Technical Note: No. SE1006 March 2014www.chromanik.co.jp

The Retention Behavior of Reversed Phase HPLC Columns
with 100% Aqueous Mobile Phase
Norikazu NAGAE*, Tomoyasu TSUKAMOTO
ChromaNik Technologies Inc., 6-3-1 Namiyoke, Minato-ku, Osaka 552-0001, Japan

Abstract
The retention behavior of the reversed phase was evaluated under 100% aqueous conditions. It is commonly said that reversed
phases, such as C18 (ODS) and C8, show a decrease in the retention time under 100% aqueous conditions. It was found that
100% aqueous mobile phase was expelled from the pores of the packing materials, so that the stationary phase in contact withthe mobile phase decreased and the retention time decreased. Some parameters, such as the pore size, length and ligand densityof the alkyl group of the stationary phase, the amount of residual silanol groups of the stationary phase, salt and ion-pairreagent concentration in the mobile phase, temperature and back pressure of the column, were shown to influence of thedecrease in retention. Furthermore, the wettability between the C18 stationary phase and water as a mobile phase was analyzed.
It was concluded that the retention behavior could be explained by capillarity, and reversed phase separation could be carriedout under 100% aqueous conditions, even if a mobile phase can not wet the stationary phase. Finally, these phenomena wereapplied to reversed phase separation using C18 stationary phase and the mobile phase with less than 70% methanol and morethan 30% water.
Keywords: Retention; Reversed phase; Water; Capillarity

1. Introduction
Currently, alkyl group bonding of carbon number 1 to 18
is widely used as packing material for the chemical bondingof reversed phase HPLC columns. Among these carbon
numbers, the number C18 (ODS) is the most commonlyused bonded phase due to its wide range of applications andsuperior durability compared to other alkyl group bonding.
It is necessary to use mobile phases containing organic
solvent as low as possible when analyzing highlyhydrophilic compounds using C18 packing material.
However, it has been considered that it is essentially
impossible to use a 100% aqueous mobile phase, mobile
phases buffered with salt or acids, or aqueous mobile
phases with a few percent of organic solvents with generalalkyl group bonded reversed phases due to the instability ofsample retentions and decrease in retention as time
progresses. These phenomena were caused by the ligand
collapse of a stearyl group (C18) and it was reported that
this collapse even changed the selectivity of stationary
phases [1-3]. In order to solve this problem, stationary
phases, such as an alkyl group including highly polar amide[4-8] or carbamate [9] groups, that were bonded to silica gel,or that bonds polar groups as an endcapping to reduce theresidual silanol groups on silica gel after stearyl groupbonding, or that is less hydrophilic achieved by reducing
the density of alkyl group bonding [10] have been used as
packing material. Essentially, it has been considered thatthe reproducibility in retention under 100% aqueous mobile
phase was achieved by having polar groups within
stationary phases and making a structure that is less likely
to cause ligand collapse of the alkyl group, such as stearylgroup. However, these stationary phases show different
separation performances due to the selectivity changes
influenced by these polar groups or lower which includes
polar groups that change selectivity due to the influence
from these polar groups or lowered durability. Occasionallyit improves separation, however, there are problems toseparate compounds caused by short retention time at thesame time.
In this paper, we confirm that retention behavior on
normal reversed phases drastically shifts [11-14] under
100% aqueous mobile phase by changing various
conditions, such as stationary phases, mobile phases andtemperature. While C18 packing material with large pore*

Corresponding author: Norikazu NAGAE
Tel: +81-6-6581-0885; Fax: +81-6-6581-0890
E-mail: nagae@chromanik.co.jp

1

size showed stable retention and reproducibility, C18
packing material with small pore size decreased in retention,and even trimethylsilyl silica (TMS, C1), which is
impossible to cause the ligand collapse of alkyl group,
reduced in retention when its pore size was 6 nm, resulting
in phenomena that are unexplainable by the previously
believed ligand collapse of the alkyl group. Based on these
phenomena, we confirm that the process of retention
decrease occurs not by the ligand collapse of alkyl groupbut capillarity [15]. We also report that reversed phases
without the previously mentioned polar groups—that is,
stationary phases which have been considered impossible touse with 100% aqueous mobile phase—can obtainreproducibility with sufficient retention by using 100%
aqueous mobile phase when using stationary phases thatsatisfy certain conditions or changing analytical conditions.
Furthermore, not only 100% aqueous mobile phase, butalso mobile phases that contain organic solvents of morethan 30% shows similar phenomena, and this mechanism
will also be described.

Fig. 1. Retention behavior of a C18 column under 100%
aqueous mobile phase conditions. Shown are (A) initial resultsand (B) results after stopping the pump for 1 h and then starting it
again. Column, 150 mm x 4.6 mm, 5 m dp C18, 10-nm pore size;
mobile phase, water; flow rate, 1.0 mL/min; column temperature,
40 ºC; detection, refractive index; applied pressure 1.7 MPa at thepostcolumn outlet. Peaks, 1 = sodium nitrite (t0), 2 = 2-propanol.

2. Problems when using 100% aqueous mobile phase
It is well known that the retention time of a C18 phase isreduced under 100% aqueous mobile-phase conditions.
Conventionally, mobile phases that contain more than 90%
of water, especially mobile phases with more than 95% ofwater, were considered to be avoided as they exhibit poor
reproducible retention. The retention behavior of sodium
nitrite and 2-propanol when using aqueous mobile phase on
a 10-nm pore size C18 phase at 40 ºC is shown in Fig. 1.
We used sodium nitrite to measure the unretained time (t0).
We changed the solvent in a C18 column to water andapplied pressure at the postcolumn outlet while retention
was measured; the reason for applying pressure will be
explained later in this report. After the column became
stable the sample was injected, and the chromatogram
obtained is shown in (A). Pumping was then stopped forone hour and restarted, and the chromatogram obtained isshown in (B). Stopping the flow for one hour caused shorter
retention times for both sodium nitrite (t0) and 2-propanol.
The difference in retention time between sodium nitrite and2-propanl became the essential retention of 2-propanol inthis case. After no flow for one hour, the t0 decreased from1.79 to 1.20 min and the retention time of 2-propanol
decreased from 7.2 to 1.61 min. As shown in Table 1, theTable 1.

essential retention of 2-propanol decreased to 7.6%
compared to the initial result. Generally, a decrease of t 0 is
unexpected.
We measured the column weight at the same time. The
column was sealed at both ends with plugs as soon as thecolumn pressure was reduced to 0 MPa to measure its
initial weight. We then removed the plugs and one hourlater sealed the column again to measure the weight after
stopping the flow for one hour. As shown in Table 1, thedecreases in t0 and column weight were 0.59 mL and 0.6 g.
The values were almost the same because the specificgravity of water is 1.0 g/mL. Furthermore, we observed that
0.6 mL of water went out of the column during the hourwhen no mobile phase flowed through the column once thepump showed 0 MPa. Out of this 0.6 mL of water,
approximately 0.3 mL flowed out in the first minute and therest, 0.3 mL, flowed out over a period of more than 10
minutes. Before obtaining the initial chromatogram,sufficient amount of mobile phase, a 70:30 (v/v)
acetonitrile/water solution was pumped each time to returnthe column to its initial state.
These results prove that the aqueous mobile phase used

Retention time and weight of a C18 column under 100% aqueous mobile phase conditions
t0

t2-propanolt2-propanol - t0

Mass of Column(min or mL)(min or mL)(min or mL)

(%)

(g)

Initial

1.79

7.20

5.41

100

62.0

After stopping

1.20

1.61

0.41

7.6

61.4

Initial - after

0.59

5.59

5.00

92.4

0.6

2

with C18 phase flows out of the column. 5 μm of C18 silica
gel is packed into the column. The water that flowed out
was either between the packing materials or in the pores ofthe packing material. As we describe later in the paper, if
capillarity is causing water expulsion, the water should becoming out of the pores of the packing material.

materials on the retention time for TMS (trimethylsilyl
silica), C8，C18 and C30 is shown in Fig. 3. Conditions
were the same as shown in Fig. 2. The horizontal axis
shows the pore diameter of each stationary phase, and thevertical axis shows the relative retention time measured one
hour after pumping stops against the initial retention time.
With all the stationary phases, the retention time decreased
as the pore diameter became smaller.

3. Influential parameters on retention reproducibility
3.1. Influences of stationary phases
3.1.1. Pore size of packing materials
The characteristics of the stationary phases examined inthis study is shown in Table 2. The alkyl group of eachstationary phase was generated with excess monofunctional
reagent with relatively high bonding density, and eachphase was endcapped with trimethylsilyl after bonding. Fig.
2 shows the retention behavior of C18 with two different
pore sizes of 10-nm and 22-nm. 10 mM of phosphate buffer(pH 7) was used as mobile phase, and the retention andelution time of thymine and sodium nitrite were measured
at the column temperature of 40 ºC. No pressure was added
to the postcolumn outlet. With the 10-nm C18, the retention
time of thymine decreased by 16% from after one hour to
19 hours later, and it was stable for 72 hours until pumping
stopped. The retention time of thymine has greatly
decreased by 58% when no pressure was added around thepacking material in the column once pumping stopped after
72 hours. Similar changes to sodium nitrite elution
time—that is, considered not retained at all—were observed.
On the other hand, the retention times of thymine andsodium nitrite were unchanged on the 22-nm C18. The only
difference between these two C18 silicas is pore size. Whenthe pore size was around 22 nm, the retention time did notchange. The influence of particle pore size of packing

3.1.2. Alkyl chain length
As shown in Fig. 3, different lengths of alkyl chain
change the relative retention time against pore diameters
and the decreasing ratio on retention time also differs.
When comparing stationary phases using the same pore sizeof 15 nm, C8 decreased in retention time by 80% and C18
by 35%. However, C30 and TMS did not decrease theretention time. The longer the length of ligand for morethan C8, the smaller the decrease in retention is withsmaller pore diameters. That is, the longer alkyl chain ismore stable on its retention even with small pore diameters.
Generally the pore diameter of approximately 10 nm is usedas packing material. However, the ligand length of C30
enables the analyses with stable retention time even using
aqueous mobile phase. Although TMS is in the methylgroup and has a short ligand, it gains high retention with apore diameter of 9 nm. However, with 7 nm of pore
diameter, the retention decreases by greater than 50%. On
the silica surface of C8, C18 and C30, the alkyl group isbonded and the silica is endcapped with trimethysilyl(TMS). However, as shown in Table 2, the ligand density of
TMS is less than 4.8 μmol/m2, and considering the theory
that the residual silanol group on the silica gel surface is 8 –9 μmol/m2, it cannot be assumed that the endcapping withTMS completely silylates residual silanol group. In other

Characteristics of the stationary phases examined in this worka.
Specific Surface
Stationary Phase
Pore Volume
Area
2(m /g)(mL/g)TMS(Trimethylslyl silica)
223
0.93
TMS
302
0.91
TMS
331
0.65
C8
128
0.90
C8
142
0.88
C8
169
0.84
C8
199
0.74
C18
113
0.80
C18
123
0.73
C18
139
0.65
C18
163
0.58
C30
176
0.60
C30
219
0.53
C30
210
0.43
a. All measurements were performed on bonded phases.
Table 2.

3

Mean Pore
Diameter
(nm)
12.9
9.3
6.5
22.4
19.0
15.1
11.3
21.9
17.9
13.9
10.4
10.4
7.2
6.2

Carbon Content

Ligand Density

(%)
4.7
5.0
6.3
5.6
7.2
8.4
10.6
11.4
12.8
13.9
18.4
18.0
19.7
21.2

(μmol/m2)
4.8
4.3
4.1
3.1
3.3
3.1
3.3
3.4
3.2
3.0
3.2
1.8
1.2
1.0

Fig. 3. Relationship between pore size and relative retention
time under 100% aqueous mobile phase. Column, C30 (○), C18( □ ), C8 (♦), and TMS (Trimethylsilyl silica) (∆); column
dimensions, 150 mm x 4.6 mm; sample, thymine. Other
conditions were the same as in Fig. 2. Relative retention time wascalculated as the ratio of the decrease in retention versus initialretention time.

Fig. 2. Effect of the pore size on retention under 100% aqueous
mobile phase conditions. Column, 150 mm x 4.6 mm, 5 μm dp
C18, 10-nm and 22-nm pore size; mobile phase, 10 mM
phosphate buffer (pH7.0); flow rate, 1.0 mL/min; columnpressure, 6.0 MPa; temperature, 40 ºC; detection, UV at 254 nm.
Sample, □ = C18 (10-nm pore size) – sodium nitrite, ○ = C18(22-nm pore size) – sodium nitrite, ■ = C18 (10-nm pore size) –thymine, ● = C18 (22-nm pore size) – thymine.

words, this is not a comparison of alkane with different
lengths of carbon chains, such as octane C8H18, octadecane
C18H38 and triacontane C30H62. Rather, this is the result
about the lengths of alkyl chains as the major constituent ofa stationary phase that is bonded on the silica surface as asingle-layer reversed phase with the thickness of 1 nm – 2nm.
The result of the influence from the pore diameters of
packing materials and the lengths of alkyl chains on
retention points out several conflicts on the theories forligand collapse that was previously believed as the cause ofretention decrease. First of all, does the ligand collapse
occur with small pore diameters and not with large
diameters? As shown in Table 2, the ligand density of
10-nm C18 and 22-nm C18 is 3.2 μmol/m2 and 3.4 μmol/m2,
which are not greatly different. Although the decreasing
ratio of retention time differs largely depending on pore
diameter, it is unlikely that ligand collapse depends on pore
diameter. Additionally, when the pore diameter is small,
TMS, which has a short alkyl group and that ligand collapse
must not physically occur with, experienced a decrease inretention time. Therefore, ligand collapse cannot be
explained by pore diameter.

Fig. 4. Relationship between C18 ligand density and relative
retention of 2-propanol under 100% aqueous mobile phase.
Column, Develosil ODS-5 (3.0 μmol/m2), Develosil ODS-K-5(2.8 μmol/m2), Develosil ODS-N-5 (2.2 μmol/m2), Develosil
ODS-P-5 (1.2 μmol/m2); column dimension, 150 mm x 4.6 mm;
temperature, ∆ = 40 ºC, ○ = 30 ºC, □ = 20 ºC. Other
conditions were the same as in Fig. 1.

pumping stopped was measured. The same series of C18
phase from Nomura Chemical was used. The ligand
densities were modified at 3.0 μmol/m2，2.8 μmol/m2，2.2μmol/m2 and 1.2 μmol/m2, and C18 was bonded on silica
gel with the same physical specification. After bonding C18
for all the silica gel, TMS was used for endcapping on

3.1.3. Ligand density
Fig. 4 shows the result of the impact to the ligand densityof alkyl group (stearyl group, C18) on retention. The
conditions were the same as shown in Fig. 1, and thedecreasing ratio of retention of 2-propanol one hour after4

Table 3.

Effect of residual silanol groupsa.
ColumnEndcapping

Hydrogen-bonding
Capacityb

Relative Amount of Residual Silanol Groups
Final Retention Time Ratioc (%)

Develosil ODS-A-5
No
1.42
Much
97.5
Develosil ODS-T-5
Single0.53
Little58.2
Develosil ODS-HG-5
Double0.38
Very little
39.9
a. Conditions as shown in Fig. 3.
b. Hydrogen-bonding capacity: separation factor of caffeine and phenol.
c. The final retention time ratio is the final retention when the retention time decreased to the initial retention time.

residual silanol by an identical method. The comparison of
influences from column temperatures at 40 ºC, 30 ºC and 20ºC will be discussed later in this paper. When the column
temperature was 40 ºC, C18 with high density, such as 3.0μmol/m2 and 2.8 μmol/m2, decreased retention by around90%. However, the C18 with 2.2 μmol/m2 showed only afew percent decrease in retention, and the C18 with 1.2
μmol/m2 did not show a decrease in retention. In short, theligand density of C18 greatly impacts reproducibility inretention, and the lower the density was, the lower thedecrease in retention was. The C18 with low density has
more TMS used for endcapping. Therefore, the stationary
phase should be considered as a mix of C18 and TMS
rather than C18, and this stationary phase is considered tohave similar behavior as 10 nm TMS that does not change
its retention.

Fig. 5. Effect of concentration of salt in the mobile phase.
Column, 150 x 4.6 mm, 5 m dp C18, 10-nm pore size; mobile
phase, ∆ = water, □ = 10 mM ammonium acetate (pH7.0), ○= 100 mM ammonium acetate (pH7.0); column temperature, 40ºC. Other conditions were the same as in Fig. 1.

3.1.4. Residual silanol groups
The effect of residual silanol groups was investigated byusing C18 with different endcapping. Table 3 shows theeffect of residual silanol groups on three types of C18
columns. One column was not endcapped, and the others
were single- and double-endcapped. Columns used wereDevelosil ODS-A-5, Develosil ODS-T-5 and Develosil
ODS-HG-5 by Nomura Chemical. The residual silanolgroup amount was measured based on the separation factor
of caffeine and phenol using a mobile phase of 30:70 (v/v)
methanol-water. The larger the retention of caffeine andseparation factor of caffeine and phenol, the more residual
silanol groups exist. The retention was measured under thesame conditions as shown in Fig. 4. It proved that C18
stationary phase with more residual silanol groups showed
fewer changes in retention time. Thus, silanol groups
stabilize retention.

pumping stops as 100%. In the first 10 minutes after
pumping stops, the retention greatly decreased and thengradually decreased over 60 minutes. When the column
temperature was at 40 ºC, more than 80% of the total
retention decrease was observed in the first 10 minutes.
Among these three kinds of mobile phases, water with 0
mM of salt concentration showed the least relative retention.
The higher the concentration of salt, the greater the increase
in relative retention and the lower retention decrease ratio.
3.2.2. Concentration of ion-pairing reagent
Fig. 6 shows the effect of the concentration of ion-pairing
reagent. Three kinds of mobile phases were prepared, whichwere 10 mM sodium phosphate (pH 7), 10 mM sodium
phosphate with 1 mM sodium octanesulfonate and 10 mM
sodium phosphate with 5 mM octanesulfonate. The
retention behavior was plotted having the horizontal axis as
pumping stop time the same as Fig. 5. Other conditions
were the same as shown in Fig. 1. With a mobile phasewithout ion-paring reagent, the relative retention decreased
to around 10% in 10 minutes. Although a mobile phase with

3.2. Effect of mobile phase
3.2.1. Concentration of salt
The retention time of three kinds of mobile phases withdifferent salt concentrations is shown in Fig. 5. Water, 10mM ammonium acetate (pH 7) and 100 mM ammonium
acetate (pH 7) were used as mobile phases. Other
conditions were the same as shown in Fig. 1. The horizontal
axis shows the time that pumping stops and the vertical axis
shows relative retention having the retention before

5Fig. 6. Effect of concentration of the ion-pairing reagent in themobile phase. Column, 150 mm x 4.6 mm, 5 μm dp C18, 10-nm
pore size; mobile phase, ∆ = 10 mM sodium phosphate (pH7.0),□ = 10 mM sodium phosphate-1 mM sodium octanesulfonate(pH7.0), ○ = 10 mM sodium phosphate-5 mM sodium
octanesulfonat (pH7.0). Other conditions were the same as inFig. 1.

Fig. 7. Effect of concentration of organic solvent in the mobile
phase. Column, 150 mm x 4.6 mm, 5 μm dp C18, 10-nm poresize; mobile phase, ♦ = methanol/water, □ = acetnitrile/water, ▲= ethanol/water, ○ = dimethylformamide (DMF), ∆ = 2-propanol;
sample, sodium nitrite. Other conditions were the same as in Fig.
1.

than 2% acetonitrile and ethanol, and less than 1%
2-propanol. Although 1% of 2-propanol shows stableretention, the polarity of 2-propanol solution and 5%
methanol solution is almost the same and therefore polar
samples such as thymine would show almost the same
retention time even using these mobile phases. Even though
the retention of 2-propanol with low concentration to 1% isstable, the separation is almost as same as 5% methanol.
Therefore, there is no merit to using 2-propanol. Put simply,
the polarity of mobile phases with any organic solvents are
the same as the concentration level of organic solvents
when mobile phases are being expelled from packing
material pores, and retention cannot be greater by changing
the kinds of organic solvents under stable retention
conditions.

1 mM ion-paring reagent showed drastic change in
retention and the relative retention decreased toapproximately 50%, it became stable after 10 minutes. On
the other hand, a mobile phase with ion-paring reagent of
normal concentration, which is 5 mM, the retention hardly
changed and showed almost 100% of relative retention.
Thus, it was confirmed that adding ion-paring reagent
reduces retention changes and there became no change in
retention at a concentration of 5 mM.
3.2.3. Concentration of organic solvent
So far, the changes in retention time using water orphosphate buffers were discussed. Now, we will discuss theconcentration of organic solvent. Fig. 7 shows different
elution time of sodium nitrite as unretained compoundusing mobile phases of water added following solvents:
methanol, acetonitrile, ethanol, dimethylformamide (DMF)
and 2-propanol. The difference of elution time on thevertical axis shows times after the elution time was being
deducted at 10% concentration from elution time of eachconcentration. The smaller values show the faster elution
times. In other words, as described in the article discussing
problems to use 100% aqueous mobile phase, the different
elution times correspond to negative values of the mobile
phase amount that was expelled from the packing material
pores. The differences of elution times, that is, the expelledamount of mobile phase from pores of packing materials,
greatly varied depending on the kinds of organic solvents.
We confirmed that mobile phases were expelled from thepacking material pores with less than 5% methanol, less3.3. Temperature
The changes in retention time of 10-nm C18 and 10-nm
C30 at different temperatures is shown in Fig.s 8 and 9.
Conditions were the same as Fig. 2 except for stationary
phases and temperatures. With 10-nm C18, the retention
time decreased at 30 ºC and 40 ºC even while pumping.
However, at 20 ºC, 10 ºC and 5 ºC, retention time did notchange while pumping and it decreased only after pumping
stopped. Furthermore, the lower the temperature was, thelower the decreasing retention time ratio. At thetemperature lower than 10 ºC, the decreasing retention time
ratio was less than 10% even after stopping the pump, andtherefore it is possible to use this phase. Moreover, 10-nm
C18, which was considered to be impossible to use, canrealize separations with high reproducibility by setting

6

Fig. 8. Effect of temperature on C18 column (10-nm pore size)
under 100% aqueous mobile phase conditions. Column
dimensions, 150 mm x 4.6 mm; temperature, ○ = 5 ºC, □ =10 º C, ■ = 20 º C, ▲ = 30 º C, ● = 40 º C. Other
conditions were the same as in Fig. 2.

Fig. 9. Effect of temperature on C30 column (10-nm pore size)
under 100% aqueous mobile phase conditions. Column
dimensions, 150 mm x 4.6 mm; temperature, ○ = 30 ºC, □ = 40
ºC, ∆ = 80 ºC. Other conditions were the same as in Fig. 2.

column temperature below 30 ºC, switching to aqueous
mobile phase and continuously pumping. This result
matches the changes in retention time at different
temperatures shown in Fig. 4. 10-nm C30 shows similar
behavior to 10-nm C18, however the temperature range
differs. 10-nm C30 did not change in retention time at 30 ºCeven after pumping stopped. At 40 ºC, the retention timedecreased by 5% after pumping stopped, and at 80 ºC, it
decreases even while pumping and showed further decrease
after pumping stopped. Compared to 10-nm C18, 10-nm
C30 showed changes in retention at the higher temperature
range of 40 ºC.

Fig. 2, 8 and 9, the retention changes over 10 hours during
pumping. Therefore, the retention was measured after
pumping more than 10 hours once the back pressure
decreased. Up to 5 MPa, the retention did not change anddecreased below 5 MPa. The condition of the aqueous
mobile phase that is inside the packing material pores
within the column is also shown in Fig. 10. It is consideredthat the retention increase and the increase of packing
material with aqueous mobile phase permeating its pores
occur at the same time. Aqueous mobile phase permeates
inside of pores at a pressure of about 16 MPa. What needs
to be looked at from this result is that greatly different
curves were obtained when increasing and decreasing backpressure. This means that an extremely large hysteresis
exists. Similarly, aqueous mobile phase sometimes
permeates packing material pores at a back pressure of 5
MPa. This is determined by the condition before a backpressure of 5 MPa is applied. When the aqueous mobile
phase permeates packing material pores, the mobile phasestays inside of the pores. On the other hand, when the
aqueous mobile phase is not inside of the packing material
pores, it does not permeate the pores even if a back pressureof 5 MPa was applied.
Hysteresis such as this exists in nature, but the hysteresis
effects on the aqueous mobile phase and C18 stationary
phase is quite large. In order to measure pore size
distribution of silica gel, an instrument, mercury intrusion
porosimeter, is used. As mercury cannot be wet by any
substance, a certain level of pressure is required to have it
permeate pores. Pore size distribution can be determined bymeasuring the pressure and volume that mercury permeates

3.4. Back pressure after the column
As aqueous mobile phase is expelled from the packing
material pores, how retention would change wasinvestigated when there is pressure around the packing
material. The result is shown in Fig. 10. The condition is
the same as Fig. 1. 10-nm C18 was used to measure theretention of 2-propanol at 40 ºC under aqueous mobile
phase. Retention has completely decreased one hour after
pumping stops. A tubing of ID 0.1mm was connected to thecolumn outlet and back pressure was applied to thepostcolumn. The back pressure was controlled by graduallyextending the tubing from 0.2 m to 3 m without stopping
pumping. The retention did not change when the backpressure was increased to 7.5 MPa after the retention
decreased once. However, the retention suddenly increased
between 10 MPa and 16 MPa, and gradually increased up to
30 MPa. The back pressure decreased from 30 MPa to 0
MPa without stopping the pump. According to the results in7

Fig. 10. Effects of the outlet pressure on the relative retention and water distribution in the column. Column, 150 mm x 4.6 mm, 5 μm
dp C18, 10-nm pore size. Other conditions were the same as in Fig. 1.

pores. This relation is known as Washuburn’s [16] equation
shown in formula (1).
Pr = -2γcosθ

(capillary) is set in liquid, the liquid surface elevates higher
than the tube or is depressed. Depending on the magnituderelationship between the cohesion of liquid molecular andadhesion between the liquid and capillary wall, the liquidsurface elevates when liquid is wetting the tube (great
adhesion) and is depressed when not wet. Capillarity can be
expressed in the following formula having the h as theheight differences in and out of the tube, r as the radius of
the tube, ρ as liquid density, γ as the surface tension ofliquid, θ as a contact angle and g as the acceleration ofgravity.

(1)

The r is radius, γ is surface tension, θ is a contact angle and
P is pressure.
In fact, the relationship between the C18 stationary phaseand water is the same as silica gel and mercury mentioned
above. Comparable curves can be obtained by shortening
the column length, making the pressure difference close tozero between the column inlet and outlet, anddifferentiating the curve when back pressure is increasing
under the same condition as shown in Fig. 10. When wemeasured the pore size distribution of silica material of
10-nm C18 stationary phase using this mercury intrusion
porosimeter, the curve showing the relationship between thevolume of mercury permeate pores and pressure wasobtained, and this curve differed when pressure wasincreasing and decreasing. Thus, hysteresis exists. Both this
hysteresis and the one shown in Fig. 10 were measured withthe pores of the same silica gels and therefore there should
not be a large difference on its pore shapes and distribution.
However, it was confirmed that the hysteresis in Fig. 10
was much larger.

h=2γcosθ/rρg(2)

As octadecane of carbon number 18 cannot solve withmethanol or water, the stearyl of C18 stationary phase isconsidered to be mixed in the methanol and aqueous mobile
phase [18-20]. Therefore, the surface of C18 stationary
phase within the packing material pores has a similar
physical property as octadecane and does not get wet withwater. As a result, a force to move out of the poresfunctions due to the capillarity. The aqueous mobile phasethat is expelled from packing material pores by capillarity is
considered to cause the retention decrease of C18 stationary
phase when using the aqueous mobile phase. The
previously mentioned Washburn equation fits the capillarity
formula by transforming it and shows the same
phenomenon. When changing the liquid explained in thiscapillarity exclusively to mercury, it applies for the
Washburn equation. Majors [21] and his associates explain
the necessary pressure for water to permeate the pores of

4. Wettability of the stationary phase and capillarity
4.1. Capillarity
Capillarity [17] is a phenomenon that when a thin tube

8

dried reversed phase Solid Phase Extraction (SPE) packing
material by using the Laplace-Young equation. Thisequation is the same as the Washburn equation when theradius r moves to the left, and shows the same as thecapillarity formula. Originally, the Laplace–Young theory
was derived from the observations of surface tension ofsubstances, and as a result, their equation is the same as
capillarity formula. Therefore, it is more appropriate toexpress the phenomenon that liquid being expelled from thepores in which the surface does not get wet as a capillaryaction is caused by capillarity rather than employing thetheory of Laplace-Young.
Based on the result shown in Fig. 10, the pressure thatwater permeates the pores of C18 stationary phase packing
material with an average pore diameter 10 nm is
approximately 16 MPa. Applying this value, the surface
tension of 69.6 dyne cm-1 at 40°and 5.2 nm as the radius ofpore diameter to the Washburn equation (1), the contact
angle θ should be 126°. The contact angles can becalculated for C8 and C30 stationary phases under similar
experiments. It is also possible to estimate contact angles
from the data shown in Fig. 3. Based on the formula (1),which is the Washburn equation, supposing that the cosine
function values of each pore diameter and contact angle areproportional when the relative retention time in Fig. 3
becomes 50%, the contact angles of the C8 and C30
stationary phases can be calculated based on the same C18
stationary phase. Pore diameters of C8, C18 and C30
stationary phases are 17.1 nm, 13.2 nm and 6.9 nm with
50% of relative retention time, and as the contact angle ofC18 stationary phase is 126°, the same for C8 and C30
stationary phases would be 140° and 108°.
Retention changes with reversed phase stationary phases
under aqueous mobile phase are considered to be due toaqueous mobile phase that comes expelled from pores ofpacking materials because of capillarity, and parameters
related to retention reproducibility can be explained basedon capillarity. More aqueous mobile phase is expelled whenpore diameters are small and less expelled whenconcentrations of salt and ion-pairing reagent are high withsmall liquid surface tension. It is also assumed that thecontact angles change depending on alkyl chain lengths,
ligand density of alkyl groups and the amount of residual
silanol groups. As temperature increases, it is consideredthat liquid density, surface tension of liquid and contact
angles change. Furthermore, it is thought that retentions
recover by increasing the back pressure at the postcolumn
outlet because higher back pressure is applied than the force
that moves liquid out of the pores due to capillarity hence
liquid moves back into the pores.
In 1992, Montgomery and his associates [2] fixed theC18 stationary phase on a glass surface and measured thecontact angle of water and the C18 stationary phase fixed

on the glass surface. They found that the contact angle was
93° and reported that water does not wet the surface of the
C18 stationary phase. However, in the discussion paragraphof the literature, they concluded that, as in previous cases,
the retention decreases of the C18 stationary phase under
100% aqueous mobile phase occurs due to the ligand
collapse of alkyl groups after all. It can be easily assumed
that water is expelled from the pores due to capillarity when
the contact angle is greater than 90°, however they did notreach this conclusion. That they have reached the sameconclusion can be considered so as the curves expressing
the relationship between pressures and retentions when thepressure around the packing material increases anddecreases to show how aqueous mobile phase moves in andout of pores is greatly different as described in the previous
section, thus there is a large hysteresis.
4.2. Retention behavior of dried C18 column
When using the mixed solvent of organic solvent andwater as a mobile phase, it is considered that the organic
solvent in the mobile phase is solvated [18, 19, 22-25] withstationary phases. Using C18 stationary phase after
removing the solvated organic solvent, the retention
reproducibility in aqueous mobile phase was investigated.
Fig. 11 shows the result. Filling the inside of a column withchloroform that is packed with 10-nm C18 opening bothcolumn ends, we evaporated the chloroform in a chamber at

Fig. 11. Typical chromatograms of nitrite and nucleic acidbases. Column, 150 mm x 4.6 mm, 5 μm dp C18, 10-nm poresize; mobile phase, water; temperature, 40 ºC; detection, UV at
254 nm; flow rate, 1.0 mL/min; sample, 1 = sodium nitrite, 2 =cytosine, 3 = uracil, 4 = cytidine, 5 = uridine, 6 = thymine; A:
C18 packing materials in a column were dried without plugs at 70ºC for 10 hours after chloroform flowed in a C18 column. Then,water as a mobile phase flowed at 40 ºC at first and sample wasinjected into a column. B: Subsequently, 23 MPa of back pressure
was added after outlet of a column without stopping flow. C:
Flow was stopped for 1 hour, then flow was restarted. D:
Successively, 23 MPa of back pressure was added after outlet of acolumn without stopping flow again.

9

70 ºC to dry the packing materials in the column. Then, we
pumped aqueous mobile phase at 40 ºC into this column
filled with dried C18 and injected the mixed samples of
sodium nitrite and 5 kinds of nucleic acid bases. The
chromatogram A shows the first injection of the sample
after the baseline became stabilized and the 6 kinds ofcomponents eluted without being separated. From thiscondition, a tubing of ID 0.1 mm was connected to thepostcolumn outlet without stopping the pump and applied
the back pressure of 23 MPa to the column outlet. It is now
assumed that aqueous mobile phase mixed with air cameout of the column and residual air that existed in pores ofthe dried C18 packing materials was expelled. The
chromatogram B shows the result of injection after thebaseline stabilized. The retention time of thymine wasapproximately 8.5 minutes and all the six different ofcomponents were completely separated. Later, we stopped
the pump for one hour and pumped again without applying
pressure at the postcolumn outlet. The obtained
chromatogram is shown as C, and the retention decreased
by around 70%. Then 23 MPa of back pressure was applied
again at the postcolumn outlet with continuously pumping.
The first time, air came out of the column, however we
could not confirm if there was air in the mobile phase thatcame out of the column the second time. Chromatogram D,
which was obtained once the baseline became stabilized,
was almost the same as chromatogram B. There was air in
the pores of the C18 packing material with chromatogram A
and approximately pressure of 6 MPa was applied at thecolumn inlet side. However, with this pressure, aqueous
mobile phase did not permeate the pores of the packing
material and it is considered that it only came out between
packing materials. Therefore, the interaction with more than
99% of C18 stationary phase in the pores did not occur andalmost all the components were eluted without being
retained. The elution time of sodium nitrite that is usually
not retained is approximately 1.8 minutes. However, it was
eluted at 1.1 minutes from chromatogram A proving theelution without permeating the pores of the packing
material based on the elution time. As chromatogram B
shows, by applying back pressure of 23 MPa, the aqueous
mobile phase permeates pores, and the retention and elution
time of sodium nitrite increased. Even if the aqueous
mobile phase permeated the pores of the packing material,
it was expelled from the pores once pumping stopped, andpressure around the packing material decreases toatmospheric pressure and the retention decreased as shownin chromatogram C. The retention increased again when 23
MPa back pressure was applied to the postcolumn outlet,
but no air was expelled. This means that no air was in the
pores of the packing material after the aqueous mobile
phase moved out. Considering that the pressure that arises
due to capillarity is more than a few MPa, it is easily

estimated that the inside of the pores is close to a vacuum,
which is -1 atmosphere. Therefore, as the water exits out ofthe pores, there is water vapor in the packing material poresand the water vapor pressure is believed to be thetemperature.
4.3. Wettability on the surface of the C18 stationary phase
The dispersion state of 10-nm C18 packing material to amixed solvent of methanol and water is shown Fig. 12.
Image C shows that 10-nm C18 packing material dispersed
at 70% methanol and was wet. As the 70% methanol wets
the surface of C18, the solvent permeates the pores due tocapillarity. Image B shows that the packing material did notdisperse by agitation only at 50% methanol but partiallydispersed by applying ultrasonic vibration. As shown in
Image A, the packing material did not disperse and wet at
30% methanol even when applying ultrasonic vibration.
From this result, we confirmed that methanol wets thesurface of C18 at more than 70% but not less than 50%.
4.4. Comparison of the C18 stationary phase that is wet bymethanol and dried
We pumped methanol to a dried C18 column for 30
minutes to wet the C18 stationary phase and applied
solutions of 10:90, 30:70, 50:50, 70:30 and 90:10 (v/v)
methanol to water as mobile phases to investigate theelution time (t0) of sodium nitrite and uracil that are
believed to not be retained under the said condition.
Sodium nitrite was used as a sample to measure each t0

Fig. 12. Photograph of C18 packing materials mixed withmethanol/water mixture solution with ultrasonic vibration.
Packing material, 5 μm dp C18, 10-nm pore size; Solution, A =methanol/water(30:70), B = methanol/water(50:50), C =methanol/water(70:30).

10

when methanol concentration of the mobile phase was 10%
and uracil was used when it was higher than 30%. In themeantime, we also investigated the elution time (t0) of
sodium nitrite and uracil by applying a solution of 10:90,
30:70, 50:50, 70:30 and 90:10 (v/v) methanol to water as
mobile phases to a dried C18 column. The amount ofmobile phase filling the spaces between particles and in thepacking material pores corresponds to the value that theelution time was multiplied by the flow rate. The relative
unretained time was sought by dividing the elution time ofthe dried C18 column by the same of the wet C18 column
with methanol. The relationship between the methanol
concentration ratio on the mobile phase and relative
unretained time is shown in Fig. 13. Once methanol was
pumped, C18 stationary phase becomes wet with methanol
and methanol permeates the packing material pores bycapillarity. When directly replacing this condition to mobile
phases of 10:90, 30:70, 50:50, 70:30 and 90:10 (v/v)
methanol to water, it is known that there is enough retention
reproducibility with each mobile phase and the retention
does not decrease as time goes by. That is, these mobile
phases are permeating the C18 packing material pores. Thatthe ratio of methanol is more than 70% and relative
unretained time is 100% means that the solvent with morethan 70% methanol that is supplied to a dried C18 column
wets the surface of C18 stationary phase and permeates thepacking material pores by capillarity. However, with thesolvent of less than 50% of methanol ratio, the relative
unretained time falls by 80%. This means that even ifsolvent with less than 50% methanol was pumped to a dried

C18 column, this solvent does not wet the C18 stationary
phase and nor permeate the packing material pores. That is,
the elution time, t0, increased and the relative unretained
time decreased as the solvent does not permeate the poresof the C18 packing materials. Furthermore, the reason why
the relative unretained time is large with a higher methanol
ratio even if it is decreasing is because there is a columnpressure of 0.1 MPa at the column inlet side even if usinglarge packing particle sizes and this pressure is believed topush more solvent with higher methanol ratio into thepacking material pores.
Once the mobile phase is expelled from the packing
material pores, it is necessary to pump the mobile phasethat wets the stationary phase, such as a methanol to water
ratio of more than 70% methanol concentration to permeate
the packing material pores again. When a mobile phasepermeates the packing material pores, a mobile phase, suchas 10:90 (v/v) methanol to water, can be replaced without
being expelled from the pores if it was under atmosphericpressure.
4.5. Capillarity of C18 stationary phase when using mixed
organic solvent and water mobile phases
As described previously, methanol-water mobile phasewith less than 50% methanol concentration does not wet theC18 stationary phase. That is, there is a force to expel themobile phase from the packing material pores. However,usually retention reproducibility is not a problem withmethanol-water mobile phase with more than 10%
methanol concentration. As shown in Fig. 10, a large
hysteresis exists and therefore a pressure of more than 16
MPa is necessary for water to permeate the C18 packing
material pores. However, when water already permeates thepores, the force to expel water is weak. Considering this
hysteresis, although a higher pressure than atmosphericpressure is necessary for methanol to water mobile phasewith less than 50% methanol concentration to permeate theC18 packing material pores, if the mobile phase wasalready in the pores, then the pressure to expel it isestimated as less than atmospheric pressure (0.1 MPa). We
made a comparison of the mobile phase weights that were
expelled from a C18 column by using a vacuum pump todecrease the pressure around the C18 column fromatmospheric pressure down to 0.01 MPa. The result isshown in Fig.s 14 and 15. The relationship between thepressure around the column and the column weight isshown in Fig. 14. We used a 4.6 x 250 mm column packed
with 10-nm C18 stationary phase. We replaced the solventin the column from methanol to 90:10 (v/v) methanol towater, left the column for 20 minutes in a vacuuming
chamber at 40 ºC and plotted the decreased column weights
on the vertical axis with negative displays. Fig. 15 shows
the weight changes against methanol concentration. Under

Fig. 13. Relationship between methanol concentration in
mobile phase and relative unretained time. Column, 150 mm x
4.6 mm, 20 μm dp C18, 10-nm pore size; mobile phase,methanol/water = (10:90), (30:70), (50:50), (70:30), (90:10);
temperature, 40 º; detection, UV at 254 nm; flow rate, 0.2
mL/min; column pressure, 0.1 MPa; sample, sodium nitrite.

11

Fig.14. Relationship between pressure around the column andcolumn weight. Column, 250 mm x 4.6 mm, 5 μm dp C18, 10-nm
pore size; enclosed solution, methanol/water = (10:90);
temperature, 40 ºC. Column weight was measured at the initialand after the pressure was reduced for 20 min.

Fig. 15. Relationship between methanol concentration in
mobile phase and column weight. Column, 250 mm x 4.6 mm, 5μm dp C18, 10-nm pore size; enclosed solution, methanol/water= (10:90), (30:70), (50:50), (70:30), (80:20); temperature, 40 ºC.
Column weight was measured at the initial and after the pressure
was reduced to 0.01 MPa for 20 min.

0.01 MPa pressure, we plotted the decrease in column
weight after 20 minutes when methanol concentration in thecolumn became 10%，30%，50%，70% and 80% as shownin Fig. 14. Fig. 14 also shows 90:10 (v/v) methanol to water
solvent was not expelled from the C18 packing materials
pores at 1 MPa, but it moved out at below 0.05 MPa.
Moreover, Fig. 15 explains that although there was nochange in the weight of the methanol concentration of morethan 70% that wets C18 stationary phase at under 0.01 MPaand solvent stayed in the pores, the column weightdecreased with the methanol concentration of less than 50%
that does not wet C18 stationary phase and solvent was
expelled from the pores. Based on this result, we confirmedthat the mobile phase containing methanol from 10% to
50% does not wet C18 stationary phase and there is a force
to expel the mobile phase from the pores due to capillarity,
but the mobile phase stays in the pores under atmosphericpressure and retentions are reproducible as this force issmaller than atmospheric pressure (0.1 MPa).
As described previously, the larger packing material
pores realize higher retention reproducibility. There are no
significant differences in the wettability of the aqueous
mobile phase against the stationary phase surfaces evenwhen the pore diameters are different, and it is believed that
wettability does not change due to the sizes of the pores.
That is, as the expel force due to capillarity decreases as thepore sizes increase and is weaker than the atmosphericpressure, it is assumed that the aqueous mobile phase stays
in the packing material pores and therefore the retention

does not decrease. Although the retention reproducibility
increases with longer alkyl chains even with the same pore
diameters, it is not that water wets C30 stationary phase butthat the contact angle between the stationary phase andwater decreases when alkyl chains get longer from C8 to
C30. However, this contact angle does not decrease to lessthan 90°, which means wettability; rather, it means that
although the contact angle is larger than 90°, it is getting
close to 90°for less wettability. It is therefore assumed thatthe aqueous mobile phase stays in the packing material
pores as the force to expel the aqueous mobile phase fromthe pores decreases and becomes less than atmosphericpressure as a result. Stationary phases that contain polargroups in its alkyl chains or reversed phase stationary
phases that high-polar endcapping was modified withrealize stable retention even under aqueous mobile phase.
This is also not because the stationary phase surfaces are
wet but because the aqueous mobile phase stays in the
pores due to atmospheric pressure being the same asstationary phases with long alkyl chain groups.
As discussed above, it is believed that the stationary
phase surfaces of most of the reserved-phase would not
become wet by aqueous mobile phase. It has been believedthat sufficient separations could not be achieved unless themobile phase wets the stationary phase. However, as shownin Fig. 11, sufficient separations could be achieved with
C18 stationary phase by applying a back pressure at thepostcolumn outlet. These mobile phases do not wet
stationary phases and are instead thought to contact each12other on the interface with a space of 0.2 nm to 0.3 nm. As
using hexane and water, which do not mix together, for theliquid-liquid partition of solute, C18 stationary phase andaqueous mobile phase can separate solute for each phase aslong as they are in contact with each other at a certain
interface. It is possible to retain and separate solute once
solute is partitioned to the stationary phase. A theory thathas been conventionally believed “separations cannot be
achieved unless the mobile phase wets the stationary phase”
should be expressed as “separations cannot be achieved
unless the mobile phase exists in the packing material
pores”.
It is believed that water does not wet the surfaces ofsilica based C18 stationary phase, however, watermolecules exist in the stationary phase including its
surfaces. For example, C18 packing materials weredispersed in a solvent of 80:20 (v/v) methanol-water. Laterit was washed by acetone and hexane and dried sufficiently
at 100 ºC, then applied to a vacuum dry at 180 ºC. During
the vacuum dry, water that corresponds to approximately
1% of the weight of the C18 packing material was trapped.
That is, it can be assumed that water molecules areadsorbed in the siloxane and silanol groups that are on thesurfaces of the roots of C18 alkyl chains. This is a micro
point of view for the inside stationary phases. In themeantime, the discussion on the wettability of C18
stationary phase is viewed with a macro issue that is theexistence condition of water in the packing material pores.
Therefore, it is possible that water exists as molecules in
C18 stationary phase but does not wet the surface of C18
stationary phase.

from them. By increasing the back pressure at postcolumn
outlet to 30 MPa and then decreasing to 5 MPa, the mobile
phase fills the packing material pores with the pumppressure increasing by only 5 MPa, hence separations withhighly reproducible retentions become possible [26].
6. Conclusion
In this study, we investigated the retention decreases
when using water and buffers as mobile phases withreversed phase stationary phases and clarified the cause,
which is that mobile phase was expelled from the packing
material pores. We also found that capillarity functions onmobile phases containing organic solvents as on aqueous
mobile phase and that there is a force to expel mobile phasefrom the packing material pores. The phenomena observedon reversed phase chromatography that were nottheoretically discussed have been recognized as empiricalrules so far. However, now many of these empirical rules
can be explained by capillarity studied in this work. We
hope that our study contributes to the analyses of highlypolar compounds with reversed phase stationary phases
which use mobile phases with no organic solvent.
References[1] R. P. W. Scott, C, F, Simpson: J. Chromatogr., 1980,
197, 11-20. DOI: 10.1016/S0021-9673(00)80531-2[2] M. E. Montgomery Jr., M. A. Green, M. J. Wirth: Anal.
Chem.,
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1170-1175.
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10.1021/ac00034a017[3] R. G. Wolcott, J. W. Dolan: LC/GC, 1999, 17,316-321.
[4] B. Buszewski, J. Schmid, K. Albert, E. Bayer: J.
Chromatogr.,
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415-427.
DOI:
10.1016/S0021-9673(01)95957-6[5] P. Kasturi, B. Buszewski, M. Jaronieic, R. K. Gilpin: J.
Chromatogr.,
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659,
261-265.
DOI:
10.1016/0021-9673(94)85067-4[6] T. Czajkowaka, I. Hrabovsky, B. Buszewski, R. K.
Gilpin, M. Jaronieic: J. Chromatogr. A, 1995, 691,
217-224. DOI: 10.1016/0021-9673(94)01034-C[7] T. L. Ascah, K. M. R. Kallury, C. A. Szafranski, S. D.
Corman, F. Liu: J. Liq. Chrom. & Rel. Technol., 1996,
19(17&18),3049-3056.
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10.1080/10826079608015125[8] T. Czajkowaka, M. Jaronieic: J. Chromatogr. A, 1997,
762, 147-158. DOI: 10.1016/S0021-9673(96)00966-1[9] J. E. O’Gara, B. A. Alden, T. H. Walter, J. S. Petersen,
G. L. Niederlander, U. D. Neue: Anal. Chem., 1995,
67, 3809-3091. DOI: 10.1021/ac00116a032[10] T. Enami, N. Nagae: Chromatography, 1998, 19,
380-381.
[11] N. Nagae, T. Enami: Bunseki Kagaku, 2000, 49,
887-893.

5. Problems using 100% aqueous mobile phase and thesolutionOnce the causes are understood, it is possible todetermine countermeasures against the problem, which isthe decrease of retention, when using aqueous mobile phaseor aquatic mobile phase containing less than 5%
concentration of organic solvent with reversed phasestationary phases. The retention decreases when using
100% aqueous mobile phase as the mobile phase is expelledfrom the packing material pores due to capillarity.
Therefore, it is effective to enlarge the packing material
pore diameters, use a stationary phase with long alkyl
chains, increase the mobile phase salt concentration, add
ion-pairing reagent, and lower the column temperature so
the pressure from capillarity decreases below atmosphericpressure. Although usable columns and mobile phasesmight be limited under these conditions, almost all kinds ofreversed phase stationary phases can solve this problem byapplying a back pressure at the postcolumn outlet. As
shown in Fig. 10, a large hysteresis exists when mobile
phase permeate the packing material pores and is expelled13[12] T. Enami, N. Nagae: Chromatography, 2001, 22,
33-39.
[13] N. Nagae, T. Emani, S. Doshi: LC/GC, 2002, 20,964-968.
[14] T. Enami, N. Nagae: American Laboratory, 2004, 36,
10-13.
[15] T. Enami, N. Nagae: Bunseki Kagaku, 53, 2004,
1309-1313.
[16] E. W. Washburn: Proc. Nail. Acad. Sci., 1921, 7,
115-116.
[17] Iwanami Physical and Chemical Dictionary Fifth
Edition.
[18] L. Zhang, L. Sun, J. I. Siepmass, M. R. Schure: J.
Chromatogr. A, 2005, 1079, 127-135. DOI:
10.1016/j.chroma.2005.03.124[19] F. Gritti, G. Guiochon: Anal, Chem., 2005, 77,4257-4272. DOI: 10.1021/ac0580058[20] J. L. Rafferty, L. Zhang, J. I. Siepmann, M. R. Schure:Anal, Chem., 2007, 79, 6551-6558. DOI:
10.1021/ac0705115[21] M. Przybyciel, R. E. Majors: LC/GC, 2002, 20,
516-523.
[22] K. Ban, Y. Saito, K. Jinno: Anal. Sci., 2004, 20,
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[23] K. A. Lippa, L. C. Sander, R. D. Mountain: Anal.
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10.1021/ac0510843[24] K. A. Lippa, L. C. Sander: J. Chromatogr. A, 2006,
1128, 79-89. DOI: 10.1016/j.chroma.2006.06.043[25] Z. Liao, J. E. Pemberton: Anal, Chem., 2008, 80,
2911-2920. DOI: 10.1021/ac702270b[26] Inventor: Norikazu Nagae, Assignee: Nomura
Chemical Co. Ltd., United State Patent, Patent No.:US6841073 B2, Date of Patent: 2005, Jan. 11.

14

Evaluation of Expelling of Mobile Phase from Pore of
C18 Packing Material under Aqueous Conditionwww.chromanik.co.jp

Norikazu Nagae* and Tomoyasu Tsukamoto
ChromaNik technologies Inc. Namiyoke, Minato-ku, Osaka Japan 552-0001*Corresponding author email: nagae@chromanik.co.jp

Abstract

It has been well known since publishing the paper by Nagae [1-5] that a mobile phase was expelled from the pore of packing materials for reversed phaseliquid chromatography under an aqueous condition by capillarity, consequently, retention decreased. Capillarity depends on the contact angle of a liquid on
the surface of a substance. In the case of more than 90 degree of the contact angle, in other words, non- wetting, the force brought by capillarity makes aliquid expel from the pore. Reversely in the case of less than 90 degree of the contact angle, in other words, wetting, the force brought by capillarity makes
a liquid permeate onto the pore. In this study, it was revealed that even if the contact angle was more than 90 degree, a liquid was not always expelledfrom the pore. Only when the contact angle was more than 90 degree, and furthermore the force brought by capillarity was more than the atmosphericpressure, a liquid was expelled from the pore. Permeating or depermeating (expelling) of a mobile phase from the pore doesn’t always depend on wetting
or non-wetting of a liquid on the surface of packing materials. The expression of dewetting has been often used when a mobile phase was expelled fromthe pore of packing materials and retention reduced. This expression, however, was considered not to be factual. It was suggested that the expression ofdepermeating should be used in such a case.
Structure of the Bonded-Phase Chains andSolvent Location

Explanation of dewetting

Decrease in retention under aqueouscondition

Mark R. Schure et al . reported theleft figure . A bonded phase collapses
under methanol:water=53/47(v/v) as
well as under water.

53% by volume

After stopping flow for 1 hour

82% by volume

For the right hand side of the upper portion of Figure 3, situation B shows a C18 phase in a 100% water
mobile phase. The C18 functionality prefers to be in a self-associated state (like prefers like) and folds uponitself in a collapsed state. The bottom portion of Figure 3 shows the situation as it actually happens. Because
most of the interactions with an LC stationary phase occur inside the pores (rather than on the outersurface), when an organic solvent is present at higher concentrations (greater than 10%), the pores are filledwith the water–organic mixture that allows the C18 bonded groups to be solvated, and everything behavesnormally. However, when the solvent within a pore becomes unfriendly (for example, very low %B or 100%A), there is a tendency to force the water out of the pore, which results in a dewetting phenomenon.

Condition
Column: C18 ,5 μm, 150 x 4.6 mm
Mobile phase: Water
Column back pressure: 6.0MPa
Back pressure post column: 1.7MPa
Flow rate: 1.0 mL/min
Temperature: 40 oC
Detection: RI
Sample: 1. Sodium nitrite, 2. 2-Propanol

N. Nagae, T. Enami and S. Doshi, LC/GC North America October 2002.

Glass tubing

h=2g cosq /(rrg)

PTFE tubingr

Mobile phase is expelled out of the column under
0.01 MPa pressure

Methanol: water=70:30hrh

Non-wetting
Non-wetting
Viewed from the side

C18 packing materials sedimented into the solvent.
Wetting(Methanol: water=70:30 solvent wet the surface of C18
packing material and permeated into the pore.)
Methanol: water=50:50

q(Methanol: water=50:50 solvent didn’t wet the surface ofC18 packing material and couldn’t permeate into the pore.)

Water

Methanol: water=30:70< 90o : wetting> 90o : non-wetting

Methanol:water(30:70)(50:50)(70:30)BC

1After dry21Adding pressure 23Mpa
4

3

6

5

After stopping pump for 1 hour

2 4
3 5 6

D

1

0

2

2

Adding pressure 23Mpa

3

4

4

6
Time / min

5

6

8

10

Typical chromatograms of nitrite and nucleic acidbases.
Column, 150 mm x 4.6 mm 5 μm dp C18, 10-nm
pore size; mobile phase, water; temperature, 40ºC; detection, UV at 254 nm; flow rate, 1.0
mL/min; sample, 1 = sodium nitrite, 2 = cytosine,
3 = uracil, 4 = cytidine, 5 = uridine, 6 = thymine.

The chromatogram A shows the first injection of the sample
after the baseline became stabilized and the 6 kinds ofcomponents eluted without being separated. From thiscondition, a tubing of ID 0.1 mm was connected to thepostcolumn outlet without stopping the pump and applied
the back pressure of 23 MPa to the column outlet. It is now
assumed that aqueous mobile phase mixed with air came outof the column and residual air that existed in pores of thedried C18 packing materials was expelled. The chromatogram
B shows the result of injection after the baseline stabilized.
The retention time of thymine was approximately 8.5 minutes
and all the six different of components were completely
separated. Later, we stopped the pump for one hour andpumped again without applying pressure at the postcolumn
outlet. The obtained chromatogram is shown as C, and theretention decreased by around 70%. Then 23 MPa of backpressure was applied again at the postcolumn outlet with
continuously pumping. The first time, air came out of thecolumn, however we could not confirm if there was air in themobile phase that came out of the column the second time.
Chromatogram D, which was obtained once the baseline
became stabilized, was almost the same as chromatogram B.
T. Enami and N. Nagae, BUNSEKI KAGAKU, 53 (2004) 1309.

C1 (TMS) shows decrease in retention
Relative retention time / %As shown in Figure, different lengths of alkyl chain change
the relative retention time against pore diameters andthe decreasing ratio on retention time also differs. When
80
comparing stationary phases using the same pore size of
C30
70
15 nm, C8 decreased in retention time by 80% and C18 by
60
35%. However, C30 and TMS did not decrease theTMS
50
retention time. The longer the length of ligand for more
40
than C8, the smaller the decrease in retention is with30
smaller pore diameters. That is, the longer alkyl chain is
C18
20
more stable on its retention even with small pore
C8
10
diameters. Generally the pore diameter of approximately
Mobile Phase: Water0
10 nm is used as packing material. However, the ligand
0
5
10
15
20
25
length of C30 enables the analyses with stable retention
Pore diameter / nmtime even using aqueous mobile phase. Although TMS is
Relationship between pore size and relative retention in the methyl group and has a short ligand, it gains highretention with a pore diameter of 9 nm. However, with 7
time under 100% aqueous mobile phase
nm of pore diameter, the retention decreases by greater
Column, C30 (○), C18 (□), C8 (♦), and TMS(Trimethylsilyl silica) (∆); column temperature, 40 oC; than 50%. No ligand collapse occurs on TMS phase. This
sample, thymine. Relative retention time wasmeans that ligand collapse has nothing to do withcalculated as the ratio of the decrease in retention
decrease in retention.
100
90

versus initial retention time.

T. Enami and N. Nagae, Chromatography, 22 (2001) 33.

C18 packing materials completely floated on the solvent(Methanol: water=30:70 solvent didn’t wet the surface ofC18 packing material and couldn’t permeate into the pore.)

-0.05

-0.1

N. Nagae, BUNSEKI KAGAKU Vol. 59, No. 3 (2010) 193.

T. Enami and N. Nagae, BUNSEKI KAGAKU, 53 (2004) 1309.

Retention behavior of dried C18 column
A 1,2,3,4,5,6

When methanol concentration in the mobile phase was 70% and80%, in other word methanol:water (70:30) and (80:20), themobile phase wasn’t expelled out of the pore. However, whenmethanol concentration in the mobile phase was less than 70%,
-0.15
-0.2
the mobile phase was expelled out of the pore and went out of-0.25
the column. As a result, the column weight decreased. If the force
-0.3
-0.35
brought by capillarity was more than 0.01 MPa, the mobile phase-0.4
was expelled out of the pore under 0.01 MPa pressure.
-0.45
When atmospheric pressure is 0.1MPa, the mobile phase with
-0.5
0
20
40
60
80
100
more than 10% of methanol concentration wasn’t expelled out of
Methanol concentration in mobile phase / %the pore as anybody knows.
0

C18 packing materials almost floated on the solvent.

Water

Column: C18, 5 μm, 250x4.6mm
Simulated atmospheric pressure: 0.01MPa
Column temperature: 40 oC
C18 column was kept in the vacuum vessel at 0.01MPa for 20 min.
The weight of the column changes if the mobile phase is expelled outof the pore.

C18 packing materials were put into each solvent and stired
with ultra sonic vibration.

g

0.5 mm i.d.
glass tubing

Mark R. Schure, et al. LCGC North America 630 Volume 31 Number 8 August 2013.

State of C18 packing materials in mixture ofmethanol and water
Viewed from aboveq: surface tension of liquid: density of liquid

Left: Snapshots from simulations in different solvents, C18 chains (gray), and alkane and alcohol
solutes (large spheres with green, red, and white indicating CHx , hydroxyl oxygen, and hydrogen,respectively). Middle and right: Density profiles of C18chains, methanol, water, and acetonitrile forpure water (W), pure methanol (M), pure acetonitrile (A), and solvent mixtures given as molepercent. T = 323 K, grafting density of 2.9 µmol/m2. Adapted, in part, from references 4 and 14.

Difference of column weight /g

Capillarity equation:

87% by volume

Methanol:water=33:67(M/M)=53:47(v/v)

Ronald E. Majors, LCGC North America, Jul 1, 2013.

Capillarity, Capillary phenomenon

60% by volume

Conclusion
Mobile phase

Wettability
on C18Structure of
the C18
chainsa
Almostcollapsed stateState of mobile phase
under 0.01MPapressure

State of mobile phase
under 0.1MPapressure

Methanol:water
Wetting
Keep in the pore
Keep in the pore(70:30)Methanol:waterNon-wetting Collapsed state Expelled out of the pore Keep in the pore
(50:50)Methanol:waterNon-wetting Collapsed state Expelled out of the pore Keep in the pore
(30:70)
Water

State of mobile Force brought byphase undercapillarity
23MPa pressure (expelling force)
Keep in the pore---

More than 0.01MPa,
Keep in the poreLess than 0.1MPa
More than 0.01MPa,
Keep in the poreLess than 0.1MPa
More than 0.1MPa,
Non-wetting Collapsed state Expelled out of the pore Expelled out of the pore Keep in the poreLess than 23MPa

a: Reported in Mark R. Schure, et al. LCGC North America 630 Volume 31 Number 8 August 2013

What does “Dewetting” mean?
A surface state changes from wetting to non-wetting due to ligand collapse? The surfaceof C18 is always non-wetting under an aqueous mobile phase with from 0% to 50% of
methanol concentration even if such a mobile phase exists in the pore, so that expressionof “dewetting” is not scientific. Depermeating is a scientific expression?
Reference[1] N. Nagae, T. Enami, BUNSEKI KAGAKU, 49 (2000) 887.

[2] T. Enami and N. Nagae, Chromatography, 22 (2001) 33[3] N. Nagae, T. Emani, S. Doshi, LC/GC, 20 (2002) 964.
[4] T. Enami and N. Nagae, BUNSEKI KAGAKU, 53 (2004) 1309.
[5] T. Enami, N. Nagae, American Laboratory, 36 (2004) 10.
[6] N. Nagae, BUNSEKI KAGAKU, 59 (2010) 193.

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