This document describes the process of ethanol determination through fractional distillation. It involves fermenting a substrate like grains to produce ethanol, then collecting alcohol fractions through distillation. The distillation unit heats a fermented sample to its boiling point, vaporizes the more volatile ethanol and condenses the vapors, collecting them in aliquots. Specific gravity measurements of the distillate, residue and original sample allow calculating the ethanol content. Temperature control is important for accuracy.

1. ETHANOL
DETERMINATION BY
DISTILLATION
By Sreermya.S
Lecturer,
Dept Of Biotechnology, Mercy College,
Palakkad

2.  Many microorganisms, notably yeasts and bacteria, extract
energy from their food (glucose) by fermentation. The overall
chemical reaction for this is: C6H12O6 2CO2 +
2CH3CH2OH (ethyl alcohol).
 It was this process which Louis Pasteur studied, leading to a
biochemical understanding of biological processes. Humans
have known about and utilized the process of fermentation for
many thousands of years. CO2 liberated by yeast cells doing
alcohol fermentation causes bread to rise.
 The Egyptians and many subsequent civilizations have
fermented grains such as barley to break the starch down to
malt (maltose), then glucose, and finally alcohol. For at least
that long, people have known that various fruits, especially
grapes, could also be fermented to produce alcoholic
beverages.

3. Distillation unit

4.  The amount of extract present in a wort or mash sample is
determined by measuring the specific gravity of the sample.
 Generally a higher specific gravity relates to a higher
concentration of the wort or mash. The, readily available,
tables of conversion relating the concentration of sucrose
(sugar) to specific gravity which brewers often rely on today is
that developed by Plato around 1900. This very exact table
replaced a less accurate one developed by Balling. Wort
concentrations in percent sugar by weight (wt/wt) derived from
Plato’s table should be called degrees Plato (°P), but
sometimes the term Balling is used even though Balling’s table
is not used for the determination.
 Other industries also use Plato’s table to calculate percent
sugar by weight and call it degrees Brix.
 For all intents and purposes Balling, Brix and Plato all mean
the same thing – percent sugar based on Plato’s table.

5.  Pycnometry: Plato measured specific
gravities by the use of a pycnometer, still a
very accurate method. Pycnometry is
covered in a separate protocol note sheet
presented below and further details are to
be found there.

6.  Specific gravity is the expression of density of an unknown
liquid compared to the density of water. Because the density
(weight per unit volume) of a liquid changes with temperature
the temperature at which weights are taken in Pycnometry has
to be specified and controlled. Most countries accept 20 0C as
the specified temperature. The specific gravity of a wort
sample is obtained by pycnometry at the specified temperature
and th corresponding percentage of extract (equivalent) to
sucrose) is determined from Plato’s table.
 The accuracy of the measurement depends on the accuracy of
the specific gravity measurement. A change in specific gravity
from 1.0151 to 1.0152 results in a change in extract of 0.03 °P
(3.85 to 3.88). A change in temperature of just 1 °C results in a
change I calculated extract of about 0.06%. Controlling the
temperature within 0.5 degrees and measuring weight to within
10 mg (0.01 g) in a 100 mL volume should give results accurate
enough for most purposes in a small brewery.

7.  Hand Refractometers for Brewing and Distilling
Operations.
 Refractometers can be used to give an indication of the
sugar content of Distiller’s mashes, and molasses
samples and also for Brewer’s worts.
 Refractometers were designed for use in the juice and
soft-drinks industry and are calibrated in Brix, which is
percent sugar, the same as °Plato (used by Brewers).
 It should always be remembered that they were
designed to measure sugar content in pure sugar
solutions and that some calibrations and factors need to
be considered when using refractometers in other
solutions such as mashes and worts.
 Nevertheless modern refractometers are easy to use can
be useful for monitoring fermentation performance and
relative sugar contents in final products.

8.  Fractional distillation (collecting the distillate in aliquots rather than the
whole solution). This is the source of liquors with higheralcohol contents.A
liquid must be brought just slightl above its boiling point before bubbl
initiation can begin to start it boiling. As a bubble of vapor appears within
the liquid, I may do one of two things: if it is below minimum size, it will
collapse because of the surface tension of the liquid, or if it is large than the
critical size, grow and rise to th surface of the liquid. If a liquid, which is free
of solid impurities or dissolved gases, I heated slowly, a temperature much
above th boiling point can be reached without any boiling actually taking
place. Thi superheating occurs because extra energy I required before
bubble formation is initiated. If a bubble should start to form in such a
superheated solution, it might suddenly growith almost explosive violence
enough to shatter the container.
 This problem, called bumping, can be overcome by adding boiling
chip, a piece of porous material, to the liquid before it is heated to the
boiling point.
 The pores act as built-in bubbles so that a June 19, 2011 10 liquid cannot
superheat. As the distillation proceeds, the air in these pores is replaced by
vapor of the distilling material, but this vapor cannot condense because the
temperature of the liquid is just slightly above its boiling

9. ARRANGEMENT OF FRACTIONAL
DISTILLATION

10. PROCEDURE
 Pour beer into 100 mL volumetric flask (use the one labeled 7 as its mark is high u on the
neck.) Use a 1 mL Tensette pipet to suck foam out of neck. Fill to the mark or just above.
Stopper and place in 20°C water bath.
 2. While beer is attemperating pull microwave oven away from wall. Place jack stand on
top of microwave oven. Elevate so that top of platform is about 8” above top of microwave.
Place 500 mL mantle on top of jack stand with control knob facing about 30° to the right of
forward.
 3. Place Cole-Parmer ringstand (with white base) to left of microwave oven. Base shoul be
behind rod and rod should carry two clamps oriented away form base i.e. towards front of
microwave.
 4. After beer has attemperated, suck out excess beer and foam so level is at the mark.
 5. Fill two 25 mL flasks with DI water to near the marks.

11.  6. Pour beer into 500 mL distillation flask. Rinse with two 25 mL portions
of DI water Add a couple of carborundum boiling stones.
 7. Place distillation flask in mantle. Stabilize with clamp on mantle rod.
 8. Place Kjeldahl trap in flask neck. Stabilize with clamp on mantle rod.
 9. Connect bridge adapter to top of trap. Be sure thermometer port is
plugged with glass
 stopper.
 10. Rinse 100 mL volumetric flask with DI water and place in 500 mL
beaker on counter
 top.

12.  11.Place straight adapter on end of 400 mm condenser and
while holding it in place lower its end into volumetric flask.
Place condenser into two clamps on Cole Parmer ringstand
without tightening. Simultaneously lower jack and position
receiver, ringstand and bridge adapter angle so that bridge
adapter mates with top of condenser. Adjust postitions so that
all glass joints are tight and so that there is a little clearance
between the straight adapter and the mouth of the volumetric
flask. Tighten the clamps.
 12. Make and check the cooling water connections.
 13. Surround the receiver with ice cubes in a little water, turn
on the cooling water and set the mantle control to 10 for 10
minutes, then lower heat setting to 6. There should be some
reflux from the neck of the flask during this time.

13.  15. Boiling should commence within a minute or so and reflux from the walls of the
Kjeldahl adapter should start. If all goes well, the alcohol in this reflux should
collapse the foam. If it doesn’t, well that’s what the trap is there for.
 16. After 20 minutes (total) foaming should be under control and alcohol should be
seen dropping from condenser tip into straight adapter. Advance heat to 7. After
stabilizing, condensate should appear at about 10 - 20 drops per minute.
 17. After 1 hour (total) raise heat to 8. Check tightness of glass joints.
 18. During distillation tare pycnometer, if pycnometer is being used, and obtain
weight
 measurement with DI water at 20°C
 19. Collect just under 100 mL of distillate. This should take about 2 and three
quarters hours.
 20. Place volumetric flask in water bath. After attemperation, make to mark with DI
water. Mix throroughly. Measure and record density, specific gravity and OIML ABV.
Find ASBC ABV from ASBC tables. Calculate ABW from grams/100 mL in ASBC
tables.
 21. Transfer residue from distillation flask to same volumetric flask. Rinse distillatio
flask with 25 mL portions of DI water. Place in water bath and make to mark with DI
water. Measure and record density, specific gravity and °P.
 22. Measure and record specific gravity, density and °P of beer.
 23. Do the sums!

14. THANK YOU