Journal of Chromatography A, 1217 (2010) 5377–5383
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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma
?-Selective stationary phases: (III) Influence of the propyl phenyl ligand density
on the aromatic and methylene selectivity of aromatic compounds in reversed
phase liquid chromatography
Paul G. Stevensona,b,1, Arianne Solivena,c,1, Gary R. Dennisa,c, Fabrice Grittid,
Georges Guiochond, R. Andrew Shallikera,c,∗
aAustralian Centre for Research on Separation Science (ACROSS), School of Natural Sciences, University of Western Sydney Node, Parramatta, NSW, Australia
bCentre for Complementary Medicine Research, University of Western Sydney, Campbelltown, NSW, Australia
cNanoscale Organisation and Dynamics Group, University of Western Sydney, Parramatta, NSW, Australia
dDepartment of Chemistry, University of Tennessee, Knoxville, TN, USA
a r t i c l e i n f o
Received 19 February 2010
Received in revised form 14 May 2010
Accepted 19 May 2010
Available online 1 June 2010
Phenyl type stationary phase
a b s t r a c t
The retention characteristics of phenyl type stationary phases for reversed phase high performance liq-
uid chromatography are still largely unknown. This paper explores the retention process of these types
of stationary phases by examining the retention behaviour of linear PAHs and n-alkylbenzenes on a
series of propyl phenyl stationary phases that have changes in their ligand density (1.23, 1.31, 1.97,
2.50?molm−2). The aromatic and methylene selectivities increased with increasing ligand density until
a point where a plateau was observed, overall the propyl phenyl phases had a higher degree of aromatic
selectivity than methylene selectivity indicating that these columns are suitable for separations involv-
ing aromatic compounds. Also, retention characteristics relating to the size of the solute molecule were
observed to be influenced by the ligand density. It is likely that the changing retention characteristics are
caused by the different topologies of the stationary phases at different ligand densities. At high ligand
densities, the partition coefficient became constant.
© 2010 Elsevier B.V. All rights reserved.
In contrast to alkyl-bonded silica stationary phases, there have
been fewer studies on the retention characteristics of phenyl type
chain length of 0, 2, 4 and 6 carbon atoms and found that aromatic
et al.  and Snyder et al. [3,4] and it was found that the retention
process of phenyl type phases was controlled by a combination
of lipophilic, ?–? and dipole–dipole interactions. No studies have
tion processes. This paper examines the retention properties of
phenyl phases and particularly of stationary phases prepared using
a propyl phenyl moiety, and more specifically, on the effect of the
ligand density on the retention processes of small molecules.
∗Corresponding author at: Nanoscale Organisation and Dynamics Group, Univer-
sity of Western Sydney, Locked Bag 1797, South Penrith Distribution Centre, NSW
1797, Australia. Tel.: +61 2 9685 9951; fax: +61 2 9685 9915.
E-mail address: firstname.lastname@example.org (R.A. Shalliker).
1Joint first authorship.
Gritti and Guiochon  found that changing the ligand density
of C18-bonded stationary phases impacted the retention process
in accord with the degree of coverage. This occurred as the topol-
ogy of the stationary phase surface changed; ligand densities with
less than 2?molm−2resulted in large voids between chains. The
chains also tended to cluster, leaving large spaces providing the
analyte and the solvent molecules with access to the end capped
in close-packed monolayers with few inter-ligand cavities. These
authors found also that as the surface coverage increases the aver-
age distance between C18 chains decreases from 15.1 to 5.5Å for
surface coverages of 0.42–3.15?molm−2, respectively.
Lork and Unger  proposed that, when a critical density of
the alkyl-bonded ligands has been reached, these ligands become
closely packed, the freedom of the chains becomes restricted,
and the ability for solute penetration into the stationary phase is
strongly reduced. They found that the critical ligand density was
not fixed, but ranged between 2.3 and 3.2?molm−2depending on
the ligand chain length and the size of the solute molecule.
Critical ligand density, and therefore, stationary phase topology
have an impact on solute retention processes. For example, as the
C18 ligand bonding density increases above 2?molm−2the space
between adjacent C18 ligands decreases, and the mechanism of
0021-9673/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
P.G. Stevenson et al. / J. Chromatogr. A 1217 (2010) 5377–5383
retention shifts towards adsorption, rather than partitioning .
Thus, retention decreases as the ligand density increases, and, once
a critical ligand density is achieved, only the outer surface of the
bonded phase is accessible for solute interactions, indicating that
the stationary phase ligands require a degree of flexibility in order
to interact effectively with the analytes [6,7]. As the surface cov-
erage increases, the exclusion of larger molecules becomes more
apparent, even though smaller molecules are still able to penetrate
into the bonded layer. At some point, however, a surface coverage
would be achieved, on which, even for small molecules, retention
is almost exclusively dominated by adsorption and the analyte can
only explore part of the length of the alkyl chain [5,8].
be derived by examining the retention behaviour of homologues
[8,9]. In this work, the focus of the study was to examine the
behaviour of phenyl stationary phases prepared using different
ligand densities. Hence, linear polycyclic aromatic hydrocarbons
(PAHs) were selected to measure the aromatic selectivity, qaromatic,
of these phases, using PAHs with systematically increasing number
of benzene rings, the aromaticity of which should engage the ?–?
selective nature of the stationary phase. The retention behaviour
of n-alkylbenzenes homologues was also examined, as systematic
changes in the length of their alkyl tail could be used to measure
the methylene selectivity, qmethyleneof the stationary phases q is
derived from Eq. (1) [8,9]:
log k = (m0+ m1nc)
where k is the retention factor, m0, m1and p are related to the
non-repeat selectivity (i.e. the selectivity of the non-repeat part of
the homologue), ncis the number of homologue repeat units (i.e.
rings for the PAHs and CH2units for the n-alkylbenzenes), ? is the
proportion of the mobile phase organic component and q is the
selectivity that relates to the nature of the homologous series.
on chromatographic surfaces, the linear solvent strength theory
[10–13] was applied. It describes the relationship between the
retention factor, k, and the mobile phase composition, ?:
log k = log kw− S?
where kw is the retention factor in pure water and the S coeffi-
cient is a measure of the number of bonding sites that can interact
directly with a solute molecule . The linear solvent strength
theory is used as an optimisation strategy for isocratic separations.
Intersections of lines between respective solutes results with co-
of these lines depend on the slope, S, plots of S vs ncillustrates the
predicted ease of optimisation for homologous series. Linearity in S
hydrophobic contact surface area increases in a manner consistent
with the addition of each subsequent repeating unit. If discontinu-
ity of the plots of S vs ncoccurs, a loss of resolution, co-elution, or
changes in elution will occur.
The objectives of the present study were to synthesise and
pack in-house a series of propyl phenyl stationary phases that
had varying degrees of surface coverage. With these columns the
aromatic and methylene selectivities (via Eq. (1)) were measured
by examining the retention behaviour of series of linear PAHs
and n-alkylbenzene homologues. This retention behaviour (Eq. (2))
also provided information about the retention process on phenyl
phases, and the influence of the ligand density.
ethylbenzene, propylbenzene, butylbenzene and hexylbenzene)
and substituted aromatics (benzene, toluene, p-cresol, anisole
and phenetole), and acetone were purchased from Sigma–Aldrich
(Castle Hill, NSW). HPLC grade methanol (MeOH), tetrahydro-
furan (THF) and dichloromethane (DCM) were purchased from
LabScan Analytical Sciences (distributed by Lomb Scientific (Aust.)
Pty Ltd, Taren Pt, NSW, Australia). Milli-Q water was obtained
in-house and filtered through a 0.2mm Teflon filter (Millipore
Australia Pty Ltd, North Ryde, NSW, Australia). The linear PAH test
probes benzene, naphthalene and anthracene were prepared in
an aqueous 80% MeOH solution. Due to poor solubility in MeOH
2,3-benzanthracene and pentacene were prepared in 100% THF.
n-Alkylbenzenes were prepared in 80% MeOH solution. All samples
were made to concentrations of 1mgmL−1and injections volumes
2.2. Preparation of stationary phases and chromatography
phenylpropyldimethylchlorosilane (propyl phenyl silane) (Gelest,
Inc., Morrisville, PA, USA) and end capped with trimethylchlorosi-
lane (TMCS) (Sigma–Aldrich, Castle Hill, NSW, Australia) with
Nucleosil 100-10 bare silica (10?m particle diameter, 100Å
pore size, 350m2g−1surface area (Alltech Associates (Aust.) Pty
Ltd, Baulkham Hills, NSW, Australia)) according to the method
refluxing for 24h. The same procedure was repeated for one propyl
reaction the silica was washed with toluene and chloroform, fol-
lowed by drying for 24h at 115◦C. The bonded stationary phases
were then end capped with TMCS by the same procedure. Ligand
density was altered by varying the amount of the propyl phenyl
silane in the reaction mixture. Also, as pyridine is used to drive
the reaction equilibrium it was withheld from the reaction mix-
ture for selected phases to reduce the ligand density (see Table 1).
The bonded stationary phases were packed into 100mm×4.6mm
stainless steel tubes (Phenomenex, Lane Cove, NSW, Australia),
using an in-house procedure described previously . The quality
of the packed chromatography columns was determined by mea-
suring the reduced plate heights (h) of phenetole using a mobile
phase of 40% MeOH . The h-data are presented in Table 1. The
void volume of each column was measured using pycnometry with
DCM and MeOH. These data are listed in Table 1. The system hold-
up volume was measured by injecting thiourea through the system
with a union connector in place of the column with 100% MeOH
at 1mLmin−1and was found to be 0.258mL. %C was measured for
both the end capped and not end capped materials at the Micro-
analytical Unit at the Australian National University with a Carlo
Erba 1106 elemental analyser. It is also reported in Table 1. Ligand
density (reported in ?molm−2) is calculated from %C of the non-
2.3. Chromatographic separations
Solvent contributions to separations need to be considered
when selecting the mobile phase environment for a separation. It
has been found that when using ?-selective stationary phases to
P.G. Stevenson et al. / J. Chromatogr. A 1217 (2010) 5377–5383
Amount of propyl phenyl silane that was used in the synthetic reaction mixture. Also, %C (total), ligand density, void volume and reduced plate height for the propyl phenyl
columns. The values for %C represent the total carbon content of the stationary phase, including the propyl phenyl ligand and the end capping agent. The ligand density is
that of only the propyl phenyl ligand.
Stationary phase Bare silica (g) Propyl phenyl silane (g)TMCS (g)%C Ligand density (?molm−2) Void volume (mL)
aReaction was completed in a solution of 20% pyridine in toluene (%, v/v).
separate aromatic solutes, solvents that contain ? electrons (such
as ACN) should be avoided. These solvents interfere with ?–?
interactions and potentially cancel aromatic selectivity from these
stationary phases [3,4,19]. Therefore, MeOH is used in this study as
the mobile phase, because this solvent contains no ? electrons and
has no detrimental effect on the separation.
Each test probe was eluted on each chromatographic column
with, ideally, a minimum of 5 different MeOH/H2O mobile phase
compositions (incrementing by 5% MeOH). Prior to the first anal-
ysis, the chromatography columns were left to equilibrate at the
mobile phase composition for 20 column volumes. To ensure that
thermal equilibrium had been reached the temperature of the
columns were maintained at 30◦C for 1h prior to analysis. A water
bath and a column jacket, including a 2mL loop prior to the injec-
tion valve, were used to maintain the temperature of the column
and mobile phase at 30◦C. All chromatographic separations were
performed on a Shimadzu LC system, incorporating a LC-10ATvp
pumping system, SIL-10ADvp auto injector, SPD-10Avp UV detec-
tor, and Shimadzu Class-VP software on a Pentium II 266MHz
2.4. Column lifespan
Stationary phase stability was tested for each column by com-
paring the retention factors of a standard test mixture of acetone
and substituted aromatics (benzene, toluene, p-cresol, anisole and
phenetole) that were analysed ideally every 200-column volumes
of solvent passage through the column. No stationary phase degra-
dation was observed, within the period of the study.
3. Results and discussion
of the stationary phases. Included in the data are the total %carbon
load and the ligand density of the phenyl moiety.
3.1. Aromatic and methylene selectivity
3.1.1. Aromatic selectivity
The aromatic selectivity of these phases was measured by fit-
ting the log of the retention factor (i.e. logk) of linear PAHs to the
number of rings of the PAH, nrings, ranging from 1 to 5 according
to Eq. (1). In total, retention information was collected at no less
than five isocratic mobile phase compositions. R2was measured
for these planes generated by Eq. (1) in the z direction only as the
x and y directions (nringsand the mobile phase composition) were
Fig. 1. Ligand density vs qaromatic.
PAHs were no less than 0.998 (see Table 2). The entire selectivity
measurements for the linear PAHs were measured in duplicate on
PP1.97 and PP2.50 to assess the reproducibility of these measure-
ments. The reproducibility of the measurements of qaromaticwere
90% for PP1.97. The qaromaticof the propyl phenyl stationary phases
are listed in Table 2, and are plotted against the ligand density in
Fig. 1. qaromaticshowed a general increase in magnitude in direct
correlation to the ligand density. It is likely that this is caused by
the increased number of aromatic rings available on the stationary
phase surface to interact with the aromatic solutes. However, it is
also possible that the increase in the ligand density may be suffi-
enhance the strength of ?–? interactions that take place between
conjugated species .
3.1.2. Methylene selectivity
The methylene selectivity was measured according to Eq. (1)
(see data in Table 3) using a series of n-alkylbenzenes with alkyl
chains ranging from 1 to 6 carbon atoms (i.e. toluene to hexylben-
zene). R2values were always greater than 0.996 (Table 3). Ligand
density vs qmethyleneis illustrated in Fig. 2 where a maximum selec-
tivity was observed at 1.97?molm−2(1.24) and a minimum at
1.23?molm−2(1.14). There was a general trend that suggests that
qmethyleneincreases with increasing ligand density over the range
although the significance of this dip has not yet been assessed.
Parameter results when logk, nc, ? data for linear PAHs are fit to Eq. (1). Aromatic selectivity is represented by q.
P.G. Stevenson et al. / J. Chromatogr. A 1217 (2010) 5377–5383
Parameter results when logk, nc, ? data for n-alkylbenzenes are fit to Eq.(1). Methy-
lene selectivity is represented by q.
Fig. 2. Ligand density vs qmethylene.
The magnitude of qmethyleneselectivity was less than that of
qaromaticselectivity, indicating that the propyl phenyl stationary
phases were more selective towards changes in the solute aro-
selectivity was greater than the range in qaromaticselectivity, indi-
cating that surface ligand density was less important for purely
aromatic species than for the mixed hydrocarbon/aromatic alkyl
3.2. Retention characteristics of phenyl type stationary phases
3.2.1. Retention comparison in constant mobile phase
Figs. 3 and 4 illustrate the relationship between logk and
the ligand density for the linear PAHs and n-alkylbenzenes,
respectively, in a single mobile phase environment (64.8% (v/v)
MeOH/H2O for the linear PAHs and 59.8% (v/v) MeOH/H2O for
the n-alkylbenzenes). For both homologue series, logk increases
Fig. 3. Plot of ligand density against logk for the linear PAHs. ? represents
benzene, ? represents naphthalene, ? represents anthracene, ? represents 2,3-
benzanthracene, and ? represents pentacene.
Fig. 4. Plot of ligand density against logk for the n-alkylbenzenes. ? represents
toluene, ? represents ethylbenzene, ? represents n-propylbenzene, ? represents
butylbenzene, and ? represents hexylbenzene.
monotonically with increasing ligand density and reaches a limit-
ing value for a critical ligand density. This is consistent with the
results of other works conducted on alkyl stationary phases, for
which the critical ligand density was found to be between 2.3 and
3.2?molm−2, consistent with the region explored here.
were also observed by plotting logk vs ? (i.e. Eq. (2)) for both linear
PAHs and n-alkylbenzenes over a range of five mobile phase com-
mobile phase compositions of both homologue test series with R2
greater than 0.965 for the linear PAHs (in most instances R2was
0.999) and R2greater than 0.987 for the n-alkylbenzenes. The mea-
surements were completed in duplicate for linear PAHs on two of
vs ? measurements.
3.2.2. Linear PAHs
The S and logkwvalues of the linear PAHs from Eq. (2) are pre-
sented in Table 4 for the propyl phenyl columns. Examination of
these plots (not shown) reveals that there is a similarity between
the slopes of these curves on all the columns that suggests similar
retention processes are at play. Upon further examination of the S
coefficients, plots of the S coefficients against the number of repeat
discontinuity between the 3 and 4 ring members (i.e. anthracene
and 2,3-benzanthracene) and the 4 and 5 rings members (i.e. 2,3-
benzanthracene and pentacene) on each stationary phase surface.
As an example S vs nringson PP1.31 is illustrated in Fig. 5, which is
Fig. 5. S vs nringsfor PP1.31.
P.G. Stevenson et al. / J. Chromatogr. A 1217 (2010) 5377–5383
List of S and logkwwhen the linear PAH data on the propyl phenyl columns are fit to Eq. (2).
PP1.23 PP1.31PP1.97PP2.50PP300 PP1.23 PP1.31PP1.97PP2.50 PP300
Angle of deviation in plots of S vs nringsfor all propyl phenyl columns.
Stationary phase Angle of deviation (◦)
First angleSecond angle
Fig. 6. S vs nringsfor PP1.23.
consistent for all phases, except with respect to the angle of devia-
tion. The angles of these deviations, measured at both positions are
listed in Table 5.
The magnitude of this angle of deviation, at both positions,
depend on the ligand density. The propyl phenyl column with the
lowest phenyl ligand density (i.e. PP1.23) had the smallest angles
of deviation (see Fig. 6). Fig. 7 illustrates that as the ligand density
increased the angles of deviation became more pronounced, until
a point where there was little difference in the angles of deviation
and a plateau was also observed.
There are numerous possibilities to explain the cause of these
deviations in S vs nrings. The two most likely relate to physical
aspects of solute retention within the phase:
(1) As the molecular size of the solute increases (as is the case with
linear PAHs), there is a concordant increase in the degree of
stationary phase reordering that must be undertaken for the
molecule to interact with the stationary phase ligand. There-
more difficult due to restrictions in the freedom of motion
of more densely bonded phases [5,8,21,22]. This explains the
plateau of the angle of deviation at the higher ligand densities.
When the critical ligand density is reached it becomes diffi-
cult for the compounds to partition into the stationary phase,
thus changing the retention behaviour (and the S coefficient)
for larger molecules. As the surface coverage increases past this
critical point there was little change in angle of deviation with
respect to molecular size. Future work may include synthesis-
ing stationary phases with higher ligand densities in order to
test if this deviation in linearity can be offset for members of
the series with less than three rings. In that way, the higher
ligand densities may make it more difficult for even the 3 ring
members to partition into the stationary phase.
(2) As the molecular size of the solute increases, there is also an
increase in the degree of solute restriction from the porous
network. Consequently larger molecules require more time
to orientate into an interaction state. As the ligand phase
density increases pore restriction should become more signif-
icant, hence the discontinuity. This was tested by synthesising
eter of 300Å, compared rather than 100Å of the other propyl
phenyl phases, was used to determine if pore access restric-
tions played a role in deviation in S vs nrings. By increasing the
pore diameter the larger solutes should have more freedom to
enter the stationary phase and interact with the same number
of stationary phase ligands as the smaller molecules result-
ing in linear S vs nringsplots and a higher degree of separation
Logk vs ? plots of the linear PAHs on the propyl phenyl 300Å
stationary phase (PP300) were linear with R2values of 0.997 and
above. The plot of S vs nringsis illustrated in Fig. 8 and the slopes
and intercepts of the linear PAHs when applied to Eq. (2) are listed
in Table 4. As with the other propyl phenyl phases there was dis-
continuity in the S vs nringsrelationship. In this instance, the first
angle of discontinuity was 46.5◦and the second angle was 33.9◦.
It is interesting to note, however, that on all the propyl phenyl
phases prepared on 100Å silica, the second angle of deviation was
always greater than the first, but for the 300Å silica phase was this
trend reversed. The reason for this has not yet been explored, but
it does at least indicate that pore restriction was not the cause of
the deviation between the 3 and 4 ring members of the series.
P.G. Stevenson et al. / J. Chromatogr. A 1217 (2010) 5377–5383
Fig. 8. S vs nringsfor PP300.
The discontinuity in the relationship between S vs nringshas an
important impact on optimisation. When a monotonic relationship
exists, separation is easy to predict as behaviour is a predictable
dictable behaviour, and the result may be a change in the elution
order, with smaller repeat members eluting after higher repeat
members of the homologue series. Whilst this seems trivial for
this small family of homologues, the complexity of the optimisa-
tion process is amplified as the peak capacity of the separation
system is approached and unrelated analyte species are the sub-
ject of the separation. For these types of analyte species, plots
of logk vs ? will not necessarily diverge as the solvent strength
decreases, but instead intersect, leading to points of co-elution and
changes in retention order. If, a complicating factor in the sep-
aration behaviour results due to solute access to the stationary
phase surface, as observed here for the PAH homologues, then the
optimisation process will become more complex. Hence, the ideal
minimal deviation in S vs ncrelationship (i.e. little to no variation
in the slope of Eq. (2)) for homologous series that are related to the
Logk vs ? plots for the series of n-alkylbenzenes were also con-
structed with a high degree of linear correlation (R2values above
0.99). The S and logkwcoefficients derived when these curves were
applied according to Eq. (2) are listed in Table 6. Plots of S vs
the substituent alkyl chain length (nCH2) are shown in Fig. 9. All
relationships were linear, showing that there is essentially no dif-
ference in retention characteristics for this series of compounds
over the range of ligand densities investigated, as evidenced by
the linear relationship. There were, however, differences in the
magnitude of S, with S increasing as the carbon load increased,
reaching a maximum for PP1.97, and then decreasing slightly for
the PP2.50 phase. There were also differences in the slopes of these
plots, shown in Fig. 10, where the slope decreases with increas-
Fig. 9. S vs nCH2for the n-alkylbenzenes on each of the propyl phenyl station-
ary phases. ? represents PP0.50, ? represents PP1.31, ? represents PP2.50, and ?
against the slopes of S vs nCH2.
ing ligand density: PP1.23–0.55, PP1.31–0.51, PP1.97–0.47, and
The n-alkylbenzene molecules have a single benzene ring with
tails of varying chain lengths. Unlike the rigid structure of the lin-
ear PAHs, the tail is free to rotate around the C–C single bond and
the size of the solute does not increase at the same rate as that
of the PAHs with respect to the increasing homologue number.
The increase in the S coefficient as a function of the carbon load is
likely to be a combination of the increased number of interactions
ary phase ligand, and partitioning of the n-alkylbenzene eventually
reaching a limiting surface load. Future work will involve perform-
ing adsorption isotherm measurements on these propyl phenyl
columns to determine what stationary phase characteristics con-
tribute to the retention process.
List of S and logkwwhen the n-alkylbenzene data on the propyl phenyl columns are fit to Eq. (2).
P.G. Stevenson et al. / J. Chromatogr. A 1217 (2010) 5377–5383 Download full-text
The ligand density plays an important role in the retention pro-
cess in HPLC. These results show that the same trends observed
by other authors on alkyl type stationary phases are also observed
on phenyl type materials at a critical ligand density. Their selectiv-
ity increases with increasing ligand density until the critical ligand
density is reached and a plateau observed for both the aromatic
and the methylene selectivities. When the ligand density increases
beyond this point, the selectivity of the propyl phenyl ligand no
The magnitude of the angles of discontinuity in the plots of S vs
nringsdepend on the ligand density, the smallest angles occurring
for the lowest ligand density. It is unlikely that this angle of devia-
tion is due to pore access restrictions as the same set of compounds
that the angle of discontinuity depends also on the critical ligand
density, as the size of the angle also plateaued at higher densities.
It is suspected that the larger molecules possesses a lower than
bers is lower than the line of best fit drawn through the 1–3 ring
these compounds to partition into the stationary phase. The angle
of deviation plateaus with the decreasing likelihood of the larger
solute entering the stationary phase.
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