01

Column Selection GuideYMC Columns ------------------------------------------- 8-9
USP ------------------------------------------------------ 10
Column selection guide (Biochromatography)------------ 11
Features of separation modes------------------------- 12-17
Column selection guide(Low molecular weight organic compounds) ------------- 18
Application -------------------------------------------- 19-21
Reversed-phase column selection guide ----------------- 22Comparison of hydrophobicity and hydrogen-bonding
capacity of various C18 columns ------------------------- 22

01

Column Selection Guide

Column Selection GuideYMC Columns
USP
Class No.

Pore size(Å)

Particle size
(μm)

C18

L1

120

1.9, 3, 5

20

C18 ExRS

L180

1.9, 3, 5

25

C8

L7

120

1.9, 3, 5

17

PhenylL11

120

1.9, 3, 5

17

PFP

L43

120

1.9, 3, 5

15

Bio C4L26

300

1.9, 3, 5

–

C18

L1

80

2.7

7

C18 BIO

L1

160

2.7

5

Product name

YMC-Triart

Meteoric Core

Pro series

C8

L7

80

2.7

5

Pro C18L1120

2, 3, 516

Hydrosphere C18

L1

120

2, 3, 5

12

Pro C18 RS

L1

80

3, 5

22

Pro C8

L7

120

3, 5

10

Pro C4

L26

120

3, 5

7

120

3, 5

17

ODS-A

L1

200

5

12

300

3, 5

7

120

3, 5

17

120

3, 5

14

Reversed-phase

ODS-AM

YMC-Pack

L1

ODS-AQ

L1

200

5

10

ODS-AL

L1

120

5

17

120

3, 5

10

C8

L7

200

5

7

C4

L26

TMS

L13

Ph

L11

UsablepH range

Page
Analytical Preparative
columncolumn
49-51

Yes

1-12

52
53

1-10

54

No

1-8

55

Yes

1-10

56

Yes

1.5-1072-74

110-114

–

1.5-9
2-8
Yes

1-10
2-7.5

78
79
80

110-11681
81

115-116110-116

82
Yes

82
83

No

2-7.5

115-116
115-116110-116
115-11683

115-11684

115-11684

115-116

300

5

4

120

3, 5

7

200

5

5

300

5

3

120

3, 5

4

85

115-11685

115-11686

115-116
115-116

120

3, 5

9

120

3, 5

7

300

5

3

Yes

CN

L10

PROTEIN-RP

L26

200

5

4

–

1.5-7.5

86

YMCbasic

L7

200

3, 5

7

Yes

2-7.5

87

–

YMC Carotenoid

L62

–

3, 5

–

–

2-7.5

87

115

88

115-116

88–

J'sphere ODS

YMC-Pack PolymerC18

8

C% Endcapping

ODS-H80

L1

80

4

22

ODS-M80

L1

80

4

14

ODS-L80

L1

80

4

9

L1

–

6, 10

–

1-9
Yes

–

2-7.5
2-13

YMC-Triart Diol-HILICSILNormal-phase

SIL-06Diol-NPYMC-Pack

CN

USP
Class No.

Pore size(Å)

Particle size
(μm)

L20

120

1.9, 3, 5

120

3, 5

60

5

L3

L20
L10

Ion exchange
GFC
GPC

SEC
HIC
Chiral separation

5

L24

120

5

120

5

NH2

L8

120

5

PA-G

–

120

5

nonporous

3, 5

nonporous

3, 5

QA

porous5

SPporous

5

250

3

SF

–

L20
L59

Diol-120
Diol-200

60
L20
L33
L59

Diol-300

YMC-GPC

120
200
300

–

3, 5

50

10

100

10

500

10

1000

10

MIX

10

–

4

Amylose-SA

L99

–

3, 5

Cellulose-SB

–

–

3, 5

Cellulose-SC

L119

–

3, 5

Cellulose-SJ

–

–

3, 5

Amylose-C Neo

L51

–

3, 5

Cellulose-C

L40

–

3, 5

300

5

NEA (R), (S)α -CD BR
β -CD BRγ -CD BR–

12

UsablepH range
2-10

2-7.5

–

–
2-9.5
2-7.5

–

–

–

–

Page
Analytical Preparative
columncolumn57–

96

115-116

96

115

97

115-116

97

–

98-99

115-116100

115-116

4-7.5

100

–

2-12

24-26

–

32-33

–

34-35

36

5-7.5

2, 3, 5

nonporous

BioPro HIC BF

YMC CHIRAL

120

L111

Diol-60CHIRAL ART

5

PVA-Sil

YMC-SEC MAB

YMC-Pack

5

PolyamineⅡ

QF
BioPro IEX

60
120

C% Endcapping

Column Selection Guide

Product name

120

5

120

5

120

5

–

–

–

–

117-118

–

–

2-12

38-39

–

62-66

62-66

2-6.5

67

–

3.5-6.5

67

–

2-9
–

–

–

–

–

9

01

Column Selection Guide

Column Selection Guide

USP
USP
Class
No.

L1

Octadecyl silane chemically bonded to porous or nonporous silica
or ceramic microparticles, 1.5 to 10 μm in diameter, or a monolithic
silica rod.

L3

Porous silica particles, 1.5 to 10 μm in diameter, or a monolithic
silica rod.

L7

Octylsilane chemically bonded to totally porous or superficiallyporous silica particles, 1.5 to 10 μm in diameter, or a monolithic
silica rod.

L8
L10

An essentially monomolecular layer of aminopropylsilane
chemically bonded to totally porous silica gel support, 1.5 to 10 μm
in diameter.
Nitrile groups chemically bonded to porous silica particles, 1.5 to10 μm in diameter.

Bondedphase

YMC productPage

YMC-Triart C18
YMC-Triart C18 ExRS
Meteoric Core C18
Meteoric Core C18 BIO
YMC-UltraHT Pro C18
YMC-Pack Pro C18
YMC-UltraHT Hydrosphere C18
Hydrosphere C18
YMC-Pack Pro C18 RS
YMC-Pack ODS-A
YMC-Pack ODS-AM
YMC-Pack ODS-AQ
YMC-Pack ODS-AL
J'sphere ODS-H80
J'sphere ODS-M80
J'sphere ODS-L80
YMC-Pack SIL
YMC-Pack SIL-06
YMC-Triart C8
Meteoric Core C8
YMC-Pack Pro C8
YMC-Pack C8
YMCbasic

49-51
52

NH2

YMC-Pack NH2

100

CN

YMC-Pack CN

86

C18

Silica

C8

72-74
78
79
80
82
82
83
83
88
96
53
72-74
81
84
87

L11

Phenyl groups chemically bonded to porous silica particles, 1.5 to10 μm in diameter.

PhenylYMC-Triart Phenyl
YMC-Pack Ph

54
85

L13

Trimethylsilane chemically bonded to porous silica particles, 3 to10 μm in diameter.

C1

YMC-Pack TMS

85
57
96
32-33

Diol

YMC-Triart Diol-HILIC
YMC-Pack Diol-NP
YMC-SEC MAB
YMC-Pack Diol-60
YMC-Pack Diol-120
YMC-Pack Diol-200
YMC-Pack Diol-300
YMC-Pack PVA-Sil

97

L20

L24

Dihydroxypropane groups chemically bonded to porous silica or
hybrid particles, 1.5 to 10 μm in diameter.

Polyvinylalcohol chemically bonded to porous silica particle, 5 μm
in diameter.

Polyvinylalcohol

L26

Butyl silane chemically bonded to totally porous silica particles, 1.5
to 10 μm in diameter.

C4

L27

Porous silica particles, 30 to 50 μm in diameter.

Silica

L33

Packing having the capacity to separate dextrans by molecular size
over a range of 4,000 to 500,000 Da. It is spherical, silica-based,
and processed to provide pH stability.

Diol

L40
L43
L51

L59

L62
L99
L111
L119

10

USP Description

Cellulose tris-3,5-dimethylphenylcarbamate coated porous silica
particles, 3 to 20 μm in diameter.
Pentafluorophenyl groups chemically bonded to silica particles by apropyl spacer, 1.5 to 10 μm in diameter.
Amylose tris-3,5-dimethylphenylcarbamate-coated, porous,
spherical, silica partilces, 3 to 10 μm in diameter.

YMC-Triart Bio C4
YMC-Pack Pro C4
YMC-Pack C4
YMC-Pack PROTEIN-RP
YMC-Pack SIL-HG
YMC-Pack Diol-60
YMC-Pack Diol-120
YMC-Pack Diol-200
YMC-Pack Diol-300

Cellulose tris-3,5CHIRAL ART Cellulose-C
dimethylphenylcarbamate
PFP

YMC-Triart PFP

Amylose tris-3,5CHIRAL ART Amylose-C Neo
dimethylphenylcarbamate
YMC-SEC MAB
Packing for the size-exclusion separation of proteins (separation by
YMC-Pack Diol-60
molecular weight) over the range of 5 to 7,000 kDa. The packing is
YMC-Pack Diol-120
Diola spherical 1.5 to 10 μm, silica or hybrid packing with a hydrophilic
YMC-Pack Diol-200
coating.
YMC-Pack Diol-300
C30 silane bonded phase on a fully porous spherical silica, 3 toC30
YMC Carotenoid
15 μm in diameter.
Amylose tris-(3,5-dimethylphenylcarbamate), immobilized on
Amylose tris-3,5CHIRAL ART Amylose-SA
porous, spherical, silica particles, 3 to 5 µm in diameter.
dimethylphenylcarbamate
Polyamine chemically bonded to porous spherical silica particles,
Polyamine
YMC-Pack Polyamine Ⅱ
5 µm in diameter.
Cellulose tris-(3,5-dichlorophenylcarbamate), immobilized on
Cellulose tris-3,5CHIRAL ART Cellulose-SC
porous, spherical, silica particles, 3 to 5 μm in diameter.
dichlorophenylcarbamate

34-35

56
81
84
86
127,132
34-35

62-66
55
62-66
32-33
34-35

87
62-66
98-99
62-66

Antibodies
Proteins
PeptidesIon
exchange

BioPro IEX Columns

For separation of charge variants

pp.24-26

Sizeexclusion

YMC-SEC MAB

For separation of aggregates and fragments of MAbs

pp.32-33

YMC-Pack Diol

For separation of proteins, sugars, and oligonucleotides

pp.34-35

Hydrophobic
interaction

BioPro HIC BF

For drug-to-antibody ratio (DAR) analysis of
antibody-drug conjugates (ADCs)

pp.38-39

Reversedphase

YMC-Triart C18/C8

For separation of biomolecules with molecularweight of up to 30,000

pp.49-51,53

YMC-Triart Bio C4

For separation of biomolecules with molecularweight of up to 150,000

p.56

YMC-Triart Diol-HILICFor separation of polar compounds with poor retention
on reversed-phase columns

p.57

HILIC

Nucleic
acids

Ion
exchange

Oligonucleotides

BioPro IEX Columns

For high resolution analysis

pp.24-26

Sizeexclusion

Oligonucleotides

YMC-Pack Diol

For separation of biomolecules by molecular weight

pp.34-35

Hydrophobic
interaction

Oligonucleotides

BioPro HIC BF

For separation of biomolecules which is difficult to be
separated by ion exchange chromatography

pp.38-39

Reversedphase

YMC-Triart C18

Nucleic acid bases
Nucleosides
Nucleotides

Hydrosphere C18

Oligonucleotides

YMC-Triart C18/C8
YMC-Triart Bio C4

HILIC

SugarsSizeexclusion

Reversedphase

HILIC

Nucleic acid bases
Nucleosides
Nucleotides

YMC-Triart Diol-HILIC
YMC-Pack Polyamine II

YMC-Pack Diol

YMC-Triart C18
Hydrosphere C18

YMC-Triart Diol-HILIC
YMC-Pack Polyamine II

Usable with 100% aqueous mobile phase

For high resolution analysis
Effective for LC/MS

For separation of polar compounds

For separation or molecular weight
determination of sugars

Usable with 100% aqueous mobile phase

For separation of polar compounds

Column Selection Guide

Column selection guide (Biochromatography)

pp.49-51
p.79

pp.49-51,53
p.56

p.57
pp.98-99

pp.34-35

pp.49-51
p.79

p.57
pp.98-99

11

01

Column Selection Guide

Column Selection Guide

Features of separation modes
Ion
exchangeSizeexclusion

Hydrophobic
interaction

Reversedphase

BioPro IEX Columns

YMC-SEC MAB
YMC-Pack Diol

BioPro HIC BF

YMC-Triart etc.

Principle ofseparation

Electric charge

Molecular weight

Hydrophobicity

Hydrophobicity

Molecular
weight range

Up to several millions

Up to about 1,000,000

Up to several millions

Up to about 150,000

Resolution

+++

++

+++

+++

Speed

++ - +++

+

+++

+++

Loading

+++

++

+++

++

Samplestability

+++

+++

+++

+ - ++

Typicalapplications

Charge variants analysis

Separation of aggregates
Separation of fragments

Drug-to-antibody ratio analysisof antibody-drug conjugates

Peptide mapping
LC/MS
Structural analysis

Reversed-phase separation of biomolecules

Molecular weight of sample

How to select reversed-phase columns

Triart C18
Triart C8(120 Å)30,000
Triart Bio C4(300 Å)150,000

Columns are selected based on molecular weights (MW) of the target
substances to separate proteins and peptides. Triart C18/C8 with poresize 120 Å provide the good separation of substances with MW of up to
30,000 under high temperature. For separation of substances with larger
molecules, wide pore columns are effective. Triart Bio C4 with pore size
300 Å can perform separation of substances with MW of up to 150,000
under high temperature.
An elevated temperature can improve efficiency and peak shape bylowering the viscosity of the mobile phase and improving mass transfer.
The appropriate MW range for a given pore size of Triart can be expanded
compared to using the same pore size at lower temperature.

Influence on separation of peptides and proteins by bonded phase and pore sizes■ Triart Bio C4 (5 μm, 300 Å)■ Triart C18 (5 μm, 120 Å)

300 Å (C4)
BSA (MW 66,000)

MW of peptides and proteins

76,00066,000β-Lactoglobulin (MW 18,363)

25,656
18,363

Column size : 150 X 3.0 mmI.D.
Eluent: A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.1)
10-95%B (0-15 min)Flow rate: 0.4 mL/min
Temperature : 40°C
Detection: UV at 220 nm
Injection: 4 µL (0.1-0.5 mg/mL)
Sample: γ-Endorphin, Insulin, Lysozyme,
β-Lactoglobulin,α-Chymotoripsinogen A,
BSA, Conalbumin

14,000Insulin (MW 5,733)

5,733
1,859
0.00

0.05

0.10

0.15

0.20

Bandwidth at half-height (min)12120 Å (C18)

The left figure shows separation of peptides and proteins
with MW from 1,859 to 76,000 with Triart Bio C4 (5 μm,
300 Å) and Triart C18 (5 μm, 120 Å). With a C18 (120 Å)column, peak broadening is observed for proteins with
MW of 10,000 or more. On the other hand, a wide pore(300 Å) C4 column provides good peak shapes evenfor proteins with high MW proteins. Thus, in general, awide pore C4 column such as Triart Bio C4 is suitable forseparation of proteins with MW of 10,000 or higher.

Effect of column temperature on separation

40℃

2
3, 4
5

F120330H

Rs (3,4)

=

1

1.97

2
3

W1/2 (1) = 0.017
W1/2 (5) = 0.039

50℃

4

1. Oxytocin(MW 1,007)
556)
2. Leu-Enkephalin(MW
3. β-Endorphin(MW 3,465)
4. Insulin(MW 5,733)
5. β-Lactoglobulin A (MW 18,400)

5F120330J

1

2

Rs (3,4) = 3.47
W1/2 (1) = 0.016

60℃

3F120330K

2

Rs (3,4) = 4.87
W1/2 (1) = 0.015
W1/2 (5) = 0.030

70℃
F120330L

0.5

1Column5

Eluent1

04

W1/2 (5) = 0.034

3

1.5

2

Flow rate
Detection

4

Column Selection Guide1

Rs (3,4) = W1/2 (1) = 0.019
W1/2 (5) = 0.046: YMC-Triart C18 (1.9 μm, 120 Å)
50 X 2.0 mmI.D.
: A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.1)
10-80%B (0-5 min): 0.4 mL/min: UV at 220 nm

5

2.5

3

min

Increasing column temperature to 70°C provides
selectivity change, sharper peaks, and therefore
improved resolution especially for larger molecules.

Separation of Antibodies/Proteins/Peptides
Charge variants analysis of monoclonal antibodies
Ion
exchange

BioPro IEX SF 5 µm, 100 X 4.6 mmI.D.

Salt gradient mode
1

2

4

3
1. Natalizumab (Humanized IgG4, pI=7.3)
2. Cetuximab (Chimeric IgG1, pI=7.9)
3. Adalimumab (Human IgG1, pI=8.4)
4. Denosumab (Human IgG2, pI=8.8)
Eluent0

10

20 minFlow rate: A) 10 mM MES-NaOH (pH 5.7)
B) 10 mM MES-NaOH (pH 5.7) containing 1 M NaCl
0-20%B (0-20 min)
: 0.6 mL/min

pH gradient mode
1

2

3

4

EluentFlow rate: A) CX-1 pH Gradient Buffer A* (pH 5.6)
B) CX-1 pH Gradient Buffer B* (pH 10.2)0-100%B (0-20 min)
: 0.6 mL/min*Purchased from Thermo Fisher Scientific Inc.

0

10

20 minJ. Pharm. Biomed. Anal., 2015, 111, 169-176.

Monoclonal antibodies were separated by ion exchange chromatography in salt gradient mode and in pH gradient mode. Sharp peaks were obtained in bothmodes with BioPro IEX columns since they have extremely low nonspecific adsorption. This shows that BioPro IEX columns are effective for separation of
charge variants and isoforms.

13

01

Column Selection Guide
Aggregates analysis of antibody-drug conjugate (ADC)

Column Selection GuideSize
exclusion

YMC-SEC MAB 3 µm, 300 X 4.6 mmI.D.

mAU
14
12
10

Eluent: 0.1 M KH2PO4-K2HPO4 (pH 7.0)
containing 0.2 M NaCl/2-propanol (85/15)
Flow rate: 0.165 mL/min
Temperature : 25°C
Detection: UV at 280 nm
Injection: 4 μL
Sample: Cysteine-conjugated ADC mimic (2.5 mg/mL)

8
6
4
2
0
-2
-4

Size exclusion chromatography is useful for separation of substanceswith different molecular weights such as MAbs, their dimers andaggregates or ADCs, their dimers and aggregates.

M180803EA

10

12

14

16

18

20

22

min

Drug-to-antibody ratio (DAR) analysis of ADC
Hydrophobic
interaction

BioPro HIC BF 4 µm, 100 X 4.6 mmI.D.

mV
30

Eluent: A) 50 mM NaH2PO4-Na2HPO4 (pH 7.0) containing
1.5 M (NH4)2SO4/2-propanol (95/5)
B) 50 mM NaH2PO4-Na2HPO4 (pH 7.0)/2-propanol(80/20)0%B (0-1 min), 0-100%B (1-15 min), 100%B (15-20 min)Flow rate: 1.0 mL/min
Temperature : 25°C
Detection: UV at 280 nm
Injection: 5 μL
Sample: Cysteine-conjugated ADC mimic (5 mg/mL)

DAR4

20
DAR6

DAR2

10

DAR8

DAR0
0

L171130C

0

5

10

15

20 minHydrophobic interaction chromatography column BioPro HIC BF
provides superior separation for ADCs and is effective for DAR
determination.

Monoclonal antibody
Reversedphase

YMC-Triart Bio C4 5 µm, 150 X 3.0 mmI.D.

mAU
1400
Good peak shapes
at 70℃ or higher

1200

Eluent1000
Adsorption

800

Adsorption

600
400

V180219D2

40℃

V180219G2

50℃

V180219K2

60℃

V180219N2

70℃

V180219R2

80℃

V180219U2

90℃

200
0
2

14

4

6

8

10

min#

Flow rate
Detection
Injection
Sample: A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.1)
30-60%B (0-15 min), 90%B (15-30 min): 0.4 mL/min: UV at 220 nm: 4 μL: Humanized monoclonal IgG1

Intact monoclonal antibody was analyzed at temperatures between
40°C and 90°C using Triart Bio C4. Good peak shapes were acquired
at 70°C and higher while there was no elution at 50°C and lower.
Highly durable Triart Bio C4 can perform stable analysis even at 90°C
and is suitable for the reversed-phase analysis of antibodies at hightemperatures.

Antibody fragments
YMC-Triart Bio C4 1.9 µm, 150 X 2.1 mmI.D.
Mobile phase containing TFA

Mobile phase containing formic acid

mAU
20

mAU
20

1. Fc/2
2. Light chain
3. Fd ’

3
15

15
1

10

2

Column Selection Guide

Reversedphase

3

10
1
2

5

5

0

0
V180705B2

V180705H2

0

1

2

3

4

5

6

7

8

9

min #

0

【Sample】

2

3

4

Eluent<TFA>

F(ab’)2
IdeS
digestion

1

Fd’

Denaturation
reduction

Light chain
Fc/2

Fc/2

Eluent<Formic acid>
Flow rate
Temperature
Detection
Injection

5

6

7

8

9

min #

: A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.1)
25-50%B (0-10 min), 90%B (10-12.5 min): A) water/formic acid (100/0.1)
B) acetonitrile/formic acid (100/0.1)
20-45%B (0-10 min), 90%B (10-12.5 min): 0.4 mL/min: 80℃: UV at 280 nm: 4 μL (0.25 mg/mL)

Above are the chromatograms of monoclonal antibody fragments analysis with mobile phase containing either TFA or formic acid. Triart Bio C4 achieves
excellent peak shape even when using mobile phase containing formic acid. Therefore it is ideal for the high sensitive LC/MS analysis.

Peptide mapping
Reversedphase

YMC-Triart C18 1.9 µm, 200 X 2.0 mmI.D. (100 X 2.0 mmI.D. two coupled)

Eluent: A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.08)
5-40%B (0-30 min)Flow rate: 0.4 mL/min
Temperature : 70°C
Detection: UV at 220 nm
Injection: 20 μL
Sample: Triptic digest of Bovine Hemoglobin

P110707A

2

24 minThe outstanding efficiency obtained by a coupling of two 100 mm
length of Triart 1.9 μm columns allows the precise separation in peptide
mapping.

Analysis of antimicrobial peptides
Reversedphase

YMC-Triart C18 1.9 µm, 50 X 2.0 mmI.D.
1. α-Defensin- 1 : A CYCRIPACIAGERRYGTCIYQGRLWAFCC MW 3,442
2. α-Defensin-2 :CYCRIPACIAGERRYGTCIYQGRLWAFCC MW 3,371
3. α-Defensin-3 : D CYCRIPACIAGERRYGTCIYQGRLWAFCC MW 3,486

mAU
60

1

40

2

3

Rs (1,2) = 1.86
Rs (2,3) = 2.47

200F120501D

0

1

2

3

4

min

Eluent: A) water/formic acid (100/0.1)
B) acetonitrile/2-propanol/formic acid (50/50/0.08)
10-25%B (0-10 min)Flow rate: 0.4 mL/min
Temperature : 70°C
Detection: UV at 220 nm
High temperature condition improves separation of peptides andproteins on reversed-phase chromatography. Since Triart columns arehighly durable at high temperatures, they are effective for analysis of
peptides and proteins under such harsh conditions.

15

01

Column Selection Guide

Column Selection Guide

Separation of Nucleic Acids
Phosphorothioate oligonucleotides
Reversedphase

YMC-Triart C8 [Metal free column] 1.9 µm, 100 X 2.1 mmI.D.

mAV
1

1. 5’-U^C^A^U^C^A^C^A^C^U^G^A^A^U^A^C^C^A^A^U-3’(RNA 20 mer All PS )2. 5’-G^U^C^A^U^C^A^C^A^C^U^G^A^A^U^A^C^C^A^A^U-3’(RNA 21 mer All PS )2

4^=Phosphorothioated3

Eluent: A) 15 mM triethylamine-400 mM HFIP*B) methanol
10-20%B (0-20 min)Flow rate: 0.42 mL/min
Temperature : 70°C
Detection: UV at 260 nm
Injection: 1 µL (each 1.25 nmol/mL)2
Rs (1,2) =1.94
1*1,1,1,3,3,3-hexafluoro-2-propanol0

Triart C8 [Metal free column] provides superior resolution of fulllength (n) and n-1 failure seaquence oligonucleotides.

AB190618B06

0

4

2

Ion
exchange

8

6

10

14

12

16

18

min

BioPro IEX QF 5 µm, 100 X 4.6 mmI.D.
Single-stranded DNA (15 mer)

mAU
10

1

5

2

1. 5’-T^T^T^T^T^T^T^T^T^T^T^T^TTT-3’(DNA 15 mer 12PS, 2PO)
2. 5’-T^T^T^T^T^T^T^T^T^T^T^T^T^TT-3’(DNA 15 mer 13PS, 1PO)
3. 5’-T^T^T^T^T^T^T^T^T^T^T^T^T^T^T-3’(DNA 15 mer All PS )3

Rs (1,2) =1.74
Rs (2,3) =1.90^=PhosphorothioatedEluent0

P180606C02

0

5

10

min

: A) 10 mM NaOH
B) 10 mM NaOH containing 1.0 M NaClO4
40-70%B (0-15 min)Flow rate: 1.0 mL/min
Temperature : 25°C
Detection: UV at 260 nm
Injection: 6 µL (each 3.3 nmol/mL)[All PS], [13PS, 1PO] and [12PS, 2PO] of DNA 15 mer wereclearly separated by BioPro IEX column.

2’-O-methyl oligonucleotides
Ion
exchangemAU

BioPro IEX QF 5 µm, 100 X 4.6 mmI.D.

1. 5’-U(M)C(M)A(M)U(M)C(M)A(M)C(M)A(M)C(M)U(M)G(M)A(M)A(M)U(M)A(M)C(M)C(M)A(M)A(M)U(M)-3’ (20 mer)
2. 5’-G(M)U(M)C(M)A(M)U(M)C(M)A(M)C(M)A(M)C(M)U(M)G(M)A(M)A(M)U(M)A(M)C(M)C(M)A(M)A(M)U(M)-3’ (21 mer)
N(M)=2’OMe RNA
2

50
1

40

Eluent: A) 10 mM NaOH
B) 10 mM NaOH containing 1.0 M NaClO4
25-55%B (0-15 min)Flow rate: 1.0 mL/min
Temperature : 25°C
Detection: UV at 260 nm
Injection: 4 μL (5 nmol/mL)

30
20
10
0

P171116A

0

16

2

4

6

8

10

12

14 min

100 mm length column of BioPro IEX QF, which has highseparation ability, is suitable for separation of oligonucleotides by
one nucleotide difference.

Oligonucleotides
YMC-Triart C18 1.9 µm, 50 X 2.1 mmI.D.

mAU

1. 5’-CAC UGA AUA CCA AU-3’ (14 mer)
2. 5’-UCA CAC UGA AUA CCA AU-3’ (17 mer)
3. 5’-UCA UCA CAC UGA AUA CCA AU-3’ (20 mer)
4. 5’-GUC AUC ACA CUG AAU ACC AAU-3’ (21 mer)

4

15

Eluent10: A) 8 mM triethylamine-200 mM HFIPB) methanol
10-20%B (0-10 min)Flow rate: 0.42 mL/min
Temperature : 65°C
Detection: UV at 260 nm
Injection: 1 μL (2-4 nmol/mL)

3
2

1
5

0

Column Selection Guide

Reversedphase

F180403J

0

2

Reversedphase

4

6

8

10 min#Triart C18 is suitable for separation of hydrophilic compounds such as
oligonucleotides.

YMC-Triart C18

Column : YMC-Triart C18 (5 μm, 120 Å)
150 X 2.0 mmI.D.
Flow rate : 0.2 mL/min
Gradient : 35-50%B (0-84 min)

mAU
20
15
10
5

Oligonucleotides d(T)2-20

00F111116H

10

20

30

40

50

60

80

70

min

: A) 10 mM di-n -butylamine-acetic acid (pH 6.0)
B) methanol
Temperature : 35°C
Detection: UV at 269 nm
Injection: 1 μL (5 nmol/mL)
EluentColumn : YMC-Triart C18 (1.9 μm, 120 Å)
50 X 2.0 mmI.D.
Flow rate : 0.8 mL/min
Gradient : 35-50%B (0-7 min)

mAU
20
15
10
5
0
0

F111109K

1

2

3

4

5

6

7 min

In the separation of oligonucleotides, 19 peaks are completely
resolved within 7 minutes using a Triart C18 1.9 µm UHPLC column.
The separation is achieved within one tenth of the analysis time of theconventional HPLC method.

Plasmid pBR322 Hae Ⅲ restriction fragments
Size
exclusion

YMC-Pack Diol-300 + Diol-200 5 µm, 500 X 8.0 mmI.D. X 2
Plasmid pBR322 Hae Ⅲ digest (8-587 bp)

mAU

Plasmid pBR322(4,361 bp)

40
30

Eluent: 0.1 M KH2PO4-K2HPO4 (pH 7.0) containing
0.2 M NaCl
Flow rate: 0.7 mL/min
Temperature : ambient (25°C)
Detection: UV at 260 nm
Injection: 10 μL

20
10
0P080617A

0

10

20

30

40

50 minIn size exclusion mode, separation is expected to be improvedby using coupled columns with different pore sizes.

17

01

Column Selection Guide

Column Selection Guide

Column selection guide (Low molecular weight organic compounds)
Pharmaceuticalproducts

Reversedphase

Agriculturalchemicals
Metabolites
Foodadditives

Normalphase

YMC-Triart C18

Suitable as the first choice column forreversed-phase separation

pp.49-51

YMC-Triart C18 ExRS, C8,
Phenyl, PFP, Bio C4

For separation of compounds of difficult by
Triart C18

pp.52-56

YMC-Pack SIL, SIL-06

Standard normal-phase column

p.96

YMC-Pack Diol-NP

Normal-phase column providing separation
characteristics different from bare silica gel

p.96

YMC-Triart Diol-HILICFor separation of polar compounds with poor
retention on reversed-phase columns

p.57

Naturalproducts
OthersHILIC

Vitamins

Reversedphase

Water-soluble
vitamins
Fat-soluble
vitamins

HILIC

Normalphase

Organic acids
Fatty acids

Phospholipids

YMC-Triart C18

YMC-Triart C18

Usable with 100% aqueous mobile phase(For separation under a buffered or ion pairing mobile phase)
Suitable as the first choice ODS column

pp.49-51

pp.49-51

YMC Carotenoid (C30)

Non-endcapped ODS, suitable for separation ofcompounds with similar structure
Separation behavior different from ODS

Water-soluble
vitamins

YMC-Pack Polyamine II, NH2

For separation of water-soluble vitamins such asvitamin C under HILIC mode

pp.98-100

YMC-Triart Diol-HILICFor simultaneous separation of water-soluble vitamins

p.57

Fat-soluble
vitamins

YMC-Pack SIL, SIL-06

For separation of fat-soluble vitamins
such as tocopherol

pp.98-99

pp.49-51

YMC-Pack ODS-AL

YMC-Pack Polyamine II

p.83
p.87

p.96

Reversedphase

YMC-Triart C18

Usable with 100% aqueous mobile phase

Normalphase

YMC-Pack SIL, SIL-06

Standard normal-phase column

Reversedphase

YMC-Triart C18

For separation of molecular species

pp.49-51

For separation of phospholipid classes

pp.96-97

p.96

YMC-Pack SIL, SIL-06

Normalphase

YMC-Pack Diol-NP
YMC-Pack PVA-Sil

Amino acids

HILIC

Free amino acids

Reversedphase

Free amino acids

YMC-Triart Diol-HILICFor simultaneous separation of amino acids
under HILIC mode

YMC-Triart C18

Usable with 100% aqueous mobile phase

pp.49-51

Hydrosphere C18

For separation of hydrophobic amino acids

p.79

YMC-Triart C18

Suitable as the first choice ODS column

YMC-Triart C18 ExRS

High-density bonding for excellent ability torecognize planar structure

Reversedphase

YMC Carotenoid (C30)
YMC-Triart C8
YMC-Triart PFPCHIRAL ART

For carotenoids separation
For separations of isomers or structural analogs
For separations of polar compounds or isomers
For separations of isomers or structural analogs

Normalphase

YMC-Pack SIL, SIL-06

Standard normal-phase column

CHIRAL ART

For separations of isomers or structural analogs

pp.62-66

For separation of chiral compounds

pp.62-67

Labeled amino acids

Structural
isomers

Chiralcompounds

18

CHIRAL ART
YMC CHIRAL NEA

p.57

pp.49-51

p.52
p.87
p.53
p.55
pp.62-66
p.96

Vitamin D

Reversed-phase

Vitamin E (Tocopherols)

Normal-phase

Separation of structurally similar compounds

Separation of tocopherol homologues

mAU

1.
H

H
H3C
CH3

1.

CH3
CH3

H

15

Column Selection Guide

Application

3

1

CH3

H3C

CH3

O

CH3

HO
CH3

CH3

CH3
CH3

CH3

α-Tocopherol

4

H

1
10

CH3

2.

CH2
HO

CH3

HO

CH3
CH3

CH3

CH3

H

2

CH3

O

β-Tocopherol

Ergocalciferol (Vitamin D2)

2
H
H3C
CH3

2.

H

5

3.

CH3

CH3

H3C

CH3

O

CH3

HO

CH3

CH3
CH3

CH3

γ-Tocopherol

H

4.

CH2
HO

0

CH3

CH3

O

CH3

HO

Cholecalciferol (Vitamin D3)
0

10

20

30
min
(P040819C)

Column: YMC-Pack ODS-AL (5 μm, 120 Å)
150 X 4.6 mmI.D.
Eluent: acetonitrile/water (95/5)
Flow rate: 1.0 mL/min
Temperature : 40°C
Detection: UV at 265 nm
Injection: 10 μL (0.01 mg/mL)

Reversed-phase

0

5

10

15 min(S970702A)

Column: YMC-Pack SIL (5 μm, 120 Å)
250 X 4.6 mmI.D.
Eluent: n-hexane/2-propanol/acetic acid (1000/6/5)
Flow rate: 1.4 mL/min
Temperature : 35°C
Detection: FLS at Ex 298 nm, Em 325 nm
Injection: 20 μL (5-20 μg/mL)

Water-soluble vitamins

Water-soluble vitamins

HILIC

Simultaneous separation of water-soluble vitamins under ion pairing mobile phase

Simultaneous separation of water-soluble vitamins under HILIC mode
O

1.

mAU

3
20
5
15

O

O

OH

NH2
N

N

Nicotinic acid

Nicotinamide
9.

4

3.

4.

OH
OH

2-O-α-D-Glucopyranosyl-L-ascorbic acid
（Ascorbic acid 2-glucoside）

NH

N

N

O

10.
N

OH

10

6

OH

5.

0
0

5

10

15

20

25

6

8

10

12

14

16

18

20
min♯

min

Eluent*Dissolve 1.4 g KH2PO4 in 800 mL water → add 26 mL 10% TBA·OH→ adjust pH 5.2 by 20% H3PO4 → add water to make 1000 mL

Flow rate: 0.8 mL/min
Temperature : 40°C
Detection: UV at 260 nm
Injection: 10 μL (5 μg/mL)

O

H2 N

H 2N

NH2
N

O

CN N
+

Co
N

O

N

NH2

6.

HO

H

O

O

N
H

O

OH

Erythorbic acid
（D-Isoascorbic acid）

O

O

O

NH2

N

O

P

(F121012A)

O

O
H2 N

Orotic acid

HO

(R100315D): YMC-Triart C18 (5 μm, 120 Å)
250 X 4.6 mmI.D.
: phosphate buffer*/acetonitrile (90/10)

11.
O

HO

Column-Cl • HCl

NH
N
H

O

4

OH

S

O

HO

2

+

Thiamine hydrochloride

0
0

N
N

Riboflavin

3

4

NH2

OH
HO

50

O

HO

O
N

78

O

O

N

12

O

OH

Pyridoxinehydrochloride

5

H

HO

• HCl

11

5

HO
HO

OH

OH

OH

100

9

O
OH

L-Ascorbic acid
8.

2.

6
7

H O

HO

Caffein

150

10

HO
HO

N
N

9

2

N

O

mAU

8

7.

N

1. Thiamine HCl (Vitamin B1)
2. Pyridoxine HCl (Vitamin B6)
3. Nicotinamide
4. Cyanocobalamin (Vitamin B12)
5. L-Ascorbic acid 2-glucoside
6. L-Ascorbic acid (Vitamin C)
7. Erythorbic acid8. Riboflavin (Vitamin B2)
9. Nicotinic acid

1

CH3
CH3

CH3

δ-Tocopherol

H

O HO N-HOOCyanocobalaminColumn: YMC-Triart Diol-HILIC (5 μm, 120 Å)
150 X 3.0 mmI.D.
Eluent: A) acetonitrile/200 mM HCOOH-HCOONH4 (pH 3.6)/water (90/5/5)
B) acetonitrile/200 mM HCOOH-HCOONH4 (pH 3.6)/water (50/5/45)
0-75%B (0-20 min)Flow rate: 0.425 mL/min
Temperature : 40°C
Detection: UV at 254 nm
Injection: 4 μL (50 μg/mL)

19

01

Column Selection Guide

Column Selection Guide

Application

Vitamin C (Ascorbic acid)

HILIC

Separation of ascorbic acid under HILIC mode

Simultaneous separation of organic acids under 100% aqueous mobile phase

1. Nicotinic acid
2. Erythorbic acid
3. L-Ascorbic acid1

Organic acids

Reversed-phase

mAU

1. Oxalic acid
2. Tartaric acid
3. Glycolic acid
4. Formic acid
5. L-Malic acid
6. Malonic acid
7. Lactic acid8. Acetic acid
9. Maleic acid
10. Citric acid
11. Succinic acid
12. Fumaric acid
13. Acrylic acid14. Propionic acid

2,3

50

12

40

2 3

4
13
30

1

56
8

9

10

20

11
14

7
10

0

10

20

0

min

0

2

4

6

8

min

10

(A970603A): YMC-Pack Polyamine II
250 X 4.6 mmI.D.
Eluent: acetonitrile/50 mM NH4H2PO4 (70/30)
Flow rate: 1.0 mL/min
Temperature : 30°C
Detection: UV at 250 nm
Injection: 10 μL (0.05-0.1 mg/mL)
ColumnColumn: YMC-Triart C18 (3 μm, 120 Å)
150 X 3.0 mmI.D.
Eluent: 20 mM phosphoric acid
Flow rate: 0.425 mL/min
Temperature : 37°C
Detection: UV at 220 nm
Injection: 2 μL (0.005-1.5 mg/mL)

Amino acids

HILIC(U111212Q)

Amino acids

Reversed-phase

Simultaneous separation of amino acids under HILIC mode

Separation of hydrophobic amino acids under highly aqueous mobile phase

pA
500

Standard solution* 1*(1.10 mg/mL L-Valine, 0.92 mg/mL L-Isoleucine, 1.84 mg/mL L-Leucine)
Phenylalanine (Phe)
Tryptophan (Trp)

400

mAU

Leucine (Leu)

Methionine (Met)
Tyrosine (Tyr)

*

3

1

Isoleucine (Ile)

**

150

Valine (Val)300

Cysteine HCl (Cys)
Proline (Pro)2

100

System suitability
requirement

Threonine (Thr)

Resolution (2, 3)

2.68

Alanine (Ala)γ-Aminobutyric acid (GABA)

200

Serine (Ser)1

Relative standard deviation of
the retention time(each of the peaks)

50

Result1.5

1.0%

Glycine (Gly)

0.02%

2

0.02%

3

0.02%

Asparagine (Asn)Glutamine (Gln)Citrulline (Cit)100

Glutamic acid (Glu)*Histidine (His)*

Arginine HCl (Arg)

0

6.0

8.0

10.0

6

2.

H

12.0 14.0 16.0 18.0 min*Chloride ion contained in the sample solution**Sodium ion contained in the sample solution

NH2

L-Valine

min
3.

H
COOH

H3C

Ornithine HCl (Orn)

4.0

4

CH3

Lysine (Lys)

2.0

2
1.

Aspartic acid (Asp)

0

0.0

0

CH3

COOH

H3C

COOH

H3C
H

NH2

L-Isoleucine

H3C: YMC-Triart Diol-HILIC (5 μm, 120 Å)
150 X 4.6 mmI.D.
Eluent: A) 100 mM HCOOH-HCOONH4 (pH 3.6)
B) acetonitrile
83-80%B (0-12 min), 80-68%B (12-20 min)Flow rate: 1.0 mL/min
Temperature : 40°C
Detection: Corona® CAD® (Charged Aerosol Detector)
Injection: 10 μL (0.1 mg/mL)
Corona and CAD are trademarks of Thermo Fisher Scientific.

20

NH2

L-Leucine(F130618A)

ColumnH(U120210A)

ColumnEluent: YMC-Triart C18 (3 μm, 120 Å)
150 X 4.6 mmI.D.
: phosphate buffer (pH 2.8)*2/acetonitrile (97/3)*2 Dissolve 31.2 g of NaH2PO4·2H2O in 1000 mL of water and adjust pH 2.8 with H3PO4

Flow rate: 0.9 mL/min (adjust the flow rate so that the retention time of L-Valine is about 2.5 min)Temperature : 40°C
Detection: UV at 210 nm
Injection: 20 μL(The Japanese Pharmacopoeia 16th; Identification)*1 Standard solution was prepared from L-Valine, L-Isoleucine and L-Leucine supplied as a reagent for laboratory use.

Reversed-phase

Pharmaceutical products

Natural products

Reversed-phase

LC-MS analysis of pharmaceutical product

LC-MS/MS analysis of fumonisin mycotoxins using metal free column
1.

1

7

COOH
COOH

CH3

O

H3C

COOH

CH3

O

H

O

OH O

OH

O

3

Fumonisin B1 (Q1/Q3 : 722/334)
OH
CH3

CH3

O

4

O

O

H3C
O

NH

NH2

COOH
O

O

OH

OH

CH3

20000

O

5

OH

CH3

H3C

2 3

+ESI MRM
854→569

6

O
O

25000

1

1.945
4645

CH3

O

Intensity cps

x102

2.

15000

O

COOH
COOH

O

1

10000

OH
CH3

H3C
H3C

OH

CH3

O

NH2

COOH

Paclitaxel

COOH

O

2

Column Selection Guide

Application

Fumonisin B3 (Q1/Q3 :706/336)

5000
3.

1

O

0
0

0

1

2

0

3

COOH
COOH

O

2

4

6

8

10
min

H3C

O

NH2

COOH
COOH

O

(R170515B)

OH

CH3

Counts vs. Acquisition Time (min)(R161118A)

OH

CH3

H3C

Fumonisin B2 (Q1/Q3 :706/336)

Courtesy of Gyeong Sang National University

Column: YMC-Triart C18 (3 µm, 120 Å)
50 X 2.0 mmI.D.
Eluent: acetonitrile/0.1% formic acid (45/55)
Flow rate: 0.3 mL/min
Temperature : 30°C
Detection: ESI positive-mode
Injection: 2 µL (1 µg/mL)

SFC

ColumnEluent: YMC-Triart C18 [Metal free column] (3 µm, 120 Å)150 X 2.1 mmI.D.
: A) water/HCOOH (100/0.1)
B) acetonitrile
25-50%B (0-5 min), 50%B (5-8 min), 50-90%B (8-10 min)Flow rate: 0.2 mL/min
Temperature : 40°C
Detection: ESI positive, Scheduled MRM
Injection: 5 µL (0.1 µg/mL)
Instrument: LC) Shimadzu Prominence UFLCMS) AB Sciex 3200 QTRAP

Steroids

Reversed-phase

Separation of steroids by SFC

Analysis of zopiclone in human hair by chiral LC/HRMS

1.
AU
1.2

OH

3.

O

O

H

2

3

Extracted ion chromatograms (m/z 389.11 > 245.022)

CH3

1.

N

HO

H

1

Chiral compounds

H
H

H

O

1
H

N

Spiked hair
(5.0 ng/mg each)O

3.78E71.0

Progesterone

O
H

N

O

N

2

Corticosterone

Cl
N

N
O

0.8
2.

4.

HO

0.6

OH
O

O
HO
H

H

0.4
H

H

O

0.2

H

H

O

Deoxycorticosterone
0.0
0.0

OH

Relative abundance(R)-Zopiclone
4

CH3

2.

N

N
O

O
H

N
N

Hydrocotisone

Cl
N

N
O

1.0

2.0

3.0

0

min

0

1

2

3

4

5 min

(X150803C)(F130618A)(S)-Zopiclone(Eszopiclone)(F170925D)

Courtesy of H. Miyaguchi, National Research Institute of Police Science.

Column: Alcyon SFC Triart Diol (5 µm, 120 Å)
150 X 4.6 mmI.D.
Eluent: CO2/methanol (80/20)
Flow rate: 3.0 mL/min
Temperature : 40°C
Detection: UV at 254 nm
Back pressure : 13.8 MPa (2000 psi)
Injection: 5 µL (0.8 mg/mL)

ColumnEluent: CHIRAL ART Cellulose-SC (3 μm) 150 X 2.0 mmI.D.
: 10 mM ammonium bicarbonate (pH 8.0 with aqueous ammonia)/acetonitrile (25/75)
Flow rate: 0.2 mL/min
Temperature : 25°C
Detection: ESI, positive
Injection: 10 µL (Hair sample extracted by micropulverized extraction)
Instrument: LC) UltimateTM 3000 liquid chromatograph(Thermo Fisher Scientific)HRMS) Q ExactiveTM mass spectrometer(Thermo Fisher Scientific)
Reference
H. Miyaguchi, K. Kuwayama, Enantioselective determination of (R )-zopiclone and (S )-zopiclone (eszopiclone)
in human hair by micropulverized extraction and chiral liquid chromatography/high resolution mass
spectrometry, J.Chromatogr . A 1519 (2017) 55-63.

21

Column Selection Guide

Column Selection Guide

Reversed-phase column selection guide
1st choice!

2nd choice!

Versatile column forseparation of bothhydrophilic andhydrophobic compounds

Triart C18 ExRS
Triart C8
YMC-Pack ODS-AL
YMC Carotenoid
Optical Isomer Separation Columns

Similar structures(Isomers)

Improvement of
resolution

01

Triart C18

Aromatic compounds(π-electron acceptor )Conjugated compounds

Triart Phenyl
Aromatic compounds(π-electron donor)
Cis-trans isomers
Polar compounds
Halogenated compounds

With typical structure or functional group

Effective for separation ofcompounds with difficulty onother C18 column!!
• Only weak retention• Poor peak shape• pH limit for mobile phase
etc.

Triart PFP

Improvement ofretention

Mobile phase
ChangeTemperature

Too much retention/resolution(Long run time)

Triart C8
Triart Bio C4
Basic compounds

Weak retention(Highly polar compounds)

Triart PFPHILIC mode
(Triart Diol-HILIC)

Comparison of hydrophobicity and hydrogen-bonding capacityof various C18 columns
0.170

Hydrophilic C18
Hydrophobicity: Low
Hydrogen-bonding capacity: High

Hydrogen binding capacity [α(Caffeine/Benzene)]

0.150

Hydrosphere C18

Atlantis dC18
ZORBAX SB-C18
Atlantis T3

0.130

CAPCELL PAK C18 AQ

TSKgel ODS-80Ts

Develosil ODS-MG

Standard C18

CAPCELL PAK C18 MGIII

0.110

Gemini C18

Pro C18

Unison UK-C18

Hydrogen-bonding capacity: Moderate

CAPCELL PAK C18 MGII

Hypersil GOLD
Gemini-NX C18

0.090

Hydrophobicity: Moderate

CAPCELL PAK C18 MG
Inertsil ODS-4

XTerra MS C18

Sunfire C18
Luna C18(2)Inertsil ODS-3
XBridge C18
Mightysil RP-18
Eternity-C18
L-column ODS

Develosil ODS-SR

Triart C18

Symmetry C18
TSKgel ODS-100S

0.070

Cadenza CD-C18
L-column2 ODS

Hydrophobic C18
Hydrophobicity: High
Hydrogen-bonding capacity: Low

ZORBAX Extend-C18

Meteoric Core C18

CAPCELL PAK C18 ACR

Pro C18 RS
InertSustain C18

0.050

Triart C18 ExRS
Eluent: methanol/water (80/20) [Amylbenzene]
methanol/water (30/70) [Caffeine, Benzene]

0.030
1.00

3.00

5.00

7.00

9.00

11.00

Hydrophobicity [k’(Amylbenzene)]

22

13.00

15.00

17.00