Evaluation of Expelling of Mobile Phase from Pore ofC18 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: WaterColumn 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)Methanol: water=70:30
C18 packing materials sedimented into the solvent.

hNon-wetting

rhNon-wetting
Viewed from the sideWetting(Methanol: water=70:30 solvent wet the surface of C18
packing material and permeated into the pore.)
Methanol: water=50:50

qC18 packing materials almost floated on the solvent.

Water(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
q < 90o : wettingq > 90o : non-wetting

Methanol:water(30:70)(50:50)(70:30)BC
D
01
1

After dry2

Adding pressure 23Mpa
4365

After stopping pump for 1 hour
2 4
3 5 6122

Adding pressure 23Mpa

3446Time / min

65810

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, 1.0mL/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 completelyseparated. 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
100
the relative retention time against pore diameters and90
the decreasing ratio on retention time also differs. When80
comparing stationary phases using the same pore size ofC30
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 with30smaller pore diameters. That is, the longer alkyl chain isC1820
more stable on its retention even with small pore
C810diameters. Generally the pore diameter of approximately
Mobile Phase: Water010 nm is used as packing material. However, the ligand
0510
152025length of C30 enables the analyses with stable retention
Pore diameter / nmtime even using aqueous mobile phase. Although TMS isRelationship 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 7time under 100% aqueous mobile phase
nm of pore diameter, the retention decreases by greaterColumn, C30 (○), C18 (□), C8 (♦), and TMS(Trimethylsilyl silica) (∆); column temperature, 40 oC; than 50%. No ligand collapse occurs on TMS phase. Thismeans that ligand collapse has nothing to do withsample, thymine. Relative retention time wascalculated as the ratio of the decrease in retention
decrease in retention.
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.)

When methanol concentration in the mobile phase was 70% and080%, in other word methanol:water (70:30) and (80:20), the-0.05
mobile phase wasn’t expelled out of the pore. However, when-0.1
methanol 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
02040
60
80
100
more than 10% of methanol concentration wasn’t expelled out ofMethanol concentration in mobile phase / %the pore as anybody knows.
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 1,2,3,4,5,6Column: C18, 5 μm, 250x4.6mm
Simulated atmospheric pressure: 0.01MPaColumn 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

Mobile phase is expelled out of the column under0.01 MPa pressure

PTFE tubing
Viewed from aboverMark R. Schure, et al. LCGC North America 630 Volume 31 Number 8 August 2013.

State of C18 packing materials in mixture ofmethanol and waterqg: surface tension of liquidr: 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 ofthe 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 23MPaa: 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.