions recombine into CH3COOH molecules. Eventually, a state is reached in which the

acid molecules ionize as fast as the ions recombine. Such a chemical state, in which

no net change can be observed (although activity is continuous on the molecular

level), is called chemical equilibrium.

Acetic acid, then, is a weak electrolyte because its ionization in water is

incomplete. By contrast, in a hydrochloric acid solution the H + and Cl- ions have no

tendency to recombine and form molecular HCl. We use a single arrow to represent

complete ionizations.

Brønsted-Lowry Acids and Bases

The definition of acids and

bases by Arrhenius are limited

because they can be only applied to

aqueous solutions. Broader

definitions were introduced by

Johannes Brønsted and Thomas

Lowry: a Brønsted acid is a proton donor while a Brønsted base is a proton acceptor.

In this definition, a proton refers to H+.

For example, HCl is a Brønsted acid because it donates a proton (H+) to water

(the Brønsted base):

HCl(aq) + H2O(l) → H3O+(aq) + Cl- (aq)

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 The hydrated proton, H3O+, is called the hydronium ion. This equation shows

a reaction in which a Brønsted acid (HCl) donates a proton to a Brønsted base (H 2O).

You might got confuse because from the previous reactions I show the

chemical equation of ionization of HCl to be:

HCl(aq) → H+(aq) + Cl- (aq)

However, keep in mind that the hydronium ion (H3O+) is really a H+ surrounded

with H2O molecules. Keep in mind that both notations represent the same species

in aqueous solution.

At the product side of the chemical equation above is conjugate acid and the

conjugate base. A conjugate acid is formed when a proton is added to a base, and a

conjugate base is formed when a proton is removed from an acid. Hence, the

conjugate acid, following Brønsted definition, is a proton donor while the conjugate

base can accept proton.

We can also illustrate these relationships by examining the reaction between

acetic acid and ammonia:

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 Acids commonly used in the laboratory include hydrochloric acid (HCl), nitric

acid (HNO3), acetic acid (CH3COOH), sulfuric acid (H2SO4), and phosphoric acid

(H3PO4). The first three are monoprotic acids; that is, each unit of the acid yields

one hydrogen (or hydronium) ion upon ionization:

HCl(aq) + H2O(l) → H3O +(aq) + Cl- (aq)

HNO3(aq) + H2O(l) → H3O +(aq) + NO3-(aq)

CH3COOH(aq) + H2O(l) ⇋ CH3COO-(aq) + H3O +(aq)
Sulfuric acid is a diprotic acid because each unit yields two hydrogen (or

hydronium) ions in two separate steps:

H2SO4(aq) + H2O(l) → H3O +(aq) + SO42-(aq)

HSO42-(aq) + H2O(l) ⇋ H3O +(aq) + SO42-(aq)
H2SO4 is a strong electrolyte or strong acid (the first step of ionization is

complete), but HSO42- is a weak acid or weak electrolyte, and we need a double arrow

to represent its incomplete ionization.

Triprotic acids, which yield three H+ ions, are relatively few in number. The

best known triprotic acid is phosphoric acid, whose ionizations are:

H3PO4(aq) + H2O(l) ⇋ H3O +(aq) + H2PO4-(aq)

H2PO4-(aq) + H2O(l) ⇋ H3O +(aq) + HPO42-(aq)
HPO42- (aq)+ H2O(l) ⇋ H3O +(aq) + PO43-(aq)
All three species (H3PO4, H2PO4-, HPO42-) are weak acids, and the double-

headed arrows are used in each ionization step. Anions such as H2PO4-and HPO42-

are found in aqueous solutions of phosphates such as NaH2PO4 and Na2HPO4.

Strong bases such as sodium hydroxide (NaOH) and barium hydroxide

(Ba(OH)2) are strong electrolytes. Thus, they also completely ionize in aqueous

solutions:

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 NaOH(s) → Na+(aq) + OH-(aq)
Ba(OH)2(s) → Ba+(aq) + 2OH-(aq)
The OH- ion can accept a proton, as follows:

OH-(aq) + H+(aq)→ H2O(l)

Hence, OH- ion is a Brønsted base.

Ammonia (NH3) is an example of Brønsted base because it can accept a proton

(H+) ion:

NH3(aq) + H2O(l) ⇋ NH4+(aq) + OH-(aq)

Ammonia is a weak electrolyte (and therefore a weak base) because only a

small fraction of dissolved NH3 molecules react with water to form NH4+ and OH- ions.

The most commonly used strong base in the laboratory is sodium hydroxide. It is

cheap and soluble. (In fact, all of the alkali metal hydroxides are soluble.) The most

commonly used weak base is aqueous ammonia solution, which is sometimes

erroneously called ammonium hydroxide

2. pH: A Measure of Acidity

Because the concentrations of H+ (and OH-) ions in aqueous solutions are

frequently very small numbers and therefore inconvenient to work with, Soren

Sorensen in 1909 proposed a more practical measure called pH. The pH of a solution

is defined as the negative logarithm of the hydrogen ion concentration (in mol/L):

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 The negative logarithm gives us a positive number for pH, which otherwise

would be egative due to the small value of [H +]. Furthermore, the term [H+] in

Equation 8.6 pertains only to the numerical part of the expression for hydrogen ion

concentration, for we cannot take the logarithm of units. Thus, like the equilibrium

constant, the pH of a solution is a dimensionless quantity (in other words, walang unit)

Because pH is simply a way to express hydrogen ion concentration, acidic and

basic solutions at 25°C can be distinguished by their pH values, as follows:

Acidic solutions: [H+]>1.0x10-7 M, pH < 7.00

Neutral solutions: [H+]=1.0x10-7 M, pH = 7.00
Basic solutions: [H+]<1.0x10-7 M, pH > 7.00

Notice that pH increases as [H+] decreases. Sometimes we may be given the

pH value of a solution and asked to calculate the H+ ion concentration. In that case,

we need to take the antilog of Equation 7.2 as follows:

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 In the laboratory, the pH of a solution can be measured with a pH meter

(Figure 7.2). Indicators like litmus paper can be

also be used. Litmus paper turns red, purple, or

blue to roughly estimate pH values (Figure 7.1). A

universal indicator is a liquid mixture that

displays a rainbow of colors. Adding a drop of a

sample to a spot of universal indicator turns it a

color that corresponds to a pH range. Other

indicators are used within several pH ranges

(Figure 7.3)).

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 Figure 7.4 shows the pHs of a number of common fluids. As you can see, the

pH of body fluids varies greatly, depending on location and function. The low pH

(high acidity) of gastric juices facilitates digestion whereas a higher pH of blood is

necessary for the transport of oxygen.

The following examples shows the application of Equation 7.1 and 7.2

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Principle behind the pH meter

A pH meter is based on potentiomery and Nernst equation. Nernst equation

derives the relation between the electric voltage with ion concentration. The Nernst

equation derived for H+ ion concentration is the basis of pH meter. The pH meter

consists of glass (also called as indicator electrode) and reference electrode. The glass

electrode consists of glass membrane, which is sensitive to hydrogen ion

concentration of test sample solution. The reference electrode is standard and has

constant potential. The reference electrode does not respond to test sample solution.

The pH meter measures and compares the potential difference between both glass

and reference electrodes.

Using the Nernst equation, the potential difference is used to measure the

hydrogen ion concentration indicating the pH of given solution. Due to the potential

difference between two electrodes, the electron flows and generates current. This

generated current is measured by voltmeter. The relationship between the potential

difference, generated current and pH has been derived. The potential difference of 1

pH is 59.16mV at 25° C and hence when there is difference of one pH unit, there will

be change in voltage by 59.16mV.

You can also watch this video:

YouTube Title Link QR code

Potentiometric pH https://youtu.be/P1wRXTl2L3I
measurement

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3. pOH: Measure of Basicity

Likewise, the hydroxide ion molarity may be expressed as a p-function, or

pOH:

The OH- ion concentration must be expressed in molarity as well. A solution

with a pOH less than 7 is a basic solution while if it is greater than 7, it is an acidic

solution. Hence, pOH is the reverse of pH. When pOH is given, OH- can be calculated

from the antilog of Equation 7.3:

The following examples shows the application of Equation 6.3 and 6.4

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Relationship of pH and pOH

The pH and pOH of a water solution at 25oC are related by the following

equation:

Hence, we can calculate pH from a given pOH, among others. Let’s see some

examples:

4. Buffers

A buffer is something that resists change. In terms of acid and base chemistry,

a buffer solution tends to resist change in pH when small to moderate amounts of a

strong acid or strong base are added. A buffer solution consists of a mixture of a

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weak acid and its conjugate base or a weak base + conjugate base. Buffer

solutions are important in most biological systems. Many biological processes

proceed effectively only within a limited pH range. The presence of buffer systems

keeps the pH within this limited range.

The Henderson–Hasselbalch equation can be used to calculate for the pH of

buffer systems:

[conjugate base]
pH = pKa + log [acid]

Let’s have an example:

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How Do Buffers Work?

Let us compare the changes in pH

that occur on the addition of equal

amounts of strong acid or strong base to

pure water at pH 7 and to a buffer solution

at pH 7. If 1.0 mL of 0.1 M HCl is added to

99.0 mL of pure water, the pH drops

drastically. If the same experiment is

conducted with 0.1 M NaOH instead of 0.1

M HCl, the pH rises drastically (Figure

7.4).

The results are different when 99.0 mL of buffer solution is used instead of

pure water. A solution that contains the monohydrogen phosphate and dihydrogen

phosphate ions, HPO42− and H2PO4−, in suitable proportions can serve as such a

buffer. The Henderson–Hasselbalch equation can be used to calculate the

HPO42−/H2PO4− ratio

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V. Activity Proper

Objectives

1. Simulation 1: To observe the pH of several substances

2. Simulation 2: To observe how the pH vary as the concentration of the
[H+] and [OH-] vary.
3. Determine the pH changes of a buffered solution

Procedure
A. pH of several substances
1. Go to this link:
https://phet.colorado.edu/sims/html/ph-scale/latest/ph-scale_en.html
2. Select MACRO

3. Determine the pH of several substances by hovering the probe to the solution.

4. Record the pH of each substance in Table A of Laboratory Report Sheet 7.

B. pH Scale
1. Go to this link:
https://phet.colorado.edu/sims/html/ph-scale/latest/ph-scale_en.html

2. Select MICRO:

3. Determine the [H+] (or H3O+) and [OH-] of each substance from the scale to the left.

4. Record all data in Table B of Laboratory Report Sheet 7.

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C. pH meter, pH pen, and buffer

1. Before using the pH meter, it is a MUST that is should be calibrated. Watch and
observe how this was done.

YouTube Title Link QR code

Calibration of pH https://youtu.be/J8O5_rxmgPg
meter

2. A Phosphate buffer was prepared by your instructor. Watch the video below and
record the necessary data in Table C and D of Laboratory Report Sheet 7.

YouTube Title Link QR code

Buffer preparation https://youtu.be/8WpzHs351fc

3. The pH of distilled water was measured using both instruments. Watch the video
and record the data in Table D of Laboratory Report Sheet 7.

4. A solution of 0.1M HCl was added to both distilled water and the buffer solution.
Then, the pH was measured using both instruments again. Watch the video below
and record the data in Table D of Laboratory Report Sheet 7.

YouTube Title Link QR code

pH measurements https://youtu.be/yjsK3ggjmPc

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VI. Laboratory Report Sheet 7

General Directions: Fill-up this table with your observations. To answer, you can
print this part and take a clear picture using a scanner app. You can also write down
the answers in a yellow pad. Upload/submit it to Lab Module 7: Performance Task 7

A. pH of Substances
Substance pH

B. [H+] and [OH-] of Substances

Substance [H+] [OH-]

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 C. Preparation of buffer
Weight of K2HPO4 (g)
Weight of KH2PO4 (g)
Volume of the buffer solution
Molarity of K2HPO4 (g)
Molarity of KH2PO4 (g)

D. pH measurements
pH meter pH pen
Buffer
Distilled water
Buffer + 0.1 M HCl
Dist. Water + 0.1 M
HCl

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VII. Post-Laboratory Questions 7

General Directions:. To answer, you can print this part and take a clear picture
using a scanner app. You can also write down the answers in a yellow pad.
Upload/submit it to Lab Module 7: Performance Task 7

1. From the simulation, which substances are acidic?

2. From the simulation, which substances are basic?

3. Is there any substance that is considered neutral?

4. From the simulation B, what is the relationship of:

a. [H+] and pH

b. [OH-] and pH

(Hint: are they directly proportional or inversely proportional)

5. Compute for the pOH of the substances from table A.

6. Research Work: Why is pH meter needed to be always calibrated?

7. Which device (pH pen or meter) you will use when you want to measure actual pH
of samples in the field? What are its advantages and disadvantages?

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VIII. Answers to Checkyourself exercises

7.1 a) pH = 8.72 b) pH=9.72 c) pH=10.72

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IX. Take Home Quiz 7

Directions: Fill up the table below. Common name of compounds is given on the 1 st
column. You are tasked to identify their chemical formula and name by researching.
Also, predict each compound’s properties. You can write down the answers in a
yellow pad. Upload/submit it to Lab Module 7: Take Home Quiz 7

1. Complete the following table by calculating the missing entries and

indicating whether the solution is acidic or basic.

pH pOH [H+] [OH-] acidic or basic

1. 4.25

2. 8.35

3. 1.6 x 10-6 M

4. 9.1 x 10-2 M

2. Calculate the [OH-] and pH of each of the following strong base solutions:
a. 0.0050 M KOH
b. 2.055 g of KOH in 500.0 mL of solution
c. 1.00 mL of 0.175 M NaOH diluted to 2.00 L.
3. Describe the color of selected substances (in Table A), if 2-3 drops of indicator is
added (look at Figure 7.3). asumming that the original substance is colorless
Substance Bromophenol Methyl red Phenolphthalein Thymol blue
blue
Battery acid
Spit
vomit
Hand soap
Chicken soup
milk

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