D E T E R M IN A T IO N O F P H O S P H O L IP ID C O N T E N T
L O W L . K . & N G C . S .
I N T R O D U C T I O N
P h o s p h o lip id s a re h y d ro ly z e d b y th e a c tio n o f p h o s p h o lip a s e . T h e a c tio n o f p h o s p h o li
p a se is u s u a lly s tro n g e r th a n th a t o f lip a se . T h e re fo re th e e x te n t o f h y d ro ly s is o f p h o s p h o lip id s is a d o p te d as an in d e x o f lip id d e te rio ra tio n .
P h o s p h o lip id c o n te n t is o b ta in e d b y u sin g c o lu m n c h ro m a to g ra p h y to s e p a ra te th e trig ly c e rid e s (n e u tra l lip id s ) fro m th e p h o s p h o lip id s . T h e p o la r p h o s p h o lip id s a re a b s o rb e d b y th e s ilic ic a c id a n d is e lu te d b y th e m e th a n o l. T h e n e u tra l lip id s w h ic h are n o t a b s o rb e d b y th e s ilic ic a c id a re fir s t e lu te d o u t b y th e c h lo ro fo rm .
I A P P A R A T U S
G la ss c h ro m a to g ra p h c o lu m n (Ø :1 -2 c m ; le n g th : 3 0 cm ) w ith T e flo n ta p . C o tto n w o o l
F ilte r p a p e r (W h a tm a n N o. 1)
P re w e ig h e d , d ry e v a p o ra tin g fla s k (50 m l c a p a c ity ) A n a ly tic a l b a la n c e
D e s ic c a to r
R o ta ry e v a p o ra to r w ith w a te r b a th (28°C) II R E A G E N T S
a) S ilic ic a c id (M a llin c h ro d t, 1 0 0 m esh) b) C e lite 5 4 5
c) M e th a n o l (a n a ly tic a l gra d e ) d) C h lo ro fo rm (a n a ly tic a l grade )
A. P R E P A R A TIO N O F T H E P A C K IN G M A T E R IA L
1. W a sh th e s ilic ic a c id a n d c e lite 5 4 5 s e p a ra te ly w ith w a rm m e th a n o l fo r 5 to 10 m in s.
2. A llo w th e m a te ria l to s e ttle a n d d e c a n t th e w a s h in g s o lu tio n . 3. R e p e a t s te p s 1. a n d 2. tw ic e .
4. W a sh w ith w a rm a c e to n e tw ic e .
5. A ir d ry a t ro o m te m p e ra tu re o v e rn ig h t.
6. T h e n o ve n d ry a t 120°C fo r 2 h o u rs.
7. C o o l a n d m ix s ilic ic a c id a n d C e lite 5 4 5 in a ra tio 2:1.
B. PR E P A R A TIO N O F C O L U M N
1. W e ig h p a c k in g m a te ria l 1 0 -1 5 tim e s th a t o f th e lip id s a m p le w e ig h t.
2. S o a k c o tto n w o o l in c h lo ro fo rm a n d p a c k in to b o tto m o f c o lu m n . E x c lu d e as m u c h a ir as p o s s ib le .
C -4 .1
3. Place 2 layers of W h atm an No. 1 filter p ap er cut into size of columns.
4. M ix the packing material in chloroform and pour gently into colum n with the aid of a glass rod.
5. Allow the packing m aterial to settle.
6. Place 2 layers of W h atm an No. 1 filter p ap er on the packing material.
7. Drain colum n of excess chloroform leaving a 1 cm high colum n of chloroform .
III P R O C E D U R E
1. Dissolve 1 g sam ple lipid in pure chloroform , making a 5 to 1 0 % solution.
2. Introduce thin sam ple onto the colum n.
3. Drain off excess chloroform till solvent level is about 1 cm abo ve th e packing m aterial.
4. Drain off with 2 5 0 ml chloroform and collect the neutral lipids in prew eighed evaporating flask (elution speed: 3 drops per second).
5. Evaporate off the solvent using th e rotary evaporator, dry up using a rotary vacuum pum p for 5 mins, cool in d esiccato r for another 3 0 min and weigh th e neutral lipids.
6. Drain off with 100 ml m ethanol and collect the phospholipids in prew eighed evaporating flask.
7. Evaporate off the solvent using th e rotary evaporator, dry up using a rotary vacuum pum p for 5 mins, cool in d esiccato r for 3 0 min and w eigh th e phospholipids.
IV C A L C U L A T IO N
Neutral lipid (% ) = (W eight of Neutral Lipid)
x 100 (W eight of PL + W t. of NL)
Phospholipid (% ) = (W eight of Phospholipid)
x 100 (W eight of PL + W t. of NL) NL = Neutral lipid
PL = Phospholipid
The phospholipid content is expressed as the percentage of phospholipid over th e total lipid present per gram of sam ple lipid.
C - 4 .2
