SunShell Core shell particle
Sunniest Fully porous particle

Biphenyl
UHPLC & HPLC column

Bentencho station

ChromaNik Technologies Inc.

Biphenyl
Trifuctional silyl-reagent used
O
O
O

Si

Feature of end-capping ofSunniest Biphenyl and SunShell Biphenyl (Sunniest End-capping)Me

MeMe MeMe Me

Double endcapping

O

Cl

Si

Si

Si

O

Cl

Hexamethyldichlorotrisiloxane

Trimethylchrolosilane

Si

OH

Si
O

O

O
Si
O

High stability

O

O

High
temperature
end-cappingOHLow bleeding

Si
O

Conversion from silanolgroups to siloxane bondFeature of BiphenylThe two benzene rings of the biphenyl group are twisted about 45 degree, andthe biphenyl group shows different separation from the phenyl group alone.
Even when an aqueous mobile phase containing no organic solvent is used,
there is no decrease in retention, and separation with high reproducibility ofretention time is achieved.
Greater retention of polar compounds such as caffeine.

1Interaction of L11 stationary phase(Phenyl, Biphenyl)
O
O Si
O

O
O Si
O

Intermolecular forces
Types of interactions

Origin of force

FeatureElectrostatic force

Charge-Charge, Permanent
dipoleWorks over long distances

van der
Waals
force

Orientation force

Permanent dipolePermanent dipole

Induced force

Permanent dipole-Induced
dipoleLondon dispersion
force

Induced dipole-induced
dipoleAttenuates at -6th power of
distance and makes animportant contribution only atshort distances.
Dispersion is dominant in nonpolar molecules.

Charge transfer interaction

Electron donor-Electron
acceptor

Coloring by charge transfer
absorption

Hydrogen bond

Functional groups such as
OH and NH

Strong specificity anddirectivity

Hydrophobic interaction

Non-polar groups in polar
solvents

Entropy domination

H
H

H

H

H
H

Electrostatic potential map ofbenzene★As shown in the figure above, benzene has the highest electron density in the center, and the phenyl and biphenylgroups of the L11 stationary phase have a dipole-dipole interaction, which is the orientation force of the van der
Waals force. It then exhibits characteristic selectivity by the π-π and CH-π interactions that are treated as part ofthem.

(ring-over-bond)

T-type stacking

Molecular arrangement
by T-type stacking(ring-over-ring)

π-π stacking
π-π interaction

CH-π interaction★π-π stacking and T-type stacking are known as benzene arrangement states as π-π interactions.It is presumed thatT-type stacking is close to the CH-π interaction, which is the interaction between the central part of benzene andhydrogen of CH.

2

Specification of Biphenyl column
Totally porous silica 5 μm
Specification of Sunniest Biphenyl
Fully porous silica

Sunniest Biphenyl

Bonded phase

Particle size
(μm)

Pore size(nm)Specific surface area
(m2/g)

Carbonloading (%)

Stationary phase

USP
L lineEnd-capping

pH range

5

12

340

11

Biphenyl

L11

Sunniest end-capping

1.5 - 9Core shell silica 2.6 μm
Specification of SunShell Biphenyl
Core shell silica

SunShell Biphenyl

Bonded phase

Particle
size (μm)

Core size(μm)

Pore size(nm)Specific surface
area (m2/g)

Carbonloading (%)

Stationary phase

USP
L lineEnd-capping

Maximumpressure

pH range

2.6

1.6

9

150

5

Biphenyl

L11

Sunniest end-capping

60 MPa1.5 - 9Specification of other Biphenyl (cited from a brochure)
Company A Biphenyl

2.6

---

10

100

11

Biphenyl

L11

TMS

60 MPa1.5 - 8.5

Company B Biphenyl

2.7

---

9

150

7

Biphenyldimethylsilane

L11

Yes

60 MPa1.5 - 8.0

Company C Biphenyl

2.7

---

9

150

7

Biphenyldimethylsilane

L11

Yes

60 MPa1.5 - 9.0

Bonding state using trifunctional reagents
O
O Si
O

★Sunniest Biphenyl and SunShell Biphenyl use biphenyltrichlorosilane reagents and are in the bonding
state as described above. Trifunctional reagents have the characteristic of improving acid stability.

Bonding state using mono-functional reagentsO Si★Other companies' A, B, and C Biphenyl all use biphenyldimethylchlorosilane reagents, and the bonding
state is as described above. Regarding acid stability, mono-functional reagents are inferior to trifunctional
reagents.

3

Superficially porous particle

Comparison of core shell Biphenyl columns
Columns for comparison
1. Kinetex Biphenyl, 2.6 μm
2. Halo Biphenyl, 2.7 μm
3. Raptor Biphenyl, 2.7 μm
4. SunShell Biphenyl, 2.6 μm

Comparison of core shell Biphenyl phases using standard samples
1 3

SunShell C18

6

4

7

5

2

Column: Company A Biphenyl, 2.6 μm 150 x 4.6 mm
13Company B Biphenyl, 2.7 μm 150 x 4.6 mm
k5=3.23

5

4

7

6

Company C Biphenyl, 2.7 μm 150 x 4.6 mm
SunShell Biphenyl, 2.6 μm 150 x 4.6 mm

Company A Biphenyl
21.8 MPa

2

Mobile phase: CH3OH/H2O=75/25
Flow rate: 1.0 mL/min, Temperature: 40 ºC

3

1

5

4

k5=3.08

Sample: 1 = Uracil, 2 = Phenol, 3 = Caffeine, 4 = Butylbenzene

7

6Company B Biphenyl
19.3 MPa

O

H3C

2

N
H

O

O

7

6

5

4

Company C Biphenyl
22.8 MPak5=3.21

CH3

N

N
CH3

CH3

2

Hydrogen bondα(Caffeine/Phenol)
3

1

CH3
N

N

5 = Amylbenzene, 6 = o-Terphenyl, 7 = Triphenylene

3

1

O

OH
NH

5

4
2

0

5

k5=5.38

6

10

SunShell C18
Company A Biphenyl
SunShell Biphenyl
Company B Biphenyl
19.2 MPa
Company C Biphenyl
SunShell Biphenyl

7

15

20

Hydrophobicity

Steric selectivityα(Amylbenzene/Butylbenzene) α(Triphenylene/o-Terphenyl)

0.39
2.69
2.52
3.07
1.82

1.60
1.39
1.37
1.38
1.44

1.46
1.45
1.46
1.45
1.28

Retention time/min
Compared with other company's core shell type Bipjhenyl. Biphenyls from companies A, B and C showed similar separation patterns. It was
confirmed that SunShell Biphenyl has a higher retention of amylbenzene No. 5 and a larger separation factor (hydrophobicity in the table) for thedifference of one carbon between butylbenzene and amylbenzene, and is more hydrophobic than other company Biphenyl. The carbon loading is
5% for SunShell Biphenyl and 7% or more for all other Biphenyls, and the high hydrophobicity of SunShell, which has the lowest carbon content,
indicates high density end-capping. Moreover, the separation of standard samples is very different for biphenyl and C18.

Comparison using pyridine

Comparison using oxine
23Company A
Biphenyl23Company A
Biphenyl

11Company A
Biphenyl

2

1
3

2

Company B
BiphenylCompany C
Biphenyl

1

3

2

1

SunShellBiphenyl

2
1

4

2

1

2

1

2

Company B
Biphenyl

32Company C
Biphenyl

2

2

1

1
3

1

3

Company B
Biphenyl210Comparison using formic acid6

8

SunShellBiphenyl

10Retention time/min
Column dimension: 150 x 4.6 mm
Mobile phase: CH3OH/H2O=30/70
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@250nm
Sample: 1 = Uracil
2 = Pyridine
3 = Phenol

Pyridine is a compound that tends to tail due toresidual silanol groups. When methanol is usedas the organic solvent in the mobile phaserather than acetonitrile, tailing of pyridine ismore likely to occur and the difference
between brands becomes clear.

0

2

4

6

8

10

Retention time/min
Column dimension: 150 x 4.6 mm
Mobile phase: CH3CN/20mM H3PO4=10/90
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@250nm
Sample: 1 = 8-Quinolinol (Oxine)
2 = Caffeine

8-Quinolinol (oxine) is a metal chelating
compound, and if there are metal impurities
in the packing material, its peak becomes atailing peak.

4

0

1

2

Company C
Biphenyl

3

SunShellBiphenyl

3

4

5

6

Retention time/min
Column dimension: 150 x 4.6 mm
Mobile phase: CH3CN/0.1% H3PO4=2/98
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@210nm
Sample: 1 = Formic acid
2 = Acetic acid
3 = Propionic Acid

Although most acidic compounds can beeluted without problems, formic acid is atypical acidic compound that easily causes
tailing. Formic acid is tailed on Company A
Biphenyl.

Superficially porous particle

Comparison of core shell Biphenyl phases using basic compounds

Company A Biphenyl

Company B Biphenyl

Company C Biphenyl

SunShell Biphenyl

Retention me/min

Retention time/min

Column dimension: 150 x 4.6 mm
Mobile phase: CH3CN/20 mM Phosphate buffer pH 7.0=60/40
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@250 nm
Sample: 1 = Uracil, 2 = Propranolol, 3 = Nortriptyline, 4 = Amitriptyline

Column dimension: 150 x 4.6 mm
Mobile phase: CH3CN/10 mM Ammonium acetate pH 6.8=40/60
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@250 nm
Sample: 1 = Uracil, 2 = Propranolol, 3 = Nortriptyline, 4 = Amitriptyline

CH 3
O

NH

CH 3

CH 3
NH

OH

N

CH 3

CH 3

Tailing of basic compounds (amitriptyline) occurs more easily in the acetonitrile/buffer mobile phase than in the methanol/buffer mobile
phase. A mixture of acetonitrile and 20 mM phosphate buffer and a mixture of acetonitrile and 10 mM ammonium acetate were used as
mobile phase to compare with core shell Biphenyls. Biphenyl from other companies had a terrible tailing for basic compounds, and it wasalso confirmed that the retention time was increased, which seems to be influenced by the adsorption to residual silanol groups.

Comparison of stability
Comparison of plate number under weakly
alkaline condition

Bleed comparison under acidic condition
80

60
50
40
30

Relative plate number/%

Absorbance/mAU

100

Peak area━ SunShell Biphenyl
100━ Company A Biphenyl 350━ Company B Biphenyl 355━ Company C Biphenyl 807

70

20

Chromatogram of a base line at
the third gradient cycle

10

80
60

● SunShell Biphenyl■ Company A Biphenyl
Company B Biphenyl▲ Company C Biphenyl

40
20

0

0

-10
5

6

7

8

9

10

11

12

0

Retention time/min

%B

0
10

5
10

10
90

100

150

200

250

300

Elution time/h

Bleed test condition
Column dimension: 50 x 2.1 mm
Mobile phase: A) 1% H3PO4 (pH 1.2)
B) Acetonitrile
Gradient programTime (min)50

Elution condition of stability test
Column dimension: 50 x 2.1 mm
Mobile phase: 20 mM Sodium phosphate pH 8.0
Flow rate: 0.2 mL/min
Temperature: 40 ºC
17
90

18
10

35
10

Measurement condition of plate number
Mobile phase: CH3CN/H2O=50/50
Flow rate: 0.2 mL/min
Temperature: 40 ºC
Detection: UV@250 nm
Sample: Butylbenzene

Flow rate: 0.3 mL/min
Temperature: 40 ºC
Detection: UV@250 nm

Stability under acidic condition compared baseline changes with gradient elution of 1% aqueous phosphate solution and acetonitrile. Biphenyl groups are cut
off from the silica surface under acidic condition and elute out of the column as the amount of organic solvent increases. Baseline variability detected cut
Biphenyl groups, with SunShell showing the least desorbed and high acid stability. In addition, under weakly alkaline condition, the silica dissolved and thecolumn-in side was dented, so the theoretical plate number of the columns were compared. Since the Biphenyl column has high reproducibility of retention
time even in a 100% aqueous mobile phase (see page 6) and is effective for separating highly polar compounds, stability comparison was performed under
the condition of pH 8 that does not contain an organic solvent. SunShell is more than twice as durable as other companies' columns.
5Comparison of stationary phases using isomers of methylhippuric acid
Methanol/buffer=25/75

Acetonitrile/buffer=13.5/86.5

p- m-

C18

α=1.039

o-

p- m-

C30

α=1.022o-p- m-

Phenyl-hexyl

α=1.033o-α=1.034o-p- m-

Phenyl-hexyl

α=1.029

o-

p- m-

C30

p- m-

C18

α=1.029

opm-

Biphenyl

α=1.047o-p-

Biphenylmo-Retention time/min

Retention time/min

2-Propanol/buffer=7/93
C18

C30

o-

p- m-

po-

Phenyl-hexyl

Tetrahydrofuran/buffer-10/90
p- m-

C18

α=1.057

α=1.077

op-

C30

m-

α=1.070po-m-

α=1.080

o-

m-

p-

Phenyl-hexyl

α=1.038m-

α=1.068o-Biphenylmo-

α=1.009

pp-

Biphenyl

α=1.066m-

α=1.050o-Retention time/min

Retention time/min

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 acid
ortho

meta
O

para
O

O

OH

NH

OH

NH

OH

NH
O

O

O
H 3C

CH3
CH3

The separation of ortho, meta and para isomers of methylhippuric acid was compared. The stationary phase was C18, C30, Phenyl-hexyl andBiphenyl, and the organic solvent in the mobile phase was methanol, acetonitrile, 2-propanol and tetrahydrofuran. The separation of metamethylhippuric acid and para-methyl hippuric acid changed depending on the organic solvent, and the separation was improved in the order ofacetonitrile, methanol, 2-propanol, and tetrahydrofuran. Biphenyl showed a particularly high retention when alcohol was used as the organic
solvent in the mobile phase, and the elution order of meta and para was reversed from that of other stationary phases. It is considered that this isdue to the high hydrogen bonding capacity obtained on comparison of separation of standard samples. When acetonitrile is used, the π-πinteraction between the solute and the stationary phase is weakened by the triple bond of CN in acetonitrile, so it is considered that thecharacteristics of Biphenyl cannot be fully exhibited. When tetrahydrofuran is used, tetrahydrofuran enters the stationary phase and a mixture of
biphenyl group and tetrahydrofuran works as a stationary phase, so it is considered that the separation behavior was different from that whenalcohol was used.
6Superficially porous particle

Comparison of stationary phases using creatinine, mandelic acid, hippuric
acid and methylhippuric acid
Mobile phase for MS detection
Column: SunShell C18 2.6 μm 100 x 2.1 mm
SunShell C8 2.6 μm 100 x 2.1 mm
SunShell Phenyl 2.6 μm 100 x 2.1 mm
SunShell Biphenyl 2.6 μm 100 x 2.1 mm
Mobile phase: 2-Propanol/20 mM Ammonium acetate pH 6.8=3/97
Flow rate: 0.4 mL/min
Temperature: 40 oC
Detection: UV@230 nm

Sample: 1 = Creatinine,

2 = Mandelic aci 3 = Hippuric acid
O

OH

NH

HN

OH

O
H3C

OH

NH

N

O

O

4 = o-Methylhippuric acid, 5 = p-Metylhippuric acid,
O

O
OH

NH
O
CH3

OH

NH
O
H 3C

6 = m-Methylhippuric acid
O
OH

NH
O
CH3

Mobile phase adding ion-pair reagent
Column: SunShell C18 2.6 μm 150 x 4.6 mm
SunShell Phenyl 2.6 μm 150 x 4.6 mm
SunShell Biphenyl 2.6 μm 150 x 4.6 mm
Mobile phase: 2-Propanol:10 mM KH2PO4, 2 mM
Sodium 1-Octanesulfonate (pH 2.5; H3PO4)=10:90
Flow rate: 1.0 mL/min
Temperature: 40 oC
Detection: UV@210 nm
Sample: 1 = Creatinine,
2 = Mandelic aci
3 = Hippuric acid
4 = o-Methylhippuric acid,
5 = p-Metylhippuric acid,
6 = m-Methylhippuric acid

Phosphate buffer was used as the mobile phase for separation of methylhippuric acid isomers on the previous page, but in this comparison of
the separation including creatinine, the ammonium acetate buffer mobile phase that can be applied to LC / MS and the mobile phase in which
an ion pair reagent were added to a phosphate buffer solution was used. 2-Propanol was used as the organic solvent. Under both mobile phaseconditions, Biphenyl showed the highest retention and the best separation. A characteristic separation of Biphenyl is that the elution order ofthe isomers meta-methylhippuric acid and para-methylhippuric acid is reversed to that of C18, C8 and Phenyl-hexyl.

7

Separation of Dinotefuran and Crossianidin
Mobile phase: Methanol/water=10/90

Acetonitrile/water=10/90

2

1

1

2

C18

1

1
2

1

2

Dinotefuran

Biphenyl2Retention time /min

Retention time /min

Crossianidin

Column: SunShell C18, Biphenyl 2.6 μm, 100 x 2.1 mm
Mobile phase: Methanol/water=10/90, Acetonitrile/water=10/90
Flow rate: 0.2 mL/min
Temperature: 45 ºC
Detection: UV@270 nm for dinotefuran, UV@265 nm for crossianidin
Injection volume: 5 μL

データ提供︓株式会社 エスコ

Biphenyl is less hydrophobic than C18, but due to the π interaction (π-π, CH-π interaction, etc.) of the Biphenyl stationary phase, Biphenyl retains more in themethanol / water mobile phase. In the acetonitrile / water mobile phase, the retention behavior was reversed because the π interaction was weakened by the πelectrons of the triple bond of acetonitrile.

Separation of Glycolic acid (organic acids)
RP-AQUA

Column: SunShell RP-AQUA, Biphenyl 2.6 μm,
150 x 4.6 mm
Mobile phase: 0.1% H3PO4
Flow rate: 1.0 mL/min
Column pressure: 18 MPa
Temperature: 40 ºC
Detection: UV@210nm
Injection volume: 1 μL
OOH
6
1 OH H O

1,2
3

5

10

7

3
1 4 56

8

9

0

1

2

3

Tartaric acid4

Retention time/min

H 3C

OH
H OH

O

7

O

O

OH

OH

2

Biphenyl

Maleic acid
Glycolic acid

12 3

5

3

OH
O

8O

OH OH
O

HO

OH

O

Citric acid

Formic acid

1

3

10

7
4

4

O

OH
O

5 86

2

3

OH

CH 3

O

4

OH

OH

O

Succinic acid

Malic acid

9
5

1

O

9

OH
OH

OH

0

OH

Acetic acid

O

1
0

OH

OH

O

Fumaric acid

Lactic acid

Retention time/min

Using alkyl-based stationary phases such as C18 and RP-AQUA, the 1st and 2nd tartaric acid and the glycolic acid peak overlap, but using Biphenyl, the 1st, 2nd
and 3rd tartaric acid, glycolic acid and formic acid are almost baseline separated. In addition, the order of elution of organic acids was significantly different.
8Comparison of stationary phases using nucleic acid bases
A) Methanol/10 mM ammonium acetate
pH6.8=20/80

B) 10 mM ammonium acetate pH6.8

3

k6=0.68223

k5=0.58
6

After stopping pump for1 hour
Decrease in retention: to 15%

3

C18

R5,6=1.0

5

0

4

4

2
2

3

0

2
2

3

4
3

6

k5=0.37

6

RP-AQUAR5,6=2.5

08

k6=4.47
5

4

1. NaNO2

6

4

1

1

2

After stopping pump for1 hour
Unchanging rate in retention: 99%

6
5

1

4

1

1

6
5

k6=4.53
5

1

2

2. Cytosine
NH 2
N

0

1

2
2

3

0

2

3,4

2

k5=0.766
5

R5,6=3.6

4
3

6

8

O

N
H

3. Uracil
After stopping pump for1 hour
Unchanging rate in retention: 96%
O

BiphenylNHk5=5.00

N
H

6
5

4

1

1

O

4. Guanine
O

0

1

2

3

0

2

Retention time/min

4

6

Retention time/min

H
N

HN

8
H 2N

N

N

5. Thymine
Column: SunShell C18 2.6 μm, 150 x 4.6 mm (pore size: 9 nm)

O
H 3C

NH

SunShell RP-AQUA 2.6 μm, 150 x 4.6 mm (pore size: 16 nm)
SunShell Biphenyl 2.6 μm, 150 x 4.6 mm (pore size: 9 nm)
Mobile phase: A) Methanol/10 mM ammonium acetate pH 6.8=20/80
B) 10 mM ammonium acetate pH 6.8
Flow rate: 1.0 mL/min
Temperature: 40 oC

N
HO

6. Adenine
N H2
N

HN
N

N

Detection: UV@250 nm
Sample: 1 = Sodium nitrite, 2 = Cytosine, 3 = Uracil, 4 = Guanine, 5 = Thymine, 6 = Adenine
Separation of nucleic acid bases was performed on C18, RP-AQUA and Biphenyl columns. Two mobile phases were used: methanol/10 mM
ammonium acetate pH 6.8=20/80 and 10 mM ammonium acetate aqueous solution. Nucleic acid bases can be separated even in the mobile
phase containing 20% methanol, but the retenLon Lme is shorter. In comparison of columns used this 20% methanol mobile phase, the retention
factor (k5) of thymine at the 5th peak was highest in Biphenyl, and the resolution of the 5th and 6th peaks of thymine and adenine (R5,6) was
also highest in Biphenyl. When an aqueous mobile phase containing no organic solvent was used, the retention factor of thymine eluting at laston Biphenyl was 5.00 and the adenine eluting at last on C18 and RP-AQUA were 4.53 and 4.47, respectively. Biphenyl showed the largest
retention factor for the compound eluted at last. The selectivity of Biphenyl is very different from that of RP-AQUA and C18, such as the order ofsample elution. It has also been confirmed that the retention time stability when using an aqueous mobile phase is as high for Biphenyl as for RPAQUA. The retention factor on C18 decreased to 15% after stopping the flow for 1 hour, confirming low reproducibility in retention.

★Biphenyl stationary phase has a high hydrogen-bonding capacity and is effective for retaining polar compounds.
Moreover, separation with high reproducibility of retention time is achieved even with 100% aqueous mobile phase.
9Comparison of Biphenyl phases using isomers of methylhippuric acid
＊Measurement condition is the same as that in page 6.

Other companies' Biphenyl and SunShell Biphenyl were compared by isomer separation of methylhippuric acid. When alcohol was used as the organic solvent forthe mobile phase, the elution order was the same, and the degree of isomer separation between meta and para was slightly different for each column. The elution
order was different for acetonitrile. SunShell Biphenyl was the best separation in tetrahydrofuran. While all other companies' Biphenyls are mono-functional
biphenyl, SunShell Biphenyl are trifunctional biphenyl, and it is presumed that this difference was greatly reflected in the separation.

１０Ordering information of SunShell Biphenyl
Packings

Inner diameter
(mm)

1.0

2.1

3.0

4.6

Length (mm)Catalog number

Catalog number

Catalog number

Catalog number

30
50
75
100
150

--------------------------

C86931
C86941
C86951
C86961
C86971

C86331
C86341
C86351
C86361
C86371

C86431
C86441
C86451
C86461
C86471SunShell Biphenyl, 2.6 μm

USP category

L11

Ordering information of Sunniest Biphenyl
Packings

Sunniest Biphenyl, 5 μm

Inner diameter(mm)
Length (mm)
50
100
150
250

2.0

3.0

4.6

10

20

USP category

Catalog number Catalog number Catalog number Catalog number Catalog number
E83241
E83261
E83271
E83281

E83341
E83361
E83371
E83381

E83441
E83461
E83471
E83481---------------E83781---------------E83881

L11

Manufacturer

ChromaNik Technologies Inc.
6-3-1 Namiyoke, Minato-ku, Osaka, 552-0001 Japan
TEL: +81-6-6581-0885 FAX: +81-6-6581-0890
E-mail: info@chromanik.co.jp
URL: http://chromanik.co.jp211211