Consideration of π-π and CH-π Interactions as
Dipole-Dipole Interactions in Biphenyl Stationary Phases
E. Shearera, N. Nagaeb, T. Tsukamotob, R. Koyamab,[a] BioNik Inc. Sugita, Fujinomiya, Shizuoka Japan[b] ChromaNik Technologies Inc. Namiyoke, Minato-ku, Osaka Japanwww.bionikinc.com

www.chromanik.co.jp

Biphenyl stationary phase is classified in L11 category as LC columns as well as phenyl stationary phase by the United States Pharmacopeial Convention. Both Biphenyl andphenyl stationary phases have a phenyl group and are said to have dipole-dipole interaction that is absent in alkyl chain stationary phases such as C18 and C8. Furthermore, itis said that there are π-π interactions, CH-π interactions, OH-π interactions, NH-π interactions, halogen-π interactions, and lone pair electron-π interactions as dipole-dipoleinteractions.
In this study, the π-π interaction and the CH-π interaction were discussed in the separation of various samples. Dinitrobenzene, trinitrobenzene and sulfonamides which have alot of double bonds are used for evaluation of π-π interactions. The isomers of methylhippuric acid, organic acids, vanillin and isovanillin, leucine and isoleucine, nbutyraldehyde-DNPH and iso-butyraldehyde-DNPH have little difference of π electrons respectively, so that they are used for evaluation of CH-π interactions. Separationcomparison of above compounds using biphenyl, phenyl and C18 stationary phases revealed each interaction. Comparing between biphenyl and phenyl stationary phases,
biphenyl stationary phase showed both stronger π-π interaction and stronger CH-π interaction as dipole-dipole interaction than phenyl stationary phase.

Separations mainly by CH-π interaction

Dipole-Dipole Interaction (π-π and CH-π interactions)【Methylhippuric acid】
Analytepm

C18

Sample: o-,m-,p-Methylhippuric acid
HydrophobicInteraction

Phenyl(ring-over-bond)
T-type stacking

OHNHOm
Dipole-dipoleInteraction

Molecular arrangement byT-type stacking

π相互作用OOHNHmOpO

OHO(ring-over-ring)

0π-π stacking

123Retention time/min

Electrostatic potential mapof benzene【Amino acids】
13

1

C18
2

3

RP-AQUA

1O+ O

31

L-Valine

1PFP

2

3

112OO

Same elution order
0121RP-AQUA

4201-

24O+ O

43NOO-+ O-NH3C+ O

121Biphenyl

123423N5O23444O5+ OO+-1+ ONO-

3Image of CH-π interaction
50411214024681012Retention time/min

141618(tR=13)

Increased
retention time

4. n-Butyraldehyde-DNPH
5. iso-Valeraldehyde-DNPH
6. n-Valeraldehyde-DNPH

Number ofhydrogen bonds

Intramolecularhydrogen bondNo intramolecularhydrogen bondIntramolecularhydrogen bond x 2

HH H０HHH2

6. Maleic acid

9. Fumaric acid8. Succinic acid3１4450510Column: Prominert C18 , Biphenyl 3.5 μm, 150 x 4.6 mm
Mobile phase: Methanol:0.1% Phosphoric acid = 25:75
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@250nm
Sample:34Stabilization by intramolecular interactions increases retention based on π electrons

RP-AQUA

53Column: Prominert C18 or Biphenyl 3.5 μm, 150 x 4.6 mm
Mobile phase: Methanol:50 mM Ammonium acetate = 20:80
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@265nm

Vanillic Acid

Isovanillic Acid

Vanillin

Isovanillin

SulfamethoxazoleSulfamerazine

Retention time/min

1Biphenyl
0Protocatechuic
Acid15２123245319674752896834

Retention time / min23

Hydrophobic＜ π Interaction
Interaction: CH-π interaction

Retention time/min

2＞ π Interaction

1524

Hydrophobic
InteractionH110105Biphenyl

158

5. Isovanillin

4. Vanillin

Sulfadiazine

06H

32

Biphenyl6H【Sulfonamides】

15C182,4,6-Trinitrotoluene*Phenyl stationary phases exert strong π-π interactions between nitro groups and phenyl orbiphenyl groups, altering the elution order of analytes. The PFP stationary phase showed thesame elution order as C18. This result implies that PFP does not have π-π interactions likephenyl groups.

54【Organic acids】

2NColumn: SunShell C18 2.6 μm, 100 x 4.6 mm
SunShell PFP 2.6 μm, 100 x 4.6 mm
SunShell RP-AQUA 2.6 μm, 100 x 4.6 mm
SunShell Phenyl 2.6 μm, 100 x 4.6 mm
SunShell Biphenyl 2.6 μm, 100 x 4.6 mm
Mobile phase: Methanol:water=60:40
Flow rate: 1.0 mL / minTemperature: 40 oC
Detection: UV@250 nm

04* In the separation of leucine, isoleucine and n-butyraldehyde-DNPH, iso-butyraldehydeDNPH in the above separation, the CH-π interaction differs depending on the position of thebranched methyl group, and the biphenyl stationary phase also showed the best separation.
Leucine and n-butyraldehyde-DNPH, which have a methyl group at the farthest position
from the functional group, were well-retained.

【Vanilin and Isovanilin】-6OC18

3

2,4-Dinitrotoluene

Retention time/min

15Column: SunShell PFP, PFP&C18, RP-AQUA, Biphenyl
2.6 μm, 150 x 4.6 mm
Mobile phase: 0.1% formic acid
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@205 nm
Sample: 1 = L-Valine, 2 = L-Isoleucine, 3 = L-Leucine

H3C14-N032Column: SunShell C18 and Biphenyl
2.6 μm, 150 x 4.6 mm
Mobile phase:
Sample:
IPA:MeOH:H2O=25:40:35
1. Acetaldehyde-DNPH
oTemperature: 40 C
2. Propionaldehyde-DNPH
Detection: UV@360 nm
3. iso-Butyraldehyde-DNPH+ O+NO2Biphenyl

Retention time/min

CH30-31(tR=7)

L-Leucine

1,3,5-Trinitrobenzene

3200Retention time/min

2

4. n-Butyraldehyde-DNPH

H3C-NO

3. iso-Butyraldehyde-DNPH

5 61,3-Dinitrobenzene

31Phenyl

3O2N

C18

3R2,3 = 2.95 3
α=1.19 2Biphenyl+

N

3O2N

3,42

L-Isoleucine

44N-N0N

Different retention

R2,3 =1.37
α=1.082HNSame retention

124NO2HN

1R2,3 =1.51
α=1.192【Components of explosive】

NO2R2,3 =1.32
α=1.13

2Separations mainly by π-π interaction
PFP&C18* As shown in the chromatogram above, the CH-π interaction is inversely proportional tothe hydrophobic interaction and is stronger on the biphenyl stationary phase than on thephenyl stationary phase. It is considered that p-methylhippuric acid has a stronger CH-πinteraction than m-methylhippuric acid, resulting in greater retention.

【Aldehyde-DNPH】
3PFP

Meta position

m-Methylhippuric acid5Column: SunShell 2.6μm 100 x 2.1mm
Mobile phase: 2-Propanol:20 mM Ammonium acetate (pH 6.8) = 3:97
Flow rate: 0.4 mL/min Temperature: 40 ℃
Detection: UV@230 nm★π-π stacking and T-type stacking are known as benzene arrangement states as π-π interaction.
It is presumed that T-type stacking is close to the CH-π interaction, which is the interaction
between the central part of benzene and hydrogen of CH.

1Para

4

H

CH3

H3C

Image of interaction

H COHNH

O

Para position

HCH3Meta

OH
N
H O

H

p-Methylhippuric acid

OO

H C

H3CO

NH

CH-π interaction

OHNH

Ortho

Biphenyl

π-π interaction

HO

CH3

pAnalyte

Image of CH-π interaction

O4* Vanillin and isocanillin were more retained on the biphenyl
stationary phase than on the C18 stationary phase. Furthermore,
the elution order of vanillin and isovanillin was reversed.
Isovanillin is thought to have a stronger CH-π interaction with theBiphenyl stationary phase than vanillin.

Column: SunShell 2.6 μm, 150 x 4.6 mm
Mobile phase: 0.1% H3PO4
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@210nm

Sample:1. Tartaric acid
2. Formic acid
3. Malic acid
4. Lactic acid

5. Acetic acid
6. Maleic acid
7. Citric acid8. Succinic acid
9. Fumaric acid

* In the case of the RP-AQUA stationary phase, the organic acids are eluted in the reverse
order of hydrophobicity, but in the case of the biphenyl stationary phase, the elution order ofthe organic acids differs from the reverse order of hydrophobicity due to the CH-πinteraction. Succinic acid with strong CH-π interaction showed relatively large retention.

Sulfamonomethoxine*Retention by the biphenyl stationary phase was significantly higher than that by the C18
stationary phase due to π-π interactions. In addition, when the organic solvent concentrationin the mobile phase was low, the π-π interaction became significant. The change in theelution order of peak numbers 3 and 4 is thought to be due to CH-π interactions.

Conclusion✓ Phenyl groups such as phenyl and biphenyl are known to have dipole-dipole interaction.
✓ Dipole-dipole interaction include not only π-π interaction but also CH-π interaction.
✓ CH-π interaction is considered to cause larger selectivity changes than π-π interactions.
✓ In the case of organic acid separations, there may not be much difference in π-π interaction
between different analytes, and CH-π interaction could explain the change in selectivity.