全 文 ：文章编号: 1007-0435( 2002) 02-0087-05
植物表皮蜡中链烷含量测定方法的研究
张英俊1, 木母村恭子, 秋山典昭, 大木规和夫
( 1. 中国农业大学草地研究所, 北京 100094; 2. 日本国家畜产草地研究所, 枥木, 329-2793)
摘要: 植物细胞壁中链烷含量测定分析研究的目的是不降低测定分析精度的同时, 节约时间和费用以便将来用于大
量样品的分析。本研究首次把链烷分析提纯过程中省略用作纯化的硅胶,直接将含有链烷但未经硅胶提纯的原提取
液注入气相色谱仪中测定,将世界通用的两种链烷分析法作为对照。结果表明 ,硅胶能否省略应根据不同牧草而定。
本实验中所用的三种牧草, 以省略硅胶而简化的链烷分析可用于多年生黑麦草和芒, 而不能用于日本结缕草中链烷
的测定。
关键词: 链烷( n-a lkane) ; 分析; 多年生黑麦草; 芒; 日本结缕草
中图分类号: Q946. 91 文献标识码: A
Study on the Determining Method on n-Alkane
in the Cuticular Wax of Plants
ZHANG
1
Ying-jun, T OGAMURA
2
Yasuko, AKIYAMA
2
Fum iaki, OT SUKI
2
Kazuo
( 1. Gr ass land In st itute, China Agricul tural U nivers ity, Beijing, 100094, C hina;
2. Nat ional Ins t itu te of Lives tock and Gras sland Scien ce, T ochigi, 329-2793, Japan )
Abstract: Based on saving cost and t ime, and increasing the turnover rate in the determ inat ion of n-alkane
concentrat ions so as to facil itate the applicat ion of the n-alkane technique in large-scale studies of her bage
intake w er e the goal o f the study on the modif icat ions of the chem ical analyt ical pro cedure to be described
in this paper. The simplif ication, eliminat ing sil ica gel and inject ing the raw ext racts into the gas
chromato graphy column, were evaluated of three pasture species: Miscanthus sinensis, Zoy sia j ap onica and
L ol ium perenne in 2000. T he simplif ied procedure of the n-alkane determinat ion w as compared w ith o ther
tw o ordinary pro cedures. T he results show ed that a simplified procedur e by el im inat ing silica gel and
inject ing the raw ex t racts into the gas chromatog raphy column could be used fo r the n-alkane determination
of M iscanthus sinensis and L olium perenne . How ever, the n-alkane concentrat ions of Zoy sia j ap onica
w ithout purificat ion by silica g el w ere not able to g et correct ly , fo r the peak of contam inated substances
interfered to the peak o f C34. T hough the samples used in this experiment w er e supposed to be insuf ficient
to the f inal conclusion, it is indicated that the pr ocedure simplif icat ion should be used caut iously in the
dif ferent herbage analysis, too .
Key words : analy tical procedure, L olium perenne , M iscanthus sinensis, n-alkane, Zoy sia j aponica.
收稿日期: 2001-10-25; 修回日期: 2002-04-04
作者简介: 张英俊,男,副教授, 1971年生,甘肃农业大学博士,日本国家畜产草地研究所博士后,现于中国农业大学草地研究所从事教学
和科研工作,专业方向为草地管理,发表论文 19篇
第 10卷 第 2期
Vo l. 10 No. 2
草 地 学 报
ACT A AGRESTIA SIN ICA
2002 年 6月
June 2002
1 Introduction
N-alkanes, saturated hydro carbons found in
the w ax cut icle of plants have been used as internal
markers fo r the est imat ion o f the animal intake and
diet composit ion [ 1～4] . T he or ig inal analyt ical
method for n-alkane determination w as developed
by M ayes et al. [ 5] which w as relatively slow ,
involving Soxhlet ex t ract ion of samples,
saponif icat ion and n-alkane ex t ract ion w ith n-
hexane. There have been many modificat ions since
1986. Vulich et al .
[ 6]
and Laredo et al.
[ 7]
modif ied
the pro cedure to the direct saponification o f f reeze-
dr ied g round samples w ith ethanolic KOH at 90℃
in 1991. Dove
[ 8]
replaced n-hexane w ith n-heptane
as so lvent for it s less hazardous to health dur ing
the w hole chemical analy sis. In 1994, ho t
ext ract ion should be alw ay s performed during the
chemical analy tical procedur e of n-alkanes by
Olivan et al .
[ 9]
, and more recent ly they
recommended that tw o internal standards, one at
each ex t reme o f the spect rum of the chain lengths
found in samples ( e. g. C22 and C34 ) , should be used
fo r the reduction of the erro rs caused by the
dif ferent ext ract ion rates of n-alkanes under the
low temper ature in the est imat ion of n-alkane
concentrat ions
[ 10] . Vulich et al. ( 1995)
[ 11]
suggested
that the evapo ration o f the sample ex t ract and
redissolving pr io r to the separ at ion of the n-
alkanes with a silica g el , could be ommited and that
the volume o f the solvent used could be reduced.
The modif icat ions of the analyt ical procedure
fo r the determinat ion of n-alkanes concentrat ion
has concentrated on increasing the accur acy, and
reducing the t ime and co st
[ 6～11] . T he object ive of
the present study w as to evaluate the simplif ied
analy tical pr ocedure fo r n-alkanes determ inat ion by
elim inat ing sil ica g el purif icat ion, and then t ime
and co st can be saved to facilitate the
implementation of the n-alkane technique in larg e-
scale studies of her bage intake.
2 Materials and Methods
2. 1 Samples
M iscanthus sinensi s and Zoy sia j ap onica, w hich
are tw o dominant species in the nat ive grassland in
Japan, w er e col lected f rom the natural g rassland at
the Nat ional Inst itute of L ivestock and Grassland
Science, Nishinasuno , Tochigi prefecture in October
1999. L ol ium p erenne was hand-plucked from the
sow n pastur e in the same Inst itute. Leaf samples of
M iscanthus sinensis, Zoysia j ap onica and L olium
p erenne were f reeze-dried and ground through
1 mm mil l.
2. 2 Experiments
T he experiment evaluated the possibility for
n-alkanes determ inat ion w ith or w ithout silica gel
separat ion. T he simplif ied analyt ical procedure
eliminating silica g el and inject ing the evaporated
r aw ex tr act into the gas chromatog raphy column
w as t reated as procedure A . The analysis by
Vulich
[ 6]
, w hich w as perfo rmed pr ev iously in this
laboratory , was t reated as procedure B, and the
analysis derived f rom Dove
[ 8]
was pr ocedure C. T he
sample w eight and amounts of reagents used were
a lit t le different f rom original analy sis in
procedures B and C, and an ident ical sample w eight
w as adopted so that three analyt ical procedures
could be compared.
Each 1. 0 g of the leaves of Miscanthus
sinensis, Zoy sia j aponica and L ol ium perenne were
w eighted into Pyr ex bot t le w ith three replicates of
each species. Dual internal standards ( 0. 2 mg C22+
0. 2 mg C34 ) , w hich w er e used to minim ize error s
due to dif ferent ial analyt ical recoveries o f n-alkanes
r elated to C-chain length during bo th the
ex tr act ion and chromatog raphic pr ocedures
[ 9, 10]
,
and 10 mL ethanolic KOH were added into the
samples. T ubes w ere capped t ightly and heated for
4. 5hr at 90℃. The ext ract ion w as per formed by
w ay of adding 7mL of heptane and 5mL o f dist illed
88 草 地 学 报 第 10卷
water, shaking vig orous and centr ifugat ion at 2000
rpm for 5 minutes. T he upper solvent layer w as
t ransferred to a g lass vial and further ext racted
w ith 7 mL of heptane ( 14 mL hexane-4 mL
distilled w ater , and an addit ional 14 mL hexane for
pr ocedure B) . T he pooled ex tr acts w ere evapo rated
w ith the compressed air st ream from an air pump
( Nisso -6000, made by Nisso Indust ry Co . Ltd) .
The r aw ex t ract in pro cedure A w as redissolved in
1 mL heptane and injected into the gas
chromato graphy direct ly. How ever , the raw ext ract
of procedur es B and C w er e redissolved in 2 mL of
hexane and heptane separately for the n-alkane
purif icat ion before applying to the column
containing 2g silica g el 60 ( 230～400 mesh, M erck)
w ith a 5 mL bed vo lume, and eluted w ith ext ra 2,
2, 4 mL hexane and heptane. Thereaf ter , the
eluates w ere evaporated to dryness, redisso lved in 1
mL hexane and heptane and injected into the gas
chromato graph.
2. 3 Gas Chromatography
Fo r all samples, 1L of f inal re-dissolv ed n-
alkane solution was injected into GC-14A gas
chromato graphy ( Shimadzu Company, Japan )
fit ted w ith a f lame ionizat ion detecto r. A T C-1 high
resolut ion capillary column, 0. 25mm in diameter
and 30m long , 0. 25m in film thickness w as used
w ith a split auto-inject ion ( split ratio 55∶1) . F low
r ate for carrier g as ( He) w as 1. 3kg cm
- 2 ( about
1. 0 mL·min- 1 ) , and 40 mL ·min- 1 for N 2 and
0. 5 kg·cm- 2 ( about 30 mL · min- 2 ) fo r H2 .
Column temperature w as set at 200℃ fo r 0. 5
m inutes init ially , and then raised sequent ially to
250℃ at 20℃ min- 1 , 300℃ at 10℃ min- 1, 324℃ at
6℃ min- 1, 350℃ at 3℃ min- 1 and down to 200℃
in 3 minutes. T he temper ature for the inject ion and
detector ports w as maintained at 350℃.
2. 4 Calibration
A standard solut ion containing a mix ture of
odd and even n-alkanes f rom C22 to C36 was
prepared w ith three dif ferent concentrat ions. T he
r esponse factors fo r each n-alkane w ere calculated
f rom peak areas and the known concentrat ions
w ith one-point o r tw o-point calibration curve
method. Ex tract ion rate was calculated from tw o
internal standards ( C22 and C34 ) . n-alkanes
concentrat ions in samples w ere calculated by
r efer ence to the difference of the ext ract ion rates of
tw o internal standards.
2. 5 Data analysis
T he data w as analy zed w ith ANOVA model to
test the ef fects of method× sample on n-alkane
concentrat ions.
Table 1 Alkane concentrations(mean±SD) (mg·kg- 1 ) in Miscanthus sinensis, Zoysia j aponica and Lolium perenne.
C29 in Zoysia j aponica and C35 in Lolium perenne were not detected because of their low concentrations.
**represents the mean value of alkane concentration diff ered signif icantly(P< 0. 01)
S ample Pr ocedu re C29 C 31 C33 C35
Miscanthus sisnensis
A 100±4 263±14 431±29 103±8
B 111±3 245±5 444±14 118±2
C 108±3 248±5 437±4 117±1
Zoy sia j ap onica
A ** 56±2 59±4 546±25
B 93±4 118±5 83±2
C 93±4 117±5 95±3
L olium p er enne
A 95±9 234±4 184±10
B 109±4 230±2 190±4
C 108±1 231±2 178±1
89第 2期 张英俊等:植物表皮蜡中链烷含量测定方法的研究
Fig. 1 The chromatographic chart analysed by Procedure
( upper) and Procedure C( lower) of Miscanthus sinensis ( M) ;
Zoysia j aponica(Z) and Lolium perenn (L) . ** ,
diff ered signif icantly between procedures(P< 0. 01)
3 Results and Discussion
The peaks o f short chain n-alkanes, less than
C29 and including C29 fo r Zoy sia j aponica, w ere no t
detected because of their low concentrat ions ( F ig.
1) . These results are consistent w ith the reports of
M olo ssini et al .
[ 12]
who found that C29 and C31 were
alw ays present in the highest relat iv e pr opo rt ions,
and C33 was followed. The concentrat ion of long
odd chain n-alkanes o f C29, C31 , C33 and C35 were
show n in Table 1. T here was no significant
difference ( P> 0. 05) for n-alkanes concentrat ions
o f M iscanthus sinensis and L olium perenne among
three pr ocedures. How ever , the concentrations of
C31 and C33 of Zoy sia j ap onica in procedure A were
significant ly ( P< 0. 01) low er than in pro cedure B
and C, and C35 was significant ly higher ( P< 0. 01) .
T his r esult could be due to the fact that the peak of
contam inated substances had interfered w ith the
determinat ion o f C34 ( F ig. 1Z ) . There were tw o
parts in the chart of 3 grasses in Figure 1. T he
low er part was the gas chr omatogr aphic charts
determined w ith procedur e C, and the upper w as
determined w ith pro cedure A . T he charts of
procedure B among 3 g rasses were similar to the
shapes of the charts determ ined by pro cedure C
( data not show n) . T her e w as no interference from
the contam inat ion in raw ex t racts of Miscanthus
sinensis and L ol ium p erenne ( F ig . 1 M , L ) . T he
peak pat ter ns o f the contaminat ion in the
chromatog raphic chart show ed the contamination
differed fr om species, and that the peak of C34 and
C35 were covered by contaminat ion in Zoy sia
j aponica. Compared to procedur e B and C, the n-
alkane concentrat ions in M iscanthus sinensis and
L olium perenne were not af fected significant ly by
the contamination in pro cedure A that could result
in t ime and cost saving s in the analyt ical
procedure. How ever , more replicates might be
r equired because of the high standard deviation
obser ved in the new simplified pr ocedure ( T able
1) .
T he results show ed the chem ical analy sis of n-
alkane w ithout purif icat ion by sil ica gel could be
used for the determ inat ion of the n-alkane
concentrat ions in M iscanthus sinensis and L olium
p erenne except for Zoy sia j aponica. How ever, the
analysis involving elim inat ion of the purification
90 草 地 学 报 第 10卷
pr ocess w ith silica gel should be used carefully,
especially in the est imat ion of diet composit ion
because the analysis of faecal samples w ould also
be required. It may not be applied to the faecal
samples w hen the g razing pasture contains the
compound botanical composit ion since many kinds
of contaminat ion ar e present . Procedure C should
be thought of as a standard analy sis o f n-alkanes in
herbage and faeces because n-heptane is a less
hazardous so lvents than n-hexane, and n-hexane
could be replaced by n-heptane in the w ho le
analy tical procedure.
In conclusion, the analysis eliminat ing silica
gel could be used for the long chain n-alkane
determinat ion of M iscanthus sinensis, L ol ium
per enne, but not fo r Zoysia j aponica.
References
[ 1] Dove H, Mayes R W . T he use of plant w ax alk anes as mar ker
substances in studies of th e n ut rit ion of herbivores : a review
[ J ] .Au st ralia J Agri Res, 1991, 42: 913～925
[ 2] Dove H, Mayes R W. Plant wax components: A new appr oach
to est imat ing intak e and ditet compos it ion in herbivores [ J ] . J
Nut rit ion, 1996, 126: 13～26
[ 3 ] Duncan A J, M ayes R W, Lam b C S, Young S A, Cast ill o I.
T he use of natur ally occurring and art if icially appl ied n-
alkanes as marker s for est imat ion of short-term diet
com posit ion and intake in sheep [ J ] . J Agri Sci , Cam bridge,
1999, 132: 233～246
[4] Mayes R W , Dove H. M easur ement of dietary nut rient intake
in free-rangin g mammalian herbivores [ J ] . Nut rit ion Res Rev,
2000, 13: 107～138
[ 5] Mayes R W , Lamb C S, Colgrove P M . T he use of dos ed and
her bage n-alkan es as markers for the deter mination of herbage
intak e[ J] . J Agri Sci, Cambridge, 1986, 107: 161～170
[ 6] Vu lich S A, oRiordan E G, Hanrahan J P. Use of the n-alkanes
for the deter mination of her bage intak e in sh eep accuracy and
precision of th e es t imates [ J ] . J Agri Sci, Cambridge, 1991,
116: 319～323
[ 7] Laredo M A, S imposon G D, M inson D J , Orpin C G. The
potent ial for us ing n-alk anes in t ropical forages as a m ark er for
the determinat ion of dry matter b y gr azin g ruminants [ J ] . J
Agri Sci, Cambr idge, 1991, 117: 355～361
[ 8] Dove H. Us ing the n-alkanes of plant cut icular w ax to est imate
the species compos ition of h erbage mix tu res [ J ] . Aus tralia J
Agri Res, 1992, 43: 1711～1724
[ 9 ] Olivn M, Osoro K, Mart ine Z. Ef fect of tem perature on
alkan es ext ract ion fr om faeces and pasture. In 45th meet ing of
European Association of Animal Produ ct ion, Edinbu rgh, 1994:
184
[10] Olivn M, Osoro K. Effect of temper ature on alkan e ext ract ion
f rom faeces and herbage[ J ] . J Agr i S ci, Cambridg e, 1999, 132:
305～312
[ 11] Vulich S A, Hanrahan J P, C rowley B A. Modif ication of the
analyt ical procedures for th e determinat ion of h erbage and
faecal n-alkanes used in the est imat ion of herbage intak e[ J ] . J
Agr i S ci, Cambridge, 1995, 124: 71～77
[ 12 ] M alossin i F, Piasent ier E, Bovolen ta S. n-Alkan e content of
s ome forages[ J ] . J Agr i S ci, Cambridge, 1990, 107: 161～170
91第 2期 张英俊等:植物表皮蜡中链烷含量测定方法的研究