Evaluation of Retention Behavior and Stabilityof Novel Trifunctional Biphenyl Phase
https://pyvot.tech/Norikazu Nagae1, Tom Toyasusukamoto1, Ryuji Koyama1, Scott Silver2
1 ChromaNik Technologies Inc. Namiyoke, Minato-ku, Osaka Japan
2 Pyvot, 1040 1st Avenue, Suite 330 New York, NY*Corresponding author email: nagae@chromanik.co.jp

Distributor in the US

Biphenyl columns are currently available from several HPLC column manufacturers. Theirbiphenyl phases are only mono-functional. In this study, tri-functional biphenyl stationary phase
was modified on a core shell silica and double end-capping was done at high reaction temperature.
Tri-functional biphenyl stationary phase was compared with mono-functional biphenyl stationary
phase not only for measurement of hydrogen bond capacity, hydrophobicity and steric selectivity
but also for a peak shape of a metal chelating compound and a basic compound. Furthermore
stability of each biphenyl stationary phase was evaluated under both acidic and basic pH
conditions. Although phenyl stationary phase shows higher hydrogen bond capacity than alkyl
stationary phases, biphenyl stationary phase showed the highest hydrogen bond capacity. Such ahigh hydrogen bond capacity leaded unique separation selectivity when separating o-, m-, pmethylhippuric acid and vanillin and DNPH-aldehydes. Proposed trifunctional biphenyl
stationary phase showed the most stable under both acidic and basic pH conditions.
Comparison of core shell Biphenyl phases using standard samples

Comparison using oxine

Methanol:0.1% Phosphoric acid=25:75

1

Core shell silica

O SiCH3

SunShell Biphenyl

1C1805105C184

31024643

15USPL line

End-capping

MaximumpressurepH range

2.61.69150

5BiphenylL11Sunniest endcapping

60 MPa1.5 - 9200 (effective)

11

Biphenyl

L11TMS

60 MPa1.5 - 8.5

Company B Biphenyl

2.7---

91357Biphenyldimethylsilane

L11Yes60 MPa1.5 - 8.0

Company C Biphenyl

2.7---

9130

7

Biphenyldimethylsilane

L11Yes60 MPa1.5 - 8.0

C18 can’t separate iso- and n-Butyraldehyde-DNPH (3 and 4).

22010+

2. Propionaldehyde

1214Relationship between concentration of IPA and log k

3. iso-Butyraldehydeα(3,4)=115 60.95

5654
30.91Biphenyl
3014684. n-Butyraldehyde

100.85Biphenyl554Retention time/min432

IPA:MeOH:H2O=25:40:35
α(3,4)=1.058

123 455. iso-Valeraldehyde

61Biphenyl
1243246810

IPA:MeOH:H2O=30:35:35
α(3,4)=1.061

3 451502C184312141618

16. n-Valeraldehyde
123 45Biphenyl
1

Retention time/minmetaOOOHNHOHNHOOColumn: SunShell C18 2.6 μm, 150 x 4.6 mm
SunShell Biphenyl 2.6 μm, 150 x 4.6 mmMobile phase: shown in figures
Flow rate: 1.0 mL/min
Temperature: 40 oC
Detection: UV@250 nm
Sample:1, Protocatechuic Acid
2. Vanillic Acid

450.55602468101223

140123 42468120

14046810

IPA:MeOH:H2O=45:20:35

OHH3C1238

4302423 54

6208Retention time/min

10Column:
SunShell C18 2.6 μm, 150 x 4.6 mm
SunShell Biphenyl 2.6 μm, 150 x 4.6 mm
Mobile phase: shown in figures
Flow rate: 1.0 mL/min
Temperature: 40 oC
Detection: UV@360 nmSample: 1. Acetaldehyde-DNPH
2. Propionaldehyde-DNPH
3. iso-Butyraldehyde-DNPH
4. n-Butyraldehyde-DNPH
5. iso-Valeraldehyde-DNPH
6. n-Valeraldehyde-DNPH

Dipole-dipole interaction with Biphenyl
affects separation of iso- and n-butyraldehyde-DNPHs.

6

When an alcohol was used as an organic solvent in the mobile phase,
Biphenyl showed long retention time and reversed an elution order ofvanillin and isovanillin to compare with C18.

15

6

Increasing the water ratio in the mobile phase
does not change the dipole-dipole interactionand increases the hydrophobic interaction.

1α(3,4)=1.069

O

When an alcohol was used as an organic solvent in the mobile phase, Biphenyl
showed the longest retention time of all stationary phases and also reversed theelution order of m- and p-methylhippuric acids.

21054,51

5. Isovanillin

O

53 4

Retention time/min

64. Vanillin

262,3α(3,4)=1.068

1α(3,4)=1.066

510

IPA:MeOH:H2O=40:25:35

CH3NH

33

16CH3para

28

IPA:MeOH:H2O= 35:30:43.3 (32.3:27.7:40)

Retention time/min

3. Isovanillic Acid

3Concentration of IPA/%
35α(3,4)0.62α(3,4)=1.064

ortho40.65

IPA:MeOH:H2O=35:30:35

Column: SunShell C18 2.6 μm, 150 x 4.6 mm
SunShell C30 2.6 μm, 150 x 2.1 mm
SunShell Phenyl 2.6 μm, 150 x 4.6 mm
SunShell Biphenyl 2.6 μm, 150 x 4.6 mm
Mobile phase: Organic solvent/25 mM Phosphate buffer pH 3.0
Flow rate: 1.0 mL/min, 0.2 mL/min for only C30
Temperature: 40 oC
Detection: UV@230 nm
Sample: o-, m-, p-Methylhippuric acidα(3,4)

Methanol:0.01% Trifluoroacetic acid=25:75

2C18Column, Biphenyl

0.75
0.7

Acetonitrile:0.01% Trifluoroacetic acid=25:75

Mobile phase, IPA:MeOH:H2O=X:(65-X):35

20.8Log k2

Biphenyl2

iso-Butyraldehyde-DNPH

68

3,4

DNPH

1. Acetaldehyde

6

IPA:MeOH:H2O=55:0:45

12C18

Stationary phase

10 (effective)α(3,4)=13,425

Methanol:0.05% Trifluoroacetic acid=25:75

2Carbonloading (%)

Comparison of stability

155Methanol:0.2% formic acid=25:75

Specific surface
area (m2/g)---IPA:MeOH:H2O=20:45:35

43Pore size (nm)2.6α(3,4)=13,45

1Core size(μm)

Company A Biphenyl

IPA:MeOH:H2O=0:65:35

14Particle size(μm)

Specification of other Biphenyl (cited from a brochure)

5Biphenyl

Bonded phase

Separation of 6 kinds of DNPH-aldehyde using C18 and Biphenyl column(2,4-Dinotrophenylhydrazine = DNPH)

223CH3231

1

Specification of SunShell

45Biphenyl

Company A, B and C Biphenyl, Monofunctional Biphenyl

2C1843OO SiO

Methanol:0.1% Trifluoroacetic acid=25:75

2C18Feature of end-capping of SunShell Biphenyl

SunShell Biphenyl, Tri-functional Biphenyl

Comparison using basic compounds

Separation of vanilin and isovanillin using C18 andBiphenyl column

Comparison of stationary phases using isomers ofmethylhippuric acidwww.chromanik.co.jp

12

As the ratio of IPA to MeOH in the mobile phase increases, the dipole-dipoleinteraction becomes stronger and separation factor (α) of iso- and n-butyraldehydeDNPH increases as the IPA increases.
IPA can control the strength of the dipole-dipole interaction with Biphenyl.

48