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ISSUE HISTORY

ISSUE Description of change Originator Effective Date

1 Initial release Kassahun Mirkena

Mulugeta Eshete

REFERENCE DOCUMENTS

DOCUMENT NUMBER Document Title

ISO 9001:2008 Clause 8.2.4 Monitoring and measurement of product

CONTENTS PAGE FOR DCC USE ONLY

0A ISSUE HISTORY 1
0B REFERENCE DOCUMENT 1
0C CONTENTS 1
PURPOSE 1
SCOPE 2
PROCESS OWNER 2
DEFINITIONS 2
CONTENT OF ANALYSIS METHODS 8

BIBLOGRAPHY 123
DIFFUSSION 123

Purpose

The purpose of this laboratory manual is to assist in standardization of the

Methara Sugar Factory Ethanol Plant laboratory practices, so that uniform
procedures are adopted by all who works in the laboratory and who uses the

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analysis results of the laboratory for process control and follow up. Besides
this, it has the merit of collecting and centralizing scattered procedures; that
has been submitted to the laboratory by different experts; within a single
manual prepared in a standard format. For the development of this manual the
KBK chemical Engineering Pvt.Ltd Laboratory manual, Integrated CASETECH
Laboratory work instructions, JP Mukherji &Association Pvt.Ltd. lab
procedures and Methara Sugar Factory Quality Control laboratory manual
were used as references. These references are believed to make the manual
complete and comparable with standard manuals. The manual is also aiming
to be a useful material for carrying out reliable analysis on different Ethanol
Plant products and Effluents.

Scope
This manual covers different analysis procedures and methods for Molasses,
Intermediate products, Effluents and the final products of Ethanol plant.

Process Owner
Ethanol plant

Definitions

Ash
Residue remaining after all the organic matter is burnt. Ash may be determined
as conductivity ash sulphited ash.

Brix
The apparent percentage of soluble solid matter (sucrose and soluble non
sucrose) determined densmetrically.

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Culture Media
Culture Media is a bacteria free liquid or semisolid state with mixed
compounds having water and nutrients for yeast or micro-organisms grow.

Counting Chamber (Haemocytometer)

The haemocytometr (so called because it was originally devised for counting
blood cells) is used for counting the fungal spores in liquid suspension. It is a
special microscope slide with furrows and with engraved squares of 0.1mm
deep, usually two squares on either side of the longitudinal furrow.

Each square has a total of 9 squares each 1x1mm engraved over it, so that the
volume of liquid over each of these squares is 0.1mm3.Put in other words, the
volume in one of the 9 squares corresponds to 1/10x1/10x1/100cm=10-4cm3
or10-4ml. Only one of such squares per field is visible under 100xmicroscope
magnification.

Ethanol
Anhydrous alcohol with strength of 99.85-99.99 %.It is also called Absolute
Alcohol.
Fermentable Sugar
Sugar converted in to alcohol in ordinary fermentation process. Examples are
Glucose and Fructose.
Fermentable Sugar = Total Reducing Sugar - Unfermentable Sugar

Fermented Wash
The liquor obtained as a result of a biochemical reaction with the involvement
of yeast cells and sugar and containing a certain percent of alcohol. The alcohol
percent of fermented wash varies from 3.5% to 11% depending upon the
concentration of sugar added and the yeast activity.

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Final Molasses
A dark heavy viscous liquid from which no further sugar can be crystallized by
normal method
Fusel oil
A High boiling point impurity and an organic compound containing mainly C3-
C5 aliphatic alcohols produced by Rectifier Column as a bottom by-product of
the distillation of ethyl alcohol from the fermentation of molasses. Fusel oil is a
relatively viscous liquid with a dark-reddish color and a very unpleasant odor.
Mother Slant
The purest form of yeast or other micro-organism kept in refrigerator on a
solid media having nutrients.
PH
PH of a solution refers to its hydrogen ion activity. It is also defined as the
logarithm of reciprocal of hydrogen ion concentration.
𝟏
𝑷𝑯 = 𝒍𝒐𝒈 [𝑯+ ] = −𝐥𝐨𝐠[𝑯+ ]

The PH can be measured either calorimetrically or electrometrically.

Pol
The apparent sucrose content of any substance expressed as a percentage by
mass and determined by a single or direct polarization method. The term is
used as if it was a real substance.
Purity
A Percentage ratio of sucrose (Pol) to the total soluble solids (or brix) in a sugar
product.
Rectified Spirit
Hydrous Ethanol Alcohol, which is the main product output of Rectifier
Column with strength of 95-96 % v/v. It is the maximum strength of alcohol
obtained by ordinary distillation process. It is either converted to Ethanol

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(anhydrous alcohol of more 99.85% v/v strength) by dehydration process or
sold to be used for production of potable liquors and other products.
Reducing Sugar
The Reducing substance in cane and its products, calculated as invert sugar.
Two familiar examples are Fructose (laevulose) and glucose (dextrose).
Sludge
A thick gummy semi fluid separated from fermented wash by sludge separators
and disposed out of fermentation process by Decanter.
Spent lease
A colorless liquid which is the under flow of the Rectifier Column and is with
no alcohol content.
Spent lees
A colorless liquid which is the under flow of the Recovery Column and is with
no alcohol content
Spent Wash
A dark brown liquid which is the under flow of the primary column and is with
no alcohol content.
Sucrose
A disaccharide which when hydrolyzed splits up in to two monosaccharaides;
𝑯𝒚𝒅𝒓𝒐𝒍𝒚𝒔𝒊𝒔
𝑪𝟏𝟐 𝑯𝟐𝟐 𝑶𝟏𝟏 + 𝐇2𝐎 → C6H12O6+C6H12O6
Sucrose Glucose + Fructose
342 parts Sucrose produce 360 parts Reducing Sugar. Original Sucrose is
equivalent to 0.95 times the Reducing Sugar formed.

Technical Alcohol/Impurity spirit

Are more volatile alcohols than ethyl alcohol, are mainly aldehydes with
acetaldehyde being the principal component. It is the main product of Aldehyde
column with of Strength 94-95 % v/v.

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Total Reducing Sugar
Molasses contain both Sucrose and Reducing Sugar. So the original Reducing
Sugar is determined first. Then the Total Reducing Sugar after hydrolysis. The
difference between the two gives the Reducing Sugar originated from Sucrose.
Multiplying this difference by the factor 0.95 will give the original Sucrose
content.

Unfermentable Sugar
Fulfills all the tests of sugar but do not convert in to alcohol in fermentation
process.

Yeast
Yeasts are very small living organisms. They are visible only under microscope.
The yeast has a major importance in fermentation of sugar to ethanol. Alcohol
fermentation is a biochemical process by which microorganisms (yeasts) oxidize
sugars in to alcohol and carbon dioxide. Prior to fermentation, the enzyme
invertase in yeast hydrolyses sucrose into glucose and fructose (6 carbon or
invert sugars). The transformation of sucrose to alcohol can be approximated
by the equation:-
𝑰𝒏𝒗𝒆𝒓𝒕𝒂𝒔𝒆
𝑪𝟏𝟐 𝑯𝟐𝟐 𝑶𝟏𝟏 + 𝑯𝟐 𝑶 → 𝑪𝟔 𝑯𝟏𝟐 𝑶𝟔 + 𝑪𝟔 𝑯𝟏𝟐 𝑶𝟔

Yeast metabolizes the invert sugars into ethyl alcohol and carbon dioxide by
the help of enzyme Zymase, releasing heat energy in the process:

Simple stoichiometry shows that if all the fermentable sugars are converted to
ethanol and carbon dioxide, the products would be 51.1% ethyl alcohol and
48.9% carbon dioxide (by wt.). Molecular mass of C6H12O6 is 180 and that of
2C2H5OH is equal to 92.

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100 𝑥 92
= 92 , ℎ𝑒𝑛𝑐𝑒 𝑥 = 180 ∗ 100 = 𝟓𝟏. 𝟏𝒌𝒈 , where x is the weight of ethanol obtained
180

from 100kg of invert sugar (glucose and fructose).

Specific gravity alcohol=0.7934, therefore 511.1KG Alcohol=511.1/0.7934 =

644.1 liters of alcohol. During fermentation, by-products like glycerin and
succinic are formed from sugar. Therefore, actually 94.5% of the total
fermentable sugars are available for alcohol conversion. Thus one Metric ton of
sugar will give 644.19x0.945 = 608.6 liters of alcohol

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Contents of Analysis Methods page

1 Yeast Preparation and Culturing ........................................................................................................ 12

1.1. Technique for Yeast Purification and Isolation ......................................................................... 12
1.2. Preparation of Solid Media for Plating/Slant Preparation ....................................................... 12
1.3. Dilution of Yeast Sample ............................................................................................................ 14
1.4. Plating Method ........................................................................................................................... 15
1.5. Preservation of Yeast Strain....................................................................................................... 15
1.5.1. Slant Preparation on Solid Media .......................................................................................... 15
1.6. Technique /Procedure for Yeast Culture Preparation .............................................................. 17
1.6.1. Laboratory Molasses Media Preparation- ............................................................................. 17
1.6.2. Procedure for Yeast Transfer ................................................................................................. 18
2. Final Molasses Analysis ...................................................................................................................... 19
2.1. Determination of Settable sludge in final molasses ................................................................. 19
2.2. Determination of sludge content of molasses .......................................................................... 20
2.3. Final molasses brix determination (1:10) .................................................................................. 20
2.4. Analysis of Higher Brix products ................................................................................................ 21
2.5. Final Molasses POL Determination ............................................................................................ 22
2.6. Determination of sucrose in final molasses .............................................................................. 22
2.7. Determination of sulphated Ash in final molasses ................................................................... 26
2.8. Determination of volatile acids in molasses ............................................................................. 27
2.9. Determination of the Reducing sugar in the given sample of final molasses .......................... 27
2.10. Determination of Total Reducing Sugar (TRS) in Final Molasses .......................................... 31
2.10.1. Method A ........................................................................................................................ 31
2.10.2. Method B ............................................................................................................................ 32
2.10.3. Method C- TRS Determination by EDTA Method................................................................ 34
2.11. Determination of UnFermentable Sugars in Molasses ......................................................... 39
2.11.1. Method A......................................................................................................................... 39
2.11.2. Method B............................................................................................................................. 39

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2.11.3. Method C ............................................................................................................................. 40
2.12. Determination of the ratio of Fermentable to non Fermentable (F/FN) ............................. 42
2.13. Estimation of Calcium Content of Molasses by E.D.T.A Method .......................................... 42
2.14. Estimation of Calcium content of molasses by Ammonium Oxalate Method ..................... 43
3. Fermented Wash Analysis .................................................................................................................. 45
3.1. Fermented wash Brix determination......................................................................................... 45
3.2. Determining PH of fermented wash .......................................................................................... 47
3.3. Temperature Determination...................................................................................................... 48
3.4. Determination of Total Organic Volatile Acids of Fermented Wash and molasses ................. 48
3.4.1. Method A ............................................................................................................................ 48
3.4.2. Method B ............................................................................................................................ 50
3.5. Determination of Residual Sugar in Fermented Wash ............................................................. 50
3.6. Determination of Sugar Concentration by Fermentation Method........................................... 51
3.7. Yeast Cell Count .......................................................................................................................... 52
3.7.1. Cell count by Heamocytometer slide (Method A) .............................................................. 52
3.7.2. Yeast Cell by counting chamber (method B) ....................................................................... 54
3.7.3. Determination of Total Yeast Count of Fermented wash Sample (Method C) ................... 55
3.8. Operating Laboratory Centrifuge............................................................................................... 58
3.9. Estimation of Volume Percent of Sedimentation in Fermented Wash .................................... 59
3.10. Performance Test of yeast separator .................................................................................... 60
3.11. Determination of total fatty matter in Turkey red oil (Defaming agent) ............................. 61
3.12. Estimation of Sugars by Arsenomolybdate Method ............................................................. 62
3.13. Estimation of Reducing Sugars by DNSA Method ................................................................. 64
4. Various Analysis for Ethanol Alcohol & Intermediate ...................................................................... 65
Products ...................................................................................................................................................... 65
4.1. Determination of Ethanol in Fermented Wash by Sykes Hydrometer ..................................... 65
4.2. Determination of Ethanol in spent wash by Sykes Hydrometer .............................................. 66
4.3. Determination of ethyl alcohol spirit by alcoholmeter (Gay-Lussac method) v/v 20 ......... 66

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4.4. Test for alcohol loss in spent less .............................................................................................. 67
4.5. Determination of ethyl alcohol content of spirit by specific Gravity method ......................... 67
4.6. Determination of ethyl alcohol content of spirit by Sike’s glass Hydrometer ......................... 68
4.7. Determination of Ethanol by Oxidation Method ...................................................................... 70
4.8. Determination of Total, fixed, and volatile acidity (as CH3COOH) of Rectified Spirit (ISI
method) .................................................................................................................................................. 71
4.9. Determination of Aldehyde (as CH3CHO) content of Rectified Spirit (ISI) ............................... 73
4.10. Determination of Aldehyde (as CH3CHO) content of spirit (AOAC method) ....................... 75
4.11. Determination of Ester (as CH3COOC2H5) content of Rectified Spirit ................................. 77
4.12. Fusel oil determination in spirit sample ................................................................................ 78
4.13. 4.13 Furfural determination in Rectified Spirit (ISI) .............................................................. 79
4.14. Determination of Cu (Copper) in the given Rectified Spirit Sample ..................................... 80
4.15. To conduct potassium permanganate (KMNO4) test for judging the quality of spirit. ....... 82
4.16. Determination of Methyl Alcohol (as CH3OH) content of Rectified Spirit............................ 83
4.17. Reduction and Blending of the Spirit ..................................................................................... 84
4.18. Estimation of Ethanol Content by Dichromate method (Spectrophotometric) ................... 88
4.19. Potassium Permanganate Time P.P Time Monitoring .......................................................... 89
4.20. Quick Tests.............................................................................................................................. 89
4.21. Alcohol Determination by Ebuliometry ................................................................................. 90
4.22. Determination of Fermentation Efficiency of Yeast growing on molasses medium ........... 92
5. Water Analysis.................................................................................................................................... 96
5.1. Determination of Hardness in Soft Water ................................................................................. 96
5.1.1.1. Method-A ........................................................................................................................ 96
5.1.2. Method B ............................................................................................................................ 98
5.2. Determination of Chlorides in Water ........................................................................................ 98
5.3. Determination of M.O Alkalinity in Water ................................................................................ 99
5.4. Determination of TDS/Suspended Matter in Water ................................................................. 99
5.5. Residual Chlorine in Water ...................................................................................................... 100
5.6. PH Determination..................................................................................................................... 101

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5.7. C.O.D Test (Chemical Oxygen Demand) .................................................................................. 103
5.8. Total Solids ............................................................................................................................... 105
5.9. Total Alkalinity.......................................................................................................................... 106
5.10. Total Alkalinity (By Potentiometric Titration Method) ....................................................... 107
5.11. Volatile Acids ........................................................................................................................ 108
6. Reagents used in the methods for factory control ......................................................................... 109
6.1. Introduction .............................................................................................................................. 109
6.2. Reagents Preparation and standardization ............................................................................. 109
7. Annexitures ...................................................................................................................................... 124

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1 Yeast Preparation and Culturing
1.1. Technique for Yeast Purification and Isolation
To isolate and purify the yeast strain following procedure is used:
In case the liquid culture from which we wish to isolate the yeast is heavily
contaminated by bacteria, acidify it to 2.5 to 3.5 PH for 2 hour retention time then use
for isolation by plating method. Antibiotics like BENZYL PENICILLIN - 3PPM,
TETRACYCLINE - 12 PPM, STREPTOMYCIN - 3 PPM can be used.
REQUIREMENTS
 INOCULATION WIRE ONE NOS
 CONOCAL FLASK 500 ML ONE NOS
 TEST TUBES 50 ML CAPACITY 10 NOS
 SOURCE OF YEAST STRAINS YEAST LIQUID CULTURE (FERMENTERS,YEAST VESSELS,
PREFERMENTERS) DRY YEAST, RIPEN GRAPES, FRUITS ETC.
 PETRE DISHES 6 PAIRS
 COTTON ROLL 500 GRAM
 TEST TUBE STAND THREE NOS ALUMINIUM OR SS
 INOCULATION CHAMBER ONE NOS
 PRESSURE COOKER ,20 LITRE ONE NOS
 ALUMINIUM FOIL ONE ROLL
 DISTILLED WATER ONE LITRE
 ETHANOL 500 ML

1.2. Preparation of Solid Media for Plating/Slant Preparation

COMPOSITION:
 YEAST EXTRACT: 0.3%
 PEPTONE: 0.5%
 AGAR AGAR: 2.0%
 DEXTROSE: 1%
 Sterilized molasses lab culture solution as medium

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Note- in case of molasses fermentation one should use molasses medium and in case
of grain fermentation malt extract medium (3%) should be used.
COTTON PLUG

150 ML SOLID MEDIA

………….
Fig.1: 500 Ml Conical Flask
PROCEDURE- weigh the above solid media components to prepare 150 ml solid
media in a 500 ml conical flask in pre sterilized and cooled lab culture molasses
media. Maintain PH 4.2 to 4.5 with Ortho-phosphoric acid or sulphuric acid. Shake
well and close the mouth of the conical flask with cotton plug; shake well and slowly
boil the solution for 5 mints taking care of vigorous foaming which may be harmful.
Then put into autoclave for 30 minutes at 1210 centigrade and 20 psi pressure in
autoclave or pressure cooker. Remove the flask at room temperature and again warm
media on water bath to melt it into liquid state so that it can be poured whenever
required.
Then pour the media while hot into petredishes up to 1/3rd depth or 3-4 mm layer in
and under flame and cover the petre dish with upper petredish in and under flame.
All this transfer should be done in laminar flow chamber taking care that top
roof of laminar flow chamber is protected by aluminum sheet from inside to
protect the plastic sheet from melting due to heat. Cool the petre dishes. After
cooling, the media will solidify and the petre dish will be ready for plating.
COTTON PLUG

UPPER PETRE DISH

………………………………………
--------------------- ..

150 ML HOT MEDIA ----…. PETREDISH WITH SOLID MEDIA

IN 500 ML CONICAL FLASK LOWER PETRE DISH
Fig. 2:- POURING HOT MEDIA ON PETRE DISHES FOR PLATIN.

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1.3. Dilution of Yeast Sample
Take 10 pre-sterilized test tubes (50 ml capacity each) ,fill each test tube with 25 ml distilled water and
then cotton plug and sterilize in autoclave for at least 30 minutes at 121 0 centigrade and 20 psi
pressure, then allow to cool while in autoclave.

… … … … …
… … … … …
… … … … …
… … … … …
…. …. …. …. ….
…. …. …. …. ….

1 2 3 4 5 6 7 8 9 10

Fig.3: Test Tubes Filled With Sterilized Water

Now take 1 ml yeast sample from yeast sample from which yeast cells/strain has to be
isolated and proceed as under flame/laminar flow chamber-
1. Add 1 ml yeast sample to test tube No.1 and shake well.
2. Then transfer 1 ml diluted sample from test tube No 1 to test tube No.2, shake
well.
3. Then transfer 1 ml diluted sample from test tube No 2 to test tube No.3, shake well.
4. Then transfer 1 ml diluted sample from test tube No 3 to test tube No.4, shake well.
5. Then transfer 1 ml diluted sample from test tube No 4 to test tube No 5, shake well.
6. Then transfer 1 ml diluted sample from test tube No 5 to test tube No 6, shake well.
7. Then transfer 1 ml diluted sample from test tube No 6 to test tube No 7, shake well.
8. Then transfer 1 ml diluted sample from test tube No 7 to test tube No 8, shake well.
9. Then transfer 1 ml diluted sample from test tube No 8 to test tube No 9, shake well.
10. Then transfer 1 ml diluted sample from test tube No 9 to test tube No10, shake
well.

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1.4. Plating Method
Now transfer 2-3 ml of diluted yeast solution from test tube no-1, 3,5,7,8 and 10 to 6
petre dishes respectively in laminar flow chamber under flame. Spread the liquid over
solid media and pour out the excess liquid carefully with partial covering of upper
petredish. Put all six petredishes in inverted position at 32-33 degree centigrade in
yeast chamber for 3-4 days. After 3-4 days cream colored 1 to 3 mm diameter yeast
colonies developed.

……….
Test tube
With diluted yeast ………………………………………
Sample ………………………………………
..

..
Adding diluted yeast sample inverted petre dish
Over solid media on petre dish

AFTER 4 DAYS YEAST COLONY

……………………………………………………………………………………………….

Fig4. Petri dish with healthy yeast colony

1.5. Preservation of Yeast Strain

1.5.1. Slant Preparation on Solid Media

COTTON PLUG
250 ML SOLID MEDIA

………….

Fig5. 1000 ML CONICAL FLASK

PROCEDURE- Weigh solid media components as given in yeast purification and isolation procedure to
prepare 250 ml solid media in a 1000 ml conical flask in pre sterilized and cooled lab culture molasses
media. Maintain PH 4.2 to 4.5 with Orthophosphoric acid or sulphuric acid. Shake well and close the

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mouth of conical flask with cotton plug shake well and slowly boil the solution for 5 mints taking care of
vigorous foaming which may be harmful. Then put into autoclave for 30 minutes at 1210 centigrade and
20 psi pressure in autoclave or pressure cooker. Remove the flask at room temperature and again warm
media on water bath to melt it into liquid state so that it can be poured whenever required. Then pour
the media while hot into 25 test tubes (50 ml capacity each) in and under flame. All this transfer should
be done in laminar flow chamber taking care that top roof of laminar flow chamber is protected by
aluminum sheet from inside to protect the plastic sheet from melting due to heat. Then while hot
media in test tubes, put test tubes in an inclined position as given in following figure and allow
solidifying and cooling to 32 o centigrade.

COTTON PLUG
SOLID MEDIA
250 ML HOT MEDIA IN SLANT POSITION
IN 1000 ML CONICAL FLASK
… 50 ML TEST TUBES
……….
…
…
.
Fig6. Solid Media
Now choose healthy colony of yeast and put it on wire loop previously sterilized on flame and transfer
to the solid media in test tube as shown in the following figure. All this operation must be done in and
under flame-

(INOCULATION)
TRANSFER OF YEAST COLONY TO SOLID MEDIA
IN TEST TUBES

………………………………………
.. …
……….
…
Yeast colonies on solid media
…
in petre dishes
.
Fig7. Yeast Strain (Mother Slant) Preserved On Solid Media in Test Tube

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Now put the yeast strain inoculated test tube at 32 degree centigrade in laminar flow chamber for 3
days till yeast is grown on solid media and then preserve the yeast slant so made in refrigerator at less
than 4 degree centigrade.
The procedure can be repeated by inoculating the yeast strain from slant into liquid media of desired
brix/alcohol concentration and temperature and again plating and preserving to solid media in test
tubes.

1.6. Technique /Procedure for Yeast Culture Preparation

1.6.1. Laboratory Molasses Media Preparation-

Prepare 10 liter molasses solution of 1.055 specific gravity or 14.0 brix in water and maintain its PH 4.5-
4.8 by sulphuric acid, then heat this solution till boiling then cool and decant off the supernatant liquor,
then add 10 gram urea, 1.5 gram magnesium sulphate, 5.1 gram zinc sulphate, 30 gram yeast extract
and Orthophosphoric acid to maintain ph 4.0-4.2. All solid ingredients should be added after proper
dissolution in water. Now transfer this solution to 10 litre,5 liter, 1 liter and 250 ml conical flasks with 5
litre,2.5 litre,0.5 liter and 125 ml volume of solution respectively.500 ml solution should also be taken in
a 1 liter flask for use in test tube culture and slant and plating use. Cotton plugs all flasks. Then wrap
with aluminum foil.

S.N SIZE OF FLASK QUANTITY OF MEDIA

1. 50 ML TEST TUBE 15 ML LIQUID MEDIA
2. 250 ML CONICAL FLASK 125 ML LIQUID MEDIA
3. 1000 ML CONICAL FLASK 500 ML LIQUID MEDIA
4. 5000 ML FLAT BOTTOM FLASK 2500 ML LIQUID MEDIA
5. 10000 ML FLAT BOTTOM FLASK 5000 ML LIQUID MEDIA

Now sterilize the above solutions by autoclaving in autoclave at 121 degree centigrade at 20 psi
pressure. In case autoclave is not available then boil the solution for 15 minutes, cool to normal
temperature and put flasks in yeast culture chamber under ultraviolet light maintaining yeast culture
chamber temperature 33-34 degree centigrade.

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1.6.2. Procedure for Yeast Transfer
NOTE: All yeast transfers will be done in bacteria free atmosphere in Laminar Flow Chamber or Under
Flame in a well cleaned Yeast Chamber.
Now take one slant add to it 15-20 ml sterilized and cooled above molasses media, put for 12 hrs at 32-
33 degree centigrade temperature After 12 hours transfer the yeast.

………. 15 ml Cotton PLUG

Sterilized Liquid
Molasses Media
AFTER 12 Hrs AFTER 12 Hrs
….. AFTER 12 Hrs 125
….. ML To 500 Ml Flask

Yeast Slant Yeast Slant with Liquid Media 250 ml Conical Flask
TEST TUBE
Fig8. Transferring yeast slant to liquid media for culturing

Culture of test tube into 250 ml conical flask which has 125 ml sterilized and cooled media. Incubate it
for 12 hours at 33-34 degree centigrade. After 12 hours transfer the yeast culture of 250 ml conical flask
into 1000 ml conical flask which has 500 ml cooled sterilized liquid media, incubate it for 12 hours at 33-
34 degree centigrade.
Cotton PLUG

From 250 Ml

Conical Flask FROM 1000 ML Conical Flask AFTER 12 HRS

500 ml AFTER 12 Hrs Transfer TO 10 lit
LITRE FLASK
Media
2500 ML Media
1000ml Conical Flask

5000 ML Flat Bottom Flask

Fig9. 500ml and 2500ml culture media

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After 12 hours transfer the yeast culture of 1000 ml conical flask into 5000 ml flat bottom flask which
has 2500 ml cooled sterilized liquid media, incubate it for 12 hours at 33-34 degree centigrade.

Cotton PLUG

Then after 12 hours, transfer the yeast

Culture of 5000 ml flat bottom flask
Into 10 liter flat bottom flask which
From 5000 ml flask has 5000 ml cooled sterilized media
Incubate it for 12 hours at 33-34 deg
Centigrade.
………………………………………
……………………………………….
5000 ML MEDIA
……………………............

Fig10. 5000ml culture media in 10 LITRE FLAT BOTTOM FLASK

Then after 12 hours transfer the yeast culture from 10 liter flask to yeast vessel no 1 which has cooled
and sterilized molasses media.

2. Final Molasses Analysis

2.1. Determination of Settable sludge in final molasses
Apparatus
 Graduated cylinder 250ml
 PH meter
 Hot stove
Reagent
 Sulfuric Acid
Procedure
 Take 100gm molasses and dilute it with 200ml water.

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 Adjust the PH4.5-4.8 by adding 10% H2SO4 acid.
 Heat up to 70 for 15minutes.keep all this mass in 250ml graduated
cylinder for settling and note down settling rate every ½ hour.

2.2. Determination of sludge content of molasses

Apparatus
 Graduated cylinder 1000ml
 Water bath
Procedure
 Prepare 1liter media of molasses with 15 brix.

 Adjust the PH 4.5 – 4.8. Sterilize and cool to room temperature.

 Put in one liter graduated cylinder for 10 hours.
 Note down the settling volume.
2.3. Final molasses brix determination (1:10)

Procedure
a) Weigh out 200grams of final molasses sample in a can and dilute
with 1800 grams of distilled water. Then mix well the diluted solution
until it mixes completely.
b) Add the prepared solution to a cylinder having a capacity of about 1
½ liters and immerse the hydrometer spindle dipper into the solution
and wait until the hydrometer stays at rest.
c) Allow sufficient time for the brix hydrometer to reach the same
temperature as the solution.
d) Read uncorrected brix at the lower meniscus of the fluid.
e) Red the temperature of the solution from the end-closed thermometer
on the brix hydrometer.

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Calculation
Example
o Uncorrected brix reading = 9.9
o Temp of the solution = 30

o Temp correction = 0.63

o Corrected brix (0.99 + 0.63) x10 = 105.3

2.4. Analysis of Higher Brix products

Brix Determination:
The determination of brix by the brix hydrometer is restricted to the limit of 30

brix. Above this value, the viscosity becomes too high and the following
problems are caused:-

 The floating hydrometer reaches very slowly or not at all to its

equilibrium.
 The air will not escape resulting in reading of a too low brix
hydrometer.
Therefore, concentrated liquid will be diluted since brix figures are percentage
of weights; the dilution therefore has to be a ratio of weights and not of
volumes.
The usual dilutions are 1:1, 1:5, 1:6 and 1:10. Therefore when using the brix
hydrometer, the samples are diluted as follows
1:3 = 600grams sample + 1200 grams water
1:5 = 400 “ “ + 1600 “ “
1:6 = 400 “ “ + 2000 “ “
1:10 = 200 “ “ + 1800 “ “

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2.5. Final Molasses POL Determination

Procedure
a) Take out 100ml diluted F-molasses solution in 100ml volumetric
flask from the solution prepared for brix determination
b) Add 2 -3 grams of lead acetate and mix the solution well.
c) Filter through what man No. 91 filter paper by covering funnel with
glass to minimize evaporation of the solution.
d) Discard the 1st 2 – 3 ml of the filtrate and collect polarization.
e) Rinse 200mm pol tube at least three times with the filtrate. Fill the
pol tube with filtrate checking that the liquid column should be free
from air bubbles and then polarize it.
Calculation
Example

 Uncorrected pol reading = 14.3

 Reading brix hydrometer = 9.9
 Corrected pol from schintztable = 3.59
 % pol of final molasses (3.59x10) = 35.9
 Corrected final molasses brix = 105.3
 Purity of final molasses = 3.59 x 100 = 34.1%
105.3
2.6. Determination of sucrose in final molasses

(Jackson and gill’s method)

Apparatus
 Beaker- 1 liter volume
 Funnel- plastic steam less, 16cm

 Watch glass- large enough to cover abase funnel.

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 Erlenmeyer flask- wide mouth, 500ml
 Fitter paper -what man No. 91
 Volumetric flask- 100ml
 Water bath- thermostatically controlled at 60± 1 ℃
 Saccharometer
 200ml pol tube
 Clerget thermometer

Reagents
 Horne’s basic lead acetate
 Kieselguhr filter aid
 Potassium Oxalate
 Sodium Chloride, 6.34 HCI
Procedure
Preparation of sample (10% w/w solution)

a) Weigh out 100 grams of molasses sample in a beaker and add

distilled water to total weight of 1000 grams
b) Mix thoroughly and homogenize
c) Determine the refractometer brix as follows.
 Take out 200ml of 10% w/w solution; add about 2 grams filter aid
and filter through a fluted what man No 91 filter paper or
equivalent.
 Reject the 1st ml of filtrate and collect sufficient filter in a beaker
 Determine the refractometer brix as close to 20 as possible.
Brix % molasses = Refractometer reading x 10
Clarification of prepared sample
a) Take out about 400ml of the 10% w/w solution to 1 liter Erlenmeyer
flask.

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b) Add minimum of dry lead acetate (about 4 grams/100ml) shaking
vigorously.
c) Filter through a fluted what man No. 91 filter paper using filter paper
aid if necessary. Cover the funnel with watch glass to minimize
evaporation.
d) Discard about 200ml in an Erlenmeyer flask

De leading of clarified sample

e) Add 5 grams of dried potassium oxalate to a 200ml of the above
filtrate. Swirl to precipitate any excess lead present.
f) Filter thorough a fluted what man No. 91 filter paper covering the
funnel with watch glass to minimize evaporation.
g) Discard the 1st 25ml of the filtrate and collect the rest.
Polarization and inversion
h) Take out two volumetric flasks of 100ml and pipette 50ml of the clear
filtrate to each flask and add 20ml distilled water.
i) To the 1st flask add 10ml sodium chloride solution from the pipette.
Mix well and keep aside.
Inversion
j) Take the 2nd flask and add 10ml standard HCL acid.
k) Swirl gently to avoid local concentration of the acid.
l) Insert a thermometer in to the flask and place the flask in to a hot
water bath at 60 1 .

m) Agitate the flask in the water bath for the 1st three minutes, then
allow to stand for a further seven minutes.
n) After a total of 10 minutes from the moment the content reach 60 ,

remove the flask from the water bath and cool rapidly under cold
running water.

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o) Remove the thermometer from the flask rinsing it carefully into the
flask with water.

Polarization
p) Collect both flasks and make up with distilled water to 100ml mark.
q) Shake well and polarize both solutions on 200mm pol tube at 20

and measure the temperature of the invert solution in the pol tube.
r) Ensure that the temperature of the two solutions do not differ by
more than 0.5 .

Note:- Note the polarimeter readings and over switch to the standard weight
for pol.

Calculation
Sucrose % molasses = 2 (P – P1) x 100 x pol factor
C – 0.53t
Example
o Brix of 1:10 w/v solution at 20 = 8.70
o P = pol before inversion (1st flask) = 6.47
o P = “ after “ (2nd flask) = 1.55
o T= Temperature of invert solution = 21.3
o From the table of Clerget divisor, at brix of 8.69 , C = 142.48

o “ “ “ of pol factor, at Bx, 8.69 = pol factor = 0.25200

 Therefore sucrose% molasses = 2 x [(6.47 – (-1.53)] x 100 x 0.25200
142.48 – 0.53 x 21.3

= 1604 x 0.25200
131.83
= 3.066 x 10
= 30.66%

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2.7. Determination of sulphated Ash in final molasses

Apparatus
 Platinum porcelain crucible (80 – 100ml)
 Analytical balance
 Desiccators
 Muffle furnace

Reagents
 Sulphuric acid

Procedure
a) Heat the crucible in the furnace at 650 for 30 minutes, then remove

and place in the desiccators for 1 hour.

b) Weigh the crucible on analytical balance and record the mass to
0.0001g
c) Place 5g of homogenized molasses in the crucible and record the mass
to 0.0001g.
d) Add 5ml concentrated sulphuric acid to the molasses.
e) Heat gently (in a fume cupboard) over a Bunsen burner until
carbonized.
f) When fuming has ceased place in the muffle furnace with free access
of air for one hour or until ashes.
g) Remove the crucible and allow to cool in protected place to prevent
draught blowing the ash away
h) When cool, add a few drops of sulphuric acid to the moisten ash
thoroughly.
i) Heat gently (in a fume cupboard) over a Bunsen burner.
j) Ignite the muffle furnace at 650 for one hour.
k) Remove the crucible. Cool in a desiccator (1 hour).

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l) Weight to 0.0001g
 Sulphated ash% molasses = Mass of ash x 100
Mass of molasses
Example

 Mass crucible + molasses = 65.4552g

 Mass of empty crucible = 60.4320g
 Mass of molasses sample = 5.0032g
 Mass of crucible after ashing = 61.1759g
 Mass of crucible (empty) = 60.4320g
 Mass of ash = 0.7439g
 Sulphated ash % molasses = 0.7439 x 100 = 14.865
50032
a. Report As 14.87%

2.8. Determination of volatile acids in molasses

Take 50 gram molasses sample, then dilute to 500 ml with distilled water, then
take 100ml of this solution and add 5ml conc. H2SO4 and 100ml water then
collect 100ml distillate and titrate with 0.1N NaOH solution.

Volatile acids in PPM = 60000 x 100 x 0.1N x 10 (dilution x volume of 0.1N NAOH used
70x100 (ML of sample taken for distillation)

2.9. Determination of the Reducing sugar in the given sample of final

molasses

Reagents
a. Neutral lead acetate solution (10%)
Dissolve 100gms of lead acetate in distilled water and make the solution up to
one liter

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b. Disodium phosphate, potassium oxalate solution
Dissolve 70gm of disodium phosphate (Na2 HPO4 2H2O) 30gms of potassium

oxalate (K2C2O4H2O) in water and make the solution up to one liter

c. Fehling’s Solution ‘A’ (Eynon and Lane)

Dissolve 34-64gms of copper sulphate (CUSO45H2O) in water and make up to
500ml

d. Fehling’s Solution ‘B’ (Eynon and Lane)

Dissolve 173gms of sodium potassium tartarate in approximately 300ml of

distilled water. Dissolve separately 50gms of sodium hydroxide (c.p.) in 50ml
of distilled water. After cooling this, add it to the solution of sodium potassium
tartarate and make up to 500ml mark with distilled water.
e. Methylene blue solution
Dissolve 0.2gms of pure methylene blue in water and make the volume up to
100ml

f. NaOH (6N)
1Normal NaOH = 40gram NaOH in 1000ml
6N NaOH = 6x40 gm. in 1000ml
= 240 gm. in 1000ml
To neutralize the acid after inversion add 10ml of 6N NaOH

g. Concentrated HCL

h. Standardization of Fehling’s Solution

Dissolve 9.5gram of sucrose in 75ml of distilled water using a measuring flask

of one liter capacity. Add 5ml of conc. Hydrochloric acid (Sp.Gr. 1.19) into the
same and leave over the mixture for about 24 hours. Then carefully neutralize
the solution adding sodium hydroxide solution, drop by drop until the reaction
is very weakly acidic. To perform the same accurately, one actually titrates

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separately 5ml of the hydrochloric acid used for inversion with a four percent
sodium hydroxide solution using methyl orange as indicator. If 45ml of the
sodium hydroxide solution is required for the neutralization of 5ml of CONC.
HCL then add only 45ml of sodium hydroxide to the invert sugar solution.
After the neutralization, make up the volume to the one liter mark. Invert
sugar solution prepared in this manner can be kept only for about five days
after which a fresh solution should be prepared again for standardization of the
Fehling’s solution.
Take 100ml of the invert sugar solution in a measuring flask of 500ml capacity
and make up the volume with water to 500ml mark.
One milliliter of this diluted solution contains;
9.5 x 20 x 100x 1_ = 2mg of invert sugar
19 500 1000

If the Fehling’s solution has been accurately made, 10ml of the same would
require 25.64 ml of invert sugar solution. In most cases the Fehling’s solution
prepared will not be very accurate and the factor should be used to get the
correct figure.
 The Factor will be = 25.64
a
Where;
‘a’ is the number of milliliters of invert sugar solution (Containing 2gm/ml)
used for the titration of the Fehling’s solution.
Procedure

Weigh accurately 12.5gms of final molasses and dilute it carefully without any
loss with approximately 100ml water. Put the diluted solution into 250ml
volumetric flask. Add 25ml of undiluted 10% lead acetate solution for
clarification purpose. Mix well and make up the volume to the 250ml mark
with distilled water. Filter after through mixing. Transfer, 50ml of the clear

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filtrate to a 250ml volumetric flask and add 10ml of disodium phosphate,
potassium oxalate solution. Make up the volume to the 250ml mark and filter
after through mixing. Use this filtrate for titration against Fehling’s solution
according to the method of Eynon and lane.

Titration Procedure;
Measure out 5ml of Fehling’s “A” and 5ml of Fehling’s “B” accurately into a
150ml Erlenmeyer flask. Fill 50ml of the filtrate into a burette. Put the
Erlenmeyer flask with the Fehling’s solution on a hot stove and pour 15ml of
the clear filtrate from the burette to the Erlenmeyer flask after addition of few
glass beads into the Erlenmeyer flask. Let the liquid boil for 15 seconds and
observe from the blue color of the solution whether the major part of copper
has been precipitated or not. If this is the case, add one milliliter of methylene
blue solution and again boil the liquid for two minutes. Add small quantities
(approximately half a milliliter) of the sugar solution and boil the liquid for
about ten seconds after each addition. Continue this until the color of the
indicator disappears (Total time of boiling being three minutes).
In case, on the addition of 15ml of sugar solution, in the beginning of the
titration and after boiling for 15 seconds, if most of the copper has not been
precipitated, add 10ml of the filtrate solution in one lump and boil for 15
seconds, and continue the process as before until most of the cupric salt has
been reduced. After this add the indicator and boil the liquid for one to two
minutes and titrate further. Keep in mind that it is necessary to add the
indicator more or less at the end of the reaction in order to obtain a good and
sharp change of color of the indictor. The change of color is from very deep blue
to the olive green and finally to the red color of the cuprous oxide. The change
of the color always shows clearest at the surface liquid against the sides of the
Erlenmeyer flask.

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Observation:- Titration Reading B.R

1 For Fehling’s factor BR __________ ml

2 For R.S in molasses BR __________ ml

Calculations
𝟐𝟓. 𝟔𝟒
𝑭𝒆𝒉𝒍𝒊𝒏𝒈 𝒇𝒂𝒄𝒕𝒐𝒓 =
𝒂

10ml of Fehling’s solution = 0.05128gms of R.S

Reducing sugar % = 0.05128 x 100
B.R x Fehling’s factor x dilution factor
Where:-
 Fehling’s factor = 1.005
 Dilution Factor = 0.01
Results
 DF = 12.5 x 50 x 50

250 250 100
 1.Fehling’s factor for given solution =

 2.Reducing sugar % in the given molasses sample =

2.10. Determination of Total Reducing Sugar (TRS) in Final Molasses

2.10.1. Method A

Reagent:-
 5N NaOH
 HCL 36%
 Phenolphthalein Indicator
 Fehling’s Solution A
 Fehling’s Solution B
 Methylene Blue 0.5%

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Procedure:-
Take 50 gram Molasses Sample
Dissolve in 450ml of water and check brix by hydrometer. Then make up
volume to 500ml and mix well. Now take 10ml in 100ml volumetric flask and
add 5ml of HCL and 20ml of water. Then heat in water bath to 69 degree for
(10-15min).Now cool it to normal temperature and neutralize with 5N NaOH
solution. Use phenolphthalein indicator 2-3 drops. End point color will be
pink. Now make up its volume to 100ml with water. Now titrate with 5ml of
Fehling solution A and 5ml of Fehling solution B, and add 20-25ml of water
using methylene blue indicator. End point just brick red color appears.
Calculation:-
TRS% = Fehling Factor x Dilution x 100
Titration value in ml

Determination of Fehling Factor

Prepare 1% w/v solution of dextrose (pre dried at 105 degree centigrade for 1
hour) then titrate this solution with 10ml Fehling solution (5ml Fehling A+5ml
Fehling B) add 15ml water using methylene blue (0.5% in water) as indicator.
Add solution through burette while Fehling solution on boiling. A brick red
color appears at the end point.
Fehling factor = Titration value in ml/100

2.10.2. Method B

Reagents
 Neutral lead acetate solution (10%)
 Disodium phosphate potassium oxalate
 Fehling’s ‘A’ and ‘B’ solution
 Methylene blue solution

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 NaOH (4%)
 Concentrated HCL
Procedure
Weigh accurately 12.5gms of final molasses and put into 250ml. volumetric
flask with the addition of distilled water. Add 25ml of undiluted 10% Neutral
lead acetate solution for clarification purpose. Make up the volume to the
250ml mark with distilled water and filter after through mixing. Take 50ml of
the filtrate in a 250ml volumetric flask and add 10ml disodium phosphate
potassium oxalate solution and filter after through mixing. Take 50ml of the
clear filtrate in a 100ml volumetric flask and add about 5ml. CONC. HCL acid
and heat to 60 in a water bath for 10-12 minutes. Cool to room temperature

and neutralize with sodium hydroxide solution, using phenolphthalein as

indicator. Make to 100ml mark with distilled water. Rinse and fill the burette
with solution and titrate with 10ml of Fehling’s solution (5ml Fehling’s ‘A’ and
5ml Fehling’s ‘B’) as usual using methylene blue as indicator. End point is the
change of color from very deep blue to olive green and finally to the red color of
the cuprous oxide.
The Fehling’s solution used has to be standardized firstly with a solution of
invert sugar containing 2gm of invert sugar per ml.

Calculation
To calculate total reducing sugar in the final molasses 10ml of Fehling’s
solution = 0.05128gms of R.S

 Total sugars as invert sugar = 0.05128 x 100

B.R x F.F x D.F
Where
 F.F = Fehling’s factor = 1.005
 D.F = Dilution factor = 0.005 x 12.5 x 50 x 50
250 250 100

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Result
The total sugars as invert sugar in final molasses %

2.10.3. Method C- TRS Determination by EDTA Method

I. Scope
The method is applicable to all samples of molasses. It may also be applied to
certain sugar syrups.
II. Field of Application
This method, which may form the basis of molasses purchasing contracts,
measure the total reducing sugars, as invert sugar, after hydrolysis of molasses
samples. It cannot be used for determining true sucrose by subtracting initial
reducing sugars and multiplying by 0.95. Where either this practice or the
direct multiplication of the total reducing sugars after hydrolysis by 0.95 to
obtain ‘Sugar’ (Sucrose) is employed, the result so obtained is only to satisfy a
commercial requirement. It is not a precise measurement of sugar (Sucrose).
The method is also used to determine ‘Sucrose’ plus ‘invert’ in refined syrups
by determining the total reducing sugars after hydrolysis. The result is usually
converted to ‘sucrose %’ by multiplying the total reducing sugars figure by
0.95.
III. Definitions
Reducing sugars: - Reducing sugars in molasses after hydrolysis are Primarily
but not exclusively, glucose and fructose.
Invert: - An equimolar mixture of glucose and fructose.
IV. Principle
The method relies upon the property of reducing sugars to reduce Fehling’s
solution under standard conditions.
The volumetric method of lane and Eynon is well known to the sugar industry
and is simple and capable of giving reproducible results under standard

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conditions. Some non-sugars, in particular calcium, can influence the results
of this determination possibly forming a complex with glucose and fructose.
This effect is eliminated by sequestering the calcium in molasses with EDTA.
With refined syrup, No need to add EDTA.
V. Reagents and Materials
Use only distilled water; Reagents should be of analytical grade or better.
a. Hydrochloric acid, concentrated, d20 1.18g/ml
b. Benzoic acid- general purpose reagent grade
c. Methylene blue solution, 1g/100ml
Dissolve 1gram of methylene blue (pure) in water and make up to a
volume of 100ml.
d. Sodium hydroxide solution, approx 2mol/L
Dissolve 80gram of sodium hydroxide in approximately 500ml of
water. Cool the solution, transfer to 1liter volumetric flask and
make up to volume with water.
e. Sodium hydroxide solution, approx., 1mol.L
Dilute the 2 mole/L reagents (5.4) 1:1 v/v
f. Hydrochloric acid, 6.34 mole/L, d20 1.1029 g/ml
Dilute 630 5ml of hydrochloric acid, d20 1.18g/ml to 1 liter with

water and adjust to exactly 6.34 mol/L after titrating 5ml with
1mol/L NaOH using methyl orange indicator.
g. Hydrochloric acid, approx., 0.5 mol/L.
Dilute 44.5 ml of the concentrated acid (5.1) to 1L.
h. Solution of disodium salt of EDTA, 40g/L.
Dissolve 20 gram of the salt in water and make the volume up to
500ml.

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VI. Apparatus
a. Volumetric glass ware - class A
b. Thermometers
c. Hot titrate /illuminator
d. Analytical balance – readable to 1mg
e. Weighing dish
f. Timer (1hour) – indicating minutes and seconds
g. Boiling flasks, Pyrex – round form or similar design 400 ml
Capacity.
h. Filter funnel
i. Water bath – maintained at 60 1 and 20 1

j. Lead rings - or other weighing devices to maintain inversion flask in

a fixed position in the water bath.

Procedure
Glass ware:-The internal surfaces of all volumetric glassware’s should be
occasionally cleaned with chromic acid mixture. The use of a special
laboratory detergent reduces the frequency of the need for chromic acid
cleaning.
Heating conditions:- The heating should be arranged so that 75ml of Water
contained in the 400ml boiling flask will Reach boiling point from 200C in
2.5min.
VII. Preparation of hydrolyzed molasses solution
First prepare a 50g/L molasses solution. Weigh as rapidly as possible into a
clean weighed beaker, 10 0.2 gram of sample and record the weight to the

nearest 0.001gram.

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Add approx. 15ml of water to the sample and mix thoroughly with a small glass
rod. Transfer the solution, without loss, to a 200ml volumetric flask. Rinse
the rod and beaker with successive additions of water and add the washings to
the flask contents.
Add sufficient water to bring the volume up to approximately 90ml and swirl
the flask to mix the solution. Finally make the volume up to 200ml at the
approximate temperature and mix to give a 50g/L solution of molasses.
Pipette 25ml of the 50g/L molasses solution in to a 250 mL volumetric flask.
Add 5ml of the 6.34 mole/L hydrochloric acid from a 25ml burette agitating the
solution gently to prevent local acid concentration in the solution. Then
immerse the flask in the water bath at 60 and swirl gently for 3 min to raise

the temperature of the acidified molasses as rapidly as possible. Place the

weighting device (lead ring) around the neck of the flask and allow to stand in
the water bath for a further 12min. During the standing period do not allow
the flask to contact any part of the heating surface of the water bath. A
suitable spacing arrangement to prevent this occurrence should be in corporate
into the water bath.
Remove the flask from the water bath and after taking off the weighting device,
cool the contents rapidly by holding the flask in cold running water. Dilute the
solution to approximately 125ml and add a few drops of phenolphthalein
solution. Then add sufficient 2mol/L sodium hydroxide solution to impart a
reddish color to the solution. During the alkali addition the solution should be
gently agitated.
Discharge the red color of the solution by the addition of a few drops of
0.5mol/L hydrochloric acid.

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Add 4.0 ml of EDTA solution mix the solution well and make up to volume with
water at 20 and mix. This solution is equivalent to 0.5 gram molasses per

100ml.
Sample titration
Pipette 20ml of Fehling’s solution in to the boiling flask. Run in 20ml of the
hydrolyzed molasses solution and add a small quantity of pumice powder.
Place the flask on the heating appliance. Allow the solution to reach boiling
point and add 4 drops of the methylene blue solution proceed with the titration
by initially adding increments of 2ml of molasses solution and progressively
reducing the addition down to 0.2ml and attempting to obtain the end point in
about 1 min from the time the solution commences boiling. The end point is
denoted by the disappearance of the blue color of the dye and the solution
assumes a faint pink color imparted by the precipitated cuprous oxide. Record
the titer (T).

VIII. Expression of Results

Calculation
The total reducing sugars concentration after hydrolysis in the molasses is
given by:-
 % total reducing sugars (as invert sugar) = 1000
CxT
Where:
 C = concentration of molasses test solution in gram per 100ml
 T = T1 = Titer (ml of molasses solution used)
if C = 0.5g/100ml, then % total reducing sugars = 2000
T
The % total reducing sugars normally lies between 50% and 60% in molasses.
The use of a 0.5g/100 ml hydrolyzed molasses solution allows for % total
reducing sugars in the range 40% (titer = 50ml) to 80% (titer = 25ml)

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Precision
The absolute difference between two results obtained under repeatability,
conditions should not be greater than 1.03% TRS in molasses other than cane
molasses and 1.51% for cane molasses.
The absolute differences between two results obtained under reproducibility
conditions should not be greater than 4.38% TRS in molasses other than cane
molasses and 2.47% for cane molasses.

2.11. Determination of UnFermentable Sugars in Molasses

2.11.1. Method A
Take 50 gram molasses add 20 gram dry yeast then make up volume to 500ml
with water and keep for 24 hours at 32 degree centigrade, then after
fermentation is complete centrifuge and titrate the solution with 10ml Fehling
solution (5ml Fehling A+5ml Fehling B) and 15ml water using methylene blue
(0.5%in water) as indicator. Add solution through burette while Fehling
solution in boiling. A brick red color appears at the end point.
Unfermentable sugars% = Fehling factor x dilution (10) x 100
Titration value in ml
Fermentable sugars = T.R.S – U.F.S

2.11.2. Method B
Apparatus
 Graduated cylinder 500ml
 Test tube 20ml
 Conical flask 500ml
 Hot stove
 Burette
 Erlenmeyer flask 150ml
Reagents/Chemicals

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 Dry Yeast or Beaker yeast
 Fehling’s “A” and “B”
 DAP
 Urea

Procedure
Take 100gm of molasses and dilute it with 400ml water. Add 2gm urea and
1gm Dap and make sure it is dissolved.
Adjust the PH of the media 4.5 – 4.8 PH. Take 10ml in the20ml test tube. Add
1 gram Beaker yeast in to the test tube. keep this for fermentation for 24 hrs.
Transfer the 390ml molasses media into 500ml conical flask. After 24hours,
transfer 10ml volume from the test tube into the 390ml media.Keep it in the
incubator at a temperature of 32-34 for 24 hours. After 24 hours make up

the volume to 500ml and do leading and de leading acording to Lynen and
Lynen procedure.

Take 50ml from 500ml dilute it to 100ml and titrate with Fehling “A” and “B”
solution.

Calculation:-
Unfermenatable sugar % = 5.128
BR x FF x DF

 Fehling Factor (FF) = 1.005

 DF = 0.1 x 100 x 400 x 50
400 500 100
 DF=Dilution Factor

2.11.3. Method C
Apparatus
 250ml conical flask
 250ml volumetric flask

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 Burette, pipette, distillation unit, etc………
Reagents
 Fehling’s “A” and “B”
 H2SO4 acid
 Urea, Dap
 Yeast
 Methylene blue indicator, etc. …..

Procedure
Take 25 grams of molasses and mix well with Distilled Water Add 2 grams of
urea and 1gram Dap to flask and adjust the PH of the flask to 4.5 with diluted
H2SO4. Make the volume of each flask to 250ml. Add 25grams of yeast to the
experimental flask. Plug it and keep it in the incubator at 32 for 24 hours.
After 24 hours, measure the volume of fermented wash in the experimental
flask, if less than that, make up the volume to 250 ml, filter the material
through ordinary filter paper. Take 25ml of the filtrate in a 100ml volumetric
flask make up the volume with water. Fill this solution in Burette and titrate
against Fehling solution “A” and “B” 5ml from each solution and use methylene
blue as indicator.
End point = Blue to brick red.

Calculation
UN Fermentable Sugar = 0.05128 x 100______
Burette Reading x FF x DF
FF = 1.005
DF = 0.025 x 25 x 25
50 100
UFS (%) = ___________
Fermentable sugars% = Total Reducing sugar - UFS

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2.12. Determination of the ratio of Fermentable to non Fermentable
(F/FN)
 TRS=Total Reducing Sugar
 RS=Reducing Sugar
 FN=Non Fermentable Sugar
 Bx=Brix of molasses

 F/FN = TRS
( Bx – (0.95 TRS + 0.05RS))

2.13. Estimation of Calcium Content of Molasses by E.D.T.A Method

Reagents:
E.D.T.A solution (Di-sodium salt of ethylene diamine tetra acetic acid) Or
Merck quality
Weigh accurately 6.6473g of E.D.T.A into beaker, dissolve the same in distilled
water and make up the solution to 1000ml to obtain exactly M/56 solution
200ml Ammonia of C.P. quality, 500gm Lead sub acetate: Horn’s dry lead,
500gm potassium Ferro cyanide (Merck quality) 100gm potassium iodide: C.P.
Quality 30ml Erichrome black T. B.D.H. Quality) weigh 0.1g of Erichrome black
T into a 100ml volumetric flask and dissolve the same in rectified spirit of
absolute alcohol. Make up the volume with the same solvent and use it as the
indicator.
Apparatus
Calibrated Brix spindle, Cylinder, Conical Flasks of 250ml, Beakers of 100 and
250ml, Funnels, Calibrated 10ml pipette.
Procedure
In case of molasses, dilute to 5 Brix. Transfer about 150ml of diluted solution
into a conical flask. Clarify the solution by basic lead sub acetate, as in Horn’s

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method. Transfer about 60ml of the clarified solution into a dry conical flask
or flask previously rinsed with the clarified solution. Add powdered potassium
Ferro cyanide little, till no further precipitate forms. Shake thoroughly and
filter. Test the filtrate for absence of lead with potassium iodide. Collect the
lead free filtrate in a conical flask, pipette out 10ml of this filtrate into a clean
conical flask, previously rinsed with distilled water and dried. Add a few 1ml of
liquor ammonia and a few drops (4-5) of the indicator solution; when a pink
red color appears, Titrate against E.D.T.A solution shaking the contents of the
flask after every addition of E.D.T.A solution. The end point is indicated by the
sharp change of color from red to olive green. Note the volume of the titrate.
Calculations
Let ‘B’ be the Brix of the solution (diluted) and ‘V’ the titrate value of E.D.T.A.
Then the amount of (Ca++ and Mg --) is expressed as
 CaO = V x 100mg/liter of juice or diluted solution

OR

= V x 100 x 100mg/Lit./100 Brix

B

2.14. Estimation of Calcium content of molasses by Ammonium Oxalate

Method

Principle
Calcium is precipitating hot in slightly acid solution as Ca-oxalate (CaCO2:H2o)

by ammonium oxalate solution and neutralized with ammonium hydroxide.

The precipitate so obtained consists of crystals, which are readily filtered. The
precipitate Ca-oxalate Hence, washing of precipitate must be done with a dilute
solution of ammonium oxalate.
Reaction

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𝑪𝒂++ + 𝑪𝟐 𝑶𝟒−𝟐 + 𝑯𝟐 𝑶 → 𝑪𝒂𝑪𝟐 𝑶𝟒 𝑯𝟐 O

During the initiation CaO4 will give CaCO3 the Ca above 500 C to 700

Reagents
5% ammonium oxalate solution, methyl red indicator, ammonium hydroxide
(1:1 diluted), 0.2% ammonium oxalate and Whitman filter paper No. 42.

Procedure
Take 10gm of molasses in silica crucible and find out the % of ash as given in
the previous experiment. Note down the weight of ash and add 2 to 3ml of
concentrated HCL to the ash in crucible. Dissolve the ash and filter through
ordinary filter paper in 100ml volumetric flask. Make the volume to 100ml and
use this filtrate for calcium estimation.
Take 100ml of sample in clean 500ml beaker and dilute it to about 100ml with
distiller water. Add two drops of methyl red indicator. Heat the solution to
boiling. To this hot solution add 5% hot solution of ammonium oxalate very
slowly with constant stirring till precipitation is completed. Mix ammonium
hydroxide drop, with constant stirring until the mixture is neutral or faintly
alkaline as shown by indicator (Red to Yellow color).
Allow the ppt. to settle down for one hour. Test the solution again for complete
precipitation with few drops of ammonium oxalate solution. Filter the
precipitate through a Whitman filter paper No. 42.

Wash the ppt. with 0.2% ammonium oxalate solution at least 5 times until the
washing does not give the test for chloride ion (by adding 2N NHO3 + AgNO3).
Dry the ppt. in oven for one hour and incinerate (Burn) the filter paper as
usual. Then transfer this in crucible. After this, set the crucible in muffle
furnace at 500 for 2 hours. Cool and weigh the crucible till constant weight
is obtained.

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Observation
1 Weight of empty silica crucible gms
2 Weight of ash taken gms
3 Weight of ash + crucible gms
4 Weight of ignited material + crucible gms
5 Weight of CaO gms

Calculations
10gm of molasses = gms of CaO
10gm of molasses = X × 10gms
Thus quantitatively;
Ca = CaO
40 = 56
56gms of CaO = 40gms Ca- -
(X x10) gms of CaO = (X x 10) x 40 Ca- -
56
Ca% in the molasses = ______________
Result: - Ca content in the given molasses sample %
3. Fermented Wash Analysis
Fermented wash samples analyzed in the laboratory include; Determination of
Brix, PH, temperature, Counting Yeast Cells; Determining Yeast cells Density
and alcohol strength in fermented wash of all fermenters. The analysis method
is the same for all fermenters. Fermented wash samples of all fermenters must
be collected from sampling line of each fermenter.
3.1. Fermented wash Brix determination
Brix determination for fermented wash is done in two methods.
a. Refractometer Brix
Apparatus
 Precision refractometer

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 Stoppard flask
 Cooling water bath
Procedure:-
a) Pour the fermented wash sample from sample can to a 100ml
flask.
b) Keep the 100ml flask in cooling water bath to bring its temperature
near 20 .
c) Switch on the precision refractometer.
d) After the sample is sufficiently cooled take out the flask from the
cooling water bath.
e) Add few drops of sample to the refractometer prism.
f) Read Brix and temperature.
g) If the Brix measurement is not made at 20 0.1 apply the

temperature correction from table.

Example
Refractomere Brix reading at 27.6℃ = 3.74, temperature correction at 27.6℃ =
0.44 from brix correction table. Therefore Brix at 20℃ = 3.74 + 0.44 = 4.18.

b. Hydrometer Brix Method

Apparatus

 Brix hydrometer (covering the required range)

 Brix hydrometer jar

Procedure

a) Fill the hydrometer jar to overflowing with Fermented Wash sample.

b) Allow the sample to stand at least 20 minutes to allow all air bubbles to
rise to surface and suspended matter settle.

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c) Insert the spindle; which must be clean and dry; in the jar and make
sure it floats freely.
d) For accurate results allow the stem to be wetted by the juice sample not
more than 5mm above the point at which the hydrometer comes to rest.
e) Take a reading after the spindle has been floating for at least 2 minutes.
f) The reading should be taken by bringing the eye level with the surface of
the liquid and noting where the meniscus intersects the scale.
g) Read the temperature immediately after the brix reading.
h) Clean the hydrometer and put it back on its stand.
i) Calculate the Brix of sample at 20 based on the hydrometer brix

reading and temperature reading using appropriate table.

Example

 Brix reading at 30 = 21.8

 Temperature correction at 30 = 0.68 table

 Corrected brix at 20°c= 21.8+0.68=22.48

Note: - The same method can be applied to determine brix of Raw and
Concentrated spent wash.

3.2. Determining PH of fermented wash

Apparatus

 PH meter
 Squat Beaker(100ml)

Procedure

a) The PH meter must be standardized with buffer solutions of PH 4, 7 and

9.

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b) No other treatment of sample is necessary
c) Rinse electrodes and beaker with a portion of the sample
d) Take sufficient sample in the beaker and immerse the electrodes in the
sample.
e) Allow the electrodes to remain in the sample for one minute before taking
the PH reading.

3.3. Temperature Determination

Temperature Readings of all fermenters and culture vessels will be taken from
the SCADA. It shall be counter checked with thermometer or other temperature
sensors as required.

3.4. Determination of Total Organic Volatile Acids of Fermented

Wash and molasses

3.4.1. Method A

Apparatus

 Round bottom flask, distillation assembly, conical flask500ml, burette,

pipette graduated, volumetric flask 250ml, glass beads, beaker 250ml.
Procedure
a. For molasses
Weigh accurately 50gm of raw molasses in a beaker, Add to it about 50ml
water. Mix and dissolve the Molasses completely and transfer carefully to a
250ml vol. flask. Rinse with 30ml distilled water. Make up volume up to mark
with distilled water.Transfer 50ml of this diluted molasses solution in 500ml
round bottom flask and adds 200ml of water and 2ml of concentrated sulfuric
acid. Put some glass beads in to the round bottom flask before boiling. Boil the
contents on heating mantle. Collect 200ml of the distillate. Transfer the

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distillate to a 500ml conical flask. Fill the burette with 0.1N NaOH. Add 2
drops of phenolphthalein indicator and titrate against 0.1N NaOH. End point
is colorless to faint pink. Note the burette reading.
b. For Fermented Wash
Take 100ml of fermented wash sample in 500ml round bottom flask and add
100ml water and 2ml of concentrated sulfuric acid. Put some glass beads into
round bottom flask before boiling. Boil the contents on heating mantle.
Collect 100ml of the distillate. Transfer the distillate to 250ml conical flask.
Fill the burette with 0.1N NaOH. Add 2 drops of phenolphthalein indicator and
titrate against 0.1N NaOH. End point is colorless to faint pink. Note the
burette reading.
Recovery Factor
To determine the recovery factor ‘f’ for a given Apparatus dilute an appropriate
volume of acetic acid stock solution to 250ml in a volumetric flask to approximate the
expected sample concentration and distill as for a sample. Calculate the recovery
factor (f).
f = a/b
Where;
 a is Volatile acid concentration recovered in distillate mg/lit and
 b is Volatile acid concentration in standard solution used mg/lit.
Calculations
Total organic volatile acids in (PPM) = B.R x N x 60000×D
Sxf

o BR = Burette Reading
o N = Normality of NaOH
o D = Dilution factor
o S = ml of sample taken
o f = Recovery factor = 0.7

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3.4.2. Method B
Take 100ml sample add 5ml CONC H2SO4 and 100ml water; then collect 100ml
distillate and titrate with 0.1N NaOH solution.

Volatile Acid in PPM = 60000 x 100x0.1N x volume of 0.1N NaOH used

0.7x 100(ml of sample taken)

3.5. Determination of Residual Sugar in Fermented Wash

Apparatus
 Measuring cylinder 5oml
 Volumetric flask 100ml
 Measuring cylinder 10ml
 Pipette 25ml
 Burette 50ml
 Glass beads
 150ml Erlenmeyer flask
 Hot stove

Reagents

 Fehling’s A and B
 Methylene Blue solution
Procedure

Take 25ml sample of Fermented Wash and put it in 100ml volumetric flask. Fill
tomark with distilled water and shake well. Take 5ml of Fehling’s solution “A”
and 5ml of Fehling’s solution “B” in 150ml Erlenmeyer flask and add 10-15ml
of distilled water to the Erlenmeyer flask. Put the Erlenmeyer flask on a hot

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stove and run about 5ml of the fermented wash from the burette to the
Erlenmeyer flask. When the solution starts boiling drop about 4 drops of
methylene blue and start titrating until the blue color changes to a faint pink
or brick red.
Calculation

Residual sugar % = Fehling Factor x Dilution

Burette Reading

3.6. Determination of Sugar Concentration by Fermentation

Method
Principle

A rapid and exact method of finding the amount of Sugar available in a

substrate is to determine the weight loss from a given volume by Fermentation.
Carbohydrates are degraded stoichiometrically according to the formula.
C6H12O6 CH3CH2OH + 2CO2
180 gram 92 grams 88gms
Glucose Ethyl-alcohol carbon dioxide
If Disaccharides or Polysaccharides provide the Fermentable carbohydrates,
water is taken up by hydrolysis, Thus:-
C12H22O11+H2O 2C6H12O6
Sucrose water Glucose Fructose
The theoretical weight loss is accordingly;
Molecular Weight of CO2 x 2
Molecular weight of carbohydrate

For Each Gram of Carbohydrate Fermented;

This gives a weight loss of 88/180gms = 0.489gm/gms of monosaccharide
fermented and 176/342 gms = 0.515gms/gms disaccharide fermented.
Apparatus

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Container closed with a fermented lock
Chemicals
Urea, Diammonium phosphate, sulfuric acid, yeasts etc.
Procedure
To 1000ml substrate containing 200gm molasses, Adjusted to PH 5.0 with
sulfuric acid, 2gm Diammonium phosphate and 10gm yeast (wet weight) is
added. Fermentation is allowed to take place in a container closed with
fermentation lock filled with concentrated sulfuric acid at 30 with agitation.

The flask is weighted at 12hrs intervals until the weight is constant. A three
double determination is carried out. The difference in sugar concentration
found when comparing the fermentation method with the official analysis
method is less than 2% (Offer and Hilden Wager 1974)

3.7. Yeast Cell Count

3.7.1. Cell count by Heamocytometer slide (Method A)

Procedure

Take 1ml sample in a 100ml measuring cylinder and add 1ml methylene blue
solution; Then makeup volume to 100ml by distilled water.Mix well. Take
heamocytometer slide and put a drop of diluted yeast culture solution on the
heamocytometer and cover with a cover slip. The viable yeast cells remains
transparent and dead cells becomes dark blue. See the viable and dead yeast
cells under compound microscope.

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Column 1 Column 2 Column 3

Column 4 Middle Column 5

1. 2

4 3

Fig11. Cell count by Heamocytometer slide (Method A)

Calculation
Column No Viable Cells Dead Cells Total Cells
1 5 NOS 1 NOS 6 NOS
2 5 NOS 0 5 NOS
3 5 NOS 1 NOS 6 NOS
4 7 NOS 1 NOS 8 NOS
5 3 NOS 1 NOS 4 NOS
Total Cells 25 NOS 4 NOS 29 NOS

Average No. of viable cells in each column = 25/5 = 5 Cells

Total no of viable cells per ML = average viable cells each column x dilution x 106
4
= 5 x 100 x 106 =
125 x 106 cells per ml
4
Average No of dead cells in each column 4/5 = 0.8 cells
Total no of dead cells per ML = Average No dead cells each column x dilution x 106
4
= 0.8 x 100 x 106
4

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= 20 x 106 cells per ml

Total Average No cells for each ML = (125+80) x 106 = 205 x 106

 % viability = 125 x 106 x 100 = 60.9%

205 x 106

3.7.2. Yeast Cell by counting chamber (method B)

Apparatus
 100ml volumetric flask
 Counting chamber (Haemocytometer)
 Pipette
 Microscope with accessories etc…
Reagents
 Methylene Blue
 Sulfuric acid
Procedure
Take 1ml of fermented wash in 100ml volumetric flask And 1ml of methylene
blue. Add 4 drops of 10% H2SO4 and make up the volume to 100ml with
distilled water. Mix well and put a drop on a counting chamber and cover it
with microscopic cover slip. Then count the cells.
 300 to 500 x 106 cell/ml of fermented Wash is the required amount

Note: Acetic Acid can be used instead of H2SO4.

Using the Haemocytometer
Count in the middle square which consists of 25 groups of 16 small squares or
we can count 5 from the middle square as is on the figure (A,B,C,D and E),
Make average of it, and multiply it by 25. Here we have to replicate our count
at least three to four times.
 Viability = Living Cell ×100

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 Viability ≥ 70% is acceptable
Total Cell
A B

C D

Figure 11: Using the Haemocytometer

3.7.3. Determination of Total Yeast Count of Fermented wash Sample (Method C)

Principle
The yeast sample is stained with methylene blue. Dead cells are stained blue
while viable cells do not take up the stain.
Apparatus
 Microscope
 Neubauer’s cell counting chamber
 10ml test tubes
 Pipette – 5ml
Reagents
I. Methylene blue solution
 Methylene blue = 0.5 grams
 95% ethanol = 15 ml
 0.2 MKH2PO4 = 235 ml

Preparation

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1. Dissolve 27.2g KH2PO4 in 1000ml flask in 500ml distilled water & dilute
to1000ml.
2. Dissolve 0.5grams methylene blue in 235ml of0.2KH2PO4.Add15.0ml of
95% Ethanol.
II. Dilution Solution
 Sucrose = 50 grams
 Ringer Powder**
 Distilled water =500ml

Note: - Make ringer solution as per directions given on the bottle.

Preparation

1. Dissolve 50 grams of sucrose and add ringer solution in 500ml distilled

water.
2. Distribute the solution in 100ml closed bottles or flask. Sterilize at 121
degree C for 15 minutes.
3. On cooling store in refrigerator or cold incubator.
4. This solution may become cloudy after sterilization, because of
precipitation of salts. Let the precipitate settle and use the clear
supernatant liquid for microscopy.
Procedure

1. Put 4.5ml diluted solution in a test tube.

2. Add 0.5ml of representative yeast sample and mix well.
3. Take 0.5ml of the diluted sample (Prepared in above procedure no. 2) and
mix well with 4.5ml of methylene blue solution in a clean test tube.
4. Put one drop of this diluted sample over each area of Neubauer’s
counting chamber and put the cover glass on.
5. Allow cell to settle for 3 to 5minutes.

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6. Count the total number of yeast cells in five of the twenty five ‘r’ squares
of the ‘R’ square;
7. Chose four ‘r’ squares at the corners and one in the center for a
representative count;
8. Count total no of cells.
“w” Square

0.25

W W
1mm 0.25

0.25

0.25

0.0625mm2or 1/16mm2
Vol 1/16 x 1/10 = 1/160mm3

‘R’ SOUARE

1mm

0.25

W W 0.25

0.25

0.25

1mm 1mm 1mm ‘r’ Square

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Depth: Distance between cover slip & chamber Vol. 1/25 x 1/10 =
1/250mm3 ‘r’ Square = 0.04mm2
square or

Figure12: Cell Count Using Neubauer’s Chamber 1/25mm2

Calculations

 Cell number = Average number of yeast cells in an ‘r’ square x chamber factor x
dilution factor
 Average number of yeast cells in an ‘r’ square = Total number of yeast cells in 5 ‘r’
squares/Five
 Chamber factor = 25 x 106
 Dilution factor = 100
Note:- Flocculation may cause problems in taking a representative cell count. If
flocculation occurs, add 2 to 8 drops of 4m H2SO4 to the diluted sample.
Example

Five ‘r’ squares show the following cell counts, for a sample diluted 100 times;
r1 = 15, r2 = 20, r3 = 12, r4 = 16 and r5 = 14
Total = (r1 + r2 + r3 + r4 + r5) = 77
Therefore, average number of cells per ‘r’ square = 77/5 = 15.4
Cell concentration = 15.4 x 25 x 104 x 100 = 15.4 x 25 x 106 = 385 million
cells/ml
Hence, the number of cells in the original sample is 385 million cells per ml.

3.8. Operating Laboratory Centrifuge

For vibration free performance:-

a) Ensure level and stability
b) Balance centrifuge tubes

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c) Insure use of rubber cushions for glass tubes
d) Bring speed knob to “Off” and increase speed gradually
e) Check carbon brushes periodically
f) Check glass fuse for non-working of unit

3.9. Estimation of Volume Percent of Sedimentation in Fermented

Wash

Principle

Sample from the fermenter containing yeast cream is centrifuged at 3500 RPM
for 10 minutes in graduated tubes. The test gives an estimation of the volume
percent of sedimentation.
Apparatus

 Centrifuge with ‘swing out’ tube holders;

 Centrifuge tubes;
 Volumetric pipette – 10ml;
 Timer
Procedure

1. Mix the sample collected from fermenter vigorously and fill two centrifuge
tubes up to 10ml with the volumetric pipette.

2. Place the centrifuge tubes in opposite tube holders and centrifuge the tubes
for 10 minutes at 3500 rpm
3. Drain the supernatant
4. Add 10ml water and re-suspend the sediment to get an accurate reading
5. Centrifuge the tubes again for 10 minutes
6. Take out the tubes and read the average sediments in volume percent. (%
v/v of sample)

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Note: - The same method may also be used to check the sludge content of
molasses and yeast concentration.

Calculation

If the sediment reading in the tube after centrifuge in the 10ml sample is a ml,
then the percent sediment of yeast concentration = a x 10 %.

Yeast concentration should be 12 – 15% If yeast concentration is >16% alcohol

production will be inhibited for the following reasons:-

a) Creates pressure on cell wall and cell goes break down.

b) High yeast concentration reduce dissolved oxygen rate
c) It generates foam

3.10. Performance Test of yeast separator

Principle

Comparing sedimentation level of Samples from fermenter # 4 sampling line,

sample from yeast cream out let of yeast separators and sample from DE
yeasted wash out let of yeast separators
Apparatus

 Centrifuge with “swing out tube holders

 Centrifuge tubes
 Volumetric pipettes – 10ml
 Timer

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Procedure
a. Separately fill the samples collected from fermenter 4 sampling line,

yeast cream out let of the yeast separator and DE yeasted fermented
wash; in different three tubes and 40ml in each tube.
b. Fill the 4th tube with water to 40ml mark
c. Centrifuge the tubes for 10 minutes with RPM of 3000-3500
d. Compare the volume of sediment in three tubes

Result

If the sediment in fermented wash sample from fermenter 4 equals the yeast

cream sample, then the yeast separator is performing well. The sediment in
the DE yeasted wash should be nearly Nil.

3.11. Determination of total fatty matter in Turkey red oil

(Defaming agent)

Turkey red oil is used as a defaming agent in distilleries. It is effective in

suppressing foam formed due to CO2 evolution during fermentation. The
quality of Turkey red oil depends on the content of total fatty material.
Chemically Turkey red oil is sulfonated castor oil.

Apparatus

 Beaker 100ml capacity, measuring cylinder 100ml cap

Reagent

 35% H2SO4

Procedure

Take 40ml of Turkey red oil sample in beaker and add 50ml of 35% diluted
sulfuric acid, mix well. Heat gently up to boiling with constant stirring. Pour
the content in 100ml measuring cylinder (glass) & let it stand for 20 – 30

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minutes foe separation of layers. Upper layer contains total fatty matter.
Count the volume of upper layer & multiply by 2 to get the total fatty matter in
percentage.
Result

 Total fatty matter % in Turkey red oil (Defaming agent)

Total fatty matter in De Foaming Agent Grade

40% - 45%-Total fatty matter IIIrd
45% - 55% Total fatty matter IInd
55% - and above Total fatty matter Ist

3.12. Estimation of Sugars by Arsenomolybdate Method

Principle

Reducing sugars will reduce copper under alkaline conditions. Reduced copper
forms a color complex with arsenomolybdate. This can be quantified
calorimetrically
Reagent

a. Na2CO3 (Anhydrous) - 35gm

 Rochelle salt - 25gm
 NaHCO3 - 20gm
 Na2SO4 (Anhydrous)
 Dilute to 1lit with distilled water.
 Store above 200C, filter to remove the sediment. This forms standing
for a few days

b. CuSO4.5H2O - 15gm,
 Dilute to 100ml with distilled water
 H2SO4 - 1-2drops

c. Aresenomolybdate reagent

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1. Ammonium molybdate - 25gm,
Dilute with 450ml of distilled water
H2SO4 - 21 drops
2. Sod Arsenate : 7H2O - 3gm
Dilute to 25ml with distilled water
Add (b) to (a) and incubate for 24 hr. at 37 , Store in a glass stopper

brown bottle.
d. Working Copper Reagent
A - 25ml
B - 1ml
e. Deporting Solution
1) ZnSO4 6H2O - 5gm,
Dilute to 100ml distilled water.
2) Ba(OH)2 - 4.142gm , dilute to 100ml distilled water
3) Standard (200Kg/ ml) 20 mg - glucose /100ml distilled water

Method
Deprotenization
 Mix 1 volume of solution with 15 volumes water and 2 volumes Ba(OH)2,
stir and after the solution turns brown add 2 volumes of ZnSO4 then stir
 Allow to stand for 10min, filter through Whitman No 1 filter.
Estimation
 Mark 10 tubes take 1.0ml or D.W in one tube
 Take 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0ml of standard and make Up to 1ml with
Distilled Water.
 Take the sample in duplicate (1ml)
 Add 1ml of working copper reagent
 Heat for 20 min in a boiling water bath

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 Cool and add 1ml of aresnomolybdate reagent to all tubes and mix.
 Dilute to the 25ml mark with distilled water
 Switch on the 25ml mark with Distilled Water
 Read after 30 min at 520 nm using the distilled Water tube (No1) as blank.
 Sensitivity = 5 – 300ug.
Precaution
 Deproteining is not required for sample lacking Protein
 For greater sensitivity, readings can be taken at 660nm (red filter)

3.13. Estimation of Reducing Sugars by DNSA Method

Reagents
DNSA:-
 Sodium hydroxide - 1gm
 Phenol - 0.2gm
 Sodium Potassium tartarate - 20gm
 Sodium sulphate - 0.05gm
 Dinitrosalicylic acid - 1gm
 Distilled water up to - 100ml
One ml of the sample mixed with 3 ml DNSA reagent. The mixture is placed in
a boiling water bath for 115min and subsequently diluted with D.W. to make
25ml. The standard curve is prepared with aqueous solution of glucose &
maltose in the range of 100-1000 kg. The absorbency is determined at 550
nm.
Result
 Estimated Reducing Sugars by DNSA method (%)

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4. Various Analysis for Ethanol Alcohol & Intermediate
Products
4.1. Determination of Ethanol in Fermented Wash by Sykes
Hydrometer

Principle
The ethanol content is determined as a function of the distilled fluid. The
distilled fluid contains some ethanol which correspondingly reduces the density
of the mixture of water and ethanol is distilled out.

Apparatus
 Lab scale distillation unit
 Sykes hydro meter
 Volumetric flasks
 Thermometer
 Measuring cylinder

Note: - Chemicals are not required for this analysis. Approx., 500 ml distilled
water should be kept ready for this analysis.

Procedure
 Measure 200 ml of sample of fermented wash in a graduated cylinder.
Again measure 200 ml of distilled water.
 Transfer the contents to a 500ml flat bottom flask
 Distill out 200ml sample using lab scale distillation unit.
 Cool to room temperature.
 Pour the distillate into a 250ml dry measuring cylinder.
 Slowly put the glass Sykes hydrometer into the cylinder.
 Note the reading on the hydrometer corresponding to the lower
meniscus.

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 Note the temperature with thermometer and find the strength from the
Sykes table.
 If strength obtained from the Sykes table is in proof, then multiply this
by 0.5714 to get strength in % v/v.

4.2. Determination of Ethanol in spent wash by Sykes Hydrometer

 Use the same procedure as 4.1.

4.3. Determination of ethyl alcohol spirit by alcoholmeter (Gay-

Lussac method) v/v 20
Apparatus
 Measuring cylinder
 Alcoholmeter
 Thermometer
Procedure

Pour the given sample of spirit in a measuring cylinder and note down the exact
temperature. Immerse the alcoholmeter in the measuring cylinder. Depress it
to the top mark on the scale. It should be free from any adherent bubbles of
air and release gently. When the alcoholmeter spindle is freely floating take the
reading of the alcohol strength. Temperature correction is needed for
temperature of the spirit above 20 . Multiply by 0.2 for each ℃ rise above 20
and subtract from the initial reading of the alcoholmeter.

Example

 Temperature of spirit = 30
 Alcoholmeter reading = 90
o 30 – 20 = 10
o 10 x 0.2 = 2

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o 90 – 2 = 88% v/v

4.4. Test for alcohol loss in spent less

Take 10ml sample and add 5ml (0.25N K2Cr2O7) solution in distilled Water,
then add 20ml concentrated H2SO4.

Result

 Yellow color - Negative for alcohol

 Green color - Positive for alcohol
o Equivalent weight of K2Cr2O7 = 49.031gm;
o [gram/liter = Equivalent weight x Normality]

4.5. Determination of ethyl alcohol content of spirit by specific

Gravity method

Apparatus
Specific gravity bottle, thermometer etc.
Procedure
Fill the pyknometer (Specific gravity bottle) with the alcoholic liquid. Dip the
thermometer into the liquid and note the exact temp.of the liquid in the
pyknometer before the performed stopper is inserted. Then weigh the
pyknometer or sp. gravity bottle with the alcoholic liquid. This fill at
t℃.Calculate the net weight in grams of the alcoholic liquid at t°c in the
pyknometer by subtracting the weight of the empty specific gravity bottle or
pyknometer. Divide the weight so obtained by the ‘Water equivalent’ that is the
weight in air of same volume of water content in the pyknometer at 15 . This
gives the specific gravity of the alcoholic liquid in air at t /15 . Record the
temp to the nearest 0.50 . Calculate specific gravity according to spirit

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preparation through use of table 1 and 2 of IS: 3506 – 1967 (Indian Standard –
Table for alcoholometry- by Pyknometer method)

Table No 1
Specific gravity conversion table from temp 15 /15 - this table gives the
specific gravity in vacuum at various temperatures (10 to 40 at intervals of
every 0.5 ) with respect to that of water at 15 for various percentage of
ethanol by volume at 15 ). With the help of this table; the specific gravity at a
particular temperature is found by pyknometer or specific gravity bottle
method; can be converted into corresponding specific gravity at 15 /15 .

Observation

S/N Description symbol Value

1 Empty weigh of Sp. Gravity (pyknometer) bottle W1 ---gms

2 Weight of Sp. Gravity bottle + distilled water W2 ----gms at temp

3 Weight of Sp,. Gravity bottle + liquid W3 ----gms at temp

Calculations
 Specific gravity
 Specific Gravity of liquid (Ethanol) = W3 – W1
W2 – W1
Result
 Ethyl alcohol content of spirit by Specific gravity method

4.6. Determination of ethyl alcohol content of spirit by Sike’s

glass Hydrometer

Apparatus

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 Measuring cylinder
 Sike’s hydrometer
 Thermometer
Procedure
Pour the given sample of spirit in the measuring cylinder and note down the
exact temperature. Select the spindle likely to be required. A too heavy spindle
may shatter itself on the bottom of the jar if released carelessly. Immerse the
selected spindle in the measuring cylinder. Depress it to the top mark on the
scale, shaking free any adherent bubbles of air and release gently. A proper
spindle will float at a point within the divisions on its stem. Bring the eye to
the level of the surface of the spirit and note down the division that is cut by
that surface when seen from below. This is “indication” of the surface of the
liquid in between any two stem divisions; the division nearest below the surface
(seen from below) is to be recorded as the indication to find out the ‘strength’ of
spirit. Refer to spirit tables for use with Sike’s hydrometer. Opposite the
indication in the table for the recorded temperature, will be found the spirit
“strength”

Example

Temperature Indication Spirit strength by table

85° 𝐹 68.2 24.9 UP

85.4 F above, so use 86 F 83.7 above so use 83.8 60.8 UP

Conversion of to F

 F = 9/5 x + 32

 ℃ = F – 32
9/5

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Example
 Conversion of degree Over proof (O.P) /Under proof (U.P) to% alcohol
 Proof = 0.5714% Ethyl Alcohol (100%v/v)
If 65 O.P. reading is obtained,

 Then add 100

 65 + 100 = 165 proof
 % alcohol = 165 x 0.5714 = 94.28% ethanol
If 25 U.P reading is obtained

 Then subtract from 100

 100 – 25 = 75 proof
 % alcohol = 75 x 0.5714 = 42.85% ethanol
Result

 % Ethyl alcohol content of Rectified Spirit = 94.28% V/V

4.7. Determination of Ethanol by Oxidation Method

Apparatus
 Round bottom flask with side tube. T. Joint, Ball tube

Reagents

 Potassium dichromate (42.49 gms/lit), 50% V/V H2SO4

 Sodium thiosulfate (2N) (248.2 gms/lit), Starch indicator

Procedure

10ml wort with maximum alcohol concentration of 1% (V/V) is poured,

together with a little pumice, into a flask with a side tube. The flask is gently
heated and ethanol vapors absorbed in a mixture of 10ml potassium
dichromate (42.59 gm/lit.) add 10ml 50% (V/V) H2SO4 contained in ‘ball tube’

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Empirically, distillation of ethanol has been found to be complete when the
content of the ball tube is hot enough to allow white fumes to emerge. The
content of the ball tube is transferred quantitatively to a 500 ml Erlenmeyer
flask, 250ml cold tap water is added together with 10ml 10% (W/V) potassium
iodide solution. The mixture is titrated with 1N Na2S2O3 (248.2 gm/lit) as
indicator a solution of starch is used.
The thiosulphate solution is normalized against 10ml of the dichromate
solution. Vol. is the amount used in this procedure. The volume of
thiosulphate used in the ethanol analysis = V

Calculation

 The Ethanol concentration in weight % = VO – V

VO
 To convert the concentration to volume % multiply by 1.26

4.8. Determination of Total, fixed, and volatile acidity (as

CH3COOH) of Rectified Spirit (ISI method)

Apparatus
 Burette, pipette, conical flasks, etc
Reagents
 Standard Sodium hydroxide solution 0.1N. Phenolphthalein indicator
Procedure
a) Total Acidity
Place 100ml of the sample in a conical flask, add 0.5ml of phenolphthalein
indicator, and titrate against Std. NaOH till you get the end point, colorless to
just pink.
b) Fixed acidity

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Place 100ml of freshly prepared distilled water with 100ml of the sample and a
few pieces of clear porous pot in a 500ml conical flask of resistance glass and
boil gently for 10 minutes. At the end of this period, close the neck of the flask
with a stopper carrying a soda lime guard tube and allow cooling. When cool,
remove the stopper, add 0.5ml of phenolphthalein indicator and examine for
alkalinity. If the sample is not alkaline, titrate with Standard NaOH solution
using a micro burette. Determine the specific gravity of the sample at room
temperature.

Observation
 Total Acidity
Burette reading = ……………. ml
 Fixed Acidity
Burette reading = …………… ml

Specific gravity of the sample

Wt. of sample. (At room temp.)

Sp. Gr. Of the sample, S = wt. Of distilled water

Calculation
Acidity (as CH3COOH) percent by weight = 0.06 V.N.
S

Where,
 V = Volume, in ml of std. NaOH Solution required for titration
 N = Normality of the std. NaOH solution used
 S = Specific gravity of the material
 Volatility of the acid = Total acidity - Fixed acidity

Result
 Volatility of the acid (as CH3COOH) % by weight in the Given Rectified
Spirit

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4.9. Determination of Aldehyde (as CH3CHO) content of Rectified

Spirit (ISI)
Apparatus
 250ml stopper flasks

a. Qualitative Test for Low Aldehyde Content

This method is suitable when the aldehyde content is expected not to exceed
o.0068 per 100ml of the material. This color reaction is based on the
resinification that takes place and the yellow color that result on treatment of
acetaldehyde with sodium hydroxide (Acetates are also included as aldehydes)
Reagents
 NaOH Solution
Dissolve 20gms of NaOH in water and dilute to 100ml with distilled water.
Procedure
Mix 10ml of the material with 5ml of NaOH solution and set aside for 5min. If
there is no yellow color formation within first 5min, then the aldehyde content
of spirit is not more than 0.0068/100ml.
b. Qualitative Test for Higher Aldehyde Content
This method is useful for material containing 0.05gm to 0.5 percent of
aldehyde.
Reagents
 Stock solution of Hydroxylamine Hydrochloride
Dissolve 20gms of hydroxylamine hydrochloride in 100ml of distilled water.

 Hydroxylamine Reagent
Dilute 10ml of the stock solution of hydroxylamine with 100ml of aldehyde free
alcohol, add 2ml of bromophenol blue solution and then add standard sodium

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hydroxide solution till the characteristic dichromic yellowish green color is
obtained.
 Metaphenylenediamine Hydrochloride
 Aldehyde free alcohol – Residual rectified: Digest at ordinary temperature
for several hours and distil slowly, rejecting the first 100ml and the last
200ml of the distillate.

 Standard sodium Hydroxide solution: - 01N

 Bromophenol blue in 1.5ml of standard NaOH solution and dilute with
water to 250ml.

Procedure
1. Take 50ml of the material in a 250ml stoppered flask, add 25ml of
hydroxylamine reagent and 25ml of distilled water.
2. Similarly prepare at blank by using of distilled water and 25ml of
hydroxylamine reagent.
3. Allow standing both the flasks for 1.5mins.
4. Titrate the blank solution with 0.1N std. NaOH by adding two drops of
bromophenol blue solution until the characteristic dichromatic yellowish
green color appears.
5. Titrate the sample solution with 0.1N standard NaOH solution until the
color matches with that of the blank solution
Calculations
Aldehyde content (as CH3CHO), gms/100ml = 0.088 (V-Vo) N
Where
 VO =Volume, in ml of std. NaOH solution required for the filtration of sample
 V = Volume, in ml of std. NaOH solution required if any in the blank
 N = Normality of Std. NaOH solution

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Result
 Aldehyde (as CH3CHO) content of Rectified Spirit (gms/100ml)

4.10. Determination of Aldehyde (as CH3CHO) content of spirit (AOAC

method)
Reagents
Sodium Thio-sulphate solution (0.05N)
Dissolve 25gms of Na2S2O3 in 1 liter water, store solution in Dark, cool place,
and do not return unused portions to stock Bottle. If solution less than 0.1N
are desired prepared by Diluting with distilled water 100ml of 0.1N Na2S2O3 +
100ml Of distilled water = 200ml 0.05N Na2S2O3
Iodine Solution (Approx., 0.05N)
Dissolve 20gm KI in 25ml distilled water and add 6.5gms resublimed iodine
and stir dissolved. Transfer to 1 liter volumetric flask and dilute to mark to get
0.05N iodine solution.

Sodium bisulfate solution (Sodium metabisulphite) 0.05N (Approx.)

Sodium disulfide
Dissolve 9.505gm of Na2S2O5 and make the volume 1 liter with distilled water
+10% alcohols.

Standardization of sodium thio-sulphite

Dissolve 2gm of KI in a 250ml stoppered conical flask in 50ml of distilled water
add (1+9) H2SO4, then 20ml of std. dichromate solution (0.025N) place in dark
for 5 minutes. Dilute to approx. 150ml and titrate with 0.05N Na2S2O3
Std. Potassium Dichromate solution (0.025N)
Dissolve exactly 1.226gms of potassium dichromate and mark the volume to
1liters with distilled water.

Procedure

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(AOAC and ENCY or Ind. Chem. Analy, Vol, & P.484)

To 200ml of sample in a 500ml distillation flask add 35ml water and a few
grains of Carborundum. Distill slowly into a100ml volumetric flask until the
distillate nearly reaches the mark. (New Whisky or Rum does not need to be
distilled unless the sample is Dirty or discolored. Place 100ml of distillate in
500ml flask (stoppered conical flask). Add 100ml of water and 25ml of freshly
prepared 0.05N aqueous sodium bisulfate solution containing 10% alcohol and
allow to stand about 30 minutes Shaking occasionally. Add 0.05N iodine
solution with stirring until the mixture remains a definite yellow color. Add a
few drops of starch solution and titrate the excess iodine with 0.05N aqueous
sodium thiosulfate solution to the colorless end points. Run a blank
containing 100ml of water, 25ml of sodium bisulfate solution add the exact
amount of iodine solution. As used for the sample. Calculate as acetyl
dehydrates the difference between titration in ml of sodium bisulfate solution
time’s 1.1 equal mg of acetaldehyde in the sample.

Observation

1 Iodine required for the sample ml

2 Sample reading ml

3 Blank reading ml

4 Difference mll

Calculations
Aldehyde (as CH3CHO) content of the given sample
= 0.022 x difference in Burette reading x Normality of Na2S2O3
= ------------ gms/100ml.

Result

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Aldehyde (as CH3CHO) content of the given sample (gms/100ml)

4.11. Determination of Ester (as CH3COOC2H5) content of Rectified

Spirit
Reagents
 Std. potassium Hydroxide solution (KOH) – 0.1 N and 0.5N
 Std. Sulphuric Acid – 0.1N
 Phenolphthalein Indicator:- Dissolve 0.5gms of phenolphthalein in
100ml of rectified spirit and carefully add std. KOH solution 0.1 N till
the color is rendered faintly pink
Procedure
1. Transfer 100ml of the sample into a 250ml round bottom tank
2. Add few drops PH phenolphthalein indicator and neutralize in the cold,
the free acid if present with 0.1N std. KOH solution.
3. Add 2ml of std. KOH solution (0.5N)
4. Attach the flask to a reflux condenser provided with a soda lime guard’s
tube and reflux the contents on water bath for at least one hour.
5. Cool the contents, pour into another flask, wash the Original Flask with
100ml of freshly prepared distilled water, add the washing to the original
liquor and then titrate with std. H2SO4 adding a few drops of
phenolphthalein indicator.
6. Carry out a blank, using 100ml of water in place of the neutralized
sample.
Calculations
Calculate the percentage of ester (as CH3COOC2H5) as follows
 Esters as Ethyl acetate (CH3COOC2H5) gms/100ml = 0.088 (V1 – V2)N
Where,
V1 = Volume, in ml of std. H2SO4 required in the blank

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V2 = Volume in ml of std. H2SO4 required with the sample
N = Normality of std. H2SO4
Result
Ester (as CH3COOC2H5) content of rectified spirit, (gms/100ml)

4.12. Fusel oil determination in spirit sample

Reagents
0.8aq. solution of salicylic aldehyde
Concentrated H2SO4
Procedure
Method A
A simple fusel oil test for grain neutral spirit consists of adding 0.5ml of 0.8
aqueous solution of salicylic aldehyde to 5ml of the sample. Then with some
minutes the color of the solution is observed.

Grade Color Range Fusel Oil PPM

1 Lemon Yellow 0 -50
2 Lemon Yellow to slightly brownish 60 – 150
3 Brown 350
4 Red color 500 (Above 500 PPM)

 Fusel oil free alcohol will remains yellow for hours

Method B

Allow 25ml of the material to evaporate in a porcelain dish Protected from dust,
until a little liquid is left over. Remove the dish away from the water bath and
allow the liquid left in the dish to evaporator without applying any external
heat till the surface of the dish is barely (just) moist. Observe if foreign odor is
perceptible.

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Add one ml of concentrated sulphuric acid. The material shall be taken as
satisfying the requirements of this test if no foreign odor is perceptible and no
red or brown color is produced on treatment with cone, sulphuric acid.
Result
 Fusel Oil in Rectified Spirit Sample (PPM)

4.13. 4.13 Furfural determination in Rectified Spirit (ISI)

Principle
Furfural in rectified spirit gives a characteristic red color with Aniline acetate
which may be used for the determination and quantitative calorimetric
determination of furfural in spirit in the presence of other aldehydes. The
temperature for test should be above 15 and time for obtaining color is 10 –

15 minutes after mixing solution. If the quantity of the furfural is too small the
color rapidly falls always after this interval the limit of sensitivity of the test
0.002 for 2 PPM of furfural.
Reagents
 Aniline
Pure redistilled, in case the material is deeply colored; yellow or reddish
redistill before use and store it in amber colored bottles.
 Acetic Acid
 Stock solution of furfural
Dissolve or dilute 1gms of furfural in 100ml of furfural free alcohol(50% vol.)
and further dilute 5ml of this solution to 100ml with furfural free alcohol (5o%
by vol.). The stock solution thus prepared contains 0.05gms for furfural per
100ml of solution.
 Standard solution of furfural
Dilute 2ml of this solution with pure furfural free alcohol of (50% by volume)
1ml of this dilute solution contains 0.02gms of furfural.

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Procedure
Take 5ml of material under test in colorless glass cylinder (25ml) Dilute with
5ml of water and after through mixing add 0.5ml of aniline preferably with a
pipette and 1ml of glacial acetic acid preferably with a burette. Gently, agitate
the mixture, till it becomes homogenous and then set aside for a period of 5
minutes at a temp above 15 . In case furfural is present (more than 2 PPM) in

the material a red color starts developing in course of few seconds and reaches
its maximum intensity in 5 – 10 minutes. If no red color either permanent or
transient develops during this time then the test for furfural content shall be
taken to have been satisfied.
Result
In Rectified Spirit sample furfural test is positive/Negative

4.14. Determination of Cu (Copper) in the given Rectified Spirit

Sample
Reagents
 Dilute H2SO4 (Approx. 10% V/V)
 Aqua-Ragia- Mixture of one volume of Concentrated HNO3 and three volume
of Conc. HCL,
 Dilute NH4OH (Approx. 10% v/v)
 Standard Copper Solution:- Dissolve 1.119gms of CuSO4 5H2O in distilled
water and make the volume to one liter. From this take 10ml and dilute to
100ml. One ml of diluted solution contains 0.02845mg of copper. Diluted
solution shall always be prepared before use only.
 Ammonium chloride
 Acetic Acid (approx., 5% by wt. v/v)
 Potassium Ferro-cyanide (approx. 4% by wt.)

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Procedure
Transfer 20ml of the material (Rectified Spirit) into Silica Crucible and add one
ml of dil. H2SO4. Heat gently in the beginning and then evaporate almost to
dryness on water bath. Ignite the crucible over smokeless flame to eliminate
the H2SO4 acid. Then cool and dissolve the residue in 2ml of water. Add four
drop of aqua-ragia solution and evaporate to dryness on water bath (by taking
care, experiment can be conducted on burner flame also)
Dissolve the residue into 2ml of diluted HCI and warm gently till the residue is
dissolved. Add 0.5gms of NH4CL and dilute to 15ml with distilled water. Add
dil. (NH4OH) till the solution comes alkaline. Boil excess of NH3 and filter into
clean Nesceller’s tube. Cool and then render the solution with acetic acid. (3-5
drops are usually sufficient). Then dilute to 40ml add 0.5ml of potassium
Ferro cyanide solution. Stir well and make up the volume to 50ml. This is
sample tube. Then prepare series of control solution, each containing in 50ml
of water.
 0.5gms of NH4CI
 3 – 5 drops of Acetic Acid (CH3COOH)
 0.5ml of K4Fe (CN6) together with increasing amount of standard
containing solution added in the control solution having nearby as
possible; the same intensity of color as that of the test solution.

Observation
Control solution containing ______ ml of Std Cu solution is nearly matching
with test solution. V = ________ ml
Calculations
Cu (Copper) gms/100ml = 0.00002845 x 12.5 x V
Result
Cu (Copper) content in Rectified Spirit sample (gms/100ml)

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4.15. To conduct potassium permanganate (KMNO4) test for judging
the quality of spirit.
Apparatus
Graduated measuring cylinder with Stopper100ml capacity, Beaker,
thermometer etc.
Reagent
Standard potassium permanganate solution – 0.01N (freshly Prepared)

Procedure
Clean thoroughly the graduated measuring cylinder first with Cone HCI, then
with water and finally with the material to be tested. Place 20ml of the
material in the cylinder; bring the temperature to 15 by placing the cylinder

in cold water. Add 1.0ml of standard permanganate solution by means of 1ml

pipette noting down the exact time as soon as addition is over. Mix the
contents at once and keep the cylinder at 15 to 16 and away from the bright

light.
The material shall be taken to have satisfied the test if the pink color does not
disappear up to 30 minutes.

Comparison Test
Reagents
 Cobalt chloride solution
Dissolve 5gms of COCl2 crystals (COCI2, .6H2O) in water and make up to 100
cc with water.
 Urenyl Nitrate Solution
Dissolve 4gms, of Urenyl nitrate crystals (UO2 (NO3)2 6H2O) in water and make
up to 100cc with water.
Procedure

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Prepare control solution by diluting to 50ml with distilled water mixture of 5ml
of CoCI2 solution and 7ml of urenyl nitrate solution. The material shall be
taken to have satisfied the test; if the time taken for the color of the reaction
solution to match with that of the control solution is not less than 30 minutes.
Note No. 1
The glass cylinders shall be thoroughly cleaned before making the test.
Cleaning with strong HCL to remove excess of manganese is recommended if
the cylinder has been previously used for this test.
Note No 2
The test shall not be performed in bright light; The temp shall be held
approximately 15 - 16℃ during the course of the reaction, and the alcohol
shall not come in contact with cork stopper.
Result
Potassium Permanganate (KMNO4) tests for Rectified Spirit (Min.)
4.16. Determination of Methyl Alcohol (as CH3OH) content of Rectified
Spirit
Reagents
1) Potassium Permanganate Solution in Phosphoric Acid
Dissolve 3gms of potassium permanganate in mixture of 15ml of phosphoric
acid containing 89% by weight of phosphoric acid (H3PO4) and 70ml of water,
and add sufficient quantity of water to produce 100 ml of the solution
Oxalic acid Solution in Sulphuric acid
Continuously add 60ml of conc. H2SO4 to equal volume of water and cool. Then
mix 100ml of this cooled dilute H2SO4 with 5gms of oxalic acid crystals.
Magenta Solution, Decolorized
Dissolve one gram of fuchsine in 600ml of water and cool in an ice bath. Add
20gms of sodium sulphite dissolve in 100ml of water, cool in an ice bath and

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further add, slowly and with constant stirring, 10ml of conc.HCL, dilute to
100ml
Decolorized solution of Magnata should be protected from light; If the resulting
solution is turbid, it should be filtered and if brown in color, should be shaken
with sufficient charcoal (0.2 to 0.3p) to render it colorless and then filtered
immediately. Occasionally, it is necessary to add 2 to 3ml of conc., HCL
followed by shaking to remove a little residual pink color. The solution
resulting from any of the foregoing modifications should be allowed to stand
overnight before use.
Procedure
1) Dilute 0.5ml of the sample with water to 5ml, and add 2.0ml of potassium
permanganate solution in phosphoric acid.Set aside for ten minutes.
Then add 2ml of oxalic acid solution in H2SO4.
2)To the above colorless solution, of Magenta and set aside at a temp between
15°𝐶to 3OOC
3) Examine the color of the above sample after 30 minutes.
4) Compare the final change in color against the blank produced by adding 5ml
of water to 5ml of Magenta solution.
5) The material shall be taken as satisfying the requirement of this test if no
color change is visible between the two solutions.

Result
In Rectified Spirit sample Methyl alcohol test is positive/Negative

4.17. Reduction and Blending of the Spirit

Apparatus
Sike’s hydrometer, measuring cylinder, beaker, thermometer etc.
Reagent
Rectified Spirit, water etc.

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Procedure
Reduction of Spirit
Definition: Reduction means the dilution of spirit with water
 Find out the proof of given sample of R Spirit.
 Fill the given sample in a perfectly vertical glass cylinder.
 Note the exact temp of the material in .

 Find out the proof of the given sample from sikes hydrometer table.
Preparation of required proof strength of the spirit
The rule is to multiply the volume of strong spirit by its proof strength and
then divide by the required proof strength and the result will be the volume of
dilute spirit, i.e. the bulk after reduction.
Shrinkage: It is a practical observation that when 49.7 volume of water and 54
volumes of alcohol are mixed together only 100 volumes of spirit being
obtained; instead of the expected 103.7 volume. This shrinkage is due to the
fact that when alcohol and water are mixed, a combination of the two
substances occurs accompanied by a distinct contraction or loss observed
during the operation is one proof of such chemical combination.
Add the shrinkage volume of the water into the original spirit to get the
required proof of the spirit. And find its proof by the above method.
Blending of Spirit
Definition: - Blending of spirits means the mixing together of two or more
spirits of different strength.
Preparation of required proof of spirit by mixing two different strength spirits.
Rules
Multiply the volume of the weak sprit by the No. of degrees. It is to be raised
& divided by the number of degrees the strong spirit will be lowered by the
mixing.

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Fix the volumes of the weak spirit and from above rule find out the volume of
strong spirit and mix well and confirm the proof strength of the blended spirit.
Example
a) Reduction
To find out degree proof of given sample of spirit
Observation
1. Temp = 23℃ = 74.4
2. Indication (hydrometer reading) = 16.5
3. Strength = 66.3 O.P (From sikes hydrometer table)
4. Proof = 100+ 66.3 =166.3
b) Preparation of required strength of spirit (70 UP) from the given
sample
1. Volume = 10ml (of 166.3 proof)
2. To prepare = 70 U.P spirit
3. Proof =100 – 70 = 30 proof
4. Volume (given) x Its proof

Required proof by Rule

= 100 x 166.3
30

= 554.33ml
= Total volume of 70 U.P of the spirit

554.33ml – 100ml = 454.33ml water should be added in 100ml

given spirit.
c) Shrinkage
 Shrinkage volume of water = 554.33ml x 1.0370ml = 574.84ml
 100 x 1.0118 =101.18ml ,(Factor for 65.7)

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 574.84ml - 101.18ml = 473.66ml is the correct amount of Water to be
added.
By rule is 554.33 – 100 = 454.33ml gives the amount contrast.
The mix gives the following Observation;
 Temp. = 29 (82.4 ) & Indication = 87.2
 Strength = 70 U.P.

d) Blending
Here you have prepared 10 O.P. spirit from 70 U.P. and 66.3 O.P. spirit
using 200ml of 70 U.P. spirit to prepare 10 O.P. means 110 proof spirit. The

weak spirit will be raised and the strong spirit will be lowered.
Weak spirit will be raised
110 – 30 = 80 proof
Strong spirit will be lowered
 166.3 – 110 =56.3 proof

 Total Volume x weak spirit raised proof

Strong spirit lowered proof

 200 x 80
56.3 = 284.19
 284.19ml of strong spirit of 66.3 O.P. strength should be added in the
200ml of 70 U.P spirit.

Observation
1 Temp. 28 (83.4℉)

2 Indication 44.6
3 Strength 12.2 O.P.

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Result
1 Given sample of Rectified spirit O.P.
strength
2 Reduction spirit sample strength U.P.

3 Blending spirit sample strength ° O.P.

4.18. Estimation of Ethanol Content by Dichromate method

(Spectrophotometric)

Principle
The procedure was based on the principle that dichromate is reduced by
measuring sepectrophotometrically, Ethanol and a chromic complex is formed,
the relative quantity of which was
Dichromate Reagent
34gms of potassium dichromate is dissolved in 500ml of distilled water in a one
liter vol. Flask. The flask is placed in a container of ice and 325ml conc. H2SO4
is added drop wise so that minimum heat is generated. The solution is
thoroughly mixed, cooled and the volume is made to 1 liter.

Estimation
One ml of fermentable broth is added to the distillation flask. Adding 25-
30mldistilledwater dilutes the sample. 50ml vol. Flask with 25ml of
dichromate reagent is used for the collection of about 20ml of the distillate.
The flask is incubated at 60 for 20 min. in a water bath, the mixture is cooled

and the volume is made to 50ml. The std. curve is prepared using 1 to10%
ethanol (v/v). The blank is prepared with distilled water. The amount of ethanol
in the test sample is determined from the std curve; At 620 nm.

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4.19. Potassium Permanganate Time P.P Time Monitoring
Reagent
N/10 – KMNO4
Weigh = 0.316gm KMNO4 in 100mlVolumetric flask and make up with
distilled water and mix well.
Procedure
1. Take 50ml clean test tube
2. Wash with conc. HCL and finally wash with sample.
3. Take 20ml of spirit sample and maintain temp = 15 degree centigrade.
4. Then add 0.1ml of N/10 KMNO4 solution.
5. Note the initial time just after adding KMNO4 which give pink color to
solution.
6. After some time pink color disappear.
7. Note the final time when pink color disappears. The difference between
initial and final time in minutes is PP time of the sample.

4.20. Quick Tests

Aldehyde Test
50ml alcohol + 2ml potassium permanganate
50ml water +“ “ “ See for color change
Potassium permanganate preparation
0.2 gm potassium permanganate in 100ml water
Fusel Oil Test
Take 20ml alcohol sample in crucible. Heat 78-80 . If there is oil, oil will

remain in the crucible

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4.21. Alcohol Determination by Ebuliometry
Ebuliometry is the most encountered procedure for determination of the
alcohol content of aqueous solutions. In principle, the analysis is based on the
Ragout’s low relationship of boiling point depression:
Where: P = h.X1
P = Vapor Pressure of Solution
H = Proportionate constant
X1 = Mole fraction of solvent
Thus, the vapor pressure of a solute (ethanol) in a solution will vary in a
regular manner as a function of its concentration. As the concentration of the
solute (alcohol) in the wine increases, the boiling point is reached at a
temperature that is low relative to the boiling point of pure water.
Ebuliometry involves a measurement of the boiling point depression caused by
the presence of alcohol in a wine sample. Mechanically, the boiling point of the
sample is measured relative to the boiling point of pure water and the
difference is related to percent ethanol. Sugar (2% p.s) is the major
interference in this determination.
Equipment
Ebuliomenter, mercury thermometer ( ), appropriate Alcohol scale. Distilled

water and cold tap water source, Micro burner or alcohol lamp
Reagents
Sodium hydroxide (1%) cleaning solution.
Procedure
a) Determination of the boiling point of water
i. Add approximately 30ml of distilled water to the boiling chamber. There
is no need to add cold tap water to the condenser at this time.

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ii. Insert the thermometer. Position the instrument over a flame. Prior to
use, inspect the thermometer, making certain that the mercury column
is not separated. If you elect to use a micro burner rather than the
alcohol lamp included with the Kit, the ebuliometer should be positioned
high enough above the flame to prevent excessive “bumping”. The later
is a sign of overheating and impedes proper boiling point determination.
iii. When the thermometer reaches a stable point allow 15 to 30 sec. For
minor fluctuations to occur. A this time, take the boiling point reading
and set he inner scale opposite 0.0% alcohol on the’ Degree Alcoholic du
Vin’ outer scale.
iv. Cool and drain the instrument.
b) Determination of the Boiling point of wine (fermented wash)
i. Rinse the boiling chamber with a few milliliters of the wine to be
analyzed and drain it.
ii. Place approximately 50ml of wine in the boiling chamber.
iii. Fill the condenser with cold tap water.
iv. Insert thermometer and place the instrument over the heat source.
Once again, check the mercury to ensure continuity of the liquid.
v. When the thermometer reaches a stable level allow 15 to 30 sec. For
changes, and take a reading.
vi. Locate the boiling point of wine on the inner “Degrees du Thermometer”
scale and record the corresponding alcohol content (% vol/vol.) on the
outer scale.
vii. Cool and thoroughly rinse the instrument.
c) Supplemental Notes:-
 Because the boiling point will vary with atmospheric pressure, it must be
determined for the distilled water reference during each day’s operation.

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It is recommended that boiling point determinations be carried out at
least twice daily and more frequently during periods of unstable weather.
 Reducing sugar levels exceeding 2% interfere with the procedure, yielding
erroneously low boiling points (hence higher apparent alcohol). The
effect of sugar may be overcome by carrying out an initial separation
(distillation) and determining the boiling point of the collected distillate.
Many laboratories simply dilute sweet wines to a level of less than 2%
reducing sugar prior to analysis. Still others use a correction factor
(reducing sugar x 0.05) and subtract it from their apparent alcohol
content.
 To prevent premature loss of alcohol, the cooling water in the condenser
must be cold (ice water preferably). Boiling point readings should be
taken per instruction. As the condenser water warms, alcohol is
volatilized and lost, resulting in erroneously high boiling points and lower
apparent alcohol.
 During extended use, debris will coat the walls of the boiling chamber,
reducing the efficiency of the unit. It is recommended that the boiling
chamber be cleaned periodically with a boiling solution of 1% NaOH.
4.22. Determination of Fermentation Efficiency of Yeast growing on
molasses medium

Apparatus
250ml conical flask, 250ml volumetric flasks, burette, pipette, Distillation unit
etc.
Chemicals
Fehling’s ‘A’ and ‘B’, H2SO4 Urea, Yeast, Dap, Methylene blue Indicator etc.

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Procedure
Take two 250ml conical flask and label them one as blank and other as
experimental. Take 25gms, of molasses and mix well. Add 2gms of Urea to
each flask and adjust the PH of the flask to 4.5 with diluted H2SO4. Make the
volume of each flask to 250ml. Put a cotton plug (non-absorbent cotton) on
both the flasks. Sterilize both these flasks at 15 lbs pressure for about 10
minutes. After sterilization cool both the flasks to room temp.
Add 2gms of yeast to the experimental flask. Plug it and keep it in the
incubator at 30℃ for 48 hours.
During sterilization there will be a slight loss in the volume hence once again
make up the volume of the blank to 250ml. Then find out the original specific
gravity by specific gravity method, and determine the initial total reducing
sugars using following procedure.
Take 50ml of the sample from the flask marked as “blank” in a 500ml
volumetric flask. Add 10ml of 10% HCL and keep the flask into the boiling
water both for about 10 minutes, cool it and make up the volume up to the
mark. Mix well. Take this solution in burette and titrate against Fehling’s ‘A’ -
5 ml and Fehling’s ‘B’ -5ml in a conical flask. Use methylene blue as an
indicator. End point is very deep to red color.
After 48 hours, measure the volume of fermented wash in the experimental
flask, if less than that make up the volume to 250ml, filter the material
through ordinary filter paper. Take 50ml of the filtrate in a 100ml volumetric
flask.
Then cool it and make up the volume up to the mark and find out the
unfermentable sugar by lane and Eynon method (Described in above
paragraph). Take 100ml of fermentable wash in 250ml round bottom flask.

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Add 15ml top water and distill of using distillation assembly. Collect the
distillate in 100ml volumetric flask, up to the mark.
Then find out the specific gravity of the distillate by specific gravity bottle.
Necessary temp corrections are applied. And find out the alcohol percentage
from the Std. table.
Actual Alcohol yield x100
Fermentation Efficiency (%) = Theoretical Yield

Observation
Blank Flask
a) Initial temp of diluted molasses = __________

b) Initial specific gravity diluted molasses = __________

 Wt. of empty specific Gr. Bottle = __________ gms
 Wt. of specific Gr. Bottle + Distilled water = ________ gms
 Wt. of specific Gr. Bottle + Diluted molasses = _____gms
Wt. of Material
Specific gravity of diluted molasses = Wt. of water

c) Dilution Factor:- for diluted molasses

50 = 1_
500 50

d) Burette Reading

1 BR ---------- ml
2 BR ---------- ml
3 BR ---------- ml

 Mean, Burette Reading = ----------- ml

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e) Fehling’s Factor =
f) Initial total reducing sugars in diluted molasses
0.05128 x 100 = __________
Burette Reading x Fehling’s Factor x Dilution Factor
Experimental Flask (After Fermentation)
1. Temp of the fermented molasses = __________
(Before dilution)
2. Specific gravity of the wash (after fermentation = _____________
3. Dilution Factor (for wash fermented) = 50/100 = 1/2
4. Burette Reading
1 BR -----------------
1 ml
2 BR ----------------- ml
3 BR ----------------- ml

5. Mean Burette reading = --------------ml

6. Fall in specific gravity = Initial gravity – Final gravity
Unfermentable Sugars(%)
U.F.S (%) = 0.05128 x 100_____________________
Burette Reading x Fehling’s Factor x Dilution Factor
Fermentable Sugars(%) = Total reducing sugar – Unfermentable Sugars

Theoretical Yield
Inversion
C12H22O11 2C6H12O6
HCL

Fermentation
C6H12O6 2C2H5OH + 2CO2
180gms 92gms 88gms
(Glucose) (Ethanol) (Carbon Di-oxide)
From the molecular formula, 100gms of sugars gives 64.4ml of Ethanol.
100 gms of sugar = 64.4ml of alcohol

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Theoretical Yield = Fermentable Sugars x 6.44 = _____ ml
Alcohol Percentage in Distillate
1. Wt. of specific Gr. Bottle = ______gms
2. Wt. of specific Gr. Bottle + Distillate = _____gms
3. Wt. of Distillate = _______ gms
4. Wt. of specific Gr. Bottle + Distilled Water =_____ gms
5. Wt. of distilled water = ______gms
6. Sp. Gravity of the Distillate = Wt. of Distillate at room temp.
Wt. of distilled water
Fermentation Efficiency, F.E. (%) = Actual % of alcohol x 100
Theoretical Yield
Result
1 Total reducing sugars in diluted molasses %
2 Unfermentable sugars in diluted molasses %
3 Fermentable sugars in diluted molasses %
4 Theoretical Yield of alcohol Ml
5 Actual Yield of alcohol in wash Ml
6 Fermentation Efficiency %

5. Water Analysis
5.1. Determination of Hardness in Soft Water

5.1.1.1. Method-A
Apparatus
 Volumetric flask 500ml
 Volumetric flask 250ml
 Graduated cylinder 250ml
 Erlenmeyer flask 250ml
 Graduated cylinder 10ml
 Burette

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 Magnetic stirrer
Reagents
 Ammonia solution (Concentrated)
 Ammonium chloride
 Eriochrome black T
 EDTA Salt

Procedure for preparation of Reagents

a) Preparation of 0.01 molar EDTA solutions
Dissolve 1,8612gram of EDTA in distilled water and dilute to 500ml flask.
b) Preparation of buffer solution (Ammonia)
Add 142ml of concentrated ammonia solution (AR) to 17.5 gram of ammonium
chloride (NH4CL) AR and dilute to 250ml with distilled water
c) Procedure for determination of Hardness in water
i. Transfer about 50ml of filtered water to a 250ml Erlenmeyer flask and
add about 5ml ammonia buffer solution and a pinch of indicator.
ii. The red coloration appears at this stage. Stir the solution with a
magnetic stirrer and titrate against standard 0.01 Molar EDTA solution
to a blue end point.
iii. If the color of the solution remains blue, report the result of zero
hardness. If the solution forms red titrate against standard 0.01Molar
EDTA solution until a blue end point is reached.
iv. Calculate
Hardness as ppm CaCO3 = titer (ml) x 20
Example titer = 7ml
Hardness = 7 x 20 = 140PPM

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5.1.2. Method B

Solutions
TITRE N/50 sodium salt of ethylene diamine tetra acetic acid
Dissolve 4.0 gram sodium salt of ethylene diamine tetra acetic acid in distilled
water. Add 0.86 gram sodium hydroxide and make up volume to 1 liter. Put
this solution in amber colored bottles.
Erichrome Black-T Indicator Solution
Dissolve 0.5 gram Erich Rome Black-T in 10ml ammonia buffer solution and
make up volume by rectified spirit to 100ml in 100ml volumetric flask.
Ammonia Buffer Solution
Dissolve 20.0 gram ammonium chloride in 100ml ammonia solution and make
up volume by distilled water to 1000ml volumetric flask.
Procedure
Take 100ml sample in a 500 ml conical flask add 2ml of ammonia buffer
solution, Then add few drops of Erich Rome black-T indicator. Then titrate with
N/50 sodium salt of Ethylene diamine tetra acetic acid solution till light blue
color appears.
Calculation
Hardness as CaCO3 in PPM = Milliliter of titer used in titration x 10

5.2. Determination of Chlorides in Water

Solutions
 0.0171 N silver nitrate solutions
Dissolve 2.905 gram silver nitrate in distilled water and make up volume to 1
liter.
 Potassium Chromate Solution K2CrO4

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Dissolve 5.0 gram potassium chromate in 75ml distilled water then add few
drops of silver nitrate solution till red precipitate appears. Filter and make up
volume to 250ml in 250ml volumetric flask.
Procedure
Take 50ml sample in a 250ml conical flask. Add few drops of K2CrO4 solution.
Then titrate with 0.0171N AgNO3 solution till light orange color appears.
Calculation
Chlorides as NaCL in PPM = Milliliter of titer used in titration x 20
Chlorides as CL = Chlorides as NaCL in PPM x 0.6066
5.3. Determination of M.O Alkalinity in Water
Solutions
 Titer N/10 H2SO4 Solution
Dissolve 2.8ml of 98% CONC H2SO4 in distilled and make up volume to 1 liter
adjust the normality by titrating with standard NaOH 0.1N solution.
 Methyl Orange Indicator Solution
Dissolve 0.5 gram methyl orange in distilled water and make up volume by
distilled water to 500ml in 500ml volumetric flask.
Procedure
Take 50ml sample in a 250ml conical flask add few drops methyl orange
indicator solution, then titrate with N/10 sulphuric acid solution.
Calculation
M.O. Alkalinity in PPM
= Milliliter of titre (N/10 H2SO4) used titration x normality of titer x 50000
Volume of sample taken in ml

5.4. Determination of TDS/Suspended Matter in Water

Procedure for TDS

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Filter 100ml sample and put in a porcelain or platinum dish. Evaporate the
water contents over a sand bath or hot plate then dry in an oven at 103

centrigrade. COOL in a desiccator and weigh.

TDS in PPM = Increase in weight in grams x 106
ML of sample taken

Procedure for suspended matter

Take 100ml unfiltered sample and put in a porcelain or platinum dish.
Evaporate the water content over a sand bath or hot plate then dry in an oven
at 103 centigrade. Cool in desiccators and weigh.

Total solids in PPM = Increase in weight in grams x 106

ML of sample taken
Suspended matter in PPM = Total dissolved solids in PPM – TDS in PPM
5.5. Residual Chlorine in Water
CL2 + H2O………HOCL + HCL
Hypochlorous Acid
Chlorine with water forms HOCL hypochlorous and which kills bacteria. For
effective chlorination it is must that treated, water must contain some residual
chlorine near to 0.5 to 1 PPM only, whereas it should be removed by passing
through activated carbon before use. The residual chlorine testing kits are
available in market but following method can also be used for determination of
residual chlorine in water. Take 10ml of 10% potassium iodide solution in a
250ml conical flask; add 50ml water sample; shake well and titrate the iodine
liberated with N/50 sodium thosulphate solution using freshly prepared 1%
starch solution as indicator, the appearance of color less solution indicates end
point in titration, run a blank also using distilled water without chlorination.

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Free chlorine in PPM = Titration Value in ML (Sample – Blank) x 35.5 x 1000
2500
Titration Chemistry
Cl2 + 2KI ……….. 2KCL + I2
I2 + 2Na2S203 ………… 2NaI + Na2S406
Sodium Thio sulphate Sodium tetra thionate

Take 10ml of 10% potassium iodide solution (10gm KI in 100ml water)

N Sodium thio sulphate solution

50
(wt. – 248.17gm)
1N = 248.17gm/liter of soln
N = 248.17/L of soln = 4.9634gm
50 50

5.6. PH Determination
Principle
PH of solution refers to its hydrogen ion activity. It is also defined as the
logarithm of reciprocal of hydrogen ion concentration.
PH =
1_____ = Log (H+)
Log (H )
+

The PH can be measured either calorimetrically or electrometrically of roughly

by PH papers.
A wide range of indicators have been used for PH determination. The
indicators are salts of week acids and bases. Each indicator has a color in
ionized form different from that of neutral molecule and undergoes various
changes with different hydrogen ion concentration. This method is less
expensive but suffers from interferences due to color, turbidity, salinity,
colloidal matter and various oxidants and reluctant. Colorimetric method is
suitable only for rough estimation.

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The electrometric method is based on the use of electrodes. The potential
developed when the electrode combination is dipped in the solution can be
measured by potentio-meter. Because of convenience the glass electrode is
commonly used. Its potential is not affected by presence of oxidizing or
reducing agents and is operative over a wide PH range. It is also free from
interferences from color, turbidity, colloidal matter or high salinity. PH meter
having temperature exerts two effects on PH measurement the PH potential
varies with temperature. The PH scale extend form ‘one’ (very acidic) ‘14’ (very
alkaline) with ‘7’ exact neutral at 25 .

Colorimetric Method
Apparatus
PH comparator (Lovibond) with indicator dish. (Phenol red)
Reagent
Indicator Phenol red.
Procedure
Place 10ml sample in each or two tubes of comparator. To one add appropriate
quantity of indicator, (0.3ml phenol red). Check that the same indicator disc is
fitted to the comparator. Place tubes in comparator and compare with color
standards and select color nearest to standard. Note PH reading.
Electrometric Method
Apparatus - PH meter
Reagents - Standard PH solutions having PH 7,4.0 or 9.2

Procedure
Standardize the PH meter by immersing the electrode in buffer solution of
know PH (7, 4.0 or 9.2). Normally standardize with buffer having a PH within
1-2 units of that of sample. Measure the temperature of buffer and set

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temperature compensator on PH meter. Read the PH and correctly adjust the
control until meter needle indicate buffer PH.
Rinse the electrode in distilled water and immerse them in sample.
Compensate temperature controller. Let the needle settle at one point. Read
the PH valve.
Significance
The determination of PH value plays an important role in control of Biological
treatment process. The most favorable range for this is from 6.5 to 8.5.
In the sludge digestion process the gaseous stage will be hindered if PH value
goes below 6.0. But for add producing bacteria a PH value of 5.6 to 6.5 is also
tolerable.
The PH value determination plays an important role in the application of
chlorine doses to sewage effluent at various stages of the treatment.

5.7. C.O.D Test (Chemical Oxygen Demand)

General Discussion
The COD test is based on the fact that all the organic compounds with a few
exceptions can be oxidized to CO2 and water by the action of strong oxidizing
agent under acidic condition. During the determination of COD test organic
matter is converted to CO2 and water regardless the biological assemblage of
substance. The oxygen consumed in the test doesn’t differ from unstable of
stable organic matter. Therefore chemical oxygen consumed values are not
directly co-related with biological oxygen demand.
The oxygen consumed in the test is of value which is estimating the strength of
certain wastes and sewages. The major advantage of COD test is that time
required for evolution is less. The determination can be made in about 3 hours
rather than 5 days required for BOD. In case of COD an excess of oxidizing

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agents must be present to ensure that all matter is oxidized completely as in
the power of reagent.

Apparatus - Reflux apparatus, round bottle, 300ml flask and Friendrictis

condensers.
Reagents
i. Conc. H2SO4
ii. Ferro in Indicator
iii. Sliver sulphate crystals
iv. Standard K2Cr2O7 (0.25N)
v. Standard ferrous ammonium sulphate soln. (0.1N)
vi. HgSO4
Procedure
i. Place the diluted 20ml sample with distilled water in round bottom flask
and add to it 10ml of standard di-chromate soln. Then carefully add
30ml of conc. H2SO4. Mix after each addition thoroughly. Add 10mg
Hg suphate and Ag SO.
ii. Attach the flask of free Drichil’s condenser and efflux the mixture for
two hrs. Then wash down the condenser with little distilled water.
iii. Dilute the to about 140ml with distilled water and titrate excess of
dichromate with standard ferrous ammonium sulphate using Ferroin
indicator. The color change is sharp, changing from bluish green to
brown (wine red).
Calculation
mg/Lit of COD = (mi of Fe (NH4)2 SO4 for blank) - (ml of Fe(NH4)2 SO4 for sample x 8000 x 0.25
Ml of sample

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Significance
COD test is extensively used for analysis of industrial waste. It is particularly
valuable to determine and control losses of sewerage systems. Results may be
obtained in relatively short time and measures are taken to correct errors on
the days they occur. The test in conjunction with BOD test is helpful in
indicating organic matter concentration. The test widely used in operation of
treatment of facilities.
5.8. Total Solids
Principle
The term total solids refer to the material left in the vessel after evaporation of
the sample and its subsequent drying in an oven at a defined temperature
(103-150 ). This term includes non-fillerable as well as filterable, non-volatile

as well as volatile residue.

Waters with high residue generally are of inferior palatability and may induce
unfavorable physiological conditions in the consumer. The water having high
percentage of solids is also unsuitable for industrial application.
Dissolved solids are obtained by filtering the given sample before evaporation.
Suspended residue is obtained by filtration. Fixed residue represents reminder
after ignition of one hour at 600
Suspended and Dissolved Solids
i. Procedure
Filter the sample through No. 42 Whitman filter paper. Take a suitable
quantity in a weighed dry dish/crucible/coming or Pyrex dish.
Evaporate to dryness Cool and weight.

Calculation
Filterable Solids, i.e Total Dissolved Solids mg/lit = mg residue x 1000
Ml Sample

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Non filterable solids i.e
Suspended Solids, i.e
Suspended solids mg/lit = Total solids (T.S)- Total Dissolved Solids (T.D.S)
Significance
The suitability of water for drinking purpose is governed by its content of
solids. High solids indicated contamination or presence of excessive amount of
matter.
The suspended solids indicate the strength of seaways and industrial waste. In
larger treatment plants suspended solids determination are used routinely as a
measure of the effectiveness of treatment units.
The determination of fixed solids is essential for the design and preparation of
sludge digestion vacuum frillier and incineration units.

5.9. Total Alkalinity

Principle
Alkalinity of natural waters is due to the presence of weak acids. There are
three types of alkalinity, hydroxide (OH), carbonate (CO3) and bicarbonate
(HCO3). The total alkalinity of water is determined by filtration with a strong
acid to the end point of mixed Bromocresol green, methyl and indicator or
methyl orange (PH4.3) and expressed in CaCO3 scale.
Reagents
1) Mixed Bromocresol green, methyl red indicator or M.O
2) Std. 0.02N H2SO4 or HCI
Procedure

Take 50ml sample in a conical flask. If sample is turbid filter through filter
paper. Add one drop of mixed indicator in the sample. Keep blank (D/W) with

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the same quantity of indicator for comparison. Titrate with 0.02N H2SO4. Note
the first light pink color change. Record the ml of H2SO4 used.
Calculation
Total Alkalinity = ml, of H2SO4 x 0.02 x 50000
Ml of sample
Significance
Information about alkalinity is useful in variety of sanitary engineering practice
e.g.
 Chemical coagulation
 Water softening
 Corrosion and corrosion control
 Industrial waste treatment
 Biological treatment

5.10. Total Alkalinity (By Potentiometric Titration Method)

Alkalinity of water is due to hydroxides, carbonates & bi-carbonates of
elements such as Calcium, Magnesium, Sodium, Potassium or Ammonia and
each expressed in CaCO3 scale. It is quantitative capacity of water to
neutralize from add to designated PH.
Reagents
H2SO4 0.1N or 0.2N
PH Meter
Procedure
Take appropriate quantity of sample in beaker. Immerse an electrode of PH
meter in it and check the PH. Go on titrating the sample with standard add
(0.2N or 0.1N) to the end point PH 3.7. Records the ml of add used.
Calculation
Total Alkalinity = ml of H2SO4 x N of H2SO4 x 50000

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ml of sample
5.11. Volatile Acids
General Discussion
Fatty acids containing 10 or fewer carbon atoms are classified as water-soluble
and those containing more than 10 carbon atoms are classified as water-
insoluble. The weight and acids classified as soluble are only ones that can be
distilled at atmospheric pressure.
The concentration of solids, volatile acids and mineral acids continuously
changes during the straight distillation process. The changes may increase the
yield of volatile acids due to hydrolysis of complex materials by increased
concentration of added mineral acids. The method is quite empirical. It is
assumed that only 70% of volatile acids will be found in the distillate.
Apparatus
Centrifuge, Distillation flask (500ml capacity), condenser
Reagents
 Sulphuric acid concentrated
 Standard NaOH soln. (0.1N)
 Phenolphthalein – Indicator
Procedure
Centrifuge 50ml of sample for 5 minutes. Pour off 4 combined supematant
liquors. Place 100ml of supematant liquor in 500ml distillation flask. Add
100ml water and 4 to 5 glass beads to prevent bumping. Add 5ml of H2SO4.
Mix so that acids do not remain at bottom of flask and distillate. Collect 150ml
of distillate in conical flask and titrate with 9.1N NaOH soln. and
phenolphthalein indicator. The end PH is faint pink coloration.
Calculation
Mg/lit of volatile acids = ml of NaOH x 0.1 x 60000
Ml of sample x 0.70

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6. Reagents used in the methods for factory control
6.1. Introduction

Reagents bottles should always be clearly labeled with the name and strength
of the reagent and its date of preparation. Distilled water or deionizer water
should be used in preparing reagents and volume should completed at a
temperature as close to 200c as possible.
6.2. Reagents Preparation and standardization
Acetic Acid (1.0M)
Measure 57ml glacial acetic acid (CH3COOH) (AR) in a measuring cylinder,
transfer to 1000ml volumetric flask, and make to the mark with distilled water.
Standardizes as follows
Apparatus
 Pipette (20ml)
 Burette (50Ml)
 Erlenmeyer flask (250Ml)
Reagents:
 Sodium hydroxide (1.0M)
 Mixed indicator
Procedure:

a) Pipettes 20ml of the prepared acetic acid solution into Erlenmeyer flask, add
a few drops of indicator solution and titrate with standardized 1.0M sodium
Hydroxide solution to the first appearance of a purple color.
Calculation
If, V1 is the volume in ml of 1.0M sodium hydroxide, then the morality (M1) of
acetic acid solution is:
M1 = V1 x 1

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20
Where,
V2 = equivalent volume of acetic acid for 1.0M
V1 = Titre of sodium hydroxide used
M1 = Actual Morality of acetic acid

Acetic acid (2M): Measure 114.5ml (120g) glacial acetic acid (CH3C00H) (AR)
in a measuring cylinder and make to the mark with distilled water in a 100 ml
volumetric flask.
Acetic Acid (30%): Measure 300ml glacial acetic acid ( CH3 OOH) (AR) in a
measuring cylinder and make to the mark with distilled water in a 1000ml
volumetric flask
Alpha Naphtol (5%): (Alcoholic solution for quantitative sugar test) weight 5g
of alphnaphtal (CH3C4H3 OH), dissolve in absolute ethanol alcohol, transfer
quantitatively in 100ml volumetric flask and make to the mark with absolute
alcohol store in amber bottle.

Alpha Naphtol (20% alcoholic solution): Weigh 20g of alpha Naphtol

(CH3C4H3OH), dissolve in absolute ethanol, transfer quantitatively in 100ml
volumetric flask and make to the mark with absolute alcohol. Store in amber
bottle.

Ammonia solution (PH 10): Add 142 ml concentrated ammonia solution (AR)
to 17.59g ammonia chloride (NH4Cl) (AR) and dilute 250ml with distilled in a
volumetric flask.
Ammonia molybdace (9%, PH= 7-8): Dissolve 10g powdered ammonium
molybdate tetra hydrate (NH4)6Mo7O24.4H2O) (AR) in a hot distilled water, cool
and make to the mark in a 100ml Volumetric flask. Filter through what man

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91 filter paper or equivalent (185ø). Add 8M Sodium hydroxide solution (Ca. 8ml)

to increase the PH to between 7 and 8.

Ammonium molybdate solution (for colorimetric determination of

phosphate)
Dissolve 10g of ammonium molybdate in distilled water to produce 100ml. Add
the solution slowly to a cooled mixture of 150ml concentrated sulphuric acid
and 150ml distilled water. (This solution keeps infinitely if stored in
borosilicate bottle and protected from light)
Barium Chloride solution (for determination of free sodium hydroxide)
Transfer 100g of barium chloride, Bacl2,2H20 (AR) to a 1000ml volumetric flask
Add about 500ml prepared distilled water, mix until dissolved and make to
volume. Mix thoroughly.
Buffer solution (PH 10) (for total hardness determination):
Add 142ml concentrated ammonia solution (AR) to 17.5g ammonia chloride
(NH4Cl)(AR), and dilute to 250ml with distilled water in a volumetric flask.

Copper Solution (knight and Allen): Add 8ml of 1M Sodium Hydroxide to

about 120ml-distilled water. Dissolve 5g anhydrous Sodium Carbonate
(Na2CO3) (AR) and 5g sodium potassium tartaratetetrahyolrate (KOOC.HCOH.
HCOH) COONa.4H2O) and either 50g disodium hydrogen phosphate
dodecahyorate (Na2HPO4 12H2O)(AR) or 14.8g of anhydrous disodium hydrogen
phosphate (NaHPO4) in about 900ml distilled water, warming finally to effect
solution. When completely dissolved, continue heating for 2 hours on a water
bath. Cool and make to 1000ml in a volumetric flask. Treat with
approximately 5g activated carbon and filter through a fluted filter paper (S
and S 613 or equivalent). Store in a dark bottle.

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EDTA (0.0025M): (Ethylenediaminetetra - acetic acid disodium salt, dihydrate
(CH2COONa)2 = N.CH2CH2N = (CH2COOH)2 .2H2O). Dissolve 0.9306g EDTA
(AR) in distilled water and dilute to 1000ml in a volumetric flask.

EDTA (0.01M): Dissolve 1.8612g EDTA (AR) in distilled water and dilute to
500ml in a volumetric flask

EDTA (Ca. 0.05M, standardized): Dissolve 18.6g EDTA (AR) in distilled water
and dilute to 1000 ml in a volumetric flask.

Standardize as follows:
Apparatus
 Pipette (20ml)
 Burette (50ml)
 Erlenmeyer flask (250ml)
 Measuring cylinder (50ml)
 Spatula (non-metallic)
Reagents
 Standard lead Nitrate Solution (0.05M)
 Hexamethylenetetramine (HMTA) (Ca. 1.0M)
 Metal indicator (solid)
 Methylene blue indicator

Procedure
a) Pipette 20ml of standard lead nitrate solution in Erlenmeyer flask
b) Add 20ml HMTA from measuring cylinder, 100mg of the solid metal
indicator and 4 drops of methylene blue indicator. Titrate with the
EDTA Solution. Until the first appearance of a green color.
Calculation:

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If, V1 is the volume in ml of EDTA solution used, then the moralities (M1) of the
EDTA solution is:
M1 = 20 x 0.05
V1
Note:
If the molarity is not 0.05M convert titers obtained using this EDTA Solution to
the equivalent volumes 0.05M. use the Formula, V2 = V1*M1
0.05

Where; V2 = Equivalent volume for 0.05m EDTA

V1 =titre of EDTA Solution used
M1 = actual molarity of standard EDTA

EDTA (4%): weigh 4g of EDTA (AR), dissolve in distilled water and dilute to
100ml in a volumetric flask.

Eriochrome Black T Indicator: Mix 0.1g Eriochrome black T (C6H4).

(C4H2OH). N = N. (C6H2OH. SO3Na). (C4H2NO2) with Ca. 30,0g potassium
chloride (KC1) or sodium chloride (Nac1). Grind the mixture in a glass mortar.

Ethanol (80%): To 800ml 98% ethanol (C2H5OH), add 200ml distilled water
and mix well.

Ethanol (Filtered): Filter 98% Ethanol through S and S 613 filter paper or
equivalent.

Ethanol (acidified): Add 100 m1 concentrated hydrochloric acid (HCl) (AR)

and 100ml distilled water to 1000 ml filtered 98% ethanol (C 2H5OH) and
shake.
Fehlings solution (I and II)

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Solution I: Dissolve 69. 278g copper sulphatepentahydrate (CuS04.5H20) (AR)
in distilled water and dilute to a 1000ml in a volumetric flask.

Solution II: Dissolve 346g sodium potassium tartrate tetra hydrate (KOOC.
HCOH.HCOH.COONa .4H2O) in 500ml distilled water. Dissolve 100g sodium
Hydroxide (NaOH) (AR) in 200ml of distilled water and cool to room
temperature. Transfer both these solutions quantitatively in to a 1000 ml
volumetric flask. Mix thoroughly and make to the mark with distilled water.
Filter though S and S 613 filter paper or equivalent.

Note: It is necessary to keep solutions II and I separate.

Standardization of Fehlings Solutions

Apparatus:
 Volumetric flask (250ml)
 Pipettes (5m1, 50ml)
 Burette (50ml
 Erlenmeyer flask (250ml)
Reagents:
 Standard invert solution
 Fehlings I solution
 Fehlings II solution
 Methylene blue indicator solution (1%)
Procedure:
a) Pipette 50ml invert solution (see under invert solution) into a 250ml
volumetric and dilute to volume with distilled water.
b) Fill the burrette with the diluted standard invert solution.

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c) Pipette 5ml of fehlings I solution and 5 ml of fehlings II solution into a
250ml Erlenmeyer flask and titrate against the standard invert
solution using the Eynon and Lane procedure.
Calculation:
If the fehlings solution is of correct strength 25.64 ml of invert solution will be
used. If less than 25.64ml of invert solution is required, the fehlings, solution
is too week and copper sulphate should be added to solution I until desired
25.64ml is used . Similarly, if more than 25.64ml is used, solution I should be
diluted until 5ml fehlings I plus 5ml Fehlings II solution corresponds to
25.64ml invert solution.

Note: The practice of using a factor when the Fehlings solution was not of the
correct strength has been found to be unsatisfactory.

Formaldehyde solution (0.2%): Dilute 5ml 40% formaldehyde to 1000ml in a

volumetric flask with distilled water.

HHSNNA Indicator: Mix 0.5g HHSNNA (C6H4).C4H. COOH.OH).N = N

(C6H.OH.SO3H). (C4H4) with 50g solid an hydrous sodium Sulphite (Na2SO4).

Hydrochloric Acid (0.05M): Pipette 10ml 1M Hydrochloric acid into a 200ml

volumetric flask and make to volume with distilled water.

Hydrochloric Acid (0.1M): Take 8.9ml of concentrated hydrochloric acid (HCl)

(AR) and dilute to 1000 ml in a volumetric flask.

Hydrochloric Acid (0.5M): Take 44.5ml concentrated hydrochloric acid (HCl)

(AR) and dilute to 1000ml in a volumetric flask.

Hydrochloric acid (1M): Take 89ml of concentrated hydrochloric acid (HCl)

(AR) and dilute 1000ml in a volumetric flask.

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Hydrochloric acid (SG = 1.103): mix together 500ml concentrated
hydrochloric acid (HCl) (AR) (SG= 1.16) and 357ml distilled water. Allow to cool
to 200c. Measure SG of the solution with a hydrometer. The SG should be
1.103. If the SG is not 1.103, then:

a. If it is less than 1.103 add 2 ml of concentrated hydrochloric acid, mix

well and measure the SG of the solution. If the SG is still too low, add
further 2ml portion of concentrated acid and after each addition, mix
thoroughly and measure the SG until the desired SG is reached. still too
low, add further 2 ml potions of concentrated acid and after each
addition, mix thoroughly and measure the SG until the desired SG is
reached

b. If the SG is more than 1. 103, add 2ml distilled water mix well and
measure the SG of the solution. If the SG is still too high, add further
2ml portions of distilled water and after each addition mix thoroughly
and measure the SG until the desired SG is reached.

Hydrochloric Acid (1:1): Mix together 500ml Concentrated hydrochloric acid

(HCl) (AR) (SG=1.16) and 500ml of distilled water.

Invert solution (standard): Dissolve 9.500g sucrose (C12H22O11) (AR) in 70 ml

distilled water in 250 ml beaker. Add 10 ml hydrochloric acid (SG = 1.103) and
stand at room temperature for 5 days. Add sufficient 4m Sodium hydroxide
solution (Ca. 32 ml) to raise the PH to about 3, using a PH meter and transfer
to a 1000 ml Volumetric flask. Dissolve 2 g benzoic acid (C6H5COOH) in
distilled water (800 ml) by boiling, cool and add to the volumetric flask. Make
to volume with distilled water. This solution Contains 1g invert Per 100 ml.
Iodine solution (0.05M): Dissolve 70 g potassium iodine (KI) (AR) in 100 ml of
distilled water. Dissolve 12.691 g iodine (I2) (AR) in this solution. Transfer

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quantitatively to a 1000ml volumetric flask and make to the mark with distilled
water. Keep in a dark bottle.

Iodine solution (0.0141m): Dissolve 25 g potassium iodide (KI) (AR) in 25 ml

distilled water in a 1000 ml volumetric flask. Add 3.6 g iodine (I2) and dissolve.
Make too the mark with distilled water. Keep in a dark bottle.

Iodine Solution (0.0165m) (ofner): Dissolve 10g of iodate free potassium

iodide (KI) (AR) in 10ml of distilled water in a 50 ml beaker. Add 2.05 g Iodide
(I2) (AR) and dissolve. Transfer quantitatively to a 500 ml volumetric flask and
make to the with distilled water. Keep this solution in a dark bottle.

Methyl Orange Indicator: Dissolve 0.05g methyl orange (NaO3S. C6H4.N. = N.

C6H4.N (CH3)2) in distilled water in 100 ml volumetric flask. Add 1.5 ml 0.1M
hydrochloric acid and make to the volume with distilled water.

Methylene blue Indicator (1%): weigh 1.0 g Methylene blue

((CH3)2.N.C6H3.(N).(S). C6H3.N(CH3)2+. Cl-) dissolve in distilled water and dilute
to 100 ml in a volumetric flask.

Methylene blue indicator (0.02%): Dissolve 40Mg Methylene blue (( CH3)2N.

C6H3 (N) (S) C6H3N(CH3+2.C1-) in distilled water, transfer to a 200ml volumetric
flask and make to the mark with distilled water.
Phenolphthalein indicator (1%): weigh 1.0g phenolphthalein (C6H4CO.O.C =
(C6H4OH)2), dissolve in ethanol and dilute to 100ml in a volumetric flask.

Phosphate (Standard Solution): To prepare a standard stock solution dissolve

0.7668g potassium dihydrogen orthophosphate (KH2PO4) (AR) in distilled
water and transfer to a 1000ml volumetric flask. Add 10ml concentrated
sulphuric acid (AR) and dilute to volume. This standard keeps indefinitely.
1ml = 0.4g P2O5

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To prepare a calibration graph, dilute 25ml of the above standard to 1000 ml.
Then 1ml = 0.01ng P2O5
Methyl red indicator: weigh 0.1g of methyl red in 18.6ml of 0.02N NaOH.
Dilute to 250ml with water.

Erichrome black T- indicator: To 30ml of distilled water add 1ml of in

sodium carbonate solution and 1.0g of Erichrome black T- and mix well. Make
to 100ml with isopropyle alcohol and mix store in amber colored bottle.

Reducing solution: Dissolve 180g sodium metabisulphite (Na2S2O5) (AR) in

1400ml distilled water. Dissolve 114.0g anhydrous sodium sulphate (NO2SO3)
and 3g 1-amino-2- naphthol-4 Sulphate acid (C6H4). (C4H. NH2OH. SO3H) in
200ml distilled water mix these two solutions and dilute to 2000ml stand
overnight and filter through glass wool or fluted what man No. 91 filter paper
or equivalent. Store in a refrigerator. This solution has a maximum useful life
of 2-months. Remove the required amount of reducing solution from the
refrigerator about 1 hour before use and allow to warm to room temperature.
Juice preservative (mercury chloride solution): prepare a saturated solution
of the salt in ethanol alcohol. Store in automatic dispenser and use at the rate
of 0.5ml per liter of juice (Dissolve 300 g of mercury chloride salt in 5 liters of
alcohol).
Rosaniline (bleached) : suspend 1g rosaniline hydrochloride (NH2.C6H4).
(C6H2.NH3 CH3). C = (C6H4 NH) HCl) in 100ml distilled water. Heat to 500c and
allow to cool with shaking stand for 48 hours, then filter the solution. The
solution may be bottled and stored for subsequent use.
Pipette 4ml of the solution into a 100ml volumetric flask, add 6ml
Concentrated hydrochloric Acid (AR), and make to volume with distilled water.
Stand for at least one hour before use.

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Sodium Carbonate solution (1N): weigh 5.3 gm of sodium carbonate, special
anhydrous, on analytical balance and transfer to 100ml Volumetric flask. Fill
to about half full with distilled water and dissolve. Complete to Volume with
distilled water and mix.

Sodium chloride solution (0.0171M) : Dry sodium chloride (NaCl)(AR) for one
hour at 1400c. Dissolve 1.000 g dry sodium chloride in distilled water. Transfer
to 1000ml ml volumetric flask, make to the mark and mix.

Sodium Hydroxide (0.1M) for Lead determination: dissolve 4g sodium

Hydroxide (NaOH) (AR) pellets in distilled water and dilute to 100 ml in a
volumetric flask.

Standardization
Apparatus
 Burette (50 Ml)
 Pipette (20 Ml)
 Erlenmeyer flask (250 Ml)
Reagents:
 Standard lead nitrate solution (0.05M)
 Mixed Indicator solution
Procedure
a) Pipette 20ml of standard lead nitrate solution in to Erlenmeyer flask.
b) Add the volume of the Ca. 0.05M EDTA obtained in the standardization
of EDTA against the standard lead nitrate
c) Add a few drops mixed indicator solution and titrate with sodium
hydroxide solution to the first appearance of Purple color.

Calculation
If, V1 is the volume in ml of the ca. 0.1m sodium hydroxide used, then
Molarity (M1) of sodium hydroxide is:

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M1 = 20 x 0.05 x 2
V1
Note:
If the Molarity is not 0.1, then to convert titers obtained using this solution of
sodium hydroxide to the equivalent volumes of 0.1M, use the formula:
V2 = V1 * Ml
0.1
Where; V2 = equivalent volume for 0.1M sodium hydroxide
V1 = titer of sodium hydroxide
M1 = molality of sodium hydroxide solution.

Sodium Hydroxide (0.05M): Pipette 10ml 1M sodium hydroxide into a 200ml

volumetric flask and make to volume with distilled water.

Sodium Hydroxide (1M): Dissolve 40g sodium Hydroxide (NaOH) (AR) pellets
in distilled water. After cooling make to volume in a 1000 ml volumetric flask.

Sodium Hydroxide (8M): weigh 80 g sodium hydroxide (NaOH)(AR) pellets and

dissolve in distilled water. Transfer into a 250 ml volumetric flask, dilute
almost to the mark, cool and make to volume.

Starch Indicator (1%): weigh 1g soluble starch into a 100ml beaker and 20ml
distilled water. Boil the solution for 1 minute. Cool and make 100 ml-distilled
water.

Sugar solution (10%): weigh 100g of refined sugar into a 1 liter beaker, add
Ca. 300ml of distilled water and stir until the sugar is dissolved. Transfer this
solution quantitatively into a 1000ml volumetric flask, mix thoroughly, and
then make to the mark.

Sugar Solution (Saturated): Add 300 g distilled water to 700 g sugar. Stir at
room temperature until no further sugar dissolves (approximately 12 hours).

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Check that the solution is saturated by measuring the brix and the sugar
solubility table. Filter the syrup through coarse Calico or equivalent filter
paper. The above quantity will make approximately 850 ml syrup.

Sulphuric Acid (0.1M): Pour 5.6ml Concentrated Sulphuric acid (H2S04) (AR)
into about 200ml distilled water with swirling cool and make to volume in a
1000ml volumetric flask.

Sulphuric Acid (0.01M): pipette 50ml 0.1M Sulphuric acid into a 500ml
volumetric flask and make to the mark with distilled water.

Sulphuric Acid (0.5M): Slowly pour 28ml concentrated Sulphuric acid (H2S04)
(AR) into about 200ml-distilled water with swirling. Keep the bleaker in cold-
water bath while adding the acid to the water. Cool and transfer to a 1000ml
volumetric flask. Make to volume with distilled water.

Total alkalinity indicator solution: Dissolve 20mg methyl red ((CH3)2 N.

C6H4.N.N.C6H4 COOH) and 100mg of sodium salt of bromocresol green (Br2OH
C6H2 C ((C6H4) SO3H) : C6H2. O. Br2) in 100ml distilled water.

Standardization of acids and alkalis Standardization of alkalis

Dry about 20g potassium hydrogen phthalate (C6H5 COOH. Cook) at 1200c for
2 hours and allow to cool in a desiccator. Weigh to + 1mg Ca. 4.1 g in pol dish
and transfer to a 250ml Erlenmeyer flask. Dissolve in about 30ml-distilled
water and titrate with 1M sodium hydroxide using phenolphthalein as
indicator. It is desirable to carry out the titration at 500c to keep out C02.
Calculation
Let the actual mass of salt taken = a g
Let the titre of NaOH = Y ml
4.0843g C6H4 COOH Cook = 20 ml of 1M NaOH

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Therefore,
Exact Morality = 20 x a
4.0843 x Y

The above titration should be done in triplicate and titres should agree with in
0.1 ml.
This standard alkali can now be used to standardization 1M Hydrochloric acid.

Standardization of acids
Pipette 20ml acid into a 250ml Erlenmeyer flask and titre against sodium
hydroxide using phenolphthalein as an indicator.

Calculation
Molarity HCl =exact molarity NaOH x titreNaOH
20
The titration should also be done in triplicate and titres should agene with in
0.1ml. For Sulphuric Acid, the calculation is:
Molarity H2SO4 =½ (exact molarity NaOH X titreNaOH)
20
Standard dextran solution (1mg/ml):
Determine the moisture content of Pharmacia dextrin 110 by weighing out 2 +
0.0001g and drying in an oven at 1050c for 3hrs. Weigh out a quantity of un
dried dextrin containing 0.100g anhydrous dextrin, i.e.
0.1000 x 100
100 - % moisture of undried dextran
Dissolve and make up 100 ml in a volumetric flask.

Trichloroacetic Acid Solution TEA (10% w/v): Take 10 + 0.1 g of

trichloroacetic Acid in a 100ml volumetric flask. Make up to the mark by

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distilled water. This solution keeps for two weeks. It also attacks protein.
Therefore don't pipette TCA by moved or no contact with skin.

Denatured absolute alcohol (for dextran determination): it is a mixture of

ed absolute ethanol and methanol (2% methanol).

Ion exchange resin mixture: this is prepared by thoroughly mixing equal

Wright of dried" Amberlite” yellow IR45 anion exchange resin and bleak IR120
cat ion exchange resin. (Normally the mixture is available in the market).

Sucrose solution (200Bx): AR sucrose (21.6 + 0.5g) is

Dissolved and diluted to 100ml.

Sucrose standardization solution (50%w/v) for dextran determination):

sucrose AR (50.0 + 0.1g) is dissolved in distilled water and diluted to 100ml.
This solution should be prepared freshly as required.

BIOLOGRAPHY

1. KBK chemical Engineering Pvt.Ltd Laboratory manual,

2. Integrated CASETECH Laboratory work instructions,
3. JP Mukherji &Association Pvt.Ltd. lab procedures
4. Methara Sugar Factory Quality Control laboratory manual

Diffusion

 Factory Operation Manager

 Ethanol Plant

 Ethanol Processing Section

Approval

Name: Tegegn Abebe Signature: Date:

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7. Annexiture

Table 7.1. Typical Analysis report of Indian cane molasses

Test Value
S/N Particulars Sample A Sample B
1 Brix 87 88

2 Moisture content (%) 21.51 16.75

3 Total suspended solids (%) 3.83 5.47
4 Total dissolved solids (%) 74.66 77.78
5 PH of molasses 4.17 4.21
6 Reducing sugars (%)by wt. 22.68 23.18
7 Total reducing sugars (%) by mass 56.26 55.66
8 Total fermentable sugars (%) by mass 50.22 50.13
9 Calcium content (gms 100 brix) 2.40 2.75

10 Fermentable Non fermentable ratio (FN) 2.05 1,81

11 Carbonated ash (%) 9.25 10.50
12 Sulphated ash (%) 12.26 13.57
13 Nitrogen (% of molasses) 0.982 0.992
14 Potassium (% of ash) 16.50 15.27
15 Sodium (% of ash) 0.93 0.93
16 Chlorides (% of ash) 13.3 14.24
17 Phosphates (%of ash) 0.32 0.26
18 Total organic volatile acids (Mg lit.) 3500 2700

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Table 7.2. Indian Standard Specifications for Cane Molasses
(I.s.I.NO.1162-1958(Reaffirmed 1976)
Requirements for- Grade
S/N Characteristics
1st 2nd 3rd
1 Density, in Brix at 27.5°𝑐 Min. 85 80 80
2 Sulphated Ash. (%) by mass (Calculated for
100 Brix), Max. 14.00 17.50 17.50
3 Total reducing matter as invert sugar (%)
by mass, Min. 50.00 44.00 40.00

Table 7.3. Indian Standard Specifications for Rectified Spirit, (IS: 323-----1959)
Requirement of Rectified Spirit
S/N Characteristics –Required for-
Grade – I Grade - II
1 Specific gravity at 15.6 /15.6 (or 60 /60 ) 0.8171 0.8171
Ethanol Content:-
2 a) Percent by volume 15.6 (or 60 ) Min. 94.68 94.68
b) Degrees Over-proof, Min.
66.00 66.00
3 Miscibility with water Miscible Miscible
4 Alkalinity NIL NIL
5 Acidity (as CH3 COOH) % by weight, Max. 0.002 0.01
6 Residue on evaporation % by weight, max. 0.005 0.01
7 Aldehyde content (as CH3CHO) gms/100ml,
max. 0.006 0.10
Ester content (as CH3COOC2H5) gms/100ml,
8 max. 0.02 --
9 Copper (as Cu), gms/100ml, max. 0.0004 --
10 Lead (as Pb), gms/100ml, Max. 0.0001 --
To Satisfy the
11 Methyl alcohol content requirement of the test --
To satisfy the
12 Fusel oil content requirement of the test --
To satisfy the
13 Furfural content requirement of the test --

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Table 7.4. Indian Standard Specification for Absolute Alcohol

-Requirement of Absolute Alcohol for-

S/N Characteristic Special
Grade Grade -1 Grade -2
1 Specific Gravity, at 15.6 /15.6 ,
Max. 0.7961 0.7961 0.7961
2 Ethanol content, percent by
volume at 15.6 , Min. 99.50 99.50 99.50
3 Miscibility with water Miscible Miscible Miscible
4 Alkalinity NIL NIL NIL
5 Acidity (as CH3COOH),
gms/100ml, Max. 0.006 0.006 0.006
6 Residue on evaporation
gms/100ml, Max. 0.005 0.005 0.005
7 Ester content (as CH3Cooc2H5)
gms/100ml, Max. 0.02 -- --
8 Aldehyde content (as CH3CHO)
gms/100ml, Max 0.10 0.006 0.10
To satisfy the
10 Methyl alcohol content -- requirement of --
the test
-- To satisfy the
11 Fuel oil content requirement of --
the test
To satisfy the
12 Ketones, isopropyl alcohol and -- requirement of --
tertiary butyl alcohol the test
13 Copper (as Cu), gms/100ml,
Max. -- 0.0004 --
14 Total sulfur and compounds of
sulfur (as S), % by mass, Max 0.001 -- --
15 Sulfur –oxide (as SO2), % by
weight, Max. 0.00005 -- --

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Table 7.5. Indian Standard Specification for Neutral Spirit of Alcoholic Drinks

S/N Characteristic Requirement

1 Relative density at 15/15 0.81245 to 0.81679

2 Ethanol Content, percent by Vol. at 15.6 94 to 95

3 Miscibility with water Miscible

4 Alkalinity NIL
5 Acidity (as CH3COOH) gm./100ml. Max 0.002
6 Residue on evaporation gm./100ml. Max 0.002
7 Esters (as CH3OOC2H5) 0.01
8 Lead (as Pb), gms/100ml, Max. NIL
9 Methyl alcohol content To passes the test
10 Furfural content To passes the test
11 Aldehyde (as CH3CHO), gms/100ml, Max. 0.004
12 Permanganate reaction time, Minutes. Min. 30
13 Copper (cu), gms/100ml, Max. 0.002
14 Isopropyl alcohol, acetone and other Ketoses To pass the test

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Table 7.6. General Characteristics of Distillery Effluent

S/N Characteristics Continuous Batch Process

Process(Biostil)
9 liters per liter 15 liters per liter
1 Quantity of spent wash production of production of
Alcohol Alcohol
2 Temperature of spent wash 100 100

3 Odor Jiggery Jiggery

4 Color Dark Brown Dark Brown
5 PH 4.3-4.6 4.3 – 4.6
6 BOD. Mg/lit. 90000 – 95000 30000 – 40000
7 COD. Mg/lit. 190000-200000 85000-95000
8 Total solids, Mg/lit. 270000-280000 80000-90000
9 Potassium (K), mg/lit. 18000-20000 8000-10000
10 Chlorides. Mg/lit. 13000-15000 5000-6000
11 Sulphates, Mg/lit. 15000-18000 2000-5000
12 Phosphates as PO4, Mg/lit. 1000-1500 800-1200
13 Calcium (Ca), Mg/lit. 2600 – 2700 500 – 600
14 Nitrogen (KTN), Mg/lit. 2000 – 2500 1000 – 1200
15 Sodium (N), Mg/lit. 300 – 500 200 - 300

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Table 7.7. Alcohol Yield of final molasses for different Fermentable sugar at
88.7% Overall efficiency
Fermentable Sugars in Molasses Alcohol Yield
% w/w Lit/ton of molasses
35 200
36 206
37 211
38 217
39 223
40 228
41 234
42 240
43 246
44 251
45 257
46 263
47 268
48 274
49 280
50 285
51 291
52 297
53 303
54 308
55 314

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