CHAPTER 3

TESTING PROCEDURE

3.1 TEST DESCRIPTION

3.1.1 FTIR

FTIR stands for Fourier Transform Infrared Spectrometry. This is the effective
method used to identify the organic and inorganic compounds present in the samples.
FTIR test relies on Infrared light rays for scanning the samples and observe the
properties. The FTIR instrument sends IR range of 10,000 - 100 cm^-1 through the
samples. After placing the samples in the setup, radiation start to flow through the
sample. Some are observed and some are passed through the sample. Then the
observed radiation is converted into vibrational energy by the molecules of the
sample. The converted vibrational energy is then detected by the detector in the FTIR
instrument, that is represented in the graph. Each of the peak in the graph represents
the particular functional group. The powdered form of antioxidant is directly used to
obtain the results in graph for each antioxidants.

Fig:

FTIR GRAPH FOR EACH ANTI OXIDANTS:

 COFFEE HORSEGRAM SEEDS

PEAS SABJA

SESAME

3.1.2 DPPH TEST

DPPH is the abbreviation of 2,2-diphenyl-1-picrylhydrazyl.This inorganic compound
is used in the DPPH assay. DPPH assay is mainly used to determine the free radical
scavenging capacity and predict the antioxidants activity. It has absorption maximum
at 517 nm.

3.1.2.1 PREPARATION OF SAMPLE SOLUTION

In DPPH Test, the control solution is first prepared by mixing 6mg of DPPH powder
with 50 ml of methanol and that will be keep in the dark room for 1 hour. Then five
sample solutions are prepared by mixing 5mg of each antioxidants with 100 ml of
ethanol in five different beakers. Magnetic stirring is done in the five different
samples by KOH pellet for 20 minutes period. After that using Wattman filter paper
each samples filtered and kept in another different beakers. After that using micro
pipette each sample solutions were taken in the concentration of 500ppm, 1000ppm,
1500ppm.Then 0.3ml of each sample solution is taken out mixed with the 2.7 ml of
DPPH control solution which is the final solution. This solution is taken into UV
spectrometry using cuvette and corresponding readings were noted.

Scavenging effect = (Acontrol - Asample)* 100 / (Acontrol)

FIG:
 FIG:

PEAS
1600
1400
1200
Concentration PPM

1000
800
600
400
200
0
30 35 40 45 50 55 60 65 70 75
DPPH. Inhibitation %
 COFFEE
1600
1400
Concentration PPM

1200
1000
800
600
400
200
0
50 55 60 65 70 75 80
DPPH. Inhibitation %

HORSE GRAM
1600
1400
1200
Concentration PPM

1000
800
600
400
200
0
60 65 70 75 80 85 90
DPPH. Inhibitation %

SABJA
1600
1400
1200
Concentration PPM

1000
800
600
400
200
0
45 50 55 60 65 70 75 80
DPPH. Inhibitation %
 SESAME
1600

1400

1200
Concentration PPM

1000

800

600

400

200

0
60 65 70 75 80 85 90 95
DPPH. Inhibitation %

DPPH SCAVENGING EFFECT(%):

3.1.3 FCR TEST

FCR is the abbreviation of Folin Ciocalteu Reagent. FCR Test is used to

determine the total phenolic content in the sample.

3.1.3.1 PREPARATION OF STANDARD AND SAMPLE SOLUTION

In this method, 1.1g of galic acid powder is mixed with 750 ml of deionized
water in 1000ml volumetric flask. This flask is then sonicated in an ultrasonic bath
until its suspended solids are dissolved. Normally 10 minutes of sonication is enough
for this method. Finally sample is dilute to the volume of the flask (1000ml) with
deionized water. Then 100mg of five antioxidant powders are mixed with 75ml of
deionized water in five different 100ml volumetric flask. These five flasks then
sonicated in water bath for 10 minutes and dilute to the flask volume (100ml) with
deionized water.5ml from these samples were taken out and poured in another five
100ml flasks where the samples were dilute to the volume of flask(100ml) with
deionized water. Then separately 20g of sodium carbonate powder is mixed with
100ml deionized water in beaker and coverage by aluminum foil.
3.1.3.2 PREPARATION OF STANDARD SOLUTION AND SAMPLE
SOLUTION FOR UV SPECTROMETRY:

First standard solution preparation is done by adding 15ml of water, 1ml of

folin-C reagent, 1ml of standard solution of different concentrations (40mg/L,
80mg/L, 120mg/L, 160mg/L, 200mg/L), 3 ml of carbonate solution in five different
beakers. Each 20 ml assays are allowed to settle for 6 minutes. Finally the absorbance
reading for galic acid is taken using UV spectrometry. After standard solution,
sample solution is prepared by adding 15 ml of water, 1 ml of folin-C reagent, 1ml of
sample solution from five different antioxidant samples, 3ml of sodium carbonate
solution in five different beakers. Each 20 ml assays are allowed to settle for 6
minutes. Then absorbance readings were noted for each antioxidant samples using
UV spectrometry. UV spectrometer is at the wavelength of 765nm.
 Formula:
y=mx+c m= y 2− y 1/ x 2−x 1

m=0.004575
y=0.004575 x +0.036

by using the above equation gallic acid conc values are calculated.

Where x = Absorbance values of samples.

A−B
∗V∗D
M
Total phenolic content= ∗100
W∗1000

A=ABSORBANCE
B=EQUATION CONSTANT
M =SLOPE
D=DILUTION FACTOR
W =WEIGHT
V =VOLUME OF SAMPLE

3.2 RESPONSE SURFACE METHODOLOGY USING

 MINITAB 17:

RANGE OF PROCESS VARIABLES FOR BOX– BEHNKEN DESIGN:

Table :
 3.2.1 INFERENCE FROM ANOVA:

• Reaction temperature influences yield to maximum extent and yield increases

till optimum reaction temperature.
• Yield largely depends on catalyst loading and increasing or decreasing of
methanol - oil ratio from optimum value affects the yield.
• YIELD = -5021 + 184.8 A + 511 B + 144.8 C - 17.46 A*A - 77.8 B*B
- 1.147 C*C-3.00 A*B - 0.125 A*C - 5.50 B*C
• Yield = 79 % for the optimum condition

3.2.1.1 SURFACE PLOT YIELD:

Fig:
Optimum condition for high yield:
Methanol - Oil ratio :- 1:5
Temperature :- 60℃
Catalyst loading :- 1.00gram
Yield=79%
3.2.1.2 CONTOUR PLOT YIELD:
Fig: