TR05A

RP + AX +CX Multi-Mode ODS column
The world's first cation and anion IEX multimode ODS column

Reversed Phase + Anion Exchange + Cation Exchange

Simultaneous analysis of both cationic and anionic compounds
ODS + Ion Exchange separation mode
Three kinds of packings with different ion exchange capacities
For polar compounds
Different selectivity from conventional ODS columns
LC-MS compatible without using ion-pair reagentsODS column consists of C18+anion+cation ligand
Purified Porous Silica / 3um Particle / 13nm Pore / ODS + Anion Exchange + Cation Exchange Ligands

ODS column consists of C18+anion+cation ligand Scherzo family columns can operate
ODS + Anion Ligand

Reversed Phase+
Anion Exchange+
Cation Exchange+
Normal Phase
ODS + Cation Ligand

without adding an ion-pairing reagent
which is required for conventional
ODS columns. In addition, both anioic
and cationic compounds are retainedon this column. The hydrophobicity of
the Scherzo family is similar to that ofa conventional ODS column, so
analysis of a compound in combination
with a Unison UK-C18 column(conventional ODS phase) will be very
effective for method development.

Three kinds of ODS with different ion exchange capacities

ODS

Strong
Anion

ODS

ODS

StrongCationLarge amount of strong ionicligands loaded onto this ODS
column.
Effective for improved retentionof zwitterions or weak ioniccompounds

Weak
Anion

ODS

ODS

Weak
CationWeak ionic ligands adequately
loaded onto this ODS column.
Designed for separation ofbasic/acidic compounds atneutral pH condition

Imtakt USA (info@imtaktusa.com)
North America

StrongAnion

ODS

StrongCationLow amount of strong ionicligands loaded onto this ODS
column. Effective for strong ioniccompound elution or basic
compounds with formic acideluentImtakt Corp./JAPAN (info@imtakt.com)
Other Countries

TR05AE-KG01

Separation modes of Scherzo C18 columns
The Scherzo C18 Family, (SS-C18, SM-C18, SW-C18), consists of not only ODS ligands, but also
anion ligands and cation ligands. It also provides reversed-phase mode, both ion exchange modes,
and normal phase mode.

Separation Mode

Stationary Phase

Properties

Reversed-Phase

Octadecyl

Increasing organic solvent composition (decreas
ing porarity of eluent) decreases retention.

CationIncreasing ionic strength (salt or acid concentration) decreases retention for acidic compounds.
Generally, low pH increases retention.

Anion

IIncreasing salt concentration decreases retention
for basic compounds. SM-C18 retains more withincreasing pH, while SS-C18 and SW-C18 retain
more at lower pH.

Anion/Cation

Polar solutes which cannot be retained with
100% aqueous eluent may be retained by using> 50% organic solvent composition due toelectrostatic interaction.

Anion Exchange

Cation Exchange

Normal Phase

Retention properties of Scherzo C18 columns (RP+AX+CX)
Multi-mode ODS Scherzo C18 columns consist of ODS ligands which have reversed-phase mode,
plus anion and cation ligands which have anion/cation exchange modes. Three kinds of Scherzo
columns have individual ion exchange capacities to find the best column for target compounds
which have different ionic properties. Moreover, hydrophobicity between Scherzo C18 and Unison
UK-C18, a conventional ODS, is similar, so these columns can be used as comparisons duringmethod development.
CH3

3
1

+

N CH3

1

2

CH3

phenyltrimethylammnonium(Cation)2

H 3C

p-toluenesulfonic acid
(Anion)

3
1

SO3H

O

3

2

C NH2

benzamide (Neutral)

The entire family, all retain bothbasic compounds (1) and acidic
compounds (2).
SS-C18 shows largest retention,
followed by SM-C18 and then
SW-C18. Retention may beaffected by compound dissociationand mobile phase pH to provide
switched retention.
Non-ionic compounds may beeluted with similar retention
between these columns.

Scherzo SS-C18, SM-C18, SW-C18, 150 x 3 mm
A: 40 mM ammonium formate
B: 100 mM ammonium formate / acetonitrile = 50 / 50
0 - 100 %B (0 - 15 min), 0.4 mL/min, 37 °C, 260 nm

31
2

At neutral pH conditons, SW-C18 has the least retention
capacity, and SM-C18 and SS-C18 have stronger retention
power. These retention differences among the columns
will be enhanced under lower initial salt concentration.

2

Affecting factors for retention on Scherzo C18 columns

0

You can find the best HPLC conditions by optimizing ionic strength or pH to improve retention /separation for ionic compounds, which is difficult on a conventional ODS column
Not only pH but also ionic strength (concentration of salt or acid) will strongly affect the retention on
Scherzo C18 columns
This effect is the same as ion-exchange columns
Scherzo C18 columns, which have RP + both ion exchange modes, require optimization of organic
solvent, pH, and ionic strength
phenyltrimethylammnonium For the Scherzo multi-mode
benzenesulfonic acid
CH3
(Cation)(Anion)ODS columns, optimizing the
SO3+
N CH3
salt concentration is as
CH3
important as optimizing theorganic solvent. In the left
Scherzo SS-C18
Scherzo SS-C18
figure, retention of both anionicand cationic compounds are
Salt
decreased
when
salt
concentration is increased. At
benzamide (Neutral)benzamide (Neutral)
the same salt concentration, SS(SS-C18, SM-C18, SW-C18)(SS-C18, SM-C18, SW-C18)
C18, which consists of a large
amount of strong ionic ligands,
Scherzo SM-C18
Scherzo SM-C18
retains strongly. SM-C18 , withweak ionic ligands has medium
retention. And finally, SW-C18,
Scherzo SW-C18
Scherzo SW-C18
which has a low amount ofstrong ionic ligands, shows thelowest retention.
20

40

60

80

100

0

20

40

60

80

100

mM HCOONH4

mM HCOONH4

benzenesulfonic acid
(Anion)SO -phenyltrimethylammnonium pH is also an important factor
CH3(Cation)for the elution of ionic

3

+

N CH3

compounds.
Retention willchange dramatically between
neutral pH (ammonium acetate
or formate) and low pH (formicacid) conditions. SS-C18 andSW-C18 consist of strong ionicligands and retention will be
increased under low pH
conditions. On the other hand,
SM-C18 consists of weak ion
ligands which cannot ionize at alow pH - therefore, retention ofbasic compounds will bedecreased due to a decrease inionization capacity at low pH.

CH3

Scherzo SS-C18

Scherzo SS-C18

pH

benzamide (Neutral)(SS-C18, SM-C18, SW-C18)

Scherzo SM-C18

Scherzo SW-C18

benzamide (Neutral)(SS-C18, SM-C18, SW-C18)

Scherzo SM-C18

Scherzo SW-C18
2

3

4

pH

5

6

7

2

3

4

pH

5

6

7

Elution strategies by Scherzo C18 columns
Non-ionichydrophobic solutes

Organic solvent composition should be optimized (similar to conventional ODS).
Peak shape is often improved by using 0.1% acetic acid.

Ionichydrophobic solutes

A combination of organic solvent and 20-100mM of salt or acid at optimal pH
should be used for separation of both acidic / basic compounds.

SS-C18 is often the best choice for weak ionic polar solutes. Ionic strength should be
Weak ionic-polar solutes increased for compounds that contain multiple ionic functional groups. Neutral pH
conditions are required for mono-carboxylic acid compounds.
SW-C18 may be useful when it is difficult to elute on SS-C18 or SM-C18. Strong

Strong ionic-polar solutes ionic compounds have strong ionic interaction, so it is recommended to use multiple
gradient elutions with ionic strength and organic solvent composition.

3

Mobile phase preparation for Scherzo C18 columns

Scherzo C18 columns consist of ODS and ionic ligands. At first this new approach is difficult for someusers to conceptualize. However, it is easier to think of the columns as an ODS column AND an ion
exchange column. The following eluent conditions are a rough guideline:

Isocratic Elution
water / acetonitrile / HCOOH = x / y / 0.1(x+y = 100)

Acidic pH Eluent

50mM ammonium acetate / acetonitrile = x / y (x+y = 100)

Neutral pH Eluent

Gradient ElutionA) water / HCOOH = 100 / 0.1
B) water / acetonitrile / HCOOH = 30 / 70 / 0.5

Gradient withAcid and Organic SolventA) 10mM ammonium acetateB) 100mM ammonium acetate / acetonitrile = 30 / 70

Gradient with
Salt and Organic SolventA) water / HCOOH = 100 / 0.1
B) 100mM ammonium formate / acetonitrile = 30 / 70

Gradient with pH,
Ionic Strength,
and Organic Solvent

Comparison of Scherzo columns: Optimized on SS-C18 (Neurotransmitter)
Each Scherzo column has different properties and target compounds depending on column choice.
SS-C18 has strong ionic ligands and is useful for zwitter-ions, as well as for separating weak ioniccompounds.

Catecholamines

O

1

H2N

CH2 CH2

CH2

C

CH2

NH2

CH2

NH

gamma-aminobutyric acid
(GABA)

2
HO

3

2

4

HO

OH

HO
CH

noradrenaline(norepinephrine)

1
5

HO

3

HO

CH

HO

4

HO

H2N
CH2 C

HO

H
C

CH3

adrenaline(epinephrine)

dihydroxy-L-phenylalanine
OH(L-DOPA)

O

1
2 4

HO

5

HO

CH2 CH2

NH2

dopamine

3
5

0

10

20

150 x 3 mm
A: water / formic acid = 100 / 0.3
B: 100mM ammonium formate / acetonitrile = 70 / 30
0-60%B (0-30 min)30 min 0.4 mL/min (9MPa), 37 °C, ELSD

Due to strong ionic ligands, SS-C18 and SW-C18 retain more than SM-C18 which has weak ionicligands. In particular, SS-C18 will provide an excellent retention performance for very polar ioniccompounds. Also, SW-C18, which interacts weakly with these compounds, may be useful for hightroughput analysis.
Formic acid is strictly required at initial conditions due to positive ion of GABA, but dopamine has astronger interaction under such acidic conditions. Finally, this application is done with a multiple
gradient mode using pH, ionic strength, and organic solvent gradients.

4Scherzo SS-C18 column applications (Zwitter-ions)Zwitter-ions which have internal salt generating iso-electric points (pI) are very polar. While it isdifficult to retain zwitter-ions on a conventional ODS column, Scherzo SS-C18 provides very effective
retention ability.
Figure 5a

Zwitter-ions

O

1

NH

O

HO P NH C N CH2 C OH
OH

CH3

creatine phosphate
150 x 3 mm
water / methanol / formic acid= 98 / 2 / 0.6
0.4 mL/min, 37 °C, ELSD

CH32

H3C N+ CH2

O
C
O-

CH3

betaine
3
0

10

2

H3C

CH2

H

N+ CH2

C

CH2

OH

O-

O

H2N

O
CH2

C

L-carnitine

20 minAcidic Eluent, Isocratic

4
creatine

3

1

5
4

Na+

5

CH3 CH2 NH C

CH2CH2 CH2

L-ornithine10

20 minFigure 5b
Na+

5
2

3
4

1

pH + Ionic Strength +Organic Solvent,Multiple Gradient
150 x 3 mm
A: water / HCOOH=100 /0.3
B: 100mM HCOONH4 /ACN
= 50 / 50
10-30 %B (0-30 min)
0.4 mL/min, 37 °C, ELSD

6

0

10

OH

H 2N
H2N

20

30 min5

O

C

L-theanine
60

CH2 CH2 C

H

C

H

O

C
OH

Zwitter-ions, as typified by amino acids are very
polar and difficult to retain on conventional ODS
columns. SS-C18 which consists of many strong
ionic ligands can retain and separate thosezwitter-ions easily.
Figure 5a shows that SS-C18 enables sufficient
retention/separation due to ionic interaction
between positive-charged zwitter-ions underacidic conditions. There are opportunities to get
optimized retention when formic acid or organic
solvent concentration is changed.
Figure 5b shows an example with multiple
gradient elution with pH, ionic strength, andorganic solvent. Zwitter ions are ionized topositive under acidic condition, so initial eluent
should have low pH conditions. In contrast,
ornithine has another amino group which
provides a positive charge and becomes a strong
cationic compound, so it is difficult to elute underacidic condition. In this case, neutral pH provides
weak ionic interaction and pH gradient withincreasing of ionic strength and organic solvent isvery effective for elution of ornithine.

Scherzo SM-C18 column applications (Salts, Vitamins)
Scherzo SM-C18, consisting of ODS ligands and both weak anion / cation ligands, is useful for awide range of ionic compounds such as salts, vitamins, acidic / basic compounds, alkaloids etc.
It is also offers different selectivity from conventional ODS like Unison UK-C18.
CH3

Simaltanious analysis of saltHO

Organic Salt

1

1

2

N
O

A: 50mM HCOOH

pH 2.5

2

1

2

1

2

A: 50mM HCOOH :50mM HCOONH4

Tipepidine hibenzate, a popular cough medicine,consists of basic and acidic components.
Conventional ODS columns can usually analyzeorganic salts, but sometimes there is a problem inseparating other compounds. By changing eluent
pH, one can change elution order (selectivity) on
SM-C18. When pH is increased, retention of basic
compounds will be increased, while acidic
compounds will be decreased. SM-C18 was
designed to show effective ionic interaction under
neutral pH conditions.
SM-C18 will provide a better solution thanconventional ODS columns when solute sensitivity
and separation is required. SM-C18 is available for
not only organic salts but also inorganic salts
separation like NaCl.

1

A: 50mM HCOOH :50mM HCOONH4

=1:9

1

2

A: 50mM HCOOH :50mM HCOONH4

pH 4.9= 5 : 95

1

2

A: 50mM HCOOH :50mM HCOONH4

pH 5.4= 2 : 98

1

2

pH 6.4

A: 50mM HCOONH4

2

4

6

Basic

Scherzo SM-C18, 50 x 3 mm
A: formate buffer, B: acetonitrile
20-60 %B (0-8min), 0.4 mL/min, 37 °C, 280 nm=2:8

pH 4.7

tipepidine

A: 50mM HCOOH :50mM HCOONH4=4:6

pH 4.2

S

Acidic=6:4

pH 3.8S

hibenzic acid

A: 50mM HCOOH :50mM HCOONH4

pH 3.5

2
OH

1

2

0

O

8 min

Water-fat soluble vitamins

532

15

Vitamin Mix
648

Scherzo SM-C18, 150 x 2 mm
A: 0.3% HCOOH aq.
B: acetonitrile
0%B (0-0.1 min)
0-30%B (0.1-10 min)
30%B (10-11 min)30-100%B (11- 12 min)
100%B (12-26 min)
0.3 mL/min, 30°C, 2uL(DMSO soln.)
ELSD (SEDEX 90LT, 40°C, 3.5Bar)
RT
Minutes31240%RSD (n=6)
LOD (S/N=3)ng (o.c.)
RT
Response

1-B1: Thiamine

0.92

0.11

2.6

5.7

2-B8: myo-Inositol

1.14

0.09

6.2

6.3

3-B6: Pyridoxine

1.44

0.16

2.7

1.8

4-C: Ascorbic acid

1.94

0.25

2.4

9.6

5-B5: Pantothenic acid

7.32

0.15

6.8

6.9

6-B9: Folic acid

9.92

0.09

3.1

8.0

7-B12: Cyanocobalamin

10.17

0.11

4.1

4.6

8-B2: Riboflavin

10.49

0.11

3.4

5.0

9-B7: Biotin

10.70

0.09

4.2

4.8

10-A: Retinol

17.64

0.06

6.7

1.7

11-K2: Menaquinone

20.87

0.07

2.1

14.2

12-D2: Ergocalciferol

22.89

0.06

1.5

47.6

13-D3: Cholecalciferol

23.26

0.06

2.6

52.6

14-E: a-Tocopherol

23.61

0.07

1.6

45.0

15-K1: Phylloquinone

25.86

0.07

1.7

41.7

10
5

5

12 14
13

78
6 9

10

11

15

20

25 minSince water-soluble vitamins are hydrophilic andfatsoluble vitamins are hydrophobic, simultaneous
analysis of both types of vitamins is a very difficult
issue.
SM-C18 succeeds in this application; gradient elution
from acidic pH aqueous conditions to acetonitrile
provides sufficient separation of various 15 vitamins.
D2 and D3 is also separated. Thiamine (basic
compound) and ascorbic acid are both retained as amulti-mode ODS column advantage.
Detecion is done by ELSD in this application, butthere is an opportunity for MS detection due to use of
a volatile mobile phase.
Data provided by Dr. Eric VERETTE, SEDERE S.A.S., France

6

Scherzo SM-C18 column applications (Separation Selectivity)
Scherzo C18 columns are designed by using the same silica and ODS ligand density as a Unison
UK-C18 column. SM-C18 may be effective in changing selectivity when it is difficult to separate
impurities on an ODS column.

1

Selectivity

H 3C

OH

1
O

2

H3C OH O

2O

mevalonolactoneHOOHmevalonic acid

150 x 3 mm
5mM ammonium formate /acetonitrile = 90 /10
0.4 mL/min (10MPa), 37 °C, ELSD

1

2

0

5 min

2.5

519

Scherzo SM-C18 and Unison UK-C18 are designed insuch a way that they provide similar retention of nonionic compounds. In addition, SM-C18 can retain bothanionic / cationic compounds under neutral pH
conditions. This means that there is an opportunity to
improve separation on SM-C18 than on a conventional
ODS by only changing columns under the same
conditions.
This comparison between SM-C18 and UK-C18 helps todetermine whether or not an unknown compound isionic.

Scherzo SW-C18 column applications (Acidic Compounds)
Scherzo SW-C18 includes a few amount of strong ionic ligands which is a novel structural idea fromtraditional ion-exchange columns. This ssurface structure offers strong ionic compounds elution.
Scherzo SW-C18 has a small amount of strong
ionic ligands which is a novel structural idea fromtraditional ion-exchange columns. This surface
structure provides better elution of strong ioniccompounds.
siRNA (small interfering RNA), around 21mer basepaired double stranded RNA, is getting lots ofattention for next generation medicine. But it isvery polar and has a lot of phosphoric acids
inside, therefore difficult to analyze in RP mode.
Figure 7a on SW-C18 shows multiple peaks ofcommercial siRNA reagents which may include
different structural compounds. SW-C18 seems torecognize the different number of nucleic bases(hydrophobic interaction) and phosphoric acids(ionic interaction). SW-C18 may be useful for
structue study of siRNA.
SW-C18 can separate not only siRNA, but also
ATP or oligo-nucleotides without adding an ionpairing reagent in the eluent. SW-C18 isapplicable for strong ionic compounds like
phosphoric compounds because there is a small
amount of ionic ligands on the surface of thestationary phase.

Figure 7a

siRNAsiRNA Universal
Negative Control(Manufacturer-S)

Control siRNA
Negative Control(Manufacturer-C)

Control siRNA
Human beta-actin(Manufacturer-N)

020

Scherzo SW-C18, 150 x 3 mm
A: 10 mM CH3COONH4
B: 200 mM CH3COONH4 / ACN = 85 / 15
0-100%B (0-45 min)
0.4 mL/min (10 MPa), 37 °C, 260 nm

40 min7

Scherzo SW-C18 column applications (Drugs, Metabolites)
Scherzo SW-C18 will provide excellent performance for basic drug compounds and ionic metabolites
between low and neutral pH conditions due to a small amount of strong anionic / cationic ligands.
SW-C18 may be useful for DMPK or metabolome studies.

Various elution conditions for basic drugs

1

HO

Basic Compounds

2 3
4[1] Acidic pH Conditions
A: water /HCOOH = 100 / 0.5
B: ACN /HCOOH = 100 / 1
20-80%B (0-15 min)

H OH

1

CCH2NHCH3

HClphenylephrineO
C NHCH2H3CN

2

CH2CH3
CH2CH3HCl

N-acetylprocainamide
HN C CH3OO

3C OCH2CH2N

3

4

1

CH2CH3
CH2CH3HClprocaineNH22[2] Multiple Conditions
A: water /HCOOH = 100 /0.1
B: 100mM HCOONH4 /ACN
= 50 /50
0-100%B (0-15 min)

4

HCl

CH2

naphazoline
Scherzo SW-C18, 150 x 3 mm
0.4 mL/min (9 MPa), 37 °C, 260 nm

314[3] Neutral pH Conditions
A:10 mM HCOONH4
B:100mM HCOONH4 /ACN
= 50 /50
0-100%B (0-15 min)

2

0

5

10

Product Information
Column Name

15 minThere are three basic ways to analyze
basic compounds on Scherzo SW-C18.
[1] Gradient with formic acid + organic
solvent concentration will retain basic
compounds as strong positive ions.
Various cationic compounds are retainedwith initial / final formic acidconcentration.
[2] Gradient between low concentrationof formic acid and high concentration ofits salt with organic solvent may separate
a wide-range of polar and strong cationiccompounds.
[3] Gradient with ionic strength at neutral
pH and organic solvent is a very usefuleasy method to analyze both basic andacidic compounds.

Spec.: purified porous silica, 3um particle, 13nm pore, ODS+anion+cation ligand
Column I.D.

Column Length

0.075mm - 0.5mm
1mm, 1.5mm, 2mm, 3mm
4.6mm, 6mm, 10mm

10mm, 20mm, 30mm
50mm, 75mm, 100mm
150mm, 250mm, 500mm

Imtakt USA (info@imtaktusa.com)
North America

Guard Column

Guard Holder
Cartridge Column

Imtakt Corp./JAPAN (info@imtakt.com)
Other Countries