Reprinted from American Laboratory October 2004

Retention Behavior of ReversedPhase HPLC Columns With 100%
Aqueous Mobile Phase
byToshiyuki Enami and Norikazu Nagae
Reversed-phase stationary phases such as octadecyl (C18)and octyl (C8) are the most widely used HPLC columns.
However, it is well-known that C18 and C8 columns
exhibit poor reproducibility of analyte retention when 98%
or higher aqueous solution is used as the mobile phase. Thisphenomenon has been thus far explained by ligand collapse, phase collapse, or the matting effect. On the contrary, the authors propose that the decrease in retention iscaused by aqueous mobile phase being expelled from themesopores of the packing materials, which decreases thesurface area on which the aqueous mobile phase can contact.1–3 Some other parameters also influence retention stability, including pore size, the alkyl chain length of the stationary phase, temperature, pressure, endcapping, andbuffer or ion-pair reagent concentration. Considering theseparameters in detail, it was concluded that the decrease inretention under 100% aqueous mobile phase conditions
was not caused by so-called phase collapse.3

Experimental

This paper describes the effect of the ligand density ofC18 stationary phases on the stability of analyte retention under 100% aqueous mobile phase conditions. Furthermore, it will demonstrate the effect of the pressure
applied in the column on analyte retention.

Distilled water was prepared in the laboratory. Other solvents and reagents, as well as test analytes, were obtained
from Wako (Osaka, Japan). To evaluate the effect of theligand density of C18 stationary phases, Develosil
ODS-5, ODS-K-5, ODS-N-5, ODS-P-5, ODS-MG-5,Table 1

Apparatus
The LC system consisted of a PU-980 pump (Jasco,Tokyo, Japan), a UV-980 or 830-RI detector (bothJasco), a model 7725 injector (Rheodyne, Cotati, CA),and an SIC 480 integrator (System Instruments,Tokyo, Japan). The column temperature of the water
bath was controlled between 20 and 40 °C using an
E5C3 temperature controller (Omron, Tokyo, Japan).
Particle size, pore size, and surface area were measured
by a Coulter Multisizer and Coulter SA-3100 surface
measurement instrument (Beckman Coulter, Fullerton, CA). The carbon content of the stationary phases
was obtained with an MT-700CN-corder (Yanaco,Kyoto, Japan).

Reagents and chemicals

Characteristics of the stationary phases examined
Silica support

Specificsurface
Stationary phasearea(m2 /g)
Develosil ODS-5a350
aDevelosil ODS-K-5350
aDevelosil ODS-N-5350
aDevelosil ODS-P-5350
aDevelosil ODS-MG-5450
ODS-(10)
302
a

Pore
volume(mL/g)
1.0
1.0
1.0
1.0
1.0
1.12

Bonded phase
Meanporediam(nm)1212121210
13.5Particlesize(µm)
555555Carbon Ligand
Porecontent densitydiam(%)b (µmol/m2 )c (nm)b
20
3.0
8.6
192.8–
172.2–
111.2–
15
1.6
7.6
18.4
3.2
10.3

Obtained from manufacturer’s literature.
Measured after end-capping.
cCalculated from specific surface area of bare silica and carbon content before end-capping.
band ODS-(10) (Nomura Chemical,Seto, Japan) were used. These silicabased stationary phases were preparedby treating porous silica gel withdimethyloctadecylchlorosilane, followed by end-capping withtrimethylchlorosilane. The physicochemical properties of the stationary
phases are listed in Table 1. The stationary phases were packed into 150 ×
4.6 mm i.d. stainless steel tubing(Sugiyama Shoji, Yokohama, Japan)
using chloroform as the slurry solvent.

Results and discussion
Figure 1 shows the effect of C18 ligand density on the relative retentionof 2-propanol at 40 °C between theinitial start of the experiment andafter stopping the supply of water into
the column for 1 hr, using water as
the mobile phase and nitrite to measure the unretained time (t0). It can Figure 1
Relationship between C18 ligand density and relative retention of 2-propanol. Colbe seen that the relative retention in umn, Develosil ODS-5, ODS-K-5, ODS-N-5, and ODS-P-5; column dimension, 150 × 4.6 mm,
the case of the 2.8- and 3.0-µmol/m2 i.d.; mobile phase, water; flow rate, 1.0 mL/min; column temperature, 40 °C; column inlet presligand densities is very low compared sure, 6 MPa; detector, RI; sample, sodium nitrite and 2-propanol.
to that achieved with other stationary phases. Actually, the retention in
the case of the 2.8- and 3.0-µmol/m2ligand densities decreased by 90%
during the 1 hr that flow was stopped
compared with the initial value. The
relative retention for the 2.2µmol/m2 ligand density remained atmore than 90% compared to the initial retention. For the 1.2-µmol/m2ligand density, the relative retention
did not vary at all. When the C18 ligand density was low, it is expectedthat more trimethylsilyl (TMS)
groups were introduced by the endcapping that followed. Since TMS isless hydrophobic than C18, the overall polarity of the stationary phasewith low C18 ligand density results ishigher than that with higher C18 ligand densities, leading to an improvement in the intrusion of water into
the pore. This result is consistent Figure 2
Effect of applied outlet pressure on relative retention of nitrite. Columns, ODS-(10)with the authors’ previous result,3 i.e., (h), Develosil ODS-5 (e), Develosil ODS-MG-5 (n); other conditions are the same as in Figure 1.
the higher the polarity, the better theobserved reproducibility of the retention time.
polar-embedded alkyl phases or low ligand density
phases with or without polar end-capping. They proMost commercially available reversed-phase stationary
vide less hydrophobic surface than common C18 staphases with high reproducibility in retention time aretionary phases, which provide poor retention repro-

ducibility under 100% aqueous
mobile phase conditions.
The mercury porosimetry technique
is one of the most useful methods ofelucidating the porous structure ofsolid materials such as silica gel,polycrystalline aggregates, androcks. The theory of mercury
porosimetry is based on the physicalprinciple that a nonreactive, nonwetting liquid does not permeate
Effects of the outlet pressure on the relative retention and water distribution in theinto pores until sufficient pressure is Figure 3column. Column, ODS-(10), 150 × 4.6 mm i.d. Other operating conditions are the same as inapplied to force its entrance. TheFigure 1.
relationship between the appliedpressure (p) and the pore size, into
which mercury intrudes, is given by the following
A certain level of outlet pressure was needed toWashburn equation:4
repermeate the water into the pores, and it wasfound that the outlet pressure required for this purp r = –2 γ cosθpose depended on the pore size. This result indicates that the present method can estimate the pore
where r is the radius of the pore into which mercury cansize of C18 stationary phases using water as theintrude, γ is the surface tension of mercury, and θ is the conmobile phase.
tact angle of mercury on the surface of a solid sample. In thecase of an ODS (C18) surface with water as a mobile phase,
Figure 3 shows the effect of outlet pressure on the relathe Washburn equation can be applied because water istive retention and water distribution in the column.
nonwetting on an ODS surface. In other words, the presAfter stopping the supply of water into the column forsure applied to the packing material keeps water in the
1 hr, the retention time was measured with increasing
pores to some degree, as expected from the above equation.
outlet pressure up to 30 MPa. Then, in the same way,after stopping the supply of water into the column for 1
Figure 2 shows the effect of applied outlet pressure onhr, the outlet pressure was kept at 30 MPa for only 2 sec
the relative retention of 2-propanol. The retention ofusing the long restrictor tubing. Without stopping flow,
2-propanol on each column was measured after the supthe outlet pressure was immediately reduced by shortply of the eluent (water) into the column was stopped
ening the restrictor tubing; subsequently, retention
for 1 hr. During the hour, the column temperature wastime was measured. It took approx. 2 min for the outlet
kept at 40 °C. The pressure was then applied to the outpressure increased up to 30 MPa because the pumping
let of the column using a restrictor tubing with 0.13 mm
system had a bumper in order to flow steadily.
i.d., and the pressure drop over the restrictor was
Hysteresis-like curves were observed for measurement
adjusted by changing its length. It should be noted thatof retention between increasing and decreasing theonce the supply of the water eluent was stopped for 1 hr,outlet pressure, as shown in Figure 3. Such a hysteretic
water was expelled from the pores, leading to decreased
phenomenon is often observed when the porous strucretention when the outlet pressure was atmospheric orture of solid materials is measured by mercury
low. The retention sharply increased at a certain outlet
porosimetry. It can be seen that water as mobile phasepressure, depending on the physicochemical property ofcannot enter into the pores of the packing materials at
the packing materials. This phenomenon is consistent
40 °C even if the outlet pressure is kept at 10 MPa.
with the Washburn equation because the pores of theWater began to permeate into the pores at pressure
packing materials for HPLC show comparatively narhigher than 10 MPa and completely permeated therow distribution. The relative retention was calculated
pores at 30 MPa, as shown in Figure 3. Once waterin Figure 2 by assuming that the relative retention was
filled the pores, it remained there unless the outlet
100% when the applied outlet pressure was 30 MPa. It
pressure was lowered to below 5 MPa. It is concluded
can be seen that the smaller the average pore diameter,that highly reproducible separations can be achieved
the higher the backpressure observed. Since the C18using a conventional C18 column under 100% aqueligand density of Develosil ODS-MG-5 phase is low, itous conditions when the outlet pressure is maintained
showed only a 10% reduction in retention, meaning litat 30 MPa. In a practical sense, a much more important
tle water was expelled from the pores.
finding is that if the outlet pressure is applied at 30

020406080

10005①⑤1530

35⑤ 11MPa

after stoppingflow

2530 MPa④ 36MPa

2016 MPa③ 22 MPa

with maintainingflow

The mobile phase moves out of the pore

The mobile phase exists in the pore

5 MPa11 MPa② 17 MPa

Outlet pressure (MPa)10②③2.5 MPa

Outlet pressure① 8.5 MPa

④Inlet pressure

Figure 3. Effects of the outlet pressure on the relative retention and the water distribution inthe column. Column, ODS (10), 150×4.6 mm I.D. Other operating conditions are the sameas in Figure 1.

Relative retention (%)

MPa for only a few seconds, reproducible retention
time can be obtained as long as the outlet pressure is
kept higher than 5 MPa. Outlet pressure of 5 MPabarely damages HPLC systems, while at 30 MPa, thepressure is too high to ensure system stability. It should
be noted that the present technique5 can be applied to
all reversed-phase stationary phases, although the outlet pressure required depends on the materials.
As proved above, control of outlet pressure allowed theauthors to recover analyte retention even under 100%
aqueous conditions. There is some thought that phasecollapse occurs on C18 stationary phases under 100%
aqueous conditions, leading to a decrease in retention,
but the results revealed here show that phase collapse
has nothing to do with decreased retention.

pores in accordance with the Washburn equation.
Reproducible separation on all reversed-phase stationary phases can be achieved under 100% aqueous mobile
phase conditions by controlling the outlet pressure.
Once outlet pressure as high as 30 MPa is applied for a
few seconds, reproducible retention is achieved unlessthe outlet pressure falls below 5 MPa. This is because
water in the pores of the packing materials remains in
the hydrophobic pores.

References
1.
2.
3.
4.
5.

Nagae N, Enami T. Bunseki Kagaku 2000; 49:887–93.
Enami T, Nagae N. Chromatography 2001; 22:33–9.
Nagae N, Enami T, Doshi S. LC·GC 2002; 20:964–72.
Washburn EW. Phys Rev 1921; 17:273.
Nagae N. Nomura Chemical Co., Ltd., Seto, Japan. Patent pending U.S. Patent Application No. 10/427,916.

ConclusionLess hydrophobic C18 stationary phases with low ligand density gave reproducible retention even under
100% aqueous mobile phase conditions. Increased outlet pressure allowed water to enter the hydrophobic

The authors are with Nomura Chemical Co., Ltd., 851 Anada-Cho,
Seto 489-0003, Japan; tel.: +81 561 48 1853; fax: +81 561 48 1434;
e-mail: nagae@develosil.net.