3.

A n a ly tic a l m e th o d s

3.6.1

S p e c tr o p h o to m e tr ic d e te r m in a tio n o f e th a n o l (C a p u ti et al 1 9 68 )
On e

m illilitre

o f th e

fe rm e n te d

w ash

w a s ta k e n

in

5 00m l p y re x

d is tilla tio n fla s k c o n ta in in g 3 0 m l o f d is tille d w a te r. T h e d is tilla te w a s c o lle c te d in

5 0 m l fla s k c o n ta in in g 25 m l o f p o ta s s iu m

d ic h ro m a te s o lu tio n (3 3 .76 8 g o f

K2C r 2O7 d is s o lv e d in 4 00 m l o f d is tille d w a te r w ith 3 25 m l o f s u lp h u ric a c id a n d

v o lu m e ra is e d to 1 litre ). A b o u t 20 m l o f d is tilla te w a s c o lle c te d in e a c h s a m p le
a n d th e fla s k s w e re k e p t in a w a te r b a th m a in ta in e d a t 6 2.5 C fo r 20 m in u te s .
T h e fla s k s w e re c o o le d to ro o m te m p e ra tu re a n d th e v o lu m e ra is e d to 5 0 m l. F iv e
m l o f th is w a s d ilu te d w ith 5 m l o f d is tille d w a te r fo r m e a s u rin g th e o p tic a l d e n s ity
a t 6 00n m u s in g a s p e c tro p h o to m e te r.
A s ta n d a rd c u rv e w a s p re p a re d u n d e r s im ila r s e t o f c o n d itio n s b y
u s in g s ta n d a rd s o lu tio n o f e th a n o l c o n ta in in g 2 to 1 2%

(v /v ) e th a n o l in d is tille d

w a te r. E th a n o l c o n te n t o f e a c h s a m p le w a s e s tim a te d a n d g ra p h w a s m a d e .
Caputi A Jr, Ueda M, Brown T (1968). Spectrophotometric determination of ethanol in wine. Am. J. Enol. Vitic. 19:
160-165.

0.7

O p tic a l d e n s ity (60 0 n m )

0.6

0.5

0.4

0.3
0.2

0.1

0
0

10

E th a n o l c o n c e n tr a tio n (% ,v /v )

F ig u r e 3.6.1 (a ) S ta n d a r d c u r v e fo r e th a n o l e s tim a tio n

12

Fermentation efficiency
It was calculated as
Actual ethanol recovery
Fermentation efficiency

100

Theoretical recovery

Theoretical recovery

= Total sugars 0.64

Actual ethanol recovery

= Actual ethanol obtained

3.6.2

Estimation of reducing sugars

The D N S method of M iller (19 59 ) was used to estimate reducing

sugars.
3.6.2 .1
1.

R eagents
Sub strate solution:

S tandard solution of 1000 g/ml concentration

was prepared by dissolving 100 mg of glucose in 100ml of distilled

water.
2.

3,5 dinitrosalicylic acid (D N S) solution: R eagent was prepared by

dissolving 10.0g of 3,5-D N S , 2.0g of phenol and 0.5 g of sodium sulphite
in 500 ml of 2% N aOH solution and then diluting it to 1 litre with distilled
water. The reagent was filled and stored in dark colored bottle.

3.

P otassium sodium tartarate (R ochelle salt) :

40 g of potassium

sodium tartarate was dissolved in distilled water and the volume was
made to 100ml.
3.6.2 .2

P rocedure
One ml of appropriately diluted solution (500-1000 g ml-1)

sample was taken in a test tube to which 3ml of D N S reagent was added. The
tubes were boiled in a boiling water bath for 15 minutes. One ml of R ochelle

salt was added to these test tubes and tubes were cooled to room
temperature and used for measuring optical density at 575 nm.
A standard curve of glucose was prepared by using 100-1000 g
concentration prepared in distilled water.

0.07

Optical density (57 5nm)

0.06
0.05
0.04
0.03
0.02
0.01
0
0

0.5

1.5

Glucose concentration(mg/ml)

Figure 3.6.2.2(a) Standard curve for estimation of reducing sugars

3.6.3

Gas chromatography

Ethanol in the fermentation broth was estimated by gas chromatography

method. A computer related Nucon series gas chromatograph eq uipped with
flame ioniz ation detector (FID) was employed for the separation and
q uantification of ethanol. A stainless steel column (5m 2mm) was fitted into the
instrument to provide on column injection. The column packing was P orapak Q .
The detector and injector temperature was maintained at 200C. The gas

chromatograph was connected to an integrator and computer system to

determine area of ethanol and internal standard peak. For analysis of ethanol the
following program has been standardized:
Nature of the column

Porapak Q

Dimensions

5m 2mm

Material

Stainless steel

Carrier G as

Nitrogen (flow rate 50 kg/cm2)

Hydrogen/oxygen ratio

2: 1

PROGRAM:

Oven 1 temperature

75C

Rate 1

10/minute

Oven 2 temperature

200C

Injection temperature

200C

Detection temperature

200C

Standard solutions of ethanol were prepared. The standards contain

2,3,4,5,6,7,8% ethanol. The standard curve was prepared with retention time 7.6
minutes. The area under peak was determined for samples and by comparing
with standard curve; the percentage of ethanol was measured.

T ime (minutes)
Fig 3.6.3(a): GC chromatogram of standard alcohol.