C H A P T E R 3

MATERIAL S AND METHODS

The Project : "Studies on chemical Compos ition

of safflower (Carthamus tinctoriousl Oil " Comprised

of the following obj e ctives

1- To survery and categorize safflower germplasm

for high and low linol ei c acid accessions.

2- To mak e a com para t ive study on the effect of

heat treatment and deep in high and low linoleic

acid o i ls .

3- To stud y the storability of high and low

linoleic acid oils under ambient conditions-

4- To study car- relati on of heat polymerization

and storage parameters with linoleic acid content

o f safflowe r s eeds .

5- To estimate oil content of seeds of various

ac cess ion s and to study their complete fatty acid

pr o file.

In order tc, ach ieve the obje ctiv e s f o 1 1 owing

materials and method s we re adopted.

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 Loc ation Q.f_ experimental site .

Kanpur is geographically sit uated at an ele v a tio n

of 125 . 9 metres from the mean sea l eve l and li es in

the subtropics at a la titude of 26' 58'N and

longitude of 88• 34'E .

The area has an annual rai nfal 1 of 6 0-ll0c m, mainly

through mon s oon rains. <Ref er table S oil a nd Cr ops >

P lan t Prote c t ion

Two spraying of Demecron.

Statistical design Q.f_ experiment : -

Rando mised Block Design .

AN ALYSIS

Seed Sampling Seed Samp l es for tw o phases were

taken separately bo th years. Each p hase involved

ana lys is of fifty one var iet ies :- The varieties

selected f o r the first ph ase were .

TM-29 , CSS 611(SVTl , A-1( IE TI 1,CTV73, CSS158 , CTV 78,

6440 !SVTl,CSSlll,499 <S Ml ,NDS - 1 CSMl, CS S611 CS Ml,

CSS 19-1 CS Ml , 6440 ( SM I , 6304CSMl , CTV 26 ,T -65 , CTH -

11 88 11
 Period Details year fertility level irriga tion time

El.!:.tl_ phase 1990 N,4 0 ;P,40; K, 20 kg/ ha 1s t De c . 1989

Second Phase 1991 N,4 0 ,P,40;K, 20 kg/ ha 1st Dec . 1990

'
✓

1,CTV9, A-l <CVT l , CTV 2 , CTV3, CTH- 2 , CS S98 <S M>,

CSS19 !SMl , CTV 84 , CTV 37 , CTV57, CTV 67 , CTV34 ,

CTV55 , CTV59,CTV79 , CVT64, Tlll , CTV74 , CTV89,

CTV72 , CTV82 , CTV86 , CTV6 8, CTV7, CTV91, CTV 7 5 ,

CTV81 , CTV97, CTV48, CTV53 , CTV85, CTV83 , CTV90 ,

CVT61.

Varieties selected for second phase were :T-65( IET -

ll, CSS98 < IET! l , TM -29 ! IET ll , CSS 111 (IETI>,

6508 (I ET ll,CTV223 , CTV221 < IET!l, CTV219 ( lETl l ,

CT V 197, CTV 205 , CTV202 , CTV 207 , CTV208 ,

CTV209,CTV206, CTV210, CTV2 11, CTV2 12,CTV213 , CTV 2 15

CTV 2 14 CTV216, CTV2 17, CTV218 , A- l(CVTl , CTV 220

< !ET 11l , HUS305 , CTV 203 ,

CTV219 , ( I ET 1 I l, CTV22 1, ( I ET I I l, 6440 , <S Ml , CS S611

( SMl CSS19 <S Ml, CSS158 ! SMl , 6508 <S M>, CSS98 (S M>,

CSSlll<S Ml ,TM29 <SM> ,CSS19 - l ( SM l, 8304(SM l ,

CSS6513!SM l , NDS-l ! SMl , 4 99! SM J , NDS-ltSVTl , TM -

29tS VT l , T-65 ! SVTl, CSS1 9 !SV T l, 6440 ! SVT l ,

152 !SV T l , CVT 187 ( AVT, A- 1 ( AVT l .

After samp ling th e seed s were analysed for physical

c hara cter.

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I

A. Analy sis Q.f_ physical characters : -

Seeds we re analys ed for test weight, hul 1 content

and K/ H ratio and a c omparative account was

prepared .

Test weight Hunde red seeds were counted from

each variety and we ig hed and represented in g / 100

seed.

Hull content _ The s eeds were soaked in water

o vernight and dried to l o osen the hul 1 . The hu lls

were remov e d ma n ually and weighed. The valu e was

expressed a s per c enta ge oftotal seed weight.

K/ H Ratio :- The kern e l percentage divided by the

Hui 1 per c e nt age g ave the K/ H ratio f o r the seed of

e ac h variet y .

Bl C hemi c a l ana ly sis

Removal of Mo i s ture :- Seed were d r i e d in ov en at

60° C for 10 hr s to r emo ve t he mo is t ure . 0i 1

co n t e nt estimation was d on e o n mo i s t ure fre e se e ds .

(1 ) Oil content. : The estima ti on of oil co n te n t

was done on oxfo rd 4000 NMR - An a l ys er o ne s in gl e

point calibration in d u p l i cate . The operational

parameters are g iv en b e l o w : -

11901I
 Instrument oxford 400 NMR an aly z er

Version NA-01 - 9 T

Mode 4
R.F l evel 350
AF Gain 5 00
Gate width 1. 5
Analytical time 10
Repeats 1
Ca libration time 10

Standard samp l e for oi l c ontent es t im a t ion was

p r epared by e x trac ting oil b y s oxhle t met h od using

J ~

40 - 6 0 Bi>.P e t r a 1 e um e th e r as s a 1 v en t .

2- Determination Qf. f atty acid p r of il e - It

i nvo l v e d t wo stages . Th e firs t is th e s am ple

p r epar ation a nd second st age is s e p ara tion of

di ff e rent fat ty acids by subject ing the s am ple

ester to Gas - liqu id chr omato gr a ph y .

2 (al Sa mp le prepa rat ion : Aoou t 4 gms seeds

were crus he d in a po rcelain pestle mortar and

C
washed with pet r ol eum ether ! 40" - 60 8 . pl to

extract the oi 1 i n co ld . Excess of solvent was

removed by kee ping the oi 1 overnig h t . Trace s of

s o lvent we r e removed by a jeat of ciir . Methyl esters

+ //91 //
 '

of the o i 1 obtained f rem above were prepared by the

method of Luddy et tl< 124 l ( 1967 l.

2 (bl Gas Chro mato grap h ic separation of fatt y

acid meth yl esters. The fol lowing operat i onal

parameters were maintained for the separation of

different fatty acid s . This gave complete

separation o f major and minor fatty acids in the

o i 1•

Instrument CIC- Dual Column Gas Chro mat ograph

Ca rrier gas Ultr a High Purity Nitrogen

Flow rate 30ml / mt.

Column Stainless steel 2m x 0.3 cm

pack ed 15% DEGS <Diethylene
glo>'c ol succ inate on chromosorb-Ul
Co 1 umn tem p. 190C ± 1C

Run I soth er mal

Fuel Gas Ultr a Hi gh P u rity Hydrogen

Detector FID IFlame Lonization Detec tor )

Recorder Poten tio metri c strip c h a rt

Sample Size IOne micro litre)

//9 2 / /
 Quantitative assessme n t of differe n t fatty acids

were made by triang u lation method depending upon

the percentage area in different peaks as fol lowing:

Percentage

of
individual
FATTY ACID Area under individual pea k
= --------- - - ---------------- X 100
Total area of al I the pea k s

The peaks were identified by the retenti on t ime of

known methyl esters of fatty acids.

Therma l Polymer i zation studi es

Twenty samples, ten of high linoleic aci d and ten

samples of low linole i c acid were sele c ted for

polymerization [Link] varieties se l ec t ed were -

High linolei c acid v arieties 6508, CTV223,

CTV205, CTV 202 , CTV210, CSS 158, CSS19 -l. TM- 29 ,

CTV 218 NDS - 1

Lo w I ino leic ac i d v erieti es : - CTV 212. CTV 2 16 ,

CTV 2 17 , SV T. NDS- 1, T- 6 5 , AVT , CVT 187 , CTV 1 97 ,

AVT. A- 1, CSS - 19 , SV T 152

(al Sample prepa r ation 0 i I was extr ac te d i n

1193 11
 0
a s e xhlet apparatus us ing 4d C 60 C B. p.

pertroleum ether as sol v en t . Ap pro xima t e ly 500 ml

of oil was extracted in c ase of ea c h variety.

(bl Cooking P ol ymeri zat i on wa s a c hieve d

at five different temperatures. v i z . 170~ 0 ,

1so• o , 1 90~ 0 a nd 200~0C , Frying of pot a to c h i ps was

d o ne i n a fr y ing pan .

Ma te ri a l Potato c hi ps

Prepara t i o n o f Pate t e c h ip s - a bout 2 - 3 mm.

thick pota to c hips wer e prepared b y a c ut t er.F ry ing

o f potat o c hips was done in a fr yi n g pan. unif o rm

he ating was achieved thr o ugh paraffi n b ath.

Samples o f thermally po ly merize d oil at different

t emperatur e wer e co lle cted s eparat ely for ea c h

variety f o r ana lys is .

(c l Quantitative De t ermina t ion of the extent

of t hermal polymerization: - Urea adducts were

pr e pa re d f o r est i mat i o n o f ex tent o f po lymerizati o n

in a l I t h e sa mples .

Ur ea ad du c t s we r e p r epar ed b y the met h o d advo c ated

by Markle y( 125 l

//94 / /
'
~

(dl Chem ical a n alysis

1. Pe rox ide value

2. Fr ee fathy acids
3. Io d ine value
Al 1 thr ee values we re ca l c u lated using method

d es cr ibe d by standar d AOCS meth od <131 1

Shelf 1 ife studies : - Ten sampl es of high linoleic

acid we re s e lected f o r sh el f 1 ife studies . The

variet i es se lec ted wer e NDS - 1. CTV 2 18 , CTV 210 ,

CTV 205, TM-29 , 6508, CS S 1 9-1, CSS 158, CTV 202,

CTV 223 About 200 ml of o il extracted by soxhlet

~ C
me th od using petrole um ethe r 40 - 60~ cas
I
solvent.

Solve n t was evaporat e d on wa t er bath and later the

oi 1 was dried by a i r bubbling by a hair drier to

re move t race s of solv ent .

Th e e xt r acted oil wa s ke pt in plastic containers of

500 ml . capacity cont a ining plastic screw. cappe d

contai n er were kept for n i ne months at a mbient

storage condi ti ons and ea c h mont h chemical analysis

for linoleic acid. FFA, Io dine value and Peroxide

value was done. The fol lowing chronological

schedu le wa s adopted for analysis.

[Link] it nati on of linoleic acid : -5 gms or ester

// 95 I
 ~
'Jr-
~

Sampling Dates and nean Wedther Data

d ur ing shelf l if e studi es

S . No . MONTH DATE OF KEEPINGS DATE OF ANAI..YS IS MO I STU RE X RE~ATIVE HUMIDI TY ~VEiAGE [Link] TE MPE RATURE
l. 0 02-04-91 14- 0 4 - 91 7 29.0 28 . 2
2. First 02-04- 91 16- 05 - 91 6 26. 7 32 . 3
3. Second 02-04---91 16- 06 - 91 7 20.1 36 . 8
4-. Third 0 2- 04-9 1 16-07 - 91 11 69 . 1 3L 2
5. Fourth 02-04-.9 1 20 - 08- 91 12 7 2.1 ~0.2
6. F I fth 0 2- 04--91 16-10- 91 11 76 . 5 31. . 2
7. S \xth 02. - 04 - 91 15 -11-9\ 7 64 . 8 26 . 1
8. Sev enth 02- 04-91 14- 12-9\ 6 68 . 5 20 . $
9. Ei gh.t 02 - 0,4--91 1:5- 01 - 92 7 72 . 7 '5.4
10 . Ni nth 0 2- 04--91 l5-0i - S2 8 Tl , 4- 15 . 2.
prepared from oil afte r each month was taken to from

ure a complex. The non complex forming fraction was

removed and •rea complex wa s broken by use of cold

benzene. The ester was distilled and heated on a

wa@ter bath to remove traces of solvent. It was

then subjected to GLC for fatty acid profile and

determination of linoleic acid percentage.

F.F.A. , I.V. were analysed by the methods described

earlier.

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 + --;.... y

Initial values of different parameters in s~mples foT

she l f l i f e stud. l es .

S . fl ll • VARIETY L I NO I EI C AC ID FF/\ % IODINE [Link] PE ROX I DE VALUE

1- NDS-1 78,8 0 . 57 147 . #t 1. 91

2- CTV 218 79.0 0 . 62 1.52.6 1.95
3- CTV 210 79 . 8 0 . 62 156 . 3 1.99
4- CTV 205 eo .2 0 . 56 152.8 2.07
5- Tl'1 - 29 80 . 4 0 . 54- 159 . 0 2 . 15
6- 6508 81. 0 0 . 51 146 . 9 2. . 05
7- css 19-1 81 . 4 0.56 153 . 6 2 . 22
8- css 158 83 . o 0 . 60 166 . 8 2.. 34
9- CTV 202 83 . 2 0 .51 l.60.5 2 . 44
10- CTV 223 67 . 6 0 . 59 165.0 2 . 60