Analytica Chimica Acta 551 (2005) 213221

An investigation on the cation-exchange and Lewis-base interactions of a

n-octadecylphosphonic acid-modified magnesiazirconia stationary phase
Hai-Bo He, Yu-Qi Feng , Shi-Lu Da, Zhong-Hua Wang
Department of Chemistry, Wuhan University, Wuhan 430072, PR China
Received 24 May 2005; received in revised form 14 July 2005; accepted 15 July 2005
Available online 24 August 2005

Abstract
The cation-exchange and Lewis-base interactions of a n-octadecylphosphonic acid-modified magnesiazirconia (C18 PZM) stationary phase
have been studied in greater detail than previous studies, which are characterized by the chromatography of some basic drugs of high pKa and
some hard Lewis base analytes, respectively. The effect of mobile phase constituents such as pH, type of buffer and buffer concentration on the
surface chemistry properties on C18 PZM are elucidated. The results show that at an intermediate pH, the cation-exchange sites are due to both
the chemisorbed phosphonate and the adsorbed buffer anion, i.e. acetate, at the hard Lewis acid sites on the C18 PZM phase; at high pH, the
cation-exchange interactions mainly result from the chemisorbed phosphonate rather than the inherent zirconol groups. The chromatography
of the hard Lewis base analytes indicates that a strong Lewis acidbase interaction still exists on C18 PZM even in the presence of Lewis base
buffer (such as acetate or phosphate) in the mobile phase or a high-pH eluents is used. Moreover, the chemisorbed n-octadecylphosphonic
acid (C18 P) is found to slowly bleed out of the C18 PZM column when exposed to a phosphate buffer. As expected, the resulting mobile-phase
phosphate modified C18 PZM column (p-C18 PZM) exhibits significantly stronger cation-exchange interactions towards the test basic solutes
than the original C18 PZM column. And the p-C18 PZM column demonstrates a tremendous improvement on the chromatography of the
Lewis base analytes over the original C18 PZM column. An approach designed to overcome the Lewis acid sites problem by attaching
methylphosphonic acid (MPA) onto the C18 PZM column, however, is not successful in improving the chromatography of the troublesome
Lewis base solutes.
2005 Elsevier B.V. All rights reserved.
Keywords: n-Octadecylphosphonic acid modification; Magnesiazirconia; Cation-exchange and Lewis-base interactions

1. Introduction
On the basis of that phosphonates are able to form very
stable coordination complexes with the hard Lewis sites on
the surface of magnesia-zirconia (ZM), an alkylphosphonate
modified ZM (APA-ZM) has been developed in our group
[1] and it is very useful for the reversed-phase separation of a
wide variety of analytes [14]. Similar to the impact of residual silanols on the separation of positively charged amines,
interactions between the hard Lewis base analytes and the
residual Lewis acid sites on ZMs surface can cause serious
chromatographic problems. Even using pure isopropanol as
the mobile phase, the hard Lewis bases, such as nitrophenols

Corresponding author. Tel.: +86 27 87867564; fax: +86 27 68754067.

E-mail address: yqfeng@public.wh.hb.cn (Y.-Q. Feng).

0003-2670/$ see front matter 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2005.07.022

and benzoic acids do not elute from the APA-ZM [5]. However, using the column of the ZM modified with fosfomycin
(sodium 3-methyl-oxiranylphosphonate) under reflux, such
Lewis base solutes are eluted under typical reversed-phase
conditions, and they are well separated on the column that
further modified by cyclodextrin [6]. Recently, a phosphonic
acid with a long hydrocarboneous chain was chosen and the
ZM particles were refluxed in the solution for a long time to
prepare a ZM-based RP-HPLC stationary phase (C18 PZM)
in our group [7], because we believed that just as identical with the modification of zirconia [8,9], both the spacial
requirement and aggressive treatment conditions are necessitated to exhaustively block the surface Lewis acid sites on
ZM as well as enhance the base stability of the modified-ZM.
In contrast to the undesirable stablity of alkylphosphonated
or silanized zirconia phases [916], the C18 PZM column

H.-B. He et al. / Analytica Chimica Acta 551 (2005) 213221

214

shows its excellent stability in the range of pH 212 under

reversed-phase chromatographic conditions, which allows a
large set of basic species to be well separated on such a
phase by a predominantly reversed-phase retention mode
at high pH [7]. More importantly, the C18 PZM phase has
demonstrated its potential for facilitating the analysis of some
basic drugs that would be troublesome on silica-based phases
[17]. Typically, a stronger Lewis base must be added to the
mobile phase to compete for the accessible Lewis acid sites
and elute such analytes on a zirconia-based stationary phase
[1821], and most interestingly, Lewis base additives such
as inorganic phosphate, fluoride and carboxylates adsorb to
the surface and impart a negative charge, which act as sites
for cation-exchange and lead to mixed-mode retention for
positively charged analytes [18,19,2124]. Just as with the
polybutadiene-coated zirconia (PBD-ZrO2 ) that has been the
most studied zirconia-based reversed-phase material [22,25],
our recent work shows that, under most chromatographic
conditions, the basic analytes undergo a reversed-phase and
cation-exchange interaction mixed-mode retention mechanism on the C18 PZM phase. And it is confirmed that the
inherent zirconol groups on ZM is unlikely to act as the
cation-exchange sites at lower mobile-phase pH, however,
either the phosphonate modification or the adsorption of
acetate would probably impart cation-exchange properties
under such condition [7]. Whereas at high mobile-phase pH,
the cation-exchange sites component is likely from both the
chemisorbed phsophonate and the inherent zirconol groups
on C18 PZM [17]. This indicates the complex surface chemistry of the C18 PZM phase due to the cation-exchange and
the Lewis acidbase interactions. To gain more understanding
of the surface chemistry on such phases, it would be necessary to make clear of the following questions: How do the
cation-exchange interactions on C18 PZM take place? How
do the mobile-phase Lewis bases affect the surface chemistry on C18 PZM? And, how does the surface chemistry on
C18 PZM affect the retention behavior of basic solutes and
Lewis base analytes? Therefore, in the present work, we
looked into the influence of the type and the concentration
of mobile-phase buffer as well as the mobile-phase pH on
the surface chemistry, i.e. the cation-exchange interactions
and the Lewis acidity on C18 PZM by its ensuing chromatography for some basic and Lewis base solutes, respectively.
Finally, a methylphposphonic acid (MPA) modified C18 PZM
column that designed to overcome the Lewis acid site prob-

lem was prepared and evaluated by the chromatography for

some Lewis base solutes.

2. Experimental
2.1. Chemicals
n-Octadecylphosphonic acid (C18 P) was synthesized according to reference [26]. Microspherical
magnesiazirconia (ZM) composite was prepared as
described previously [27] with a mean particle diameter of
57.5 m. Methylphosphonic acid (MPA) was purchased
from Sigma (St. Louis, MO). The physical properties about
the bare ZM and the resultant modified ZM particles used
in this study are summarized in Table 1. Tetrahydrofuran,
trihydroxymethylaminomethane (Tris) and acetate were
purchased from Shanghai General Chemical Reagent Factory (Shanghai, China). Ammonium acetate and ammonium
phosphate monobasic were purchased from Shanghai General Chemical Reagent Factory (Shanghai, China). Quinine
was obtained from Shanghai Chemical Co. Ltd. (Shanghai,
China). Metoprolol and timolol were purchased from Sigma
(St. Louis, MO). Other basic solutes were supplied by the
National Institute for the Control of Pharmaceutical and
Biological Products of China (NICPBP, Beijing, China).
All other compounds and reagents were commercially
available and, unless noted otherwise, were analytical
reagent grade. Double distilled water was used for all experiments, and was boiled to remove carbon dioxide before
use.
2.2. Preparation of n-octadecylphosphonic
acid-modied magnesiazirconia stationary phase
The octadecyl-ZM stationary phase was prepared on the
basis of the method of chemisorption of C18 P to the Lewis
acid sites on ZMs surface [7]. Briefly, pretreated ZM (16 g)
was placed in 250 ml round-bottomed flask and suspended in
120 ml tetrahydrofuran containing 0.4 g of C18 P. The particles were sonicated under vacuum to allow the solution
to fully infiltrate the pores before heating. After 48 h at
reflux, the particles were allowed to settle, and the C18 P
solution was decanted. Then, the particles were filtered and
washed in sequence with tetrahydrofuran and methanol. Sub-

Table 1
Physical properties of ZM and modified-ZM particles used in this studya
Designation

Surface area (m2 /g)

Pore volume (mL/g)

Pore diameter (nm)

Carbon contentb (%)

Surface coverage (mol/m2 )

ZM
C18 PZM
MPAC18 PZMd

59.76
48.57
37.18

0.110
0.086
0.072

4.65
4.01
4.01

0.0
1.603
0.506

0.0
1.274c

a
b
c
d

Data obtained by nitrogen adsorption analysis unless noted otherwise.

Data obtained by elemental analysis.
Surface coverage of the chemisorbed n-octadecylphosphonic acid.
MPAC18 PZM obtained by modification of C18 PZM with MPA as in Section 3.3.

H.-B. He et al. / Analytica Chimica Acta 551 (2005) 213221

215

sequently, the obtained C18 PZM was dried in a vacuum

oven at 100 C overnight and stored in capped bottles for
packing.
2.3. Chromatography
The liquid chromatographic system comprised a Shimadzu LC-10AT pump, a Rheodyne Model 7125 injector with 20 l loop, a Shimadzu SPD-10A variable
wavelength UV-detector attached Echrom 98 chromatographic data system (Dalian Elite, Dalian, China). The
n-octadecylphosphonic acid-modified ZM microspheres
(C18 PZM) were packed into stainless-steel columns
(150 mm 4.6 mm i.d.) by using slurry technique. The samples were dissolved in methanolwater (1:1, v/v). Typically,
2.0 l injection volumes of solutes were injected. The mobile
phases were prepared by first dissolving buffers in water,
adjusting the pH with dilute NaOH or HCl solutions, then
mixing the aqueous solution with methanol, and finally filtered through a G4 fritted glass funnel with 34 m pore size
and degassed prior to use. The flow rate of the mobile phase
was 1.00 ml min1 . The wavelength used for detection was
254 or 220 nm. The void volume was determined using acetone for k calculation. All chromatographic results were the
means of at least duplicate injections at ambient temperature
(27 2 C).

3. Results and discussion

The column efficiency of the C18 PZM phase used in
this study was determined with methanolwater (60:40,
v/v) as mobile phase. The number of theoretical plates
was 13198 m1 using biphenyl as solute at a flow rate of
1.00 ml min1 . The Lewis acidity on the C18 PZM phase
was preliminarily evaluated by eluting some Lewis bases
under a variety of typical reversed-phase conditions. Unfortunately, analytes such as nitrophenols and benzoic acids do not
elute from C18 PZM using the mixtures of methanolwater as
mobile phases, suggesting that the addition of Lewis bases,
such as acetate or phosphate to the eluent is needed to elute
such troublesome solutes. However, due to the high population of accessible Lewis acid sites on C18 PZM, the chemistry
of C18 PZM could in turn be affected by the buffer Lewis
base constituents. As, thus, the effect of the type of buffer
constituents on the surface chemistry of C18 PZM, i.e. the
cation-exchange interactions and the Lewis acidity was studied by the chromatography for some basic and Lewis base
solutes, respectively.
3.1. Effect of the type of mobile-phase buffer
Three buffers (acetate, Tris and phosphate) were choosen
in this experiment because acetate and phosphate are very
common buffers for RPLC, and since Tris does not have a
hard Lewis base functionality and it is not expected to binding

Fig. 1. Effect of mobile-phase pH on the retention factors (k ) of basic solutes

on C18 PZM. Column: 150 mm 4.6 mm. Mobile phase: methanolbuffer
(60:40, v/v) containing 10 mM HAc and 50 mM NaCl for pH 4.0 and 6.1,
and containing 10 mM Tris and 50 mM NaCl for pH 8.0 and 10.1. Flow rate:
1.00 ml min1 ; temperature: 27 2 C; UV: 220 nm for tetracaine, 254 nm
for all the other solutes. Solutes: (
) diphenhydramine; () quinine; ()
tetracaine; () doxepin; (+) verapamil.

to the ZMs surface via Lewis acidbase interactions, Tris

buffer was chosen to test the cation-exchange behavior on
C18 PZM due to the chemisorbed phosphonate other than any
of the adsorbed Lewis base anionic buffer constituents on the
stationary phase.
3.1.1. Testing in methanolacetate buffer mobile phase
Fig. 1 shows that the retention of five basic solutes on
the C18 PZM phase reach a maximum at pH 6.1 in the range
of pH 4.08.0, which is evidence of the cation-exchange
interactions at pH 6.1 as proposed in our recent work [17].
It is well-known that the eluents buffer concentration is a
crucial factor affecting on solute retention in ion-exchange
chromatography. Since the cation-exchange properties for
some bases on C18 PZM at pH 6.1 using [Na+ ] as counter
ion is well-recognized in our recent work [7], this time 5, 10
and 20 mM ammonium acetate buffers rather than sodium
acetate buffers were choosen to examine how changes in
buffer concentration can affect retention on C18 PZM. As
can be seen from Fig. 2, the solutes retention decreases as
the buffer concentration is increased, which confirms the
cation-exchange interactions under this condition. As stated
in our another work [7], the inherent zirconol groups are not
expected to behave as cation-exchange sites at such low pH,
and the adsorbed acetate on accessible ZM surface sites is
more likely to impart the cation-exchange properties than the
chemisorbed phosphonate. Accordingly, a tridentate bonding mode between the phosphonoic groups and the ZM
support was proposed as the primary form for the modification under rigorous conditions (as shown in Fig. 3A). It
seems reasonable according to Randon et al. [28] because

216

H.-B. He et al. / Analytica Chimica Acta 551 (2005) 213221

Fig. 4. Chromatography of Lewis base analytes on C18 PZM with 70%

methanol10 mM HAc buffer adjusted to pH 6.1 as mobile phase. Other
experimental conditions: 1.00 ml/min; 254 nm. Solutes: (1) m-nitrophenol;
(2) p-nitrophenol; (3) o-nitrophenol.

Fig. 2. Plot of log k for basic solutes vs. logarithm of acetate buffer concentration in mM on C18 PZM. Mobile phase: 60% methanol + NH4 Ac buffer
adjusted to pH 6.1. Other experimental conditions are the same as in Fig. 1.
Solutes: (
) diphenhydramine; () quinine; () tetracaine; () doxepin; (+)
verapamil.

the P O and P OH stretches at 1220 and 950 cm1 , respectively, are not presented in the FTIR spectra for the C18 PZM
[7]. However, Reven and co-workers [29] considered that
the absence of these two bands is hardly as evidence for
a tridentate mode since the ranges for the different P O
stretching peaks greatly overlap and depend on the degree of
hydrogen bonding or metal binding. As, thus, the other two
likely adsorption form of C18 P on the support, as shown in
Fig. 3B and C, respectively, were proposed. And the definite
assignment of the cation-exchange source would be difficult
because both the monodentated (Fig. 3B) and the bidentated
(Fig. 3C) bonded phosphonate would act as negative charged
sites.
It should be pointed out that the o-, m-, p-nitrophenol
isomers are eluted within 60 min when 10 mM acetate is presented in the mobile phase. However, the resulting peaks
are obviously not acceptable (see Fig. 4) and none of the
benzoic acids is eluted from the C18 PZM column. This indicates the effective competition of the dissolved acetate for
unblocked Lewis acid sites on the C18 PZM phase, but the
Lewis acidbase interactions between surface and the analytes are still very strong.

Fig. 3. Proposed models for the adsorption forms of n-octadecylphosphonic

acid on ZM.

3.1.2. Testing with mobile phase excluding Lewis base

from buffer
As displayed in Fig. 5, all the solutes have very small retentions at both pH 4.0 and 6.1 when Tris is used as mobile-phase
buffer. In contradistinction, Fig. 1 shows that when acetate
buffer was used in the mobile phase, the retention of the same
solutes, sharply increases from pH 4.0 to 6.1, and reaches a
maximum at pH 6.1 in the range of pH 4.08.0. This indicates
that the cation-exchange component from the chemisorbed
octadecylphosphonate is suppressed, even does not survive
the TRIS conditions. One may argue that it is not correct to
use TrisHCl as buffer at low pH 4.0 and 6.1 because it is
out of the buffer range of Tris. However, we observed a good
repeatability for retention of the test solutes under such conditions. This might due to very small retention for bases at
such pH. As stated in our recent work [17], this implied the
tridentate bonded form as shown in Fig. 3A and excluded
the chemisorbed C18 P from the cation-exchange behavior.
Nonetheless, it is in fact not a solid evidence for excluding
the octadecylphosphonate from the cation-exchange behavior that stated in our recent work, because Tris has pKa above

Fig. 5. Effect of mobile-phase pH on the retention factors (k ) of basic solutes

on C18 PZM. Mobile phase: methanolbuffer (60:40, v/v) containing 10 mM
Tris and 50 mM NaCl adjusted with HCl for pH 4.0, 6.1 and 8.0, and with
NaOH for pH 10.1. Other experimental conditions are the same as in Fig. 1.
Solutes: (
) diphenhydramine; () quinine; () tetracaine; () doxepin; (+)
verapamil.

H.-B. He et al. / Analytica Chimica Acta 551 (2005) 213221

Fig. 6. Effect of Tris buffer concentration on the retention factors of basic

solutes on C18 PZM. Column: Mobile phases: 60% methanol + 5, 10, 20,
35 mM Tris buffer adjusted to pH 6.1, respectively. Other experimental conditions are the same as in Fig. 1. Solutes: (
) diphenhydramine; () quinine;
() tetracaine; () doxepin; (+) verapamil.

8.0 (20 C) [30] and at intermediate pH, it is expected to be

protonated and interact with the dissociated octadecylphosphonate. The decrease of solutes retention upon increasing
the concentration of Tris buffer at pH 6.1 (as shown in Fig. 6)
confirms the coulombic interactions between the Tris and the
octadecylphosphonate, and which is analogous to stating that
the chemisorbed C18 P is not at all excluded from imparting
the cation-exchange properties. Therefore, the adsorption of
C18 P onto ZM support may be in terms of forms as shown in
Fig. 3B and C rather than that in Fig. 3A, and accounting the
adsorbed acetate for the major source of the cation-exchange
interactions is under question. However, as indicated in Fig. 7,
the solutes retention at pH 6.1 becomes obviously much larger
when acetate is added into the Tris buffer. A possible explanation is that the adsorption of acetate on the accessible Lewis
acid sites of ZM leads to an increase in cation-exchange
interactions between solutes and support. Alternatively, the

Fig. 7. Comparison of the retention factors (k ) of basic solutes on C18 PZM.
Mobile phases: 60% methanol + pH 6.1 buffer containing (
) 5 mM Tris,
( ) 5 mM Tris + 10 mM HAc, and () 10 mM HAc, respectively. Other
experimental conditions are the same as in Fig. 1.

217

coexistenceof Tris and acetate in mobile phase results in ionpair effect, which makes solutes more retained. However, the
retenion is much higher in acetate buffer in comparison with
Trisacetate mixed buffer with the same pH, that is, solutes



retained in the increased order of kTris
 kTris+HAc
< kHAc
,
suggesting that our first explanation is the more reasonable
one. Consequently, the cation-exchange properties at intermediate pH should be ascribed to both the adsorbed acetate
and the chemisorbed phosphonate.
Our recent work [17] showed that basic solutes with
high pKa values experienced a predominantly reversed-phase
retention mode at high pH on C18 PZM. Nonetheless, one
ought not too lightly dismiss the cation-exchange contributions to the total retention at high pH such as pH 10.1.
As discussed above, the cation-exchange sites due to the
chemisorbed phosphonate is well-recognized. However, at
such high pH, whether the inherent zirconol groups would
result in the cation-exchange properties or not is unclear. On
the basis of the two-site retention model for basic analytes
in RP-HPLC [23,3133], we obtained a quantitative estimate
of the individual contributions of reversed-phase and cationexchange process to retention by the analysis of the plots of
k versus 1/[Na+ ] at pH 10.1 on C18 PZM [17] (see Table 2).
According to Carr and co-workers [23], in the present work

we assessed the dependence of log kIEX
on log [Na+ ] to test
whether the two-site model and the evaluation method of the

 are accurate. Fig. 8 shows
relative amount of kIEX
to kRP

the excellent linear relationship between log kIEX
for eight
+
basic solutes of high pKa and log [Na ] on the C18 PZM column. It is clearly that the slopes of the plots are very close
to 1, the theoretical value (see Table 3). This validates
the proposed two-site model for such analytes on C18 PZM
wherein the retention involved in a pure reversed-phase and
a pure cation-exchange process. A control experiment (see
Table 4) using bare ZM, i.e. ZM without modified with
C18 P but otherwise identical conditions clearly indicates that
such solutes are nearly eluted at the column dead volume. The
complete lack of retention for these solutes implies that the
inherent zirconol groups could hardly act as cation-exchange
sites on the C18 PZM phase at least at pH 10.1, which further corroborates the high pHpzc of ZM as measured by
our earlier work [34]. Since the two-site model for these
solutes is validated as discussed above, it therefore suggests
that the cation-exchange properties mainly comes from the
chemisorbed phosphonate on C18 PZM; the adsorption form
of C18 P on ZM that proposed as in Fig. 3B and C is more
reasonable.
It is worth noting that both the nitrophenols and benzoic
acid are allowed to elute from the C18 PZM column using 70%
methanol10 mM Tris for pH 10.1, but this approach failed
to separate these Lewis bases due to very poor efficiency and
a change in overall retention behavior over the course of the
test. This suggests that such high-pH mobile phase effectively
reduces the strength of the ligand-exchange retention, but
it is very troublesome to completely eliminate Lewis base
analytes access to the ZM.

H.-B. He et al. / Analytica Chimica Acta 551 (2005) 213221

218
Table 2
Analysis of plots of k vs. 1/[Na+ ]
k at [Na+ ] (mM)a

Solute

Tramadol
Timolol
Metoprolol
Ephedrine
Diphenhydramine
Quinine
Doxepin
Promethazine
a

 )b
Intercept (kRP

 /k  [Na+ ] (mM)c
%kIEX
at

15

25

35

50

1.19

15

25

35

50

1.93
2.55
2.88
4.95
4.93
3.63
7.42
11.74

1.70
1.95
2.09
2.99
4.22
2.91
6.38
9.94

1.54
1.78
1.78
2.41
3.70
2.75
5.65
9.43

1.37
1.43
1.42
1.36
3.55
2.41
5.36
8.70

1.04
0.86
1.28
2.91
1.95
4.45
1.19
7.48

38.73
59.4
70.3
74.1
41.0
46.3
40.0
36.3

30.2
46.7
59.1
57.2
31.0
33.0
30.2
24.8

23.0
41.6
52.0
46.9
21.2
29.1
21.2
20.7

13.6
27.5
39.7
5.9
18.0
19.2
16.9
14.0

MethanolTris buffer containing 15, 25, 35, 50 mM NaCl at pH 10.1 (60%, v/v).
 ) = Intercept of k vs. 1/[Na+ ] (mM).
Intercept (kRP

 /k  = 100
Relative contribution of ion-exchange interactions based on the two site model [23]: %kIEX


k kRP
.
k

3.1.3. Testing in methanolphosphate buffer mobile

phase
Another common used Lewis base buffer for RPLC, i.e.
ammonium phosphate, was also chosen to investigate its
effect on the surface chemistry of the C18 PZM phase. In
view of the competition for ZMs Lewis acid sites between
the small mobile-phase phosphate and the adsorbed C18 P
with large hydrocarbonaceous chain, the stability safety of
the C18 PZM column is to be concerned first when exposed to
phosphate. Fig. 9 indicates that the retention factors for the
neutral probes kept decreasing against the purged eluents volume, implying that the chemisorbed C18 P slowly desorbed
from the Lewis acid site and bled out of the column when
subjected to eluents containing phosphate. As thus, eleunts
containing any phosphate buffer should be avoided when
using the C18 PZM phase due to the column bleed under this
condition. However, as for preventing the deleterious Lewis
acidbase interactions between the surface and the analytes, a
phosphate modified C18 PZM column (p-C18 PZM) is likely
more optimistic than the original C18 PZM column. This
necessitates a further investigation on the surface chemistry
of the p-C18 PZM column.


Fig. 8. Dependence of log kIEX
vs. log [Na+ ] for basic solutes on C18 PZM.
Mobile phases: 60% methanol + 10 mM Tris containing 15, 25, 35 and
50 mM NaCl adjusted to pH 10.1, respectively. UV: 220 nm for timolol,
metoprolol and ephedrine. Other experimental conditions are the same as in
Fig. 1. Solutes: (
) tramadol; () timolol; () metoprolol; () ephedrine;
() diphenhydramine; () quinine; () doxepin; (+) promethazine.

Table 3

Regression data of log kIEX
vs. log [Na+ ] for basic solutes on C18 PZM as shown in Fig. 8
[Na+ ] (mM)

(8.3)e

Tramadol
Timolol (8.8)e
Metoprolol (9.7)e
Ephedrine (10.3)e
Diphenhydramine (9.0)e
Quinine (8.5)e
Doxepin (9.0)e
Promethazine (9.1)e
a
b
c
d
e

 a
log kIEX

15

25

35

50

0.128
0.180
0.306
0.695
0.305
0.226
0.472
0.630

0.288
0.040
0.092
0.476
0.116
0.018
0.285
0.391

0.450
0.129
0.034
0.383
0.104
0.096
0.078
0.290

0.733
0.406
0.248
0.108
0.196
0.334
0.042
0.087

Logarithm of the retention due to pure cation-exchange interactions as referred to Table 2.


r for correlation coefficient of the regression of log kIEX
vs. log [Na+ ] in Fig. 8.
Slope of the linear regression equation of log k vs. log [Na+ ] in Fig. 8.
Standard deviation of the overall linear regression.
pKa s [35] of the test basic drugs.

rb

Slopec

S.D.d

0.975
0.981
0.995
0.982
0.989
0.988
0.995
0.995

1.131
1.068
1.038
1.072
1.001
1.026
1.015
1.009

0.071
0.058
0.028
0.057
0.040
0.044
0.030
0.027

H.-B. He et al. / Analytica Chimica Acta 551 (2005) 213221

Table 4
Retention factors of basic solutes on C18 PZM and Bare-ZMa
Solute

Tramadol
Timolol
Metoprolol
Ephedrine
Diphenhydramine
Quinine
Doxepin
Promethazine

C18 PZM

Bare-ZM

k

 b
kIEX

k

1.93
2.55
2.88
6.32
4.93
3.36
7.42
11.74

0.75
1.51
2.03
4.95
2.02
1.68
2.97
4.26

0.04
0.08
0.07
0.28
0.02
0.02
0.02
0.01

a Mobile phase: 60% methanol + 10 mm Tris containing 15 mM NaCl

adjusted to pH 10.1. Other experimental conditions are the same as in Fig. 8.
b Retention due to pure cation-exchange interactions based on the two site

 , k  = intercept of k vs. 1/[Na+ ] (mM).
model [16]: kIEX
= k kRP
RP

3.2. Surface chemistry characterization of p-C18 PZM

The p-C18 PZM column in this experiment was obtained by
flushing the original C18 PZM column with approximately
4000 ml of 60% methanol20 mM ammonium phosphate
monobasic buffer for pH 7.0. It is believed that the mobilephase phosphate does not displace all the chemisorbed C18 P
from the Lewis acid sites on the ZMs surface, as still a
decreasing trend shown in Fig. 9 after purging with 1600 column volumes of the mobile phase. It should be pointed out
that the p-C18 PZM column was just randomly prepared in
this experiment, and such a p-C18 PZM column in HPLC was
in fact unrealistic as for preparation reproducibility. What we
are really interested in is not its application in HPLC but the
surface chemistry on a pseudo C18 PZM phase that modified by a strong hard Lewis base.
Seven basic solutes were used to characterize the surface
charge, i.e. the cation-exchange character of p-C18 PZM. A
pH 7.0 mobile phase was chosen to ensure that all of the

219

solutes are positively charged because each of the solutes has

pKa above 8.0. As displayed in Fig. 10A, the retention factors
for all probe solutes are significantly higher on p-C18 PZM
than on C18 PZM under the same chromatographic conditions.
Since the hydrophobicity of p-C18 PZM is undoubtedly much
lower than C18 PZM due to the column bleed of C18 PZM, the
higher retention of the probe solutes should be a result of
increased cation-exchange interactions with the negatively
charged phosphate on the surface. A similar study was conducted on the same p-C18 PZM with pH 10.1 mobile phase.
As identical to the results with pH 7.0 mobile phase, the
retention of the probe solutes is obviously much higher on
p-C18 PZM than on C18 PZM at this pH (see Fig. 10B). This
should also be attribute to the much stronger cation-exchange
interactions on p-C18 PZM than on C18 PZM. Drawing a comparison between Fig. 10A and B, it is observed that increasing
the mobile-phase pH to 10.1 increases the solutes retention
on C18 PZM whereas decreases that on p-C18 PZM. This is
reasonable because these solutes become deprotonated upon
increasing mobile-phase pH, the cation-exchange interactions between the solutes and the surface are reduced while
hydrophobic interactions are increased. This indicates predominantly reversed-phase retention on C18 PZM whereas
substantially cation-exchange retention on p-C18 PZM.
The effect of the phosphate modification of C18 PZM on
the chromatography of Lewis base analytes was also investigated. Fig. 11 shows that the hard Lewis bases readily
eluted from the p-C18 PZM column without the use of any
mobile-phase additives. Moreover, much better peak shapes
for these Lewis base solutes were achieved in Fig. 11 as
compared to Fig. 4. This implies that the free Lewis acid
sites on the ZMs surface are likely to be maximally blocked
by attaching a small size of phosphate or phosphonate on
C18 PZM. Separation of hard Lewis base analytes on ZMbased reversed-phases seems feasible by attaching a densely
adsorbed layer of phosphate or phosphonate on ZM.
3.3. Modication of C18 PZM with MPA

Fig. 9. Stability investigation of the C18 PZM column in the presence of mobile phase containing phosphate buffer. Mobile phase: 60%
methanol20 mM NH4 H2 PO4 buffer adjusted to pH 7.0; UV: 254 nm. Other
experimental conditions are the same as in Fig. 1. Solutes: (
) benzene; ()
toluene; () naphthalene; () biphenyl; () fluroene; () phenathrene.

As suggested above, the residual Lewis acid site on

C18 PZM would be likely to be effectively shielded by a
phosphonic acid with less steric hindrance. Therefore, a
methylphosphonic acid (MPA) endcapped C18 PZM stationary phase was prepared by attaching MPA onto the C18 PZM
column. This approach was designed to overcome the Lewis
acid site problem and conducted as following: 500 ml of
the solution of 10% methanol10 mM MPA was prepared
and circulated through a new original C18 PZM column
at 0.3 ml min1 for 68 h. As can be seen from Table 1, the
MPA modification reduces the surface area, pore volume
and the carbon content of the C18 PZM, suggesting that
the dissolved MPA is much more mobile and can find free
Lewis acid sites more readily than the desorbed C18 P. To
quantify any differences in chromarographic efficiency due
to endcapping effects, biphenyl and Lewis bases such as
nitrophenols and benzoic acids were injected on the resulting

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H.-B. He et al. / Analytica Chimica Acta 551 (2005) 213221

Fig. 10. Comparison of retention factor (k ) for basic solutes between C18 PZM and p-C18 PZM with (A) 60% methanol10 mM NH4 Ac buffer adjusted to pH
7.0 and (B) 60% methanol10 mM Tris buffer adjusted to pH 10.1 as mobile phase, respectively. Other experimental conditions are the same as in Fig. 8. (*)
Promethazine do not elute from the p-C18 PZM column for more than 1 h.

MPAC18 PZM column under the same conditions as on the

original C18 PZM column. Less retention but relatively
higher chromatographic efficiency (15373 m1 ) for biphenyl
on the MPAC18 PZM as compared to the C18 PZM was
observed; the nitrophenols were found to be eluted from
the MPAC18 PZM without any Lewis base additives but
using 70% methanolwater as eluent. The results implies
the effective shielding of some residual Lewis acid sites
by MPA. The benzoic acids, however, could not be eluted
under the same condition. The chromatogram shown as in
Fig. 12 indicates that athough the benzoic acid was allowed
to elute from the MPAC18 PZM column when acetate was
added in the mobile phase, no substantial improvement in
the chromatographic efficiency but just less retention for
nitrophenols was observed as compared to Fig. 4. It seems
that unless a prohibitively thick deposition of phosphate or
coating of polymer is applied, it is impossible to completely
eliminate the strong Lewis acidbase interactions between
analytes and the accessible hard Lewis acid sites on C18 PZM.

Fig. 11. Chromatography of Lewis base analytes on p-C18 PZM. The

experimental conditions are the same as in Fig. 4. Solutes: (1) pbenzenedicarboxylic acids; (2) p-nitrophenol; (3) phenol; (4) m-nitrophenol;
(5) o-nitrophenol.

Fig. 12. Chromatography of Lewis base analytes on MPAC18 PZM. The

experimental conditions are the same as in Fig. 4. Solutes: (1) m-nitrophenol;
(2) p-nitrophenol; (3) o-nitrophenol; (4) benzoic acid.

4. Conclusions
This work demonstrates a complex retention characteristics including hydrophobic, cation-exchange and Lewis
acidbase interactions on the C18 PZM stationary phase. And
the experimental results show that, the cation-exchange interactions between the test basic solutes and the C18 PZM are
due to both the phosphonate modification and the adsorption
of Lewis base buffer anion constituents, i.e. acetate, on accessible Lewis acid sites; the inherent zirconol groups is unlikely
to behave as cation-exchange sites even using mobile phase
with pH 10.1. As similar to the impact of residual silanols
on the separation of positively charged amines, the residual
Lewis acid sites on ZMs surface are rather problematic in the
separation of analytes containing hard Lewis base moieties. A
mobile-phase Lewis base additive, such as acetate is required
to elute the hard Lewis base analytes such as nitrophenols, but
the resulting peak and efficiency are very unacceptable and
none of the benzoic acids analytes are eluted under this condition. A few benzoic acid derivatives such as nitrobenzoic
acids and benzenedicarboxylic acids are allowed to elute only
after the C18 PZM column exposed with the phosphate buffer.
Unfortunately, the C18 PZM column is permanently modified by the adsorption of the mobile-phase phosphate which

H.-B. He et al. / Analytica Chimica Acta 551 (2005) 213221

results in excessive cation-exchange retention with sacrificing hydrophobicity. Exploring the chromatography of some
Lewis base analytes on a methylphosphonic acid endcapped
C18 PZM column suggests that, a more densely coverage is
required to overcome the Lewis acid sites problem on ZMbased phases.

Acknowledgements
The authors gratefully acknowledge National Nature Science Foundation of China (Grant: 20475040), the Excellent
Young Teachers Program of MOE, P.R.C.

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