Ionisation in the absence of
high voltage using SFC-MS:
a route to greater sensitivity
Mohini Thite
Email: mat@
soto
n.ac.uk
BMSS M
eeting -11th Sept 2007
2
Outline
•W
hat is SF
C?
•Couplin
g SF
C-M
S
•Exper
imen
tal Des
ign
•Det
ection
•ESI-A
PCI
•SF
C conditions
•Pre
ssure
•Modifier
•Flow rate
BMSS M
eeting -11th Sept 2007
3
What is SF
C?
•Su
per
critical fluid chro
matogr
aphy
•Mobile
phas
e in super
critical state
•Mobile
phas
e: non polar ca
rbon dioxide
•Modifier
: polar org
anic solven
t to
impro
ve
chro
matogr
aphy
•Polar stationar
y phas
es
•e.
g. silica
, am
ine, 2-E
P
•Consider
ed as norm
al phas
e te
chnique
BMSS M
eeting -11th Sept 2007
4
SFC-M
S Couplin
g
Ref
: M. G
arzo
tti,
M. H
amda
n, J
. Chr
omat
ogr
B,7
70
(2
00
2)
53
-61
.
Pump port
Knauer
Detector
Fluid control
module
Connec
ted to
hea
t ex
chan
ger
CO
2MeO
H
Chec
k va
lve
Autosampler
10uL loop
10mm cell
Column port
TCM-
Heater
control
module
Peak
detection
module
Mixer
Make-up
from
HPLC
Pump
Mass
Spectrometer
Valco
Tee
Valco
Tee
PEEK tubing
BMSS M
eeting -11th Sept 2007
5
MS Det
ection
•Initial studies utilis
ed ESI
•O
ptimisation o
f API fo
r SF
C
•O
bse
rvation o
f sa
mples ionising in the ab
sence
of
high voltag
es
•Sp
ecific te
st compounds an
alys
ed to pro
be th
is
ionisation m
echan
ism
BMSS M
eeting -11th Sept 2007
6
What is ca
using ionisation?
•API so
urc
e co
nfig
ura
tion dep
enden
t?
•ESI c
f.APCI
•Chro
matogr
aphic sys
tem spec
ific?
•HPLC c
f.SF
C
BMSS M
eeting -11th Sept 2007
7
Is ionisation related
to?
•Char
ge res
idual m
odel?
•Addition o
f ac
id
•Ther
mosp
ray?
•Addition o
f am
monium ace
tate
•So
nic spra
y ionisation?
•Pre
ssure
chan
ge
•% m
odifier
chan
ged
•Neb
ulis
er gas
Ref
: C. Das
s, Principles an
d Pra
ctice of Biologica
l Mas
s Sp
ectromet
ry, Jo
hn W
iley & Sons, Inc., New
York
, 2001.
BMSS M
eeting -11th Sept 2007
8
Exper
imen
tal co
nditions
OH
O O
N
STD2. Oxybutynin
O
N
STD1. Diphenhydramine
O
OOO
O
NN H
O
O
OO
STD4. Reserpine
HO
N
HO
STD3. Terfenadine
H
H
H
H
H
H
43
21
HV: 0kV
CV: 0V
HV: 0kV
CV: 20V
HV: 3.5kV
CV: 0V
HV: 3.5kV
CV: 20V
No make-up
HV: 3.5kV
CV: 0V
HV: 3.5kV
CV: 0V
HV: 3.5kV
CV: 0V
HV: 0kV
CV: 20V
HV: 0kV
CV: 20V
HV: 0kV
CV: 20V
HV: 0kV
CV: 0V
HV: 3.5kV
CV: 20V
Methanol + 0.1%
HCOOH
HV: 0kV
CV: 0V
HV: 3.5kV
CV: 20V
Methanol + 0.1%
NH4OAc
HV: 0kV
CV: 0V
HV: 3.5kV
CV: 20V
Pure methanol
BMSS M
eeting -11th Sept 2007
9
ESI res
erpine HV o
n/o
ffReserpine (ESI)
Make-up solvent
MeOH
+NH4OAc
+HCOOH
None
Peak area
01e+4
2e+4
3e+4
4e+4
5e+4
6e+4
HV = 3.5kV
HV = 0kV
4No m
ake-up
solvent(M
+H)+
HV =
3.5kV
(M+H)+
HV =
0kV
BMSS M
eeting -11th Sept 2007
10
ESI and A
PCI
Reserpine (ESI)
Make-up solvent
MeOH
+NH4OAc
+HCOOH
None
Peak area 01e+4
2e+4
3e+4
4e+4
5e+4
6e+4
HV = 3.5kV
HV = 0kV
Resepine (APCI)
Make-up solvent
MeOH
+NH4OAc
+HCOOH
None
Peak area
02e+2
4e+2
6e+2
8e+2
1e+3
1e+3
HV= 3.5kV
HV= 0kV
NH4OAc
NH4OAc
No m
ake-up
solvent
No m
ake-up
solvent
BMSS M
eeting -11th Sept 2007
11
Summar
y of ESI and A
PCI
•Compar
able/Impro
ved ionisation in abse
nce
of high
voltag
e
•Io
nisation suppre
ssed
with addition o
f am
monium
acet
ate
•Fo
rmic acid does
not en
han
ce ionisation
•Data fo
r APCI so
urc
e co
mpar
able w
ith ESI sourc
e
•Io
nisation indep
enden
t of ca
pillar
y type
BMSS M
eeting -11th Sept 2007
12
Pre
ssure
and m
odifier
(b) Pressure : 100 Bar
% M
odifier
10% M
eOH20% M
eOH50% M
eOH
Peak Area 0.0
2.0e+3
4.0e+3
6.0e+3
8.0e+3
1.0e+4
1.2e+4
1.4e+4
HV : On
HV : Off
(a) Pressure : 75 Bar
% Modifier
10% M
eOH20% M
eOH50% M
eOH
Peak Area 0.0
2.0e+3
4.0e+3
6.0e+3
8.0e+3
1.0e+4
1.2e+4
1.4e+4
(c) Pressure: 150 Bar
% Modifier
10% MeOH20% M
eOH50% M
eOH
Peak Area 0.0
2.0e+3
4.0e+3
6.0e+3
8.0e+3
1.0e+4
1.2e+4
1.4e+4
HV : On
HV : Off
HV : On
HV : Off
•Mixtu
re o
f te
rfen
adine, o
xyb
utynin and ery
thro
myc
in
analys
ed at va
rying pre
ssure
and m
odifier
per
centage
•O
ptimum conditions fo
r max
imum sen
sitivity in
pre
sence
or ab
sence
of high voltag
e:Pre
ssure
100bar
and m
odifier
30% M
eOH
BMSS M
eeting -11th Sept 2007
13
Oxybutynin
Flow rate in m
L/m
in
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Peak Area
02e+5
4e+5
6e+5
8e+5
HV = 3.5kV
HV = 0kV
Nicotinanilide
Flow rate in m
L/m
in
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Peak Area
01e+5
2e+5
3e+5
4e+5
HV = 3.5kV
HV = 0kV
Mebendazole
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
01e+5
2e+5
3e+5
4e+5
5e+5
HV = 3.5kV
HV = 0kV
Peak Area
Flow rate in m
L/m
in
Reserpine
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
02e+5
4e+5
6e+5
8e+5
HV = 3.5kV
HV = 0kV
Peak Area
Flow rate in m
L/m
in
Oxybutynin
Flow rate in m
L/m
in
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Peak Area
02e+5
4e+5
6e+5
8e+5
HV = 3.5kV
HV = 0kV
Flow rate
BMSS M
eeting -11th Sept 2007
14
High flow rate (5
mL/m
in)
•Im
pro
vemen
t in sen
sitivity upon incr
easing th
e distance
bet
wee
n the ESI nee
dle tip and the sa
mple cone
Mebendazole
Distance of needle and sampling cone
default
plus 1 mm
plus 2 mm
Peak Area
02e+4
4e+4
6e+4
8e+4
1e+5
1e+5
1e+5
HV = 3.5kV
HV = 0kV
BMSS M
eeting -11th Sept 2007
15
Modifier
effec
tComparison of acetonitrile and m
ethanol modifiers
Oxybutynin
Nicotinanilide
Mebendazole
Reserpine
Peak Area 01e+5
2e+5
3e+5
4e+5
HV: On (MeCN)
HV: Off (MeCN)
HV: On (MeOH)
HV: Off (MeOH)
BMSS M
eeting -11th Sept 2007
16
Modifier
effec
t
•Incr
ease
d ionisation w
ith M
eOH m
odifier
compar
ed
to M
eCN
•Is the ionisation efficiency
related
to pre
form
ed ions
in solution?
Comparison of acetonitrile and m
ethanol modifiers
Oxybutynin
Nicotinanilide
Mebendazole
Reserpine
Peak Area 01e+5
2e+5
3e+5
4e+5
HV: On (MeCN)
HV: Off (MeCN)
HV: On (MeOH)
HV: Off (MeOH)
BMSS M
eeting -11th Sept 2007
17
High voltag
e pro
file
•Not elec
trosp
ray
•CRM -
Ions fo
rmed
in solution o
r during sp
ray pro
cess
•CRM plus oth
er pro
cesses
, so
nic spra
y
oxybutynin
HV (kV)
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Peak area
02e+5
4e+5
6e+5
8e+5
1e+6
BMSS M
eeting -11th Sept 2007
18
Conclusions
•Compounds ionise in the ab
sence
of high voltag
e∴
not elec
trosp
ray ionisation
•Addition o
f am
monium ace
tate
does
not aid
ionisation ∴
not th
ermosp
ray ionisation
•Su
gges
ts ions ar
e fo
rmed
in solution o
r during th
e su
per
sonic jet
expan
sion
BMSS M
eeting -11th Sept 2007
19
Conclusions
•MeC
Nas
mak
e-up solven
t or as
modifier
does
not
assist in ionisation, unlik
e MeO
H ∴
supporting CRM?
•Io
nisation o
n FIA
-ESI-M
S with H
V turn
ed o
ff but not as
go
od as SF
C-M
S
•API-MS re
sponse
dep
enden
t on m
odifier
conce
ntration;
this should be take
n into
acc
ount fo
r gr
adient elution
studies
BMSS M
eeting -11th Sept 2007
20
HV: Off
HV: On
Conclusions
For sm
all molecu
le analys
isif se
nsitivity is an
issue :
Turn
ing off the elec
trosp
ray high voltag
e co
uld
impro
ve LO
D-
e.g.
oxyb
utynin, re
serp
ine data
21
Ack
now
ledg
emen
ts
Gla
xoSm
ithK
line,
CA
SS, N
ew
Fron
tiers
Nor
th, H
arlo
w,
CM
19
5A
W,
UK
Pfiz
er G
loba
l Res
earc
h an
d D
evel
opm
ent,
Sand
wic
h,
CT
13
9N
J, U
K
Laure Hitzel
Frank Pullen
Met
tler-
Tol
edo
Aut
oche
m, 1
30
E
xecu
tive
Drive
, Sui
te 2
A, N
ewar
k,
DE
19
70
2, U
SA
Clare Paterson
Bob Boughtflower
Paul Oakley
Prin
ceto
n C
hrom
atog
raphy
Inc
., C
ranb
ury,
NJ
08
51
2, U
SA
Jeff Caldwell
G. John Langley
Julie Herniman
Amaury Cazenave Gassiot
Scho
ol o
f C
hem
istr
y,U
nive
rsity
of
Sout
ham
pto
n,SO
17
1B
J, U
K
Gla
xoSm
ithK
line,
Gun
nels
Woo
d R
oad,
Ste
vena
ge, S
G1
2N
Y, U
K
Steve Lane
Evo
tec,
15
1 M
ilton
Pa
rk, A
bing
don,
O
X1
4 4
SD, U
K
Stefan Richardson
22
Tha
nk Y
ou