EXPERIMENTS 1-3

PH AND BUFFERS
BIOCHEMICAL PROCESSES
TEST FOR CARBOHYDRATES
pH and Buffers
pH
 pH is commonly expressed as –log[H+]

It approximates the negative log (base 10) of the molar concentrations

of hydrogen ions H+ (really hydronium ions H30+) in solution

So a solution of HCl with a pH of 2.0 has a concentration of hydronium

ions of 1x 10-2 (1/100!!)

Compared to a more dilute solution of HCl with a pH of 5.0, which has

a hydronium ions concentration of 1 x 10-5 (1/100,000).
pH
 pH is commonly expressed as –log[H+]
 Pure water has [H+]=10-7 and thus pH=7.
pH
 pH is commonly expressed as –log[H+]
 Pure water has [H+]=10-7 and thus pH=7.
 Acids have a high [H+] and thus a low pH.
 Bases have a low [H+] and thus a high pH.
Bases contribute –OH ions when they dissociate. These bind to the H+ ions
produced when water dissociates. Thus, these OH ions “suck up” the H+
ions in solution, reducing their concentration.

NaOH with a pH of 12.0 contributes so many –OH ions that almost all the H+
ions are bound into water molecules, reducing the free H+ (and hydronium)
ion concentration to 1 x 10-12 (1,000,000,000,000 = 1/trillion)
pH
Acid Normality pH
Acetic N 2.4
Acetic 0.1 N 2.9
Acetic 0.01 N 3.4
Hydrochloric N 0.1 How do normality
Hydrochloric 0.1 N 1.1 and molarity relate
to pH??
Hydrochloric 0.01 N 2.0
Sulfuric N 0.3
Sulfuric 0.1 N 1.2
Sulfuric 0.01 N 2.1

Molarity is the fractions of a mole in solution; normality is a

measure of the concentration of reactive groups which may
affect pH.
Ways to measure pH
 pH meter
 Electrode measures H+ concentration
 Must standardize (calibrate) before using.
Actually measuring a voltage – a
charge differential – between a
control solution and the external
fluid.
Ways to measure pH
 Indicator dyes and test strips
 Less precise
 Each indicator is only good for a small pH range (1-2
pH units)
 But may be good for field usage, or measuring small
volumes, or dealing with noxious samples.
INDICATORS

INDICATOR ACID pH Range Base

Methyl orange Red 3-5 Yellow
Phenolphthalein Colorless 8-10 Red
Bromothymol Yellow 6-7.8 Blue
blue
Litmus paper Red 6-8 Blue
 Why is pH important in
biology?

 pH affects solubility of many substances.

[A] (mol/L) 1 10−1 10−2 10−3 10−4 10−5 10−6 10−7 10−10

Initial pH 0.00 1.00 2.00 3.00 4.00 5.00 6.00 6.79 7.00

Final pH 6.75 7.25 7.75 8.14 8.25 8.26 8.26 8.26 8.27

Dissolved
CaCO3 (g
50.0 5.00 0.514 0.0849 0.0504 0.0474 0.0471 0.0470 0.0470
per liter of
acid)

More calcium carbonate dissolves as pH drops

Increased CO2 increases the ocean’s acidity

Increases in H+ causes cation

displacement and the dissolution
of Calcium Carbonate (shell,
limestone, etc.)
Drives equilibria and reversible states of compounds

Carbonic Acid Bicarbonate Carbonate

Why is pH important in
biology?

 pH affects solubility of many substances.

 pH affects structure and function of most proteins -
including enzymes.
Why is pH important in
biology?

 pH affects solubility of many substances.

 pH affects structure and function of most proteins -
including enzymes.
 Many cells and organisms (esp. plants and aquatic
animals) can only survive in a specific pH
environment.
Why is pH important in
biology?

 pH affects solubility of many substances.

 pH affects structure and function of most proteins -
including enzymes.
 Many cells and organisms (esp. plants and aquatic
animals) can only survive in a specific pH
environment.
 Important point -
 pH is dependent upon temperature
Buffers

 Definition: a solution that resists change in pH

 Typically a mixture of the acid and base form of a chemical
 Can be adjusted to a particular pH value

Blood: pH = 7.35-7.45

Too acidic? Increase respiration rate expelling CO2, driving

reaction to the left and reducing H+ concentration.

Excretory system – excrete more or less bicarbonate

Buffers

 Definition: a solution that resists change in pH

 Typically a mixture of the acid and base form of a chemical
 Can be adjusted to a particular pH value

pH below 7.4 in rats – CaCO3 in BONE dissociates, carbonates soak up

extra H+ to buffer blood. But bones weakened.
Buffers

 Definition: a solution that resists change in pH

 Typically a mixture of the acid and base form of a chemical
 Can be adjusted to a particular pH value
 Why use them?
 Enzyme reactions and cell functions have optimum pH’s for
performance
 Important anytime the structure and/or activity of a
biological material must be maintained
How buffers work
 Equilibrium between acid and base.
 Example: Acetate buffer
 CH3COOH  CH3COO- + H+
 If more H+ is added to this solution, it simply
shifts the equilibrium to the left, absorbing H+,
so the [H+] remains unchanged.
 If H+ is removed (e.g. by adding OH-) then
the equilibrium shifts to the right, releasing H+
to keep the pH constant
Limits to the working range of
a buffer
 Consider the previous example:
 CH3COOH  CH3COO- + H+
 If too much H+ is added, the equilibrium is
shifted all the way to the left, and there is no
longer any more CH3COO- to “absorb” H+.
 At that point the solution no longer resists
change in pH; it is useless as a buffer.
 A similar argument applies to the upper end
of the working range.
TWO FACTORS THAT DERTERMINES
THE EFFECTIVENESS OF BUFFER

 Molar concentration (directly proportional to

the concentration of the buffer components
 Ratio of conjugate base to the concentration
of the weak acid
Titration is used to determine the concentration of an acid or base
by adding the OTHER and finding an equivalency point…
Titration is used to determine the concentration of an acid or base
by adding the OTHER and finding an equivalency point…

Suppose you have a KOH

solution, and you want to
know its concentration
(molarity).

Slowly add an acid (HCl) with

a known concentration (0.1
M) and find the equivalency
point…in this case it will be
at pH = 7… and we use an
indicator that changes color
at that pH determine when
that point has been reached.

So, suppose it takes 10ml of

0.1 M HCl to buffer 50 ml of
the KOH.
Titration is used to determine the concentration of an acid or base
by adding the OTHER and finding an equivalency point…

So, suppose it takes 10ml of

0.1 M HCl to buffer 50 ml of
the KOH.

The original concentration of

the base =

Vol Acid x conc. Of acid

Volume of Base

10 ml x 0.1 M
50 ml = 0.02 M
Chemistry of buffers
 Ka = equilibrium constant for H+ transfer…
also described as the dissociation
constant…the tendancy of an acid to
dissociate. AH  A- (base conjugant) + H+
 Ka = [A-] [H+]/ [AH] = [base] [H+] / [acid]
 Weak acids have low values… contribute few
H+ ions…
 Because we are usually dealing with very
small concentrations, log values are used…
 The log constant =
Chemistry of buffers
 Ka = [A-] [H+]/ [AH] = [base] [H+] / [acid]
 Weak acids have low values… contribute few
H+ ions…
 Because we are usually dealing with very
small concentrations, log values are used…
 The log constant =
 SO! Since pK is the negative log of K, weak
acids have high values … (-2 – 12).
 HCl = -9.3 – very low ~complete dissociation
Chemistry of buffers
 First rearrange the first equation and solve for
[H+]
 [H+] = Ka x [acid]/[base]
 Then take the log of both sides
 log10[H+] = log10Ka + log10 [acid]/[base]

-pH -pKa
Chemistry of buffers
 -pH = -pKa + log10 [acid]/[base]
 Multiply both sides by –1 to get the
Henderson-Hasselbach equation
 pH = pKa - log10 [acid]/[base]
Chemistry of buffers
 What happens when the concentration of the acid
and base are equal?
 Example: Prepare a buffer with 0.10M acetic acid and
0.10M acetate
 pH = pKa - log10 [acid]/[base]
 pH = pKa - log10 [0.10]/[0.10]
 pH=pKa
 Thus, the pH where equal concentrations of acid and base
are present is defined as the pKa

 A buffer works most effectively at pH values that are

+ 1 pH unit from the pKa (the buffer range)
equilibrium pKa value
H3PO4 H2PO4− + H+ pKa1 = 2.15
H2PO4− HPO42− + H+ pKa2 = 7.20
HPO42− PO43− + H+ pKa3 = 12.37
Drives equilibria and reversible states of compounds

Carbonic Acid Bicarbonate Carbonate

Factors in choosing a buffer
 Be sure it covers the pH range you need
 Generally: pKa of acid ± 1 pH unit
 Consult tables for ranges or pKa values
 Be sure it is not toxic to the cells or
organisms you are working with.
 Be sure it would not confound the experiment
(e.g. avoid phosphate buffers in experiments
on plant mineral nutrition).
What to report when writing
about a buffer:
 The identity of the buffer (name or chemicals)
 The molarity of the buffer
 The pH of the buffer
 Examples:
 “We used a 0.5M Tris buffer, pH 8.0.”
 “The reaction was carried out in a 0.1M boric acid
– sodium hydroxide buffer adjusted to pH 9.2.”
Three basic strategies for
making a buffer
1. Guesswork – mix acid and base at the pH
meter until you get the desired pH.
 Wasteful on its own, but should be used for final
adjustments after (2) or (3).
2. Calculation using the Henderson-
Hasselbach equation.
3. Looking up recipe in a published table.
Calculating buffer recipes
 Henderson-Hasselbach equation
 pH = pKa - log10 [acid]/[base]
 Rearrange the equation to get
 10(pKa-pH) = [acid]/[base]
 Look up pKa for acid in a table. Substitute
this and the desired pH into equation above,
and calculate the approximate ratio of acid to
base.
 Because of the log, you want to pick a buffer
with a pKa close to the pH you want.
Example
 You want to make about 500 mL of 0.2 M
acetate buffer (acetic acid + sodium acetate),
pH 4.0.
 Look up pKa and find it is 4.8.
 10(4.8 - 4.0) = 100.8 = 6.3 = [acid]/[base]
 If you use 70 mL of base, you will need 6.3X
that amount of acid, or 441 mL. Mix those
together and you have 511 mL (close enough).
Seatwork
 Calculate the pH of the following
 0.04M HCl
 0.02M NaOH
 Blood has a [H=] of 5x10-7. What is the pH?
Henderson-Hasselbach
 widely used by many scientists especially
chemists, biologists and pharmacists.

pH = pKa + log ([A–]/[HA])

or
pH = pKa + log [(salt)/(acid)]
or
pH = pKa + log ([ionized]/[unionized])
Unknowns to be calculated
 The pH of the solution- The pH of a solution
is a measure of the acidity and basicity of the
solution.
 The pKa of a chemical in solution- The pKa of
a solution determines the acidity of the
compound. The pKa also determines the
amount of ionization of a molecule in a
solution of particular pH.
Unknowns to be calculated
 The [A-] or salt concentration or the amount
of ionized chemical and the [HA] or acid
concentration or the amount of unionized
chemical- Knowing the ratio of the ionized
and unionized forms of the molecule is
important for certain reactions as well as for
pharmaceutics related problems as a lot of
biological functions such as absorption and
excretion of drugs depends upon the
ionized/unionized state of the drug.
Example 1
 Calculate the pH of a buffer composed of
0.1M acetic acid (CH3COOH) and 0.6M
acetate (CH3COO-) knowing that the acid
dissociation constant Ka is 1.8 x 10-5
Example 1:Given
 [HA] = [CH3COOH] = 0.1M (unionized
species)
 [A-] = [CH3COO-] = 0.6M (ionized species)
 Ka = 1.8 x 10-5
Example 1: Computation
pH = pKa + log ([A–]/[HA])
pH = 4.7 + log ( 0.6 / 0.1)
pH = 4.7 + log 6
pH = 4.7 + 0.78
pH = 5.48
Seatwork
 Calculate the pH of a buffer solution
prepared by dissolving 363 mg of Tris in
10 mL of 0.2M HCl and diluting to 100 mL
with water. [Tris: mw 121 g/mol and pKa =
8.08 for the conjugate acid]
Example 2
 Aspirin (acetylsalicylic acid) has a pKa of
3.5. (i) Calculate the ratio of
ionized/unionized of the drug in the
stomach where pH is 1. (ii) Calculate the
ratio of ionized/unionized in the intestine
where pH is 6. (iii)Based on these
calculations- where is aspirin absorbed
within the body?
Example 2: Given
 pKa of aspirin = 3.5
 pH of stomach = 1
 pH of intestine = 6
 Unknown = log [ionized]/[unionized].
Example 2: Computation
pH of stomach = pKa of aspirin + log
[ionized]/[unionized]
1 = 3.5 + log [ionized]/[unionized]
log [ionized]/[unionized] = 1-3.5
log [ionized]/[unionized] = -2.5
thus, [ionized]/[unionized] = Antilog (-2.5)
[ionized]/[unionized] = 0.00316 (Answer)
Seatwork
 pH of intestine?
Biochemical
Processes
Diffusion
Osmosis
Dialysis
Surface tension
Diffusion and
Osmosis
 Functions of Membranes
1. Protect cell
2. Control incoming and outgoing
substances
3. Maintain ion concentrations of various
substances
4. Selectively permeable - allows some
molecules in, others are kept out
5. ALL THIS MAINTAINS HOMEOSTASIS
(internal balance)
Phospholipid Bilayer
Fluid Mosaic Model
Methods of Transport Across
Membranes
1. Diffusion

2. Osmosis

3. Facilitated Diffusion

4. Active Transport
 Polar heads
love water
& dissolve.

Non-polar
tails hide
from water.
Carbohydrate cell
markers

Proteins
Types of Cellular Transport
Weeee!!
 Passive Transport !

cell doesn’t use energy

1. Diffusion
high
2. Facilitated Diffusion
3. Osmosis low

 Active Transport
This is
cell does use energy gonna
1. Protein Pumps be hard
work!!
high
2. Endocytosis
3. Exocytosis
low
Diffusion
 Movement of molecules from an area of
high concentration to an area of low
concentration.

 Movement from one side of a

membrane to another, un-facilitated
Diffusion
Passive Transport:
Diffusion
Diffusion: random movement of
particles from an area of high
concentration to an area of
low concentration.
(High to Low)
 Diffusion continues until all
molecules are evenly spaced
(equilibrium is reached)-Note:
molecules will still move around
but stay spread out.
http://bio.winona.edu/berg/Free.htm
 Passive Transport:
Osmosis
 Osmosis: diffusion of
water through a
selectively permeable
membrane
 Water moves from high to
low concentrations
•Water moves freely
through pores.
•Solute (green) too
large to move across.
Osmosis
Tonicity is a relative term
 Hypotonic Solution - One solution has
a lower concentration of solute than
another.
 Hypertonic Solution - one solution has
a higher concentration of solute than
another.
 Isotonic Solution - both solutions have
same concentrations of solute.
Plant and Animal Cells put into
various solutions
 Hypotonic Solution
Hypotonic: The solution has a lower concentration of
solutes and a higher concentration of water than
inside the cell. (Low solute; High water)

Result: Water moves from the solution to inside the

cell): Cell Swells and bursts open (cytolysis)!
 Hypertonic Solution
Hypertonic: The solution has a higher concentration
of solutes and a lower concentration of water than
inside the cell. (High solute; Low water)

shrinks

Result: Water moves from inside the cell into the

solution: Cell shrinks (Plasmolysis)!
 Isotonic Solution
Isotonic: The concentration of solutes in the solution
is equal to the concentration of solutes inside the cell.

Result: Water moves equally in both directions and

the cell remains same size! (Dynamic Equilibrium)
Types of Transport
 Passive Transport:
Facilitated Diffusion A B
Facilitated diffusion:
diffusion of specific particles
through transport
proteins found in the
membrane
a. Transport Proteins are Facilitated Diffusion
specific – they “select” only diffusion (Lipid
certain molecules to cross (Channel Bilayer)
the membrane Protein)
b. Transports larger or
charged molecules

Carrier Protein
 Passive Transport: Facilitated Diffusion
Glucose
molecules
Cellular Transport From a-
High
High Concentration
• Channel Proteins
animations

Cell Membrane

Protein
Low Concentration channel
Low
Transport
Through a 
Go to
Protein
Section:
Types of Active
Transport
 Endocytosis: taking bulky
material into a cell
• Uses energy
• Cell membrane in-folds
around food particle
• “cell eating”
• forms food vacuole &
digests food
• This is how white blood
cells eat bacteria!
 Types of Active Transport
Exocytosis: Forces
material out of cell in bulk
• membrane surrounding the
material fuses with cell
membrane
• Cell changes shape –
requires energy
• EX: Hormones or
wastes released from
cell
 DIALYSIS AND
SURFACE TENSION
 INTRODUCTION
Solution is a homogenous mixture of two
or more substances (solid, liquid or gases)
distributed uniformly among each other.
Solvent is the component of a solution
which forms larger portion of that solution
e.g. water.
Solute is the component of a solution
which form minor portion of that solution e.g.
sugar.
 INTRODUCTION
Example: 0.9% NaCl solution. Here water is
solvent (represent major portion) & NaCl is
solute (represent minor portion of solution).
Homogenous mixture is the mixture of
two or more substances of same
composition.
Heterogeneous mixture is the mixture of
two or more substances of different
composition in different parts of the total
mixture.
 INTRODUCTION
True solution is a homogenous mixture of solute
and solvent. Here solutes are crystalloid and exist in
the solution as molecules or ions.
Molar/Molal solution is the
solution 1 mole of solute per liter solution/
containing
per kg solvent.
Normal solution (1 Eq/L) is the
solution containing 1 Eq of solute per liter
solution.
Osmolar/Osmolal
containing 1 osmole solution of soluteis per theliter
solution kg
s o lution/per
13-Feb-18

solvent.
 INTRODUCTION
Saturated solution is the solution containing
the maximum concentration of solute. Additional
solute will not dissolve in a saturated solution.
Supersaturated solution is a solution that has
more solute than it can dissolve at a given
temperature e.g. when salt is added to water
until the rock salt is formed in water.
Unsaturated solution is the solution which can
dissolve more solute at a given
5
 t e mperature.
1 3-F eb-18
 SURFACE
TENSION
Surface tension is the force with which the
molecules on the surface are held together. It’s
expressed as dynes/cm.
Surface tension decreases with increasing
temperature i.e. Surface tension 1/α Temperature.
SURFACE
TENSION
 SURFACE
TENSION
 Application of surface tension:
 Fat digestion- Bile salts decrease the surface
tension of fat and emulsify big fat globules into
minute particles for effective digestion.
 Surfactant & Lung function- Surfactant
decreases surface tension of alveoli and keeps
the alveoli in expand state for efficient exchange
of gases in lungs.
 Surfactant &
Lung function
 DIALYSIS
Dialysis comes from Greek word ‘dialusis’ means
dissolution. It is the process of separation of
colloids and crystalloids from their mixture
through a semi-permeable membrane.
Dialysis is a process for removing waste and
excess water from the blood. Used as artificial
replacement for lost kidney function.
 DIALYSIS
Principle of dialysis: Crystalloid substances in
solution can pass through semipermeable
membrane while colloid particles cannot. In dialysis,
semipermeable membrane is called dialyzing
membrane or dialyzer, usually in the form of an
elongated tube or of a bag.
The goal of dialysis is to remove accumulated
fluid and toxins [by diffusive transport (based on
countercurrent flow of blood and dialysate) &
convective transport (solvent drag with
ultrafiltration)] to maintain their concentrations
below the levels at which they produce uremic
13-Feb- SOLUTIONS- DR. SUBIR
18 KUMAR
 MODALITIES OF
DIALYSIS
1) Peritoneal dialysis
2) Intermittent hemodialysis
3) Hemofiltration
4) Continuous renal replacement therapy
 Decision of modality determined by catabolic rate,
hemodynamic stability, and whether primary goal
is fluid or solute removal.
General tests for
Carbohydrates
Introduction
 Carbohydrates are the key source of energy used by
living things.
 Also serve as extracellular structural elements as in cell
wall of bacteria and plant.
 Carbohydrates are defined as the polyhydroxy
aldehydes or polyhydroxy ketones. H O
C

H C OH

 Most , but not all carbohydrate have a formula

HO C H

H C OH
 (CH2O)n (hence the name hydrate of carbon) H C OH
 In human body, the D-glucose is used. CH OH 2

 Simple sugars ends with –ose D-glucose

Classification
1-Simple sugar: (one unit)
Monosaccharides contain one
monosaccharide unit.
2-Complex sugar (more than one):
Disaccharides contain two monosaccharide
units.
- Oligosaccharides contain 3-9
monosaccharide units.
- Polysaccharides can contain more than 9
monosaccharide units.
Monosaccharide
 They can be classified by the number of carbon atoms
 trioses (C-3)
 tetroses (C-4)
 pentoses (C-5)
 hexoses (C-6)
 heptoses (C-7)
 also be classified as ketoses or aldoses.
 A ketose contains a carbonyl group attached to
two R groups having one or more hydroxyl groups. H
C
O

H C OH

HO C H

H C OH
 An aldose contains terminal aldehyde group in
addition to R group containing -OH. H C OH

CH2OH

D-glucose

aldose
Solubility
 Monosaccharide and disaccharide can be
dissolved freely in water because water is a
polar substance, while polysaccharide cannot be
dissolved easily in water, because, it has high
molecular weight , which give colloidal solutions
in water soluble.
Reducing and non reducing
sugars
 Reducing and non reducing sugar :If the oxygen on the
anomeric carbon of a sugar is not attached to any other
structure, that sugar can act as a reducing agent and is
termed a reducing sugar.

v
v Anomeric
carbon

reducing
Non-
reducing
Molisch test
 This test is specific for all carbohydrates.
Monosaccharide gives a rapid positive test,
Disaccharides and polysaccharides react
slower.

 Objective: To identify the carbohydrate from

other macromolecules lipids and proteins.
 Principle: The test reagent(H2SO4) dehydrates
pentose to form furfural and dehydrates hexoses to form
5- hydroxymethyl furfural.
 The furfural and 5- hydroxymethyl furfural further react
with α-naphthol present in the test reagent to produce a
purple product.
α-naphthol Purple color

furfural

α-naphthol
Purple color

5- hydroxymethyl furfural
Anthrone test
Principle:
 Anthrone test is also another general test for
all carbohydrates. In this test also,
carbohydrate gets dehydrated when react
with conc. H2SO4 to form furfural. This
furfural reacts with anthrone to give bluish
green colored complex.
Anthrone test

positive test: all carbohydrate give test

positive
 Benedict's test
 Benedict's reagent is used as a test for the presence of
reducing sugars.
 All monosaccharides are reducing sugars; they all have a
free reactive carbonyl group.

 Some disaccharides have exposed carbonyl groups and

are also reducing sugars. Other disaccharides such as
sucrose are non-reducing sugars and will not react with
Benedict's solution.

 Large polymers of glucose, such as starch, are not

reducing sugars
 Objective: To distinguish between the reducing and non-
reducing sugars.
Benedict's test
 Principle: The copper sulfate (CuSO4)
present in Benedict's solution reacts with
electrons from the aldehyde or ketone group of

e
sucros
lactose
the reducing sugar in alkaline medium.

e
glucos
 Reducing sugars are oxidized by the copper
ion in solution to form a carboxylic acid and a
reddish precipitate of copper oxide.

reddish precipitate of copper

Barfoed’s Test
 This test is performed to distinguish between reducing
monosaccharides, reducing disaccharides and non
reducing disaccharides.
 Objective: To distinguish between mono- , di- and poly
saccharides.
 Principle: Barfoed’s test used copper (II) ions in a slightly
acidic medium
 Reducing monosaccharides are oxidized by the copper ion
in solution to form a carboxylic acid and a reddish
precipitate of copper (I) oxide within three minutes.
Reducing disaccharides undergo the same reaction, but do
so at a slower rate.
 The nonreducing sugars give negative result.
 Barfoed’s reagent, cupric acetate in acetic acid ,
so in acidic medium , disacchride is a weaker
reducing agent than monosacchride, so mono
sacchride will reduce the copper in less time.
Moore’s test
 Principle: based on the liberation of
aldehydes subsequently polymerizes to form
resinous substance.
 test for reducing sugar except sucrose
 yellow/orange/dark brown
 liberating caramel odor
Fehling’s test
 Principle: Fehling’s test is one of the
sensitive test for detection of reducing
sugars. Fehling’s solution A is aqueous
copper sulphate and Fehling’s solution B is
alkaline sodium potassium tartarate (
Rochelle salt). Rochelle salts (sodium
potassium tartarate) present in the reagent
acts as the chelating agent in this
reaction.These two solution are mixed in
equal amount before test.
Fehling’s test
 Principle: Fehling’s test is one of the
sensitive test for detection of reducing
sugars. Fehling’s solution A is aqueous
copper sulphate and Fehling’s solution B is
alkaline sodium potassium tartarate (
Rochelle salt). Rochelle salts (sodium
potassium tartarate) present in the reagent
acts as the chelating agent in this
reaction.These two solution are mixed in
equal amount before test.
Fehling’s test
 Positive Fehling’s test: reddish brown ppt
(glucose, fructose, lactose)
 Negative Fehling’s test: No red ppt
(sucrose, starch)
Nylander’s test
 consists of bismuth nitrate, potassium sodium
tartrate and potassium hydroxide, is added to
a solution with reducing sugars, a black
precipitate of metallic bismuth is formed.
Picric acid test
 Picric acid (2,4,6-trinitrophenol) or TNP
reacts with reducing sugars
 Red color appears (picramic acid)
mahogany red
Tollen’s test (‘silver mirror’)
 Tollen’s Phloroglucin reaction
 Differentitae aldehyde from ketone
 Oxidizes both aliphatic and aromatic
aldehyde to carboxylic acids to produce silver
mirror effects
Thank you! 
Godbless.