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High Value Note

Mole Concept and Problem

Solving Approaches
in Life Sciences.
Dr. Aditya Arya | Dr. Amit Kumar
High Value Note: Drawing Pin Publishing

L i fe Sc i e n c e s

New Delhi, India

High Value Note: Mole Concept and Problem Solving Approaches in Life Sciences.

1st Edition - Mar 2016.

1st revised reprint – Aug 2018

Copyright © 2018. Drawing Pin Publishing

All rights reserved. No part of this publication including text, tables, and
illustrations may be reproduced or transmitted in any form or by any means,

electronic or mechanical, including photocopying, recording, or any other
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All the copyright-related queries may be directly sent to the publisher at

Acknowledgements O
This article has been prepared for the academic purpose and data,
information has been retrieved from authentic research articles of various
researchers, guidelines and definitions from the International Union of Pure

and Applied Chemistry (IUPAC), National Institute of Standard (NIST). We

sincerely acknowledge the efforts of researchers who unraveled a lot of on
mole. The questions at the end of the booklet were obtained from some of

the national fellowship exams for research in India which are now available in
the public domain. However, the solutions were developed by drawing pin


Author or Publisher do not take any responsibility of any kind of personal or


financial loss due to any inadvertent or unavoidable errors.

Plagiarism declaration

This article was checked for plagiarism and contains >70% unique test, certified by
Drawing Pin Publishing.

Human Hours

128 Human hours were devoted in preparing this article.

Printed in India

1 Arya A, Kumar A. (2018). High Value Note, Drawing Pin Publishing, 04: 1-
High Value Note: Drawing Pin Publishing

Mole Concept and Problem

Solving Approaches
in Life Sciences.
Aditya Arya*, Ph.D., Amit Kumar, Ph.D.
27th Apr 2018

©2018. All Rights Reserved. Drawing Pin Publishing.

Cite as: Arya A, Mole concept and problem solving approaches in life sciences (2018). High Value Note 04, 1e, Drawing Pin Publishing.

If we are told that a guy is 6’2” tall, or we dispense 20 liters of waters in the toilet at one time, or
somebody ate a dozen bananas, a perception about the quantity is immediately built in our minds. We
immediately respond that ohh! 6’2” means really tall guy, or a dozen bananas at a time means too
much! But how would you respond to a number of moles of oxygen entering into your body with each
breath or molarity of sugar in lemonade you prepare? Perhaps our mind stops responding as quickly as
previous answers. The primary reason is that we have never brought mole as a unit into our daily lives,
and thus it became increasingly difficult to comprehend. Mole as a fundamental unit like meter, litre,

second etc. is equally important. Although it is indispensable for chemists, but highly valuable for
biologists as well. A poor understanding and difficulty in realization of mole concept can lead to

enormous errors and wastage of resources while performing scientific experiments, due to erroneous
calculations. Experiments like biochemical assays, animal cell culture, polymerase chain reactions,
spectrometry, and routine buffer preparation and most of the analytical techniques require a very good
understanding on mole concept. This high values note brings you a highly organized set of information
of mole concepts, recent updates in the definition, and various problem solving approaches in mole

concept and concentration terms.

1.1 Why Mole Concept is Needed?

A very simple question that is generally not answered to students at K-12 (or high school) level is why at all
we require mole concept and need to calculate the number of moles and use concentration terms. Is mass
not enough as a unit of quantity? If we understand this question, we would gradually realize that mole is a
really important quantity both for chemists and biologists. In the following subsections we will emphasize
on realizing this fact that knowing number of molecules of a reaction mixture is important and also a quantity
that depicts specific number of molecules is needed.

1.1.1 Need of knowing the numbers of molecules

We often go to market for buying various goods, vegetable fruits etc. and use various units like gram,
kilogram, dozen etc. to represent quantity of various goods. Similarly, in chemistry or biology, when we are
observing a reaction between two or more type of molecules, we need to represent their quantities in some
units. Although we already have a well-established unit of mass called gram (g), which can be used to

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represent the quantities of various reacting species. Now, why do we need units like mole instead of gram
to represent quantities of matter or reacting molecules? Let us understand with the help of following

If we perform a reaction between five grams of sodium and five grams of chlorine to make sodium chloride
under suitable conditions so that NaCl can be formed, what is the maximum possible amount of sodium
chloride that can be formed? If you wonder that 10 grams of sodium chloride would be formed, you are
wrong (for calculation of exact amount see practice solved practice question in later part of section 1.3). The
reason behind why 10 grams will not be correct answer can be understood with the help of a simple analogy
described below.

Suppose you bought 1 kg apples and 1 kg grapes and you are asked to make some decorative fruit salad
that contain 1 apple and 1 grape fixed using a tooth pick (As shown in figure 1), only few structures could
be made until, all apples are used up, because apples are larger and therefore smaller in number and a
number grapes will be spare. But if we buy exactly equal number of grapes and apples there will be no spare
grapes. Now consider apples analogous to one reacting elements and grapes analogues to another element.
We know that all the atoms are not of same size and mass (due to different number of protons, neutrons

and electrons). Some of the atoms are small (light) and some are big (heavy), so when they are mixed in the
equal volumes or equal mass, one that is with larger mass will be consumed earlier than one with smaller
size, because the combination of atoms to form molecules occurs according to their numbers but not as per
mass. Hence, in case we take two chemical ingredients in terms of their mass, one of them will always be

in excess and wasted. So in order to allow both the components to be consumed completely, one must take
equal number of atoms (or molecules) of both. So if a reaction is being carried out in small scale in lab or on
a larger scale in industries, estimating the number of atoms or molecules is more important than merely
knowing the weight, this justifies the need of knowing the number of atoms or molecules in a given mixture.
Figure 1 illustrates the difference between considering equal mass and equal numbers with aforesaid
analogy with apple and grapes.

Equal Weights Equal Numbers Equal Weights Equal Numbers


1 kg Grapes 1 kg Apples 1 kg Grapes 1 kg Apples Element 1 Element 2 Element 1 Element 2


Light Heavy Light Heavy


Compounds Compounds
Grape-Apple structures Grape-Apple structures
Spare elements No spare elemets
No spare grapes
Spare Grapes

© 2018. All Rights Reserved. Drawing Pin Publishing.

Fig 1. Illustration representing that chemical reactions occur in terms of numbers but not in terms of mass and also the importance of knowing
number of particles in chemical reactions can prevent the wastage or excess of one of the substrates.

Practice Question

Q. What if mole concept would not have emerged and industries were using equal masses of two reactants to prepare a
Solution: If two reacting species were combines in equal masses then , one with the larger molecular weight would have had smaller number
of molecules (or atoms) in given mass and other would have had higher number of molecules (or atoms). So, one of the substrates would have
exhausted earlier and other one would remain unused. In this was a lot of reactants would be wasted.

In stricter context of chemistry, the generalization we made above, that chemical reactions occur in terms of

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number of atoms or molecules was a well-proven scientific observation and known to us from over two to
three centuries ago. Some of these scientific theories include, law of definitive proportion (Joseph Proust,
1765), Law of multiple proportion (Dalton, 1803), Dalton’s atomic theory (Dalton, 1808), Gay Lussac’s law of
gaseous volumes (Gay Lussacs, 1808) Avogrado’s Law (Avogadro, 1811). But before discussing these laws
let us ask a simple question, that do we need a machine or should we use a machine to count atoms or

1.1.2 Do we need a machine to count atoms or molecules?

In previous section we learnt that it is convenient to take two reacting species in equal numbers (or in the
ratio of molecularity, like for making water hydrogen and oxygen atoms should in in the ratio 2:1). So next
important question is how to exactly count the atoms? It’s no joke. The atoms are almost 1 millionth of a
millimetre. We may not be able to see it even with light microscope. So, let us imagine a machine that can
calculate the number of atoms or molecules before any chemical reaction. If the counting speed of a machine
is about a billion particles per second, which is quite fast compared to other counting machine known today.
Do you know how much time it would take to count 6.022x1023 atoms (we will define this number later)?

6.022 × 1023
𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑡𝑡𝑡𝑡 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑎𝑎 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 = = 1.91 × 107 𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦
109 × 60 × 60 × 24 × 365

Astonishingly long time, almost 10 times than the age of evolution of humans! So, it would never be a good

idea to develop such a machine. Then how to count the number of atoms or molecules in a given mass of
matter? It actually a child’s play. The counting of number of molecules or atoms has been simplified by
development of a law called Mole concept.
1.1.3 Fundamental laws which built foundations of Mole Concept
The mole concept is a simple relationship that enables us to count the number of atoms or molecules just

by weighing or measuring volume, without the requirement any sophisticated costly machines. Hence, it is
very much possible to count the number of water molecules in a mineral water bottle of one litre, the number

of oxygen atoms we breathe every minute, and the number of alcohol molecules in a Beer can of 500 ml,
without even actually visualizing the molecules or atoms.

Law of definitive proportions states that “a given compound always contains exactly the same proportion
of element by weight”. This supports the idea that during the formation of a compounds atoms combine in
a definite ratio, which does not change by nature of synthesis.

Law of Multiple proportions states that “if two elements can combine in more than one form to form more
than one compound, then masses of one element that combines with the fixed weight of other are in the
simple ration of small whole numbers”. This further strengthens the idea that combination of atoms is in
whole numbers.

Dalton’s Atomic Throy established the existence of an atom and stated that “chemical reactions involve
reorganization of atoms, and compounds are formed when atoms of different elements combine in fixed
ratio”. This theory supported the above two laws, but could not answer these rules for gases.

Gay Lussac’s law of gaseous volumes states that “ when gases combine or are produced in a chemical
reaction they do so in a simple ration by volume, provided all gases are at same temperature and pressure”.
This augmented the missing links between previous laws and Dalton’s theory.

Avogadro’s law established the relationship between the volumes and number of moles of gaseous
molecules, it states that “equal volumes of all the gases at the same temperature and pressure should
contain equal number of molecules”. This lead to the famous gas equation (PV = nRT) that enables us to
calculate number of a moles of a gas under given conditions, or to compare the number of moles of two

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gases or same gas at two conditions (P1V1 = P2V2)

These laws eventually lead the foundation of what we know today as mole concept. The Avogadro’s law
was much closer to the concept of determination of number of particles, and proposed enough in his article
published in Journal de Physique in 1810, but it not was not recognized well until 1960, when it was
discussed extensively in an international chemistry meeting at Karlsruhe, Germany. The mole concept is a
simple relationship that enables us to count the number of atoms or molecules just by weighing or measuring
volume, without the requirement any sophisticated costly machines. Hence, it is very much possible to
count the number of water molecules in a mineral water bottle of one litre, the number of oxygen atoms we
breathe every minute, and the number of alcohol molecules in a Beer can of 500 ml, without even actually
visualizing the molecules or atoms.

1.2 Definition of A Mole and Formulation of Mole concept

The development of mole concept and definition of one mole has taken several decades. In relative context
we can imagine that the formulation of fundamental laws was the founding step, which was succeeded by

development of some naïve definition of mole. The term and unit mole had been in use in chemistry since
roughly the start of the 20th century, however, it was incorporated into the SI as a base unit, along with the
base quantity amount of substance only in 1971. However an old textbook by Selwood (1964) states that
“The word ‘mole’ is a collective noun like flock (of birds) or galaxy (of stars). But mole has the added meaning of a very

definite number of particles, namely, Avogadro’s number”. Another book by reads “….the weight of a single
molecule in atomic mass units is the same, numerically, as the weight of a mole in grams. The mole is a
convenient package, like a dozen or a gross; but numerically it is much larger.” However, these primitive
definitions of mole were replaced by standard definition set for by the IUPAC in 1971 as SI unit. The standard
definition of SI unit recommended by IUPAC was “The mole is the amount of substance of a system
which contains as many elementary entities as there are atoms in 0.012 kilogram of carbon 12; its
symbol is “mol”.

However, some recent textbooks also have slightly modified versions. Gilbert et al. (2015), in their book

define mole as an amount of material (atoms, ions, or molecules) that contains Avogadro’s number (NA =
6.022 × 1023) of particles.” Silberberg (2013) uses the current SI definition and states that Mole is the SI

base unit for amount of a substance. The amount that contains a number of objects equal to the number of
atoms in exactly 12 g of carbon-12 (which is 6.022 × 1023).

Later recently, the IUPAC draft of 2103 has revised definition of mole, which has finally been accepted in
2018. The current definition is as follows:

The mole, symbol mol, is the SI unit of amount of substance of a specified elementary entity, which
may be an atom, molecule, ion, electron, any other particle or a specified group of such particles; its
magnitude is set by fixing the numerical value of the Avogadro constant to be exactly 6.02214129 ×
1023 when it is expressed in the SI unit mol−1.
The changes and new definition has been re-iterated and emphasized in section 1.2.2. Fig 2 illustrates a brief
timeline of the development of mole concept and updates.

Pound Mole: Chemical engineers use the mole concept extensively, but the unit is rather small for industrial use. For convenience in avoiding conversions
in the Imperial (or American Customary Units), some engineers adopted the pound-mole (notation lb-mol or lbmol), which is defined as the number of entities
in 12 lb of 12C. One lb-mol is equal to 453.59237 mol.

5 Arya A, Kumar A. (2018). High Value Note, Drawing Pin Publishing, 04: 1-
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1765 1811 1971 2018
Law of definitive Avogadro’s Law Standard definition Revised definition
proportion of gaseous of mole was set up of mole accepted
by Joseph Proust volumes by IUPAC by the IUPAC

Dalton’s Theory First use of term Avogadro’s New definition

and Gay Lussac’s number and estimation of proposed in draft
law for gases its value by Jean Baptiste of IUPAC
1808 1909 2013

Fig 2. Timeline of events associated with mole concept

1.2.1 Revision in definition of mole in 2018

The new definition was proposed in 2013 and published after rigorous discussion and accepted in January
2018 by IUPAC. The new definition emphasizes that the quantity ‘amount of substance’ is concerned with
counting entities rather than measuring the mass of a sample. As per the new definition one mole contains
exactly 6.022 14076 × 10 23 elementary entities. This number is the fixed numerical value of the
Avogadro constant, N, when expressed in mol, and is called the Avogadro number. This new definition

is in contrast to the current definition adopted in 1971 which relies on the mass of the kilogram. The new SI
will be linking all seven base units to fundamental physical constants. In November 2018, revised definition
of the kilogram, ampere, kelvin and mole are expected to be approved by the CGPM and the revised
definition are expected to come into force on World Metrology Day, 20 May 2019.
1.2.2 The mole concept: Mass number relationship
The mole concept is all about establishment of the mass-number relationship. As described in the definition

of mole, it is possible to predict the number of particles in a given mass of substance. In general, any
substance when weighed in grams in a weighing balance equal to its molecular weight represents one mole.
For example, molecular weight of sodium chloride is 58.5 u, hence if we weigh 58.5 grams of this salt in

weighing balance we will have 1 mole quantity of sodium chloride. Same is true for water, molecular weight
of water is 18 u, and hence 18 gram of water (almost a large spoonful) would represent 1 mole. Unlike
solids, it is not easy for us to measure the mass of gases, hence they are related with volumes, but volumes
change with pressure and temperature, hence a standard temperature and pressure is considered for

defining one mole. Hence, for gases, it is 22.4 liters of any gas at STP (25oC and 1 atm pressure), that
represents one mole quantity.

International Mole Day: International mole day is celebrated annually on October 23 from 6:02 a.m. to 6:02 p.m. to commemorate Avogadro's number
(6.02 x 1023), which is a basic measuring unit in chemistry. Mole Day was created as a way to foster interest in chemistry. Schools throughout the United
States and around the world celebrate Mole Day with various activities related to chemistry and/or moles.

1.2.3 Defining mole on a lighter note: Chemist’ dozen

If you still find these textbook definitions difficult, just relax. Again go back to your daily life experiences and
imagine how you would ask for bananas. How much for a dozen? Right. So if it would have been to a shop
of atoms and molecules and you may have to buy say 6.022 x 1023 atoms or molecules, you could ask, how
much for a mole? So for just like you use dozen for 12, a mole can be used for 6.222 x1023 items. It may be
these many sand grains, these many hairs, these many rice grains or anything. Perhaps this was the reason
that Nivaldo Tro, in his chemistry textbook introduced mole as chemist’s dozen.
Moreover, a confusion may arise if number of moles of hydrogen atoms are to be determined in one mole
of water or moles of electron needs to be determined in 1 mole of oxygen gas! These question are analogous
to asking the number of legs in chairs. Suppose there are 20 chairs in a classroom and each chair has 4 legs,
so total number of legs would be 4 x 20. Similarly, if one water molecule has two hydrogen atoms, n moles

Arya A, Kumar A. (2018). High Value Note, Drawing Pin Publishing, 04: 1-20. 6
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will have n x 2 moles of hydrogen.

Practice Question

Q. You are given 1 mole of potassium permanganate (KMnO4), and asked to calculate the number of moles of each type of atom
and total number of moles of all the atoms present.
Solution: Each molecule of potassium permanganate has one potassium, one manganese, and four oxygen. Hence one mole of potassium
permanganate would have 1 mole of potassium, one mole of manganese, and four moles of oxygen. And total moles will be six (1+1+4).

Now before we try some tricks on calculations related to mole concept, let us ask a few more question.
Why the value of one mole is 6.22 x 1023, who determined this value and how?

1.3 How the Avogadro’s number was determined?

Contrary to the beliefs of generations of chemistry students, Avogadro’s number was not discovered by
Amadeo Avogadro (1776-1856), but it was named in his honor by Perrin. The first person to estimate the
actual number of particles in a given amount of a substance was Josef Loschmidt, an Austrian high school

teacher who later became a professor at the University of Vienna. In 1865 Loschmidt used kinetic molecular
theory to estimate the number of particles in one cubic centimeter of gas at standard conditions. This
quantity is now known as the Loschmidt constant, and the accepted value of this constant is 2.6867773 x
1025 m-3. He actually calculated the Loschmidt number, a constant that measures the same thing as

Avogadro’s number, but in different units (ideal gas particles per cubic meter at 0◦C and 1 atm). When
converted to the same units, his number was off by about a factor of 10 from Avogadro’s number. The next
estimates were made by van der Waals, Roentgen, Rayleigh, Plank, Wilson, Thomson, and Einstein between
1873 and 1908 using a variety of methods.

The term "Avogadro’s number" was first used by French physicist Jean Baptiste Perrin. In 1909 Perrin

reported an estimate of Avogadro’s number based on his work on Brownian motion--the random movement
of microscopic particles suspended in a liquid or gas. Perrin was also awarded Nobel Prize in Physics for
1926 for his seminal contribution.

1.3.1 Perrin’s method for determination of Avogadro’s number


Perrin calculated Avogadro’s constant in several different ways using his own experimental data and
Einstein’s theories of Brownian motion. In fact he used three different methods, first was based on the
exponential distribution of particles with gravity, second was based on measurement of Brownian
movement and the diffusion constant and the third one was based on measurement of rotational motion in

Brownian systems. Perrin prepared tiny spheres'of gamboge, a resin, all of uniform size and density, by
elaborate fractional centrifuging. He dispersed about 17000 particles of the resin in a water column of height
0.1 mm. Using a microscope with a resolution of a quarter micron, he measured the vertical distribution of
colloidal particles and calculated the value of Avogadro’s number.

1.3.2 Millikan’s method for determination of Avogadro’s number

Accurate determinations of Avogadro’s number require the measurement of a single quantity on both the
atomic and macroscopic scales using the same unit of measurement. This became possible for the first time
when American physicist Robert Millikan measured the charge on an electron. The charge on a mole of
electrons had been known for some time and is the constant called the Faraday. The best estimate of the
value of a Faraday, according to the National Institute of Standards and Technology (NIST), is 96,485.3383
coulombs per mole of electrons. The best estimate of the charge on an electron based on modern
experiments is 1.60217653 x 10-19 coulombs per electron. If we divide the charge on a mole of electrons by
the charge on a single electron we obtain a value of Avogadro’s number of 6.02214154 x 1023 particles per

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1.3.3 X-ray diffraction based method for the determination of Avogadro’s number
The most accurate way to-date to measure the Avogadro constant is using x-ray diffraction. When x-rays
pass through a crystal lattice, they are scattered in a predictable way. The scattering depends on the type of
atoms, the arrangement of the atoms, and the spacing between them. By measuring the pattern of the
scattered x-rays, scientists can reconstruct exactly how the atoms are arranged in the crystal and the
distances between the atoms. With this very exact data, they can count how many atoms there are in a unit
cell of the crystal, which gives them the number density (atoms/volume) of the material. Silicon crystals are
the purest, most perfect crystals that we can make so they are used in measuring Avogadro’s constant.

Besides this there are several other methods which have been attempted by a number of scientists to prove
the validity of Avogadro’s number. Some of the methods include black body radiation method (used by
Plank), Counting of alpha particles, electrochemical methods (Townsend in 1897) and mass spectrometric

1.4 Problem Solving approach: The Love triangle of Mole

“Love triangle of the mole” can be considered as a trick to interconvert mole, mass and volumes of matter
and solve problems based on mole concept. If you love this triangle, mole concept would become easier.


Fig 2. The love triangle of the mole

This would help us to understand the problem solving approaches basic tricks to quickly interconvert number
of particle to mole to mass or volume. Usually, some of the physical properties of matter such as mass can
be measured using weighing balance, and using Avogadro’s relation. However, it would be really difficult to
measure the mass of gases, hence their volume can be measured. Also, it must be noted that volume of
the gases is affected greatly by pressure and temperature, hence these two components must be standard.
Let us try to understand each inter-conversion individually.

Problem solving approach: Always look for what is given in the question and what is asked and then pick up a suitable corner of mole triangle. Molecular
mass may not be always given as it can be calculated using molecular formula. But either of the three things actual mass of matter, actual volume of a gas,
or number of molecules must be provided to determine the number moles. Once you know number of moles you can determine either mass or volume (in
case of gas). Never use volume- mole relationship for solids, or liquids. You may also remember a trick called GK. i.e. Given divided by known for determining
mole from number, mass or volume, i.e. given number/Avogrado’s number, given mass/molecular mass or given volume/22.4.

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1.4.1 Mole to mass inter-conversion
Mass and mole can be interconverted by using the fundamental statement of mole concept. Simply, divide
the given molecular weight of the sample by its molecular weight (Molecular weight of a molecule can be
calculated by adding the atomic mass/mass number of each atom of the molecule).
𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 𝑖𝑖𝑖𝑖 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔
𝑁𝑁𝑜𝑜 𝑜𝑜𝑜𝑜 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 =
𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 𝑖𝑖𝑖𝑖 𝑎𝑎𝑎𝑎𝑎𝑎

𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑜𝑜𝑜𝑜 𝑎𝑎 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 = 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑥𝑥 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑖𝑖𝑖𝑖 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔

Practice Question

Q. You went to friend’s house and due to scorching summer, u felt thirsty. Your friend brought a one liter bottle containing
mineral water. If you consumed exactly half the water, how many molecules of water entered into your body? How many
hydrogen atoms were there in total?
Solution: Given mass of water =? , volume is given – 500 ml, Mass = volume x density (density of water is 1 g/ml if not specified)
i.e. mass of water consumed = 500 (ml) x 1 = 500 g; molecular weight of water – 18.
Using the relation n = given weight/molecular weight = 500/18 = 27.78 moles of water
Number of molecules = Avogrado’s number x number of moles = 27.78 x 6.022 x 1023 =167.29 x 1023 = 1.6729 x 1025 Molecules.

Number of hydrogen atoms in 500 ml water = twice the number of molecules (each molecule has two atoms of hydrogen),
i.e 2 x 1.6729 x 1025 = 3.3458 x 1025 atoms of hydrogen.

1.4.2 Mole to number inter-conversion

The number of moles can also be achieved we have clues about the number of molecules in a given sample
also, the other way out, number of particles (atoms or molecules) may be calculated if we are aware about O
the number of moles of a given matter.
𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛 𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝
𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 = (𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 𝑡𝑡𝑡𝑡 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎, 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖)
𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝑜𝑜 ′ 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑏𝑏𝑏𝑏𝑏𝑏

𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑖𝑖𝑖𝑖 𝑎𝑎 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 = 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑥𝑥 𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝑜𝑜′ 𝑠𝑠 𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛

Practice Question

Q. How many moles of DNA are present in a human sperm cell? What will be the number of DNA molecules if someone has 2

picogram of human DNA (our genome has 3 x 109 bp)

Solution: In this question, we can use the mole-number relationship, as it is convenient to know the number of molecules of DNA per cell.
Each chromosome is a molecule of DNA. So total molecules per cell will be 46 in somatic cells, while sperm cell is haploid so it will have 23
molecules of DNA. Now, in another part of this question, no of moles of DNA can be calculated by using given weight and molecular weight
of human DNA. Molecular weight = 650 x 3 x 109 = 1.95 x1012 Da.

Number of moles of DNA in 2 picogram = given weight/ Mol wt = 2 x 10-12/ 1.95 x1012 = 1.02 moles. Number of DNA molecules = moles x
Avogadro’s number = 6.022 x 1023 molecules.

1.4.3 Mole to volume inter-conversion

As mentioned above that the measurement of mass of gases is a difficult and challenging aspect, hence,
the volume of gases is often considered as a convenient choice for representing their quantity. Moreover,
the fundamental laws of chemistry also establish a relationship between volume and number of moles. It
was observed that any gas at STP (standard temperature and pressure, 25oc and 1 Atm) occupying 22.4
litres of volume would contain 1 mole of constituent units. i.e. 22.4 litres of hydrogen, helium oxygen or
nitrogen would have same number of molecules which will equal to Avogadro’s number. We can make
inter-conversion using following relationships.

𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝑜𝑜𝑜𝑜 𝑔𝑔𝑔𝑔𝑔𝑔 (𝑖𝑖𝑖𝑖 𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙)

𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑜𝑜𝑜𝑜 𝑔𝑔𝑔𝑔𝑔𝑔 (𝑎𝑎𝑎𝑎 𝑆𝑆𝑆𝑆𝑆𝑆) =

𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉 𝑜𝑜𝑜𝑜 𝑔𝑔𝑔𝑔𝑔𝑔 (𝑖𝑖𝑖𝑖 𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙) = 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑥𝑥 22.4

9 Arya A, Kumar A. (2018). High Value Note, Drawing Pin Publishing, 04: 1-
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This can further we extended to conversion of volume to mass or vice versa using previous relationship.

Practice Question

Q. Calculate the number of moles of oxygen breathed in by a normal human being in one day (24 hours) under STP conditions, if
we breathe about 16 times per minutes and 500 ml of air is normally breathed in (tidal volume) during effortless breathing.
(Consider the percentage of oxygen in air as 20%)
Hint: Amount of oxygen breathed in 1 breath = 20% of 500 ml = 20 x 500/100 = 100 ml or 0.1 Litre
Provided we respire at STP. The number of molecules of oxygen entering into our body per minute = 0.1/22.4 = 0.00446
In one day no of moles entering our body = 24 x 60 x 16 x 0.00446 = 102.84 moles.

1.4.4 Importance of mole concept in life sciences

Life Sciences, in particular biochemistry and molecular biology calculation involve the usage of mole concept
at several instances. Preparation of buffers to pharmacological preparations require the knowledge of mole

concept and concentration terms. Moreover, basic understanding of mole concepts and concentration terms
also help us in various analytical techniques such as proteomics, enzyme assays, spectrophotometry, kinetic
assays, polymerase chain reaction, metabolomics, photochemistry etc. Extending as an analogy we can
represent variety of clinical and biochemical terms in moles, such as concentration of sugar in blood for a
clinician could be 80-120 mg/dl, the same quantity is often represented as 4.5 to 7.5 mM in biochemistry

text book. The amount of ions, neurotransmitters and other metabolites are mostly represented in terms of
moles. Therefore a sound understanding of mole concept can help a better comprehension of concepts in
life sciences as well as error free calculation for preparation of various reagents in scientific experiments.
Mole concept may also be applied to the measurement of quantity of specific metabolites in fluids as well
as intracellular compartments (See practice questions in the end).

Useful information for calculations in Life Sciences: Average molecular weight of free amino acid – 128 Da, Average molecular weight of amino acid
residue in protein – 110 Da. Calculating molecular weight of protein when number of amino acids is n , MW = n x 110 + 18. Average molecular weight of

a nucleotide (dNTP) – 325 Da, (NTP) – 330. Average molecular weight of a nucleotide pair (dNTP) – 650 Da, (NTP) – 660. Molecular weight of a given
molecule of DNA = number of nucleotide pairs x 650, Molecular weight of a given piece of RNA = number of nucleotides x 330. Molecular weight of glucose
180 Da. Most of the intracellular compartments or cell when considered spherical, the volume = 4/3 πr3, when considered cylindrical, volume – πr2h. Most

of the volumes in cells can be easily converted into mass by considering them as aqueous environments and using the density of water (1 m3 = 1000 L, or 1
cm3 = 1 ml or 1 mm3 = 1 µl). Remember, these volume and mass interconversion apply only for aqueous systems and if the specific density is not provided
separately. Dalton (Da) is equivalent to gram molecular weight.

1.5 Concentration Terms and Their Relative Usage


Concentration represents the quantity of one of the component with respect to other, in most cases quantity
of solute with respect to overall solvent or solution. There a number of different terms for representing the
concentration some of which are well known such as molarity, molality, normality etc. Besides learning the
basic definition of the each of the concentration terms it is important to learn that when to use a specific
type of concentration term or how to make a choice?

The choice of concentration term depends upon the need of the experimenter and kind of the experimental
conditions. Usually the concentration terms that are used for pharmaceutical purpose or displayed on labels
of food need to be simple and easy to understand, therefore, most of them are percentage concentration
terms. While those which are used for the laboratory purpose need to display the number of moles or
particles as one need to know these parameters for knowing the stoichiometry of the reaction. The third
class of concentration terms is for very small magnitude and especially used for environmental applications
or toxicological studies, such as representing the level of pollutants. IUPAC recommends Molarity as a
standard concentration term. Figure 4 outlines the categories of concentration terms on the basis of usage.

Arya A, Kumar A. (2018). High Value Note, Drawing Pin Publishing, 04: 1-20. 10
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Classification of Concentration Terms

colo gic at or y u n mental

ma b or iro



En v

u se
Ph a

Simple to comprehend Simple to comprehend Very small concentration
Easy to calculate Easy to calculate terms

w/w w/v v/v

ur e de p re i ndep
rat atu
Te mp e

Tem per


Molarity | Formailty | Normality Molality | Mole fraction

© 2018. All Rights Reserved. Draw ing Pin Publishin

Fig 4. Chart of various concentration terms (Note that this is not a recommendation but the commonly used practice and therefore in order to make the choice simpler

1.5.1 Concentra�on terms for pharmacological applica�ons

author chose to categories them

Terms which are used for pharmacological purpose and food industry are simple to express and
comprehend, so that just a glimpse of value can provide the relative amount of solute in the given solution.
Most of the medicines and food products contain their ingredients displayed in such simple units. Most
commonly used simple concentration term in this category is percentage. The percentage simply means
parts of solute in 100 parts of solution. Percentage terms can be represented in three different ways,

percentage w/w, w/v and v/v.

Percentage weight by weight (% w/w) is used when both solute and solvent are solids such as percentage

of starch in potato;
• Percentage weight by volume (% w/v) is used when solute is solid but solution is liquid e.g. 0.9% Saline, which means 0.9 parts of sodium chloride in 100
ml of solution.
• Percentage volume by volume (% v/v) is used when both solute and solution are liquid, for example concentration of alcohol in alcoholic beverages (5% v/v
alcohol means 5 ml alcohol in 100 ml solution or 95 ml water).

As stated above, the logic for making a choice between three types of percentage terms is based on the
state of solute and solvent applies in day to day life and can be used to label constituents in
food/pharmaceutical products. However, in chemistry, the most preferred percentage term is w/w, which is
also known as weight percentage or mass percentage. The reason for this preference is easy conversion of
w/w into molarity and other concentration terms, while w/v or v/v cannot be converted into molarity/molality
if we do not know density of the solution.

Proof as concentration term for alcoholic beverages: A special type of concentration term is used for representing the concentration of alcohol in
alcoholic beverages called proof. Proof is just half of the percentage v/v. So an alcoholic drink marked 30 proof represents 15% alcohol by volume. A bottle
of vodka marked 60 proof indicates 30% v/v alcohol in it.

Practice Question

Q. What is the percentage composition, by mass, of oxygen in H2O?

Solution: overall molecular weight of water = 18, molecular weight of oxygen is 16 hence, oxygen is nearly 88.8% of water.
(16/18 x 100 = 88.88)

11 Arya A, Kumar A. (2018). High Value Note, Drawing Pin Publishing, 04: 1-
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1.5.2 Concentration Terms for Laboratory Applications : Temperature dependent
Concentration terms for the laboratory application must be indicating the number of particles (atoms or
molecules) in the solution, so that while carrying out chemical reactions, a relative estimate of constituents
can be made (recall that chemical reactions occur in terms of number of molecules and not mass). Some of
the concentration terms are affected by temperature i.e. their values changes if temperature is altered.

Molarity: The Molarity or Molar concentration, (M) is defined as the number of moles of solute present in
one liter of the solution. Now in order to calculate the molarity we may need to calculate two things one the
number of moles and volume of solution.

𝑾𝑾𝑩𝑩 𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏
Formula: 𝑴𝑴 = 𝑴𝑴𝑩𝑩
; WB - given weight of solute, MB - Molecular weight of solute, V – volume in ml.
When to use: Always (unless specified) prefer this unit, also recommended by IUPAC.

Practice Question

Q. What will happen to the molarity/Formality/Normality of a solution if the aqueous solution is heated or cooled?

Hint: On heating above 4oC the volume will increase or cooling below 4oC volume will increase but the amount of solute in a solution will
remain constant. So, a decrease in concentration will be observed. The density of water is maximum at 4oC and therefore volume of aqueous
solution will increase above and below this temperature, an increase in volume will cause decrease in concentration].

Normality: Normality (N) is a measure of concentration equal to the gram equivalent weight per liter of
solution. Gram equivalent weight is the measure of the reactive capacity of a molecule. The solute's role in
the reaction determines the solution's normality. Normality is also known as the equivalent concentration of
a solution.
𝑾𝑾𝑩𝑩 𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏
Formula: Formula: 𝑭𝑭 = 𝑴𝑴𝑩𝑩
; WB - given weight of solute, MB - Molecular weight of solute, V – volume in ml, Interco version of with
molarity – Molarity = formality x feq (feq -basicity of acid or acidity of base)
When to use: Especially when the strength of acid or base is needed in a reagent.

Practice Question

Q. Calculate the normality of 1 M – HCl, H2SO4, H3PO4, NAOH, Mg(OH)2 and Al(OH)3
Hint: Normality depends on number of gram equivalents (moles of effective constituents such as H+ or OH- here). In this case gram equivalents
of HCl, H2SO4, H3PO4, are 1, 2 and 3 respectively, as their one mole would produce 1, 2, 3 moles of H+ ions respectively. Hence they have
normality of 1N, 2N and 3N respectively. Similarly gram equivalents of NaOH, Mg(OH)2 and Al(OH)3 are 1, 2 and 3 respectively, as their
one mole would produce 1,2, 3 moles of OH- ions respectively. Hence they have normality of 1N, 2N and 3N respectively.

Formality: Formality is the number of formals (formula weights) present in one liter of the solution. The
difference between formal and molar concentrations is that the formal concentration indicates moles of the
original chemical formula in solution, without regard for the species that actually exist in solution. Molar
concentration, on the other hand, is the concentration of species in solution. To understand it better, if we
represent the concentration of acetic acid as 1 M, what would be mean from it? As there will be CH3COOH,
CHCO- and H+, so 1M is the concentration of which species. In such situations formality represents the
molar sum of all the species present in the solution, i.e. everything that is present in the solution. So formality
and molarity of a solution that does not ionize (like glucose) would be the same.

𝑵𝑵𝑵𝑵𝑵𝑵𝑵𝑵𝑵𝑵𝑵𝑵 𝒐𝒐𝒐𝒐 𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇 𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆𝒆

Formula: Formula: 𝑭𝑭 = 𝑽𝑽𝑽𝑽𝑽𝑽𝑽𝑽𝑽𝑽𝑽𝑽 𝒊𝒊𝒊𝒊 𝒍𝒍𝒍𝒍𝒍𝒍𝒍𝒍𝒍𝒍𝒍𝒍
; WB - given weight of solute, MB - Molecular weight of solute, V – volume in ml,
When to use: Especially when the compound in solution is partially ionized and concentration is represented in terms of total ions. (Generally
the use of Formality is discouraged by IUPAC).

Molanity: Molanity is comparatively new concentration term which means number of moles of solute present in one kilogram of the solution (and not solute
unlike molality).

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Practice Question

Q. What will happen to the molarity/Formality/Normality of a solution if the aqueous solution is heated or cooled?
Hint: On heating above 4oC the volume will increase or cooling below 4oC volume will increase but the amount of solute in a solution will
remain constant. So, a decrease in concentration will be observed. The density of water is maximum at 4oC and therefore volume of aqueous
solution will increase above and below this temperature, an increase in volume will cause decrease in concentration].

1.5.3 Effect of temperature on concentration terms

As a matter of fact, the density of water is maximum at 4oC, therefore any aqueous solution would occupy
minimum volume at the 4oC. So, if we increase the temperature of a solution above 4 or cool below 4, the
density will decrease and hence the volume would increase (if you take 1 litre mineral water bottle and
freeze it, the bottle will swell due to increased volume, same would happen if u keep it in the Sun). As we
could note from previous section that the aforesaid concentration terms were represented as amount of
solution in given volume of solution, hence any change in temperature the volume would change, but
amount of solute would remain same hence the concentration would also change. So those terms are not

suited for representation when a process is being performed over a range of temperature. A context from
life sciences, such as molarity of magnesium ions in PCR master mix would not be same at denaturation
step in the PCR, also molarity of a buffer prepared in lab would not be same when it at 121oC inside
autoclave. So, there are some concentration terms which are effective in such conditions and do not change

due to change in temperature.

Decreasing temperature 4oC Increasing temperature

Volume Mass
Mass Volume Mass Exceeds 1 Kg
1 L mark
1 Kg Exceeds 1 Kg
1 litre
1 litre

Solute remains Solute remains


the same the same

Effect on < 1 Molar - decreased 1 Molar solution < 1 Molar - decrreased

Molarity (No of moles per litre reduced) (1 mole NaCl in 1litre water) (No of moles per litre reduced)

Effect on 1.062 molal - unchanged 1.062 molal 1.062 molal - unchanged

Molality (1.06 moles NaCl in 1 kg) (1.06 moles NaCl in 1 kg) (1.06 moles NaCl in 1 kg)
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Fig 5. Effect on temperature on concentration terms


1.5.4 Concentration terms for laboratory applications : Temperature independent

Two commonly used terms are molality and mole fraction are not affected by change in temperature and
hence considered temperature independent concentration terms.

Molality: Molality (m) is defined as the number of moles of solute present in one kilogram of solvent. So,
this term is different from molarity and other terms in two aspects. First, it represents the number of solute
in terms of solvent (not in terms of solution) and second, mass of solute but not volume is required to
calculate molality. For this reason, if one doesn’t have volumetric flask or measuring cylinder, a solution of
desired molality can be prepared just by using weighing balance. Also, as it does not involve volume, the
term is unaffected by temperature.

𝑾𝑾𝑩𝑩 𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏
Formula: 𝒎𝒎 = 𝑴𝑴𝑩𝑩 ×𝑾𝑾𝑨𝑨
; WB - given weight of solute, MB - Molecular weight of solute, V – volume in ml, WA is the mass of solvent
in kg. Inter-conversion of with molarity – molarity = 1000 x molality x density / 1000 + molality x mol mass of solute
Usage: Especially when the reaction that is being carried out is over a long range of temperatures and also, if volume measuring
devices/equipments are not available.

Mole fraction: mole fraction is defined as fraction of number of moles of one of the component of a solution

13 Arya A, Kumar A. (2018). High Value Note, Drawing Pin Publishing, 04: 1-
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with respect to the total number of moles of the solution. The mole fraction is sometimes denoted by the
lowercase Greek letter χ (chi) instead of a Roman x. For mixtures of gases, IUPAC recommends the letter χ
eg. If there are 2 solute components in an aqueous solution - component A, component B, then mole fraction
of each of these component will be defined as:

Formula: 𝝌𝝌𝑨𝑨 = ; χA –Mole fraction of component A, nA – number of moles of A and nB, number of moles of B. (A and B may be solute
and solvent in some cases). Also sum of mole fractions all components in a mixture is 1. i.e. (χB = 1- χA).
Usage: Especially when relative concentration of more than one ingredients of the solution is required or the solution is made up of more than
one ingredients and concentration needs to be represents as per each one of them.

1.5.5 Concentration terms for environmental/toxicological applications

Concentration terms used to depict the toxicological doses of heavy metals, pesticides or any other
compounds are usually depicted by certain concentration terms which are simple to comprehend like
percentage, but also very smaller relative to molarity. Such concentration terms are parts per million (ppm),
parts per billion (ppb), parts per trillion (ppt) and parts per quadrillion (ppq), which represents the relative
parts of solute in 106, 109, 1012 and 1015 parts of solution. PPQ is roughly equivalent to a drop of ink in a

medium-sized lake.

1.6 Sample Dilutions

Sometimes, the solutions that are prepared have concentration higher than the desired value within the
analytical range and therefore, they need to be diluted. Most of the commonly used buffers in the life
sciences research as well as chemistry are prepared in much higher concentration and can be diluted at the
time of use. Similarly, a number of experiments in pharmacology (related to drug doses), clinical biochemistry
and microbiology require dilution of samples as the concentration of analyte is usually much higher than the
detection level. Experiments in molecular biology such as polymerase chain reaction requires preparation of

master mix containing a number of ingredients, preparation of DNA-SYBR green mix form concentrated
stocks all need basic rules of dilution. Based on the extent of dilution, the methods are broadly grouped into

two types, one called step dilution or simple dilution and second called as serial dilution. We will now discuss
both types with examples.

Stock volume of stock (V 1= 0.1 ml)
20 ml (Desired volume - V )2

(V 1 = 0.005 ml)
5 ml (V 2)

(0.005 ml buffer (0.1 ml buffer

remaining water) remaining water)

1 M NaCl 1 mM NaCl 5 mM NaCl

(Stock conc. - C )1 (Desired conc - C )2 (Desired conc - C )2
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Fig 6. An illustration representing step dilution of a solution

1.6.1 Step dilutions

Step dilution is simply one time dilution of more concentrated solution into relatively less concentrated
solution by dispensing the required amount into the solvent. The more concentrated solution is represented
as stock and the dilute sample is known as working. A very common formula in use for the calculation of
amount of volume needed to pick up from stock to obtain desired concentration is described below:

Arya A, Kumar A. (2018). High Value Note, Drawing Pin Publishing, 04: 1-20. 14
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𝐶𝐶1 𝑉𝑉1 = 𝐶𝐶2 𝑉𝑉2

Where C1 is the concentration of stock solution, V1 is the volume of stock solution (usually required to be
calculated), C2 is the concentration of desired solution (working solution) and V2 is the volume of desired
solution. It must be noted that there is not restriction on the type of units of concentration terms and volumes
to be used in this formula, but all the units should be same (a requirement to avoid errors).
This is the reason that sometimes this formula is also represented as 𝑀𝑀1 𝑉𝑉1 = 𝑀𝑀2 𝑉𝑉2 or 𝑁𝑁1 𝑉𝑉1 = 𝑁𝑁2 𝑉𝑉2 where M
and N terms representing molarity and normality respectively.

Practice Question

Q. If a buffer is labelled 10X used for running a SDS-PAGE gel (running buffer), need to be prepared? In case you require 2 litres
of running buffer. How much stock buffer should be mixed with water to prepare a working solution?
Solution: 10X represents the strength of stock compared to working, which means that the solution needs to be diluted 10 times before it
becomes equivalent to working concentration. Hence we can use simple relation C1V1= C2V2, and calculate V1, using C1 as 10 and C2 as 1,
and V2 as 2 litres. The amount of buffer thus required is 0.2 litre or 200 ml, remaining 1800 ml will be water.

1.6.2 Serial Dilution
Serial dilution reefers to dilution of a sample through a series of small dilution steps. The basic requirement

of serial dilution is for the purpose of dilution to several fold, which lies beyond the dispensable limits of
existing liquid handling devices (micropipettes). To emphasize more we can consider an example, that if 1M
solution of sugar has to dilution to 1 picomolar, in 1 ml volume. Using formula stated for step dilution, one
has to dispense a picolitre of stock solution into 1 ml solvent. Dispensing 1 picolitre is error prone and not
possible using conventional pipettes. Hence, in such case a series of dilution steps may be performed to
achieve a high degree of dilution. Serial dilution is utilized at many places in life sciences. In pharmacology

dilution of concentration phytoextracts, organic drugs, nanomaterials etc., to a scale of micro molar or
nanomolar, serial dilution is required. In molecular methods like ELISA, serial dilution may be used to obtain
desired antibody dilution.

Practice Question

Q. Researchers have recently found that specific type of metal oxide nanoparticles (0.1 M) can confer protection against
chemotherapy, In order to test this a cancer cell culture grown in 6-well culture plate (having around 2 ml culture media) were
used. If the researcher want to test the effect of 2 micro molar and 2 nanomolar concentrations of nanoparticles on cell culture,
how should (s)he proceed with experiment?

Hint: In order to dilute a nanoparticle formulation from given concentration of stock to the working level of 2 micromolar and 2 nanomolar,
one has to prepare a lower dilution from stock i.e. in mill molar range, which will be assigned as secondary stock. And then using relation
C1V1= C2V2, calculation of desired volume (V1) can be made considering that final volume would be the volume of each well of a 6-well plate,
i.e. 2 ml.

In microbiology, if one wish to culture and count the microbes in a sample of sewage water one may have
to dilute the water to several million or billion fold to achieve distinct colonies, following which counting of
microbes may be done. In serial dilution, the overall dilution of the sample is represented by the product of
dilution at each step. So if we dilution a sample by 1/10 over 5 steps the overall dilution will be 10-5 of the
stock. Fig 7 illustrates the meaning and application of serial dilution.

Note: A ten-fold dilution for each step is called a logarithmic dilution or log-dilution, a 3.16-fold in each step dilution is called a half-logarithmic dilution or
half-log dilution, and a 1.78-fold in each step dilution is called a quarter-logarithmic dilution or quarter-log dilution.

15 Arya A, Kumar A. (2018). High Value Note, Drawing Pin Publishing, 04: 1-
High Value Note: Drawing Pin Publishing
10 µl 10 µl 10 µl 10 µl 10 µl
sample sample sample sample sample

Sewage water 990 µl 990 µl l 990 µl 990 µl 990 µl

sample autoclaved autoclaved autoclaved autoclaved autoclaved
water water water water water
Dilution factor 1/100 1/104 1/106 1/10 8 1/10 10

Bacterial Lawn
Decreasing number of bacterial colonies
© 2018. All Rights Reserved. Draw ing Pin Publishin

Fig 7. An illustration representing serial dilution of a soil sample for the purpose of microbial culture

Practice Question

Q. A sample of sewage water was obtained from a nearby drain in University of Delhi, and diluted to 1 million fold by serial
dilution. The number of colonies obtained when the diluted water was spread on an LB –Agar plate were 45? What was the
number of colourable bacteria per ml of the sewage water?

Solution: As the water was diluted to 1 million times (i.e. 10-6 of the original), hence the number of colonies of bacteria from this diluted water
represent 1 million fold lower than the actual concentration. So in actual water the concentration of bacteria would be – number of bacterial
cells on plate x dilution factor i.e. 45 x 106 or 4.5 x 107.
Finally, it can be concluded that mole concept is not a term of topic to be scared of, rather it should be brought
into the real life just as meter, seconds, litre etc. and should be realized and estimated in with similar capabilities.
So next time when you for lunch, don’t see 1 litre water bottle as 1 litre rather see it as 3.34 x 1025 molecules of



Q1. The genome of bacterium is composed of single molecule of DNA , so if there were 6.022 x 1023 per cell , it
DNA molecule which is 109 bp long, how many moles of would have been 1 mole But there are just 46. So the number of
genomic DNA is present in the bacterium. (Consider moles = 46/ 6.022x 1023 = 7.63 x 10-23 Moles
Avogrado no. 6.022 x 1023) [CSIR NET 2015]

Q3. The mass of Glycine required to make 250 ml of a

1. 1/6 x 10-23 0.015 solution is [IIT JAM 2014]
2. 1/6 x 10-14 [Hint: Masses in amu H – 1, C – 12, N -14, O – 16]
3. 1/6 x 1023 1. 1.13 g
4. 1/6 x 1010 2. 0.4 g
3. 0.56 g
Solution: (Correct answer – 1) The number of DNA molecules 4. 0.28 g
in bacterial cell =1 (ignore the size of the genome). As number
of moles may be derived using the number of molecules. If there Solution: (Correct answer – 4) Sample text here - ref Q 89
would have been 6.022 x 1023 molecules, it would have been one chopater 2 MCQ Molecular weight of glycine may be calculated
mole. But there is just one molecule , hence no. of moles = by recalling its structure (NH2- CH2- COOH), and it turns
1/6.02 x 10-23 . out to be 75 daltons. So in order to make 1 L of 0.015 molar
solution one needs 0.015x 75 = 1.125 g. However, for 250 ml
Q2. What is the number of moles of DNA present in a only one fourth amount will be needed. i.e. 1.125/4 = 0.281.
Human diploid cell? [CSIR NET 2014]
Q4. . The molecular weight of ethanol is 46 and its
1. 7.63 x 10-23 Moles density is 0.8 g/cm3 . What would be the molar
2. 1 mole concentration of ethanol in a 23% v/v wine. [TIFR 2012]
3. 6.022 x 1023 moles
4. 0.22 moles 1. 4 molar
2. 0.5 molar
Solution: (Correct answer – 1) Similar to previous question,, 3. molar
human cells have 46 chromosomes and each chromosome is a 4. None of the above

Arya A, Kumar A. (2018). High Value Note, Drawing Pin Publishing, 04: 1-20. 16
High Value Note: Drawing Pin Publishing
Solution: (Correct answer – 1). If the density is 0.8 g/cm3 and would be closest to : (use avogadro’s number – 6.022 x
the amount of ethanol in wine is 23% v/v the weight of ethanol 1023)
in wine = (0.8 x 23)/100 = 0.184 g per ml (same as cm3) or 184 1. 24
g per liter. In terms of moles 184 g is equal to 184 (given wt) /46 2. 3
(mol wt) = 4 moles/ liter. 3. 2400
4. 300
Q5. You are given 10 ml of 1 M salt solution asked to
make 10 ml of 1x 10-3 M salt solution The best way to do Solution: (Correct answer – 2). Given the pH of endosome as 5,
this is [TIFR 2014] we can estimate the concentration of protons. As pH represents
negative log of H+ ion concentration. Hence, H+ = antilog (-5) =
1. Do three serial dilutions of 1 ml in 9 ml water,
10-5 molL-1. As the concentration in molarity represents
starting with 1M solution
number of moles per litre, in order to dermine exact number in
2. Take 1 ml of 1 M solution and add 999 ml of
the endosome we must calculate the volume of vesicle. (given the
diameter = 100 nm, radius = 50 nm or 5 x10-8 m)
3. Take 10 μl of the 1 M solution and add to 9990
Using the formula for volume of sphere V = 4/3 πr3 = 1.33 x
μl of water
3.14 x (5 x 10-8)3= 5.2381 x 10-22 m3
4. Take 0.1 ml of 1 M solution and add to 9.9 ml
Considering the aquesous ennvironment, the desnity would be 1
of water
m3 = 1000 L, therefore volume in litres = 5.2381 x 10-19 Litres.
Solution: (Correct answer – 3). Although, all methods to dilute Now we can calculate the exact no of protons by using conc. and
the solution are correct, but it is error prone to dispense lower volume
No of moles of protons = Conc (molL-1)/ Vol (L) =10-5/5.2381 x

volumes hence, serial dilutions are recommended for very large
dilutions (such as a million fold or more). Therefore, option 1 is 10-19 = 5.2381 x 10-24
correct. However, if one uses precisely calibrated pipettes option No of protons = moles x avogadro’s number = 6.022 x 1023 x
3 may also be preferred. 5.2381 x 10-24 = 3.19 x 100 = 3 protons.

Q6. The molarity of water in a water: alcohol mixture

(15:85 v/v) is approximately [GATE 2011]

1. 0.85 MolL-1
Q 9. How many molecules of DNA does the Drosophila
cell nucleus contains [TIFR 2014]
1. 2 x 109
2. 5.55 MolL-1 2. 46
3. 8.5 MolL-1 3. 8
4. 55.5 MolL-1 4. 0

Solution: (Correct answer – 3). The molarity of 100% water is

Solution: (Correct answer – 3). Note that the number of
55.5 (calculated by dividing 100g from 18), In this case water is
molecules of DNA in a cell is equal to the number of
just 15% of the total solution, and hence 15% of 55.5 is 8.5
chromosomes. (Each chromosome is a molecule).

Q7. The number of water molecules present in 300

residues soluble protein of spherical shaper (diameter : Q 10. The number of electrons in 18 ml of H2O is [TIFR

2nm) having 20% v/v water closest to : Consider desnity 2016]

of water 1000 kg/m3, Avogadro’s number = 6 x 1023 1. 60.23 x 1023
1. 224 2. 6.023 x 1023
2. 9 3. 3.011 x 1023
3. 140 4. 0.6023 x 1023

4. 28
Solution: (Correct answer – 1). The number of electrons in each
Solution: (Correct answer – 4). As the peptide adopts spherical water molecule are 2+8 = 10 (one in each hydrogen and 8 in
configuration, the overall volume of the peptide can be oxygen)
calculated using the mathematical relation, V = 4/3 πr3 , And 18 ml of water is equivalent to 18 gram which means 1
provided the diameter is 2 nm, hence readius = 1 nm or 10-9 m mole of water . So one mole of water has 6.023 x 1023 molecules
V= 1.33 x 3.14 x (10-9)3= 4.19 x 10-27 m3. and each molecule has 10 electrons. So total no. of electrons =
Additionally, it provided in the qeustion that volume of water 60.23 x1023 .
associated with the peptide is 20% of the total protein volume,
i.e. Volume of associated water = 20/100 x 4.19 x 10-27 = 8.38 x Q 10.Calculate the number amount of sodium cholide
10-26 m3. formed if 5 g of sodium was added to 5 of chlorine and
Considering the density of water as 1000 kg/m3 , the overall reasction was conducted in suitable environemnet untill
mass of associated water = 8.38 x 10-23 kg or 8.38 x 10-20 g. completion?
Number of molecules of water = (given wt /mol wt) x 1. 5g
Avogadro’s number = (8.38 x 10-20/18) x 6.022 x 1023=28.03 2. 10g
molecules. 3. 8.24 g
4. 4g

Q8. Aquesous environment in a spherical endosome –a

Solution: (Correct answer – 3). If 5 g of sodium shall be added to
closed vesicle of 100 nm in diameter has a pH 5.0. This
5 g chlorine to form sodium chloride, chlorine will be limiting
is required to denature and hydrolyse the material by a
reagent and thus moles of NaCl formed will be 5/35.5
cell. The number of free protons in the endosonme
Mass of 35.5 moles of NaCl = (5/35.5) x 58.5 = 8.239 g.

17 Arya A, Kumar A. (2018). High Value Note, Drawing Pin Publishing, 04: 1-
High Value Note: Drawing Pin Publishing
1. Avogadro's Constant, Nancy Eisenmenger- http://www.scienceline.ucsb.edu/images/AvogadroConstant.pdf
2. Experimental determination of Avogadro’s number, S. Ramashesha – Resonance. - http://www.scienceline.ucsb.edu/images/AvogadroConstant.pdf
3. How was Avogadro's number determined? – Scientific American - https://www.scientificamerican.com/article/how-was-avogadros-number/
4. Physical Review Letters, 1974, 33, 464 – X-ray diffraction based method for determination of Avogadro ’s number. – Various methods of avogadro’s number
5. https://iupac.org/new-definition-mole-arrived/ - Update on the definition of mole
6. Nivaldo J. Tro, Chemistry in Focus: A Molecular View of Our World, 2018. 5e. Cengage YouBook Series.
7. Carmen J. Giunta, The Mole and Amount of Substance in Chemistry and Education: Beyond Official Definitions:. J. Chem. Educ. 2015, 92, 1593−1597.
8. https://www.moleday.org/ - Website describing more on mole day.
9. https://chem.libretexts.org/Textbook_Maps/Analytical_Chemistry/Book%3A_Analytical_Chemistry_2.0_(Harvey)/02_Basic_Tools_of_Analytical_Chemistry/2.
2%3A_Concentration –
10. Atkins, Peter, and Jones, Loretta (2002). Chemical Principles , 2nd edition. New York: W. H. Freeman and Company.
11. Lide, David R., ed. (2000). The CRC Handbook of Chemistry & Physics , 81st edition. New York: CRC Press.
12. Masterton, William L., and Slowinski, Emil J. (1977). Chemical Principles , 4th edition. Philadelphia: W. B. Saunders Company.



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