7/24/2019 Quantitative Gas Cromatography Analisys of Volatiles Fatty Acids in Spent Culture Media http://slidepdf.com/reader/full/quantitative-gas-cromatography-analisys-of-volatiles-fatty-acids-in-spent-culture 1/8 Vol. 23, No. 3 OURNAL OF CLINICAL MICROBIOLOGY, Mar. 1986 p. 523-530 0095-1137/86/030523-08 02.00/0 Copyright C 1986, American Society for Microbiology Quantitative Gas Chromatographic Analysis of Volatile Fatty Acids in Spent Culture Media and Body Fluids ANTHONY E. VAN DEN BOGAARD, MATHEW J. HAZEN, AND CEES P. VAN BOVEN Department of Medical Microbiology, University of Limburg, 6200 MD Maastricht, The Netherlands Received 10 September 1985/Accepted 19 November 1985 Gas chromatographic analysis of volatile fatty acids for identification of obligately anaerobic bacteria and for presumptive diagnosis of anaerobic infections is now widely practiced. However, it is difficult to compare data because only a qualitative analysis is done or only chromatograms ar e presented instead of quantitative data on volatile fatty acid production. We compared three stationary phases for volatile fatty acid analysis of aqueous solutions and four methods of pretreating samples for ga s chromatography. Quantitative analysis could be done accurately by using Carbowax as the stationary phase after pretreatment of spent culture media with Dowex columns. If only qualitative analysis is required (e.g., for presumptive diagnosis of anaerobic infections), ether extraction and headspace analysis ar e equally suitable. The overall variation coefficient for volatile fatty acid production by four reference strains of obligately anaerobic bacteria after 24 h ofincubation was approximately 1 0 . In the 25 years since gas chromatography (GC) was developed, many workers have used this sensitive technique for taxonomy and classification of microorganisms. For species identification of obligately anaerobic bacteria GC analysis of the volatile fatty acids (VFAs), which are the principal metabolic products of interest in these organisms, is often necessary. The recent renaissance of medical anaerobic bacteriology has increased the use of GC techniques in clinical bacterio- logical laboratories. Because of improvements in isolation and culture methods and a greater awareness of the clinical importance of these methods among clinicians and bacteri- ologists, the rate of isolation of obligately anaerobic bacteria from clinical infections has increased during the last 10 years. In addition, the development and availability of antibiotics that are active especially against obligately anaerobic bacteria have caused an increasing demand for rapid laboratory diagnosis of anaerobic bacterial infections. Unfortunately, isolation and cultivation of obligately anaerobic bacteria are laborious and time consuming. Often critically ill patients are involved, and anaerobic bacterial infections m ay run a fulminating course, so an early pre- sumptive diagnosis is mandatory. Several authors have shown that detection of VFAs by direct GC analysis of clinical specimens (4-6, 12, 16-18, 21) and blood cultures (20) is a rapid and reliable method for detection of anaerobic bacterial infections. This procedure allows a presumptive diagnosis within 60 min at a relatively low cost once the necessary capital investments have been made. Moreover, the technique is not hampered by preex- isting antibiotic therapy, inadequate sampling, or delayed transport to thelaboratory. For these reasons GC analysis of VFAs has become an important technique in clinical anaerobic bacteriology in medical and veterinary laborato- rnes. Because GC is no w widely used, it would be very useful if results from different laboratories could be compared. How- ever, this is difficult since most laboratories perform only qualitative or semiquantitative analyses of VFAs and so far only chromatograms, which are influenced by the variety of * Corresponding author. the different GC systems used, have been published. This is especially a disadvantage if data on VFA production are used for identification and taxonomy. The use of an internal standard m ay facilitate the comparison of chromatograms obtained in different laboratories (8), but to be really able to compare results from different laboratories it is desirable to obtain quantitative data on VFA production by obligately anaerobic bacteria. Quantitative GC analysis of VFAs has been shown to be feasible for other purposes (3). The goal of this study w as to compare different pretreat- ment methods and different stationary phases for quantita- tive analysis of VFAs in aqueous solutions. The influence of different inoculum sizes and different incubation periods on the production of VFAs by four reference strains of obligately anaerobic bacteria w as also determined. The results obtained with each stationary phase were analyzed in terms of separation efficiency, analysis time, sensitivity, and variability. The pretreatment methods were compared for usefulness, level of recovery, and variability. Calibration curves were made for each VFA (C2 through C6) with one selected pretreatment method and stationary phase. MATERIALS AND METHODS Stationary phases. The following three stationary phases for packed columns were used: (i) Carbowax (Supelco Inc., Hilversum, The Netherlands), which contained 0.3 Carbopack C, 20 M Carbowax, and 0.1 H3PO4; (ii) Chromosorb 101 (Chrompack B.V., Middelburg, The Netherlands), a styrene divinylbenzene polymer; and (iii) SP 1200 (Supelco), which contained 10 SP 1200, 1 H3PO4, and acid-washed 80/100 Chromosorb W. The three stationary phases were selected because of suitability (according to the manufacturers) for analysis of VFAs in aqueous solutions. Standard solutions. To test the stationary phases, we used an aqueous standard solution gravimetrically blended con- taining 10 mmol of each of the following VFAs per liter: formic acid (C1), acetic acid (C2), propionic acid (C3), isobutyric and n-butyric acids (C4), isovaleric and n-valeric acids (CO), isocaproic and n-caproic acids (C6), and n- heptanoic acid (C7). Three standard solutions, which con- tained 10 mmol of each gravimetrically blended VFA (C2 through C6) per liter in water, in GLC-broth (Biotrading, 523
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7/24/2019 Quantitative Gas Cromatography Analisys of Volatiles Fatty Acids in Spent Culture Media
http://slidepdf.com/reader/full/quantitative-gas-cromatography-analisys-of-volatiles-fatty-acids-in-spent-culture 1/8
V o l .
2 3 , N o . 3
OURNAL
O F C L I N I C A L
M I C R O B I O L O G Y ,
M a r . 1 9 8 6
p . 5 2 3 - 5 3 0
0 0 9 5 - 1 1 3 7 / 8 6 / 0 3 0 5 2 3 -0 8 0 2 . 0 0 / 0
C o p y r i g h t
C
1 9 8 6 ,
A m e r i c a n S o c i e t y
f o r M i c r o b i o l o g y
Q u a n t i t a t i v e
G a s
C h r o m a t o g r a p h i c
A n a l y s i s
o f
V o l a t i l e
F a t t y
A c i d s
i n
S p e n t
C u l t u r e
M e d i a a n d
Body F l u i d s
ANTHONY
E . VAN
DEN BOGAARD,
MATHEW J . HAZEN,
AND
CEES P . VAN
BOVEN
D e p a r t m e n t o f M e d i c a l
M i c r o b i o l o g y ,
U n i v e r s i t y
o f
L i m b u r g , 6 2 0 0 MD
M a a s t r i c h t ,
T h e N e t h e r l a n d s
R e c e i v e d
1 0
S e p t e m b e r 1 9 8 5 / A c c e p t e d
1 9
November
1 9 8 5
G a s c h r o m a t o g r a p h i c
a n a l y s i s o f v o l a t i l e f a t t y
a c i d s f o r
i d e n t i f i c a t i o n
o f o b l i g a t e l y a n a e r o b i c b a c t e r i a
a n d
f o r
p r e s u m p t i v e
d i a g n o s i s o f a n a e r o b i c
i n f e c t i o n s
i s now
w i d e l y p r a c t i c e d .
H o w e v e r , i t
i s
d i f f i c u l t t o compare
d a t a
b e c a u s e
o n l y a q u a l i t a t i v e
a n a l y s i s
i s
d o n e
o r o n l y
c h r o m a t o g r a m s
ar e
p r e s e n t e d
i n s t e a d o f q u a nt i t at i v e
d a t a
o n v o l a t i l e
f a t t y
a c i d
p r o d u c t i o n .
We
c o m p a r e d
t h r e e s t a t i o n a r y
p h a s e s f o r v o l a t i l e f a t t y
a c i d a n a l y s i s
o f
aqueous
s o l u t i o n s a n d f o u r
m e t h o d s o f
p r e t r e a t i n g
s a m p l e s
f o r ga s c h r o m a t o g r a p h y . Q u a n t i t a t i v e
a n a l y s i s
c o u l d
b e
d o n e a c c u r a t e l y b y u s i n g
C a r b o w a x
as
t h e s t a t i o n a r y
p h a s e a f t e r
p r e t r e a t m e n t o f s p e n t
c u l t u r e
m e d i a
w i t h Dowex c o l u m n s .
I f o n l y
q u a l i t a t i v e
a n a l y s i s i s r e q u i r e d
( e . g . , f o r p r e s u m p t i v e
d i a g n o s i s
o f a n a e r o b i c
i n f e c t i o n s ) ,
e t h e r e x t r a c t i o n
a n d h e a d s p a c e
a n a l y s i s
ar e e q u a l l y s u i t a b l e .
The o v e r a l l
v a r i a t i o n c o e f f i c i e n t
f o r
v o l a t i l e f a t t y a c i d
p r o d u c t i o n b y
f o u r
r e f e r e n c e s t r a i n s
o f o b l i g a t e l y
a n a e r o b i c
b a c t e r i a
a f t e r 2 4 h
o f i n c u b a t i o n
was a p p r o x i m a t e l y
1 0 .
I n
t h e
2 5
y e a r s
s i n c e
g a s
c h r o m a t o g r a p h y ( G C )
w a s
d e v e l o p e d , many w o r k e r s
h a v e
u s e d
t h i s
s e n s i t i v e
t e c h n i q u e
f o r t a x o n o m y
a n d c l a s s i f i c a t i o n
o f
m i c r o o r g a n i s m s .
F o r
s p e c i e s
i d e n t i f i c a t i o n
o f
o b l i g a t e l y
a n a e r o b i c b a c t e r i a
GC
a n a l y s i s o f t h e v o l a t i l e
f a t t y
a c i d s ( V F A s ) ,
w h i c h a r e
t h e
p r i n c i p a l
m e t a b o l i c p r o d u c t s
o f i n t e r e s t
i n t h e s e o r g a n i s m s ,
i s o f t e n
n e c e s s a r y .
T h e r e c e n t r e n a i s s a n c e
o f m e d i c a l
a n a e r o b i c
b a c t e r i o l o g y
h a s i n c re a se d
t h e u s e
o f GC
t e c h n i q u e s i n
c l i n i c a l
b a c t e r i o -
l o g i c a l
l a b o r a t o r i e s .
B e c a u s e o f i m p r o v e m e n t s
i n i s o l a t i o n
a n d
c u l t u r e m e t h o d s a n d a
g r e a t e r
a w a r e n e s s
o f t h e
c l i n i c a l
i m p o r t a n c e
o f t h e s e m e t h o d s
among
c l i n i c i a n s
a n d b a c t e r i -
o l o g i s t s ,
t h e
r a t e
o f
i s o l a t i o n
o f o b l i g a t e l y a n a e r o b i c
b a c t e r i a
f r o m c l i n i c a l
i n f e c t i o n s h a s i n c r e a s e d
d u r i n g
t h e
l a s t
1 0
y e a r s .
I n a d d i t i o n ,
t h e d e v e l o p m e n t
a n d a v a i l a b i l i t y
o f
a n t i b i o t i c s t h a t
a r e
a c t i v e e s p e c i a l l y a g a i n s t o b l i g a t e l y
a n a e r o b i c
b a c t e r i a h a v e
c a u s e d
a n
i n c r e a s i n g
d e m a n d f o r
r a p i d
l a b o r a t o r y d i a g n o s i s
o f a n a e r o b i c b a c t e r i a l
i n f e c t i o n s .
U n f o r t u n a t e l y ,
i s o l a t i o n
a n d c u l t i v a t i o n
o f
o b l i g a t e l y
a n a e r o b i c
b a c t e r i a a r e l a b o r i o u s a n d
t i m e
c o n s u m i n g .
O f t e n
c r i t i c a l l y
i l l
p a t i e n t s
a r e i n v o l v e d ,
a n d
a n a e r o b i c
b a c t e r i a l
i n f e c t i o n s
may
r u n
a f u l m i n a t i n g c o u r s e ,
s o a n e a r l y
p r e -
s u m p t i v e
d i a g n o s i s
i s
m a n d a t o r y .
S e v e r a l a u t h o r s h a v e
s h o w n t h a t d e t e c t i o n
o f
VFAs
b y
d i r e c t GC a n a l y s i s
o f c l i n i c a l s p e c i m e n s
( 4 - 6 , 1 2 ,
1 6 - 1 8 , 2 1 )
a n d b l o o d
c u l t u r e s
( 2 0 )
i s
a
r a p i d
a n d
r e l i a b l e
m e t h o d
f o r
d e t e c t i o n o f
a n a e r o b i c
b a c t e r i a l
i n f e c t i o n s .
T h i s
p r o c e d u r e
a l l o w s
a
p r e s u m p t i v e
d i a g n o s i s
w i t h i n
6 0
m i n
a t
a r e l a t i v e l y
l o w c o s t
o n c e
t h e n e c e s s a r y
c a p i t a l
i n v e s t m e n t s
h a v e
b e e n
m a d e .
M o r e o v e r ,
t h e
t e c h n i q u e
i s
n o t h a m p e r e d
b y
p r e e x -
i s t i n g
a n t i b i o t i c
t h e r a p y ,
i n a d e q u a t e s a m p l i n g ,
o r
d e l a y e d
t r a n s p o r t
t o
t h e l a b o r a t o r y .
F o r
t h e s e
r e a s o n s
GC
a n a l y s i s
o f
VFAs h a s
b e c o m e
a n
i m p o r t a n t
t e c h n i q u e
i n c l i n i c a l
a n a e r o b i c
b a c t e r i o l o g y
i n
m e d i c a l a n d
v e t e r i n a r y
l a b o r a t o -
r n e s .
B e c a u s e
GC
i s no w
w i d e l y u s e d ,
i t w o u l d
b e
v e r y
u s e f u l
i f
r e s u l t s f r o m
d i f f e r e n t
l a b o r a t o r i e s
c o u l d
b e
c o m p a r e d .
How-
e v e r ,
t h i s i s d i f f i c u l t
s i n c e
most
l a b o r a t o r i e s
p e r f o r m
o n l y
q u a l i t a t i v e
o r s e m i q u a n t i t a t i v e
a n a l y s e s
o f
VFAs
a n d s o
f a r
o n l y
c h r o m a t o g r a m s ,
w h i c h a r e
i n f l u e n c e d
b y
t h e
v a r i e t y
o f
*
C o r r e s p o n d i n g
a u t h o r .
t h e
d i f f e r e n t
GC
s y s t e m s
u s e d ,
h a v e b e e n
p u b l i s h e d .
T h i s
i s
e s p e c i a l l y
a d i s a d v a n t a g e
i f
d a t a
o n VF A
p r o d u c t i o n
a r e
u s e d f o r
i d e n t i f i c a t i o n
a n d t a x o n o m y .
T h e u s e o f
a n
i n t e r n a l
s t a n d a r d may
f a c i l i t a t e
t h e c o m p a r i s o n
o f c h r o m a t o g r a m s
o b t a i n e d
i n
d i f f e r e n t l a b o r a t o r i e s
( 8 ) ,
b u t t o
b e
r e a l l y
a b l e t o
c o m p a r e
r e s u l t s f r o m
d i f f e r e n t l a b o r a t o r i e s i t
i s d e s i r a b l e
t o
o b t a i n
q u a n t i t a t i v e
d a t a
o n VFA p r o d u c t i o n
b y o b l i g a t e l y
a n a e r o b i c
b a c t e r i a .
Q u a n t i t a t i v e
GC
a n a l y s i s o f
VFAs h a s
b e e n
s h o w n
t o
b e f e a s i b l e f o r o t h e r
p u r p o s e s ( 3 ) .
T h e
g o a l o f
t h i s s t u d y
was t o c o m p a r e d i f f e r e n t
p r e t r e a t -
m e n t
m e t h o d s
a n d
d i f f e r e n t
s t a t i o n a r y p h a s e s f o r
q u a n t i t a -
t i v e a n a l y s i s
o f VFAs
i n
a q u e o u s
s o l u t i o n s .
T h e i n f l u e n c e
o f
d i f f e r e n t
i n o c u l u m
s i z e s a n d d i f f e r e n t
i n c u b a t i o n p e r i o d s
o n
t h e
p r o d u c t i o n
o f VFAs b y
f o u r r ef e r en ce
s t r a i n s
o f
o b l i g a t e l y
a n a e r o b i c
b a c t e r i a
was a l s o
d e t e r m i n e d .
T h e
r e s u l t s
o b t a i n e d
w i t h
e a c h
s t a t i o n a r y
p h a s e
w e r e a n a l y z e d
i n
t e r m s
o f
s e p a r a t i o n
e f f i c i e n c y ,
a n a l y s i s
t i m e ,
s e n s i t i v i t y ,
a n d
v a r i a b i l i t y . T h e p r e t r e a t m e n t
m e t h o d s
w e r e c o m p a r e d
f o r
u s e f u l n e s s ,
l e v e l
o f r e c o v e r y ,
a n d v a r i a b i l i t y .
C a l i b r a t i o n
c u r v e s
w e r e
m a d e
f o r
e a c h VF A
( C 2
t h r o u g h
C 6 ) w i t h
o n e
s e l e c t e d
p r e t r e a t m e n t
m e t h o d a n d
s t a t i o n a r y
p h a s e .
MATERIALS
AND
M E T H O D S
S t a t i o n a r y
p h a s e s .
T h e
f o l l o w i n g
t h r e e
s t a t i o n a r y p h a s e s
f o r
p a c k e d
c o l u m n s
w e r e u se d :
( i )
C a r b o w a x
( S u p e l c o
I n c . ,
H i l v e r s u m ,
T h e N e t h e r l a n d s ) ,
w h i c h
c o n t a i n e d
0 . 3
C a r b o p a c k
C ,
2 0
M
C a r b o w a x ,
a n d
0 . 1
H 3 P O 4 ;
( i i )
C h r o m o s o r b
1 0 1
( C h r o m p a c k
B . V . , M i d d e l b u r g ,
T h e
N e t h e r l a n d s ) ,
a
s t y r e n e
d i v i n y l b e n z e n e
p o l y m e r ;
a n d
( i i i )
S P
1 2 0 0
( S u p e l c o ) ,
w h i c h
c o n t a i n e d
10
SP
1 2 0 0 ,
1
H 3 P O 4 ,
a n d
a c i d - w a s h e d
8 0 / 1 0 0
C h r o m o s o r b
W.
T h e
t h r e e
s t a t i o n a r y
p h a s e s
w e r e s e l e c t e d
b e c a u s e
o f
s u i t a b i l i t y
( a c c o r d i n g
t o
t h e
m a n u f a c t u r e r s )
f o r
a n a l y s i s
o f VFAs
i n
a q u e o u s
s o l u t i o n s .
S t a n d a r d
s o l u t i o n s .
To
t e s t t h e
s t a t i o n a r y p h a s e s ,
w e u s e d
a n
a q u e o u s
s t a n d a r d
s o l u t i o n
g r a v i m e t r i c a l l y
b l e n d e d
c o n -
t a i n i n g
1 0
mmol
o f
e a c h
o f t h e
f o l l o w i n g
VFAs
p e r
l i t e r :
f o r m i c
a c i d
( C 1 ) ,
a c e t i c
a c i d
( C 2 ) ,
p r o p i o n i c
a c i d
( C 3 ) ,
i s o b u t y r i c
a n d
n - b u t y r i c
a c i d s
( C 4 ) ,
i s o v a l e r i c
a n d
n - v a l e r i c
a c i d s
( C O ) ,
i s o c a p r o i c
a n d
n - c a p r o i c
a c i d s
( C 6 ) ,
a n d
n -
h e p t a n o i c
a c i d
( C 7 ) .
T h r e e
s t a n d a r d
s o l u t i o n s ,
w h i c h c o n -
t a i n e d
1 0
mmo l
o f
e a c h
g r a v i m e t r i c a l l y
b l e n d e d
VFA
( C 2
t h r o u g h
C 6 ) p e r
l i t e r
i n
w a t e r ,
i n G L C - b r o t h
( B i o t r a d i n g ,
5 2 3
7/24/2019 Quantitative Gas Cromatography Analisys of Volatiles Fatty Acids in Spent Culture Media
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5 2 4
VAN D EN B OGA AR D
ET A L .
T A B L E
1 .
GC
c o n d i t i o n s
f o r t h e
s t a t i o n a r y p h a s e s
e x a m i n e d
C o l u m n
O v e n
t e m p
( ° )
I n j e c t o r
D e t e c t o r
N .
c a r r i e r
A n a l y s i s
S a m p l e
S t a t i o n a r y
p h a s e l e n g t h
t e m p
t e m p
g a s
f l o w
t i m e
s i z e
( m )
I n i t i a l
F i n a l
( O C )
( 0 C )
r a t e
( m i n )
( p 1 )
C h r o m o s o r b
1 0 1 1 . 8 3 2 0 0
2 0 0 2 0 0
2 4 0
1 0
2 2
1
S P 1 2 0 0
1 . 8 3
1 2 5
1 2 5
2 0 0 2 0 0
4 0 1 2
1
C a r b o w a x
0 . 9 1
1 2 0 1 2 0
2 0 0
2 0 0
2 0
2 0
1
C a r b o w a x
0 . 9 1
1 1 0 1 9 0 1 5 0
2 0 0 2 0
1 0
1
W i l n i s , T h e
N e t h e r l a n d s ) ,
a n d i n
c o n c e n t r a t e d human
b u f f y
c o a t c e l l s i n
p l a s m a ,
w e r e
u s e d i n t h e
s e c o n d s e r i e s o f
e x p e r i m e n t s t o t e s t p r e t r e a t m e n t
m e t h o d s . T h e
b u f f y
c o a t
c e l l s
s u s p e n d e d
i n
p l a s m a w e r e u s e d
t o m i m i c
b o d y
f l u i d s
a n d a r e r e f e r r e d
t o
a s p u s
b e l o w .
G C .
T h e
GC
s y s t e m
c o n s i s t e d
o f
a
P a c k a r d - B e c k e r
m o d e l
4 3 3
g a s c h r o m a t o g r a p h
e q u i p p e d
w i t h
a
d u a l - c o l u m n
s y s t e m
a n d
f l a m e i o n i z a t i o n
d e t e c t o r s
i n
c o n j u n c t i o n
w i t h
a
d i g i t a l
p r o c e s s o r . T h e s e n s i t i v i t y o f
t h e d e t e c t o r s
w a s 1 0 - 1 4
A ,
a n d
t h e
d e t e c t o r s
w e r e
c o n n e c t e d t o a GC c o n t r o l
s t a t i o n
e q u i p p e d
w i t h
a p r i n t e r
p l o t t e r ( m o d e l 4 3 3 c o n t r o l
s t a t i o n ;
P a c k a r d - B e c k e r ,
D e l f t ,
T h e
N e t h e r l a n d s ) .
G l a s s
c o l u m n s
w e r e
u s e d
( 1 . 8 3 m b y 2 mm
f o r C h r o m o s o r b 1 0 1
a n d
S P 1 2 0 0
a n d
0 . 9 1 m
b y 2 mm
f o r
C a r b o w a x ) .
A l l
c o l u m n s
w e r e
c o n d i t i o n e d
o v e r n i g h t
w i t h t h e e f f l u e n t
e n d d e t a c h e d
f r o m
t h e
d e t e c t o r
a n d
a t
a c a r r i e r
g a s ( N 2 )
f l o w
r a t e
o f
2 0
m l / m i n .
T h e
S P
1 2 0 0
a n d
C a r b o w a x c o l u m n s w e r e c o n d i t i o n e d
a t
1 7 5 ° C , a n d
t h e
C h r o m o s o r b
1 0 1
c o l u m n
w a s c o n d i t i o n e d a t
2 5 0 0 C .
A f t e r
c o n d i t i o n i n g ,
t h e
c o l u m n s
w e r e t r e a t e d w i t h
w a t e r
t o
s a t u r a t e
t h e
s t a t i o n a r y p h a s e s .
T h i s w a s d o n e
b y i n j e c t i n g
5
, u l o f w a t e r
i n t o t h e
C h r o m o s o r b c o l u mn 2 0
t i m e s . T h e S P
1 2 0 0
c o l u m n
w a s p r e t r e a t e d
b y
i n j e c t i n g
1
, u l o f w a t e r 1 0
t i m e s .
W i t h t h e
C a r b o w a x
c o l u m n
5
R I
o f
f o r m i c
a c i d
( 0 . 1
[ v o l / v o l ]
i n
w a t e r ) w a s
i n j e c t e d
a t
1 7 5 ° C s ev e n t i m e s ;
t h e
t i m e
i n t e r v a l
b e t w e e n i n j e c t i o n s w a s
a p p r o x i m a t e l y
1 0
m i n .
A n a l y s e s w e r e c a r r i e d
o u t
b y u s i n g
t h e
GC c o n d i t i o n s
r e c o m m e n d e d
b y
t h e
m a n u f a c t u r e r s
o f
t h e
s t a t i o n a r y
p h a s e s
( T a b l e
1 ) .
T h e
C a r b o w a x
c o l u m n w a s
u s e d
b o t h
i s o t h e r m a l l y
a t
1 2 0 ° C
a n d
w i t h
t h e
f o l l o w i n g
t e m p e r a t u r e
p r o g r a m :
a n
i n i t i a l
o v e n
t e m p e r a t u r e
o f 1 1 0 ° C ;
a f t e r 1 . 5
m i n t h e o v e n
t e m p e r a t u r e w a s
r a i s e d
2 0 ° C / m i n
t o
1 9 0 ° C ; a n d t h e f i n a l
t e m p e r a t u r e w a s h e l d
f o r
4 . 5
m i n . I n
a d d i t i o n t o
g o o d
p e a k
s e p a r a t i o n , p e a k
s y m m e t r y ,
a n d l a c k o f
t a i l i n g
o n t h e
c h r o m a t o g r a m s ,
t h e
c o l u m n s
w e r e t e s t e d
f o r
g h o s t i n g b y
r e p e a t e d
i n j e c t i o n s w i t h
t w i c e - d i s t i l l e d w a t e r
a n d 0 . 1 f o r -
m i c
a c i d .
S e n s i t i v i t y w a s
d e t e r m i n e d
b y
m e a s u r i n g t h e
m i n i m u m
l e v e l s a t
w h i c h e a c h o f
t h e
VFAs
( C 2
t h r o u g h C 6 )
c o u l d
b e m e a s u r e d .
T h i s
w a s d o n e
b y i n j e c t i n g
1 - p l I
p o r t i o n s
o f
t w o f o l d
d i l u t i o n s o f
t h e
a q u e o u s s t a n d a r d
s o l u t i o n u n t i l
we
i d e n t i f i e d t h e l o w e s t
d i l u t i o n a t
w h i c h a l l
VFAs ( C 2
t h r o u g h C 6 )
c o u l d b e
m e a s u r e d q u a n t i t a t i v e l y a t
t h e h i g h e s t
p o s s i b l e d e t e c t o r
s e n s i t i v i t y .
F o r m i c
a c i d ,
w h i c h
i s
l o g i c a l l y
g r o u p e d
w i t h t h e
V F A s , w a s n o t
m e a s u r e d b e c a u s e f l a m e
i o n i z a t i o n
d e t e c t o r s
d o n o t r e s p o n d
t o i t .
V a r i a t i o n w a s
c a l c u l a t e d a f t e r 1
, u l
o f
t h e
a q u e o u s
s t a n d a r d
s o l u t i o n w a s
i n j e c t e d
i n t o e a c h
s t a t i o n a r y
p h a s e 1 0 t i m e s
c o n s e c u t i v e l y .
T h e
v a r i a t i o n
c o e f f i c i e n t
f o r e a c h VFA
w a s e s t i m a t e d
f r o m
t h e
r e s u l t s
o f
1 0 i n j e c t i o n s
a n d w a s c a l c u l a t e d
a s
f o l l o w s :
( s t a n d a r d
d e v i a t i o n / m e a n )
x
1 0 0 . T h e
v a r i a t i o n
c o e f f i c i e n t o f
a
c o l u m n
w a s
c o n s i d e r e d
t o b e t h e s u m
o f t h e
v a r i a t i o n
c o e f f i c i e n t s
o f
t h e
VFAs ( C 2
t h r o u g h
C 7 ) d i v i d e d b y
t h e
n u m b e r
o f
v a r i a t i o n
c o e f f i c i e n t s ( n
=
9 ) .
T h e
v a r i a t i o n i n
t h e
r e t e n t i o n
t i m e s f o r
e a c h
VFA ( C 2 t h r o u g h
C 7 ) w a s c a l c u l a t e d
i n
t h e same
w a y .
A f t e r e a c h
i n j e c t i o n
t h e
s y r i n g e
was
w a s h e d w i t h
g l a s s - d i s t i l l e d
w a t e r ,
e t h a n o l ,
a n d
e t h y l
e t h e r
a n d d r i e d
b y
s u c t i o n .
S a m p l e p r e t r e a t m e n t . I n a s e c o n d s e r i e s o f
e x p e r i m e n t s
w e
c o m p a r e d
t h e
f o l l o w i n g
f o u r
p r e v i o u s l y d e s c r i b e d
m e t h o d s
f o r
p r e t r e a t m e n t
o f
s p e n t
c u l t u r e
m e d i a
a n d
b o d y
f l u i d s
p r i o r
t o
GC
a n a l y s i s
o f
V F A s :
vacuum
d i s t i l l a t i o n
( 2 2 ) , m o l e c u l a r
s i e v i n g
( 1 , 1 5 ) , h e a d s p a c e
a n a l y s i s
( 7 ) ,
a n d
e t h e r e x t r a c t i o n
( 1 2 , 1 3 ) .
A l l
c o nc en t ra t i o ns o f
f o r m i c a c i d
g i v e n b e l o w
a r e
v o l u m e
p e r c e n t a g e s
i n
w a t e r ,
a n d a l l
GC a n a l y s e s w e r e
d o n e
b y
u s i n g C a r b o w a x
a s t h e
s t a t i o n a r y
p h a s e
a n d
t h e
t e m p e r -
a t u r e
p r o g r a m
d e s c r i b e d
a b o v e .
( i )
Vacuum
d i s t i l l a t i o n . A 2 - m l
s a m p l e
was a c i d i f i e d w i t h
2 0 0
R I
o f
0 . 1 f o r m i c
a c i d ,
a n d 1
d r o p
o f
a n t i f o a m i n g
a g e n t
3 3 1 5 1
B DH
C h e m i c a l s
L t d . ,
P o o l e ,
U n i t e d
K i n g d o m ) was
a d d e d . T h e
s p e c i m e n f l a s k
( 2 2 ) w a s
c o n n e c t e d
w i t h
a r e -
c e i v e r
t u b e ,
w h i c h
w a s
c o o l e d
i n
l i q u i d
n i t r o g e n . T h i s
r e c e i v e r
t u b e
w a s
t h e n
e v a c u a t e d
w i t h
a
w a t e r j e t
pump
a n d
k e p t e v a c u a t e d
b y
c o n t i n u o u s
s u c t i o n , w h i l e t h e
c o n t e n t s o f
t h e
s p e c i m e n
f l a s k
w e r e
m i x e d w i t h
a m a g n e t i c
s t i r r e r a n d
s l o w l y
h e a t e d
o v e r a p er i o d o f 2 0 m i n
f r o m
room
t e m p e r a -
t u r e
t o 1 2 0 ° C i n
a n o i l
b a t h . A f t e r t h i s t h e
s p e c i m e n
was
r e d u c e d t o
c o m p l e t e
d r y n e s s ,
a n d a f t e r
t h e
d i s t i l l a t e
w a s
t h a w e d a t room
t e m p e r a t u r e ,
i t
w a s
d i r e c t l y i n j e c t e d
i n t o
t h e
GC
a p p a r a t u s .
( i i )
M o l e c u l a r
s i e v i n g .
P a s t e u r
p i p e t t e s w e r e f i l l e d w i t h
1 m l
o f
p a c k e d
c a t i o n - e x c h a n g e r e s i n i n
t h e
h y d r o g e n f o r m
( A G
5 0
W - X 4 ; 2 0 0 - 4 0 0
m e s h ;
B i o - R a d
L a b o r a t o r i e s ,
R i c h m o n d ,
C a l i f . )
o n
p h o s p h o r i c
a c i d - t r e a t e d
g l a s s
w o o l
( c a t a l o g n o .
2 - 0 3 8 3 ;
S u p e l c o ) . A f t e r t h e f l u i d w a s
d r a i n e d ,
t h e s e
D o w e x
c o l u m n s w e r e
r e a d y
f o r
u s e . B e f o r e t h e
c o l u m n s
w e r e
f i l l e d ,
t h e
r e s i n w a s
c o n v e r t e d
t o
t h e
h y d r o g e n
f o r m
b y
w a s h i n g i t
w i t h d i s t i l l e d
w a t e r
u n t i l t h e
s u p e r n a t a n t w a s
c l e a r ;
t h i s
w a s
f o l l o w e d
b y
w a s h i n g w i t h 1
N
NaO H f o r
2 4 h .
A f t e r
n e u t r a l i z a t i o n
t o
pH 7
b y r e p e a t e d
w a s h i n g
w i t h
d i s t i l l e d
w a t e r ,
t h e
r e s i n w a s w a s h e d w i t h
4 N H C l
f o r 2
h .
B e f o r e
u s e
t h e r e s i n w a s
a d j u s t e d t o
pH 7 . A 1 - m l
p o r t i o n
o f
a
s a m p l e
w a s a c i d i f i e d
w i t h
1 0 0
p u l
o f
0 . 1
f o r m i c a c i d ,
a n d t h i s
p r e p a r a t i o n
w a s
p a s s e d
t h r o u g h t h e
r e s i n ;
i n
a d d i t i o n , t h e
r e s i n
c o l u m n
w a s r i n s e d
t w i ce w i t h
0 . 5
m l o f
0 . 1 f o r m i c
a c i d . A l l
o f t h e e f f l u e n t s
f r o m t h e
c o l u m n
w e r e c o l l e c t e d i n
a
t e s t t u b e a n d a n a l y z e d
b y G C .
( i i i )
H e a d s p a c e
a n a l y s i s .
S a m p l e s
( 3
m l )
w e r e
a c i d i f i e d
w i t h
3 0 0
p u l
o f 0 . 1
f o r m i c
a c i d ,
a n d
t h e s e
p r e p a r a t i o n s
w e r e
t r a n s f e r r e d t o
1 0 - m l
a m p o u l e s
c o n t a i n i n g 2 g o f
a n h y d r o u s
N a 2 S O 4 .
T h e
f l a s k s w e r e s e a l e d
g a s
t i g h t w i t h
r u b b e r s t o p -
p e r s
a n d
p r o t e ct e d w i t h a l u m i n u m
c r i m p c a p s b e f o r e
b e i n g
h e a t e d i n a w a t e r
b a t h a t
7 5 ° C f o r 1 0 m i n w i t h
o c c a s i o n a l
s h a k i n g .
T h e n
2
m l
o f
t h e
g a s
a t m o s p h e r e i n t h e
b o t t l e s w a s
w i t h d r a w n
i n t o a
H a m i l t o n
g a s - t i g h t
s y r i n g e a n d
i n j e c t e d
i n t o t h e
GC
a p p a r a t u s . T h e
s y r i n g e
w a s
p r e h e a t e d t o 8 0 ° C t o
a v o i d
c o n d e n s a t i o n
o f
t h e
s a m p l e
c o m p o n e n t s d u r i n g
i n j e c -
t i o n .
( i v )
E t h e r
e x t r a c t i o n . An
a c i d i f i e d 1 - m l
s a m p l e w a s e x -
J . C L I N . M I C R O B I O L .
7/24/2019 Quantitative Gas Cromatography Analisys of Volatiles Fatty Acids in Spent Culture Media
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GC
ANALYSIS
O F VFAs
5 2 5
T A B L E
2 .
R e p r o d u c i b i l i t y
o f GC
s y s t e m s ,
i n c l u d i n g t h e
i n j e c t i o n
p r o c e d u r e s
V a r i a t i o n
c o e f f i c i e n t s o f p e a k
a r e a s
M % Y '
f o r VFA
V a r i a t i o n
c o e f f i c i e n t s o f
r e t e n t i o n t i m e s ( ) f o r VFA
Stationary ~~~~~~~~~C2
,
S t a t i o n a r y
C 2
C 3
i s o - C 4 n - C 4 i s o - C 5
n - C s
i s o - C 6 n - C 6
C 7 t h r o u g h
C .
C ,
i s o - C 4 n - C 4
i s o - C 5
n I C 5
i s o - C 6
1 - C 6
C 7
t h r o u g h
C h r o m o s o r b 3 . 3
4 . 3 1 . 9 3 . 3
2 . 9 4 . 9 2 . 6
3 . 7
5 . 6
3 . 7
0 . 7 0 . 4 0 . 2 0 . 3
0 . 2
0 . 3 0 . 3 0 . 3
0 . 4 0 . 3
1 0 1
S P
1 2 0 0 3 . 0 2 . 9 2 . 5 2 . 5 2 . 2 2 . 4 9 . 1 5 . 3 3 . 7 3 . 7
6 . 0 4 . 0 3 . 6 3 . 1 2 . 1 1 . 8
1 . 2 0 . 9 0 . 5 2 . 6
C a r b o w a x
1 . 8
2 . 7
2 . 1 3 . 1 2 . 3
2 . 6
1 . 4
1 . 1
1 . 4
2 . 0
1 . 1
0 . 9 0 . 5 0 . 2
0 . 1
0 . 1
0 . 1
0 . 1
0 . 1
0 . 4
T h e
r e s u l t s
a r e
t h e
m e a n s o f 1 0 i n j e c t i o n s .
b
C a l c u l a t e d a s f o l l o w s :
( s t a n d a r d
d e v i a t i o n / m e a n ) x 1 0 0 .
t r a c t e d
w i t h
1 m l
o f e t h e r
t h o r o u g h l y
m i x e d u s i n g
a V o r t e x
m i x e r a n d t h e n c e n t r i f u g e d t o b r e a k t h e e m u l s i o n . A 1 - , u l
p o r t i o n
o f t h e e t h e r
l a y e r
w a s i n j e c t e d
d i r e c t l y i n t o
t h e GC
c o l u m n .
T h e
t e s t t u b e s c o nt a i ni n g e t h er w e r e
k e p t o n i c e
t o
p r e v e nt e v ap o r a t i o n o f t h e e t h e r .
B e c a u s e
s w a b s a r e
o f t e n u s e d
t o c o l l e c t c l i n i c a l
m a t e r i a l
s u c h a s
p u s
f o r b a c t e r i o l o g i c a l i n v e s t i g a t i o n s ,
t h e f o l l o w i n g
f o u r
t y p e s o f s w a b s w e r e t e s t e d : c o t t o n
s w a b s , c h a r c o a l -
c o a t e d s w a b s ,
f e t a l
c a l f
s e r u m - c o a t e d s w a b s ,
a n d
p o l y e s t e r
s w a b s ( A l b i c B . V . , M aa ss l u i s,
T h e
N e t h e r l a n d s ) .
T h e c h a r -
c o a l - c o a t e d
a n d f e t a l c a l f
s e r u m - c o a t e d
s w a b s w e r e
p r e p a r e d
b y
s o a k i n g
c o t t o n s w a b s i n
a n
a q u e o u s 1 c h a r c o a l
s u s p e n -
s i o n
a n d
i n
f e t a l c a l f
s e r u m , r e s p e c t i v e l y . A f t e r d r y i n g a n d
a u t o c l a v i n g t h e s w a b s w e r e
i m m e r s e d i n t h e s t a n d a r d p u s
s o l u t i o n ,
d r a i n e d o f
e x c e s s
f l u i d , r e p l a c e d
i n a
h o l d e r ,
a n d
s t o r e d a t r o o m t e m p e r a t u r e
f o r 2 h
t o
s i m u l a t e t h e d e l a y
b e t w e e n
c o l l e c t i o n
a n d
p r o ce ss i ng o f c l i n i c a l
s p e c i m e n s .
T h e
a m o u n t o f t h e
a b s o r b e d
s t a n d a r d w a s m e a s u r e d
b y w e i g h i n g
t h e s w a b
b e f o r e
a n d i m m e d i a t e l y a f t e r i m m e r s i o n i n t o t h e
s t a n d a r d
s o l u t i o n
a n d d r a i n i n g
o f
t h e
e x c e s s
f l u i d . F o r
VFA
a n a l y s i s
t h e s w a b
h e a d
w a s
s n a p p e d f r o m
i t s s t i c k
i n t o
a
m i c r o t e s t
t u b e .
E t h e r ( 1 m l ) a n d 1 0 0
, u l
o f 0 . 1 f o r m i c
a c i d
w e r e a d d e d .
T h e
m i c r o t e s t
t u b e w a s c a p p e d a n d
v o r t e x e d
f o r
a p p r o x i m a t e l y
2 0 s . A f t e r
c e n t r i f u g a t i o n
a t
2 5 0
x
g f o r
2
m i n ,
1
1 . l
o f
t h e
e t h e r
l a y e r
w a s
i n j e c t e d i n t o t h e
GC
a p p a r a t u s .
A f t e r
p r e t r e a t m e n t e ac h s a m p l e
w a s
s p i k e d
w i t h
2
, i l
o f
h e p t a n o i c a c i d ( a n a l y t i c a l
g r a d e ;
c a t a l o g n o .
A - 9 3 7 8 ; S i g m a
C h e m i c a l
C o . ,
S t .
L o u i s , M o . )
p e r
m l a s
a n
i n t e r n a l s t a n d a r d
a n d
a n a l y z e d
f o r VFAs
b y
G C . T h e
v a r i a t i o n
c o e f f i c i e n t a n d
r e l a t i v e e r r o r ( i . e . ,
1 0 0
x
[ e r r o r / t r u e v a l u e o f e a c h p r e t r e a t -
m e n t
m e t h o d
i n
c o m b i n a t i o n
w i t h t h e C a r b o w a x c o l u m n ] )
f o r
e a c h
VFA
( C 2
t h r o u g h
C 6 )
w e r e
c a l c u l a t e d .
C a l i b r a t i o n c u r v e s .
C a l i b r a t i o n
c u r v e s
f o r
q u a n t i t a t i v e
a n a l y s e s
o f
C 2 t h r o u g h
C 6
VFAs i n
a q u e o u s
s o l u t i o n s
w e r e
o b t a i n e d
a f t e r p r e t r e a t m e n t b y
m o l e c u l a r
s i e v i n g
a n d
GC
a n a l y s i s .
S i x
s a m p l e s o f
f o u r s t a n d a r d s o l u t i o n s
c o n t a i n i n g
0 . 1 , 1 , 1 0 ,
o r
2 0
mmol
o f e a c h
VFA
( C 2 t h r o u g h
C 6 )
p e r
l i t e r
i n
GL C b r o t h
s u p p l e m e n t e d
w i t h
c o o k e d m e a t
w e r e
p r e -
t r e a t e d
b y
m o l e c u l a r
s i e v i n g
o v e r
D o w e x
c o l u m n s .
A
c a l i -
b r a t i o n l i n e was
f i t ,
a n d
t h e c o r r e l a t i o n c o e f f i c i e n t
was
c a l c u l a t e d .
I n o c u l u m
s i z e a n d
i n c u b a t i o n
p e r i o d .
The i n f l u e n c e o f
i n o c u l u m s i z e a n d i n c u b a t i o n
p e r i o d
o n
t h e
p r o d u c t i o n
o f
VFAs b y o b l i g a t e l y
a n a e r o b i c
b a c t e r i a
i n a s t a n d a r d m e d i u m
w a s m e a s u r e d b y i n o c u l a t i n g o n e , t h r e e ,
a n d f i v e c o l o n i e s
a n d a
s w a b
o f
c o n t i n u o u s
g r o w t h
f r o m
a c u l t u r e
a f t e r
4 8
h
o f
i n c u b a t i o n o n
W e n s i n c k
a g a r ( 1 9 )
i n t o
t u b e s
c o n t a i n i n g
5 - m l
p o r t i o n s
o f
GL C
b r o t h
s u p p l e m e n t e d
w i t h
c o o k e d
m e a t . A l l
i n o c u l a t i o n s
w e r e
d o n e
i n
t r i p l i c a t e .
A f t e r
2 4 a n d 4 8
h
o f
i n c u b a t i o n a t
3 7 ° C
i n
a n
a n a e r o b i c
g l o v e
b o x ,
t h e amounts
o f
C 2
t h r o u g h
C 6
VFAs
p r o d u c e d w e r e m e a s u r e d b y
t e m p e r a -
t u r e - p r o g r a m m e d
g a s - l i q u i d
c h r o m a t o g r a p h y
w i t h
C a r b o w a x
a f t e r
p r e t r e a t m e n t b y m o l e c u l a r
s i e v i n g .
B a c t e r o i d e s
f r a g i l i s
N C T C 8 5 6 0 , F u s o b a c t er i u m v a r i u m A T C C 8 5 0 1 , C l o s t r i d -
i u m
d i f f i c i l e
A T C C 9 6 8 9
a n d P e p t o st r e p t o c o c cu s a n ae r o b i u s
A T C C 2 7 3 3 7
w e r e
u s e d
a s
r e f e r e n c e
s t r a i n s .
RESULTS
AND
DISCUSSION
S t a t i o n a r y p h a s e s .
T h e v a r i a t i o n
c o e f f i c i e n t s
f o r
p e a k
a r e a s
a n d r e t e n t i o n
t i m e s
f o r
e a c h
VFA
( C 2 t h r o u g h
C 7 ) i n t h e
s t a t i o n a r y
p h a s e s
t e s t e d a r e s h o w n i n
T a b l e
2 .
C h r o m o s o r b
1 0 1 w a s n o t c o m p l e t e l y s a t i s f a c t o r y .
T h e
p e a k s o f
t h e
c h r o m a t o g r a m c o u l d
n o t
b e c o m p l e t e l y s e p a -
r a t e d b e c a u s e
o f
t a i l i n g ( F i g .
1 A ) . R e p e a t e d r e c o n d i t i o n i n g ,
o v e r l o a d i n g
t h e c o l u m n s w i t h
w a t e r ,
a n d
t e m p e r a t u r e p r o -
g r a m m i n g d i d
n o t i m p r o v e
t h e
c h r o m a t o g r a m s
o f
a q u e o u s
s t a n d a r d s o l u t i o n s
o f
V F A s ,
n o r d i d
s a t u r a t i o n o f t h e c a r r i e r
g a s
w i t h
f o r m i c a c i d . D e s p i t e t h i s , q u a n t i t a t i v e a n a l y s i s w a s
p o s s i b l e .
T h e
a n a l y s i s
t i m e
w i t h t h i s
c o l u m n w a s
l o n g ( > 2 0
m i n ) .
N o
g h o s t i n g o c c u r r e d ,
a n d t h e minimum
l e v e l o f
d e t e c t i o n f o r
C 2 t h r o u g h C 7
VFAs
w a s 0 . 0 4 m m o l / l i t e r .
S P 1 2 0 0
w a s
l e s s
s a t i s f a c t o r y .
T h e
p e a k s
o f t h e c h r o m a t o -
g r a m s h o w e d t a i l i n g , a n d c o m p l e t e s e p a r a t i o n
w a s n o t
p o s s i b l e
( F i g .
1 B ) .
R e c o n d i t i o n i n g o f
t h e
c o l u m n
was
n e c e s -
s a r y
a f t e r
e a c h
1 0
t o
1 2
s a m p l e s .
A t
h i g h
d e t e c t o r
s e n s i t i v i t y
t h e
p e a k s
o f
t h e
c h r o m a t o g r a m
b e c a m e
d e f o r m e d ,
a n d
i n j e c t i o n
o f
t w i c e - g l a s s - d i s t i l l e d
w a t e r
p r o d u c e d
g h o s t
p e a k s .
T h i s m a d e i t
i m p o s s i b l e
t o
m e a s u r e
t h e
l o w e s t
d e t e c t i o n
l e v e l .
I n o u r h a n d s S P
1 2 0 0
w a s
n o t
s u i t a b l e f o r
q u a n t i t a t i v e a n a l y s i s
o f VFAs
i n
a q u e o u s
s o l u t i o n s .
C a r b o w a x
p e r f o r m e d
t h e b e s t a n d w a s
w e l l s u i t e d f o r
q u a n t i t a t i v e a n a l y s i s
o f VFAs
i n
d i l u t e
a q u e o u s
s o l u t i o n s .
R e p e a t e d
i n j e c t i o n s
o f
0 . 1
( v o l / v o l )
f o r m i c
a c i d
s t a b i l i z e d
t h e
c o l u m n ,
a n d
t h e
c h r o m a t o g r a m
s h o w n
i n
F i g . 1D
was
m a d e
a f t e r m o r e t h an 5 0 0
s a m p l e s
h a d b e e n
i n j e c t e d
i n t o
t h e
c o l u m n .
When i s o t h e r m a l
a n a l y s i s
was
u s e d ,
t h e
a n a l y s i s
t i m e w a s r a t h e r
l o n g ,
a n d
d e f o r m a t i o n o f t h e
p e a k s
l a t e i n t h e
c h r o m a t o g r a m
o c c u r r e d
( F i g .
1 C ) .
W i t h
t e m p e r a t u r e
p r o -
g r a m m i n g
t h e
a n a l y s i s
t i m e
was
l e s s
t h a n
1 1
m i n ,
a n d
t h e
c h r o m a t o g r a m ( F i g . 1 D ) i m p r o v e d c o n s i d e r a b l y ,
w i t h b e t t e r
s e p a r a t i o n
o f
t h e
e a r l y p e a k s .
A
d i s a d v a n t a g e
o f
C a r b o w a x
w a s
t h a t a f t e r
e t h e r e x t r a c t i o n
q u a n t i t a t i v e
a n a l y s i s
o f a c e t i c
a c i d
a n d
p r o p i o n i c
a c i d was
d i f f i c u l t ,
b e c a u s e
t h e
p e a k s
o f
t h e s e
a c i d s
o v e r l a p p e d
w i t h t h e e t h e r
p e a k .
T h e v a r i a t i o n
c o e f f i c i e n t s
o f t h e
p e a k
a r e a s a n d
t h e r e t e n t i o n
t i m e s w ere
s m a l l e r
t h a n t h o se
f o r b o t h o t h e r
s t a t i o n a r y
p h a s e s
( T a b l e 2 ) ,
a n d
t h e
minimum l e v e l o f
d e t e c t i o n f o r
C 2 t h r o u g h C 7
VFAs
w a s
0 . 0 2
m m o l / l i t e r .
P r e t r e a t m e n t
m e t h o d s .
T a b l e
3 s h o w s
t h e
v a r i a t i o n c o e f f i -
c i e n t s ,
r e l a t i v e
e r r o r s ,
a n d
r a t e s o f
r e c o v e r y
f o r
t h e f o u r
V O L .
2 3 ,
1 9 8 6
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5 2 6 VAN
DEN BOGAARD
ET AL.
A
A
c
c
C
P
iB
B
i V
V
i C
B
D
VI
i v
B
A
i c
H
I
~~~~~~~~~~~~IIII
1 5 1 0
m i n u t e s
minutes
F I G .
1 .
C h r o m a t o g r a m s o f a s t a n d a r d s o l u t i o n c o n t a i n i n g
1 0 m m o l o f
e a c h VFA
( C 2
t h r o u g h
C 6 )
pe r
l i t e r i n
w a t e r ,
a n a l y z e d
w i t h
Chromosorb 1 0 1
( A ) ,
SP 1 2 0 0
( B ) ,
Carbowax
( i s o t h e r m a l l y ) ( C ) ,
a n d Carbowax
( t e m p e r a t u r e
p r o g r a m m e d )
( D ) . A ,
A c e t i c
a c i d ;
P ,
p r o p i o n i c
a c i d ;
i B , i s o b u t y r i c
a c i d ; B , n - b u t y r i c a c i d ;
iV,
i s o v a l e r i c
a c i d ; V ,
n - v a l e r i c
a c i d ; i C ,
i s o c a p r o i c
a c i d ; C , n - c a p r o i c
a c i d ; H , n - h e p t a n o i c
a c i d .
d i f f e r e n t
p r e t r e a t m e n t m e t h o d s
w i t h
s a m p l e s
o f
t h e t h r e e
s t a n d a r d
s o l u t i o n s ,
a n d
F i g . 2
s h o w s
t h e
c h r o m a t o g r a m s
o b t a i n ed w i t h a
s t a n d a r d
s o l u t i o n
c o n t a i n i n g
1 0 m m o l o f
e a c h
VFA
( C 2 t h r o u g h
C 6
p e r
l i t e r i n
pus
a f t e r
p r e t r e a t m e n t .
( i ) Vacuum d i s t i l l a t i o n . Vacuum
d i s t i l l a t i o n
was
a l a b o r i -
o us
m e t h o d ;
a p p r o x i m a t e l y 3 0
m i n o f
t r e a t m e n t
per
s a m p l e
was
r e q u i r e d .
T h e r a t e s o f r e c o v e r y
w i t h t h i s m e t h o d w e r e
b e t w e e n a p p r o x i m a t e l y 8 0 a n d 1 1 0
i n
s p e n t c u l t u r e
m e d i a
a n d
pus.
T h i c k a n d s t i c k y pus
a n d
even t i s s u e c o u l d
b e
p r e t r e a t e d
b y
t h i s
m e t h o d .
( i i ) M o l e c u l a r
s i e v i n g .
T h e
Dowex
c o l u m n s were
p r e p a r e d
i n
a d v a n c e a n d s t o r e d a t 4 ° C u n t i l use. L a r g e
n u m b e r s
o f
s a m p l e s
c o u l d
b e
p r e t r e a t e d
i n
a
s h o r t
t i m e
b y
t h i s
m e t h o d .
T h e r a t e s
o f r e c o v e r y
were
b e t w e e n
a p p r o x i m a t e l y
9 0 a n d
1 1 0 .
The
v a r i a t i o n
c o e f f i c i e n t s f o r
t h i s
m e t h o d
were
o f
t h e
same o r d e r
o f
m a g n i t u d e
as t h e v a ri a ti o n
c o e f f i c i e n t s
f o r
t h e
u n p r e t r e a t e d
aqueous
s t a n d a r d w i t h t h e
Carbowax c o l u m n .
A
d r a w b a c k o f
t h i s
m e t h o d was t h a t
o n l y
l e s s
v i s c o u s
s a m p l e s
c o u l d
b e
a s s a y e d .
The r e l a t i v e erro rs were <10 .
( i i i ) H e a d s p a c e
a n a l y s i s .
B e c a u s e i s o a c i d s ar e more v o l a t i l e
t h a n
t h e
c o r r e s p o n d i n g s t r a i g h t - c h a i n a c i d s , t h e y
p r o d u c e d
much
l a r g e r
p e a k s
i n
t h e
c h r o m a t o g r a m s
( F i g .
2 D ) .
The l e v e l
o f r e c o v e r y
o f
t h e
l a r g e r
VFAs
was
c o n s i d e r a b l y
l o w e r
t h a n
t h e
l e v e l o f
r e c o v e r y
o f t h e o t h e r
a c i d s b e c a u s e o f
t h e i r
h i g h e r
b o i l i n g p o i n t s .
T h i s d e c r e a s e d l e v e l
o f r e c o v e r y o f
t h e
l a r g e r
VFAs
m i g h t
h a v e
b e e n d u e
t o c o n d e n s a t i o n
i n
t h e
T A B L E
3 .
C o m p a r i s o n
o f
f o u r
d i f f e r e n t p r e t r e a t m e n t
m e t h o d s . R a n g e o f
t h e v a r i a t i o n c o e f f i c i e n t s , l e v e l s
o f
r e c o v e r y ,
a n d r e l a t i v e e r r o r s
a f t e r
p r e t r e a t m e n t
a n d
GC
a n a l y s i s
w i t h
C a r b o w a x
o f a n
a q u e o u s
s t a n d a r d
s o l u t i o n ,
a
G L C - b r o t h
s t a n d a r d
s o l u t i o n ,
a n d
a
p u s s t a n d a r d
s o l u t i o n
c o n t a i n i n g
1 0
mmol
o f
e a c h
VFA
( C .
t h r o u g h
C 7 )
p e r
l i t e r
V a r i a t i o n
c o e f f i c i e n t s
( )
L e v e l s
o f
r e c o v e r y ( )
R e l a t i v e
e r r o r s
( )
P r e t r e a t m e n t
m e t h o d
A q u e o u s
P u s
b r o t C
A q u e o u s
P u s
GL C
A q u e o u s P u s
G r o L t C
s t a n d a r d s t a n d a r d s t a n d a r d s t a n d a r d
b r o t h
s t a n d a r d s t a n d a r d
s o l u t i o n s o l u t i o n
s t a n d a r d
s o l u t i o n s o l u t i o n
s t a n d a r d
s o l u t i o n s o l u t i o n
s t a n d a r d
s o l u t i o n
s o l u t i o n
s o l u t i o n
M o l e c u l a r
s i e v i n g 0 . 5 - 2 . 6 1 . 6 - 4 . 3 0 . 4 - 2 . 3
9 2 . 6 - 1 0 6 . 6
8 8 . 7 - 1 0 9 . 5
9 6 - 1 0 9 1 . 2 - 7 . 4 0 . 8 - 1 1 . 3 2 - 1 0 . 3
H e a d s p a c e a n a l y s i s 4 5 . 4 - 6 0 . 4 2 2 . 4 - 8 0 . 9 1 1 . 8 - 1 4 2
6 . 5 - 1 0 8 5 . 3 - 9 1 1 6 . 5 - 1 7 0 . 5 5 - 9 3 . 5
9 - 9 4 . 7
2 6 . 1 - 8 3 . 5
Vacuum
d i s t i l l a t i o n
1 . 5 - 4 . 9 2 . 2 - 1 8 . 2
1 . 7 - 1 1 . 4
9 0 . 6 - 9 5 . 8 8 0 . 6 -1 0 9 . 4 7 9 . 4 - 1 0 9 . 8 4 . 2 - 9 . 4
0 - 1 9 . 4 1 . 8 - 2 0 . 6
E t h e r
e x t r a c t i o n
3 . 5 - 5 . 5 2 . 1 - 6 . 2
3 . 1 - 8 . 7 1 0 0 . 8 - 1 1 3 . 5
1 1 4 - 1 2 4 1 0 8 - 1 4 7 0 . 8 - 1 3 . 5
1 4 - 2 4
8 - 4 7
E a c h s t a n d a r d s o l u t i o n w a s
p r e t r e a t e d
a n d
a n a l y z e d
t h r e e
t i m e s .
I
c
J . C L I N .
M I C R O B I O L .
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GC
A N A L YSIS O F
VFAs
5 2 7
A
A
L
V
C
jc
H
10
m i n u t e s
B
0 5
10
minutes
D
6
5
m i n u t e s
F I G . 2 . Chromatograms
o f a
s t a n d a r d s o l u t i o n
c o n t a i n i n g
1 0
mmol
o f
e a c h
VFA ( C 2 t h r o u g h
C6
pe r
l i t e r
i n
pus
a f t e r
p r e t r e a t m e n t
b y
vacuum
d i s t i l l a t i o n
( A ) , e t h er e xt r ac t i on
( B ) ,
m o l e c u la r
s e i v i n g
( C ) , a n d
h e a d s p a c e a n a l y s i s
( D ) .
GC
a n a l y s i s
was
p e r f o r m ed b y
u s i n g
Carbowax as
t h e s t a t i o n a r y
p h a s e
a n d
t e m p e r a t u r e
p r o g r a m m i n g .
F o r an
e x p l a n a t i o n
o f
a b b r e v i a t i o n s ,
se e t h e l e g e n d t o
F i g .
1 .
s y r i n g e
a n d
a b s o r p t i o n
t o
g l a s s o f
t h e
l a r g e r
VF A m o l e c u l e s .
F o r t h e s e reasons
h e a d s p a c e
a n a l y s i s
i s u n s u i t a b l e
when
q u a n t i t a t i v e
a n a l y s i s
i s r e q u i r e d .
The minimum
l e v e l s
o f
d e t e c t i o n o f
C 3 t h r o u g h C 6
VFAs
c o u l d
n o t b e
d e t e r m i n e d
f o r
t h i s m e t h o d .
( i v )
E t h e r e x t r a c t i o n .
W i t h C a r b o w a x
as t h e s t a t i o n a r y
p h a s e ,
a c e t i c a c i d
a n d
p r o p i o n i c
a c i d
c o u l d n o t
b e p r o p e r l y
m e a s u r e d
q u a n t i t a t i v e l y ,
as t h e p e a k s
o f
C 2 a n d
C 3 a c i d s
o n
t h e
c h r o m a t o g r a m s
were n o t s e p a r a t e d
f r o m
t h e e t h e r
p e a k s .
The
l e v e l s
o f
r e c o v e r y f o r
t h e o t h e r
VFAs
( C 4
t h r o u g h
C 6 )
were
b e t w e e n 1 0 0
a n d 1 4 7 .
Q u a l i t a t i v e
a n a l y s e s o f
p us
s a m p l e s
t a k e n
w i t h c h a r c o a l - c o a t e d
s w a b s
p r o v e d
t o b e
more
s e n s i t i v e
t h a n
a n a l y s e s o f
s a m p l e s
t a k e n
w i t h o t h er
s w a b s . T h i s
was d u e t o
t h e
f a c t
t h a t
t h e
c h a r c o a l - c o a t e d
s w a b s a b so r b e d
more s t a n d a r d pus
s o l u t i o n ( 0 . 3 4
±
0 . 0 5 g )
t h a n
n y l o n
s w a b s ( 0 . 1 2
±
0 . 0 5 g ) .
S e r u m - c o a t e d
s w a b s
a n d
p l a i n
c o t t o n
s w a b s
a b s o r b e d
a l m o s t n o t h i n g .
C a l i b r a t i o n
curves.
The
r e s u l t s o f
an
a n a l y s i s
o f
GLC
b r o t h
s t a n d a r d s c o n t a i n i n g
d i f f e r e n t
c o n c e n t r a t i o n s
o f
C 2
t h r o u g h C 6
VFAs ar e
shown i n T a b l e
4 . A
t y p i c a l e x a m p l e
o f
T A B L E 4 . L e v e l s
o f
r e c o v e r y
o f
C . t h r o u g h
C ,
VFAs
b y
GC
a n a l y s i s
f o l l o w i n g
m o l e c u l a r
s i e v i n g
o f a GL C
b r o t h
s t a n d a r d
s o l u t i o n
c o n t a i n i n g
d i f f e r e n t c o n c e n t r a t i o n s
o f VFAs
C o n c n
No o f
L e v e l s
o f
r e c o v e r y
( m m o l / l i t e r )
( m m o l /
l i t e r )
s a m p l e s
C 2
C )
i s o - C 4
n - C 4
i s o - C s
n - C s i s o - C 6 s
n - C 7
0 . 1 3
0 . 0 8
+
0 . 0 1 0 . 0 4
+
0 . 0 2 0 . 0 6
+
0 . 0 1
0 . 0 6
+
0 . 0 1
0 . 0 7
±
0 . 0 1
0 . 0 8
+
0 . 0 1 0 . 0 9
+
0 . 0 1
0 . 1 6
+
0 . 0 2
( 8 0 )
( 4 0 )
( 6 0 )
( 6 0 )
( 7 0 )
( 8 0 ) ( 9 0 )
( 1 6 0 )
1
3
0 . 8 7
+
0 . 2 1 0 . 7 4
±
0 . 1 4
0 . 7 4
+
0 . 0 9
0 . 8 0
+
0 . 0 8
1 . 0 7
+
0 . 0 8 1 . 0 4
+
0 . 1 0 1 . 1 2
+
0 . 0 6 1 . 2 0
±
0 . 0 8
( 8 7 )
( 7 4 ) ( 7 4 )
( 8 0 )
( 1 0 7 )
( 1 0 4 )
( 1 1 2 )
( 1 2 0 )
1 0
3
1 0 . 5 5
+
0 . 5 6
1 0 . 1 1
±
0 . 4 6
9 . 9 0
+
0 . 5 7 1 0 . 0 3
±
0 . 5 1 9 . 9 8
±
0 . 5 5
1 0 . 1 9
±
0 . 4 8 9 . 9 1
±
0 . 4 6 9 . 9 2
±
0 . 4 0
( 1 0 6 )
( 1 0 1 )
( 9 9 )
( 1 0 0 )
( 1 0 0 )
( 1 0 2 ) ( 9 9 )
( 9 9 )
2 0
3 2 0 . 3 6
+
0 . 6 6
2 1 . 7 9
±
0 . 3 2 2 1 . 8 2
±
0 . 2 2
2 2 . 5 7
±
0 . 5 6
2 2 . 3 3
±
0 . 2 3 2 3 . 3 6
±
0 . 2 3 1 9 . 6 0
±
0 . 3 5
2 0 . 4 8
±
0 . 1 7
( 1 0 2 )
( 1 0 9 )
( 1 0 9 )
( 1 1 3 )
( 1 1 2 )
( 1 1 7 )
( 9 8 )
( 1 0 2 )
aMean
±
s t a n d a r d d e v i a t i o n .
Th e n u m b e r s
i n
p a r e n t h e s e s
a r e
p e r c e n t a g e s .
c
V O L .
2 3 ,
1 9 8 6
v
i
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5 2 8
VAN D EN B O G A A R D
ET
A L .
- E
2
~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Y
1 , 0 1 4 8 X + 0 , 0 4 7 1
r
0 , 9 9 9 5
0
5
1 0 1 5
2 0
m M / L
s t a n d a r d
s o l u t i o n
c
F I G . 3 . S t a n d a r d
c u r v e o f c ap r o i c
a c i d .
Z
2 )
a c a l i b r a t i o n c u r v e
o f
o n e VFA
( c a p r o i c a c i d )
i s s h o w n
i n
F i g .
3 .
W i t h
t h e
e x c e p t i o n s
o f
a c e t i c a n d
p r o p i o n i c
a c i d s t h e
0
v a r i a t i o n c o e f f i c i e n t s
o f t h e
o t h e r
VFAs
w e r e
a p p r o x i m a t e l y
0
10
o r
l e s s a t
c o n c e n t r a t i o n s
b e t w e e n
1
a n d
2 0 m m o l / l i t e r .
I n o c u l u m s i z e
a n d
i n c u b a t i o n p e r i o d .
T h e r e s u l t s o f
a
C 2
t h r o u g h
C 6
VFA a n a l y s i s
a f t e r 2 4
h
o f
i n c u b a t i o n
a r e
s h o w n
i n T a b l e
5 . T h e i n o c u l u m
s i z e s e e m e d
n o t
t o
b e
v e r y
c r i t i c a l ,
c
a s
t h e s t a n d a r d d e v i a t i o n s
d i d
n o t
s i g n i f i c a n t l y
i n c r e a s e
i f t h e
r e s u l t s
o b t a i n e d
a f t e r
i n o c u l a t i o n
o f
o n e ,
t h r e e ,
o r
f i v e
c o l o n i e s
w e r e c a l c u l a t e d
t o g e t h e r
o r
s e p a r a t e l y .
H o w e v e r ,
a f t e r
i n o c u l a t i o n
o f t h e b r o t h c u l t u r e
w i t h a
s w a b o f
c o n t i n -
u o u s
g r o w t h ,
t h e
s t a n d a r d
d e v i a t i o n s
t e n d e d
t o
i n c r e a s e
i n
some
o r g a n i s m s
a n d f o r
s p e c i f i c
V F A s .
A f t e r
i n o c u l a t i o n
o f
o n e , t h r e e ,
o r
f i v e
c o l o n i e s
t h e v a r i a t i o n c o e f f i c i e n t s
w e r e
b e t w e e n 1 a n d 1 5 .
A f t e r 4 8 h
o f i n c u b a t i o n
t h e
s t a n d a r d
d e v i a t i o n s i n c r e a s e d .
We c h o s e q u a n t i t a t i v e
a n a l y s i s
o f
VFAs i n
a q u e o u s
s o l u -
°
t i o n s
b e c a u s e
t h i s m e t h o d
s i m p l i f i e s
a n d
s p e e d s
u p p r e t r e a t -
m e n t o f s a mp l es , b u t m o s t l y
b e c a u s e o r g a n i c
e x t r a c t i o n
m e t h o d s ,
s u c h
a s e t h e r
e x t r a c t i o n ,
d o
n o t
e x t r a c t a l l VFAs
e q u a l l y ,
w h i c h m a k e s q u a n t i t a t i v e
a n a l y s i s
m o r e
d i f f i c u l t .
Q u a n t i t a t i v e
a n a l y s e s
o f
VFAs i n
a q u e o u s
s o l u t i o n s w i t h
C h r o m o s o r b 1 0 1 a n d S P 1 2 0 0 p r o v e d t o
b e
d i f f i c u l t ,
m a i n l y
b e c a u s e
o f t a i l i n g .
T h i s
c a u s e d
i n c o m p l e t e
s e p a r a t i o n
o f
t h e
0
p e a k s
a n d
was
d u e
t o t h e
i n s t a b i l i t y
o f
t h e s e
c o l u m n s
i f
. °
a q u e o u s
s o l u t i o n s w e r e i n j e c t e d .
E
D e s p i t e r e s u l t s o b t a i n e d b y o t h e r
w o r k e r s ( 2 , 1 0 , 1 1 ) ,
0
s a t u r a t i n g
t h e
c a r r i e r
g a s
w i t h f o r m i c
a c i d d i d
n o t
i m p r o v e
t h e
p e r f o r m a n c e
o f
t h e
C h r o m o s o r b 1 0 1
c o l u m n
a t
a l l , b u t
p r o v e d
t o
b e d e l e t e r i o u s
t o o u r GC
e q u i p m e n t . T h e a n a l y s i s
t i m e w i t h t h i s
c o l u m n
w a s r e l a t i v e l y l o n g ( > 2 0 m i n ) .
W i t h
t h e
C a r b o w a x
c o l u m n
w e u s e d t e m p e r a t u r e p r o g r a m m i n g
t o
i m p r o v e
t h e
c h r o m a t o g r a m
a n d t o
s h o r t e n
t h e e l u t i o n
t i m e .
V FA
c o n c e n t r a t i o n s
a s l o w a s
0 . 0 2
m m o l / l i t e r
c o u l d t h e n
b e
m e a s u r e d i n
a q u e o u s s o l u t i o n s .
T h e q u a l i t y o f
t h e c o l u m n
i m p r o v e d
w h i l e
i t w a s
u s e d .
T h e s e p a r a t i o n
o f
t h e p e a k s w a s
c o m p l e t e ,
t h e o v e r a l l
v a r i a t i o n
c o e f f i c i e n t
w a s 2 ( r a n g e ,
1
t o
3 ) ,
a n d
t h e e l u t i o n
t i m e
w a s s h o r t
( l e s s t h a n 1 2 m i n ) .
T h e
l a t t e r
i s o f p r i m e i m p o r t a n c e when
many s a m p l e s
a r e t o b e
0 e
0-
00
.
E
U
&
8
_ e
0
c
_ e
0-
0 0 c
Q E 8
_ e
C e
_ :
c
2
0 - c
0 0
QE
~
U - e
U t c Q
c
C .
8
_ O
00
2
o
. 0
< :
0
ON
O N
( - I
re )
o o 00
0000
+ 1
+ 1
+ 1 + 1
' I C
N C
' l
' I C
C = C =
D 6
~. ON.
+ l
+ l
NC
0 0
'I
00
r 000
0000
+ 1 + 1 + 1 + 1
r
0 0
NC
N C
'I
t l + l
+ l
+ l
1 +
£ o No
0
NC
NC000
t l
t l N C t l + l
ON
) ci
r' f
N
N I C
0 = -
0000
+ 1 + 1
+ 1
+ 1
N r NC
I N - -
00
NNC
o
_ ;
+ 1 + 1
<,
N
NC
N
0 0
N I C
C I I
=
0 0 0
00
r ' I
1
r 5 n
S )
ooo
+ 1 + 1 + 1
+ 1 + 1
+ 1
N l C
C C
ON
O N l
0
C I A
N C
T
C
~ ~
0 0 0
0 0
N
N
0
N C
t
N , C
0 -
+ 1 + 1 + 1 + 1 + 1 + 1
+ 1
+ 1
N°C
- 4
N
- 4
N°
t r
)
CC
CC ~
~0 0
44
5' ) Fc-
. 0 .
. 0 . . 0 .0
a
a aE
a
z
. 2
.
_
t
5
* _ z S z
t . :
i
.6
J .
C L I N .
M I C R O B I O L .
s
. 0
.u
>
Q
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ci
3
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0
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C
;
m u
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t
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GC
A NA LY SI S O F
VFAs 5 2 9
a n a l y z e d . F o r q u a l i t a t i v e a n d q u a n t i t a t i v e a n a l y s i s o f
VFAs
i n
a q u e o u s s o l u t i o n s C a r b o w a x
a p p e a r e d
t o b e t h e b e s t
c o l u m n
m a t e r i a l
i n
o u r h an d s . H o w e v e r ,
i f
e t h e r e x t r a c t s
w e r e i n j e c t e d i n t o t h e C a r b o w a x c o l u m n , t h e p e a k s o f a c e t i c
a n d
p r o p i o n i c
a c i d s
c o u l d n o t
b e
r e a d i l y s e p a r a t e d f r o m t h e
s o l v e n t
p e a k
i n
t h e c h r o m a t o g r a m . H o w e v e r , l o w e r i n g t h e
i n i t i a l
c o l u m n
t e m p e r a t u r e
t o
6 0 ° C
a n d c h a n g i n g t h e t e m p e r -
a t u r e
p r o g r a m
c o u l d h a v e s o l v e d t h i s
p r o b l e m , b u t
a t
a c o s t
o f
a
c o n s i d e r a b l y
l o n g e r
e l u t i o n t i m e .
An e x t r a p e a k
w a s
f o u n d
j u s t
b e f o r e t h e
i s o v a l e r i c a c i d
p e a k .
T h i s
p e a k w a s
f o u n d i n
a l l
o f t h e
s t a n d a r d s
a n d
a f t e r
i n j e c t i o n
o f
p u r e
a n a l y t i c a l g r a d e i s o v a l e r i c a c i d
s o l u t i o n s .
M a s s s p e c -
t r o g r a p h i c a n a l y s i s
r e v e a l e d t h a t t h i s
p e a k w a s c a u s e d b y
2 - m e t h y l - b u t y r i c a c i d . T h i s p e a k w a s
a l s o
o f t e n
p r e s e n t i n
s p e n t
c u l t u r e m e d i a
a n d
i n
c l i n i c a l s a m p l e s .
D i r e c t GC
a n a l y s i s
o f t h e
s u p e r n a t a n t s
o f a c i d i f i e d
s p e n t c u l t u r e
m e d i a
h a s
b e e n
p r o p o s e d ( 1 4 ) . T h i s i s e a s y t o p e r f o r m b u t h a s t h e
c o n s i d e r a b l e
d i s a d v a n t a g e
t h a t
n o n v o l a t i l e c o m p o u n d s t e n d
t o
b l o c k t h e i n j e c t o r a n d t h e
c o l u m n .
P r e t r ea t m en t e x t en d s
t h e
l i f e - s p a n
o f
GC
c o l u m n s
a n d , b y r e m o v i n g i n t e r f e r i n g
s u b s t a n c e s ,
i m p r o v e s
t h e
q u a l i t y
o f
c h r o m a t o g r a m s .
T h i s i s
e s p e c i a l l y i m p o r t a n t f o r q u a n t i t a t i v e a n a l y s i s .
T h e
vacuum d i s t i l l a t i o n
p r e t r e a t m e n t g a v e g o o d r e s u l t s .
T h e
d i f f e r e n c e s
i n
t h e
l e v e l s
o f r e c o v e r y
a m o n g
t h e
VFAs
m i g h t
h a v e b e e n d u e
t o
a b so r p ti o n o f l a r g e r
V FA
m o l e c u l e s
t o t h e
g l a s s
w a l l
o f
t h e
c o n n e c t i n g t u b e . B e c a u s e v a c u u m
d i s t i l l a t i o n i s l a b o r i o u s a n d t i m e
c o n s u m i n g ,
i t d o e s n o t
s e e m
v e r y s u i t a b l e
a s
a
p r e t r e a t m e n t
m e t h o d f o r l a r g e n u m b e r s o f
s a m p l e s .
T h i c k ,
s t i c k y p u s
a n d e v e n t i s s u e c o u l d b e
p r e -
t r e a t e d
b y
t h i s
m e t h o d
W i t h
h e a d s p a c e
a n a l y s i s
o n l y
v a p o r i z e d
m a t e r i a l i s
i n t r o -
d u c e d i n t o
t h e
GC
a p p a r a t u s ,
b u t
v o l a t i l e
c o m p o u n d s
o t h e r
t h a n
VFAs
i n
t h e
s a m p l e
a r e
a l s o
v a p o r i z e d
a n d
may
c a u s e
i n t e r f e r i n g p e a k s
o n t h e
c h r o m a t o g r a m ;
t h i s m a k e s
i d e n t i f i -
c a t i o n o f
t h e
VFAs d i f f i c u l t a n d
s o m e t i m e s
e v e n
i m p o s s i b l e .
B e c a u s e t h e
p e a k
a r e a s
d o
n o t
p r o p o r t i o n a l l y r e p r e s e n t
t h e
t r u e
c o nc en t ra t i o ns o f
t h e VFAs
p r e s e n t
i n a
s a m p l e ,
q u a n -
t i t a t i v e
a n a l y s i s
i s
n o t p o s s i b l e .
E t h e r
e x t r a c t i o n i s t h e m o s t o f t e n u s e d m e t h o d o f e x t r a c -
t i o n f o r
VFAs.
N e v e r t h e l e s s ,
a p p r e c i a b l e
a m o u n t s o f a c e t i c
a n d
p r o p i o n i c
a c i d s r e m a i n
i n
t h e
a q u e o u s
p h a s e
a f t e r
e x t r a c t i o n , r e s u l t i n g
i n
r e l a t i v e l y
p o o r
l e v e l s o f
r e c o v e r y
o f
t h e s e
a c i d s .
T h e
r e c o v e r y
r a t e f o r t h e o t h e r VFAs
i s
b e t w e e n
1 0 0
a n d 1 4 5 .
F o r
q u a n t i t a t i v e a n a l y s i s
e t h e r e x t r a c t i o n i s
m o r e l a b o r i o u s t h a n m o l e c u l a r
s i e v i n g ,
b e c a u s e
s p e c i a l
p r e c a u t i o n s
m u s t
b e t a k e n
t o
p r e v e n t e v a p o r a t i o n
o f
t h e
e t h e r . An
a d v a n t a g e
o f
t h i s
m e t h o d
i s t h a t
m o r e
v i s c o u s
s a m p l e s
c a n
b e
r e a d i l y p r e t r e a t e d ,
a n d
e v e n s w a b s
m o i s t -
e n e d w i t h
p u s
c a n b e
u s e d . T h i s i s
i m p o r t a n t
a s o f t e n
j u s t
a
s w a b
d i p p e d
i n t o
p u s
i s d e l i v e r e d
a s a
s p e c i m e n
t o
t h e
l a b o r a t o r y , d e s p i t e
t h e
f a c t
t h a t
s e n d i n g
s w a b
s p e c i m e n s
f o r
i s o l a t i o n
o f
o b l i g a t e l y
a n a e r o b i c
b a c t e r i a s h o u l d b e
s t r o n g l y
d i s c o u r a g e d .
C h a r c o a l - c o a t e d
s w a b s
c a n a d s o r b
e n o u g h
VFAs f r o m
b r o t h c u l t u r e s
a n d
p u s
f o r
q u a l i t a t i v e a n a l y s i s
o f
t h e
VFAs
b y G C .
As
c o t t o n
a n d
wood
may
c o n t a i n
V F A s ,
w o r k e r s
s h o u l d
t e s t e a c h
b a t c h
o f
s w a b s b e f o r e u s e .
S w a b s
f o r
GC
a n a l y s i s
s h o u l d a l s o b e
t r a n s p o r t e d
i n t h e
d r y
s t a t e .
T r a n s p o r t
medium
d o e s
n o t i n t e r f e r e
w i t h
GC
a n a l y s i s ,
b u t
e x t e n s i v e
d i l u t i o n
i n t o t h e
m e d i u m
o f
t h e VFAs
d r a s t i c a l l y
r e d u c e s
t h e r a t e
o f d e t e c t i o n o f
V F A s ,
w h i c h
m i g h t
o n l y
p a r t l y
b e
c o m p e n s a t e d
f o r
b y
e v a p o r a t i n g
t h e e t h e r
t o
c o n c e n t r a t e t h e
VFAs
p r e s e n t . H i g h - m o l e c u l a r - w e i g h t
ma-
t e r i a l
( e . g . ,
e x t r a c e l l u l a r
l i p i d s )
p r e s e n t
i n e t h e r
e x t r a c t s
may
c a u s e
t a i l i n g , may a p p e a r
o n
t h e
c h r o m a t o g r a m
i n a
l a t e r
a n a l y s i s ,
o r
may
e v e n
b l o c k
t h e GC
c o l u m n s .
A
s i m p l e ,
r a p i d ,
a n d
r e l i a b l e m e t h o d
i s
p r e t r e a t m e n t
o f
s a m p l e s w i t h c o l u m n s p a c k e d
w i t h c a t i o n - e xc h an g e
r e s i n .
T h i s n o t o n l y r e m o v e s l a r g e
m o l e c u l e s a n d
d e b r i s
f r o m
t h e
s a m p l e b y
( m o l e c u l a r )
s i e v i n g , b u t a l s o r e m o v e s o t h e r m o l -
e c u l e s a n d
i o n s b y c a t i o n e x c h a n g e .
The
l e v e l
o f
r e c o v e r y
o f
t h e
VFAs
f r o m
p u s
a n d
t h e
GL C
b r o t h s t a n da rd
w a s
b e t w e e n 9 0
a n d
1 1 0 , w i t h a v a r i a t i o n
c o e f f i c i e n t o f o n l y
3 ±
1
w i t h
t h e
GL C b r o t h
s t a n d a r d . T h i s
i s
o n t h e s a m e
o r d e r
o f m a g n i t u d e a s
t h e
v a r i a t i o n c o e f f i c i e n t
o f
t h e C a r b o w a x
c o l u m n
u s e d
f o r
a n a l y s i s .
A
d i s a d v a n t a g e
o f
t h i s p r e t r e a t -
m e n t
m e t h o d
i s
t h a t v i s c o u s
c l i n i c a l
s p ec i m e n s h a v e t o b e
d i l u t e d .
C a r e s h o u l d b e t a k e n
t o
p r e v e n t
e m u l s i f i c a t i o n
d u r i n g d i l u t i o n .
T h e c a l i b r a t i o n
c u r v e s
s h o w
t h a t
a t
c o n c e n -
t r a t i o n s b e t w e e n 1 a n d
2 0 m m o l / l i t e r q u a n t i t a t i v e a n a l y s i s o f
C 2 t h r o u g h C 6
VFAs i s
f a i r l y
a c c u r a t e
a n d
make
i t
f e a s i b l e
t o
p r e s e n t
q u a n t i t a t i v e d a t a
o n VFA
p r o d u c t i o n
b y o b l i g a t e l y
a n a e r o b i c b a c t e r i a i n s t e a d
o f
c h r o m a t o g r a m s .
T h i s
i s i m p o r -
t a n t
f o r
i d e n t i f i c a t i o n
a n d t a x o n o m i c
p u r p o s e s .
D e s p i t e
t h e
f a c t
t h a t
t h e i n o c u l u m
s i z e d i d n o t
s e e m t o
b e
v e r y
c r i t i c a l i f VFA p r o d u c t i o n w a s
m e a s u r e d
a f t e r
2 4 h o f
i n c u b a t i o n , s t a n d a r d i z a t i o n o f c u l t u r e m e d i a ,
c u l t u r e c o n d i -
t i o n s ,
a n d i n o c u l a i s
n e c e s s a r y
t o m a k e s u r e
t h a t d i f f e r e n t
l a b o r a t o r i e s
o b t a i n
t h e s a m e r e s u l t s w i t h t h e
s a m e
s t r a i n
o r
s p e c i e s .
C a r b o w a x
i s
t h e
p a c k e d
c o l u m n
m a t e r i a l
o f c h o i ce
f o r
q u a l i t a t i v e
a n d q u a n t i t a t i v e a n a l y s i s o f
VFAs
i n b i o l o g i c a l
a q u e o u s s o l u t i o n s .
I n
o u r
h a n d s , m o l e c u l a r s i e v i n g
w i t h
D o w e x
c o l u m n s
i s
t h e b e s t
p r e t r e a t m e n t
m e t h o d when
q u a n t i t a t i v e a n a l y s i s
i s
r e q u i r e d ( e . g . ,
f o r
i d e n t i f i c a t i o n a n d
t a x o n o m i c p u r p o s e s ) . T h e c o m b i n a t i o n o f
a
C a r b o w a x c o l -
um n a n d
p r e t r e a t m e n t
o f
s p e n t c u l t u r e
m e d i a
b y m o l e c u l a r
s i e v i n g
w i t h
a
D o w e x c o l u m n m a k e s
i t
f e a s i b l e
t o p r e s e n t
q u a n t i t a t i v e
d a t a
o n
VFA
p r o d u c t i o n
o f
o b l i g a t e l y a n a e r o b i c
b a c t e r i a l
s p e c i e s
i n s t e a d o f
c h r o m a t o g r a m s .
F o r
q u a l i t a t i v e
a n a l y s i s
( e . g . , p r e s u m p t i v e d i a g n o s i s
o f
a n a e r o b i c b a c t e r i a l
i n f e c t i o n s )
e t h e r e x t r a c t i o n
a n d
h e a d s p a c e
GC a r e
g o o d
a l t e r n a t i v e s
f o r
p r e t r e a t m e n t
o f
s a m p l e s .
ACKNOWLEDGMENT
We
a r e
g r a t e f u l
t o
H .
W.
T h i j s s e n
f o r
v a l u a b l e
a d v i c e a n d
h e l p f u l
d i s c u s s i o n s
d u r i n g p r e p a r a t i o n
o f
t h e
m a n u s c r i p t .
LI TE RA T U RE
CITED
1 .
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1 9 7 3 .
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f o r
i d e n t i f i c a t i o n
o f b a c t e r i a l m e t a b o l i c
p r o d u c t s . A p p l .
M i c r o b i o l .
2 5 : 2 8 7 - 2 8 9 .
2 .
C o c h r a n e ,
G . C .
1 9 7 5 . A
r e v i e w
o f
t h e
a n a l y s i s
o f
f r e e
f a t t y
a c i d s
( C ' - C 6 ) .
J .
C h r o m a t o g r .
S c i . 1 3 : 4 4 0 - 4 4 6 .
3 .
G e h r k e ,
C .
W . ,
a n d W. M. L a m k i n . 1 9 6 1 .
Q u a n t i t a t i v e
d e t e r -
m i n a t i o n
o f s t e a m - v o l a t i l e
f a t t y
a c i d s
b y
g a s - l i q u i d c h r o m a t o g -
r a p h y . A g r i c .
F o o d
C h e m .
9 : 8 5 - 8 8 .
4 .
G o r b a c h ,
S .
L . ,
J .
W.
M a y h e w ,
a n d
J .
G .
B a r t l e t t . 1 9 7 6 .
R a p i d
d i a g n o s i s
o f
a n a e r o b i c
i n f e c t i o n s
b y
d i r e c t
g a s - l i q u i d
c h r o m a -
t o g r a p h y
o f
c l i n i c a l
s p e c i m e n s .
C l i n .
I n v e s t .
5 7 : 4 7 8 - 4 8 4 .
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M.
G . ,
D . A .
E s c h e n b a c h ,
C .
A .
S p e i g e l - B r o w n ,
a n d
K . K .
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1 9 8 2 .
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o f
a m n i o t i c - f l u i d i n f e c t i o n
b y
g a s - l i q u i d
c h r o m a t o g r a p h y .
N .
E n g l .
J .
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G .
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H .
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G .
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a n d
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i n f e c t i o n s
b y g a s -
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J .
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7 .
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P .
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G .
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o f
a l c o h o l s
a n d v o l a t i l e
f a t t y
a c i d s
b y h e a d s p a c e g a s c h r o m a t o g -
r a p h y
i n i d e n t i f i c a t i o n
o f a n a e r o b i c
b a c t e r i a .
J .
C l i n . M i c r o b i o l .
7 : 2 3 - 2 7 .
8 .
M a y h e w ,
J .
W . ,
a n d
S .
L .
G o r b a c h .
1 9 7 9 .
I n t e r n a l s t a n d a r d s
f o r
g a s c h r o m a t o g r a p h i c
a n a l y s i s
o f
m e t a b o l i c
e n d
p r o d u c t s
f r o m
a n a e r o b i c
b a c t e r i a .
A p p l .
E n v i r o n .
M i c r o b i o l .
3 3 : 1 0 0 2 - 1 0 0 3 .
9 .
N o r d ,
C .
E . 1 9 7 7 .
D i a g n o s i s
o f a na er o b i c
i n f e c t i o n s
b y g a s -
l i q u i d c h r o m a t o g r a p h y .
A c t a
P a t h o l .
M i c r o b i o l .
S c a n d . S e c t .
B
VOL.
2 3 ,
1 9 8 6
7/24/2019 Quantitative Gas Cromatography Analisys of Volatiles Fatty Acids in Spent Culture Media
http://slidepdf.com/reader/full/quantitative-gas-cromatography-analisys-of-volatiles-fatty-acids-in-spent-culture 8/8
5 3 0 VAN D EN B O G A A R D
ET A L .
2 5 9 : 5 5 - 5 9 ,
1 0 . O t t e n s t e i n , D . M . ,
a n d D .
A .
B a r t l e y .
1 9 7 1 . S e p a ra t io n o f f r e e
a c i d s C 2 - C 5
i n d i l u t e a q u e o u s s o l u t i o n
c o l u m n t e c h n o l o g y . J .
C h r o m a t o g r .
S c i . 9 : 6 7 4 - 6 8 1 .
1 1 .
P a c k e t t , L . V . , a n d R . W. M a c C u n e .
1 9 6 5 .
D e t e r m i n a t i o n
o f
s t e a m - v o l a t i l e
o r g a n i c
a c i d s
i n
f e r m e n t a t i o n
m e d i a
b y
g a s - l i q u i d
c h r o m a t o g r a p h y . A p p l . M i c r o b i o l .
1 3 : 2 2 - 2 7 .
1 2 . P h i l l i p s ,
K .
D . ,
P . V . T e a r l e ,
a n d A . T . W i l l i s .
1 9 7 6 .
R a p i d
d i a g n o s i s o f a n a e r o b i c
i n f e c t i o n s b y
g a s - l i q u i d
c h r o m a t o g r a p h y
o f
c l i n i c a l
m a t e r i a l .
J .
C l i n .
P a t h o l .
2 9 : 4 2 8 - 4 3 2 .
1 3 . R e e d , P . J . , a n d
P .
J . S a n d e r s o n .
1 9 7 9 . D e t e c t i o n o f
a n a e r o b i c
w o u n d
i n f e c t i o n b y a n a l y s i s o f p u s
s w a b s f o r
v o l a t i l e
f a t t y a c i d s
b y g a s - l i q u i d
c h r o m a t o g r a p h y . J .
C l i n .
P a t h o l .
3 2 : 1 2 0 3 - 1 2 0 5 .
1 4 . R o g o s a ,
M . , a n d
L .
L . L o v e .
1 9 6 8 . D i r e c t q u a n t i t a t i v e g a s
c h r o m a t o g r a p h i c
s e p a r a t i o n o f
C 2 - C 6 f a t t y a c i d s ,
m e t h a n o l ,
a n d
e t h y l a l c o h o l
i n
a q u e o u s
m i c r o b i a l
f e r m e n t a t i o n
m e d i a . A p p l .
M i c r o b i o l .
1 6 : 2 8 5 - 2 9 0 .
1 5 .
S a s a k i ,
N . ,
a n d I . T a k a z o e .
1 9 7 4 . S u b g r o u p i n g
o f B ac t er o i de s
m e l a n i n o g e n i c u s
f r o m t h e p a t t e r n o f v o l a t i l e f a t t y
a c i d
p r o d u c -
t i o n . B u l l . T o k y o D e n t .
C o l l . 1 5 : 1 2 5 - 1 3 2 .
1 6 .
T h a d e p a l l i ,
H . , a n d
P . K . G an g o p a d h y a y .
1 9 8 0 . R a p i d
d i a g n o s i s
o f a na er o b i c empyema
b y d i r e c t
g a s - l i q u i d
c h r o m a t o g r a p h y
o f
J . C L I N .
M I C R O B I O L .
p l e u r a l f l u i d . C h e s t
7 7 : 5 0 7 - 5 1 3 .
1 7 .
v a n d e n B o g a a r d ,
A . E .
J .
M . ,
a n d M. J .
H a z e n . 1 9 8 3 . T h e
d e t e c t i o n o f
o b l i g a t e
a n a e r o b i c b a c t e r i a
i n s w i n e a b s c e s s e s . A
c o m p a r i s o n b e t w e e n
g a s - l i q u i d c h r o m a t o g r a p h y a n d b a c t e r i o -
l o g i c a l
c u l t u r i n g m e t h o d s .
V e t .
M i c r o b i o l .
8 : 3 8 9 - 3 9 6 .
1 8 .
W a t t ,
B . ,
P .
A .
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G r e e n a n ,
S .
K .
N a p i e r ,
a n d
A .
M i t c h e l l . 1 9 8 2 . G a s - l i q u i d
c h r o m a t o g r a p h y i n t h e d i a g n o s i s
o f
a n a e r o b i c
i n f e c t i o n s :
a t h r e e
y e a r e xp e r i en ce .
J . C l i n .
P a t h o l .
3 5 : 7 0 9 - 7 1 4 .
1 9 .
W e n s i n c k ,
F . ,
a n d
J .
G . H . v a n
R u s e l e r - E m b d e n .
1 9 7 1 .
T h e
i n t e s t i n a l
f l o r a o f c o l o n i s a t i o n
r e s i s t a n t m i c e .
J .
H y g .
6 9 : 4 1 3 - 4 2 1 .
2 0 . W u s t , J .
1 9 7 7 . P r e s u m p t i v e
d i a g n o s i s o f a n a e r o b i c
b a c t e r e m i a
b y
g a s - l i q u i d
c h r o m a t o g r a p h y o f
b l o o d
c u l t u r e s .
J .
C l i n . M i c r o -
b i o l . 6 : 5 8 6 - 5 9 0 .
2 1 .
W u s t , J .
1 9 8 0 .
S c h e l l d i a g n o s e
a n a e r o b e r I n f e c t i o n e n
m i t d e r
d i r e c t e n
g a s
c h r o m a t o g r a p h i s c h e n
U n t e r s u c h u n g v o n
K l i n i s c h e
M a t e r i a l .
S c h w e i z .
M e d .
W o c h e n s c h r . 1 1 0 : 3 6 2 - 3 6 8 .
2 2 .
Z i j l s t r a ,
J . B . ,
J .
B e u k e m a ,
B . G .
W o l t h e r s ,
B . M. B y r n e ,
A .
G r o e n ,
a n d
T . D a n k e r t .
1 9 7 7 . P r e t r e a t m e n t
m e t h o d s
p r i o r
t o
g a s
c h r o m a t o g r a p h i c a n a l y s i s
o f v o l a t i l e f a t t y
a c i d s
f r o m f a e c a l
s a m p l e s .
C l i n . C h i m .
A c t a
7 8 : 2 4 3 - 2 5 0 .
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