Buffers and

Buffer
Preparation
 What is a Buffer?

• A buffer is a solution that can resist a

marked change in pH on the addition of
small quantities of acid or bases
• They are important in maintaining the
pH of living systems.
Buffering Capacity
• A buffer or buffering capacity is the efficiency of a buffer in
maintaining a constant pH when a small quantity of an acid or base is
added.
• It is a measure of the magnitude of resistance to pH change on the addition of
an acid or a base.
• Buffer capacity is also referred to as buffer value, buffer efficiency,
buffer index, or buffer coefficient.
• A buffer in its buffering zone can resist a pH change up to ±1 of its
buffering zone
• Buffering capacity = Amount of acid or base added
change in pH
 Applications of buffer
• In the Laboratory
• To enable in vitro analysis to occur as they would in biological system by maintaining
the normal pH of different physiological conditions
• In the Pharmaceutical industry
• Buffers are added in drug preparation to minimize drug degradation
• Drugs applied through injections are maintained at an ideal pH of 7.4, same as that
of blood
• In the Cosmetic industry
• Use to ensure stability of creams and ointments
• They are used in shampoos to counteract the basicity of the detergents present in
the shampoo. This is because alkaline pH affects hair fibre surface leading to cuticle
damage.
• In the Textile Industry
• Used to provide the appropriate conditions for dyeing fabrics
• The colour strength of dyes is related to a particular pH which is maintained
by using different buffer system.

• In the Food Industry

• Set up appropriate conditions for the fermentation process
• Used to maintain the acidity of foods in order to preserve its flavour and
appearance
Buffer Preparation
Units of concentration used in Buffer
Preparation
• Concept of strength (x) and stock
• Molarity (M)
solutions
• Normality (N)
• Preparing buffers from stock
• Parts solutions
• Part per hundred (Percentage)
• Parts per thousand (milligram/mL)
• Part per million (microgram/mL)
• Parts per billion (microgram/µL)
 Molarity (Molar concentration)
• The molarity of a solution is the gram
molecular weight of a solute per liter of 
solution. 
• Solute + solvent = Solution
• A one molar solution is a solution that contains
the gram molecular weight of the solute in a
litre (1dm3) of the solution
• Unit is g/dm3 (M, mM, µM)
• Gram molecular weight is also referred to as
gram molecular mass or gram formular weight
• The molecular mass is always written on the
container of every solute and solvent with its
unit as g/mol.
• The molecular weight can also be calculated if the chemical
composition of the compound is known
• Examples
• Hydrochloric acid – HCl has one Hydrogen ion and and a chloride ion. Their
mass number is 1 and 35.5 respectively. To calculate the molecular mass of
HCl = (1 x 1) + (1 x 35.5) = 36.5g/mol
• Sodium Hydroxide (NaOH) [Na = 23, O = 16, and Hydrogen = 1]. The molecular
mass of NaOH = (1 x 23)+ (1 x 16) + (1 x 1) = 40g/mol
• It is advisable to always check the molecular weight on the container
of the reagent and only attempt to calculate if the information is not
available or supplied.
• NaCl = (1 x 23) + (1 x 35.5) = 58.5
 Preparing buffers with molar concentration
1. Prepare a buffer containing 1M
Tris • Molecular weight
2. Prepare a buffer containing • Tris = 121.14g/mol
100mM Tris
• Na2 EDTA = 372.24g/mol
3. Prepare 100mls of 100mM Tris
• NaCl = 58.44g/mol
4. Prepare 100mL of a buffer
containing 50mM Tris and 10mM NOTE
EDTA
The result should always be written in prose,
5. Prepare 250mL of TE buffer describing the preparation process.
(10mM Tris, 1mM EDTA) [pH 8.0) When preparing a buffer, use a volumetric flask
to make up the volume
 Normality (Normal concentration)
• The normality of a solution is the gram equivalent weight of a solute per
liter of solution.
• A one normal solution is a solution that contains the gram equivalent
weight of the solute in a litre of the solution
• Gram equivalent weight = gram molecular weight
number of replaceable hydrogen or hydroxide ion
• Unit is eqg/dm3 (N, eq/L, or meq/L)
• Like Molarity, the reference volume for normality is also 1 litre
• It is important to note the relationship between molar
concentration and normal concentration
• Normal concentration = Molar concentration
number of replaceable hydrogen or hydroxide ion
• For HCl, normality = molarity
• For H2S04, normality = molarity divided by 2.
• Molecular weight of H2S04 = (2 x 1) + (1 x 32) + (4 x 16) = 98g/mol
• Equivalent weight of H2S04 = 98 ÷ 2 (since H2S04 has 2 replaceable hydrogen
ion) = 49g/mol
• Hence 1N H2S04 = 0.5M H2S04
Part per hundred (Percentage)
• PPH is also referred to as percentage
• Calculations involving concentration in parts does not require using
the gram molecular weight or the gram equivalent weight
• The reference volume for percentage is 100 millilitres
• Hence to prepare a 2% solution. You weight out 2g of the solute and
dissolve and make up to 100mL in a volumetric flask
Part Per Thousand (PPT)
• PPT is also known as milligram per millilitre (mg/mL)
• The reference volume for mg/mL is 1 millilitre (1000 microlitres)
• For a 20mg/mL solution. You weigh out 20mg and dissolve and make
up to 1000µL
PPM and PPB
• Part per million (microgram/mL)
• Parts per billion (microgram/µL)
• Note
• Preparing buffers with PPM and PPB can be quite dauting without an
analytical weighing balance that can weigh micrograms (6 decimal
place)
• It may be necessary to prepare the buffer as a higher stock
concentration (strength) and dilute to the working concentration
when needed.
 Concept of Stock Solutions (X solutions)
• Concentrated solutions, that may then be diluted as needed for laboratory applications, are
often stored. These are referred to as stock solutions
• Concentrated stocks of buffer solutions are prepared to save time and space.
• These concentrated stocks can last for a long period of time (if stored properly) and can be
easily diluted for use.
• Typical stock solutions include 1mTris [pH 8.0], 0.5M EDTA [pH 8.0], and 5M NaCl
• When a buffer contains more than one component, then the unit of concentration is the X
factor. The X-factor indicates that the solution is concentrated (x = concentration times the
number indicated).
• In preparing such solutions, you need to measure N times the amount of each solute to come
up with a highly concentrated stock solution.
• X factors solutions are commonly labeled as 10X, 5X, 100X etc. and must be diluted usually
with water to a 1X concentration for use.
• Examples include 10x TBE, 50x TAE, 100x TE buffer.
 Preparing buffers from Stock Solution
Tris SDS
• C1V1 = C2V2 C1 = 1M = 1000mM, V1 = ?, C1 = 10% = 1000mM, V1 = ?,
• Where C1 is the stock concentration. C2 = 10mM, V2 = 1L = 1000mL C2 = 1%, V2 = 1L = 1000mL
• V1 is the volume required from the C1V1 = C2V2 C1V1 = C2V2
stock V1 = (C2V2) / C1 V1 = (C2V2) / C1
• C2 is the concentration of the stock V1 = (10 x 1000) / 1000 V1 = (1 x 1000) / 10
solution. V1 = 10mL V1 = 100 mL
• V2 is total volume needed for the
experiment. • Answer
• Example - Make 1 Litre of a solution • Mix 10 mL of 1M Tris and 100 mL
which contains 10mM Tris and 1% of 10% SDS and bring to 1 L with
SDS, using a 1 M Tris and a 10% SDS ddH20
stock.
Examples
1. Prepare 100mL of a buffer containing 0.1 M Tris-Cl (pH 8.0), 0.01 M
EDTA (pH 8.0), and 1 M NaCl
2. Prepare 500 mls of TENS buffer
• (TENS = 50mM Tris-Cl (pH 8.0), 10mM EDTA (pH 8.0), 1 N NaOH, and 2% SDS)
3. Prepare the above buffer from a 1M Tris-Cl [pH 8.0], 0.5 M EDTA [pH
8.0], 2N NaOH, and 10% SDS)
4. Prepare 10X TE buffer (TE buffer = 10mM Tris [pH 8.0] and 1mM
EDTA [pH 8.0])