1.

Saponification : Saponification is the hydrolysis of fats or oils under basic

conditions to afford glycerol and the salt of the corresponding fatty acid.

2. Saponification value : The saponification number is the number of milligrams

of potassium hydroxide required to neutralize the fatty acids resulting from the
complete hydrolysis of 1g of fat.
3. Indicator : phenolphthalein
4. Ratio of fat and KOH in saponification : 1:3
5. Calculation of Saponification value :
1 mole of fat = 3 mole of KOH
80 g = 3 × 56 g of KOH
1 g of fat = 3 × 56 / 306 g of KOH
SV = 3 × 56 × 1000/ 306 μg of KOH
6. Significance of saponification value :
● It is important to the industrial user to know the amount of free fatty acid
present, since this determines in large measure the refining loss.
● The amount of free fatty acid is estimated by determining the quantity of
alkali that must be added to the fat to render it neutral.
● It is also considered as a measure of the average molecular weight (or
chain length) of all the fatty acids present.
● The long chain fatty acids found in fats have low saponification value
because they have a relatively fewer number of carboxylic functional
groups per unit mass of the fat and therefore high molecular weight.
7. Ester hydrolysis (only in alkaline condition) : Fats are not soluble in
aqueous solution. Only soluble in a mixture of alcoholic solution.
8. Molecular weight of Oxalic acid : 126 g/mol OR 90 g/mol
9. Formula of Saponification value :

10. Why do we use oxalic acid to standardize KOH?

 The standard solution of oxalic acid is used to find the strength of solutions of
alkalies like NaOH, KOH whose standard solutions can not be made by direct
weighing.
11. Iodine value : Iodine value or number is the number of grams of iodine
consumed by 100g of fat.

12. Wij’s reagent (ICl)in glacial acetic acid (13g of ICl in 1 liter glacial acetic acid)
13. Indicator : Starch
14.Significance of Iodine value :
● Method for checking the unsaturation level in fatty acids
● Higher iodine value indicates a higher degree of unsaturation
15.Calculation of Iodine number :
1 mole of fat = 3 mole of I2 or 6 mole of I
864 g = 6 × 127 g of I
1 g = 6 × 127/864 g of I
IV = 6 × 127 × 100/864 g of I
16.Drying, non-drying and semi-drying oils :
● Drying oils contain more than 50% of polyunsaturated acids. They are
quickly absorbed and leave no greasy layer on oily skin.
 ● Semi-drying oils contain 20%-50% of polyunsaturated acids. These
natural oils are suitable for normal and combination skin but may clog the
pores in oily skin.
● Non-drying oils contain less than 20% of polyunsaturated fatty acids.
Non-drying oils are recommended for dry and mature skin which has lost
firmness but is free from blackheads.
17. Iodine value of mustard and coconut oil :
Mustard oil = 96-112
Coconut oil = 6-11
18.Why do we use an iodine flask to determine iodine value?
The iodine flask prevents the iodine vapors from escaping as iodine is volatile in
nature, improving the accuracy of analysis.
19.Formula of iodine value :

20. Molecular weight of Iodine : 126.90447 g/mol

21. Chromatography : a technique for the separation of a mixture by passing it in
solution or suspension through a medium in which the components move at
different rates.
22. Principle of Chromatography : Chromatography is based on the principle
where molecules in mixture applied onto the surface or into the solid, and fluid
stationary phase (stable phase) is separating from each other while moving with
the aid of a mobile phase.
23. Thin layer chromatography : Thin Layer Chromatography can be defined as
a method of separation or identification of a mixture of components into
individual components by using finely divided adsorbent solid / (liquid) spread
over a plate and liquid as a mobile phase.
24. Difference between paper and thin layer chromatography :
25. Electrochromatography : Electrochromatography is a chemical separation
technique in analytical chemistry, biochemistry and molecular biology used to
resolve and separate mostly large biomolecules such as proteins. It is a
combination of size exclusion chromatography (gel filtration chromatography)
and gel electrophoresis.
26. Stationary phase in paper chromatography : Uniform absorbent paper
27. Stationary phase in thin layer chromatography : Silica gel or alumina
28. Disadvantages of paper chromatography :
● Large quantities of sample cannot be applied on paper chromatography.
● In quantitative analysis paper chromatography is not effective.
● Complex mixture cannot be separated by paper chromatography.
29. Among paper chromatography and thin layer chromatography,
which one is better and why?
Thin layer chromatography has certain advantages over paper chromatography.
A huge advantage, which is provided by thin layer chromatography, is that it is
responsible for saving a lot of time. On the other hand, paper chromatography is
responsible for taking numerous hours to develop the plate.
30. Retention factor : Rf value of a compound is equal to the distance traveled by
the compound divided by the distance traveled by the solvent front (both
measured from the origin). It is unitless.
31.Why can Rf not be more than 1 ?
Rf values are a ratio of the distance a spot has traveled to the total distance of the
solvent front. Since the distance of the front is always greater than the distance of
a spot, the Rf value is always less than 1.
32. Spraying agent in chromatography : Ninhydrin for amino acids, and aniline
hydrogen phthalate for sugars
33. Reducing and non-reducing sugars, with examples :
34. If the sugar is reduced, will it show maturation? Why?
The compounds having hemiacetal in their structure give mutarotation.
Generally, all monosaccharides (glucose, fructose, ribose, arabinose) and
disaccharides (lactose, maltose) give mutarotation, except sucrose. Sucrose,
despite being a disaccharide, fails to give mutarotation because it doesn't contain
hemiacetal.
35. Test for sugars and their procedures :
(a) Molisch’s Test:
Take 2 ml of the given sample solution in a clean test tube.
Add 2-3 drops of Molisch reagent slowly.
Now add concentrated sulfuric acid along the sides of the test tube.
The acid layer forms a layer at the bottom.
Note the junction of the two layers.
If there is a formation of the violet ring then the presence of carbohydrate is
confirmed.
(b) Fehling’s Test:
Take 2 ml of given sample solution in a clean test tube.
Add 2 ml of Fehling’s solution A and Fehling’s solution B to it.
Keep the solution in a boiling water bath for about 10 minutes.
If there is the formation of red precipitate then the presence of carbohydrate is
confirmed.
(c) Benedict’s Test:
Take the given sample solution to be tested in a clean test tube.
Add 5 ml of Benedict’s reagent to it.
Boil the solution for about 2 minutes.
Cool the solution and observe the solution.
If there is formation of green, red or yellow precipitate then there is presence of
reducing sugars.
(d) Tollen’s Test:
 Take the given sample solution in a clean test tube.
Add 2-3 ml of tollen’s reagent to it.
Keep the test tube in a boiling water bath for 10 minutes.
If there is, the appearance of a shiny silver mirror confirms the presence of
reducing sugars.
36. Role of sulphuric acid in molisch test : dehydration
37. Why can’t we store tollen’s reagent? Poor shelf life
38. Complex of tollen’s reagent : [Ag (NH3)2]NO3
39. Why blank test of tollen's is performed?
40. Among Fehling's test and Benedict's test, which one is better and
why?
Fehling's solution is made up out of two separate solutions, is caustic, and doesn't
keep well. Benedict's solution is more stable, is a single solution, and has no
caustic properties, making it easier to handle.
41.Why does fructose give tollen’s test?
Tollen's reagent is used to identify an aldehyde group −CHO because tollen's
reagent oxidizes aldehyde. Fructose is a carbohydrate that does not contain the
aldehyde group but fructose shows keto-enol tautomerism and thus isomerizes to
glucose due to which it gives positive Tollen's reagent test and silver mirror.
42. Starch molecules are glucose polymers linked together by the alpha-1,4 and
alpha-1,6 glycosidic bonds. An unbranched, single chain polymer of 500 to 2000
glucose subunits with only the alpha-1,4 glycosidic bonds is called amylose. On
the other hand, the presence of alpha1,6 glycosidic linkages results in a branched
glucose polymer called amylopectin.
43. Amylase activity : Amylase is an enzyme that catalyzes the breakdown of
starch into sugars
44. Enzyme activity is usually measured in terms of the activity unit (U) which is
defined as the amount which will catalyze the transformation of 1 micromole of
the substrate per minute under standard conditions. Another unit of enzyme
activity is the katal (kat) which is defined as the amount which will catalyze the
transformation of one mole of substance per second (1 kat = 60 000 000 U).
45. α- amylase : an enzyme of glycoside hydrolases mainly produced in the salivary
glands and pancreas
46. Functions of α- amylase :
● Elevated levels of α-Amylases in serum can be used as markers for clinical
diagnosis of diseases.
● When the pancreas is diseased or inflamed, amylase is released into the
blood.
● Chloride is the allosteric effector of vertebrate pancreatic and salivary
α-amylases.
47. How can we determine the activity of α-amylase using the standard
curve (DNS method) ?
The α -amylase activity is measured using a colorimetric method with
3,5-dinitrosalicylic acid (DNS) reagent. In this method, starch by α –amylase is
converted into maltose. Maltose released from starch is measured by the
reduction of 3,5-dinitrosalicylic acid. Maltose reduces the pale yellow coloured
alkaline 3, 5-Dinitro salicylic acid (DNS) to the orange- red color. The intensity of
the color is proportional to the concentration of maltose present in the sample.
This intensity change in color is measured using a spectrophotometer as the
absorbance at 540 nm wavelength. Wave length is set to 540 nm because it is the
region where orange-red color absorbs. DNS binds only to reducing sugars (eg.
glucose, maltose) and cannot bind to non-reducing sugars (eg.sucrose). During
the incubation period of this experiment, enzymes act upon substrates to give
products which are reducing sugars. Then you add DNS. Then, the next step is
heating the reaction mixture which is responsible for 2 steps, one for heat
inactivation of enzyme and other for efficient binding of reducing sugars to DNS
to give ANS which shows maximum absorbance at 540 nm.

48. Define turnover number (unit of enzyme).

The turnover number of an enzyme, or the kcat, is the maximal number of
molecules of substrate converted to product per active site per unit time when the
enzyme is saturated with substrate
49. Effect of temperature on enzyme activity, before and after
optimum temperature.
Each enzyme performs best at an optimum temperature. Extremely high or low
temperature leads to loss of enzymatic activity. Too high temperature leads to
denaturation of α-amylase enzymes as a result its activity gets decreased.
Therefore the activity of α-amylase will increase with temperature and after
reaching a certain temperature its activity gets decreased i.e. nonfunctional.
α-amylase works best at a temperature of 37ºC and above this its activity
decreases significantly.
At low temperature (4°C) catalytic activity of salivary amylase is either slow or nil
due to lack of heat and energy.
50. Kinetics of Michaelis-menton reaction

51.What is the Km value of enzymes?

Km (Michaelis Menten) indicates that substrate concentration attains half its
maximum velocity when enzymes catalyze the chemical reaction. Km values
generally lie between 10-1 to 10 -6M.
52. Factors on which enzyme activity depends and their respective
graphs.
The factors affecting enzyme activity are:
1. Temperature: An enzyme activity is maximum within a narrow range of
temperature. The temperature at which an enzyme shows its maximum activity is
called optimum temperature. The optimum temperature for most of the enzymes
is between 25-35°C. Temperature above and below this range affects the enzyme
activity. High temperature above 50°C results in the destruction of enzymes by
causing their denaturation, and very low temperature preserves the enzymes in
their inactive state.
2. Enzyme Concentration: The rate of enzymatic reaction increases with
increased enzyme concentration up to a point called saturation point. Above this
limit, there is little effect on enzyme activity.

3. pH: Enzymes work at their optimum pH. A rise or fall in pH reduces the
activity of enzymes. Most of the intracellular enzymes function near-neutral pH
except for several digestive enzymes that are active either at acidic or alkaline pH
ranges. Change in pH causes alteration in the structure of the enzyme, including
its active site. Under extreme pH, denaturation of enzymes occurs.

4. Substrate concentration: Initially, the rate of enzymatic reaction increases

with the increase in substrate concentration. In the beginning, the velocity of the
reaction is high, but later it does not increase progressively with the increase in
substrate concentration. It happens because enzyme molecules get fully
saturated, and no more active sites are left free to bind additional substrates.
53. Dinitrosalicylic acid (DNSA)
54. Phosphate buffer saline (PBS)
55. Sørensen formål titration (SFT) invented by S. P. L. Sørensen in 1907 is a
titration of an amino acid with sodium hydroxide in the presence of
formaldehyde.
56. Glycine estimation by formylation method
Amino acids e.g. glycine exist in zwitter ionic form and cannot be titrated directly
with alkali. Thus, amino groups of amino acids are blocked by reaction with
formaldehyde. But formaldehyde does not react with the charged amino groups
(-NH3 + ), thus first the amino acid reacts with sodium hydroxide solution to give
NH2CHRCOO¯, which condenses with formaldehyde to give a stable anion.
Thus, amino acids react with formalin to form methylene amino acids.

57.Why is it difficult to titrate glycine with NaOH?

An amino-acid such as glycine, NH2CH2COOH, cannot be estimated by direct
titration with standard alkali solution, owing to the opposing effects of the basic
and the acidic groups.
58. Amino acids are organic compounds containing both an amino group (-NH3) and
a carboxyl group (-COOH). When amino acid is dissolved in water, it exists in
solution as the dipolar ion, or “zwitterion”. A zwitterion can act as either an acid
 (proton donor) or a base (proton acceptor). Substances having this dual nature
are amphoteric.
Amino acids in aqueous solution exist predominantly in isoelectric form. The
characteristic pH at which the net electric charge is zero is called the isoelectric
point (pI). So, an amino acid has a net negative charge at any pH above its pI, and
has a net positive charge at any pH below its pI. Each amino acid has its own pI
value. The ionizable groups of amino acids act as weak acids or bases, giving off
or taking on protons when the pH is altered. Simply, common amino acids are
weak polyprotic acids.

59. Henderson-Hasselbalch equation

60. Titration curve : Titration curves are produced by monitoring the pH of given
volume of a sample solution after successive addition of acid or alkali. Titration
curves are usually plots of pH against the volume of titrant added or more
correctly against the number of equivalents added per mole of the sample.
61. Aspirin : Aspirin, also known as acetylsalicylic acid (ASA), is a nonsteroidal
anti-inflammatory drug (NSAID) used to reduce pain, fever, and/or
inflammation, and as an antithrombotic.
62. Synthesis of aspirin

63. Why is H2SO4 added to it?

 To break intra-hydrogen bonds
64. DNA isolation :
Steps in DNA isolation -
1. Cell Lysis and homogenization
2. Deproteinization
3. Cooling
4. Filtration
5. Precipitation
6. Spooling
65. Cell lysis chemicals : sodium dodecyl sulfate (SDS), sodium chloride (NaCl),
sodium acetate, and ethylenediamine tetraacetic acid (EDTA)
66. Structure of DNA :
DNA is composed of two linear strands that run opposite to each other, known as
antiparallel strands; these strands twist together to form a double helix. Each
strand is composed of a long chain of monomer nucleotides. The nucleotide of
DNA consists of a deoxyribose sugar molecule to which is attached a phosphate
group and one of four nitrogenous bases: two purines (adenine and guanine) and
two pyrimidines (cytosine and thymine). The nucleotides are joined together by
covalent bonds between the phosphate of one nucleotide and the sugar of the
next, forming a phosphate-sugar backbone from which the nitrogenous bases
protrude. The two strands, though identical, run in opposite directions as
determined by the orientation of the 5′ to 3′ phosphodiester bond. One strand is
held to another by hydrogen bonds between the bases; the sequencing of this
bonding is specific—i.e., adenine bonds only with thymine, and cytosine only with
guanine.
67. Components of homogenization solution :
● The detergent-like action of SDS helps to dissolve cell membranes and
denature proteins.
● EDTA is a chelating agent which binds to divalent (Mg2+ and Ca2+) which
is needed for membrane stability.
● The NaCl and Sodium acetate stabilizes the DNA by forming a Na+ shell
around the negatively charged phosphates of the DNA.
● Sodium acetate causes cellular debris to precipitate for easy filtration.
68. Protease enzyme Papain breaks peptide bonds attached to DNA
69. Why do we isolate DNA from onions?
We are using onion to isolate its DNA because it has low starch content and does
not have a lot of cellulose fibers. Enough DNA can be extracted and can be seen
with the unaided eye.
SIGNIFICANT STEPS IN DNA ISOLATION
1. Cell Lysis and homogenization: DNA is found in the nucleus of membrane bound
cells. These membranes are made up of lipids and proteins. Cell membranes must be
broken to release DNA. Cell lysis and homogenization involves breaking down and
removing cell walls and membranes using homogenization solutions.

2. Deproteinization: It involves adding a protease enzyme Papain which breaks

peptide bonds attached to DNA. This makes the molecule flexible and easy to pool.

3. Cooling: this brings down denaturation which happens by the action of DNAse.

4. Filtration: it traps the precipitated cell debris while the soluble DNA passes through
it and goes into the filtrate.

5. Precipitation: DNA is insoluble in alcohol and therefore can be used to precipitate

DNA.

6. Spooling: The DNA will gather at the interface of filtrate and ethanol and can be
spooled with a glass rod. DNA clings to the glass because the negative charge of DNA is
attracted to the positive charge of glass silica.