Preparation of Solutions and Buffers

Once you enter into a laboratory, the first thing what you are going to do is, you are going to prepare different types of solutions or
reagents. Whether it is a reagent for performing the SDS-PAGE or the agarose gel
electrophoresis or whether it is a reagent for doing some of the cell biology, immunology or the
molecular biology experiments.

All these reagent preparation requires a definite training as well as the precautions what you have
to take. So, in today's lecture, we are going to discuss about all these precautions, how to prepare
the solutions and in addition, we have also going to discuss how to prepare the buffers. Because
most of these solutions are made up of a buffers, so that it does not change the pH of that
particular solution while you are doing the reactions.

So, we will start the lectures with the understanding how to prepare the different types of
solutions, how you can what are the precautions you should take while you are preparing the
solutions and what are the different ways in which you can be able to prepare the solutions.
(Refer Slide Time: 02:10)

So, as the name suggests, the solution means a liquid mixture in which the minor component that
is the solute or the powder is uniformly distributed within the major component that is the
solvent or the liquid. Which means a solution is the summation of the solute which is actually the
powder + the solvent which is actually the liquid part. So, solvent is going to be the major
component, the solute is going to be the minor component.

But when you prepare the solutions in a life sciences lab or in a chemistry lab, you can actually
prepare the solution by 2 ways. Either you take a solute which is actually going to be in the form
of powder and then you dissolve that into a solvent and that actually is going to give you a
solution. In the other way you can also have the liquid reagents like glycerol like for example.

And then you can add that to a solvent the, so there are 2 ways in which you can be able to
prepare the solutions. Either you take the powder and mix it with the solvent and that actually is
going to give you a solution or you can be able to just mix the 2 different liquids and that also is
going to give you the solutions. So, either of this way the solution can be prepared in different
ways.
(Refer Slide Time: 03:34)

So, you can prepare the solution in the molar terms. So, when you prepare a solution, you can
use the different types of unit to prepare the solution. For example, you can prepare the molar
solutions like, so the molarity of a solution depends on the number of moles of the solute per litre
of the solutions it can be millimolar. That is the millimolar means 10 to power -3 molar, it can be
a micro molar, which means 10 to the power - 6 molar or a nanomolar solution which is actually
going to be 10 to power - 9 solutions.

So, let us see how to prepare a molar solution. So, for example if I ask you to prepare the 1 molar
glucose 100 ml solutions. So, the information what you require, if you want to prepare a molar
solution is that you require a molecular weight of the molecule. So, in this case, the molecular
weight of the glucose is 180 and the volume what you require, so volume is 100 ml. So, to
prepare the hundred ml 1 molar glucose, what you need to do is, you need to weigh the 18 grams
of glucose and transfer it to a 1 litre volumetric flask.

Then you add the 700 to 800 ml of purified water and you allow that to swirl to dissolve and then
you can add water. So, that the bottom of the meniscus is at the line of the flask then you can use
the stopper and mix well. The flask must be labeled with the solution consideration that is the 0.1
molar glucose which is 100 millimolar glucose. And date prepared and the name of the preparer.

This means the molarity what you can prepare is simply with this formula that is the w into 1000
divided by the molecular weight into the volume of that particular solution. So, if you put like 1
molar, for example, so in this case you put 1 here and weight you have to calculate and the
molecular weight is 180 whereas the volume is 100. So, if I do that and if you do a math the w is
going to be 18 grams in this case.

So, this is very easy to do because the molecular weight of a compound if it is dissolved in 1
litre, it is actually going to give you a 1 molar solutions. So, that you only have to remember, if
you require to prepare the 0.1 molar solution then you just divide the molecular weight by one
tenth. If you want to prepare the 1 molar solution but the volume is 100 ml then you just divide.

So, because when you prepare the solution in the lab you cannot do this kind of extensive
calculations and you know. So, that is why it is very easy to understand that the molecular
weight of a compound dissolved in 1 litre of solution or dissolved in solvent is actually going to
give you 1 molar solution. If you dissolve the milligrams of solution and dissolve it into the 1 ml
of solution or 1 ml of solvent that actually is going to give you the millimolar concentrations.

So, that is actually the way you have to calculate, so that when you are actually preparing these
solutions, you should do very quickly. Because if I ask you 50 millimolar Tris pH 8.0 then you
should not take time because you know the calculation for 18 millimolar or suppose 100
millimolar NaCl So, you know the NaCl molecular weight is 58.5, so you just divide that number
by 10, so 5.8 grams is what required for 100 millimolar NaCl solution for 1 litre.

So, that is the way you have to do it in your lab, so initially when you are a new student in the
lab, you might have to do calculation every time. But when you are slightly experience and then
you know that what is the trick. The trick is you should remember if I have to prepare a 1 molar
solution, I have to just take the molecular weight, I have to just go with the molecular weight
which is actually being given on to the level of that particular bottle.

And then I have to just divided according to the volume. Similarly you can prepare the normality
solution. So, normality is the number of equivalents of the solute per litre of the solutions. The

way that you prepare the molar solution the same way you have to prepare the equivalent
solutions, a normality solution. The only difference is that you have to take the equivalent
molecular weights of the equivalents.

Then you can also prepare the percentage by the weight which means the consultation based on
the number of grams of the solute per 100 grams of solutions or per 100 grams of solvent. So,
weight percentage is very, very difficult to do because you know in the case of solvent, how you
are going to calculate the weight actually. So, if you want to calculate the weight, actually you
have to take the formula of the water.

For example if I am using the water as for the weight, then the water molecular weight is actually
the 18 grams. So, if 18 grams of water is actually going to give you so that is a way you have to
calculate the molecular weight of your solvent. And the molecular weight the weight of your
compound is anyway going to be in powder anyway. Then percentage by the volume which
means the weight percentage by volume, so concentration based on the number of grams of
solute per 100 ml of solutions.

Then you have the weight per volume which means the percentage based on the number of
grams of or milligrams or the micrograms of solute per unit volume. For example the milligram
per ml, gram per litre or milligram per 100 ml.
(Refer Slide Time: 09:14)

When you are preparing a solution you might have to do 2 processes one you can have to do a
dilution of the strong acid or the base. So, when you are preparing a when you diluting a strong
acid or a strong base. For example, if I am diluting like sulfuric acid which is actually a strong
acid or if I am diluting the NaOH which is actually a strong base then I have to take a lot of
precautions.
Because when you are diluting a strong acid you have to dilute the acid in such a way that you
have to take the acid. And then you take that acid and drop wise you have to add that acid into
the solvent system. Because you want to avoid the exothermic reaction, when you are actually
diluting a strong acid it actually is a exothermic reactions. So, because of that the solution is
going to be very, very hot, so if I take acid and if I start adding the water the exothermic reaction
is going to be even bigger.

And that actually will sometime can actually damage the vessel where you have kept the acid.
Number 2, it can sometime actually cause the injury because if the acid is very if the exothermic
reaction occurs and that actually going to break the glass vessel or flask it actually can cause the,
you know acid bond or the injury. The same is true for the base also. So, base when you dilute
you have to be very, very careful because the base is also going to give you the exothermic
reactions.

Similarly you might have to dilute like viscous thick solvent like for example the glycerol. So, if
I have to take a glycerol and I want to dilute for example when you are going to prepare the
sample buffer for the electrophoresis. The sample buffer actually contains the 40% glycerol
which means you have to take the glycerol from the 100 ml from the 100% glycerol from the
bottle and then you dilute it to 40%.

In that case taking out the viscous material like thick glycerol is actually quite a lot of
precautions. Because of the only reason that these are thick solutions, so actually they are
sucking through the pipette is going to be very, very slow. And they are also going to be deposit
onto the outer surface of the tip. For example, if I am drawing the glycerol what happened is the
glycerol role is going to attach to the outer surface of this tip.

As well as the movement of glycerol is going to be very, very small because the suction pressure
is same whether t is the water or it is a glycerol. So, in that case what we normally do or what is
recommended is that you cut the tip that top surface of the tip. And because of that the lower end
of the tip is going to have the bigger diameter and then the sucking is going to be faster, you do
not have to worry that you know I have removed the some part of the tip the accuracy of the
volume what I am withdrawing is also going to be different.

That is not going to different because what the volume what you are going to withdraw is
actually be proportional to the amount of vacuum what you have created in the pipettes, not to
the tip shape or the size of the tip actually. So if I cut it actually I am going to suck the glycerol
into much quicker. And on top of that because you are going to have large amount of glycerol on
top of this tip, you also have to wipe this tip or across the, you know the bottle.

Because if that we would do, that you are going to remove all the excess glycerol what is present
in. And then when you are dispensing this liquid into the next solvent, then also you have to be
very careful. Because you have to keep pressing the pipette and you have to remain in that
situation for very, very long time, so that the last drop of the glycerol is also going to be removed
from your pipette tip.

Because as I said you know, the sucking of the thick liquid is also going to be a problem that the
dispensing of that particular liquid is also going to be having the same trouble because it will
take more amount of time for this liquid to come out from the pipette tip. And so that is why it is
recommended yet you have to be very, very careful when you are handling the viscous thick
liquids.

So, now once you have prepared the solutions, the solutions are actually been made in such a
way that the solutions are actually going to have one component which actually going to resist
for the change in pH. Because in most of the buffer most of the solutions you are actually going
to add the buffer components. So, that when you are doing the reactions it should not change the
pH of that particular solution because you want to keep the pH of that particular solution to be
remained intact as the same.

For example if I am running the SDS-PAGE and if I am using the 1.5 molar Tris which 8.8, I
want to ensure that the pH of this particular solution remain 8.8, it should not go like 6.8 or 9.5.
Because if that happens then the resolution of that particular gel is going to be affected, the way
the solutions are the way the molecules are going to be resolved onto the SDS-PAGE also may
get affected. If there will be a change in pH.

So, that is why the change in pH is very, very crucial for performing the reactions as far as the
you are talking about the biological reactions or the in the case of biochemistry.
(Refer Slide Time: 15:03)

So, what is mean by the pH is that pH is actually a scale which actually measures the
concentration of the hydrogen ion concentration within the solutions. So, you can understand that
when a water is present it actually get ionized in the form of H + and OH -. So, if you take the 2
water molecules, it actually going to give you a hydronium ion as well as the OH -. Say if I ask
write the equilibrium constant of this particular reactions, what I will do is, I will write the
equilibrium constant of H +, OH - and the H2O.

So, the concentration of the pure water, so if I put the values of all these things. So, the
concentration of the pure water is 55.5 molar and the K equilibrium for this reaction is actually
1.8 into 10 to the power - 16. So, if I put these values what will happen is that if you calculate the
H + into the OH- is going to be 1.8 into 10 to the power - 16 divided by 55.5. So, if you solve all
these and put the value of this that you are going to get the K w.

So, see the multiplication of H + and OH - is actually going to give you a value which is called
as K w. And the K w is actually called is the value of the K w is 10 to power - 14 M squared. So
K w is the ion product of the water at 25 degrees Celsius which means the product of the H + and
OH -. This means in the pure water, the H + is going to be the underscore of K w and that is
going to be underscore of 10 to power - 14.

So, if you solve that the H + is going to be equivalent to the OH - and that actually is going to
give you a value of 10 to power - 7 M. So, ion product of a water is a constant and that allow us
to calculate the H + in case the OH - is known or vice versa. So, this means the H +, the product
of H + and OH - is actually going to be 10 to power - 14. So, if I have H + I can calculate the OH
-, if I have the OH - I can calculate the H +.

For example if I have a solution of 10 to power - 3 and it is HCL actually, so then what will
happen let us see, right. So if I have to calculate the OH -, OH - is going to be 10 to power - 11
because you can just divide this and that actually going to give you the OH -. So, hence it is use
to develop a pH scale to define the concentration of H + OH - in any aqueous solution. This
means if I know the H + I can calculate the OH -.

And that is why this ion product is giving you a scale where on one side you are going to have
the acidic range. On other side you are going to have the basic range and in the center you are
going to have the neutral center. So, that is why see so it is developed a pH scale where actually
you are going to have the - 7 on one side and + 7 on the other side. And the pH is defined as the
negative log of the hydrogen ion concentration, so it is ranges from 1 to 14.

So, this is what you have to see that the pH scale, where you have a 0 scale which is actually the
pH 07. And on this side you are going to have the acidic range, on this side you are going to have
the basic range which means on this side you are going to have more and more acid and this side
you are going to have more and more bases. For example, I have given you an example of
different types of products what we use in a daily life.

For example you have the HCL solution which is actually going to have a pH of 1 then you have
the lime which is actually or lemon which is actually going to have a pH 2. Then you have the
apples which are actually going to have the pH 3, then you have tomato which is pH of 4. Then
banana, then potato and then you have the water which is actually of the pH 7 which is actually
the neutral pH.

Similarly on the basic side also you have lot of solutions like you have the detergent powders
and you have the acids and you have the NaOH solution which is actually going to give you a pH
of 14. So, these are, so pH is a very, very important scale to measure the acidity or the alkalinity
of a particular solution. So, if the pH is less than 7 it is actually going to be acidic, if pH is more
than 7 then pH going to be alkaline solutions.
(Refer Slide Time: 20:01)

Now the question comes why you actually have required a buffer and what is mean by the buffer.
So, buffer solutions are used as a mean of keeping pH at a nearly constant value in a wide range
of chemical applications. In nature there are many systems that use the buffering for pH
regulations. One of the classical example is the bio carbonate buffering system, so bicarbonate
buffering system actually utilizes the 3 component, the carbonic acid H 2 CO 3 bicarbonate ions
H 2 CO3 -.

And then the carbon dioxide which is actually been present into the air. So, by using these 3
molecules under the equilibrium the bicarbonate buffer is actually maintaining the pH of the
blood to support the proper metabolic reactions. And you have many conditions or many
situations where the slight change in the pH of the blood. For example pH of the blood is ranging
from 7.35 to 7.4.

So, if you have slight change in the pH, for example if you have a pH of 7.2 you are going to
have the acidosis problems like where the person is going to have the lot of problem in the
breathing difficulties and all those kind of thing. Because what you remember is if it goes into
the acidic range indirectly you are actually going to affect the carbon assimilations or carbon
transport within the system, which means you are indirectly going to affect the oxygen transport
as well.

So, if it is actually more and more as carbon dioxide is going to be associated with the body it is
actually going to reduce the amount of oxygen within the body as well. So, that is actually is
because the carbon dioxide is under the equilibrium within this particular buffering system. And
that is how the carbon dioxide is been transported from one part of the body to another part of
the body.

And eventually it reaches to the lung where the carbon dioxide is been removed from the body
and the oxygen is been transported back. But if you actually going to change the pH of the blood
and that actually if allows the accumulation of carbon dioxide within the blood, then it is actually
going to change the overall respiratory activities and other kind of activities. Because the whole
body depends on the respiration to perform the all the functions.

For example, even if it a liver which does not mean directly attached with the lungs and other
places but it requires the oxygen to perform the functions. So, if there will be any change in
blood of the pH that eventually going to accumulate the carbon dioxide into the liver or it
actually not going to provide the enough oxygen for the liver to respire to produce the energy
and to perform all the metabolic reactions. So, that is how the maintaining a crucial pH is very
important for the normal physiology of a human being as well as for the other animals.
(Refer Slide Time: 23:09)

Apart from the physiology, let us see how the pH is also changing the other biological processes.
For example, the enzymes or proteinous in nature and they are made up of individual amino acid
with the ionizable side chain such as the histidine. In addition the active side of enzyme also has
the amino acids. A particular enzyme is important for substrate binding formation of catalytic
intermediates and the release of product.

For example the pepsin is a serine protease present in a stomach and has the optimum pH of 1.5.
Whereas the trypsin has a pH optima of 7.4. So, that is why most of the enzymes are actually
having the ionizable groups, they have the active side residues and all these active side residues
are have to be present in a particular you know valency state and as well as the ionization states.
So, because of that the particular pH of that particular value or the place where these enzymes
are present it has to be in a perfect order.

So, that it should these enzymes are actually going to work very efficiently. One of the classical
example is the pepsin which is actually present in the stomach and it requires a pH of 1.5 to
digest the food. Whereas the trypsin which is another protease, require a pH of 7.4 to function.
Similarly in many pathological conditions such as diabetes body utilizing stored food as an
alternative energy process.

The similar condition exists in the case of starvation or fasting and under these conditions a large
amount of acid like the beta hydroxy butyric acid from fat is generated leading to the lowering of
blood pH to cause the acidosis. It disturbs the activity of several enzyme present in the blood and
ultimately leads to the headache nausea and the convulsions. So, as I think we already discuss
about the role of the blood pH. So, here are the few example like if a person is suffering from the
diabetes and instead of using the glucose, if it starts using the fat and other kind of stored food
material.
Then eventually it is actually going to produce lot of acidic bio byproducts like metabolic
byproducts. And that actually is going to lower down the pH of the blood and that condition is
called as the acidosis. And acidosis is directly going to affect the first organ that is the brain
actually. So, if the acidosis is there, it actually going to affect the brain because the brain is going
to deprived of oxygen.
And ultimately it is going to cause initially with the minor mild symptoms, it is going to cause
the development of headache. But if the conditions continued and there will be no change in the
pH of that particular blood. So, that there will be no supply of oxygen, then the headache is
going to be turned into the nausea and convulsions. Similarly the pH blood pH is maintained by
the bicarbonate buffer and it plays a vital role in respirations.
So, that means the buffer is very important for the enzymatic activity as well as the normal
physiology for the body. Here are few examples where I have given you a table of showing that
what is the pH optima of different enzyme. For example, even if you have a same enzyme you
see the same enzyme is present in 3 different locations like the lipase which is present in
pancreas having a pH optima of 8.
Whereas if the lipase is present in stomach has a pH optimal of 4 to 5, and if the lipase is present
in the castor oil that if the plant the pH optima is 4.7. Then the pepsin which is the pH optima of
1.5, the trypsin pH optima of 7.8, urease 7, invertase, maltase, amylase and catalase. And in general what you see is if you plot the pH based activity of an enzyme what you will see is. It is
actually having a biphasic behavior which means at this side you are actually having the
optimum pH.
This is the place where the enzyme is going to work optimally on the both the side whether you
were go on to the acidic side or whether you go on to the basic side you are actually going to
affect the activity of these enzymes, why you are going to change the activity of enzyme.
Because you are actually changing the ionization status of those amino acids which are either
present into the crucial points where either they are crucial for stabilizing the structures or they
are important for catalyzing the reactions.
So, either of these situations the change in pH is actually going to affect the ionization stage of
the side chains. And eventually it is going to disrupt some of the, you know the electrostatic
interactions or Vander wall interactions or the salt bridge interactions with the neighboring
residues. For example, if you have a lysine and it is making a interaction with the glutamate and
if you change the pH either of these pair are actually not going to be in a proper ionization state.
And that is how that particular interaction is going to be broken down. And once these
interactions are going to be broken down, it eventually leads to either that particular portion of
the enzyme is going to be moved or there will be a conformational changes in the enzyme. And
that eventually is going to make the enzyme less efficient compared to that when it was present
in the optimal pH conditions.
So, that is why the pH is very important and that is why the buffer is also very important that
why to maintain a pH. Now the question comes how the buffer is actually maintaining the pH.
So, the buffer is actually a mixture of weak acid and a conjugate base or the vice versa. So, you
can imagine that you have a condition like HA + B and that actually is ionizing to give you A-
and HB. So, in this ionization reactions, the HA and A - are actually being part of the one
conjugate acid base pair. Whereas the B - and the HB are actually making another conjugate acid
base pair which means, the HA and B is making one pair and the B - and HB is making another
pair.

This means the HA can that is why the HA is weak acid which is been associated with a strong
base and that actually is going to give you the buffer. So, HA is going to be ionized like HA H+
and A -, so that is the ionization of the HA which is a weak acid and with a strong base. So, if
you add the strong acid like if you add the H + so what will happen is the HA + H +, so there
should be increase in H +.

Because you are increasing the H + but what happen is the H + whatever you are adding is
actually combining with A -. And that is how you are actually getting the more amount of HA
instead of getting the more amount of + H +, which means the H + remained constant and that is
the resultant of the change in pH. Because when you calculate what is the pH of the solution you
are actually going to consider only the H + or ionizable H+ present in this particular solutions.
So, even if you have added the acid which is a strong acid and that actually is going to combine with the strong base and that is how it is actually going to be get neutralized. Now imagine that if
I have added these strong base OH -, then what will happen the OH- is going to be added to the
HA which is actually a buffer. Then what will happen OH - should have increased the pH.

But what happen is the OH- is going to combine with H+ and it is actually going to form the
water and the A- will remain the same which means the base component will remain the A -
which is actually been responsible for that particular pH of that particular solution. Since you are
adding the base you expect that the A - should go up because that is the base component of that
particular solution.

But instead of that it actually been neutralized by the acid component and that is how it is
actually going to be you know remain the same pH. That is how in a buffer you have a
combination of the acid as well as the base. So, if you add the acid the base is going to react and
neutralize the acid. If you add the base the acid is going to react and neutralize the base.

And that is how it is actually going to maintain the pH of that particular solution but how long
that buffer is going to maintain the pH. So, that is in always been measured with a definition
called as the buffering capacity or the buffer capacity. So, buffer capacity is a quantitative
measure of resistance to the change of pH of a solution containing a buffering agent with respect
to a change of acid or the alkaline consideration.

Because you can imagine the even we are adding the H + and that H + is acting neutralized by A
- that is actually going to be equal or proportional to the amount of A + or A - what you have in
the solution. So, once the A - are going to be exhausted which means keep adding the acid
eventually what will happen is that the A - is going to be exhausted, which means there is no
longer the A - is going to be available to take care of the H + what you are adding from the acid.

And in that situation, if you add another drop of H + that H + is not going to be neutralized and
at that actually is going to lower down the pH of that particular solution. So, that is all the buffer
is going to maintain the pH until you have some form of ionizable bases or some form of
ionizable acid present in that particular solution. And that is actually decides the buffering
capacity of that particular buffer solutions.

And that can be measured simply by quantitatively if you titrate a buffer solutions with the acid
and the base. And that actually is going to give you that value, what is the buffering capacity of
that particular solution. And is it advisable that you should work with the buffer within it is
buffering capacity, which means you cannot work beyond that buffering capacity. Because if you
work beyond the buffering capacity then as soon as you are actually going to have any change in
the H + or OH- concentration.

Or if there will be any generation of H + or OH - within the solution it is actually going to
change the pH of the solution because you are working beyond the buffering capacity of that
particular buffer.
Now, let us see how you can be able to do a titration. So, if you titrate a weak acid, for example
in this case, I have taken an example of acetic acid and with a strong base l