クロマトグラフィーを中心とする機器分析の実際（株）日東技術情報センター

掘正典

1．はじめにクロマトグラフィーは化学に関するあらゆる分野で利用されている
分離・分析技術です。機器の取り扱いに不慣れな人や、この手法に習熟していない分析者が試みても、何らかの有益な情報を得ることができる
簡便さとパワフルさの両方を備えています。しかし一見、表面的には簡単そうに見えるこの技術も目的に合った情報を得るためのプロセスは複雑な場合が多く、クロマトグラフィーと別の機器を組み合わせ複合技術として扱う場合も少なくありません．
今回はクロマトグラフィーとその周辺の技術を使って得られた分析
結果について、て(1）GC を用いる発生ガス分析、（2）はによる天然油脂の分子種解明、を主題として実際の分析例を中心に紹介します。
2．GC を用いる発生ガス分析
ある環境下で試料から発生する化学物質を分析することは、それはど単純ではなく現在では種々の方法が考案、開発されています。その理
由のーつは試料のおかれている環境すなわち温度、放置している時間、試料の回りの雰囲気（空気中不活性ガス中あるいは真空中等）が分析目的によって異なるため、ーつの分析手法だけで包括してしまうことが事実上不可飴である事があげられます．我々は市販の装置や比較的安価に製作でさるサンプリング装置を製作しこれらに対処してきましたので以下に紹介します．
2．1 フィルム中の残存溶剤の定量
従来から樹脂やフィルム中の低沸点成分の分析にはスタティツクヘッ
ドスペース法やパージアンドトラップを用いるダイナミックヘッドスペ
ース法が用いられてきました。後者の方法は検出感度がスタテック法に
比ベ 10〜100 倍向上すると言われその代表的な装置が JHS−100 ですが、我々はキュリーポイントパイロライサーを用いてこの種の分析も試みてきました．
図 1 にフィルム中に残存している高沸点溶剤 NMP（N−メチルピロリドン）を、キュリーポイントパイロライサー（機種 JHS-3S 型）GC により分析した時のクロマトグラムを示しました。試料は 315℃で 5 分間加熱され、その間カラムを 50℃に保って生成したガスをカラム内に吸着させ、その後カラム温度を昇温し NMP を溶出させています。分離カラムは TenaxGC を用いていますが、これは同時に吸着管としても作用します。試料の加熱
温度は試料があまり分解しない温度でかつ効率よくガスを発生する条件を見いだす必要がありますが、一度条件が決まれば溶媒抽出と比べ簡便で高感度な方法です。本法の場合発生したガスを室温付近で吸着させる
ため沸点があまり低すぎたり、吸着能力の弱い物質の定量精度は悪くなりますが、この手法が適用できる物質は比較的多く存在しています。
図 2 には同様な方法で樹脂中のトリクロロエチレンを検出した例を示してあります。試料はパイロホイルに包まず直接石英の試料管に入れ150℃で 10 分間加熱しました。検出器に質量分析計を用いていますので試料の同定がより確実にできマスクロマトグラムから定量も可能です。
この試料の場合 ppb レベルでの検出が可能でした。
2.2 害虫防除フエロモンデイパイスからのフエロモン放出速度の測定
農薬による害虫駆除一辺倒から抜け出し作物の被害を許容し得る水準以下に抑えるように害虫の密度を管理する害虫防除が、最近積極的に進められています。その一端を担うものとして性フエロモンを用いる生物的駆除があります。これを実用化させるにはフエロモンを一定期間分解する事なくほぼ一定の量で放出させるフエロモンデイパイスが必要となりますが、デイパイスの評価としてフエロモンの揮散量すなわちフエ
ロモン放出速度を測定することが必要です。
フエロモンは図 3 に示したように酢酸エステルやアルデヒド、アルコ
ール、ケトン等の脂肪族化合物が多くまた高沸点を有するものがほとんどです。従って放出速度を正確に求めるには、これら高沸点物を効率よく捕集するシステムが必要となります。我々はフエロモンを温度や風速などが制御された環境下で吸着剤に捕集しそれを溶媒抽出して GC 分析す
ることにより放出速度を直接的に測定する手法を、デイパイス評価の一つとして用いてきました（図 4 参照）。検出下限はチャノコカクモンハマ
キの性フエロモンの一種である Z−11−テトラデセニルアセテート（Z−11−
TDA）で、捕集時間を 5 時間として 50ng／day と求まっています。図 5 には実際のデイパイスから得られたガスクロマトグラムの一例を示しましたが、

この手法を使えば野外の環境をシュミレーションしておおよそのフエロ
モン放出速度を見積もることも可能です。また吸着管を JHS−100 に装着
し分析すれば感度は約 1000 倍は向上すると予想されるので、捕集環境を整えれば虫体一匹から放出されるフエロモンの定量も比較的容易にできるかもしれません．
3．LC による天然油脂の分子種解明
トリグリセライド（TG）は天然油脂の主成分で動物の皮下脂肪や植物の種子などに多く存在しています。この分子構造は図 6 に示したように 3個の脂肪酸が 1 個のグリセリンにエステル結合したものです。したがって理論上、油脂が n 種の脂肪酸で構成されているとき異性体を全く考慮しなくても（N3+3n2+2n）／6 個の分子種が考えられます．天然油脂は通常数種類の脂肪酸よりなっているので、その組み合わせよりなる分子種は非常に複雑になります．
これらの分子種解明に最近では逆相系の LC を使うことが多くなっています。これは脂肪酸の総炭素数と二重結合の総数から定義される
Partition Number（PN）とよばれる分離因子と TG の保持時間の対数が経験的にはぼ linear な関係になることが発見されたからです（図 7）．この法則と LC/MS を組み合わせれば天然油脂の組成解明に非常に有効であることは既に示したことがあります（試料 1 参照）。しかし脂肪酸組成の複雑な海産油の解明には今のところそのままでは適用が困難であるのが現状です．図 8、図 9 に一例として高度不飽和脂肪酸（PUFA）を多く含む魚油のLC/MS のクロマトグラムと構成脂肪酸のガスクロマトグラムを示しました。魚油に含まれる個々の TG 分子種の存在と量的関係を明らかにするた
めには少なくとも理論段救が数十万以上のはカラムが必要と言われています．従って現在ではその分析が困難なことから分子種に関する知見がほとんどありません．図 8 のクロマトグラムの画分Ⅰ、Ⅱは油旨のなかでも最も極性の高いフラクションですが、同じ PN を多く含みかつ完全分離が不可億なため分析をさらに進めるうえでは LC/MS のデータの他に分取が必要です。図 10、図 11 に LC−908 を用いて得られたリサイクルはのクロ
マトグラムと分取した後の分析データを示しました．完全に単離できな
いまでも精製と分取が同時にできるリサイクルはのメリットは後の工程を考えれば大きいものがあります。

4．結語
クロマトグラフィーは 1906 年、TsWett らが色素混合物を分離したことより始まりましたが、今現在でも分離、精製の中核をなしています．
クロマトグラフィーの技術は今後さらに発達して行くものと思いますがその中でもパイロライサーやリサイクル LC により、今まで限界であった
ことが少しづつ克服されていくようです．

資料 1

Determination of Molecular Species of Triacylglycerols
by Reversed Phase Liquid Chromatography/
Double Focussing Mass Spectrometry
with a Frit-CI Interface
Masanori Hori. Kenji Sugiura.Yuko Sahashi,I, and Seiji Koike[質量分析. voI. 39, No. 3, p, I33〜140(1991). 別冊]

MASS SPECTROSCOPYREGULAR PAPERS

Vol. 39. No. 3.June 1991

Determination of Molecular Species of Triacylglycerols
by Reversed Phase Liquid Chromatography/
Double Focussing Mass Spectrometry
with a Frit-CI Interface
Masanori Hori. Kenji Sugiura.Yuko Sahashi,I, and Seiji Koike(Received January 26, 199l)
This paper demonstrates the application of a combined system of reversed phase liquid
chromatography and chemical ionization mass spectrometry with a frit interface (frit-CI LC/MS) for identification of molecular species of triacylglycerols in natural oils. From the studies
of fundamental operating conditions of this LC/MS system. we found that the chemical
ionization using ammonia as a reagent gas yielded readily detectable ammonium adduct andprotonated ions as well as characteristic fragment ions. The intensities of these ionsdepended considerably on the ion source temperature. The ion source temperature operated
from 350 to 400℃ was suitable for both the resolution of chromatograms obtained by the LC/
MS and the relative intensities of the molecular ion species. This method enabled to get theidentifiable mass spectrum of trieicosapentaenoin (20 : 5-20 : 5-20 : 5 MW=944). while it has
been difficult to analyze by the GC/MS. This system was also applied to the compositional
analysis of evening primrose oil composed of triacylglycerols having total carbon number of
52 and 54. Mass chromatographic technique as well as mass spectra provided detailed
information on the structures of triacylglycerols in the oil.

1. Introduction
Triacylglycerols, main components innatural oils, are esters formed from one
glycerol combined with three fatty acids
and exist mainly in subcutaneous tissuesof animals and seeds of plants. Bacause
many kinds of fatty acids exists innature,it is expected that there are many molecular species of triacylglycerols in naturaloils. Based on a theoretical calculation,1)
when the oil is constructed of n kinds offatty acids, the number of molecular species of triacylglycerols is (n3+3n2+2n)/6
without thinking of isomers. Therefore

the experimental determination of theirdetailed structure remains the most formidable problem in lipid chemistry. Gas
chromatography/mass spectrometry (GC/MS) has been used to study the composition of these complicated oils.2) However,
it is generally not easy to analyze by GC/
MS when they have the high boilingpoints. Reversed phase liquid chromatography/mass spcctrometry equippedwith a direct liquid inlet3〜5) or a thermospray interfaces) has been recently used
for the studies of lipids. The advantage of
LC/MS of triacylglycerols is that it provides not only the mass spectra and mass

chromatograms but also information of
the partition number (PN)7) defined bythenumber of double bonds (DB) and that of
total carbons (TC) of a triacylglycerol (PN=TC-2DB).
In this paper some Preliminary results
are reported on the frit-CI LC/MS of authentic triacylglycerols and evening primrose oil to get information on molecularspecies of triacylglycerols in natural oils.
2. Experimental
2.I Materials
Triolein and trilinolein from Gasukuro
Kogyo lnc. were used without further
purification. Evening primrose oil waspurchased from a commercial source andit had a purity better than 95%.
Trieicosapentaenoin (tri-EPA) was synthesized bylipase esterification of eicosapentaenoic
acid and glycerol. Resulting tri-EPA was
purified by a FIorisil column. All thesamples were stored in closed containers
at 〜20cC. Tetrahydrofuran of HPLC
grade was used within a day or two after
openning the bottle. The other chemicals
were of analytical reagent grade.
2.2 Apparatus
The HPLC analyses were performed on
a Hewlett Packard model l090L liquid
chromatograph with a reversed phasecolumn, ODS-Hypersil(100 mmx2.1 mm).

The mobile phase composed of acetonitrile and tetrahydrofuran was used in ahelium atmosphere. The column was
operated at a flow-rate of 0.l5 ml/min anda column temperature of 40℃. Triacylglycerols were dissolved in the mobile
phase at 5〜50mg/ml. and 1〜3μl portions were injected into the column. The
eluent via a UV detector was introduced
into a JOEL JMS-AX505H double focussing mass spectrometer equipped with a
firt-CI interface (shown in Fig. I). A pneumatic splitter was used to reduce theflow-rate of the eluent into the ion source
to as little as a few A/min. While the reduced eluent permeated through the frit.
triacylglycerols and the solvents were
evaporated by the ion source heaters. Triacylglycerols were then chemically ionized with the solvent vapor or ammonia
as the reagent gas.
All the positive ion mass spectra wereobtained at 250 eV of the electron energy
and at +3kV of the ion accelerating
voltage. A post acceleration detector
using a conversion dynode at -10kV
was used in order to detect the high mass
region sensitively. Full mass spectra (50〜l500 mass unit) were recorded every 3
see over the entire elution profile, and thedata were processed using a JEOL
JMA-DA5000 data system. A Hewlett
Packard model 5890A gas chromatograph with a columun DB-23 (30 mxO.25
mm df=0.25pm) was used for GC analysis. and a Hitachi model M-80A double
focussing mass spectrometer was used for
FD mass spectrometry.
3. Results and Discussion
3.I Fundamental operating conditions of LC/MS
The LC/MS equipped with a direct
liquid inlet operated in chemical ioniza-

tion mode using an elution solvent as thereagent gas have been demonstrated tobesuitable for the identification of triacylglycerols by Kuksis et al.3) In the mass
spectra obtained by this system. the[MH-RCOOH] + ion of the base pat wasused to determine the fatty acid composition of triacylglycerols. However, theother characteristic ions including the
RCO', [RCO+74] +, and [M+H] + ionswere observed with unsufficient intensity
for analysis of the mass spectra. Murata8)
employed gas chromatography/chemical
ionization mass spectrometry (GC/CIMS)
with ammonia to determine the composition of triacylglycerols. By this method,the [MH-RCOOH]+ and [M+NH4] + ionswere recorded intensely enough to be
used for the structural study. Therefore.,
it is expected that the liquid chromatography in combination with ammonia CI
mass spectrometry can provide more information than use of an elution solvent
as the reagent gas.
Figure 2 shows the ammonia CI mass
spectrum of triolein measured by the fritCI LC/MS at 390℃ of the ion source
temperature. When ammonia was intro-

duced into the ion source, the ammonium
adduct ion [M+NH4]+ and the protonated ion [M+H] + were observed as themolecular ion species. In the GC/CIMS
using ammonia Murata8) reported that
only the [MHNRCCN3H] + ion was observed as the fragment ions, and no ionswere recorded in the low mass region.
However under the LC/MS conditions inFigure 2, the [M+NH4] + ion at m/I 902
and the fragment ions such as the RCO'
at m/I 265 and the [RCO+74] + at m/I
339 were recorded intensely enough toanalyze the mass spectrum. The basepeak was the [MH-RCOOH] + ion at m/I
603 and the [M+NH4] + ion was recordedwith relative intensity of 59% against thebase peak as shown in Table I. On theother hand, in case of using the elution
solvent as the reagent gas, the mass spectrum of triolein showed only the [M+H] +ion as molecular ion specis with relative
intensity of ca. 3% against the [MHRCWH] + ion of the base peak ion. The
other characteristic ions observed weresimilar to those of the ammonia CI mass
spectrum.
Figure 3 shows the ammonia CI mass

spectra of trilinolein measured by thefrit-CI LC/MS at 300 to 450℃ of the ion
source temperatures. When the ion

source temperature was 300℃, the basepeak was the [M+NH4]+ ion at m/I 896.
and the fragment ions were recorded with

relative intensity of less than 10%
againstthe base peak. The [M+H]' ion at m/I
879 was also observed more intenselycompared with triolein and the intensity
showed a maximum at 400T. An increase in the ion source temperature wasaccompanied with an increase inintensityof the fragment ions, and a decrease inintensity of the [M+NH.] + ion. These
results indicate that pyrolysis of triacylglycerols on the frit and/or the cleavageof the [M+NH.]+ ion are accelerated
with an increase of the ion source temperature. The temperature more than
350T was necessary to get the fragment
ions of intensity enough to determine thestructure of trilinolein. In case of using
the elution solvent as the reagent gas at
an ion source temperature of 390℃, the[M+H] + ion was observed with relative
intensity of 35% against the base pear atm/z 599 as shown in Table I. The intensity was also larger than that of triolein,and enough for the structural study.
Above mentioned the intensity of the[M+H] + ion was affected by the numberof double bonds of the triacylglycerol aswell as the ion source temperature.

Figure 4 shows the reconstructed ion
chromatograms (RIC). from m/z 200 to1000 of evening primrose oil measured by
the frit-CI LC/MS to investigate the dependence of the resolution of RIC on theion source temperature. From the GC
analysis after hydrolysis and methyl esterification, evening primrose oil wascomposed of palmitic acid (l6:0), stearicacid (18 : 0), oleic acid (l8 : 1 n-9),
Iinolenic
acid (l8: 3 n-6), and linoleic acid of main
component (l8 : 2 n-6. 73% in fatty acids).
The carbon number of the triacylglycerols in this oil were estimated to be 52 and
54 from the FD mass spectrum. The RICs
shown in Fig. 4 indicate that the ion
source temperature more than 350℃ is
necessary to get better resolution for thisoil. The resolution of RIC at 350℃ was
similar to that of the chromatogram givenby the UV detector.
The results mentioned above lead to an
unambiguous conclusion that the firt-CI
LC/MS using ammonia operated at an ion
source temperature of 350 to 400℃ is
suitable for the structural study of triacylglycerols. The mass spectrum andthe RIC of tri-EPA (20 : 5-20 : 5-20 : 5 MW

=944) measured under the similar condition were shown in Fig. 5. Since the triEPA having fifteen's double bonds is notstable when exposed to high temperature
for a long time. it is difficult to analyze by
the GC/MS. The frit-CI LC/MS enabled to
get identifiable mass spectrum of
tri-EPA,however the mass spectrum became more
complicated than those of triolein andtrilinolein.
3.2 Compositional analysis of evening primrose oil
This LC/MS system was applied to theidentification of triacylglycerols in theevening primrose oil. A linear gradient of
0〜30% (0〜30 min) of tetrahydrofuran inacetonitrile was employed. The gradientanalysis provided sharper peaks andmorecomplete detection of the low degree unsaturated species than any of theisocraticsolvent system. Furthermore. this gradient system apperas to be applicable todiacylglycerol and triacylglycerol mixtures.
The total ion chromatogram and mass
chromatograms of the evening primrc6e
oil recorded by the RCO+ ion at m/I 263.
the [RCO+74]+ ion at m/z 313 and [MH-RCOOH]+ ions at m/z 575. 601. 599 areshown in Fig. 6. The peaks of m/z 575
and 60l in the mass chromatograms haveslightly different retention time in thepeak 5. This result indicates that thepeak5 contains triacylglycerols more than two
species. The mass chromatograms andthe LC/MS spectrum (shown in Fig. 7)of the peak 5 represent 18: I-18: 2-18: 2of triacylglycerol as indicated by thefollowing ions: the [MH-RCCX]H]+ ions at
m/I 599(l8:2-18:2)and mz 60l (18:118:2),the RCO+ ions at m/z 263 (l8:2)

and 265 (l8:I), the [M+H]+ion at m/I
88landthe [M+NH4] + ion at m/I 898.
The presence of l6:018:2-l8:2 of triacylglycerol is also indicated by thefollowing ions: the [MH-RCCK]H] + ion atm/z 575 (16:0-l8:2), the[M+H]' ion at
m/I 855 and [M+NH.] + ion at m/I 872.
Mass chromatograms and mass spectra
given by the frit-CI Lt/MS provide usefull information on the possible combination of fatty acids in each triacylglycerol component. As shown in Fig. 6, thedistribution of fatty acids in the oil could
be also determined by recording mass
chromatograms of the RCO+ ion or [RCO+74] + ion. For example, the presence oflinoleic acid was confirmed in the peaksof
2, 3, 5, and 6 by monitoring the RCO' ion
at m/I 263. Since the saturated acid suchas palmitic acid had a weak intensity of
the RCO' ion, the [RCO+74] + ion wasmonitored to investigate the distribution.
Table 2 showed the fatty acid composition of each peak analyzed in a similarmanner as described above. We could
identify all the major triacylglycerols in
the evening primrose oil by means of thefrit-CI LC/MS. However. it was difficult
to distinguish positional isomers such as
POL, OLP, and LPO where P. O, and L

of quasi-molecular and fragment ionswere found to vary with the degree of
unsaturation and the ion source temperature.

represent palmitic. oleic, and linoleic
acid.
respectively. These results given by thefrit-CI LC/MS also coincide with resultsfrom the partition number. The
quantities
of individual species were estimated fromthe peak area of RIC from m/I 200 to
1000. However. better chromatographic
separation and determination. of appropriate calibration factors are necessary
for accurate quantitation since the yields