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New Approaches To Interfacing Liquid Chromatography And Mass Spectrometry
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NEW APPROACHES T O INTERFACING L I Q U I D CHROMATOGRAPHY AND MASS SPECTROMETRY S. Tsuge Department o f S y n t h e t i c C h e m i s t r y , F a c u l t y of E n g i n e e r i n g , Nagoya U n i v e r s i t y , Nagoya 464, J a p a n
INTRODUCTION There h a s been a n i n c r e a s i n g i n t e r e s t i n t h e d i r e c t c o u p l i n g of l i q u i d chromatography (LC) w i t h mass s p e c t r o m e t r y (MS), a f t e r t h e g r e a t s u c c e s s o f t h e d i r e c t c o u p l i n g o f g a s c h r o m a t o g r a p h y (CC) w i t h MS. The p o t e n t i a l a b i l i t y of LC/MS i s v e r y l a r g e b e c a u s e of t h e v e r s a t i l e s e p a r a t i o n c a p a b i l i t y o f r e c e n t h i g h - p e r f o r m a n c e LC (HPLC) even f o r v a r i o u s t h e r m a l l y l a b i l e , p o l a r o r g a n i c compounds. Among r e c e n t d e v e l o p m e n t s i n t h i s f i e l d a r e (1) t h e d i r e c t i n t r o d u c t i o n method [l-111 ( 2 ) t h e p r e l i m i n a r y e v a p o r a t i o n method [12211 and ( 3 ) t h e m e c h a n i c a l t r a n s f e r method [22-30J.
The f o r m e r two
methods a r e s i m i l a r t o e a c h o t h e r s i n c e t h e y b o t h u s e t h e s o l v e n t vapour as t h e r e a g e n t gas f o r chemical i o n i z a t i o n ( C I ) (Table 1). Although some of t h e s e a r e now c o m m e r c i a l l y a v a i l a b l e , t h e y a r e m o s t l y s t i l l u t i l i z e d by t h e d e v e l o p e r s of t h e s y s t e m .
T h u s , new
t e c h n i q u e s e n a b l i n g enough s t a b l e a p p l i c a t i o n s t o a v a r i e t y of n o n - v o l a t i l e p o l a r compounds have b e e n e x p e c t e d t o a p p e a r f r o m v a r i o u s f i e l d s where HPLC i s r e c o g n i z e d a s a n i n d i s p e n s a b l e a n a l y t i c a l t o o l . A vacuum n e b u l i z i n g i n t e r f a c e f o r micro-LC-MS
coupling has
been d e v e l o p e d by m o d i f y i n g a j e t s e p a r a t o r f o r GC-MS [12J. T h i s i n t e r f a c e was l a t e r improved by t h e u s e of a v e r y s h o r t c o u n t e r n o z z l e s o t h a t i t c o u l d be a p p l i e d t o v a r i o u s l e s s v o l a t i l e compounds s u c h as g l u t a m i c a c i d . s t e r o i d s , a r o m a t i c a m i n e s and c h l o -
218
TABLE 1 V a r i o u s i n t e r f a c i n g methods f o r LC/MS Interfacing method
Ionieation mode
Research group
~~
(1)Direct introduction
McLafferty
CI
method
(2)Preliminary evaporation method (3)Mechanical transfer
et al.
[ 7 1 81
Henion
et al.
[9-11]
Tsuge
thermo-spray
Vestal
CI, EI
McFadden
[12-191
et al. et al.
et al.
Games
et al.
Bennighoven
r20, 211
L22-251
126, 271
Scott et al. SIHS
[1-6]
Arpino
CI (EI)
method
et al.
128, 291 et al.
[30]
(4)Others (API, Membrane, Heated-wire, etc.)
rine-containing insecticides micro-LC-MS
[LA].
Furthermore, a d i r e c t l y coupled
s y s t e m was c o n s t r u c t e d by u s i n g b a s i c a l l y t h e same
t y p e o f vacuum n e b u l i z i n g i n t e r f a c e a n d a small d o u b l e - f o c u s i n g MS
[l5].
I n t h e s e r e f e r e n c e s , t h e p o t e n t i a l a b i l i t y o f t h e vacuum neb-
u l i z i n g i n t e r f a c e was d e m o n s t r a t e d e m p i r i c a l l y . However, s i n c e vacuum n e b u l i z a t i o n o f t h e L C - e f f l u e n t i s a h i g h l y h e a t - a b s o r b i n g phenomenon, h e a t e n e r g y h a s t o be s u p p l i e d , a t l e a s t t o t h e t o p of t h e n e b u l i z i n g n o z z l e . Depending on t h e nebul i z i n g c o n d i t i o n s s u c h as t h e n a t u r e a n d t h e amount o f n e b u l i z i n g g a s , t h e t e m p e r a t u r e of t h e n e b u l i z i n g t i p and e v a c u a t i o n speed f o r t h e n e b u l i z e r , e x c e s s h e a t energy s u p p l i e d around t h e t o p of t h e n e b u l i z e r c a n s o m e t i m e s c a u s e u n d e s i r a b l e t h e r m a l decomposit i o n o f t h e t h e r m a l l y u n s t a b l e components i n t h e e f f l u e n t b e f o r e
i t r e a c h e s t h e t o p of t h e n e b u l i z e r .
219
Considering the above-mentioned factors, a small water cooling jacket was first of all incorporated into the vacuum nebulizing interface to prevent the introduced LC-effluent from over-heating, causing either boiling up o r thermal decomposition of labile components in the effluent. Then, a bubble saturator was inserted in the line of the nebulizing gas supplier, which enabled the adjustment of the nature of the nebulizing gas saturating a desired solution [16].
In addition, further modification of the nebulizer
was made by changing the nebulizing nozzle from a coaxial capillar y to a fused silica capillary with about 12 B m i.d. which enabled
self-spouting o f the LC-effluent at the top of the nebulizing nozzle 1171. These improvements extend the applicability of the microLC-MS system to fairly non-volatile polar organic compounds.
PRINCIPLE OF NEBULIZING INTERFACE FOR LC/MS Figure 1 shows a general flow diagram of directly coupled chromatograph (CC o r LC)/MS. Conventional GC o r LC is operated with Carrier (Gas o r L i q . I
Sample Inlet
LC
or Gc
Separation column
of excess carrier
Fig. 1. General flow diagram of directly coupled chromatograph (GC or LC)/MS.
220
t h e column e x i t a t one a t m o s p h e r e (760 t o r r ) , w h i l e a MS must m a i n t a i n t h e a n a l y z e r vacuum a t l e a s t as low a s 10-4-10-5 t o r r
[31].
T h e r e f o r e , t h e i n t e r f a c e s h o u l d f u n c t i o n as a remover of ex-
c e s s c a r r i e r o r a s a n e n r i c h e r o f t h e s o l u t e component. I n a GC/MS, both t h e s o l u t e (sample) and t h e s o l v e n t ( c a r r i e r ) a r e t r e a t e d i n t h e g a s e o u s p h a s e , w h i l e i n a LC/MS t h e column e f f l u e n t i s a k i n d of s o l u t i o n i n which a t h e r m a l l y l a b i l e s o l u t e ( s a m p l e ) i s u s u a l l y dissolved i n t h e c a r r i e r solvent. Therefore, i n t h e l a t t e r c a s e t h e L C - e f f l u e n t h a s t o be v a p o r i z e d i n t h e i n t e r f a c e p r i o r t o t h e removal o f e x c e s s c a r r i e r ( e n r i c h m e n t ) . F i g u r e 2 i l l u s t r a t e s t h e e f f e c t i v e n e s s of a vacuum n e b u l i z a t i o n f o r LC/MS i n t e r f a c i n g . I n o r d e r t o e v a p o r a t e a s o l u t e i n s o l u t i o n , (11Whm the bulk solusion i a b a s d under atmepheric prearurr:
F i g . 2. P r i n c i p l e o f vacuum n e b u l i z a t i o n .
221
we u s u a l l y a p p l y h e a t e n e r g y t o t h e s o l u t i o n . When t h e s o l u t e i s v o l a t i l e enough a t a t m o s p h e r i c p r e s s u r e , s i m p l e h e a t i n g i s s a t i s f a c t o r y t o v a p o r i z e t h e s o l u t e as w e l l as t h e s o l v e n t . However, i n t h e c a s e of a n o n - v o l a t i l e s o l u t e , f o r example, s u c r o s e i n w a t e r , when t h e b u l k s o l u t i o n i s h e a t e d u n d e r a t m o s p h e r i c p r e s s u r e ( A ) , t h e s e l e c t i v e e v a p o r a t i o n of t h e s o l v e n t (water) p r o c e e d s u n t i l t h e f i n a l c o n d e n s a t i o n i s f o l l o w e d by t h e t h e r m a l d e c o m p o s i t i o n o f t h e s o l u t e . On the other hand, when t h e s o l u t i o n i s n e b u l i z e d by a p p l y i n g h e a t i n v a c u o ( B ) , t h e r e s u l t i n g a e r o s o l m o s t l y c o n s i s t s of t h e s o l v e n t v a p o u r , small p a r t i c l e s o f t h e s o l v e n t and small p a r t i c l e s of t h e s o l u t i o n . Of c o u r s e , t h e d i s t r i b u t i o n o f t h e p a r t i c u l a t e s d e p e n d s on t h e n e b u l i z a t i o n c o n d i t i o n s , s u c h as t h e f l o w r a t e s of t h e s o l v e n t and t h e n e b u l i z i n g g a s , t e m p e r a t u r e , t h e d e g r e e of vacuum, e t c . However, i f a s t e a d y s t a t e i s a t t a i n e d f o r t h e n e b u l i z a t i o n , a c e r t a i n p o r t i o n of t h e s o l v e n t i s always d i v i d e d i n t o d r o p l e t s which c o n t a i n a few s u c r o s e m o l e c u l e s . When t h e s e small d r o p l e t s a r e e x p o s e d t o h i g h t e m p e r a t u r e s i n v a c u o , t h e s e l e c t i v e evaporation of t h e s o l v e n t (water) f i n a l l y causes t h e f o r m a t i o n of t h e s o l u t e m o l e c u l e ( s o l u t e v a p o u r ) . H e r e , o u r
LC/MS c o u p l i n g s y s t e m was d e s i g n e d by r e p l a c i n g t h e d i l u t e s o l u t i o n c o n t a i n e r mentioned i n F i g . 2 w i t h a micro-LC.
INSTRUMENTAL AND EXPERIMENTAL Flow diagram o f LC/MS s y s t e m F i g u r e 3 shows a s c h e m a t i c f l o w d i a g r a m of t h e micro-LC-MS
sys-
tem u t i l i z e d i n t h i s work. The whole s y s t e m c o n s i s t s o f t h r e e main components s u c h as a micro-LC,
a n e b u l i z i n g i n t e r f a c e and a quadru-
p o l e mass s p e c t r o m e t e r , and two a u x i l i a r y s u b - s y s t e m s f o r n e b u l i z i n g g a s and c o o l i n g water, r e s p e c t i v e l y . E i t h e r a micro-LC, F a m i l i c - 1 0 0 o r F a m i l i c - 3 0 0 (JASCO) w a s used m o s t l y a t a f l o w r a t e a b o u t
222
I
Y
.
F i g . 3 . S c h e m a t i c f l o w d i a g r a m o f micro-LC/MS vacuum n e b u l i z i n g i n t e r f a c e [16] 20 p l / m i n .
system u s i n g a
A PTFE m i c r o packed column ( 0 . 5 m m i . d . x 1 4 . 5 cm l o n g )
c o n t a i n i n g SS-10-ODs-B (JASCO) was u s e d when s e p a r a t i o n -
needed.
The whole e f f l u e n t from t h e d e t e c t o r was i n t r o d u c e d i n t o t h e vacuum n e b u l i z i n g i n t e r f a c e e i t h e r t h r o u g h a c o a x i a l s t a i n l e s s - s t e e l capi l l a r y tube (0.d.
0.31 m m , i . d .
0.13 mm w i t h c o r e - w i r e 0 . 1 1 mm d i a -
m e t e r ) o r a f u s e d s i l i c a c a p i l l a r y w i t h c a . 1 2 pm i . d .
A quadrupole
mass s p e c t r o m e t e r , JMS-Q1OA (JEOL) was u s e d i n a c h e m i c a l i o n i z a t i o n
( C I ) mode t o t a k e t h e mass f r a g m e n t o g r a m s a n d / o r t h e mass s p e c t r a of t h e n e b u l i z e d s a m p l e components where t h e s o l v e n t v a p o u r s were u t i l i z e d a s t h e r e a g e n t g a s e s f o r C I . C o o l i n g w a t e r was s u p p l i e d t o t h e n e b u l i z e r by t h e c o o l i n g w a t e r s y s t e m a t a c o n s t a n t r a t e o f up t o 5 ml/min
( m o s t l y , 3 ml/min was u s e d ) . The n e b u l i z i n g g a s s y s t e m
was s o m o d i f i e d t h a t a b u b b l e s a t u r a t o r was i n s e r t e d , t o c h a n g e t h e n a t u r e of t h e g a s by u s i n g v a r i o u s s o l u t i o n s ( m o s t l y , a b o u t 1 0 0 min o f h e l i u m (NTP) was u s e d as t h e main n e b u l i z i n g g a s ) .
nil/
223
Nebulizing i n t e r f a c e A s c h e m a t i c d i a g r a m of t h e n e b u l i z i n g i n t e r f a c e i s shown i n F i g .
4 , t o g e t h e r w i t h t h e m a g n i f i e d p o r t i o n of t h e n e b u l i z i n g t i p . The h o u s i n g c a s e of t h e c o o l i n g w a t e r j a c k e t was made of a low h e a t c o n d u c t i v i t y g l a s s c e r a m i c ( M a c o r ) . I n a d d i t i o n , t o minimize t h e h e a t p e n e t r a t i o n , a 0 . 5 mm t h i c k h e a t - r e s i s t a n t p o l y m e r ( p o l y m i d e ) f i l m was i n s e r t e d between t h e h o u s i n g c a s e and t h e w a t e r j a c k e t . On t h e o t h e r h a n d , a 2 m m t h i c k p i e c e o f c o p p e r d i s k was a t t a c h e d t o t h e top of t h e nebulizing nozzle t o c o l l e c t t h e h e a t energy necessary f o r t h e n e b u l i z a t i o n of t h e LC-effluent.
Thus, t h e LC-
e f f l u e n t c o n d u c t e d a t t h e t o p o f t h e n e b u l i z e r was f i n e l y n e b u l i z e d by a j e t s t r e a m of n e b u l i z i n g g a s w h i c h was s u p p l i e d t h r o u g h t h e
0.63 mm. i . d .
g a p between a s t a i n l e s s - s t e e l s h e a t h ( 0 . d .
1 0 mm l o n g ) and t h e n e b u l i z i n g t u b e from t h e micro-LC.
0.33 m m ,
The d e t a i l -
ed s t r u c t u r e s o f t h e c o a x i a l ( T y p e - I ) and t h e f u s e d s i l i c a c a p i l -
'"D i s k \
Rotary PumD
Cooling Water Jacket
Mac0 r I
4
L
L
I
0
2cm
F i g . 4 . S c h e m a t i c d i a g r a m of vacuum n e b u l i z i n g i n t e r f a c e f o r LC/ MS [16].
224
l a r y (Type 11) n o z z l e a r e i l l u s t r a t e d i n F i g . 5. The Type-I1 i s a m o d i f i c a t i o n of Type-I by s e t t i n g a f u s e d s i l i c a c a p i l l a r y w i t h c a . 1 2 pm i . d .
t o enable t h e s e l f - s p o u t i n g of t h e LC-effluent a t t h e
t o p of t h e n o z z l e . TYPE-I( COlJXlOl
/
Stalnless-steel Cap1I larv
\\
Core-W1 re
-
0
0.5
F i g . 5. D e t a i l e d s c h e m a t i c d i a g r a m s o f t h e n e b u l i z i n g t i p s [17].
Temperature d i s t r i b u t i o n s a r o u n d t h e n e b u l i z e r
tie
F i g u r e 6 shows t h e t e m p e r a t u r e d i s t r i b u t i o n s a r o u n d t h e n e b u l i z i n g t i p which were e s t i m a t e d s e m i - e m p i r i c a l l y .
Here, a s o l u t i o n
c o n t a i n i n g a small amount of s a c c h a r i d e was c o n s i d e r e d a s a t y p i c a l example a t 2OoC (room and c o o l i n g water t e m p e r a t u r e ) . When t h e e f f l u e n t i s n e b u l i z e d u n d e r c o n d i t i o n s o f h e a t e r - o f f and c o o l i n g water-off,
t h e t e m p e r a t u r e i n t h e i m m e d i a t e v i c i n i t y o f t h e nebu-
l i z i n g t i p r e a c h e s w e l l below z e r o b e c a u s e o f t h e l a r g e amount of l a t e n t e n e r g y a b s o r b e d by t h e r a p i d a d i a b a t i c e x p a n s i o n o f t h e e f f l u e n t (curve A ) .
I f p u r e water i s n e b u l i z e d u n d e r t h e same c o n d i -
t i o n s , we c a n even see e i t h e r snow or t h i n t h r e a d s o f i c e . I n t h i s c a s e , any s p e c i f i c i o n s o f t h e s o l u t e a r e n o t o b s e r v e d a t a l l . Howe v e r , i f t h e n e b u l i z i n g h e a t e r i s t u r n e d on t o r a i s e t h e t e m p e r a -
226
[SOTHEWS
.
Fi 6. T e m p e r a t u r e d i s t r i b u t i o n a r o u n d n e b u l i z i n g n o z z l e [16]. ( A T heater-off, without cooling water; (B) heater-on. with cooling w a t e r ; ( C ) h e a t e r - o n , w i t h o u t c o o l i n g w a t e r ; T: t e m p e r a t u r e (T1 < T2 < T3 < Tq.. .)
.
t u r e a t t h e t o p of t h e n o z z l e w i t h o u t a n y c o o l i n g water f l o w i n g , t h e t e m p e r a t u r e p r o f i l e around t h e t i p might resemble curve C . I n t h i s c a s e , even i f t h e t e m p e r a t u r e a t t h e t i p is a d j u s t e d t o j u s t below t h e b o i l i n g t e m p e r a t u r e o f w a t e r (lOO°C) by a dynamic comp r o m i s e o f t h e s u p p l i e d h e a t e n e r g y from t h e h e a t e r a n d t h e h e a t absorption through t h e nebulization, t h e temperatures a t t h e inner p a r t s o f t h e c o n d u c t i o n c a p i l l a r y t u b e would e x c e e d 100°C, c a u s i n g u n d e s i r a b l e b o i l i n g up o r t h e thermal decomposition of t h e t h e r mally u n s t a b l e s o l u t e i n t h e c a p i l l a r y tube. Consequently, a fav o u r a b l e t e m p e r a t u r e p r o f i l e ( c u r v e B) f o r l e s s v o l a t i l e compon e n t s t o be s t a b l y n e b u l i z e d t o y i e l d t h e m o l e c u l a r - r e l a t e d i o n s c o u l d be o b t a i n e d when a s u i t a b l e amount o f c o o l i n g w a t e r i s s u p -
226
p l i e d t o t h e w a t e r j a c k e t w h i l e h e a t e n e r g y i s c o n t i n u o u s l y supp l i e d t o t h e n e b u l i z i n g t i p . I n t h i s c a s e , t o o much w a t e r s u p p l y sometimes hampers t h e s t a b l e n e b u l i z a t i o n , w h i l e t o o l i t t l e w a t e r causes undesirable b o i l i n g o r t h e thermal decamposition of t h e component i n t h e c o n d u c t i n g c a p i l l a r y t u b e . I n t h e f o l l o w i n g , s i n c e t h e t o l e r a n c e r a n g e f o r t h e s e c o n d i t i o n s c h a n g e s d e p e n d i n g on t h e t h e r m a l s t a b i l i t y a n d / o r t h e v o l a t i l i t y of t h e sample m o l e c u l e , t h e optimum h e a t i n g t e m p e r a t u r e and t h e f l o w r a t e of c o o l i n g w a t e r were e m p i r i c a l l y d e t e r m i n e d . A h y p o t h e t i c a l i s o t h e r m a r o u n d t h e n e b u l i z i n g t i p f o r t h e c a s e of c u r v e B i s shown a t t h e b o t t o m of F i g . 6 where t h e t e m p e r a t u r e o r d e r i s T 1 < T 2 < T3
The e f f e c t of s e l f - s p o u t i n g and t h e n e b u l i z i n g gas I n o r d e r t o a v o i d t h e c l o g g i n g of t h e n e b u l i z i n g t i p e i t h e r w i t h t h e r e s i d u e of n o n - v o l a t i l e s o l u t e s o r w i t h t h e r m a l l y decomposed p r o d u c t s of t h e s o l u t e , s e l f - s p o u t i n g o f t h e L C - e f f l u e n t a t t h e t i p was i n c o r p o r a t e d i n t o t h e s y s t e m . F i g u r e 7 shows t h e r e l a t i o n s h i p between t h e s e l f - s p o u t i n g h e i g h t o f t h e L C - e f f l u e n t
(methanol)
and t h e f l o w r a t e ( o r l i n e a r v e l o c i t y ) of t h e e f f l u e n t f o r t h e fused s i l i c a c a p i l l a r y t i p with 1 2 pm i . d .
(Type I1 i n F i g . 5 ) un-
d e r a t m o s p h e r i c p r e s s u r e . The e x t r a p o l a t e d c u r v e t e l l s u s t h a t when t h e l i n e a r v e l o c i t y e x c e e d s a b o u t 3 m/sec ( 2 0 pl/mi.n),
self-
s p o u t i n g can b e o b s e r v e d f o r m e t h a n o l . With w a t e r , t h e t h r e s h o l d v e l o c i t y s h i f t s t o t h e higher value because of i t s h i g h e r viscosity
.
F i g u r e 8 i l l u s t r a t e s t h e e f f e c t of t h e n e b u l i z i n g g a s on t h e s t a b l e sample i n t r o d u c t i o n . Here, 1 p 1 of a m e t h a n o l sample solut i o n c o n t a i n i n g 1 pg of a m i n o p y r i n w a s i n j e c t e d u s i n g m e t h a n o l as t h e c a r r i e r s o l v e n t a t 30 p l / m i n . H e r e , t h e t e m p e r a t u r e o f t h e 0
n e b u l i z e r was m a i n t a i n e d a t 200 C and t h e (M t H ) '
of aminopyrin
227
F i g . 7. R e l a t i o n s h i p between s e l f - s p o u t i n g h e i g h t of t h e e f f l u e n t a t t h e n o z z l e w i t h 1 2 pm i . d . a n d i t s f l o w r a t e ( l i n e a r v e l o c i t y )
~ 7 1 .
,'.:,.:.:I-. .
.
. . .. . . .... . . , ,.>. .. .... .. ... .. ..:- u-... ;. .,, , . ..-.. , . . '
-
i
2mln 0
Without Nebulizing Gas (He)
Z
O
With Nebulizing Gas (He)
Aminooyrln (MeOH Soln.) SIN F i g . 8. E f f e c t of n e b u l i z i n g g a s on sample i n t r o d u c t i o n [17].
a t m/z = 232 was m o n i t o r e d by means o f s e l e c t e d i o n m o n i t o r i n g (SIM). A s shown i n t h i s f i g u r e , when t h e n e b u l i z i n g g a s is used,
t h e L C - e f f l u e n t i s more f i n e l y n e b u l i z e d a n d t h e r e s u l t i n g mass fragmentogram becomes v e r y s t a b l e , w h e r e a s when t h e g a s flow is
228
stopped, t h e n e b u l i z a t i o n has wider d i s t r i b u t i o n s both i n t h e part i c l e s i z e a n d t h e w i d t h , a n d t h e r e s u l t i n g mass f r a g m e n t o g r a m r e f l e c t s t h e d i s t u r b e d s a m p l e i n t r o d u c t i o n . T h i s t e n d e n c y be c om e s more s e r i o u s f o r l e s s v o l a t i l e co mp o u nds.
APPLICATIONS AND DISCUSSION R e p e a t a b i l i t y o f t h e met h o d The r e p r o d u c i b i l i t y o f t h e met h o d i s shown i n F i g . 9 u s i n g a m i n o p y r i n a s a s a m p l e . The m e t h a n o l s o l u t i o n (1 ~ 1 c)o n t a i n i n g 1 p g o f t h e s o l u t e was i n t r o d u c e d r e p e t i t i v e l y s i x times i n t o t h e n e b u l i z e r by a c o n s t a n t f l o w o f m e t h a n o l ( 3 0 u l / m i n ) a t t h e n e b u l i z i n g t e m p e r a t u r e o f 20OoC. The mass f r a g m e n t o g r a m s w e r e t a k e n by SIM a t m/z = 23 2 . I n t h i s c a s e , t h e r e p r o d u c i b i l i t y of t h e o b s e r v e d d a t a was a b o u t 3% i n C V . A n o t h e r e x a m p l e i s i l l u s t r a t e d i n F i g . 10, w h e r e 1 p 1 o f m e t h a n o l s o l u t i o n c o n t a i n i n g 1 ug of d i p h e n y l a m i n e , n a p h t h y l a m i n e a n d n i c o t i n a m i n e w e r e s i m u l t a n e o u s l y m e a s u r e d by
SIM a t t h e ( M t H )
t
p e a k o f t h e co mp one nts, r e s p e c t i v e l y , f o u r t i m e s .
These d a t a s u g g e s t t h a t i f
L
m o n i t o r i n g peak i s n o t i n t e r f e r e d w i t h
L
Fig. 9. R e p r o d u c i b i l i t y of t h e i n t r o d u c t i o n sample: 1 ug of SIM a t m/z=232 1171. a m i n o p y r i n (MW=231), d e t e c t i o n :
229 C A: Dlmenvlaalne
8: N O d l t h V l d N
c:
nlCOtlPollllde
Tlrn +
b
f
i
a
l
n
Fig. 10. Repetitive measurements of mass fragmentograms [17]
t h e o t h e r fragment. peaks.
.
t h e a s s o c i a t e d component can be d e t e r m i n -
ed w i t h o u t any chromatographic s e p a r a t i o n from t h e m a t r i x .
Mass spectra of the developing solvents Methanol, water and their mixtures were mostly used as the developing solvents for LC. The nebulizing gas (He) was usually saturated with the same solvents by using the bubble saturator to stabilize the nebulization of the LC-effluent and to prevent the deposition of the sample solute at the heated tip of the nebulizing nozzle. Figure 11 shows the spectra of the solvents, water, methanol, and methanol by use of ammonia-enriched nebulizing gas. In the last case, a small amount of 15N aqueous ammonia was added to the solvent in the bubble saturator. In these mass spectra, the base peaks are commonly (2M t H) t , and additionally ( M t H)' H)
t
and (3M t
peaks are also observed. In spectrum (c), two peaks at m/z = 18 t and m/z = 50 (CH30H t
of (NH4)
NH ) t can be seen in addition to
4
those observed for (b). In the following study, these reagent ions formed from the solvent vapours were used for CI of the sample components in the LC-effluent.
230
11. Mass spectra of the developin solvents in CI-mode [16]. (aT'Water; (b) methanol; (c) methanol fin the presence of N H 3 ) .
Fi
Various applications to less volatile compounds Figure 12 shows a mass spectrum of cholesterol and its mass fragmentograms, using methanol as the solvent. The temperatures of the nebulizing heater and the ion source were both 25OoC. In the mass spectrum (a), (M quasi-molecular ion (M
-
OH)'
-
H)'
at m/z
=
369 is the base peak and the
at m/z = 385 is observed to be about 10%
in the relative intensity. Two successive mass fragmentograms were measured by SIM using 1 p 1 of the methanol solution containing 1 p g of cholesterol at a fixed mass of m/z = 369. The symmetrical and sharp figures of the mass fragmentograms indicate that very stable sample introduction is attained. Figure 13 gives adenosine as an example, using water as the s o l -
231
100
IM-HI'
(a1
Ij
0I7 1 01 m2i n
lbl
I
Fi 1 2 . C I mass s p e c t r u m and mass f r a g m e n t o g r a m s f o r c h o l e s t e r o l [ l b j . ( a ) Mass s p e c t r u m ; ( b ) r e p e a t e d mass fragmentograms a t m/z=369.
0- I1 c
TS
min
Fi 13. CI mass s p e c t r u m and mass f r a g m e n t o g r a m s f o r a d e n o s i n e [ l b j . ( a ) Mass s p e c t r u m ; ( b ) r e p e a t e d mass f r a g m e n t o g r a m s a t m/z=136. v e n t . The n e b u l i z i n g h e a t e r and t h e i o n s o u r c e were m a i n t a i n e d a t
2OO0C and 19OoC, r e s p e c t i v e l y . I n t h i s c a s e , t h e b a s e p e a k a t m/z = 1 3 6 was ( a d e n i n e
+ H)'.
The q u a s i - m o l e c u l a r p e a k o f (M
+ H)'
at
m/z = 268 was v e r y s e n s i t i v e t o t h e t e m p e r a t u r e s o f t h e n e b u l i z e r a n d t h e i o n s o u r c e , a n d d i s a p p e a r e d a t e l e v a t e d t e m p e r a t u r e s . The mass fragmentograms r e p e a t e d l y measured by SIM a t m/z 136 a l s o s u g g e s t f a i r l y s t a b l e n e b u l i z a t i o n . T a b l e 2 summarizes t h e C I mass s p e c t r a of v a r i o u s n u c l e o s i d e s t a k e n by t h i s method.
232
TABLE 2
CI mass s p e c t r a of n u c l e o s i d e s [18] nucleoside
adenosine
molecular weight
nebulizing gas
267
relative intensity [HtH] [BasetH]
He
100
50
100
86
100
10
100
83(69).96(72) ,100(40)
31
100
132(16)
4
100
130(14).132(13),125(11)
38
100
9 9 ( 1 9 ) ,113(11), 1 1 6 ( 2 8 ) .135(14)
33
100
98(70) ,109(24),ll6(79)
180-C 4 H 10
60
100
96(12).98(66) ,116(70)
He
18
100
132(21)
CH4
36
100
132(24)
49
100
CH4 243
He CH4 IEO-C~H~~
thymidine
242
He
CH4 244
uridine
Iao-C H
4 10 ~~~~
Sample
1 ug/ul
Chamber Temperature
o t h e r main peaks m/z ( R . I . I )
94
Ieo-C4Hl0 cytieine
(Z) +
Solvent 200°C
H20
112(30) 112(15)
~
Carrier Flow Rate
1 6 ul/min
N e b u l i s i n g Temperature 200°C
Cooling Water
3 ml/min
F u r t h e r examples a r e shown i n F i g s . 14 and 1 5 f o r t r y p t o p h a n and glycyl-glycyl-glycine,
r e s p e c t i v e l y . The e x p e r i m e n t a l c o n d i -
t i o n s were b a s i c a l l y t h e same as t h o s e f o r a d e n o s i n e e x c e p t t h a t t h e t e m p e r a t u r e s of t h e n e b u l i z i n g h e a t e r and t h e i o n s o u r c e were b o t h 24OoC. The mass f r a g m e n t o g r a m s were o b t a i n e d by SIM a t t h e
14. C I mass s p e c t r u m and mass f r a g m e n t o g r a m s f o r t r y p t o p h a n [l%j. ( a ) Mass s p e c t r u m ; ( b ) r e p e a t e d mass f r a g m e n t o g r a m s a t
Fi
m/z=205.
233
NH21CH CONH12CH2COOH 15
IHIHI'
- i --r-
I-
la1
'
min
(bl
F i g . 1 5 . C I mass s p e c t r u m and mass f r a g m e n t o g r a m s f o r g l y c y l - g l y c y l - g l y c i n e [16]. ( a ) Mass s p e c t r u m ; ( b ) r e p e a t e d mass f r a g m e n t o grams a t m/z=115. a s s o c i a t e d b a s e p e a k s . T a b l e 3 summarizes t h e C I mass s p e c t r a o f
22 k i n d s of amino a c i d s u s i n g w a t e r ( 1 6 p l / m i n ) a s t h e s o l v e n t . H e r e , t h e starred peak f o r g l u t a m i c a c i d was o b s e r v e d u s i n g NH
3 en-
r i c h e d nebulizing gas. F i g u r e 1 6 shows t h e r e s u l t s f o r d - g l u c o s e ( m o n o s a c c h a r i d e ) u s i n g methanol a s t h e s o l v e n t . Both t e m p e r a t u r e s o f t h e n e b u l i z i n g h e a t e r and t h e i o n s o u r c e were 180°C. B e f o r e a d d i n g ammonia t o t h e s o l v e n t i n t h e b u b b l e s a t u r a t o r , t h e main p e a k s were (MH a t m/z = 163, (MH
-
2H20)t a t m/z = 145, and (MH
-
3H20)'
-
H201t a t m/z =
1 2 7 and t h e q u a s i - m o l e c u l a r p e a k was n o t o b s e r v e d a t a l l . On t h e o t h e r hand, when t h e ammonia-enriched n e b u l i z i n g g a s was u s e d , a d d i t i o n a l q u a s i - m o l e c u l a r p e a k s s u c h as (M (M
-
+
NH ) + a t m/z = 198 a n d
4
H20 t NH ) a t m/z = 180 were a l s o o b s e r v e d . Thus, t h e o b s e r v -
4
e d mass fragmentograms by SIM a t m/z = 198 a l s o s u g g e s t f a i r l y s t a b l e n e b u l i z a t i o n . H e r e , t h e a d d i t i o n of ammonia t o t h e n e b u l i z i n g g a s p r o v e d t o be a v e r y e f f e c t i v e improvement t o t h e s o f t n e s s of C I , b e c a u s e o f t h e s t r o n g p r o t o n a f f i n i t y of ammonia. F i n a l l y ,
F i g . 1 7 shows t h e s p e c t r a f o r m a l t o s e ( d i s a c c h a r i d e ) . The e x p e r i -
234
TABLE 3
C I mass s p e c t r a of amino a c i d s [16] amino acid
relative intensity ( 5 )
molecular weight
[Mtd'
glycine
75
100
alanine
89
100
valine
117
100
29
norvaline
117
83
100
leucine
131
58
100
norleucine
131
63
100
isoleucine
131
100
67
phenylalanine
16 5
16
'78
t.yrosi ne
181
100
39
threonine
119
100
27
42
serine
105
100
50
69
proline
[M-OH']'
[M-COOK]'
115
21
hydroxyproline
131
100
8
50
tryptophan
204
100
9
9
methionine
149
100
14
38
cystine
240
asparagic acid
133
glutsmic aicd
147
glutamine
146
lysine
146
arginine
174
histidine
155
100
12
100
100
4
11
29
19
34
100
10
6
3
15
100
14
44
other main peaks
m/z(R.I.)
236
IC)
(b)
Fig. 16. CI mass spectra and mass fragment0 rams for d-glucose [16]. (a) Mass spectrum before adding NH ; Tb) mass spectrum after adding NH (c) repeated mass fragientograms at m/z=198 on mass spectrui' (b)
-
2
.
100
127
145
I
la1
lb)
Fi 17. CI mass spectrum and mass fragmentograms for maltose [l:]. (a) Mass spectrum in the presence of NH3; (b) repeated mass fragmentograms at m/z=180. mental conditions were the same as those for d-glucose except for the solvent (methanol/water
=
3/1). In this case, even by the use
of the ammonia-enriched nebulizing gas, any quasi-molecular peaks were not observed. However, the shapes of the mass fragmentograms taken by SIM at the base peak of m/z introduction is still fairly stable.
=
180 suggest that the sample
236
Applications t o f r e e f a t t y a c i d s F i g u r e 18 shows methanol-CI mass s p e c t r a o f e n a n t h i c a c i d ( C 7 ) , m y r i s t i c a c i d (Cl,+) and b e h e n i c a c i d (C22). F a i r l y s t r o n g common q u a s i - m o l e c u l a r p e a k s s u c h as ( M t H t CH30H
-
t
H20)
-
H20)
t
,
(M t H)'
and (M t H
a r e o b s e r v e d . I n t h e s p e c t r u m of e n a n t h i c a c i d ,
some a d d u c t p e a k s s u c h as ( M t H . t CH30H)t a n d ( M t H t 2CH30H)'
4 summarizes t h e methanol-CI mass s p e c t r a
a r e a l s o observed. Table
of v a r i o u s f r e e f a t t y a c i d s . ( C 7 1 M W 00
MYRISTIC
ACID
m/z
0EHENlC ACID (C 22) M W 340
350
300
400
mli
F i g . 18. Methanol C I mass s p e c t r a of e n a n t h i c a c i d , m y r i s t i c a c i d and b e h e n i c a c i d [19].
TABLE 4 Methanol-CI mass s p e c t r a of f r e e f a t t y a c i d s . [19]
e n a n t h i o .=id (07)
130
9
100
19
95
c s p r y l i c w i d ICE)
144
7
100
24
97
19111)
pelmrnonio .old
158
9
92
30
100
20512)
c a p r i c acid (C10)
172
6
100
33
86
undsoanoio a d d I C l l )
186
4
100
37
83
lmuric maid (C12)
200
3
85
37
100
m y r i a t i a m i d (Cl4)
228
100
62
96
p a l m i t i o w i d (Cl6)
256
58
21
100
(C9)
0.2
margarlo acid lC17)
270
61
100
59
mtemria a c i d (Cl8)
284
87
47
100
312
100
41
99
340
100
55
91
mraohidio .old
11720)
k h - n i o s o i d (022) 8mple. I.bulisinn
1 vrlul
1.mp.rmtur..
Oolrmnt. 185OO
CHjOH
0arri.r
? l o r Rat.,
Chambar I e m p r a t u r o .
16 l l m i n
185%
cool in^
177(1)
Nobuliainn ,as. Yahr.
3 mllmin
H.
I70 nl/min)
237 On t h e o t h e r h a n d , s i n c e some f r e e f a t t y a c i d s h a v e b e t t e r s o l u b i l i t y i n benzene t h a n i n m e t h a n o l , benzene-CI mass s p e c t r a were a l s o examined. F i g u r e 1 9 shows t h e r e s u l t i n g benzene-CI mass s p e c t r a o f some f a t t y a c i d s . T a b l e 5 summarizes t h e benzene-CI mass s p e c t r a o f v a r i o u s f r e e f a t t y a c i d s . I n most cases, f a i r l y s t r o n g q u a s i - m o l e c u l a r p e a k s s u c h a s (M t H) t and ( M
+ H
-
H20) t a r e ob-
served. N a t u r a l f a t s mainly e x i s t as t r i g l y c e r i d e s of v a r i o u s f a t t y acids. Therefore, i n order t o determine t h e f a t t y a c i d content i n f a t s , t h e s a p o n i f i c a t i o n of f a t s h a s t o be c a r r i e d o u t . I n t h e
100 [M+IIl+
80 [M+H-H20]+
-60
I
.
a 40 20
I
86
/I
1 1
Stearlc Acld (C18) [M+H1+ (m.w, 284) Solvent C ~ H ~
,
d,
~
,
.,
.
,
,[M;H-y:+,
I
2 220
.
100
80
240
260
Arachldlc Acld, ( C 2 0 ) (m,w. 312) Solvent C6H6
280
,
~
. ,
320
300
[M+HI
.
+
F i g . 1 9 . Benzene CI mass s p e c t r a of p a l m i t i c a c i d , s t e a r i c a c i d and a r a c h i d i c a c i d 1191.
238
TABLE 5 Benzene-CI mass spectra of free fatty acids fatty acid
molecular
relative intensity 1 othsr main peaks
weight
[M+H]',
palmitic acid (C16)
256
100
margario acid (C17)
270
100
stsaric aoid (C18)
284
100
olsio aoid (ClSil)
282
23
linolsia acid (Cl8:Zj
280
100
linolsnic acid (C18i3)
278
100
arachidlc acid (C20)
312
100
behsnic acid (C22)
340
100
Sampls
1 ug/pl
Nsbuliring gas
Solvsnt
06H6
He (70 ml/min)
Chamber Tsnpsrature
17OoC
1191
[M+H-H20]*
m/z ( R . I .
X)
12
Carrier Flow Rate
16 ul/min
Nebuli6ing Temperature Cooling watsr
19OoC
3 nl/min
following applications,bean oil and palm oil were used as fat samples. About 1 . 5 g of a fat sample was saponified in 1 5 m l of 1N NaOH in methanol for 1 hr at 6OoC. After the evaporation of methanol, 30 m l of 1N HC1 was added. The C % C l 3 extract of the reactants was used as the specimen for LC/MS. The micro HPLC separation was carried out by a microcolumn (0.5 mm i.d.xl4.5
cm long) packed
with SS-10-ODs-B (JASCO) using a developing solvent (methanol/water = 911) at a flow rate of 16 ul/min. The UV-detection was made at 210 nm and the mass fragmentographic de-ction
was carried out
by a multi-ion detection.system (MID) at the (M t H t CH30H
-
H20)
t
239
of the corresponding fatty acids. Figures 20 and 21 are t h e resulting chromatograms of bean o i l , and palm oil, respectively. I n the
w
10
0
mi".
F i g . 20. Chromatograms of bean oil by UV-detection and MID.
I 0
MID
10
20.
.In.
F i g . 21. Chromatograms of palm oil by U V and MID.
240
f o r m e r case, t h e components which a p p e a r a t a b o u t 1 5 min, a p p e a r
a s one peak by U V - d e t e c t i o n . However, d e t e c t i o n by M I D t e l l s U S t h a t t h e r e a r e - t w o o v e r l a p p i n g p e a k s s u c h as o l e i c a c i d (C18
with
one d o u b l e bond) a n d p a l m i t i c a c i d (C16 s a t u r a t e d ) . F u r t h e r m o r e , s i n c e palm o i l c o n t a i n s m o s t l y s a t u r a t e d f a t t y a c i d s , t h e UV-det e c t i o n g i v e s a v e r y p o o r chromatogram, w h i l e M I D g i v e s q u i t e c h a r a c t e r i s t i c peaks of t h e s a t u r a t e d f a t t y a c i d s .
REFERENCES
1
M.A.
Baldwin and F.W.
2
P.J.
A r p i n o , B.G.
3
P. A r p i n o , B.G. Dawkins and F.W. M c L a f f e r t y , Biomed. Mass Spectrom., 1 (1974) 80-82. F.W. M c L a f f e r t y , R . K n u t t i , R . V e n k a t a r a g h a v a n , P . J . A r p i n o and B.G. Dawkins, Anal. Chem., 47 (1975) 1503-1505. F.W. M c L a f f e r t y and B.G. Dawkins, Biochem. SOC. T r a n s . , 3 ( 1 9 7 5 )
4 5
6 7
a 9
10
1111-1112.
1 2 ~ ~ 7 574-5a8. 4 )
M c L a f f e r t y , Org. Mass SpeCtrom.,
Dawkins and F.W.
7 (1973)
M c L a f f e r t y , Chromatogr. SOC.,
856-858.
B.G. Dawkins, P.J. Arpino and F.W. M c L a f f e r t y , Biomed. Mass Spectrom., 5 ( 1 9 7 8 ) 1-6. J . M . S c h m i t t e r , P . J . Arpino and G . Guiochon, J . Chromatogr. 167
(1978) 149-158.
P.
115-121.
11 J . D . Henion and T . Wachs, Anal. Chem., 53 (1981) 1963-1965. 1 2 S. Tsuge, Y . H i r a t a and T . T a k e u c h i , Anal. Chem., 5 1 (1979)
166-169. 17 Y . H i r a t a , T . T a k e u c h i , S. Tsuge and Y . Y o s h i d a , Org. Mass S p e c t r o n . , 1 4 (19751) 126-128. 1 4 Y. Yoshida, n . Yoshida, S . Tsuge, T. T a k e u c h i a n d K . M o c h i z u k i , High Res. Chromatogr. Chromatogr. Commun., 3 (1980) 16-20. 1 5 S. Tsuge, Y . Y o s h i d a , T . T a k e u c h i , K . Mochizuki, N . Kokubun and K . H i b i , Chem. Biomed. E n v i r o n . I n s t r . , 10 (1980) 405-418. 1 6 H . Y o s h i d a , K . Matsumoto, K. I t o h , S. T s u g e , Y . Hirata. K. Mochizuki, N. Kokubun and Y. Y o s h i d a , F r e s e n i u s Z . A n a l . Chem., 711 (1382) 674-680. 1 7 S . Tsuge, K . Matsumoto, K . O h t a , K. Yasuda and H . C . L i u , t o be
21
published. K . Matsumoto, H. Y o s h i d a , K . I t o h and S . Tsuge, t o be p u b l i s h e d . K . Matsumoto, H . Y o s h i d a , K . Ohta and S. Tsuge. t o be p u b l i s h e d . C . R . R l a k l e y , J .J. Carmody a n d M .L. V e s t a l , J . Am. Chem. SOC., 102 (1980) 5931-5933. C.R. B l a k l e y , J . J . Carmody and M.L. V e s t a l , Anal. Chem., 52
22
W.H.
18
19 20
(1980) 1636-1641.
McFadden, H.L.
(1976) 389-396.
S c h w a r t z and S. E v a n s , J . Chromatogr..
122
241
23 24 25 26 27 28
29 30 31
W . A . D a r k , W.H. McFadden a n d D.C. B r a d f o r d , J . C h r o m a t o g r . S c i . , 1 5 ( 1 9 7 7 ) 454-460. W.H. McFadden. D.C. B r a d f o l d . D.E. Games a n d J . L . Gower, A m . Lab., 9 ( 1 9 7 7 ) 5 5 - 6 4 . W . A . Dark a n d W.H. McFadden, J . C h r o m a t o g r . S c i . , 1 6 ( 1 9 7 8 ) 289-293. R.P.W. S c o t t , C . G . S c o t t , M. Muroe a n d J . Hess J r . , J . Chrom a to99 ( 1 9 7 4 ) 3 9 5 - 4 0 5 gr., R.P.W. S c o t t , Nat. Bur. S t a n d . ( U . S . ) , S p e c . P u b l . , 519 ( 1 9 7 9 ) 637-645. D.E. Games, J . L . Gower, M.G. Lee, I.A.S. L e w i s , M.E. Pugh a n d M . R o s s i t e r , P r o c . Anal. Div. Chem. S c i . , 1 5 ( 1 9 7 8 ) 101-105. D.E. Games, Chem. P h y s . L i p i d s , 2 1 ( 1 9 7 8 ) 389-402. A . B e n n i n g h o v e n , A . E i c k e , M. J u n a c k , W . S i c h t e r m a n n , J . K r i z e k , H . P e t e r s , Org. Mass S p e c t r o m . , 9 ( 1 9 8 0 ) 459-462. W.H. McFadden, J . C h r o m a t o g r . S c i . . 1 7 ( 1 9 7 9 ) 2 - 1 7 .
NEW APPROACHES T O INTERFACING L I Q U I D CHROMATOGRAPHY AND MASS SPECTROMETRY S. Tsuge Department o f S y n t h e t i c C h e m i s t r y , F a c u l t y of E n g i n e e r i n g , Nagoya U n i v e r s i t y , Nagoya 464, J a p a n
INTRODUCTION There h a s been a n i n c r e a s i n g i n t e r e s t i n t h e d i r e c t c o u p l i n g of l i q u i d chromatography (LC) w i t h mass s p e c t r o m e t r y (MS), a f t e r t h e g r e a t s u c c e s s o f t h e d i r e c t c o u p l i n g o f g a s c h r o m a t o g r a p h y (CC) w i t h MS. The p o t e n t i a l a b i l i t y of LC/MS i s v e r y l a r g e b e c a u s e of t h e v e r s a t i l e s e p a r a t i o n c a p a b i l i t y o f r e c e n t h i g h - p e r f o r m a n c e LC (HPLC) even f o r v a r i o u s t h e r m a l l y l a b i l e , p o l a r o r g a n i c compounds. Among r e c e n t d e v e l o p m e n t s i n t h i s f i e l d a r e (1) t h e d i r e c t i n t r o d u c t i o n method [l-111 ( 2 ) t h e p r e l i m i n a r y e v a p o r a t i o n method [12211 and ( 3 ) t h e m e c h a n i c a l t r a n s f e r method [22-30J.
The f o r m e r two
methods a r e s i m i l a r t o e a c h o t h e r s i n c e t h e y b o t h u s e t h e s o l v e n t vapour as t h e r e a g e n t gas f o r chemical i o n i z a t i o n ( C I ) (Table 1). Although some of t h e s e a r e now c o m m e r c i a l l y a v a i l a b l e , t h e y a r e m o s t l y s t i l l u t i l i z e d by t h e d e v e l o p e r s of t h e s y s t e m .
T h u s , new
t e c h n i q u e s e n a b l i n g enough s t a b l e a p p l i c a t i o n s t o a v a r i e t y of n o n - v o l a t i l e p o l a r compounds have b e e n e x p e c t e d t o a p p e a r f r o m v a r i o u s f i e l d s where HPLC i s r e c o g n i z e d a s a n i n d i s p e n s a b l e a n a l y t i c a l t o o l . A vacuum n e b u l i z i n g i n t e r f a c e f o r micro-LC-MS
coupling has
been d e v e l o p e d by m o d i f y i n g a j e t s e p a r a t o r f o r GC-MS [12J. T h i s i n t e r f a c e was l a t e r improved by t h e u s e of a v e r y s h o r t c o u n t e r n o z z l e s o t h a t i t c o u l d be a p p l i e d t o v a r i o u s l e s s v o l a t i l e compounds s u c h as g l u t a m i c a c i d . s t e r o i d s , a r o m a t i c a m i n e s and c h l o -
218
TABLE 1 V a r i o u s i n t e r f a c i n g methods f o r LC/MS Interfacing method
Ionieation mode
Research group
~~
(1)Direct introduction
McLafferty
CI
method
(2)Preliminary evaporation method (3)Mechanical transfer
et al.
[ 7 1 81
Henion
et al.
[9-11]
Tsuge
thermo-spray
Vestal
CI, EI
McFadden
[12-191
et al. et al.
et al.
Games
et al.
Bennighoven
r20, 211
L22-251
126, 271
Scott et al. SIHS
[1-6]
Arpino
CI (EI)
method
et al.
128, 291 et al.
[30]
(4)Others (API, Membrane, Heated-wire, etc.)
rine-containing insecticides micro-LC-MS
[LA].
Furthermore, a d i r e c t l y coupled
s y s t e m was c o n s t r u c t e d by u s i n g b a s i c a l l y t h e same
t y p e o f vacuum n e b u l i z i n g i n t e r f a c e a n d a small d o u b l e - f o c u s i n g MS
[l5].
I n t h e s e r e f e r e n c e s , t h e p o t e n t i a l a b i l i t y o f t h e vacuum neb-
u l i z i n g i n t e r f a c e was d e m o n s t r a t e d e m p i r i c a l l y . However, s i n c e vacuum n e b u l i z a t i o n o f t h e L C - e f f l u e n t i s a h i g h l y h e a t - a b s o r b i n g phenomenon, h e a t e n e r g y h a s t o be s u p p l i e d , a t l e a s t t o t h e t o p of t h e n e b u l i z i n g n o z z l e . Depending on t h e nebul i z i n g c o n d i t i o n s s u c h as t h e n a t u r e a n d t h e amount o f n e b u l i z i n g g a s , t h e t e m p e r a t u r e of t h e n e b u l i z i n g t i p and e v a c u a t i o n speed f o r t h e n e b u l i z e r , e x c e s s h e a t energy s u p p l i e d around t h e t o p of t h e n e b u l i z e r c a n s o m e t i m e s c a u s e u n d e s i r a b l e t h e r m a l decomposit i o n o f t h e t h e r m a l l y u n s t a b l e components i n t h e e f f l u e n t b e f o r e
i t r e a c h e s t h e t o p of t h e n e b u l i z e r .
219
Considering the above-mentioned factors, a small water cooling jacket was first of all incorporated into the vacuum nebulizing interface to prevent the introduced LC-effluent from over-heating, causing either boiling up o r thermal decomposition of labile components in the effluent. Then, a bubble saturator was inserted in the line of the nebulizing gas supplier, which enabled the adjustment of the nature of the nebulizing gas saturating a desired solution [16].
In addition, further modification of the nebulizer
was made by changing the nebulizing nozzle from a coaxial capillar y to a fused silica capillary with about 12 B m i.d. which enabled
self-spouting o f the LC-effluent at the top of the nebulizing nozzle 1171. These improvements extend the applicability of the microLC-MS system to fairly non-volatile polar organic compounds.
PRINCIPLE OF NEBULIZING INTERFACE FOR LC/MS Figure 1 shows a general flow diagram of directly coupled chromatograph (CC o r LC)/MS. Conventional GC o r LC is operated with Carrier (Gas o r L i q . I
Sample Inlet
LC
or Gc
Separation column
of excess carrier
Fig. 1. General flow diagram of directly coupled chromatograph (GC or LC)/MS.
220
t h e column e x i t a t one a t m o s p h e r e (760 t o r r ) , w h i l e a MS must m a i n t a i n t h e a n a l y z e r vacuum a t l e a s t as low a s 10-4-10-5 t o r r
[31].
T h e r e f o r e , t h e i n t e r f a c e s h o u l d f u n c t i o n as a remover of ex-
c e s s c a r r i e r o r a s a n e n r i c h e r o f t h e s o l u t e component. I n a GC/MS, both t h e s o l u t e (sample) and t h e s o l v e n t ( c a r r i e r ) a r e t r e a t e d i n t h e g a s e o u s p h a s e , w h i l e i n a LC/MS t h e column e f f l u e n t i s a k i n d of s o l u t i o n i n which a t h e r m a l l y l a b i l e s o l u t e ( s a m p l e ) i s u s u a l l y dissolved i n t h e c a r r i e r solvent. Therefore, i n t h e l a t t e r c a s e t h e L C - e f f l u e n t h a s t o be v a p o r i z e d i n t h e i n t e r f a c e p r i o r t o t h e removal o f e x c e s s c a r r i e r ( e n r i c h m e n t ) . F i g u r e 2 i l l u s t r a t e s t h e e f f e c t i v e n e s s of a vacuum n e b u l i z a t i o n f o r LC/MS i n t e r f a c i n g . I n o r d e r t o e v a p o r a t e a s o l u t e i n s o l u t i o n , (11Whm the bulk solusion i a b a s d under atmepheric prearurr:
F i g . 2. P r i n c i p l e o f vacuum n e b u l i z a t i o n .
221
we u s u a l l y a p p l y h e a t e n e r g y t o t h e s o l u t i o n . When t h e s o l u t e i s v o l a t i l e enough a t a t m o s p h e r i c p r e s s u r e , s i m p l e h e a t i n g i s s a t i s f a c t o r y t o v a p o r i z e t h e s o l u t e as w e l l as t h e s o l v e n t . However, i n t h e c a s e of a n o n - v o l a t i l e s o l u t e , f o r example, s u c r o s e i n w a t e r , when t h e b u l k s o l u t i o n i s h e a t e d u n d e r a t m o s p h e r i c p r e s s u r e ( A ) , t h e s e l e c t i v e e v a p o r a t i o n of t h e s o l v e n t (water) p r o c e e d s u n t i l t h e f i n a l c o n d e n s a t i o n i s f o l l o w e d by t h e t h e r m a l d e c o m p o s i t i o n o f t h e s o l u t e . On the other hand, when t h e s o l u t i o n i s n e b u l i z e d by a p p l y i n g h e a t i n v a c u o ( B ) , t h e r e s u l t i n g a e r o s o l m o s t l y c o n s i s t s of t h e s o l v e n t v a p o u r , small p a r t i c l e s o f t h e s o l v e n t and small p a r t i c l e s of t h e s o l u t i o n . Of c o u r s e , t h e d i s t r i b u t i o n o f t h e p a r t i c u l a t e s d e p e n d s on t h e n e b u l i z a t i o n c o n d i t i o n s , s u c h as t h e f l o w r a t e s of t h e s o l v e n t and t h e n e b u l i z i n g g a s , t e m p e r a t u r e , t h e d e g r e e of vacuum, e t c . However, i f a s t e a d y s t a t e i s a t t a i n e d f o r t h e n e b u l i z a t i o n , a c e r t a i n p o r t i o n of t h e s o l v e n t i s always d i v i d e d i n t o d r o p l e t s which c o n t a i n a few s u c r o s e m o l e c u l e s . When t h e s e small d r o p l e t s a r e e x p o s e d t o h i g h t e m p e r a t u r e s i n v a c u o , t h e s e l e c t i v e evaporation of t h e s o l v e n t (water) f i n a l l y causes t h e f o r m a t i o n of t h e s o l u t e m o l e c u l e ( s o l u t e v a p o u r ) . H e r e , o u r
LC/MS c o u p l i n g s y s t e m was d e s i g n e d by r e p l a c i n g t h e d i l u t e s o l u t i o n c o n t a i n e r mentioned i n F i g . 2 w i t h a micro-LC.
INSTRUMENTAL AND EXPERIMENTAL Flow diagram o f LC/MS s y s t e m F i g u r e 3 shows a s c h e m a t i c f l o w d i a g r a m of t h e micro-LC-MS
sys-
tem u t i l i z e d i n t h i s work. The whole s y s t e m c o n s i s t s o f t h r e e main components s u c h as a micro-LC,
a n e b u l i z i n g i n t e r f a c e and a quadru-
p o l e mass s p e c t r o m e t e r , and two a u x i l i a r y s u b - s y s t e m s f o r n e b u l i z i n g g a s and c o o l i n g water, r e s p e c t i v e l y . E i t h e r a micro-LC, F a m i l i c - 1 0 0 o r F a m i l i c - 3 0 0 (JASCO) w a s used m o s t l y a t a f l o w r a t e a b o u t
222
I
Y
.
F i g . 3 . S c h e m a t i c f l o w d i a g r a m o f micro-LC/MS vacuum n e b u l i z i n g i n t e r f a c e [16] 20 p l / m i n .
system u s i n g a
A PTFE m i c r o packed column ( 0 . 5 m m i . d . x 1 4 . 5 cm l o n g )
c o n t a i n i n g SS-10-ODs-B (JASCO) was u s e d when s e p a r a t i o n -
needed.
The whole e f f l u e n t from t h e d e t e c t o r was i n t r o d u c e d i n t o t h e vacuum n e b u l i z i n g i n t e r f a c e e i t h e r t h r o u g h a c o a x i a l s t a i n l e s s - s t e e l capi l l a r y tube (0.d.
0.31 m m , i . d .
0.13 mm w i t h c o r e - w i r e 0 . 1 1 mm d i a -
m e t e r ) o r a f u s e d s i l i c a c a p i l l a r y w i t h c a . 1 2 pm i . d .
A quadrupole
mass s p e c t r o m e t e r , JMS-Q1OA (JEOL) was u s e d i n a c h e m i c a l i o n i z a t i o n
( C I ) mode t o t a k e t h e mass f r a g m e n t o g r a m s a n d / o r t h e mass s p e c t r a of t h e n e b u l i z e d s a m p l e components where t h e s o l v e n t v a p o u r s were u t i l i z e d a s t h e r e a g e n t g a s e s f o r C I . C o o l i n g w a t e r was s u p p l i e d t o t h e n e b u l i z e r by t h e c o o l i n g w a t e r s y s t e m a t a c o n s t a n t r a t e o f up t o 5 ml/min
( m o s t l y , 3 ml/min was u s e d ) . The n e b u l i z i n g g a s s y s t e m
was s o m o d i f i e d t h a t a b u b b l e s a t u r a t o r was i n s e r t e d , t o c h a n g e t h e n a t u r e of t h e g a s by u s i n g v a r i o u s s o l u t i o n s ( m o s t l y , a b o u t 1 0 0 min o f h e l i u m (NTP) was u s e d as t h e main n e b u l i z i n g g a s ) .
nil/
223
Nebulizing i n t e r f a c e A s c h e m a t i c d i a g r a m of t h e n e b u l i z i n g i n t e r f a c e i s shown i n F i g .
4 , t o g e t h e r w i t h t h e m a g n i f i e d p o r t i o n of t h e n e b u l i z i n g t i p . The h o u s i n g c a s e of t h e c o o l i n g w a t e r j a c k e t was made of a low h e a t c o n d u c t i v i t y g l a s s c e r a m i c ( M a c o r ) . I n a d d i t i o n , t o minimize t h e h e a t p e n e t r a t i o n , a 0 . 5 mm t h i c k h e a t - r e s i s t a n t p o l y m e r ( p o l y m i d e ) f i l m was i n s e r t e d between t h e h o u s i n g c a s e and t h e w a t e r j a c k e t . On t h e o t h e r h a n d , a 2 m m t h i c k p i e c e o f c o p p e r d i s k was a t t a c h e d t o t h e top of t h e nebulizing nozzle t o c o l l e c t t h e h e a t energy necessary f o r t h e n e b u l i z a t i o n of t h e LC-effluent.
Thus, t h e LC-
e f f l u e n t c o n d u c t e d a t t h e t o p o f t h e n e b u l i z e r was f i n e l y n e b u l i z e d by a j e t s t r e a m of n e b u l i z i n g g a s w h i c h was s u p p l i e d t h r o u g h t h e
0.63 mm. i . d .
g a p between a s t a i n l e s s - s t e e l s h e a t h ( 0 . d .
1 0 mm l o n g ) and t h e n e b u l i z i n g t u b e from t h e micro-LC.
0.33 m m ,
The d e t a i l -
ed s t r u c t u r e s o f t h e c o a x i a l ( T y p e - I ) and t h e f u s e d s i l i c a c a p i l -
'"D i s k \
Rotary PumD
Cooling Water Jacket
Mac0 r I
4
L
L
I
0
2cm
F i g . 4 . S c h e m a t i c d i a g r a m of vacuum n e b u l i z i n g i n t e r f a c e f o r LC/ MS [16].
224
l a r y (Type 11) n o z z l e a r e i l l u s t r a t e d i n F i g . 5. The Type-I1 i s a m o d i f i c a t i o n of Type-I by s e t t i n g a f u s e d s i l i c a c a p i l l a r y w i t h c a . 1 2 pm i . d .
t o enable t h e s e l f - s p o u t i n g of t h e LC-effluent a t t h e
t o p of t h e n o z z l e . TYPE-I( COlJXlOl
/
Stalnless-steel Cap1I larv
\\
Core-W1 re
-
0
0.5
F i g . 5. D e t a i l e d s c h e m a t i c d i a g r a m s o f t h e n e b u l i z i n g t i p s [17].
Temperature d i s t r i b u t i o n s a r o u n d t h e n e b u l i z e r
tie
F i g u r e 6 shows t h e t e m p e r a t u r e d i s t r i b u t i o n s a r o u n d t h e n e b u l i z i n g t i p which were e s t i m a t e d s e m i - e m p i r i c a l l y .
Here, a s o l u t i o n
c o n t a i n i n g a small amount of s a c c h a r i d e was c o n s i d e r e d a s a t y p i c a l example a t 2OoC (room and c o o l i n g water t e m p e r a t u r e ) . When t h e e f f l u e n t i s n e b u l i z e d u n d e r c o n d i t i o n s o f h e a t e r - o f f and c o o l i n g water-off,
t h e t e m p e r a t u r e i n t h e i m m e d i a t e v i c i n i t y o f t h e nebu-
l i z i n g t i p r e a c h e s w e l l below z e r o b e c a u s e o f t h e l a r g e amount of l a t e n t e n e r g y a b s o r b e d by t h e r a p i d a d i a b a t i c e x p a n s i o n o f t h e e f f l u e n t (curve A ) .
I f p u r e water i s n e b u l i z e d u n d e r t h e same c o n d i -
t i o n s , we c a n even see e i t h e r snow or t h i n t h r e a d s o f i c e . I n t h i s c a s e , any s p e c i f i c i o n s o f t h e s o l u t e a r e n o t o b s e r v e d a t a l l . Howe v e r , i f t h e n e b u l i z i n g h e a t e r i s t u r n e d on t o r a i s e t h e t e m p e r a -
226
[SOTHEWS
.
Fi 6. T e m p e r a t u r e d i s t r i b u t i o n a r o u n d n e b u l i z i n g n o z z l e [16]. ( A T heater-off, without cooling water; (B) heater-on. with cooling w a t e r ; ( C ) h e a t e r - o n , w i t h o u t c o o l i n g w a t e r ; T: t e m p e r a t u r e (T1 < T2 < T3 < Tq.. .)
.
t u r e a t t h e t o p of t h e n o z z l e w i t h o u t a n y c o o l i n g water f l o w i n g , t h e t e m p e r a t u r e p r o f i l e around t h e t i p might resemble curve C . I n t h i s c a s e , even i f t h e t e m p e r a t u r e a t t h e t i p is a d j u s t e d t o j u s t below t h e b o i l i n g t e m p e r a t u r e o f w a t e r (lOO°C) by a dynamic comp r o m i s e o f t h e s u p p l i e d h e a t e n e r g y from t h e h e a t e r a n d t h e h e a t absorption through t h e nebulization, t h e temperatures a t t h e inner p a r t s o f t h e c o n d u c t i o n c a p i l l a r y t u b e would e x c e e d 100°C, c a u s i n g u n d e s i r a b l e b o i l i n g up o r t h e thermal decomposition of t h e t h e r mally u n s t a b l e s o l u t e i n t h e c a p i l l a r y tube. Consequently, a fav o u r a b l e t e m p e r a t u r e p r o f i l e ( c u r v e B) f o r l e s s v o l a t i l e compon e n t s t o be s t a b l y n e b u l i z e d t o y i e l d t h e m o l e c u l a r - r e l a t e d i o n s c o u l d be o b t a i n e d when a s u i t a b l e amount o f c o o l i n g w a t e r i s s u p -
226
p l i e d t o t h e w a t e r j a c k e t w h i l e h e a t e n e r g y i s c o n t i n u o u s l y supp l i e d t o t h e n e b u l i z i n g t i p . I n t h i s c a s e , t o o much w a t e r s u p p l y sometimes hampers t h e s t a b l e n e b u l i z a t i o n , w h i l e t o o l i t t l e w a t e r causes undesirable b o i l i n g o r t h e thermal decamposition of t h e component i n t h e c o n d u c t i n g c a p i l l a r y t u b e . I n t h e f o l l o w i n g , s i n c e t h e t o l e r a n c e r a n g e f o r t h e s e c o n d i t i o n s c h a n g e s d e p e n d i n g on t h e t h e r m a l s t a b i l i t y a n d / o r t h e v o l a t i l i t y of t h e sample m o l e c u l e , t h e optimum h e a t i n g t e m p e r a t u r e and t h e f l o w r a t e of c o o l i n g w a t e r were e m p i r i c a l l y d e t e r m i n e d . A h y p o t h e t i c a l i s o t h e r m a r o u n d t h e n e b u l i z i n g t i p f o r t h e c a s e of c u r v e B i s shown a t t h e b o t t o m of F i g . 6 where t h e t e m p e r a t u r e o r d e r i s T 1 < T 2 < T3
The e f f e c t of s e l f - s p o u t i n g and t h e n e b u l i z i n g gas I n o r d e r t o a v o i d t h e c l o g g i n g of t h e n e b u l i z i n g t i p e i t h e r w i t h t h e r e s i d u e of n o n - v o l a t i l e s o l u t e s o r w i t h t h e r m a l l y decomposed p r o d u c t s of t h e s o l u t e , s e l f - s p o u t i n g o f t h e L C - e f f l u e n t a t t h e t i p was i n c o r p o r a t e d i n t o t h e s y s t e m . F i g u r e 7 shows t h e r e l a t i o n s h i p between t h e s e l f - s p o u t i n g h e i g h t o f t h e L C - e f f l u e n t
(methanol)
and t h e f l o w r a t e ( o r l i n e a r v e l o c i t y ) of t h e e f f l u e n t f o r t h e fused s i l i c a c a p i l l a r y t i p with 1 2 pm i . d .
(Type I1 i n F i g . 5 ) un-
d e r a t m o s p h e r i c p r e s s u r e . The e x t r a p o l a t e d c u r v e t e l l s u s t h a t when t h e l i n e a r v e l o c i t y e x c e e d s a b o u t 3 m/sec ( 2 0 pl/mi.n),
self-
s p o u t i n g can b e o b s e r v e d f o r m e t h a n o l . With w a t e r , t h e t h r e s h o l d v e l o c i t y s h i f t s t o t h e higher value because of i t s h i g h e r viscosity
.
F i g u r e 8 i l l u s t r a t e s t h e e f f e c t of t h e n e b u l i z i n g g a s on t h e s t a b l e sample i n t r o d u c t i o n . Here, 1 p 1 of a m e t h a n o l sample solut i o n c o n t a i n i n g 1 pg of a m i n o p y r i n w a s i n j e c t e d u s i n g m e t h a n o l as t h e c a r r i e r s o l v e n t a t 30 p l / m i n . H e r e , t h e t e m p e r a t u r e o f t h e 0
n e b u l i z e r was m a i n t a i n e d a t 200 C and t h e (M t H ) '
of aminopyrin
227
F i g . 7. R e l a t i o n s h i p between s e l f - s p o u t i n g h e i g h t of t h e e f f l u e n t a t t h e n o z z l e w i t h 1 2 pm i . d . a n d i t s f l o w r a t e ( l i n e a r v e l o c i t y )
~ 7 1 .
,'.:,.:.:I-. .
.
. . .. . . .... . . , ,.>. .. .... .. ... .. ..:- u-... ;. .,, , . ..-.. , . . '
-
i
2mln 0
Without Nebulizing Gas (He)
Z
O
With Nebulizing Gas (He)
Aminooyrln (MeOH Soln.) SIN F i g . 8. E f f e c t of n e b u l i z i n g g a s on sample i n t r o d u c t i o n [17].
a t m/z = 232 was m o n i t o r e d by means o f s e l e c t e d i o n m o n i t o r i n g (SIM). A s shown i n t h i s f i g u r e , when t h e n e b u l i z i n g g a s is used,
t h e L C - e f f l u e n t i s more f i n e l y n e b u l i z e d a n d t h e r e s u l t i n g mass fragmentogram becomes v e r y s t a b l e , w h e r e a s when t h e g a s flow is
228
stopped, t h e n e b u l i z a t i o n has wider d i s t r i b u t i o n s both i n t h e part i c l e s i z e a n d t h e w i d t h , a n d t h e r e s u l t i n g mass f r a g m e n t o g r a m r e f l e c t s t h e d i s t u r b e d s a m p l e i n t r o d u c t i o n . T h i s t e n d e n c y be c om e s more s e r i o u s f o r l e s s v o l a t i l e co mp o u nds.
APPLICATIONS AND DISCUSSION R e p e a t a b i l i t y o f t h e met h o d The r e p r o d u c i b i l i t y o f t h e met h o d i s shown i n F i g . 9 u s i n g a m i n o p y r i n a s a s a m p l e . The m e t h a n o l s o l u t i o n (1 ~ 1 c)o n t a i n i n g 1 p g o f t h e s o l u t e was i n t r o d u c e d r e p e t i t i v e l y s i x times i n t o t h e n e b u l i z e r by a c o n s t a n t f l o w o f m e t h a n o l ( 3 0 u l / m i n ) a t t h e n e b u l i z i n g t e m p e r a t u r e o f 20OoC. The mass f r a g m e n t o g r a m s w e r e t a k e n by SIM a t m/z = 23 2 . I n t h i s c a s e , t h e r e p r o d u c i b i l i t y of t h e o b s e r v e d d a t a was a b o u t 3% i n C V . A n o t h e r e x a m p l e i s i l l u s t r a t e d i n F i g . 10, w h e r e 1 p 1 o f m e t h a n o l s o l u t i o n c o n t a i n i n g 1 ug of d i p h e n y l a m i n e , n a p h t h y l a m i n e a n d n i c o t i n a m i n e w e r e s i m u l t a n e o u s l y m e a s u r e d by
SIM a t t h e ( M t H )
t
p e a k o f t h e co mp one nts, r e s p e c t i v e l y , f o u r t i m e s .
These d a t a s u g g e s t t h a t i f
L
m o n i t o r i n g peak i s n o t i n t e r f e r e d w i t h
L
Fig. 9. R e p r o d u c i b i l i t y of t h e i n t r o d u c t i o n sample: 1 ug of SIM a t m/z=232 1171. a m i n o p y r i n (MW=231), d e t e c t i o n :
229 C A: Dlmenvlaalne
8: N O d l t h V l d N
c:
nlCOtlPollllde
Tlrn +
b
f
i
a
l
n
Fig. 10. Repetitive measurements of mass fragmentograms [17]
t h e o t h e r fragment. peaks.
.
t h e a s s o c i a t e d component can be d e t e r m i n -
ed w i t h o u t any chromatographic s e p a r a t i o n from t h e m a t r i x .
Mass spectra of the developing solvents Methanol, water and their mixtures were mostly used as the developing solvents for LC. The nebulizing gas (He) was usually saturated with the same solvents by using the bubble saturator to stabilize the nebulization of the LC-effluent and to prevent the deposition of the sample solute at the heated tip of the nebulizing nozzle. Figure 11 shows the spectra of the solvents, water, methanol, and methanol by use of ammonia-enriched nebulizing gas. In the last case, a small amount of 15N aqueous ammonia was added to the solvent in the bubble saturator. In these mass spectra, the base peaks are commonly (2M t H) t , and additionally ( M t H)' H)
t
and (3M t
peaks are also observed. In spectrum (c), two peaks at m/z = 18 t and m/z = 50 (CH30H t
of (NH4)
NH ) t can be seen in addition to
4
those observed for (b). In the following study, these reagent ions formed from the solvent vapours were used for CI of the sample components in the LC-effluent.
230
11. Mass spectra of the developin solvents in CI-mode [16]. (aT'Water; (b) methanol; (c) methanol fin the presence of N H 3 ) .
Fi
Various applications to less volatile compounds Figure 12 shows a mass spectrum of cholesterol and its mass fragmentograms, using methanol as the solvent. The temperatures of the nebulizing heater and the ion source were both 25OoC. In the mass spectrum (a), (M quasi-molecular ion (M
-
OH)'
-
H)'
at m/z
=
369 is the base peak and the
at m/z = 385 is observed to be about 10%
in the relative intensity. Two successive mass fragmentograms were measured by SIM using 1 p 1 of the methanol solution containing 1 p g of cholesterol at a fixed mass of m/z = 369. The symmetrical and sharp figures of the mass fragmentograms indicate that very stable sample introduction is attained. Figure 13 gives adenosine as an example, using water as the s o l -
231
100
IM-HI'
(a1
Ij
0I7 1 01 m2i n
lbl
I
Fi 1 2 . C I mass s p e c t r u m and mass f r a g m e n t o g r a m s f o r c h o l e s t e r o l [ l b j . ( a ) Mass s p e c t r u m ; ( b ) r e p e a t e d mass fragmentograms a t m/z=369.
0- I1 c
TS
min
Fi 13. CI mass s p e c t r u m and mass f r a g m e n t o g r a m s f o r a d e n o s i n e [ l b j . ( a ) Mass s p e c t r u m ; ( b ) r e p e a t e d mass f r a g m e n t o g r a m s a t m/z=136. v e n t . The n e b u l i z i n g h e a t e r and t h e i o n s o u r c e were m a i n t a i n e d a t
2OO0C and 19OoC, r e s p e c t i v e l y . I n t h i s c a s e , t h e b a s e p e a k a t m/z = 1 3 6 was ( a d e n i n e
+ H)'.
The q u a s i - m o l e c u l a r p e a k o f (M
+ H)'
at
m/z = 268 was v e r y s e n s i t i v e t o t h e t e m p e r a t u r e s o f t h e n e b u l i z e r a n d t h e i o n s o u r c e , a n d d i s a p p e a r e d a t e l e v a t e d t e m p e r a t u r e s . The mass fragmentograms r e p e a t e d l y measured by SIM a t m/z 136 a l s o s u g g e s t f a i r l y s t a b l e n e b u l i z a t i o n . T a b l e 2 summarizes t h e C I mass s p e c t r a of v a r i o u s n u c l e o s i d e s t a k e n by t h i s method.
232
TABLE 2
CI mass s p e c t r a of n u c l e o s i d e s [18] nucleoside
adenosine
molecular weight
nebulizing gas
267
relative intensity [HtH] [BasetH]
He
100
50
100
86
100
10
100
83(69).96(72) ,100(40)
31
100
132(16)
4
100
130(14).132(13),125(11)
38
100
9 9 ( 1 9 ) ,113(11), 1 1 6 ( 2 8 ) .135(14)
33
100
98(70) ,109(24),ll6(79)
180-C 4 H 10
60
100
96(12).98(66) ,116(70)
He
18
100
132(21)
CH4
36
100
132(24)
49
100
CH4 243
He CH4 IEO-C~H~~
thymidine
242
He
CH4 244
uridine
Iao-C H
4 10 ~~~~
Sample
1 ug/ul
Chamber Temperature
o t h e r main peaks m/z ( R . I . I )
94
Ieo-C4Hl0 cytieine
(Z) +
Solvent 200°C
H20
112(30) 112(15)
~
Carrier Flow Rate
1 6 ul/min
N e b u l i s i n g Temperature 200°C
Cooling Water
3 ml/min
F u r t h e r examples a r e shown i n F i g s . 14 and 1 5 f o r t r y p t o p h a n and glycyl-glycyl-glycine,
r e s p e c t i v e l y . The e x p e r i m e n t a l c o n d i -
t i o n s were b a s i c a l l y t h e same as t h o s e f o r a d e n o s i n e e x c e p t t h a t t h e t e m p e r a t u r e s of t h e n e b u l i z i n g h e a t e r and t h e i o n s o u r c e were b o t h 24OoC. The mass f r a g m e n t o g r a m s were o b t a i n e d by SIM a t t h e
14. C I mass s p e c t r u m and mass f r a g m e n t o g r a m s f o r t r y p t o p h a n [l%j. ( a ) Mass s p e c t r u m ; ( b ) r e p e a t e d mass f r a g m e n t o g r a m s a t
Fi
m/z=205.
233
NH21CH CONH12CH2COOH 15
IHIHI'
- i --r-
I-
la1
'
min
(bl
F i g . 1 5 . C I mass s p e c t r u m and mass f r a g m e n t o g r a m s f o r g l y c y l - g l y c y l - g l y c i n e [16]. ( a ) Mass s p e c t r u m ; ( b ) r e p e a t e d mass f r a g m e n t o grams a t m/z=115. a s s o c i a t e d b a s e p e a k s . T a b l e 3 summarizes t h e C I mass s p e c t r a o f
22 k i n d s of amino a c i d s u s i n g w a t e r ( 1 6 p l / m i n ) a s t h e s o l v e n t . H e r e , t h e starred peak f o r g l u t a m i c a c i d was o b s e r v e d u s i n g NH
3 en-
r i c h e d nebulizing gas. F i g u r e 1 6 shows t h e r e s u l t s f o r d - g l u c o s e ( m o n o s a c c h a r i d e ) u s i n g methanol a s t h e s o l v e n t . Both t e m p e r a t u r e s o f t h e n e b u l i z i n g h e a t e r and t h e i o n s o u r c e were 180°C. B e f o r e a d d i n g ammonia t o t h e s o l v e n t i n t h e b u b b l e s a t u r a t o r , t h e main p e a k s were (MH a t m/z = 163, (MH
-
2H20)t a t m/z = 145, and (MH
-
3H20)'
-
H201t a t m/z =
1 2 7 and t h e q u a s i - m o l e c u l a r p e a k was n o t o b s e r v e d a t a l l . On t h e o t h e r hand, when t h e ammonia-enriched n e b u l i z i n g g a s was u s e d , a d d i t i o n a l q u a s i - m o l e c u l a r p e a k s s u c h as (M (M
-
+
NH ) + a t m/z = 198 a n d
4
H20 t NH ) a t m/z = 180 were a l s o o b s e r v e d . Thus, t h e o b s e r v -
4
e d mass fragmentograms by SIM a t m/z = 198 a l s o s u g g e s t f a i r l y s t a b l e n e b u l i z a t i o n . H e r e , t h e a d d i t i o n of ammonia t o t h e n e b u l i z i n g g a s p r o v e d t o be a v e r y e f f e c t i v e improvement t o t h e s o f t n e s s of C I , b e c a u s e o f t h e s t r o n g p r o t o n a f f i n i t y of ammonia. F i n a l l y ,
F i g . 1 7 shows t h e s p e c t r a f o r m a l t o s e ( d i s a c c h a r i d e ) . The e x p e r i -
234
TABLE 3
C I mass s p e c t r a of amino a c i d s [16] amino acid
relative intensity ( 5 )
molecular weight
[Mtd'
glycine
75
100
alanine
89
100
valine
117
100
29
norvaline
117
83
100
leucine
131
58
100
norleucine
131
63
100
isoleucine
131
100
67
phenylalanine
16 5
16
'78
t.yrosi ne
181
100
39
threonine
119
100
27
42
serine
105
100
50
69
proline
[M-OH']'
[M-COOK]'
115
21
hydroxyproline
131
100
8
50
tryptophan
204
100
9
9
methionine
149
100
14
38
cystine
240
asparagic acid
133
glutsmic aicd
147
glutamine
146
lysine
146
arginine
174
histidine
155
100
12
100
100
4
11
29
19
34
100
10
6
3
15
100
14
44
other main peaks
m/z(R.I.)
236
IC)
(b)
Fig. 16. CI mass spectra and mass fragment0 rams for d-glucose [16]. (a) Mass spectrum before adding NH ; Tb) mass spectrum after adding NH (c) repeated mass fragientograms at m/z=198 on mass spectrui' (b)
-
2
.
100
127
145
I
la1
lb)
Fi 17. CI mass spectrum and mass fragmentograms for maltose [l:]. (a) Mass spectrum in the presence of NH3; (b) repeated mass fragmentograms at m/z=180. mental conditions were the same as those for d-glucose except for the solvent (methanol/water
=
3/1). In this case, even by the use
of the ammonia-enriched nebulizing gas, any quasi-molecular peaks were not observed. However, the shapes of the mass fragmentograms taken by SIM at the base peak of m/z introduction is still fairly stable.
=
180 suggest that the sample
236
Applications t o f r e e f a t t y a c i d s F i g u r e 18 shows methanol-CI mass s p e c t r a o f e n a n t h i c a c i d ( C 7 ) , m y r i s t i c a c i d (Cl,+) and b e h e n i c a c i d (C22). F a i r l y s t r o n g common q u a s i - m o l e c u l a r p e a k s s u c h as ( M t H t CH30H
-
t
H20)
-
H20)
t
,
(M t H)'
and (M t H
a r e o b s e r v e d . I n t h e s p e c t r u m of e n a n t h i c a c i d ,
some a d d u c t p e a k s s u c h as ( M t H . t CH30H)t a n d ( M t H t 2CH30H)'
4 summarizes t h e methanol-CI mass s p e c t r a
a r e a l s o observed. Table
of v a r i o u s f r e e f a t t y a c i d s . ( C 7 1 M W 00
MYRISTIC
ACID
m/z
0EHENlC ACID (C 22) M W 340
350
300
400
mli
F i g . 18. Methanol C I mass s p e c t r a of e n a n t h i c a c i d , m y r i s t i c a c i d and b e h e n i c a c i d [19].
TABLE 4 Methanol-CI mass s p e c t r a of f r e e f a t t y a c i d s . [19]
e n a n t h i o .=id (07)
130
9
100
19
95
c s p r y l i c w i d ICE)
144
7
100
24
97
19111)
pelmrnonio .old
158
9
92
30
100
20512)
c a p r i c acid (C10)
172
6
100
33
86
undsoanoio a d d I C l l )
186
4
100
37
83
lmuric maid (C12)
200
3
85
37
100
m y r i a t i a m i d (Cl4)
228
100
62
96
p a l m i t i o w i d (Cl6)
256
58
21
100
(C9)
0.2
margarlo acid lC17)
270
61
100
59
mtemria a c i d (Cl8)
284
87
47
100
312
100
41
99
340
100
55
91
mraohidio .old
11720)
k h - n i o s o i d (022) 8mple. I.bulisinn
1 vrlul
1.mp.rmtur..
Oolrmnt. 185OO
CHjOH
0arri.r
? l o r Rat.,
Chambar I e m p r a t u r o .
16 l l m i n
185%
cool in^
177(1)
Nobuliainn ,as. Yahr.
3 mllmin
H.
I70 nl/min)
237 On t h e o t h e r h a n d , s i n c e some f r e e f a t t y a c i d s h a v e b e t t e r s o l u b i l i t y i n benzene t h a n i n m e t h a n o l , benzene-CI mass s p e c t r a were a l s o examined. F i g u r e 1 9 shows t h e r e s u l t i n g benzene-CI mass s p e c t r a o f some f a t t y a c i d s . T a b l e 5 summarizes t h e benzene-CI mass s p e c t r a o f v a r i o u s f r e e f a t t y a c i d s . I n most cases, f a i r l y s t r o n g q u a s i - m o l e c u l a r p e a k s s u c h a s (M t H) t and ( M
+ H
-
H20) t a r e ob-
served. N a t u r a l f a t s mainly e x i s t as t r i g l y c e r i d e s of v a r i o u s f a t t y acids. Therefore, i n order t o determine t h e f a t t y a c i d content i n f a t s , t h e s a p o n i f i c a t i o n of f a t s h a s t o be c a r r i e d o u t . I n t h e
100 [M+IIl+
80 [M+H-H20]+
-60
I
.
a 40 20
I
86
/I
1 1
Stearlc Acld (C18) [M+H1+ (m.w, 284) Solvent C ~ H ~
,
d,
~
,
.,
.
,
,[M;H-y:+,
I
2 220
.
100
80
240
260
Arachldlc Acld, ( C 2 0 ) (m,w. 312) Solvent C6H6
280
,
~
. ,
320
300
[M+HI
.
+
F i g . 1 9 . Benzene CI mass s p e c t r a of p a l m i t i c a c i d , s t e a r i c a c i d and a r a c h i d i c a c i d 1191.
238
TABLE 5 Benzene-CI mass spectra of free fatty acids fatty acid
molecular
relative intensity 1 othsr main peaks
weight
[M+H]',
palmitic acid (C16)
256
100
margario acid (C17)
270
100
stsaric aoid (C18)
284
100
olsio aoid (ClSil)
282
23
linolsia acid (Cl8:Zj
280
100
linolsnic acid (C18i3)
278
100
arachidlc acid (C20)
312
100
behsnic acid (C22)
340
100
Sampls
1 ug/pl
Nsbuliring gas
Solvsnt
06H6
He (70 ml/min)
Chamber Tsnpsrature
17OoC
1191
[M+H-H20]*
m/z ( R . I .
X)
12
Carrier Flow Rate
16 ul/min
Nebuli6ing Temperature Cooling watsr
19OoC
3 nl/min
following applications,bean oil and palm oil were used as fat samples. About 1 . 5 g of a fat sample was saponified in 1 5 m l of 1N NaOH in methanol for 1 hr at 6OoC. After the evaporation of methanol, 30 m l of 1N HC1 was added. The C % C l 3 extract of the reactants was used as the specimen for LC/MS. The micro HPLC separation was carried out by a microcolumn (0.5 mm i.d.xl4.5
cm long) packed
with SS-10-ODs-B (JASCO) using a developing solvent (methanol/water = 911) at a flow rate of 16 ul/min. The UV-detection was made at 210 nm and the mass fragmentographic de-ction
was carried out
by a multi-ion detection.system (MID) at the (M t H t CH30H
-
H20)
t
239
of the corresponding fatty acids. Figures 20 and 21 are t h e resulting chromatograms of bean o i l , and palm oil, respectively. I n the
w
10
0
mi".
F i g . 20. Chromatograms of bean oil by UV-detection and MID.
I 0
MID
10
20.
.In.
F i g . 21. Chromatograms of palm oil by U V and MID.
240
f o r m e r case, t h e components which a p p e a r a t a b o u t 1 5 min, a p p e a r
a s one peak by U V - d e t e c t i o n . However, d e t e c t i o n by M I D t e l l s U S t h a t t h e r e a r e - t w o o v e r l a p p i n g p e a k s s u c h as o l e i c a c i d (C18
with
one d o u b l e bond) a n d p a l m i t i c a c i d (C16 s a t u r a t e d ) . F u r t h e r m o r e , s i n c e palm o i l c o n t a i n s m o s t l y s a t u r a t e d f a t t y a c i d s , t h e UV-det e c t i o n g i v e s a v e r y p o o r chromatogram, w h i l e M I D g i v e s q u i t e c h a r a c t e r i s t i c peaks of t h e s a t u r a t e d f a t t y a c i d s .
REFERENCES
1
M.A.
Baldwin and F.W.
2
P.J.
A r p i n o , B.G.
3
P. A r p i n o , B.G. Dawkins and F.W. M c L a f f e r t y , Biomed. Mass Spectrom., 1 (1974) 80-82. F.W. M c L a f f e r t y , R . K n u t t i , R . V e n k a t a r a g h a v a n , P . J . A r p i n o and B.G. Dawkins, Anal. Chem., 47 (1975) 1503-1505. F.W. M c L a f f e r t y and B.G. Dawkins, Biochem. SOC. T r a n s . , 3 ( 1 9 7 5 )
4 5
6 7
a 9
10
1111-1112.
1 2 ~ ~ 7 574-5a8. 4 )
M c L a f f e r t y , Org. Mass SpeCtrom.,
Dawkins and F.W.
7 (1973)
M c L a f f e r t y , Chromatogr. SOC.,
856-858.
B.G. Dawkins, P.J. Arpino and F.W. M c L a f f e r t y , Biomed. Mass Spectrom., 5 ( 1 9 7 8 ) 1-6. J . M . S c h m i t t e r , P . J . Arpino and G . Guiochon, J . Chromatogr. 167
(1978) 149-158.
P.
115-121.
11 J . D . Henion and T . Wachs, Anal. Chem., 53 (1981) 1963-1965. 1 2 S. Tsuge, Y . H i r a t a and T . T a k e u c h i , Anal. Chem., 5 1 (1979)
166-169. 17 Y . H i r a t a , T . T a k e u c h i , S. Tsuge and Y . Y o s h i d a , Org. Mass S p e c t r o n . , 1 4 (19751) 126-128. 1 4 Y. Yoshida, n . Yoshida, S . Tsuge, T. T a k e u c h i a n d K . M o c h i z u k i , High Res. Chromatogr. Chromatogr. Commun., 3 (1980) 16-20. 1 5 S. Tsuge, Y . Y o s h i d a , T . T a k e u c h i , K . Mochizuki, N . Kokubun and K . H i b i , Chem. Biomed. E n v i r o n . I n s t r . , 10 (1980) 405-418. 1 6 H . Y o s h i d a , K . Matsumoto, K. I t o h , S. T s u g e , Y . Hirata. K. Mochizuki, N. Kokubun and Y. Y o s h i d a , F r e s e n i u s Z . A n a l . Chem., 711 (1382) 674-680. 1 7 S . Tsuge, K . Matsumoto, K . O h t a , K. Yasuda and H . C . L i u , t o be
21
published. K . Matsumoto, H. Y o s h i d a , K . I t o h and S . Tsuge, t o be p u b l i s h e d . K . Matsumoto, H . Y o s h i d a , K . Ohta and S. Tsuge. t o be p u b l i s h e d . C . R . R l a k l e y , J .J. Carmody a n d M .L. V e s t a l , J . Am. Chem. SOC., 102 (1980) 5931-5933. C.R. B l a k l e y , J . J . Carmody and M.L. V e s t a l , Anal. Chem., 52
22
W.H.
18
19 20
(1980) 1636-1641.
McFadden, H.L.
(1976) 389-396.
S c h w a r t z and S. E v a n s , J . Chromatogr..
122
241
23 24 25 26 27 28
29 30 31
W . A . D a r k , W.H. McFadden a n d D.C. B r a d f o r d , J . C h r o m a t o g r . S c i . , 1 5 ( 1 9 7 7 ) 454-460. W.H. McFadden. D.C. B r a d f o l d . D.E. Games a n d J . L . Gower, A m . Lab., 9 ( 1 9 7 7 ) 5 5 - 6 4 . W . A . Dark a n d W.H. McFadden, J . C h r o m a t o g r . S c i . , 1 6 ( 1 9 7 8 ) 289-293. R.P.W. S c o t t , C . G . S c o t t , M. Muroe a n d J . Hess J r . , J . Chrom a to99 ( 1 9 7 4 ) 3 9 5 - 4 0 5 gr., R.P.W. S c o t t , Nat. Bur. S t a n d . ( U . S . ) , S p e c . P u b l . , 519 ( 1 9 7 9 ) 637-645. D.E. Games, J . L . Gower, M.G. Lee, I.A.S. L e w i s , M.E. Pugh a n d M . R o s s i t e r , P r o c . Anal. Div. Chem. S c i . , 1 5 ( 1 9 7 8 ) 101-105. D.E. Games, Chem. P h y s . L i p i d s , 2 1 ( 1 9 7 8 ) 389-402. A . B e n n i n g h o v e n , A . E i c k e , M. J u n a c k , W . S i c h t e r m a n n , J . K r i z e k , H . P e t e r s , Org. Mass S p e c t r o m . , 9 ( 1 9 8 0 ) 459-462. W.H. McFadden, J . C h r o m a t o g r . S c i . . 1 7 ( 1 9 7 9 ) 2 - 1 7 .