Download Column Selection Guide

01
Column
Selection Guide
USP-----------------------------------------------------12
Column selection guide
(Biochromatography)------------------------------13
Comparison of separation mode--------- 14~16
Reversed-phase separation of peptides
and proteins------------------------------------ 17~20
Column selection guide
(Low molecular weight organic compounds)-----21
Application 1
(Fat-soluble vitamins, Water-soluble vitamins)--22
Application 2
(Water-soluble vitamins, Organic acids, Amino acids)---23
Reversed-phase column selection guide----24
01
Column Selection Guide
Column Selection Guide
USP
USP
CLASS
No.
USP Description
Functional
group
YMC product
YMC-Triart C18
YMC-Triart C18 ExRS
Meteoric Core C18
Meteoric Core C18 BIO
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.
C18
page
59~61
62
72~75
YMC-UltraHT Pro C18
83
YMC-UltraHT Hydrosphere C18
83
YMC-Pack Pro C18
84
Hydrosphere C18
85
YMC-Pack Pro C18 RS
86
YMC-Pack ODS-A
87
YMC-Pack ODS-AM
87
YMC-Pack ODS-AQ
88
YMC-Pack ODS-AL
88
J'sphere ODS-H80
J'sphere ODS-M80
89
J'sphere ODS-L80
L3
Porous silica particles, 1.5 to 10 μm in diameter, or a monolithic
silica rod.
L7
Octylsilane chemically bonded to totally porous or superficially
porous silica particles, 1.5 to 10 μm in diameter, or a monolithic
silica rod.
Silica
YMC-Pack SIL
YMC-Pack SIL-06
YMC-Triart C8
C8
104
63
Meteoric Core C8
72~75
YMC-Pack Pro C8
96
YMC-Pack C8
97
YMCbasic
100
L8
An essentially monomolecular layer of aminopropylsilane
chemically bonded to totally porous silica gel support, 1.5 to 10 μm
in diameter.
NH2
YMC-Pack NH2
108
L10
Nitrile groups chemically bonded to porous silica particles, 1.5 to
10 μm in diameter.
CN
YMC-Pack CN
99
L11
Phenyl groups chemically bonded to porous silica particles, 1.5 to
10 μm in diameter.
Phenyl
YMC-Triart Phenyl
64
YMC-Pack Ph
98
L13
Trimethylsilane chemically bonded to porous silica particles, 3 to
10 μm in diameter.
C1
YMC-Pack TMS
98
L20
Dihydroxypropane groups chemically bonded to porous silica or
hybrid particles, 1.5 to 10 μm in diameter.
Diol
YMC-Triart Diol-HILIC
66
YMC-Pack Diol-NP
104
YMC-Pack Diol-60
YMC-Pack Diol-120
YMC-Pack Diol-200
45, 46
YMC-Pack Diol-300
12
L24
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.
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.
L40
Cellulose tris-3,5-dimethylphenylcarbamate coated porous silica
particles, 5 to 20 μm in diameter.
L43
Pentafluorophenyl groups chemically bonded to silica particles by a
propyl spacer, 1.5 to 10 μm in diameter.
L51
Amylose tris-3,5-dimethylphenylcarbamate-coated, porous,
spherical, silica partilces, 5 to 10 μm in diameter.
YMC-Pack PVA-Sil
105
YMC-Pack Pro C4
96
YMC-Pack C4
97
YMC-Pack PROTEIN-RP
Silica
YMC-Pack SIL-HG
99
130, 135
YMC-Pack Diol-60
Diol
YMC-Pack Diol-120
YMC-Pack Diol-200
45, 46
YMC-Pack Diol-300
Cellulose tris-3,5CHIRAL ART Cellulose-C
dimethylphenylcarbamate
PFP
YMC-Triart PFP
Amylose tris-3,5CHIRAL ART Amylose-C
dimethylphenylcarbamate
26~29
65
26~29
YMC-Pack Diol-60
L59
Packing for the size-exclusion separation of proteins (separation by
molecular weight) over the range of 5 to 7,000 kDa. The packing is
a spherical 1.5-to 10-μm, silica or hybrid packing with a hydrophilic
coating.
Diol
L62
C30 silane bonded phase on a fully porous spherical silica, 3 to
15 μm in diameter.
C30
YMC-Pack Diol-120
YMC-Pack Diol-200
45, 46
YMC-Pack Diol-300
YMC Carotenoid
100
Proteins
Peptides
Ion exchange
Size exclusion
Reversedphase
Molecular weight
5,000 or less
Molecular weight
5,000 or more
HILIC
Nucleic
acids
Ion exchange
Oligonucleotides
Nucleic acids
Size exclusion
Oligonucleotides
Nucleic acids
Reversedphase
Nucleic acid bases
Nucleosides
Nucleotides
YMC-BioPro
For separation of biomolecules by
the difference in surface charge
P.37~39
YMC-Pack Diol
For separation of biomolecules by molecular weight
P.45~47
YMC-Triart C18
Meteoric Core C18
Suitable as the first choice ODS column
Core-shell column with ultra fast analysis
P.59~61
P.72~75
YMC-Triart C18
For separation of biomolecules with molecular
weight of up to 30,000 using high temperature
P.59~61
Meteoric Core C18 BIO
Core-shell column for separation of biomolecules
with molecular weight of up to 30,000
P.72~75
Wide-Pore Columns
Column with wide pore size useful for
separation of macromolecules
P.17
YMC-Pack PROTEIN-RP
Specialized column with excellent acid resistance
for separation of proteins or peptides
P.99
YMC-Triart Diol-HILIC
For separation of polar compounds with
poor retention on reversed-phase columns
P.66
YMC-BioPro
For separation of biomolecules by
the difference in surface charge
P.37~39
YMC-Pack Diol
For separation of biomolecules by molecular weight
P.45~47
YMC-Triart C18
Hydrosphere C18
Usable with 100% aqueous mobile phase
P.59~61
P.83, 85
Usable with 100% aqueous mobile phase
P.59~61
P.83, 85
YMC-Triart C18
Oligonucleotides
Hydrosphere C18
Wide-Pore Columns
HILIC
Sugars
Size exclusion
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
Column with wide pore size useful for
separation of macromolecules
For separation of polar compounds
For separatiion or molecular weight
determination of sugars
Usable with 100% aqueous mobile phase
For separation of polar compounds
Column Selection Guide
Column selection guide (Biochromatography)
P.17
P.66
P.106, 107
P.45~47
P.59~61
P.83, 85
P.66
P.106, 107
13
01
Column Selection Guide
Comparison of separation mode
Column Selection Guide
Separation of proteins by different mode
Human serum
Ion exchange
YMC-BioPro QA 5 μm, 50 X 4.6 mmI.D.
YMC-Pack Diol-300 + Diol-200 5 μm, 300 X 8.0 mmI.D. X 2
Size exclusion
Albumin
IgG
mAU
mAU
Albumin
Transferrin
150
Transferrin
10
100
IgG
50
0
0
N080313E
0
10
20
30 min
N080616A
0
Eluent
: A) 20 mM Tris-HCl (pH 8.6)
B) ‌20 mM Tris-HCl (pH 8.6) containing 0.5 M NaCl
0-30%B (0-15 min), 30-100%B (15-30 min)
Flow rate
: 0.5 mL/min
Temperature : 25°C
Detection
: UV at 280 nm
Injection
: 20 μL (100 μL/mL)
10
20
30
40
50
60 min
Eluent
: 0.1 M KH2PO4-K2HPO4 (pH 7.0) containing 0.2 M NaCl
Flow rate
: 0.5 mL/min
Temperature : ambient (25°C)
Detection
: UV at 280 nm
Injection
: 20 μL (100 μL/mL)
Proteins in human serum are separated by the difference in the surface charge on ion exchange chromatography (IEC) and by the difference in the molecular
weight on size exclusion chromatography (SEC).
Mouse monoclonal IgG1 anti-human IgG4 (Purified by DEAE chromatography, containing NaN3)
Ion exchange
Size exclusion
YMC-BioPro QA-F 5 μm, 30 X 4.6 mmI.D.
NaN3
YMC-Pack Diol-200 5 μm, 300 X 4.6 mmI.D.
mAU
mAU
4
Mouse monoclonal IgG1
(Anti-human IgG4)
40
Mouse monoclonal IgG1
(Anti-human IgG4)
5
NaN3
30
3
20
2
10
1
0
0
P080220A
0
2
4
6
8
10
12
14
16
min
Eluent : A) 20 mM Tris-HCl (pH 8.1)
B) 20 mM Tris-HCl (pH 8.1) containing 0.5 M NaCl
10-25%B (0-18 min)
Flow rate : 1.0 mL/min
Temperature : 25°C
Detection : UV at 220 nm
Injection : 10 μL (0.1 mg/mL)
P080530A
0
5
10
15
20
25
30 min
Eluent : 0.1 M KH2PO4-K2HPO4 (pH 7.0)
Flow rate : 0.17 mL/min
Temperature : ambient (25°C)
Detection : UV at 220 nm
Injection : 10 μL (0.05 mg/mL)
Mouse monoclonal antibody against human IgG4 is analyzed on ion exchange chromatography (IEC) and size exclusion chromatography (SEC). Several peaks
possibly derived from isoform of antibody are observed in ion exchange mode, while a single peak is detected in size exclusion mode.
Mouse IgG Fc fragment (Prepared from normal serum)
Size exclusion
YMC-Pack Diol-200 5 μm, 300 X 8.0 mmI.D.
1. Rabbit IgG
2. Mouse IgG Fc fragment
1
50
Reversed-phase
YMC-Pack C4 (300 Å) 5 μm, 150 X 4.6 mmI.D.
mAU
2
mAU
Mouse IgG Fc fragment
30
20
10
25
0
0
R080619B
R080623D
0
5
10
15
20 min
Eluent : 0.1 M KH2PO4-K2HPO4 (pH 6.9) containing 0.2 M NaCl
Flow rate : 1.0 mL/min
Temperature : ambient (27°C)
Detection : UV at 220 nm
Injection : 5 μL (0.5 mg/mL)
0
5
10
15
20
25
30
35
40 min
Eluent : A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.1)
25-45%B (0-40 min)
Flow rate : 1.0 mL/min
Temperature : 37°C
Detection : UV at 220 nm
Injection : 5 μL (1.0 mg/mL)
Size exclusion chromatography (SEC) is useful for separation of substances which have distinct differences in molecular weight, like between IgG and its
fragments. On the other hand, reversed-phase chromatography (RPC) is suitable for a precise analysis of peptides and proteins with a molecular weight of less
than 100 kDa such as IgG Fc fragment.
14
Tryptic digests of BSA
Ion exchange
Eluent : A) 20 mM Tris-HCl (pH 8.6)
B
) 20 mM Tris-HCl (pH 8.6) containing 0.5 M NaCl
0-15%B (0-30 min), 15-60%B (30-60 min)
Flow rate : 0.5 mL/min
Temperature : 25°C
Detection : UV at 220 nm
Injection : 20 μL
YMC-BioPro QA 5 μm, 50 X 4.6 mmI.D.
mAU
20
10
*
0
Column Selection Guide
Separation of proteins by different mode
N080422B
0
Size exclusion
10
20
30
40
50
60 min
Calibration curve of peptides and proteins
YMC-Pack Diol-120 + Diol-60 5 μm, 500 X 8.0 mmI.D. X 2
5
10 MW
mAU
60
1
4
50
10
2
40
3
103
30
4
20
10
5
*
0
Reversed-phase
10
20
30
102
101
N080623L
0
1. Myoglobin 2. Insulin (Bovine) 3. Neurotensin 4. Tetraglycine 5. Glycine 40
50
60 min
(MW 17,000)
(MW 5,700)
(MW 1,672)
(MW
246)
(MW
75)
Eluent : 0.1 M KH2PO4-K2HPO4 (pH 7.0)
containing 0.2 M NaCl/acetonitrile (70/30)
Flow rate : 0.7 mL/min
Temperature : ambient (25°C)
Detection : UV at 220 nm
Injection : 5 μL
YMCbasic 5 μm, 150 X 2.0 mmI.D.
mAU
40
30
*
20
10
0
N080318B
0
10
20
30
40
50
60 min
* undigested BSA
Eluent : A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.1)
5-35%B (0-50 min), 35-45%B (50-55 min),
45%B (55-60 min)
Flow rate : 0.2 mL/min
Temperature : 37°C
Detection : UV at 220 nm
Injection : 1 μL
These chromatograms show separation of tryptic digests of BSA (MW: 66,000) in ion exchange chromatography (IEC), size exclusion chromatography (SEC)
and reversed-phase chromatography (RPC). The molecular weight of the digests is estimated to be approximately from 100 to 20,000 by SEC chromatogram.
IEC and RPC chromatograms show many peaks of fragments which are separated by the difference in structure, charge and hydrophobicity.
Separation of sugar chains by different mode
Pyridylamino (PA) -Sugar chains
Reversed-phase
YMC-Pack ODS-A (120 Å) 5 μm,
150 X 4.6 mmI.D.
Normal-phase
1.PA-Sugar Chain 014
YMC-Pack Polyamine II 5 μm,
150 X 4.6 mmI.D.
2.PA-Sugar Chain 001
3.PA-Sugar Chain 002
0
10
20 min
G910607A
Eluent : methanol/20 mM NH4H2PO4 (5/95)
Flow rate : 1.0 mL/min
Temperature : 37°C
Detection : FLS at Ex. 320 nm, Em. 400 nm
Injection : 2 μL (3.3 pmol/mL)
Sample : PA-Sugar Chain Series,
manufactured by TAKARA BIO INC.
0
10
20 min
G910620A
Eluent : methanol/20 mM NH4H2PO4 (80/20)
Flow rate : 1.0 mL/min
Temperature : 37°C
Detection : FLS at Ex. 320 nm, Em. 400 nm
Injection : 3 μL (3.3 pmol/mL)
Sample : PA-Sugar Chain Series,
manufactured by TAKARA BIO INC.
Pyridylamino (PA) sugar chains are often analyzed for structural determination of sugar chain in glycoproteins and glycolipids. Separations of PA sugar chains
in reversed-phase (RP) mode and normal-phase (NP) mode are shown. Two dimensional HPLC combining two different modes, such as RP mode and NP
mode, is useful tool for structural determination of sugar chain.
15
01
Column Selection Guide
Comparison of separation mode
Column Selection Guide
Separation of nucleic acids by different mode
DNA fragments 1 Kb DNA ladder (75 - 12,216 bp)
Ion exchange
Eluent : A) 20 mM Tris-HCl (pH 8.1) containing 0.7 M NaCl
B
) 20 mM Tris-HCl (pH 8.1) containing 1.0 M NaCl
0-100%B (0-30 min)
Flow rate : 0.5 mL/min
Temperature : 25°C
Detection : UV at 260 nm
Injection : 20 μL
YMC-BioPro QA-F 5 μm, 100 X 4.6 mmI.D.
mAU
100
50
0
N080212G
0
10
20
30 min
DNA fragments are analyzed with YMC-BioPro QA-F ion exchange column. 100 mm length column of YMC-BioPro QA-F is ideal for high-resolution analysis of
nucleic acids.
Plasmid pBR322 restriction fragments
Ion exchange
Eluent YMC-BioPro QA-F 5 μm, 100 X 4.6 mmI.D.
Plasmid pBR322 Hae III digests (8-587 bp)
mAU
40
Plasmid pBR322 (4,361 bp)
30
: A) 20 mM Tris-HCl (pH 8.1)
B
) 20 mM Tris-HCl (pH 8.1) containing 1.0 M NaCl
70-85%B (0-20 min), 85%B (20-25 min)
Flow rate : 0.5 mL/min
Temperature : 35°C
Detection : UV at 260 nm
Injection : 10 μL
20
10
0
N080610F
0
5
Size exclusion
10
15
20
25 min
YMC-Pack Diol-300 + Diol-200 5 μm, 500 X 8.0 mmI.D. X 2
Plasmid pBR322 Hae III digests (8-587 bp)
mAU
Plasmid pBR322
(4,361 bp)
40
30
Eluent 20
10
0
P080617A
0
10
20
30
40
50 min
: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
The separation of plasmid pBR322 restriction fragments (8-857 bp) is compared between in ion exchange mode and size exclusion mode. Ion exchange
chromatography (IEC) is applicable to identification of each fragment requiring high resolution and size exclusion chromatography (SEC) is usable for
characterization of molecular weight distribution.
Oligonucleotide (mi RNA)
YMC-Triart C18 3 μm, 150 X 2.0 mmI.D.
5’-pUGG AGU GUG ACA AUG GUG UUG-3’
5’-pUGG AGU GUG ACA AUG GUG UUG U-3’
UV
mAU
20
(21 nt, MW 6890.1)
(22 nt, MW 7196.3)
21 nt
22 nt
10
0
0.0
MS
2.5
5.0
7.5
5
10.0
12.5
15.0
21 nt
0
0.0
17.5
min
MIC
(m/z 1721.5, m/z 1798.1)
2.5
5.0
7.5
10.0
12.5
15.0
22 nt
17.5
min
Courtesy of M.Yamada, SHIMADZU CORPORATION
16
Eluent
: A) 10 mM DBAA* (pH 7.5)
B) 10 mM DBAA* (pH 7.5)/acetonitrile (50/50)
62-72%B (0-20 min)
Flow rate
: 0.2 mL/min
Temperature : 30°C
Detection
: UV at 260 nm and ESI-negative mode
Injection
: 4 μL (5 nmol/mL)
Instrument : LC) Shimadzu Prominence
MS) Shimadzu LCMS2020
* di-n-butylamine-acetic acid
This figure shows LC/MS analysis of oligonucleotides in
reversed-phase mode. YMC-Triart C18 columns are useful for
oligonucleotides and they can achieve excellent separation by onenucleotide difference and sufficient intensity in UV and ESI-MS.
Reversed-phase separation of peptides and proteins
Functional
group
Molecular
weight
of sample
C18
C8
C4
Pore size
120 Å
5,000
200 Å
Column Selection Guide
How to select reversed-phase columns
To separate proteins or peptides, it is important to select columns based on the molecular
weight of the compounds to be separated. As shown in the table on the right, the C18 column
with 120 Å pore size is generally suitable for small peptides up to MW 5,000. In the case
of large peptides or small proteins up to MW 20,000, the C8 column with 200 Å pore size
often gives the best column efficiency. Furthermore, most of proteins are eluted effectively
by the C4 column with 300 Å. Separation may also be influenced by the hydrophobicity of
the analyte and the type of the functional group as well as molecular weight. If the sufficient
separation is not achieved with columns marked with a double circle, perform optimization as
indicated by the arrows shown in the table. In addition to columns C18, C8, and C4 shown in
the table, PROTEIN-RP and CN type columns with different selectivity are also useful.
20,000
300 Å
100,000
Separation of peptides (MW 574 - 3,465)
Excellent peak shapes for basic peptides
Hydrosphere C18 (120 Å) 5 μm,
150 X 4.6 mmI.D.
1
4
2
6
5
3
4
5
23
7
6
1
5
10
15
A000313D
20 min
0
5
10
15
(MW 1,425)
(MW 587)
(MW 1,746)
(MW 574)
(MW 588)
(MW 1,899)
(MW 3,465)
Eluent : A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.1)
20-40%B (0-15 min),
40%B (15-20 min)
Flow rate : 1.0 mL/min
Temperature : 37°C
Detection : UV at 220 nm
7
A000308B
0
1. BAM-12P
2. [D-Ala2,Met5]-Enkephalinamide
3. α-Endorphin
4. Met-Enkephalin
5. [D-Ala2,Met5]-Enkephalin
6. γ-Endorphin
7. β-Endorphin
Brand E2 (100 Å) 5 μm,
150 X 4.6 mmI.D.
(ODS column for hydrophilic compounds)
20 min
Generally, the conventional C18 column with 120 Å pore size is suitable for analysis of small peptides up to 5,000 in molecular weight. Especially Triart and
Pro series ODS columns, which are processed with advanced endcapping technology, are ideal for separation of basic peptides. As shown in the above,
Hydrosphere C18, a Pro series column, exhibits excellent separations and superior peak shapes of basic peptides (peak 1 and 7), in contrast to the commercial
ODS column for hydrophilic compounds, Brand E2.
Separation of peptides and proteins (MW 4,300 - 17,000)
Comparison of separation on columns with different pore size and functional group
Pore size
C8, 120 Å
1
2
Functional group
1
C18, 200 Å
4
5
3
2
6
4
3
5
6
04072910.D
04073006.D
1
C8, 300 Å
4
2
1,2
C4, 200 Å
6
4
6
5
3
3
5
10
15
04080204.D
04073002.D
0
5
10
15
min
0
5
1. Cytochrome c
2. Insulin (Bovine)
3. Amyloid β-protein
4. Lysozyme
5. α-Lactalbumin
6. Myoglobin
Optimized combination of pore size and functional group
C8, 200 Å
1
6
4
2
5
3
04082002.D
0
5
10
15
min
min
(MW 12,400)
(MW 5,700)
(MW 4,300)
(MW 14,300)
(MW 14,100)
(MW 17,000)
Column : 5 μm, 150 X 4.6 mmI.D.
Eluent : A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.1)
25-60%B (0-20 min)
Flow rate : 1.0 mL/min
Temperature : 37°C
Detection : UV at 220 nm
For proteins and peptides with molecular weight of 4,300 to 17,000, separation characteristics are compared using columns with different pore size and
functional group. In accordance with the table above, the suitable column is C8, 200 Å for groups of compounds with a molecular weight within this range. If
either pore size or functional group of the packing material is not optimized, peak broadening and poor resolution are observed. By using the most suitable
column (C8, 200 Å) for the target compounds, sharp peak shapes and excellent separation are achieved.
17
01
Column Selection Guide
Reversed-phase separation of peptides and proteins
Column Selection Guide
Separation of proteins (MW 66,000 - 96,000)
Optimization of eluent conditions (C4, 300 Å)
1
1
1. BSA
(MW 66,000)
2. Conalbumin (MW 77,000)
3. Lipoxidase (MW 96,000)
2
2
2-propanol
addition
3
3
05011405.D
0
5
10
15
Column : YMC-Pack C4 (5 μm, 300 Å)
150 X 4.6 mmI.D.
Gradient : 30-75%B (0-15 min),
75%B (15-20 min)
Flow rate : 1.0 mL/min
Temperature : 37°C
Detection : UV at 220 nm
04093007.D
min
0
5
10
15
min
A）water/TFA (100/0.1)
B）acetonitrile/2-propanol/TFA (50/50/0.1)
A）water/TFA (100/0.1)
B）acetonitrile/TFA (100/0.1)
Gradient elution of water and acetonitrile containing TFA are often employed in an analysis of proteins and peptides. In some cases, addition of a “third
solvent” is effective for change in selectivity and separation. The above example shows the resolution between highmolecular weight proteins (peak 1 and 2) is
improved by adding 2-propanol into the standard mobile phase of acetonitrile/water/TFA.
Comparison of separation on columns with different pore size and functional group
1
1. BSA
(MW 66,000)
2. Conalbumin (MW 77,000)
3. Lipoxidase (MW 96,000)
C4, 300 Å
Column Eluent : 5 μm, 150 X 4.6 mmI.D.
: A) water/TFA (100/0.1)
B
) acetonitrile/2-propanol/TFA (50/50/0.1)
30-75%B (0-15 min), 75%B (15-20 min)
Flow rate : 1.0 mL/min
Temperature : 37°C
Detection : UV at 220 nm
2
3
04093007.D
0
4
8
12
16
min
Pore size
Functional group
1
C4, 200 Å
12
2
3
C8, 300 Å
3
05011711.D
05011708.D
C4, 120 Å
1
2
12
3
C18, 300 Å
3
05011714.D
0
4
8
12
16
min
05011802.D
0
4
8
12
16
min
Separation characteristics of proteins with molecular weight of 66,000 to 96,000 are compared using columns with different pore size and functional group. The
columns with smaller pore size, which have the same C4 functional groups, provide broader peak shapes and poor separations. In comparison among the 300
Å pore columns with different functional groups, the longer alkyl chain such as C18 and C8 results in poor resolution. It is important to choose optimal pore size
and functional group depending on molecular weight of proteins for better peak shapes and resolutions. Proteins with molecular weight of 20,000 to 100,000
are separated effectively by the C4 column with 300 Å pore size.
18
Column Selection Guide
Effect of column temperature on separation of peptides and proteins
Condition A (MW 500-18,400)
mAU
60
21.8-25.8 MPa
(3,160-3,740 psi)
50
40˚C
1
Condition B (MW 14,300-25,700)
mAU
Peak capacity
= 230
60
2
50
40
37.0-37.5 MPa
(5,370-5,440 psi)
1
2
40
4, 3
30
3
30
5
20
20
10
10
0
F111228B
0.0
0.5
1.0
1.5
2.0
mAU
60
2.5
1
15.8-18.5 MPa
(2,290-2,680 psi)
50
70˚C
Peak capacity
= 67
3.0
3.5
0
min*
F111226B
0.0
0.5
1.0
1.5
2.0
mAU
Peak capacity
2 = 279
60
50
40
3.0
3 4
min*
3
30
5
20
4.0
2
40
30
3.5
Peak capacity
= 107
1
27.4-28.4 MPa
(3,970-4,120 psi)
2.5
20
10
10
0
F111228A
0.0
0.5
Analytes
Condition A
1. Oxytocin
2. Leu-Enkephalin
3. β-Endorphin
4. Insulin
5. β-Lactoglobulin A
Condition B
1. Lysozyme
2. α-Chymotrypsinogen
3. β-Lactoglobulin A
1.0
1.5
2.0
2.5
MW
Peak width ½（min ）
40°C
70°C
1,007
0.017
0.014
556
3,465
5,733
18,400
0.015
0.043
0.015
0.016
0.015
0.030
14,300
25,700
18,400
0.069
0.080
0.080
0.044
0.049
0.048
3.0
3.5
min*
Column Eluent Gradient Flow rate Detection Injection System 0
F111226A
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
min*
: 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) - condition A
B) acetonitrile/2-propanol/TFA (50/50/0.1) - condition B
: 10-80%B (0-5 min) - condition A
30-60%B (0-5 min) - condition B
: 0.4 mL/min
: UV at 220 nm
: 1 μL (50 μg/mL) - condition A
1 μL (250 μg/mL) - condition B
: Agilent 1200SL
PC (peak capacity) = 1 + (gradient time/peak width*)
*peak width = 2W0.5h average
The effect of temperature on separation of peptides and proteins with a variety of molecular weight (MW) is estimated. The separations at 40°C and 70°C are
compared.
By increasing column temperature to 70°C, selectivity change is observed, and peaks become sharper. Thus, improved resolution especially for larger
molecules is obtained. Generally, larger molecules diffuse very slowly compared to small molecules. An elevated temperature can improve efficiency and peak
shape by lowering mobile phase viscosity and improving mass transfer.
Temperature is a simple and effective tool to increase resolution in separation of proteins and peptides.
19
01
Column Selection Guide
Reversed-phase separation of peptides and proteins
Column Selection Guide
Improvement of resolution by increasing column temperature and coupling of 1.9 μm columns
Peak capacity = 365
40˚C
1.9 µm, 100 X 2.0 mmI.D.
15 min gradient
46.5-48.5 MPa (6,740-7,030 psi)
mAU
30
20
10
0
P110705A
0
70˚C
2
4
6
8
10
12
Peak capacity = 450
30
27.6-28.6 MPa (4,000-4,150 psi)
20
10
Coupling of
two columns
9
0
58.1-61.6 MPa (8,430-8,930 psi)
2
4
6
8
10
12
10.2
10.6 min
14
15 min
30
20
10
19
20
21
min
P110707A
0
20
9.8
25mAU
Peak capacity = 630
0
Column Eluent Flow rate Detection Injection Sample System 9.4
P110728A
0
mAU
Two coupled
1.9 µm, 100 X 2.0 mmI.D.
30 min gradient
min
25mAU
mAU
1.9 µm, 100 X 2.0 mmI.D.
15 min gradient
15
14
: YMC-Triart C18 (1.9 μm, 120 Å)
: A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.08)
5-40%B (0-15 min) for a single column
5-40%B (0-30 min) for two coupled columns
: 0.4 mL/min
: UV at 220 nm
: 10 μL for a single column
20 μL for two coupled columns
: Tryptic digest of Bovine Hemoglobin
: Agilent 1290
4
8
12
16
20
24
28
30 min
23% more peaks can be resolved by increasing the column temperature to 70°C in the
separation of tryptic digest of Hemoglobin.
The outstanding efficiency obtained by a coupling of two 100 mm length of Triart 1.9
μm columns reduces co-elution peaks and allows the precise separation in an analysis
of complicated samples, such as peptide mapping.
Pharmaceutical
products
Reversedphase
YMC-Triart C18
YMC-Triart
(C18, C18 ExRS, C8, PFP, Phenyl) Effective for method screening with 5 chemistries
Agricultural
chemicals
Metabolites
Suitable as the first choice column for
reversed-phase separation
P.59~61
P.59~65
Normalphase
YMC-Pack SIL, SIL-06
Standard normal-phase column
P.104
YMC-Pack Diol-NP
Normal-phase column providing separation
characteristics different from bare silica gel
P.104
Others
HILIC
YMC-Triart Diol-HILIC
For separation of polar compounds with poor
retention on reversed-phase columns
Vitamins
Reversedphase
Food
additives
Natural
products
Water-soluble
vitamins
Fat-soluble
vitamins
HILIC
Normalphase
Organic acids
Fatty acids
Water-soluble
vitamins
Fat-soluble
vitamins
Reversedphase
Normalphase
Phospholipids
Reversedphase
YMC-Triart C18
P.59~61
P.59~61
YMC Carotenoid (C30)
YMC-Pack Polyamine II, NH2
For separation of water-soluble vitamins such as
P.106~108
vitamin C under HILIC mode
YMC-Triart Diol-HILIC
For simultaneous separation of water-soluble vitamins
YMC-Pack SIL, SIL-06
For separation of fat-soluble vitamins
such as tocopherol
YMC-Pack ODS-AL
YMC-Pack Polyamine II
YMC-Triart C18
Usable with 100% aqueous mobile phase
YMC-Pack SIL, SIL-06
Standard normal-phase column
YMC-Triart C18
For separation of molecular species
P.88
P.100
P.66
P.104
P.106, 107
P.59~61
P.104
P.59~61
For separation of phospholipid classes
P.104, 105
YMC-Pack Diol-NP
HILIC
Free amino acids
Reversedphase
Free amino acids
YMC-Triart Diol-HILIC
For simultaneous separation of amino acids
under HILIC mode
YMC-Triart C18
Usable with 100% aqueous mobile phase
Hydrosphere C18
For separation of hydrophobic amino acids
P.59~61
P.83, 85
YMC-Triart C18
Suitable as the first choice ODS column
P.59~61
P.66
Reversedphase
YMC Carotenoid (C30)
YMC-Triart C8
YMC-Triart PFP
CHIRAL ART
High-density bonding for excellent ability to
recognize planar structure
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
P.26~29
Reversedphase
CHIRAL ART
YMC CHIRAL NEA
For separation of optical isomers
P.26~30
Normalphase
CHIRAL ART
For separation of optical isomers
P.26~30
YMC-Triart C18 ExRS
Optical
isomers
(For separation under a buffered or ion pairing mobile phase)
Suitable as the first choice ODS column
Non-endcapped ODS, suitable for separation of
compounds with similar structure
Separation behavior different from ODS
YMC-Pack PVA-Sil
Labeled amino acids
Structural
isomers
Usable with 100% aqueous mobile phase
P.66
YMC-Pack SIL, SIL-06
Normalphase
Amino acids
YMC-Triart C18
Column Selection Guide
Column selection guide (Low molecular weight organic compounds)
YMC CHIRAL NEA
P.62
P.100
P.63
P.65
P.26~29
P.104
21
01
Column Selection Guide
Application 1 (Fat-soluble vitamins, Water-soluble vitamins)
Column Selection Guide
Vitamin D
Reversed-phase
Vitamin E (Tocopherols)
Normal-phase
Separation of structurally similar compounds
Separation of tocopherol homologues
1.
mAU
H
H
1.
CH3
H3C
CH3
CH3
H
15
CH3
CH3
O
CH3
HO
3
1
H3C
α-Tocopherol
CH3
CH3
4
CH3
2.
CH3
HO
1
10
HO
3.
H
2
Ergocalciferol (Vitamin D2)
4.
CH3
H
5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
O
CH3
HO
2
H
H3C
CH3
2.
H3C
CH3
β-Tocopherol
CH3
CH2
CH3
CH3
CH3
O
H
CH3
γ-Tocopherol
CH3
CH3
O
CH3
HO
δ-Tocopherol
H
CH2
HO
0
H
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 mm I.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
1
3
20
5
15
O
O
OH
NH2
N
N
Nicotinic acid
Nicotinamide
9.
4
3.
• HCl
4.
OH
OH
2-O-α-D-Glucopyranosyl
-L-ascorbic acid
（Ascorbic acid 2-glucoside）
NH
N
O
10.
10
6
N
OH
5.
0
5
10
15
20
25
4
6
8
10
12
14
16
18
20
min*
min
Column : YMC-Triart C18 (5 μm, 120 Å)
250 X 4.6 mmI.D.
Eluent : phosphate buffer*/acetonitrile (90/10)
*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)
22
11.
O
O
H2 N
H 2N
NH2
N
Orotic acid
O
CN N
+
Co
N
O
N
NH2
6.
HO
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
HO
(R100315D)
-
Cl • HCl
NH
N
H
O
2
OH
S
O
HO
0
+
Thiamine hydrochloride
0
0
N
N
Riboflavin
3
4
NH2
OH
HO
50
O
HO
O
N
OH
O
O
N
78
O
OH
N
12
5
H
HO
Pyridoxine
hydrochloride
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 acid
8. Riboflavin (Vitamin B2)
9. Nicotinic acid
O HO N
-
HO
O
Cyanocobalamin
Column : 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)
Application 2 (Water-soluble vitamins, Organic acids, Amino acids)
Organic acids
Reversed-phase
Separation of ascorbic acid under HILIC mode
Simultaneous separation of organic acids under 100% aqueous mobile phase
1. Oxalic acid
2. Tartaric acid
1. Nicotinic acid
2. Erythorbic acid
3. L-Ascorbic acid
1
3. Glycolic acid
mAU
2,3
4. Formic acid
50
5. L-Malic acid
6. Malonic acid
7. Lactic acid
12
40
2 3
8. Acetic acid
4
9. Maleic acid
13
30
1
Column Selection Guide
Vitamin C (Ascorbic acid)
HILIC
10. Citric acid
11. Succinic acid
56
8
12. Fumaric acid
9
10
20
13. Acrylic acid
11
14. Propionic acid
14
7
10
0
10
20
0
min
0
2
4
6
8
min
10
(A970603A)
Column : 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, 0.16 AUFS
Injection : 10 μL (0.05~0.1 mg/mL)
HILIC
(U111212Q)
Column : 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
Amino acids
Reversed-phase
Simultaneous separation of amino acids under HILIC mode
Separation of hydrophobic amino acids under highly aqueous mobile phase (JP method)
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)
γ-Aminobutyric acid (GABA)
200
Serine (Ser)
2.68
1
Relative standard deviation of
the retention time
(each of the peaks)
50
Result
1.5
Alanine (Ala)
1.0%
Glycine (Gly)
0.02%
2
0.02%
3
0.02%
Asparagine (Asn)
Glutamine (Gln)
Citrulline (Cit)
100
Glutamic acid (Glu)
Histidine (His)
*
Aspartic acid (Asp)
Lysine (Lys)
0
Ornithine HCl (Orn)
2.0
4.0
0
2
6.0
8.0
10.0
12.0 14.0 16.0 18.0 min
*Chloride ion contained in the sample solution
**Sodium ion contained in the sample solution
(F130618A)
4
1.
6
2.
H
COOH
H3C
H
NH2
L-Valine
min
3.
CH3
Arginine HCl (Arg)
*
0.0
0
CH3
COOH
H3C
COOH
H3C
H
NH2
L-Isoleucine
H3C
H
NH2
L-Leucine
(U120210A)
Column : 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)
Column : YMC-Triart C18 (3 μm, 120 Å)
150 X 4.6 mmI.D.
Eluent : 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)
Corona and CAD are trademarks of Thermo Fisher Scientific.
*1 Standard solution was prepared from L-Valine, L-Isoleucine and L-Leucine supplied as a reagent for laboratory use.
23
01
Column Selection Guide
Reversed-phase column selection guide
2nd choice!
Versatile column for
separation of both
hydrophilic and
hydrophobic compounds
Similar structures
(Isomers)
Improvement of
resolution
Column Selection Guide
1st choice!
Triart C18
Triart C18 ExRS
Triart C8
YMC-Pack ODS-AL
YMC Carotenoid
Optical Isomer Separation Columns
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 of
compounds with difficulty on
other C18 column!!
• Only weak retention
• Poor peak shape
• pH limit for mobile phase
etc.
Triart PFP
Improvement of
retention
Mobile phase
Change
Temperature
Too much retention/resolution
(Long run time)
Triart C8
Basic compounds
Weak retention
(Highly polar compounds)
Triart PFP
HILIC mode
(Triart Diol-HILIC)
Comparison of hydrophobicity and hydrogen-bonding capacity of various columns
0.170
Hydrophilic ODS
Hydrophobicity: Low
Pro C4
Hydrogen-bonding capacity: High
Hydrogen binding capacity [α(Caffeine/Benzene)]
0.150
Develosil C30-UG
Atlantis dC18
Hydrosphere C18
ZORBAX SB-C18
Atlantis T3
0.130
CAPCELL PAK C18 AQ
TSKgel ODS-80Ts
Develosil ODS-MG
Standard ODS column
CAPCELL PAK C18 MGIII
Pro C8
0.110
Gemini C18
Pro C18
Unison UK-C18
Gemini-NX C18
0.070
Hydrogen-bonding capacity: Moderate
CAPCELL PAK C18 MGII
Hypersil GOLD
0.090
Hydrophobicity: Moderate
CAPCELL PAK C18 MG
Inertsil ODS-4
XTerra MS C18
Meteoric Triart C8
Core C8
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
Cadenza CD-C18
L-column2 ODS
Hydrophobic ODS
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]
(S150318A)
0.030
1.00
3.00
5.00
7.00
9.00
11.00
Hydrophobicity [k’(Amylbenzene)]
24
13.00
15.00
17.00