Mass spectrometry labelling technique - acid catalyzed deuterium and oxygen 18 exchange by gas-liquid chromatograph

Burlingame, A. L.

Richter, W. J.

Senn, M.

English

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UUSA CR O R  TMX O R  AD NUMBER1 
11 653 July 65 
CONVENIENT LABELLING TECHNIQUE FOR MASS SPECTROMETRY: 
A C I 9  CATALYZED DEUTERIUM AND OXYGEN-18 EXCHANGE V I A  
GAS-LIQUID CHROMATOGRAPHY 
-
Wilhelm J. Richter, Martin Senn and A. L. Burlingame 
Department of Chemistry and Space Sciences Laboratory, 
University of California, Berkeley, California 94720 
The technique of prime importance in the elucidation of 
electron-impact -induced fragmentation mechanisms and rearrange- 
ment processes in an organic molecule is the selective 
substitution of deuterium for hydrogen at exchangeable sites. 
The preparation of oxygen-is iatjellee zompott?ds is 'of 
additional value in providing complementary mechanistic 
information. A recent review (1) concerning the exchange of 
enolizable hydrogen for deuterium by "wet" chemical means 
emphasizes the wide variety of conditions (solvent, catalyst 
temperature, time, purification, etc.) employed routinely to 
effect labelling of milligram quantities of sample with high 
isotopic purity. 
F 
Recently the exchange of enolizable hydrogen 
for deuterium by a single passage of sample through a pre- 
deuterated alkaline GLC (gas-liquid chromatographic) column 
has been reported ( 2 ) .  The average isotopic purity oi' the 
resulting labelled ketones was shown to be 96% by mass 
spectrometric analysisj whereas, amides, aldehydes and esters 
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https://ntrs.nasa.gov/search.jsp?R=19660012964 2020-03-16T22:55:22+00:00Z
displayed l e s s  s a t i s f a c t o r y  r e s u l t s .  
I 
i Current r e s u l t s  employing ac id i c  exchange conditions 
on a tandem GLC column ( 3 )  have extended the scope of appl i -  
c a b i l i t y  of the GLC labelling method i n  severa l  major aspects .  
Ketones which incorporated an average of 96% deuterium under 
a lka l ine  conditions (2), exchanged w i t h  analogous ease under 
the  prescribed ac id i c  conditions.  The compounds l i s ted  In  
Table 1 were chromatographed a t  the physical parameters indicated,  
i n  each case a s i n g l e  passage of sample through the column 
w a s  performed, and the co l lec ted  sample analyzed mass spectro-  
met r ica l ly  ( 4 )  . Carbonyl compounds which were precluded 
previously by the  nature of addi t iona l  ac id i c  func t iona l i ty ,  
e . g .  carboxyl and phenolic hydroxyl groups, showed e s s e n t i a l l y  
quan t i t a t ive  uptake o f  deuterium under a c i d i c  conditions.  
A l s o  the excharige of p l y f u n c t i o n a l  corr?pounds containing 
e a s i l y  saponif iable  groups and/or a l k a l i  r eac t ive  functions,  
e .g. ke to  e s t e r s  and aldehydes, respect ively,  were labelled 
s a t i s f a c t o r i l y  as indicated i n  Table  1. 
In addi t ion t o  the augmented scope f o r  deuterium labelling, 
the  ac id i c  medium has allowed extension of this method t o  
oxygen-18 l a b e l l i n g  f o r  the compounds discussed above, see 
Table 1 f o r  comparison of deuterium and oxygen-18 incorporation. 
A f u r t h e r  point t o  be mentioned concerns molecules which undergo 
reac t ion  under ac id i c  conditions t o  y i e ld  ac id i c  funct ional  
groups, e .g .  acid chlorides and anhydrides, thereby incorporating 
oxygen-18 i n  the process.  
i n  Table 1, the  effect iveness  of the ‘oxygen l a b e l l i n g  is  qu i t e  
A s  demonstrated by the 0-18 data 
s a t i s f a c t o r y .  
TABLE 1 
Deuteration Data Oxygen-18 Dataa 
Total GLC $ Total GLC * 
Exchange Conditionsb Exchange Conditions 
Labelled Compound 
methyl -n-nonyl - ketone 
3-tetradecanone 
valerqhenone 
.4O 
43 
45 
51 
49 
isovalerophenone 
c aprophenone 
7 
alpha-naphthylmethyl- 
ketone 200/11 51 =5/3 
beta-naphthylmethyl- 
kel;one 49 
39 n-butyl levullnate 
o-hydroxyacetophenone 
- 
45 *5 
50 
60 
m-ni t roace tophenone 
n -iiaiyl a1 de hyde dlme r - 
55 - n-tetradecaldehyde 
o -tolualdehyde 
p - tolualdehyde 
benzoic acidC 
- beta-naphthoic acidC 
acetic acide 
65 
60 
29d 
67 
31 
a A l l  compounds were labelled with 71% enriched %OI8 (Weizmann 
b Column temperature (OC)/retention time listed in column; 
Institute). 
carrier gas flow rate was 40-50 ml ./min. 
on the column. 
c Gbtained from t h e  cQrresponding I C i d  c h l o r i d e s  - via hydrnlysia 
d 31% enriched %0l8 was used. 
e Obtained from column hydration of acetic anhydride. 
I 
. 
It should be noted that this tandem column arrangement 
preserves all purification advantages of the GLC labelling 
approach (2),  by allowing separation on a wide selection of 
proper liquid phases, i.e. phases which are otherwise 
incompatible with phosphoric acid at elevated temperatures, 
The ease and rapidity with which exchange of enolizable 
hydrogens and carbonyl oxygens can be quantitatively achieved, 
. emphasizes the practical utility of the tandem GLC column. 
The method is well suited for the high quality preparation of 
pure labelled samples of synthetic and natural products 
available in only minute amounts. 
Acknowledgement: This work was supported in part by the 
National Aeronautics and Space Administration Grants NsG 
101-61 and NsG 243-62. 
REFERENCES 
1. For a comprehensive review of deuteration procedures, see: 
H. Budzikiewicz, C, Djerassi and D. H. Williams, Structure 
Elucidation of Natural Products by Mass Spectrometry, 
Volume I: Alkaloids, GoIden-Day E c T a n  Krancisco, 1964, 
Chapter 2. . 
2. M. Senn, W. J, Richter and A. L. Burlingame, J. Am. Chem. 
3 .  
- - -  - SO~. , 3, 0000 (1965). 
The tandem OM: column consisted of two 1/4" x 5' coiled 
stainless steel columns linked with a Swagelock union. 
The column packing in the first section (exchanger) 
consisted of Chromosorb W(45/60 mesh) coated with 55% 
phosphoric acid; in the second section (separator'), a 
liquid phase was selected depending on the chromatographic 
parameters required for the type of compound, e.g. SE-30, 
Apiezon L, Carbowax 2OM, etc. Preparation of this tandem 
column for use required: (1) for deuteration, conditioning 
by injection of approximately 300 microliters of %O and 
stabilization for 5-10 houryb (2)  for oxygen-18 exchange, 
sequential injection of H20 
within 10 seconds. were used per 
milligram of compound. 
followed bylghe sample 
In general, 4 mg. 50 
4 
( I .  .. 
4.  A l l  mass spec t ra  were determined on a modified C.E.C. 
21-1032 mass spectrometer ( f o r  de ta i l s  of modifications 
and performance, see F. C . Walls and A .  L. Burlingame, 
Anal. Chem., i n  preparation) equipped w i t h  a heated g l a s s  
mt -em operated at 2 0 O o C .  
recorded a t  ionizing voltage 70 e .v., ionizing current  
10-50 pa and 160-180 v o l t s  per  stage on the mul t ip l i e r .  
Representative samples were determined emp1,oying a d i r e c t  
Most spec t ra  were 
i n l e t  system (see, A .  L. Burlingame, i n  Advances I n  Mass 
Spectrometry, vol. 111, Pergamon Press, 'in p repa rX%~on)  
and scanning the  en t i re  spectrum i n  30 seconds. 


Convenient Labelling Technique for Mass Spectrometry - Acid Catalyzed Deuterium and Oxygen-18 Exchange via Gas-liquid Chromatography

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