Chapter 29
Micro Liquid Chromatography-Mass Spectrometry Combination
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Application to Allelochemical Compounds Hans Alborn and Gunnar Stenhagen Department of Chemical Ecology, University of Göteborg, Kärragatan 6, S-431 33 Mölndal, Sweden
Many nonvolatile and thermally labile allelochemicals can be well separated by liquid chromatography (LC). Identification of the separated components on-line by mass spectrometry (MS) is of great value. Fused-silica LC columns of 0.22 mm ID packed with small-particle material are used in the described LC/MS system. The shape of the column end allows direct connection to a electron impact ion source of a magnetic sector mass spectrometer. Separations by LC are reported and LC/MS mass spectra are shown for monoterpenes, diterpene acids, phenolic acids and cardiac glycosides. The LC/MS system provides identification capability and high-efficiency chromatography with a universal detector. Allelochemicals found in extracts of such botanical materials as plant leaves can often be well separated by liquid chromatography (LC). Identification of the separated components on-line by mass spectrometry (MS) is of great value because LC has the a b i l i t y to deliver samples into the ion source of the spectrometer with low or no thermal decomposition. A mass spectrometer adapted for chemical ionization can tolerate a solvent flow rate into the vacuum system of 5-10 yL/min. This suggested the use of a LC column operating at this low flow rate. A micropacked fuseds i l i c a column (1-3) is well suited for this application. In this paper we describe how this type of column can be very simply interfaced to a mass spectrometer with an electron impact ionization source. We also report experimental results obtained with compounds of a l l e l o chemical interest. 0097-6156/87/0330-0313$06.00/0 © 1987 American Chemical Society
In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
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LC/MS System Fused-silica tubing (20-50 cm χ 0.22 mm ID) is filled with 3-or 5- ym h i g h - p e r f o r m a n c e l i q u i d chromatography (HPLC) p a c k i n g m a t e r i a l by u s i n g a h i g h p r e s s u r e slurry packing technique (3). In o r d e r t o e f f e c t a s i m p l e and e f f i c i e n t c o u p l i n g o f t h e column t o t h e i o n s o u r c e o f t h e mass spectrometer, the column ( f o r r e a s o n s described below) must end w i t h a f i n e t i p . F i g u r e 1 and 2 shows two a l t e r n a t i v e d e s i g n s of the columns. Figure 1: The column t u b e end i s drawn t o a fine tip. To prevent f o u l i n g the i o n source with packing material a small amount o f c o a r s e r HPLC material is placed i n s i d e t h e t i p . By c o a t i n g w i t h high-temperature epoxy glue (EPO-TEK 353ND) t h e tip is mechanically protected. This design g i v e s the best chromatographic results. A 30-cm p a c k e d column w i t h 3 ym S p h e r i s o r b ODS particles g i v e s almost 5 χ 10 theoretical plates (3). The separation impedance (unretained solute time per plate multiplied by the pressure drop per plate and divided by the viscosity of the eluent) has been c a l c u l a t e d as 750. Figure 2: In t h e end o f t h e column a 50- ym ID f u s e d - s i l i c a t u b e i s cemented w i t h h i g h - t e m p e r a t u r e epoxy g l u e . A g l a s s f i b r e f i l t e r f r i t i n f r o n t of the tube h o l d the chromatographic bed. A f t e r t h e p a c k i n g , t h e end t u b e i s drawn o u t t o a f i n e t i p and p r o t e c t e d w i t h epoxy g l u e . Higher mechanical s t r e n g t h o f t h e t i p and n o t so high p a c k i n g p r e s s u r e i s needed w i t h t h i s d e s i g n . A s t a n d a r d HPLC pump ( S p e c t r a P h y s i c s 8700) i s u s e d in c o n s t a n t - p r e s s u r e mode and p u l s e - f r e e f l o w r a t e s from 1 t o 5 yL/min a r e o b t a i n e d w i t h o u t any m o d i f i c a t i o n s of t h e pump. A 0.5-yL syringe-loaded micro i n j e c t o r (Rheodyne 7520) i s u s e d f o r sample i n j e c t i o n . To minimize the dead volume (< 20 nL) t h e fused silica column is connected d i r e c t l y t o the i n j e c t o r b l o c k . The column i s l e d i n t o the ion source through a ball v a l v e and a 0.5-mm ID s t a i n l e s s - s t e e l t u b e (Figure 3). The t u b e t e r m i n a t e s a b o u t 15 mm f r o m one o f t h e f o u r sample p o r t s i n the ion source b l o c k . The high vacuum seal c o n s i s t s o f t h e v e s p e l ( t r a d e m a r k o f DuPont) f e r u l e mounted on t h e b a l l v a l v e . During o p e r a t i o n the t i p of t h e column can be e a s i l y adjusted for optimal signal-ton o i s e r a t i o . Exchange o f LC c o l u m n s o r c h a n g e - o v e r t o t h e gas c h r o m a t o g r a p h i c s y s t e m can be done i n a few m i n u t e s . Ions are produced i n a s t a n d a r d e l e c t r o n impact i o n source (VG 70-70). The o n l y m o d i f i c a t i o n n e c e s s a r y to obtain t r u e e l e c t r o n impact s p e c t r a was t o w i d e n t h e LC i n l e t h o l e and u n c o v e r t h e o t h e r t h r e e i n l e t p o r t s . Vaporization i s governed by four factors: the electrostatic f i e l d between t h e column t i p and the ion source block, t h e c o n f i g u r a t i o n o f t h e column t i p , t h e pressure in the i o n source housing, and the ambient temperature. The e l e c t r o s t a t i c f i e l d between t h e t i p and 4
In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on November 21, 2015 | http://pubs.acs.org Publication Date: January 8, 1987 | doi: 10.1021/bk-1987-0330.ch029
29.
ALBORN AND STENHAGEN
Micro
LC-MS
Combination
F i g u r e 1. Column t i p , f i r s t v e r s i o n . Column I.D. 0.22 outlet I.D. 40 ym; 1 mm o f t h e column c o n s t r i c t i o n f i l l e d w i t h c o a r s e p a c k i n g m a t e r i a l (30-50 ym).
315
mm; is
1 5
1mm
Figure 2. Schematic d i a g r a m o f column t i p , second version. 1, F u s e d - s i l i c a column I.D. 0.22 mm; 2, chromat o g r a p h i c b e d ; 3, g l a s s f i b e r f i l t e r f r i t ; 4, d r a w n - o u t 50 ym fused-silica tube; 5, h i g h temperature epoxy coating
F i g u r e 3. S c h e m a t i c d i a g r a m o f t h e LC/MS c o n n e c t i o n . 1, Ion s o u r c e ; 2, s t a i n l e s s - s t e e l tube; 3, b a l l v a l v e ; 4, h i g h vacuum s e a l ; 5, LC c o l u m n ; 6, GC i n l e t ; 7, window. Modified from r e f . 3.
In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on November 21, 2015 | http://pubs.acs.org Publication Date: January 8, 1987 | doi: 10.1021/bk-1987-0330.ch029
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the i o n source i s t h e major f a c t o r f o r vaporization. Owing t o t h i s t h e e l u a t e from t h e column l e a v e s t h e tip i n t h e form o f v e r y s m a l l i n v i s i b l e d r o p l e t s . To p r e v e n t electrical spark t h e e n d p a r t ( a t l e a s t 10 mm) of the column must be e l e c t r i c a l l y i s o l a t e d from g r o u n d . A fine column t i p c a n be u s e d w i t h l o w e r e l e c t r i c f i e l d t h a n a c o a r s e t i p . Normal t i p p o s i t i o n i s shown i n F i g u r e 3 and the e l e c t r o s t a t i c f i e l d obtained with the acceleration v o l t a g e (3-5 kV) i s s u f f i c i e n t . The mass s p e c t r o m e t e r i s a l a r g e - r a d i u s magneticsector instrument b u i l t i n o u r d e p a r t m e n t (3) f o r b o t h GC/MS and LC/MS work. The c a p a c i t y o f t h e h i g h vacuum system i s s i m i l a r t o t h a t o f o r d i n a r y mass spectrometers adapted t o chemical ionization. Results M o n o t e r p e n e s a r e o f t e n a n a l y z e d by c a p i l l a r y g a s chromatography. LC/MS o f f e r s a new method f o r t h e s e p a r a t i o n and i d e n t i f i c a t i o n o f members o f t h i s c l a s s o f compounds ( F i g u r e 4 ) . The s e p a r a t i o n o c c u r s i n a c o l d l i q u i d p h a s e , which eliminates the risk of thermal decomposition. S p e c t r a o b t a i n e d from t h e LC/MS s y s t e m ( F i g u r e 5) c a n be i n t e r p r e t e d by c o m p a r i s o n w i t h GC/MS r e f e r e n c e s p e c t r a . P h e n o l i c a c i d s a r e o f t e n found i n p l a n t t i s s u e , and have been i m p l i c a t e d i n many c a s e s o f a l l e l o p a t h y ( 4 ) . Figure 6 shows a separation of three free phenolic a c i d s and Figure 7 shows mass spectra obtained from these compounds. T h e s e s p e c t r a g i v e b o t h m o l e c u l a r w e i g h t and structural information. Phenolic a c i d s can e a s i l y be t h e r m a l l y d e c a r b o x y l a t e d . The h e i g h t o f t h e m o l e c u l a r i o n peak v a r i e s owing t o i o n s o u r c e t e m p e r a t u r e . The v a r i a tion depends a l s o t o some e x t e n t on t h e c o m p o s i t i o n of t h e LC e l u e n t , and t h i s w i l l be f u r t h e r e x a m i n e d . Chlorogenic acid, t h e most widespread depside i n the p l a n t kingdom, h a s o f t e n been a s s o c i a t e d w i t h r e s i s t a n c e of plants t o fungal attack (5). Most p h e n o l i c s o c c u r i n plant i n w a t e r - s o l u b l e form as g l y c o s i d e s but hydroxycinnamic acids differ from most other phenols in o c c u r r i n g most f r e q u e n t l y a s q u i n i c a c i d e s t e r s ( 6 ) . The mass s p e c t r u m o f c h l o r o g e n i c a c i d i s shown i n F i g u r e 8. The peak a t m/z 354 r e p r e s e n t s t h e m o l e c u l a r i o n and o n l y small peaks ( e . g . M-18 a t m/z 336) a r e p r e s e n t i n t h e high-mass r e g i o n . The p r o m i n e n t p e a k s a t m/z 180 a n d 163 are r e l a t e d t o the aromatic ( c a f f e i c acid) part of the structure and o n l y s m a l l f r a g m e n t p e a k s f r o m t h e q u i n i c acid part are present. Resin a c i d s . To o b t a i n good mass s p e c t r a o f r e s i n a c i d s it h a s p r e v i o u s l y been n e c e s s a r y t o e s t e r i f y t h e a c i d s . Figure 9 shows a chromatogram o f a c r u d e resin sample f r o m P i n u s montana. The s m a l l p e a k s a t the beginning of the chromatogram r e p r e s e n t s m o n o t e r p e n e s and t h e main
In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on November 21, 2015 | http://pubs.acs.org Publication Date: January 8, 1987 | doi: 10.1021/bk-1987-0330.ch029
29.
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Micro LC-MS
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F i g u r e 4. LC s e p a r a t i o n o f a t e r p e n e m i x t u r e . 1, Thymol; 2, p-cymene; 3, γ - t e r p i n e n e ; 4, α-terpinene; 5, 3 pinene; 6, α - p i n e n e . Column: 30 cm χ 0.22 mm I.D. 3-um Spherisorb ODS. Mobile phase: methanol-water (80:20) D e t e c t i o n : T I C ( i o n s o f m/z ζ
π c r Η c
2
> Ο
70 Ο
Ο ο PC m § η >
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29.
ALBORN AND STENHAGEN
Micro
LC-MS
Combination
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10 MIN
F i g u r e 6. Separation of free phenolic acids. 1, C a f f e i c a c i d ; 2, p - c o u m a r i c a c i d ; 3, s i n a p i c a c i d . Column 18 cm χ 0.22 mm I.D. 3-ym S p h e r i s o r b ODS. M o b i l e p h a s e : m e t h a n o l w a t e r - a c e t i c a c i d (20:75:5). D e t e c t i o n : T I C ( i o n s o f m/z
