Characterization of sn-2 alk-1'-enyl ethers of glycerol from rice bran oil.

Abstract

Ether lipids have biological applications which would dissipated as an important constituent in cell membranes. These are mostly found in animal tissues and rare in plant origin. Alk-1'-enyl ethers are class of ether lipid forming aldehydes on cleavage of ether bonds. The present study enrolled the presence of aldehyde in unsaponifiable matter of rice bran oil (RBO) and hence the identification of source of aldehydes in RBO was conducted. With respect to the earlier reports the investigation turned to major lipid constituents such as triacylglycerols, diacylglycerols etc. Using the column chomatographic method lipid fractions are separated, recolumned, purified and analyzed by spectrochemical methods such as FT-IR, (1)HNMR, (13)CNMR, Mass spectrometry and confirmed the presence of ether lipids. The sn-2 position was confirmed by enzymatic hydrolysis using pancreatic lipase. Moreover the formation of aldehyde from these ether lipids was also confirmed by spectrometric methods.

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521
Journal of Oleo Science
Copyright ©2010 by Japan Oil Chemists’ Society
J. Oleo Sci. 59, (10) 521-526 (2010)
Characterization of sn-2 Alk-1'-Enyl Ethers of
Glycerol from Rice Bran Oil
L. S. Afi nisha Deepam and C. Arumughan＊
National Institute for Interdisciplinary Science and Technology (CSIR) (Thiruvananthapuram, Kerala, India, Pin 695019)
Abstract: Ether lipids have biological applications which would dissipated as an important constituent in
cell membranes. These are mostly found in animal tissues and rare in plant origin. Alk-1'-enyl ethers are
class of ether lipid forming aldehydes on cleavage of ether bonds. The present study enrolled the presence of
aldehyde in unsaponifi able matter of rice bran oil (RBO) and hence the identifi cation of source of aldehydes
in RBO was conducted. With respect to the earlier reports the investigation turned to major lipid
constituents such as triacylglycerols, diacylglycerols etc. Using the column chomatographic method lipid
fractions are separated, recolumned, purified and analyzed by spectrochemical methods such as FT-IR,
1HNMR, 13CNMR, Mass spectrometry and confirmed the presence of ether lipids. The sn-2 position was
confi rmed by enzymatic hydrolysis using pancreatic lipase. Moreover the formation of aldehyde from these
ether lipids was also confi rmed by spectrometric methods.
Key words: alk-1'-enyl ethers, fatty aldehyde, rice bran oil.
1 INTRODUCTION
Lipids having bond with carbon atom of glycerol and ox-
ygen of an alkyl chain, forming an ether bond is called
ether lipids. Plasmalogens are one of the classes of ether
lipids having vinyl ether bond at sn-1 position and a phos-
pholipid at one of the carbon atom of glycerol1）.The other
major group includes alkyl and alk-1-enyl glyceryl ethers
having saturation and unsaturation on the alpha-beta car-
bon of O-ether linkage with glycerol2）. The attached alkyl
chain usually includes hexadecanal, octadecanal, octadece-
nal etc3）.These alkyl chain get cleaved on hydrolysis there-
by yielding aldehydes4）. Ether lipids are mostly found in
micro organisms such as fungus and bacteria, and in mus-
cles of shark, adipose tissues of rat, liver of dog fi sh etc5）.
Ether lipids are rare in plant origin.
Plasmalogens are abundant in cell membrane of nervous
tissues and play a major role as cellular antioxidants. It act
against singlet oxygen, peroxyl radical etc due to the pres-
ence of vinyl ether bond which is a target of oxidative at-
tack by radicals and thereby protecting the diacyl-glycero-
phospholipid part from oxidative damage6）. It has specifi c
function in the cholesterol transport pathways. It reduces
radiation damage, suppress tumor growth, increase haemo-
poesis, accelerate wound healing, prevent cataract etc7）. It
is found to be a major constituent of synaptic vesicle mem-
branes, in alveolar surfactants, indicating involvement in
＊Correspondence to: C. Arumughan, National Institute for Interdisciplinary Science and Technology (CSIR), Thiruvananthapuram,
Kerala, India, Pin 695019.
E-mail: carumughan @yahoo.com
Accepted March 28, 2010 (recieved for review December 18, 2009)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
http://www.jstage.jst.go.jp/browse/jos/
the expedite process of synaptic transmission and mem-
brane dynamics during breathing cycles8）. Since they have
endogenous antioxidant capacity it is used in human medi-
cines3, 7）. It has been reported that dietary intake of ether
glycerolipids helps in the synthesize of membrane plasmal-
ogen in most tissues and there by increase the biological
functions9）. Its defi ciency causes diseases such as Zellwe-
ger syndrome, Alzheimer’ s, Down syndrome etc10-12）.
Usually the refi ned oil is devoid of aldehyde since it un-
derwent drastic refining conditions which push away the
volatile matter from oil. So a study was conducted to light-
en the path of being forming aldehydes from rice bran oil.
Regarding the earlier reports13）the investigation turned to
major lipid constituents such as Triacylglycerols （TG） , dia-
cylglycerols and the objective of the study was certained to
fi nd out the unique primary source of aldehyde viz: identi-
fi cation, characterization and confi rmation and also the for-
mation of secondary product, aldehyde by various spectral
techniques such as 1HNMR, 13CNMR, FT-IR, and Mass spec-
trometry analysis. The second objective is to quantify alde-
hyde by analytical method since it contributing in USM.
The isolation, identifi cation and characterization by various
spectroscopic techniques are discussed as follows.
Abbreviations: CRBO, crude rice bran oil; DIP, direct inlet probe;
MG, monoacylglycerols; RRBO, refined rice bran oil; RBO, rice
bran oil; TG, triacylglycerols; USM, unsaponifi able matter

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L.S. Afi nisha Deepam and C. Arumughan
J. Oleo Sci. 59, (10) 521-526 (2010)
522
2 EXPERIMENTAL
The analytical and chromatographic grade solvents pur-
chased from Merck Chemicals （Mumbai, India） . Standard
compounds such as TG, diacylglycerols, monoacylglycerols
（MG） , fatty acid and heptadecanoic acid methyl ester were
obtained from Sigma Aldrich （Steinheim, Germany） . Fatty
aldehydes were isolated from USM of refined RBO and
used as reference standards. Crude Rice Bran Oil （CRBO）
was obtained from Chakkiyathumoodu Solvent Extractors,
Angamali, Kerala, India. Physical refi ning of CRBO was car-
ried out in the laboratory14）and thus obtained Refi ned Rice
Bran Oil （RRBO） was used for the study.
2.1 Isolation of ether lipid from RRBO
RRBO （10 g） was adsorbed in silica gel, 60-120 mesh （350
g） and packed in a glass column （60 cm×3.5 cm） . The col-
umn was eluted starting with hexane （300 mL） , followed by
hexane/diethyl ether in the ratio 95:5 （300 mL） and fi nally
with 80:20 ratio. The third fraction is evaporated and re-
column in different ratios of hexane and diethyl ether. Six
fractions were collected viz: 100:0, 94:6, 92:8, 90:10, 88:12
and fi nally eluted with chloroform. All fractions were evap-
orated and subjected to 1H NMR, 13CNMR, FTIR and Mass
spectrometry analysis.
2.2 Isolation of aldehyde from TG of RRBO
RRBO （500 mg） dissolved in chloroform was adsorbed in
silica gel and transferred to a silica gel column （75 g silica
gel of mesh 60-120） of 45 cm length and 2.5 cm i.d. The sil-
ica gel was eluted first with 150 mL hexane （fraction Ⅰ）
and followed by 200 mL of 2％ diethyl ether （fraction Ⅱ）
and 200 mL of 5％ diethyl ether in hexane （fraction Ⅲ） .
The fraction Ⅲ was evaporated to dryness and separated
on preparative TLC using the solvent system toluene :
chloroform in the ratio 7:3. The separated band at Rf 0.74
（TG） was collected as it is confi rmed by the TG standards
and extracted from silica gel using chloroform. TG thus ob-
tained was saponified15）and subjected to various spectral
studies FTIR, 1HNMR, 13CNMR etc.
2.3 Separation of fatty aldehydes by GC and quantitation
The fatty aldehyde obtained by saponifi cation of TG was
converted into dimethyl acetal esters by refl uxing it with
2％ anhydrous methanolic sulfuric acid for 3 h. Heptadeca-
noic acid methyl ester （1 mL） was added as internal stan-
dard （1 mg/mL） for quantification. The reaction mixture
was cooled, extracted with 25 mL hexane （3 times） and
washed with 3％ sodium bicarbonate solution （3×10 mL）
followed by distilled water （3×10 mL） and evaporated to
obtain dimethyl acetals of fatty aldehyde. The aldehyde es-
ters were separated in gas chromatography （GC-2010） Shi-
madzu, Japan using a DB-23 capillary column （30 m length,
0.32 mm id （wide bore） with 0.25 μm fi lm thickness）
lent Technologies, USA） . The initial temperature was fi xed
（Agi-
as 180 ℃ for 2 min and then increased to 200 ℃ at the rate
of 5 ℃ /min and held at 200 ℃ for 5 min. The carrier gas
fl ow was 1.2 mL/min and the FID detector used was set at
300 ℃.
2.4 Spectral studies
Spectral studies of compounds were done using the fol-
lowing instruments. FT-IR: Perkin Elmer Spectrum 100 FT-
IR instrument （UK） , 1H and 13CNMR （CDCl3） on Bruker
AVANCE DPX-300 spectrometer＆500 MHz （Germany） at
room temperature. Mass analyzed by GC-17A Gas Chro-
matograph equipped with QP-5050 （Quadruple）
mass spectrometer by Direct Inlet Probe （DIP） method.
（Japan）
2.5 sn-2 Positional analysis of ether linked TG in RBO
by pancreatic lipase-catalyzed hydrolysis and TLC
fractionation
sn-2 Positional analysis was done by pancreatic lipase-
catalyzed hydrolysis and the method16）in brief was as fol-
lows. 1 M tris buffer （2 mL） , 2.2％ CaCl2（2 mL） , 0.05％ bile
salt （0.5 mL） were added to 100 mg TG and equilibrated at
40℃ in a water bath for one min before adding standard-
ized amount of 5 mg pancreatic lipase. The amount of pan-
creatic lipase was optimized so as to occur 35％ hydrolysis
of the TG （partial hydrolysis） . The mixture was shaken vig-
orously at 40℃ using mechanical shaker for 2-4 min. The
reaction was stopped by the addition of 1 M ethanol fol-
lowed by the addition of 6 M HCl （mL） . The whole mixture
was extracted with 10 mL diethyl ether for 3 times and
Fig. 1 Schematic representation of identification of
aldehyde from TG.

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Ether Lipids in Rice Bran Oil
J. Oleo Sci. 59, (10) 521-526 (2010)
523
pooled. The solvent layer was washed with distilled water
and dried over anhydrous sodium sulphate. The solvent
was then evaporated. The sample thus obtained was sepa-
rated on preparative TLC （0.2 mm） using solvent system
hexane / diethyl ether/acetic acid in the ratio, 8:2:0.1 using
silica gel coated TLC glass plates. The bands correspond-
ing to TG, free fatty acids, diacylglycerols and MG were
collected after identifi cation of band using reference stan-
dards. Schematic presentation for the identifi cation proce-
dure of ether linked TG shown in Fig. 1.
2.5.1 FT-IR and GC analysis of 2-monoacylglycerols ob-
tained by pancreatic lipase hydrolysis
2-Monoacylglycerol obtained was analyzed by FT-IR for
the confi rmation of characteristic peaks and also after hy-
drolysis in alkaline media. The aldehyde obtained by hy-
drolysis was converted to dimethyl acetals as explained
above and analyzed in GC to compare the alkyl groups in
2-monoacylglycerols （lipase hydrolyzed） and that obtained
from USM.
3 RESULTS
Ether lipids are mostly of animal origin. In our earlier
fi ndings aldehydes contributed a very good percentage in
the unsaponifi able matter of RBO which prompted to trace
its origin. The results gathered from this study are detailed
in the following section to establish the occurrence of ether
lipid in RBO.
3.1 Isolation and characterization of ether lipid from RBO
The preliminary step of isolation and characterization of
ether lipid was achieved by repeated column chromato-
graphic purifi cation of RRBO, using different ratios of hex-
ane and diethyl ether. Each fraction was subjected to spec-
tral analysis and found that chloroform fraction obeying
the spectral data of ether bonded TG. The spectral data are
presented as follows.
1HNMR: 1HNMR （500 MHz, CDCl3） of the alk-1’ -enyl glyc-
erol ethers （s, d, t and m indicate singlet, doublet, triplet
and multiplet） :δ/ppm: 5.83 （1H, d, J＝15Hz, H-1’ ） , 5.09 （t,
J＝10Hz, CH＝CH） , 4.23 （1H, q, J＝6Hz, J＝6Hz, H-2’ ） ,
4.11 （1H, m, H-2） , 3.91 （2H, dd, J＝3.5Hz, J＝5Hz, H-3a,
3b） , 3.71 （2H, d, J＝5.5Hz, H-1a, 1b） , 2.06 （m, CH2CH＝CH-
CH2） , 1.62 （m, CH2） ,0.97 （2H, q, J＝6.5Hz, J＝7Hz） .
13C NMR δ: 173 （C-1） , 129 （-CH＝CH-） , 77 （C-O-C） , 33
-CH2） ,29 （-CH3） .
FT-IR: vmax＝2854 and 2923 （CH stretching） , 1743 （C＝
O） , 1459 （-CH2） , 1377 （-CH3） , 1161 （OC-O） .
3.2 Mass spectra
Mass spectra of ether lipid by Direct Inlet Probe method
showed m/z of fragment ions at 601, 575, 337, 339, 313 and
285 which showed the presence of myristic （C14:0） , palmitic
（C16:0） , palmitoleic （C16:1） , stearic （C18:0） , oleic （C18:1） and lin-
olenic （C18:2） as the bounded fatty acid in the 1,3 position of
TG with ether bond at the second position and the data is
in agreement with a previous report regarding HPLC-APCI
mass spectrometric analysis of oil samples17）.
3.3 Isolation and characterization of fatty aldehydes from
TG fraction of RBO
Alk-1’ -enyl glycerol ethers yield aldehyde as the hydro-
lyzed product. The experiment was designed to character-
ize the hydrolyzed products of ether linked TG. After sa-
ponifi cation of TG, it was extracted with petroleum ether,
water washed, evaporated and subjected to spectral stud-
ies. The results are discussed as under:
1HNMR: 1HNMR showed a singlet signal at 9.92 δ charac-
teristic of aldehydic proton, olefinic protons at δ 5.36,
methylene protons and at 2.77 （br t, J＝10Hz） , methylene
protons adjacent to carbonyl carbon 2.4 （t, 2H） and with a
strong aliphatic region.
13C NMR δ : 180 （C＝O） , 129 （-CH＝CH-） , 34 （-CH2） , 29
（-CH3） .
FTIR: FTIR spectra showed the characteristic peak of al-
dehyde at 1717 cm－1 corresponding to C＝O vibration, two
moderately intense bands at 2851 cm－1 and 2929 cm－1 cor-
responds to aldehyde C-H stretching and at 1465 cm－1C-H
bending.
GC-MS: GC-MS of fatty aldehydes showed ［M］
m/z, 212, 240, 268, 266 and 264 and the presence of peaks
corresponding to ［M-18］
tence of aldehyde function of long chain C14, C16, C18, C18:1,
C18:2 respectively.
GC analysis of isolated aldehydes: The values obtained in
GC analysis showed relative ％ of C18:1 was more in CRBO
USM and C16 in RRBO USM. Contribution of C18:1 get de-
creased in refi ned due to its volatile nature which get elimi-
nated during refi ning process. Moreover C16 get increased
from 30.66％ （CRBO） to 61.73％ （RRBO） .The dimethyl ace-
tal composition of the fatty aldehydes in CRBO and in
RRBO USM was shown in Table 1 and its chomatogram in
Fig. 2.
＋ peaks at
＋, ［M-44］
＋ suggesting the exis-
Table 1 Relative percentage by GC analysis of fatty
aldehydes obtained from USM of CRBO, RRBO
and 2-MG from pancreatic lipase hydrolysis.
AldehydeRt
Area % a
CRBO USM RRBO USM
1.21±0.01
30.66±0.53
3.71±0.18
47.42±0.93
17.00±0.36
2-MG
C14
C16
C18
C18:1
C18:2
1.83.36±0.40
61.73±0.81
6.67±0.06
25.79±0.86
2.44±0.28
5.44
2.8 55.32
4.27.42
4.522.94
4.98.86
a Mean ± SE (n=3)

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L.S. Afi nisha Deepam and C. Arumughan
J. Oleo Sci. 59, (10) 521-526 (2010)
524
Pancreatic lipase hydrolysis: Having established the
presence of ether linked TG this experiment was conduct-
ed to confi rm the position of ether bond with glycerol. Pan-
creatic lipase cleaves primary ester bond preferentially
when subjected to partial hydrolysis of TG18）. The TG sepa-
rated from RBO by column chromatography subjected to
digestion with pancreatic lipase so as to occur 35％ hydro-
lysis. After hydrolysis with lipase each fractions was col-
lected preparatively and further analyzed. Since partial hy-
drolysis occurred 2-MG which does not undergo hydrolysis
get concentrated. Spectral data of individual fractions
showed that among all fractions 2-MG showed an intense
peak at 1215 cm－1 in FTIR that corresponds to the asym-
metrical C-O-C stretching of vinyl ether19）. For the subse-
quent confi rmation of ether linkage the same fraction was
hydrolyzed and subjected to spectral analysis for the con-
firmation of formation of aldehydes. After hydrolysis the
extracted compound showed an intense peak at 1709 cm－1
in FT-IR, which is a characteristic peak of aldehyde C＝O
and the peak at 1215 cm－1 get vanished. This substantiate
the release of aldehyde on hydrolysis. The FTIR spectra
shown in Fig. 3 confirms the formation of 2-MG having
ether bond from TG after enzyme hydrolysis and further
2-MG subjected to hydrolysis yielding aldehyde and the
standard MG. The structure of ether linked triacylglycerol
is shown in Fig. 4.
4 DISCUSSION
Ether lipids are major constituents of animal tissues and
have biological properties, now characterized from RBO.
The spectral data confi rm the presence of ether linked tria-
cylglycerols and also the formation of aldehydes by hydro-
lysis of these lipids. The particular characteristic of the
identifi ed lipid was the unsaturation adjacent to the ether
bond. Because of the presence of unsaturation it yields al-
dehyde on hydrolysis. The presence of mono ether moiety
was substantiated by spectroscopic data. 1HNMR showed
characteristic peaks which can be explained as follows.
The H-2 proton showed a multiplet at 4.11 δ and a double
doublet at 3.91 δ for H-3a and 3b protons. At 3.71 δ a dou-
blet corresponds to H-1a, 1b protons. A doublet at 5.83 δ
was assigned to H-1' proton and quartet at 4.23 δ to the
H-2' proton. The alkyl chain attached have unsaturation
and thereby a triplet at 5.09 δ indicating the presence of
unsaturation20, 21）. 13C NMR showed the carbonyl stretching
Fig. 2 Gas chromatogram of dimethyl acetals of fatty
aldehydes.
1. C14, 2. C16, 3. C17 (internal standard), 4. C18, 5.
C18:1, 6. C18:2 obtained from USM of RBO.
Fig. 3 FTIR spectra of MG.
a) Fatty aldehyde obtained after saponifi cation of
enzyme hydrolyzed MG showing characteristic
peak of aldehyde b) standard MG c) MG obtained
after enzyme hydrolysis showing peak of ether
linkage.
Fig. 4 Structure of ether linked lipid in RBO

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Ether Lipids in Rice Bran Oil
J. Oleo Sci. 59, (10) 521-526 (2010)
525
at 173 followed by 129ppm corresponds to-CH＝CH- and
C-O-C at 77 ppm. Finally the FT-IR spectral data proves
the characteristic functional groups a sharp symmetric
stretching at 1743 cm－1 of carbonyl group, 1459 cm－1 of
acyl CH2 scissoring, terminal CH3 bending mode at 1377 cm－1.
The C-O-C stretching corresponds to 1161 cm－1. Mass
spectral analysis of ether lipids substantiate the ionization
of glyceride molecule in such a way that a positive ion is
formed at the second position there by cleaving the ether
linked alkyl group. Further it was evidenced that the other
two positions of glycerol occupied by myristic （C14:0） , pal-
mitic （C16:0） , stearic （C18:0） , oleic （C18:1） and linolenic （C18:2）
groups showing fragmentation ions at 601, 575, 339, 313
and 285. The present results were in well agreement with a
previous report regarding the triacylglycerols composition
of plant oils by HPLC-APCI mass spectrometric analysis17）.
Aldehydes are the hydrolyzed product of ether lipids as
evidenced from the previous reports1）. Presently its sub-
stantiation was done by hydrolysis of ether lipids in pres-
ence of alkali25）. Mild alkaline hydrolysis does not cleave
the ether bonds but the previous reports envisage that al-
kali metals are highly induce to the heterolytic cleavage of
carbon-oxygen bond by chemical mode of cleavage. In ad-
dition to this presence of -CH＝CH- adjacent to the ether
bond stabilize the ion formed by electron transfer there by
forming aldehyde22, 23）.
The formation of aldehydes was substantiated by spec-
troscopic data. 1HNMR showed characteristic peak for al-
dehydic protons at 9.92 δ and olefi nic protons at 5.36 with
a strong aliphatic region. The carbonyl carbon showed
peak at 180 ppm in 13C NMR and also for -CH＝CH-, methy-
lene and methyl carbon at 129, 34 and 29 respectively. FT-
IR analysis showed peak at 1717 cm－1 corresponding to C
＝O vibration, two moderately intense bands at 2851 cm－1
and 2929 cm－1 corresponds to aldehyde C-H stretching
and at 1465 cm－1 C-H bending. Moreover the presence of
C14, C16, C18, C18:1, C18:2 alkyl chains are confi rmed by GC-MS
analysis as due to the presence of ［M］
240, 268, 266 and 264 respectively followed by ［M-18］
and ［M-44］
The pancreatic lipase hydrolysis confi rmed the sn-2 po-
sition of ether linkage as it hydrolyses 1 and 3 position
only. Figure 3 substantiate the formation of aldehyde after
hydrolysis of enzyme hydrolyzed 2-MG with that of pure
2-monoacylglycerol （enzyme hydrolyzed） and standard MG.
Moreover from this figure the vinyl ether characteristic
peak at 1215cm－1 was depleted in the same sample after
hydrolysis which was converted to aldehyde C＝O peak at
1717cm－1.
USM of RBO contains sterols, oryzanols, tocopherols, to-
cotrienols etc. Comparing with other edible oils, the USM
percentage in RBO was greater. Our previous investigation
on each constituents in USM light out the presence of alde-
hydes. Earlier reports pursue the presence of free alde-
＋ peaks at m/z, 212,
＋
＋ peaks.
hydes in CRBO24） but its presence in refi ned oil was not re-
ported. On following the various steps of refi ning especially
under high temperature in presence of vacuum, aldehydes
get removed and so refined oil was devoid of aldehydes.
But at present, the contribution of aldehydes was found in
USM of CRBO and RRBO. Most of the ether glycerolipids
contain a monounsaturated vinyl alk-1-enyl chain and
cleavage of the ether bond lead to the formation of fatty al-
dehydes. Such type of lipids was found out in fungus, bac-
teria and in various animal sources. With regard to earlier
reports, studies on major lipid components in RBO was
carried out and in this point of view triacylglycerols isolat-
ed from RBO was fractionated by column chromatography
and characterized by spectrochemical techniques and
ether linked triacylglycerols was confirmed by spectral
analysis. The TG thus obtained on hydrolysis yield alde-
hyde by the heterolytic cleavage of carbon and oxygen
bond in presence of alkali which favors reductive cleavage
of ethers. Ether bonds are very reactive to alkali25） and
here during the saponifi cation concentration of alkali was
very high （50％ ethanolic potassium hydroxide） which lead
to the fast cleavage of ether bonds. Regarding the solubility
of aldehydes in water, its solubility generally decreases as
the length of the non polar part （the alkyl part） increased.
At about fi ve carbon atoms aldehydes are only slightly sol-
uble and less when the number of carbon atom is increased
and so lose of aldehyde in water during water washing in
the course of USM preparation can be wiped out.
The results obtained from GC analysis of dimethyl ace-
tals of fatty aldehydes showed 2.24％ sn-2 alk-1'-enyl ether
lipid in CRBO and 0.99％ in RRBO. The percentage of fatty
aldehydes in CRBO is 1.25％ greater than in RRBO that
supports the presence of free aldehydes before refi ning as
reported earlier. Volatile nature of aldehydes causes the re-
moval of free aldehydes during refining in deacidification
and deodorization steps which lead to the absence of free
aldehydes in RRBO. In conclusion the source of aldehyde
in RRBO is from ether linked TG and in CRBO it is from
both.
ACKNOWLEDGMENTS
The first author is grateful to CSIR for the Senior Re-
search Fellowship granted during the period of this work.
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