10
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Microcomputer-Assisted Retention Prediction in Reversed-Phase Liquid Chromatography Kiyokatsu Jinno and Kazuya Kawasaki School of Materials Science, Toyohashi University of Technology, Toyohashi 440, Japan
A computer-assisted system for predicting retention of aromatic compounds has been investigated in reversed-phase liquid chromatography. The basic retention descriptions have been derived from the studies on quantitative structure-retention relationships. The system was constructed on a 16-bit microcomputer and then evaluated by comparing the retention data between measured and predicted values. The excellent agreement between both values were observed on an octadecyls i l i c a stationary phase with acetonitrile and methanol aqueous mobile phase systems. This system has been modified to give us the information for optimal separation conditions in reversed-phase separation mode. The approach could also work well for any other reversed-phase stationary phases such as octyl, phenyl and ethyl silicas. Although
liquid
chromatography
has
been
widely
used
the
last
decade.
chromatography into
their
tively. sive the
as
With
(LC),
phenomena utility
efficiency
of
the
of
history
LC h a s
been
of
for
instrument
the
selection
is
ration for find
a major
condition,
their the
phase. the
s t i l l
best
analysts
respective that,
approach
is
difficult
involves
systematic
one
In
as
order the
with
the
impres-
modern
detection well
as
to
set
data
the
are
and
sepa-
about
and
i t
then
stationary
performed
purposes. An i m p r o v e d
computer
condi-
best
information articles,
high
systems,
separation
phase
their
and t i m e - c o n s u m i n g .
optimization
qualitamost
of
mobile for
and the
development
experiments
condition
liquid separated
Furthermore,
on-line
survey of
of
it during
chemistry.
i n many p u b l i s h e d
and e r r o r
separation
is
satisfactory
LC.
combination
trial
optimal very
in
readily
ago,
only
performance
operations, of
generally
purposes
most p r o m i s i n g After
problem
the
and s p e c i f i c
and m i c r o p r o c e s s o r s tions
by
years
quantitatively
analytical
handling.
However,
high
technique
catalyzed
sensitive
of
80
techniques
c a n now b e
analyzed
this
about
analytical
mixtures
i n the
columns,
discovered
development
and then
ascent
was
efficient
complex
components
The r a p i d
an
to
find
This approach
techniques
0097-6156/ 86/ 0297-0167$06.25/ 0 © 1986 American Chemical Society
Ahuja; Chromatography and Separation Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1986.
such
as
C H R O M A T O G R A P H Y A N D SEPARATION CHEMISTRY
168 "Window useful
Diagram" It
necessitates
In
i t
is
practice,
can create
alternate
attempt
In
tion
experimental
attained
directed
At present,
theories LC.
to
describe
solute
According to this anticipated
surface and
area,
that
sed-phase
LC.
solubility parameters
been
determined
based
deal
of
effort
promising
have
method
measure
of
made,
is
to predict
based
on t h e p r e m i s e parameters
solute
as s t r u c t u r e ,
its
retention, in
such
i f
k
is
1
which t
and been
derive
is
a l .
(29).
factor
this
benzenes,
as
coef-
good
parameter
(22-28).
in
This
quanidea
i n reversed-phase exist
electronic
LC c a n
between
the
properties states
The b a s i c
of
e t c . , and concept
is
i t
defined
and t
Q
is
k
i n various
the equation
1
= ( t
r
- t
0
) / t
0
,
t h e column v o i d - t i m e Such approach has
relationships
o n t h e QSRR s t u d i e s ,
equation-1
as
of the solute.
structure-retention
contribution,
we w i l l
one w i l l
(QSRR)
be a b l e
reversed-phase
obtained,
retention
describe
p r e d i c t i o n system
p o l y c y c l i c aromatic
application
of
by
to
LC
retention
of
predicted.
benzenes,
system
of a solute time
by u s i n g
the retention
between
a
i n reversed-phase L C .
are available.
parameters
Based
such
And t h e n , c a n be
stituted
is
(1)
the retention
equations
In struct
great most
of partition
the molecular
and/or
have
a
LC as t h e
which
(QSAR)
rever-
r e t e n t i o n and
(Pi)
as q u a n t i t a t i v e
et
solutes
system
phases
in
F o r example,
relationships
shape
i t can
solute's
parameters
an important
representing
parameters
i s physicochemical
systems.
The
= f
1
a capacity
r
named
Horvâth
is
model,
equation-1: k
in
such
between
of logarithm
retention
that
reversed-phase
as
to use reversed-phase
to say, prediction of retention
the
where
correlations
et
consistent
two i m m i s c i b l e
a n d some o f t h e s e
this
reten-
spectroscopy
the retention
relationships.
i t s hydrophobicity:
physicochemical
shown
with
i n 1-octanol/water
QSAR c a n b e a p p l i e d
in
such
between
structure-activity relationships
That be
made
much
i n t r o d u c e d by Horvâth
parameters
for the determination
o f a compound
in
on those
been
ficient titative
such
understanding
of solute
driven interaction
may c o r r e l a t e exist
pre-
procedure
years,
and/or
d i s t r i b u t i o n phenomena
In p r a c t i c e ,
physicochemical
That
can be
a s one o f t h e most
its partition coefficient
i t s aqueous
disadvantage,
(6-10).
clear
t h e mechanism
theory"
physicochemical
materi-
and t h e r e -
this
recent
by chromatography
entropically
standard
optimization
During
acknowledged
major
time.
investigate
"solvophobic
i s generally
very-
available.
of solutes
o f any s o l u t e , a
i s needed.
LC b o t h
be
toxic
To overcome
retention
is
i t has one
by the authors
i n a short
to
because
condition,
retention
mechanism
i n reversed-phase (19-21)
If
approach
of interest
this
proposed
prediction.
to predict
has been
(11-18). al.
be
easily
order
This
however,
or are highly
new p o l l u t i o n p r o b l e m s .
the retention
effort
to achieve available
approach has been
at appropriate
c a n b e more of
difficult
i s retention
dicted
(1-5).
conditions;
a l l materials
may n o t b e c o m m e r c i a l l y
fore an
methods
separation
limitation. als
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or "Simplex"
to get optimal
based
hydrocarbons
on t h e use o f
and physicochemical
has been
constructed
for optimization
the basic
approach
i n reversed-phase such
(PAHs)
parameters
on a 1 6 - b i t
of separation
and polar
established
con-
alkyl-
group
sub-
relationships
of these
microcomputer, conditions
to
LC f o r
will
compounds. and the be
strated.
Ahuja; Chromatography and Separation Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1986.
demon-
Microcomputer-Assisted
10. JINNO A N D KAWASAKI
Retention Prediction
169
Experimental The 2
liquid
Co.,
100III
ultraviolet
set
at
210
620
(Tokyo,
(2)
C-phenyl
The
mobile
methanol water.
A l l
Prior
the
(10
test
it
was
mobile There
have
topics
in
37),
we
LC.
elution
centration All
solutes not
reproducibility
c.a.
0.5
Seto,
Japan),
C2
(10
Japan)
a
few
and
4
from
the as
or
in
many
uL/min. each
retention
solutions
hundred parts
soluble
De-
urn).
available
analyzed
(5
(3)
equilibrated with
were
cm
and p u r i f i e d
always for
12 C18
χ
FineSIL
acetonitrile
obtained
of
mm i . d .
Jasco
was
of
publications subject
the
methanol on
is
and b a s i c
of
in
per
mobile
depending
used
capacity at
each
t
are
LC. in
0
mobile
factors
least
e a c h r u n was
as
for
one
on
than
hot
subject(36,
good
this
of
the
solutes
Therefore, work.
the Its
con
compounds
with
phase.
aromatic
in triplicates.
better
of
on t h i s
and sodium n i t r a t e
ppm i n
made
determination
s t i l l
study
i n reversed-phase
100
of
were
of
were
easily
and t h i s
our knowledge
was
LC systems
of
(1)
i n pure a c e t o n i t r i l e
a number
measurements
various
was
s o d i u m n i t r i t e was
used
a K o m a t s u DW
(Tokyo,
was
concentration
sodium n i t r i t e
of
in
FineSIL
phase
columns
test
column v o i d - t i m e
time
placed
commercially
The
(30-35)
From
decided
measure
the
detector
use.
been
void-volume
the
0.1°C.
Jasco
were
Uvidec-
as
tubings
mobile
a Microfeeder MFpump a n d a
Japan)
HPLC g r a d e
value
a
were +
Chemicals
the
a solute
in
column
to
Kanto of
dissolved
phase
(4) of
constant
in
If
(PTFE
and
substances
a
phase,
(ppm).
phase,
Cp)
measurements until
20
slurry technique;
consisted
rate
at
of
the
Tokyo,
The columns kept
as
urn, N o m u r a C h e m i c a l s ,
jmm,
phases
consisted
Japan)
(Jasco,
columns
(5
substance.
mobile
million the
of
phase
test
each
C8
The f l o w
to
mobile
nm.
the
purchased from
sources.
the
p r e p a r e d by
Develosil
velosil
260
thermostat
reversed-phase
were
urn),
Japan)
used
L t d . , Tokyo,
spectrometer
nm a n d / o r
Four long)
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chromatographic system
(Azuma E l e c t r i c ,
1.0
The
average
% relative
standard
deviation. Multiple 800
computer
regression
analyses
(Mitsubishi
computer
system
NEC
(Nippon E l e c t r i c ,
9801
were
written
Results In
the
18
PAHs
and
for
retention
i n BASIC
study,
a n d 28
polar
the
and
acetonitrile-water.
reversed-phase order
to
the
followings
were
log
is
π
;
V ,
A ;
it
w
features
the
use
o f aMELCOM
Japan).
16-bit
Japan),
The
microcomputer and the
programs
the
with mobile
i t
the
they
of
here
20
were
phases
relationships
molecular
logarithm of
and the
is
of
values
benzenes
of
between
we c a l l
properties
alkylbenzenes, determined
retention
and
"descriptors", of
a
on
methanol-water
solute,
as
the
selected:
the
water the w
columns
establish
factor
substituted
physicochemical parameters,
describing ;
a
L t d . , Tokyo,
capacity
groups
four
Ρ
p r e d i c t i o n was
Co.,
by
L t d . , Osaka,
language.
the
In
performed
Co.,
Discussion
present
various
were
Electric,
measure
composing
partition coefficient
the
hydrophobicity of
hydrophobic substituent
hydrophobicity of are
the of
calculated a molecule
the
substituted
from the (40).
constant
van V
w
is
der the
as
group Waals van
in
the
1-octanol/
measure
a molecule
radii der
in
a molecule
of
Waals
Ahuja; Chromatography and Separation Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1986.
the
(38). of (39).
atoms
volume
and
C H R O M A T O G R A P H Y A N D SEPARATION CHEMISTRY
170 A F
;
c
is
w
t h e van d e r Waals
correlation bonds)
+
factor,
(number
non-aromatic σ
η
HA,HD
;
Hammett's
;
they
surface is
c
area.
calculated
as Fc=(number
o f primary and secondary
ring
carbons)
of
double
-
0.5
for
(41,42).
constant.
are parameters
HA i s is
F
equal
equal
to
to
showing
t h e number
t h e number
the extent
o f hydrogen
o f hydrogen
of
substituted
acceptor
groups;
groups
donar groups i n a
a n d HD
molecule
(39).
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From
the basic
groups
substituted
highly
promising to
the
four
and
l o g Ρ a r e good
C18
a n d Cp c o l u m n s ,
ively. good
different
ability
equations
of
analyses tions
i n Table
for retention
are only
compositions,
the
f o r each form
were
regression
compound's as
multiple group
X=X
2
log k'
= a Pi
X=X
n
l o g k»
= a Pi
and system
2
n
t h e volume
+
c^
+ b P2
+
c
+ b P
+ c
2
2
n
fraction
2
of
and a and b a r e the c o e f f i c i e n t s respectively
and c
experimental
conditions.
is
where
m is
cients and each
the
regression The
mobile
equa phase
by
The r e l a t i o n
described
i n the
= f !
(X)
=
jjj
diX
b
= f
2
(X)
=
1
e ^
c
= f
3
(X)
=
i
fjX*
t h e number
χ
x
less to
5 c a n be d e r i v e d ,
modifier
and η i s as
i n the mobile
descriptors, t h e number
functions
the following
regression
a
corresponding
(2·)
n
organic
correlated,
by t h e m u l t i p l e
log
c a n be
mobile
be o b t a i n e d
analysis.
corresponding
b and c c a n be e x p r e s s e d
compounds'
for η different should
2
the intercept,
and X - c are h i g h l y obtained
ah are
follows: + biP
be
are
hydrogen
π and
compounds.
c
respect
results,
the fixed
descriptions
with
= aiPi
X-b
at
η equations
1
a,
the
by t h e m u l t i p l e
the following
log k
If
indicates
aromatic
F
with
π a n d (HA-HD)
For phenols,
descriptions
1
X is
of
and PAHs,
retention
By t h e above
obtained
description
X=X
where
(HA-HD)
are
LC w i t h
C8 a n d C2 c o l u m n s ,
molecule.
the retention
same p r o c e d u r e s
ship
i n reversed-phase
phenol's,
columns.
and p o l a r
follows
however.
compositions,
general
I
the retention
To g e n e r a l i z e phase
except
instance,
the four
PAHs
as
for alkylbenzenes
and l o g Ρ with
w
stating
for describing their
t h e whole
with
listed
phases:
benzenes
In t h i s
descriptors
the retention
descriptors and A
alkylbenzenes,
the descriptors
describe
For substituted
accepting
o n QSRR f o r
stationary
descriptors.
good
studies benzenes,
of X, three
of
namely,
(3) (4) (5)
than η and d ^ ,
the following
= f (X)P 1
1
can
analyses:
1
" i " powers
of X.
e^
and f i
If
such
equation-6
are the
equations
will
+ f (X)P 2
2
+ f (X) 3
,
i f X-a,
equations
1
2
examined
coeffi as
be o b t a i n e d
group. k'
phase
Ρχ and P
(6)
Ahuja; Chromatography and Separation Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1986.
3,
4
for
10.
Table
I.
Retention
f o r aromatic
ship
is
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by m u l t i - c o m b i n a t i o n
compounds,
where
o f two
the following
171
descrip
relation
k
= a
1
Ρ
+ b P
λ
+ c
2
(2)
a n d PAHs
and mobile
phase*-*-
P^
P
a
2
b
c
r*2
C18
A
log Ρ F
c
0.187
0.029-0.552
0.990
C18
M
log Ρ F
c
0.230
0.056
0.989
-0.847
C8
A
A
w
l o g Ρ 0.070
0.067
-0.331
0.983
C8
M
A
w
l o g Ρ 0.107
0.095
-0.634
0.989
Cp
A
log Ρ F
c
0.235-0.022
-0.213
0.995
Cp
M
log Ρ F
c
0.325-0.016
-0.663
0.996
C2
A
A
w
l o g Ρ 0.049
0.090
-0.284
0.979
Ç2
M
A
w
l o g Ρ 0.010
0.056
-0.829
0.982
polar
groups
column
(c)
Retention Prediction
assumed:
alkylbenzenes column
(b)
descriptions
tors
log (a)
Microcomputer-Assisted
JINNO A N D KAWASAKI
substituted
and mobile
benzenes
phase
P i
p
except
phenols
a
2
b
c
r
C18
Â
π (HA-HD)
0.136-0.049
-0.007
C18
Μ
π (HA-HD)
0.130-0.048
-0.050
0.925
C8
Α
(HA-HD)
0.240-0.031
0.215
0.965
C8
Μ
π (HA-HD)
0.283-0.045
0.117
0.978
Cp
Α
(HA-HD)
0.178-0.025
0.222
0.976
Cp
Μ
0.191
0.005
0.106
0.982
C2
Α
(HA-HD)
0.234-0.017
0.212
0.950
C2
Μ
π (HA-HD)
0.273-0.010
-0.098
0.982
π π
π (HA-HD) π
0.959
phenols column
and mobile
phase
*2 r :
p
2
a
D
c
r
C18
Α
π
S*3
0.182
0.182
-0.368
0.952
C18
Μ
π
S
0.166
0.175
-0.343
0.974
C8
Α
π
S
0.294
0.080
-0.192
0.952
C8
Μ
π
S
0.343
0.099-0.235
0.979
Cp
A
π
S
0.220
0.131
-0.221
0.960
Cp
M
π
S
0.191
0.158
-0.401
0.970
C2
A
π
S
0.302
0.115
-0.103
0.953
C2
M
π
S
0.312
0.152
-0.171
0.956
*1 A : a c e t o n i t r i l e : w a t e r M:
i
p
methanol:water correlation
* 3 S = πσ, h
=75
=65 :
:
35.
25.
coefficient.
(1- π),
Ahuja; Chromatography and Separation Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1986.
C H R O M A T O G R A P H Y A N D SEPARATION CHEMISTRY
172 This modifier are
given,
mined of
equation in
for
the
the the
any
column has The column
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Table
III
focused
were is
used were
factors
equations
in
capacity
factors 10
column other
suggests
for
to
structed
on t h e
RPS i s
the
shown
interactive
computer;
(1)
solutes,
(2)
fraction
of
the
mobile When
computer
and then phase
used
Then,
=
or
agree
to
use
the
the
the
measuredones
with
agreement the
C18
retention
on
RPS a s
(mobile
column of
the on
are the
formula
phase
phase
to
input
with
CRT o f
of
(M),
(X),
wascon
thisfunction
system
data
function
chemical
computer-
C18
flow-chart
of
mobile
using
predicted
good
the
the
following
the
C18
columns.
for
The
the
themeasured
the
fairly
predict
and
for
and
values
C18
II
compounds.
that
with
this
factor
the
conditions in
Figure 1
the
Table
p r e d i c t e d by
procedures,
accessing
names
or
suitable
capacity
input the
can be
in
(RPS)
the
theC18
the
interesting
volume
flow
rate
of
(F)). chemical
factors are
values
of
(k')
F,
the
mobile
Ν =
3482 F
2
the
input
input
solutes
to
RPS,
the
compounds' at
selected
theoretical
though
plate
modified
generally
Ν is
number, for a
Ν
is
thecolumn
function
of
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