Abstract Number: 2080 - 13P

Study of State of Alkyl Chainsfor Reversed-Phase:Collapse or Brush up
Norikazu Nagae Ph.D, Kouji Yamamoto, Chiaki Kadota
ChromaNik Technologies Inc.
Email: info@chromanik.co.jp
Website: http://chromanik.co.jp
1

Abstract
It had commonly been said that ligand collapse maderetention decrease under 100% aqueous conditions forreversed-phase liquid chromatography. However Nagae etal. reported at 2000 that reducing retention under 100%
aqueous conditions had nothing to do with ligand collapse
and its cause was that a mobile phase like water expelledfrom the pore of the packing materials by capillarity.
Solvation volume of an organic solvent in a mixture oforganic solvent and water as a mobile phase to alkyl chain
as a stationary phase was measured directly by retention ofa organic solvent under lower concentration of organic
solvent in a mobile phase, furthermore, indirectly by changeof stationary volume obtained from measurement of elution2

Abstract 2
volume of uracil as an non-retained substance under higher
concentration of organic solvent in a mobile phase. Solvation
volume of tetrahydrofuran (THF), acetonitrile and methanol
were measured to be 0.35mL, 0.15mL and 0.06mL
respectively on 0.35 mL of C18 stationary phase in the column
in dimension of 150 x 4.6 mm i.d. THF solvated 6 times morethan methanol on the C18 stationary phase, while acetonitrilesolvated 2.5 times. Solubility of octadecan (C18H38) to not
only water but also methanol is almost zero, so that C18 alkyl
chain is considered to agglomerate on the surface even if
100% methanol is used as a mobile phase. These results
suggested that THF solvated in the whole alkyl chain and alkyl
chain brushed up while methanol solvated on the surface ofagglomerating C18 phase.
3

State of octadecane in organic solvent
Octadecan （C18H38) doesn’t
dissolve in methanol.

Octadecane dissolves inTerahydrofuran（THF).

It is considered that octadecane aggregates inmethanol and contact surface area is the least.

4

Chromatograms of Acetonitrile
using Acetonitrile/Water mobile phases
20% Acetonitrile
Systemvolume0.291 min

Column: C18 150x4.6mm

t0 (Sodium nitrite) Flow rate: 0.5 mL/min Temp.: 40℃

10% Acetonitrile
5% Acetonitrilets (Vacant peak of acetonitrile)

2% Acetonitrile
1% Acetonitrile

01234TIIME (min)5678Acetonitrile retains using a mixture of acetonitrile and water as a mobile phase.
In other word, acetonitrile partitons into a stationary phase. This patition
volume is assumed to be solvation volume.
5

Retention time, retention and partition
volume to stationary phase using
acetonitrile/water as mobile phasek = (ts-t0) / (t0-system volume) k = S / M

S=kxM

Column: C18(%C: 16%) 150x4.6mm. About 1.5 g packings exists in a column. The C18 stationary
phase in its packings is about 0.28 g or o.35 mL.

0.8

60.101mL

Retention time of acetonitrile

50.7

40.5

Elution time of sodium nitrite (t0)0.4

3210.059mL
Retention of acetonitrile（ｋ）
0.033mL
0.017mL

Partition volume of acetonitrile to stationary phase

0.3

Retention (k)Time (min)

0.6

0.2
0.1

0.011mL

000%

5%

10%

15%

Concentration of acetonitrile in mobile phase (%)

20%
6Solvation volume in stationary phase (mL)

Partition (solvation) volume of acetonitrile on
C1, C18 and C28 stationary phases
0.16

TMS (C1)
Column: TMS (C1, %C: 5%)

C18
0.12

C18 (%C: 16%)C28

C28 (%C: 16%)150x4.6mm
0.08Partition volume of acetonitrile
on each stationary phase isalmost same.

0.04

005101520

Concetration of organic solvent (%)

257

Schematic diagram of solvation ofacetonitrile under acetonitrile/water
mobile phase
CH3CN/H2O
C28: Acetonitrile

C18
C1 (TMS)

8

Partition (solvation) volume into C18
Column: C18，（C28) 150x4.6mm

0.2Solvation volume in stationary phase (mL)Tetrahydrofuran THF(C28)
Tetrahydrofuran THF

１）1.5 g of C18 packing material ispacked into a column.

Acetonitrile

0.15

Ethanol２）Carbon content of C18: 16%

Methanol

Carbon content of C28: 16%

0.1

Volume of octadecyl group in acolumn is calcurated to be ca. 0.35mL.

0.05

Methanol, acetonitrile and THF aresolvated 0.04 mL, 0.10 mL and 0.15
mL respectivily at 20% concentrationof each organic solvent.

0051015

Concentration of organic solvent (%)

209Schematic diagram of solvation onC18 stationary phase under 20%
organic solvent concentration
Methanol

Acetonitrile

Solvation of methanol and acdetonitrile occurred on
the surface of agglomerated C18 stationary phase.

Tetrahydrofuran THF

THF has swollen C18
Stationary phase.

10

Solvated organic solvent works as apart of stationary phase
Chromatograms of Acetonitrile using Acetonitrile/Water mobile phases

Column: C18 150x4.6mm
20% Acetonitrile t0 (Sodium nitrite) Flow rate: 0.5 mL/min
Systemvolume0.291 min

Temp.: 40 oC

10% Acetonitrile
5% Acetonitrilets (Vacant peak of acetonitrile)

2% Acetonitrile
1% Acetonitrile
01234TIIME (min)5678

Elution time of sodium nitrite (t0) reduces as Organic solvent concentration inmobile phase increases. This result leads that solvatd organic solvent works apart of stationary phase, so that volume of stationary phase grows larger and t0decreases. In other words, the variation of t0 equals to that of stationary phasevolume.
11

Variation of t0 among methanol,
acetonitrile and THF
751 3224 64512321 30744Mobile
phase:
50% THF

Column: C18, 5μm, 150 x 4.6 mm
Mobile phase :6 7
THF/water (50:50), acetonitrile/water
74% Acetonitrile (74:26), methanol/water (82:18)
5682% Methanol
Detection: UV @ 250 nm.

6810(mL)

Elution volume of Uracil(to)Sample:1=Uracil, 2=Caffeine, 3=Phenol,
4=Butylbenzene, 5=o-Terphenyl,
6=Amylbenzene, 7=Triphenylene

Volume of stationary phase under
50%THF is larger than that under 82%methanol.
Variation of volume of stationary phasebetween 50% THF and 82% methanol
6is 0.254 mL.

201.64mL
1.80mL1.89mL

12Solvation volume of organic solvent
in stationary phase
Solvation volume in stationary phase (mL)

0.55

Column: C8, C18, C28

0.5

150 x 4.6 mm

0.45

THF (C28)

0.4

THF (C18)

0.35

THF (C8)

0.3

Acetonitrile (C18)0.25

Ethanol (C18)

0.2

Methanol (C18)

0.15
0.1
0.05
0020

40

60

80

Concentration of orgainc solvent (%)

100

It was assumed that solvation volumeof methanol under 82% methanol
mobile phase was 0.06 mL. As thisvalue was the criterion, the othervalues were determined bymeasurement of elution volume ofUracil.
13Separation of standard samples
using methanol and THF
Methanol

2 31Sunniest C28 ％C:16%

2Sunniest C18 ％C:16%

121436754345675075Sunniest C8 ％C:10%

6101520
Retention time/min

25

30

35

THFSunniest C28 ％C:16%

71 2536471 2Sunniest C18 %C:16%

534167

2Sunniest C8 %C：10%

5340Column: Sunniest C18, C28, C8, 5μm
4.6x150 mm
Mobile phase:
CH3OH/H2O=75/25
THF/H2O=50/50
Flow rate: 1.0 mL/min
Temperature: 40 ºCSample: 1 = Uracil,
2 = Caffeine,
3 = Phenol,
4 = Butylbenzene,
5 = o-Terphenyl,
6 = Amylbenzene,
7 = Triphenylene

561015Retention time/min

Peak 5
2025

Peak7
14

Schematic diagram of solvation on
C28, C18 and C8 stationary phases
C28

C18

CH3OH/H2O=75/25
C8C28: Methanol

C18

THF/H2O=50/50

: THF

C815

Volume of C28, C18 and C8
stationary phases undermethanol/water mobile phase
CH3OH/H2O=75/25
tR (amylbenzene): 21.5 mintR (amylbenzene): 21.6 min

C28

C18: MethanoltR (amylbenzene): 10.8 min
C8Volume of stationary phase is almost same.
Volume of stationary phase is different.
16Volume of C28, C18 and C8stationary phases under THF/watermobile phasetR (amylbenzene): 20.8 min

C28tR (amylbenzene): 14.5 mintR (amylbenzene): 12.2
min

C18

THF/H2O=50/50

: THF

C8

Volume of stationary phase is different.

17

Almost same retention time1

Conventional C18%C:18%

Column size: 4.6x250 mm
Mobile phase: 97/3=CH3OH/H2O
Flow rate: 1.0 mL/min
Temperature: 25 ºC

2, 34C18Detection: UV at 295nm
05101520retention time / min

2530Sample: 1=δ-Tocopherol
HOOSunrise C28 %C:18%

2=γ-Tocopherol

1C28HOO

3=β-Tocopherol

2HO43O

4=α-Tocopherol
HO05101520retention time / min

253035O

Each stationary phase isalmost same volume.
18

Two times longer retention time: Chloroform
C18
1 2

12Column size: 4.6x250 mm
Mobile phase:80/20=CH3CN/CHCl3
Flow rate: 1.0 mL/min

Conventional
C18 %C:18%

Temperature: 30 ºC
Detection: UV at 450nm

2 times larger
C28Sample: 1=α-Carotene

Sunrise C28 %C:18%2=β-Carotene
05101520253035

Retention time / min19Conclusion• Solvation volume in stationary phase wasobtained by measuring both retention of organic
solvent and elution time of Uracil.
• Solvation volume of methanol and acetonitrile isalmost same on the any stationary phases suchas C8, C18 and C28.
• The longer an alkyl chain, the larger Solvation
volume of THF.
• It is considered that alkyl chains are brush up
under THF/water mobile phase, especially athigh concentration of THF.
20