TR03A
Imtakt's 10th anniversary product
The world 's first multi-mode ODS column
Anion Exchange + Cation Exchange + Normal Phase + Reversed Phase

Simultaneous analysis for both cationic and anionic compounds
ODS + IEX or Reversed Phase + Normal Phase
For polar compounds
LC-MS compatible without using ion-pair reagent
Purified Porous Silica / Particle Size 3um / Pore Size 13nm / ODS + Anion Exchange + Cation Exchange Ligands

Appearance of revolutionary multi-mode ODS column
ODS + Anion Ligands

RP+
Anion Ex.
+
Cation Ex.
+(NP)

RP + CX + (NP) mode

ODS + Cation Ligands

Mode

RP + AX + (NP) mode

Imtakt has developed a revolutionary multi-mode ODS column.
Many biomaterials / metabolites are ionic and cannot be retained on conventional ODS
columns. Recent solutions for this issue have the following limitations:* Ion-pairing RP chromatography is not compatible with LC-MS* Aqueous NP (HILIC) fails to separate polar & non-polar compounds
Scherzo SM-C18 can solve many of these problems. SM-C18 contains ODS+cation+anion
ligands and can be used to separate highly polar compounds. Unlike other mixed-mode RP
columns, Scherzo SM-C18 contains ODS ligands, and shows similar behavior to conventional
ODS columns for non-ionic compounds. In addition, the IEX ligands on SM-C18 are highlypolar and provide normal phase mode. This allows for both RP + NP separation mode.
This next generation (multi-mode) ODS column expands the separation possibilities for the
ODS phase.

Imtakt USA (info@imtaktusa.com)
North America

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

TR03AE-IH06

Separation mechanism on multi-mode ODS (RP + Anion Ex.+ Cation Ex.)
Scherzo SM-C18 is a multi-mode ODS column that provides the following modes of separation:
reversed-phase, anion exchange, and cation exchange. These interactions enable the separationof water-soluble vitamins without the use of ion-pairing reagents.
Water-soluble vitamins

1

2

HOCH2

C

O

1

HO

CH2OH
CH2OH

H3 C

N

OH

HO

O

H

RP + Anion Ex. + Cation Ex.

pyridoxine (B6)OH

3

L-ascorbic acid (C)OCnicotinamide areretained by weakcation exchangeinteraction.
O

CH2CH

CH

CHCH2OH

H 3C

N

O

H 3C

N

C

OH

6

N

4 nicotinic acid (B3)
Carboxylgroup innicotinic
acid
interacts byanion
exchange
mode.

2

N

NH
O

4

3

7

5

riboflavin (B2)

Riboflavin is a strong
basic compound(pKa=10.2). Under
these conditions, highionic strength in theeluent decreases
cation exchangeinteraction.
Therefore retention isalmost completely
derived fromhydrophobicity(similar toconventional ODS).

Scherzo SM-C18

NH2
N

Nicotinic acidcontains pyridine
and a carboxylgroup. At neutral
pH, intramolecularinteractions canoccur betweent h e s e t w oionizable groups.
This results inbroad peak shapeon conventional
ODS.

OH

7

2 Pyridoxine andAscorbic acidis difficult
to retain on
ODS.
Retention(without ionpairing reagent) is
possible on
SM-C18 due toanion exchangeinteraction.

OH

nicotinamide (B3)

N

1

OH

NH2

H3 C

N

CH2

N+

5

S

CH3
CH2CH2OH

thiamine (B1)3
5Thiamine
contains an
ionized thiazole
group and iswell retained bystrong cationexchange.

O
H 3C

N

O

6

6 caffeine
Caffeine is
N

CH3

N
N
CH3

7

4

retained byhydrophobic andelectrostaticinteraction on
ODS phase.
Scherzo SM-C18's
design provides
the same ODS
ligand density as
conventional ODS,
resulting insimilarretention.

Unison UK-C18
0

5

10

15

150 x 3 mm (Scherzo SM-C18, Unison UK-C18)
A: 3mM ammonium formate
B: 25mM ammonium formate /acetonitrile = 80 / 20
0 - 100%B (0 - 15 min)
0.4 mL/min (8-9MPa), 37 deg.C, 260 nm
2 uL (0.08-1.5ug, diluted with 0.1M ammonium formate)2

20 minThe mobile phase contains low salt concentration anddoes not require ion-pairing reagents. A dual
gradient consisting of organic solvent strength (RP)and ionic strength (IEX) is effective at producing abalanced separation for these water-soluble
vitamins.
It is sometimes necessary to add salt to a sample
solution containing both anions and cations. Thiscan help to prevent solute intermolecularinteractions.

Separation mechanism on multi-mode ODS (RP + Anion Ex.+ Cation Ex.)
Conventional ODS columns can struggle to retain polar ionic compounds. But Scherzo SM-C18
will be useful in separating both anionic and cationic compounds.
ODS column comparison
1 2

1

CH3
+

Cl-

N CH3

3

2

3
SO3H

O
C NH2

CH3

benzenesulfonic acidphenyltrimethylammnonium

Scherzo SM-C18
1

3

Strong ionic compounds, such as quaternary amines orsulfonic acids, can be difficult to retain / separate on
conventional ODS. Mixed-mode RP columns have a single
ionic ligand (anion or cation) and struggle to retain bothacidic and basic compounds. Separation of both acids andbases require two different methods with two different
mixed-mode RP columns. In contrast, the multi-mode ODS
column, Scherzo SM-C18, consists of both anionic andcationic ligands. Separation of both cations and anions is
possible using one column and one method.

2

ODS + WAX
2

3
1

ODS + WCX
2 1

3

Unison UK-C18
0

5

10

benzamide

150 x 3 mm
50 mM ammonium formate /acetonitrile = 85 /15
0.4 mL/min (8-9MPa), 40 deg.C, 260nm

min

pH dependency to retention
1.5

Scherzo SM-C18

SO3H

benzenesulfonic acid

log k

1

benzamide on Scherzo
0.5

benzamide on Unison
0

Unison UK-C18

-0.5

2

3

4

pH

5

6

7

benzamide on Scherzo

0.5

CH3

benzamide on Unison

+

0.3

N CH3

log k

CH3
0.1

phenyltrimethylammnonium

Scherzo SM-C18

-0.1

-0.3

Unison UK-C18
-0.5

2

3

4

5

6

7

pH
(50mM HCOOH - 50mM HCOONH4) /acetonitrile = 85 /15, 40 deg.C

3

Because Scherzo SM-C18 is a multi-mode
column consisting of ODS+cation+anion
ligands, retention for ionic compounds canbe affected by eluent pH. In the figures to theleft, strong ionic solutes like sulfonic acid andquaternary amine have poor retention onconventional ODS column (regardless ofeluent pH). However, retention for theseionic compounds on Scherzo SM-C18 (IEX) is
dependent upon eluent pH (due to changes
in ionic interaction). The data shows that
retention for acidic compounds is highest
using low pH conditions, highest for basic
compounds using high pH conditions, andconstant for neutral compounds (regardless ofeluent pH).
Scherzo SM-C18 provides hydrophobic
interaction and ion exchange interaction(including both anion and cation exchange).
Changing organic strength and ionic strength
on Scherzo SM-C18 can provide different
selectivity for ionic compounds. This multimode ODS column provides different
selectivity than conventional ODS, and will
be a powerful tool for separation scientists.

Effect of pH, organic solvent, salt concentration, and temperature on solute elution
Elution properties may be affected by eluent pH, organic solvent composition, salt concentration,and temperature.
Organic solvent dependency to retention

1

CH3
+

Cl-

2

C NH2

SO3H

N CH3
1.2

O

3

CH3

benzenesulfonic acidphenyltrimethylammnonium

1benzamide

0.8

One of the benefits to using Scherzo SM-C18 is its ability toretain ionic compounds. Elution for ionic compounds isdependent upon many parameters.

O

0.6

C NH2
0.4

The figure to the left shows the relationship between organic
solvent concentration and retention. The substituted
benzene rings have the following properties: base(quaternary amine), acid (sulfonic acid), and neutral (amide).
Each solute has unique ionic properties. As a result, elution
for these ionic compounds changes with decreasing ionicstrength (due to increasing organic solvent composition).

log k

0.2

0

CH3
+

SO3H

N CH3

-0.2

CH3
-0.4

-0.6

-0.8

0

10

20

30

40

% acetonitrile
50mM HCOONH4 / acetonitrile, 40 deg.C

The figure to the lower left shows retention vs. salt
concentration. Benzamide (neutral) is unaffected by salt
concentration. However, retention for the ionic compounds
is affected by salt concentration. The data to the lower rightshows retention vs. temperature. These three solutes havehydrophobic properties and therefore show decreasing
retention with increasing temperature.

Salt concentration dependency to retention

Temperature dependency to retention
0.6

0.75

O

0.65

0.5

C NH2

O

0.55

C NH2

0.4

0.45

0.35

log k

log k

0.3

SO3H

SO3H
0.2

0.25

0.1
0.15

CH3
+

CH3

0.05

N CH3

0

CH3

+

N CH3
CH3
-0.05

-0.1

-0.15

0

20

40

60

80

-0.2

100

25 30

mM HCOONH4

40

50

60 65

temperature (°C)
50 mM HCOONH4 / acetonitrile = 85 / 15

HCOONH4 / acetonitrile = 85 / 15, 40 deg.C

4

Separation mechanism on multi-mode ODS (Reversed-phase)
The main mode of interaction for Scherzo SM-C18 is hydrophobicity (via the ODS ligand). Scherzo
SM-C18 is designed to get similar results as conventional ODS column (Unison UK-C18) forhydrophobic neutral compounds.
Hydrophobicity
Hydrophobic interaction is an importantinteraction for the ODS phase. Scherzo SMC18
hassimilarhydrophobicity
asconventional ODS column. The figure to theleft shows the relationship between retention
and alkyl carbon number (n) of alkylbenzenes.
The slope (log k / CH2) indicateshydrophobicity; the data shows hydrophobicity
for Scherzo SM-C18 is similar to that of Unison
UK-C18. Therefore, using SM-C18 and UKC18 (under the same experimental condtions)
may be useful for tracking elution behavior ofionic compounds.

2.0
Unison UK-C18

Scherzo SM-C18

log k

1.5
Unison UK-C8

1.0
Unison UK-Phenyl

0.5

0
1

2 3 4 5 6 7 8
Alkyl Carbon Number (n)water /acetonitrile = 40 /60, 1 mL/min, 37 °C
2
4
1
3
5

a(1/4) = 0.52
a(2/4) = 0.69
a(3/4) = 0.81a(5/4) = 1.36

1

1 nitrobenzene
2 benzene
3 ethylbenzoate
4 toluene
5 naphthalene

1 uracil
2 methylparaben
3 ethylparaben
4 isopropylparaben
5 propylparaben
6 isobutylparaben
7 butylparaben

2

3
Rs = 2.0
45N(3) = 11700

Scherzo SM-C18

N(3) = 23900

Scherzo SM-C18
6 7

2

4

1
3
5

a(1/4) = 0.50a(2/4) = 0.68
a(3/4) = 0.81a(5/4) = 1.34

75 x 4.6 mm
water /ACN /AcOH= 40 /60 /0.1
1mL/min (5-6MPa)37 deg.C, 260nm

2

150 x 4.6 mm
water /ACN /AcOH = 60 /40 /0.1
1mL/min (10-11MPa), 37 deg.C, 260nm
3
Rs = 1.9
45

N(3) = 11300

Unison UK-C18

N(3) = 24500

Unison UK-C18
6 7

0

2

4 min

0

5

10

15 minComparison of hydrophobic compounds shows This data shows similar results for separation of isomers
similar elution for SM-C18 and UK-C18. on SM-C18 and UK-C18. This indicates that molecular
Resolution and column performance is similar. recognition of hydrophobic compounds on SM-C18 isThis indicates that neutral hydrophobic the same as conventional ODS columns. This excellent
compounds are not affected by ionic ligands on performance is observed on all Imtakt 3um particle
SM-C18.
products (including multi-mode ODS).

5Separation mechanism on multi-mode ODS (Ion Exchange)
A multi-mode ODS column, Scherzo SM-C18, can be used as an ion-exchange column whenstrong ionic compounds do not interact with reversed-phase ligands.

Anion exchange separationO

Scherzo SM-C18

Ba + NaHOH2C

NaO P OH2C
ONa

1

HO

OH

OH

HO

OH

2

OH

1) D- glucose-6-phosphate(G-6-P)

CH2OH
OO
OH

1, 2

HO

150 x 3 mm
water / formic acid = 100 / 0.1
0.4 mL/min (9MPa)
37deg.C
ELSD, 0.8 uL (2.6-5.3ug)

Unison UK-C18
5

10

15 minO
CH2O P O
2+O Ba

2) D- fructose-1-phosphate(F-1-P)NO2
p-nitrophenyl-beta-glucoside(PNPG)

OH

0

OH

O

O

Scherzo SM-C18 retains strong ionic compounds.
The figure to the left shows that glycophosphates (low pKa
values) interact strongly with cation ligands on the stationary
phase. These compounds are retained under 100% aqueousconditions. Highly polar compounds, which are retained
via ionic interaction (and do not interact with non-polar
ligands), can also elute under normal phase conditions (seefigure below).

Temperature dependency to retention

Organic solvent dependency to retention
1.6

1

1.4

0.95
1.2

PNPG
0.9

log k

0.8

log k

F-1-P

1

G-6-P

0.85

F-1-P

0.6

0.8

0.4

PNPG

G-6-P

0.75

0.2

0.7
0

-0.2

0.65

0

10

20

30

25

% acetonitrile

30

35

40

45

50

55

60

65

70

temperature (°C)
water /acetonitrile /HCOOH = 93 /7 /0.1

water /acetonitrile /HCOOH = A /B /0.1, 37 deg.C

PNPG contains a phenol group and shows
decreasing retention with increasing organic solvent.
In contrast, sugar phosphates (highly polar anioniccompounds) seem to exhibit normal phase behavior.
This can be useful for LC-MS applications where theaddition organic solvent improves ionization
efficiency.

PNPG shows decreasing retention at elevatedtemperatures due to hydrophobicity. In contrast,
sugar phosphates show no loss in retention atelevated temperatures.
Therefore, column
temperature can be tuned to provide a balanced
separation between polar and hydrophobic
compounds.

6

Separation mechanism on multi-mode ODS (Reversed-phase + Normal phase)
The main mode of separation for Scherzo SM-C18 is reversed-phase. But normal phase mode
may also be useful for highly polar compounds.

Relationship between organic solvent and retention
2.5

tocopherol
2

1.5

log k

ascorbic acidibuprofen

1

serine0.5

caffeine

0

-0.5

-1

0

20

40

60

80

100

% acetonitrile

The figure to the left shows the relationship between
acetonitrile concentration and retention on Scherzo
SM-C18.
Tocopherol is a non-polar compound and requires highorganic solvent composition for elution. Ibuprofen, an
acidic and middle-polar compound, also shows
reversed-phase elution.
Caffeine is polar, but does have some hydrophobicity;retention is reduced as acetonitrile is increased up to
60% (RP), but increases as acetonitrile goes past 80%(NP).
Ascorbic acid is highly polar; retention is reduced asacetonitrile is increased up to 40% (RP), but increases
as acetonitrile goes past 50% (NP).
Serine is highly polar (zwitter ion) and is difficult toretain on conventional ODS. Retention on SM-C18
increases over 50% organic (NP).
Low polarity compounds are retained on SM-C18 via
reversed-phase mode. In contrast, polar compounds
may be retained via normal phase mode.

Example for RP + NP mode at the same time
Vitamin formulation often includes both Vitamin C andVitamin E. The polarity of these compounds is quite
different and difficult to analyze with one method.

ascorbic acid (Vitamin C)
Normal phase

tocopherol (Vitamin E)
Reversed phase

0

2

4

6

The figure to the left shows both compounds are
separated by using reversed-phase + normal phase. As
mentioned above, tocopherol elutes via reversed-phase,
but ascorbic acid is retained via normal phase at highorganic composition. Therefore, separation is possible
under isocratic elution with optimized eluent
composition. This method will be useful for vitamin C
and E analysis at quality control laboratories.

8 min

Scherzo SM-C18, 50 x 3 mm
water /acetonitrile /formic acid = 5 /95 /0.1
0.4 mL/min, 37 deg.C, 270 nm

7Application for polar compounds
The multi-mode ODS column, Scherzo SM-C18, will increase separation possibilities overconventional ODS phase by combining basic RP + anion + cation + NP mode.
NeurotransmittersCl-

Neurotransmitters, including catecholamines, arehighly polar and require ion-pairing on conventional
ODS column. The figure to the left shows successful
separation for GABA, glutamic acid, acetylcholine, andcatecholamines on Scherzo SM-C18 without ionpairing reagent.

Scherzo SM-C18
3
7
1 2

4 5

6

1) 4-aminobutyric acid (GABA)
2) glutamic acid3) acetylcholine hydrochloride
4) noradrenaline
5) adrenaline
6) dopamine hydrochloride
7) serotonin hydrochloride

1,Cl-

Unison UK-C18

6
7

4
2
3,5

0

5

10

150 x 3 mm
A: 3mM ammonium acetate
B: 80mM ammonium acetate /acetonitrile = 80 /20
0-100%B (0-12min)
0.4mL/min (9MPa), 37deg.C, ELSD
15 min 3uL (0.65-2.6ug)

Nicotine and metabolites
1

2

Nicotine and its metabolites are basic and difficult
compounds to separate on conventional ODS column.
Scherzo SM-C18 can separate these compounds withisocratic elution (neutral pH).

Scherzo SM-C18

OH

3
N
N

1

N

1) hydroxycotinine

CH3

N

2

O

O

2) cotinine

CH3

Unison UK-C18
N

3
N

0

5

3) nicotine

CH3

150 x 3 mm
50mM ammonium acetate /acetonitrile = 85 /15
0.4mL/min (9-10MPa), 37deg.C, 260nm
0.4uL (0.25ug)

10 min8

Application for polar compounds
Steroid hormone and metabolites
1

Sulfate or glucuronide metabolites are highly polar
compounds with anionic groups and are difficult toretain on conventional ODS columns. In contrast, SMC18 interacts with the dissociated forms of thesemetabolites via IEX mode. This means there is an
opportunity to improve both retention and LC-MS
sensitivity for these metabolites by using SM-C18.

1

OH
HO
OH

2

O
NaOOC

1) b-estradiol 17-(b-D-glucuronide)

Scherzo SM-C18

2

3

H3C

O

H3C

OH
HO

3

2) 17b-estradiol
HO
H3C

OSO3K

3) b-estradiol 3,17-disulfate
KO3SO

50 x 3 mm
A: 5mM ammonium acetate
B: 125mM ammonium acetate /acetonitrile = 20 /80
30-100%B (0-5min)
0.5mL/min (5-9MPa), 37deg.C, 280nm
1uL (0.5-2.5ug)

Unison UK-C18
0

2

6 min

4

Melamine and cyanuric acid1

2

Melamine (cation) and cyanuric acid (anion) are bothpolar compounds and difficult to retain on
conventional ODS columns and mixed-mode RP
columns. Scherzo SM-C18 has anion / cation ligands
and can retain both compounds via IEX mode.
Inaddition,
normal
phase
mode
can
be
used
to
retain5mM ammonium formatethese polar compounds.

Anion Exchange+ Cation Exchange

1
NH2

OH

2

HO

Normal Phase

N

N

N

N

N

OH

1) cyanuric acid

H 2N

N

2) melamine

5mM ammonium formate /acetonitrile = 7 / 93

05

Scherzo SM-C18, 150 x 3 mm
0.4mL/min (4-9MPa), 37deg.C, ELSD
3uL (1.5ug, 2.5%NH4OH)

10 min9

NH2

Recommendations for Scherzo SM-C18
Scherzo SM-C18 consists of ODS, anion, and cation ligands. The stationary phase interacts with ionicsolutes which can sometimes lead to fluctuating retention and separation (due to electric charge
between eluent and stationary phase). The following recommendations should be considered for
reproducibility and ruggedness.

Confirm the reproducibility by repeat-injections

Variable retention / separation can be observed during repeat injections due to variable dissociation of ligands
on the stationary phase. To confirm optimized condition, several injections are recommended. If retention is
not reproducible, it is recommended to either increase ionic strength in eluent or change to gradient elution.

Salt concentration in sample solution

Acidic and basic compounds in sample solution may interact with each other forming complexes which canhave a detrimental effect on peak shape due to undesirable dispersion within the column. It is therefore
recommended to add salt, acid, or alkali to the sample solution in order to avoid this problem.

Do not use pH meter. Buffer in eluent should be prepared by mixing of acid and base solutions

Buffers prepared with pH meters (e.g. titrate to desired pH) are not reproducible. Precision for [salt] and pH is
crucial for buffers used with HPLC. It is strongly recommended to prepare buffers by measuring appropriate
volumes of (equal concentration) acid and base solutions. Please refer to the relationship between acid/basevolume ratio and pH table below. Buffer concentration should be optimized for each method.
(room temp.) pH values in the table to the left were

Acetate Buffer
20mM CH3COOH
20mM CH3COONH4

pH

10 9 8 7 6 5 4 3 2 1 0
0 1 2 3 4 5 6 7 8 9 10
3.2 3.8 4.1 4.3 4.6 4.7 4.8 5.0 5.3 5.6 6.8

Formate Buffer
50mM HCOOH
50mM HCOONH4

pH

10
8
6
4
2
1 0.5 0.2 0.1 0
0
2
4
6
8
9 9.5 9.8 9.9 10
2.5 3.1 3.5 3.8 4.2 4.7 4.9 5.4 6.1 6.4

measured under room temperature and fixed
concentration. These pH values will varyslightly due to differences in temperature andconcentration. Appropriate volumes of (equal
concentration) acid and base solutions
should be measured and mixed in order toachieve desired pH. After mixing of buffered
solution, appropriate volume of organic
should be added in order to achieve desiredaqueous / organic composition.

Ordering Information
Particle: 3um Silica, Pore Size: 13nm, Ligand: ODS + Anion Exchange + Cation Exchange
Length
(mm)
10
20
30
50
75
100
150
250
500

Product Code

1 mm I.D.

1.5 mm I.D.

2 mm I.D.

3 mm I.D.

4.6 mm I.D.

6 mm I.D.

10 mm I.D.

SM011
SM012
SM013
SM014
SM015
SM016
-

SM071
SM072
SM073
SM074
SM075
SM076
-

SM020
SM029
SM021
SM022
SM023
SM024
SM025
SM026
-

SM030
SM039
SM031
SM032
SM033
SM034
SM035
SM036
-

SM000
SM009
SM001
SM002
SM003
SM004
SM005
SM006
SM007

SM061
SM062
SM063
SM064
SM065
SM066
-

SM0P1
SM0P2
SM0P3
SM0P4
SM0P5
SM0P6
-

Guard Holder( Column coupler enclosed )Guard Cartridge SM-C18

Separation column ID

Product CodeSeparation column ID

Product CodeNote

for 1 - 6 mm I.D.

GCH01Sfor 10 mm I.D.

GCH02Mfor 1-1.5 mm I.D.
for 2 - 6 mm I.D.
for 10 mm I.D.

GCSM0CGCSM0SGCSM0M

5 x 1 mm, 3 pcs
5 x 2 mm, 3 pcs
10 x 8 mm, 2 pcs

Imtakt USA (info@imtaktusa.com)
North America

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