Polar molecule separation
by COSMOSIL PBr
Technical Note
11
COSMOSIL PBr is a reversed-phase column suitable for separating hydrophilic compounds, with high retention for compounds
not sufficiently retained on ODS columns. Below are some featured applications for several classes of hydrophilic compounds.
Contents
Category
Applications
1 Nucleotide-related compounds
2 Nucleic acid base-related compounds
3 Other aromatics and heterocycles
Page
Nucleotides
AMP / ADP / ATP
1
Sugar nucleotides
UDP-galactose / UDP-glucose
1
Pyrimidine / purine rings
Nucleic acid bases
2
Halogenated compounds
Uracil derivatives
2
Catechol rings
Catecholamines
2
Triazine rings
Melamine derivatives
3
4 Peptide-related compounds
Peptides of different chain lengths
Peptides
3
5 Aminoglycoside antibiotics
Gentamicin C complex
Gentamicin
3
Applications
1. Nucleotide-related compounds
● Nucleotides (AMP / ADP / ATP)
Nucleotide analysis is a challenge due to the phosphate groups, which are highly polar. COSMOSIL PBr achieves baseline analysis
without ion-pair reagents.
5C18-PAQ
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
PBr
4.6mmI.D.-150mm
Methanol/ 20mmol/l Phosphate buffer(pH7.0) = 10/90
1.0 ml/min
30°C
UV260nm
1; Adenosine 5’-triphosphate
(0.5mg/ml)
2; Adenosine 5’-diphosphate
(0.8mg/ml)
3; Adenosine 5’-monophosphate (0.8mg/ml)
1.0µl
Sample:
Inj.Vol.:
NH2
N
O
HO
O
N
O
P O P O P O
OH
OH
N
k (Peak.1)
0.01
k (Peak.1)
0.52
N
O
OH
Adenosine 5'-triphosphate
OH
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OH
● Sugar nucleotides (UDP-galactose / UDP-glucose)
Due to the hydrophilicity of sugars, retention times are very close on C18 columns. However, COSMOSIL PBr was able to separate the
below compounds under simple conditions.
5C18-PAQ
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
4.6mmI.D.-250mm
100mmol/l Phosphate buffer(pH7.0)
1.0 ml/min
30°C
UV260nm
Sample:
1; Uridine-5'-diphosphate
(0.8mg/ml)
2; Uridine-5’-diphosphogalactose (0.8mg/ml)
3; Uridine-5’-diphosphoglucose (0.8mg/ml)
1.0µl
Inj.Vol.:
O
OH OH
O
HO
PBr
OH
O
O
O
P
P
O OH O OH
O
OH
O
OH OH
O
NH
N
OH
2.Uridine-5’-diphosphogalactose
O
OH
OH
NH
N
O
O
O
P
P
O OH O OH
O
O
OH
OH
3.Uridine-5’-diphosphoglucose
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2. Nucleic acid base-related compounds
● Pyrimidine / purine rings
COSMOSIL PBr strongly retains analytes containing pyrimidine and purine rings, such as nucleobases. Compounds with more rings are
retained more strongly.
5C18-PAQ
PBr
Column:
Column size: 4.6mmI.D.-150mm
Mobile phase: Methanol/ 20mmol/l Phosphate
buffer(pH7.0) = 10/90
Flow rate:
1.0 ml/min
Temperature: 30°C
Pyrimidine Rings
Detection:
UV260nm
Sample:
1; Cytosine
2; Uracil
3; Thymine
4; Guanine
5; Adenine
1.0µl
Inj.Vol.:
O
NH2
(0.05mg/ml)
(0.05mg/ml)
(0.05mg/ml)
(0.05mg/ml)
(0.05mg/ml)
O
NH
N
N
H
N
H
O
1. Cytosine
NH
O
N
H
2.Uracil
O
3.Thymine
Purine Rings
O
N
NH2
N
NH
N
H
N
N
H
NH2
4.Guanine
N
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N
5.Adenine
● Halogenated compounds (uracil derivatives)
COSMOSIL PBr offers stronger retention of halogenated analytes.
5C18-PAQ
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
4.6mmI.D.-150mm
20mmol/l Phosphate buffer(pH2.5)
1.0 ml/min
30°C
UV210nm
1; 5,6-Dihydrouracil
2; Uracil
3; 5-Fluorouracil
4; 5-Bromouracil
10µl
Sample:
Inj.Vol.:
(0.005mg/ml)
(0.005mg/ml)
(0.005mg/ml)
(0.005mg/ml)
O
O
O
NH
NH
N
H
PBr
N
H
O
O
NH
N
H
O
2.Uracil
1.5,6-Dihydrouracil
F
Br
O
3.5-Fluorouracil
NH
N
H
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O
4.5-Bromouracil
3. Other aromatics and heterocycles
● Catechol rings (catecholamines)
Catecholamines are challenging due to their highly polar amino group. COSMOSIL PBr achieves baseline analysis without ion-pair
reagents.
5C18-PAQ
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
4.6mmI.D.-150mm
20mmol/l Phosphate buffer(pH2.5)
1.0 ml/min
30°C
UV280nm
1; L-Noradrenaline
2; L-Adrenaline
3; Dopamine
4; L-DOPA
1.0µl
Sample:
Inj.Vol.:
HO
PBr
NH2
NH2
HO
k (Peak.1)
0.05
O
OH
OH
2.L-Adrenaline
k (Peak.1)
0.81
OH
NH2
N
H
OH
OH
1.L-Noradrenaline
(0.5mg/ml)
(0.5mg/ml)
(0.5mg/ml)
(0.5mg/ml)
OH
OH
OH
3.Dopamine
OH
4.L-DOPA
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● Triazine rings (melamine derivatives)
Melamine derivatives are highly polar compounds due to the amino groups bound to the triazine ring. COSMOSIL PBr retains these
analytes longer than C18.
5C18-PAQ
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sample:
4.6mmI.D.-250mm
20mmol/l Phosphate buffer(pH7.0)
1.0 ml/min
30°C
UV225nm
1; Ammelide
2; Cyanuric Acid
3; Ammeline
4; Melamine
1.0µl
Inj.Vol.:
HN
N
N
HO
(0.1mg/ml)
(0.5mg/ml)
(0.1mg/ml)
(0.1mg/ml)
O
OH
N
PBr
NH2
1; Ammelide
O
k (Peak.1)
0.12
NH2
NH
N
H
N
O
2; Cyanuric Acid
NH2
N
N
HO
k (Peak.1)
0.48
N
NH2
H2N
3; Ammeline
N
N
4; Melamine
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NH2
t0: Retention time of the mobile phase
4. Peptide-related compounds
● Peptides of different chain lengths
Glycine is not retained well by C18 or PBr. However, longer peptide chains, such as glycylglycine, were retained more strongly by PBr.
5C18-PAQ
Column:
Column size:
Mobile phase:
Flow rate:
Temperature:
Detection:
Sample:
4.6mmI.D.-250mm
0.1%TFA-H2O
1.0 ml/min
30°C
UV220nm
1; Glycine
2; Glycylglycine
3; Glycylglycylglycine
4; Glycylglycylglycylglycine
1.0µl
Inj.Vol.:
PBr
OH
H2N
O
H
N
H2N
O
Glycine
k (Peak.1)
0.00
(25mg/ml)
(5mg/ml)
(5mg/ml)
(5mg/ml)
k (Peak.2)
0.05
k (Peak.1)
0.05
k (Peak.2)
0.20
O
OH
Glycylglycine
NACALAI TESQUE, INC
5. Aminoglycoside antibiotics
● Gentamicin C complex
The positions of the amino and methyl groups are different for gentamicin C2 and C2a. COSMOSIL PBr was able to separate them.
Column:
PBr
Column size: 2.0mmI.D.-150mm
Mobile phase: 0.1% Pentafluoropropionic Acid
-Acetonitrile/ H2O = 10/90
Flow rate:
0.2 ml/min
Temperature: 40°C
Detection:
ESI-MS, Positive, SIM
Sample:
Gentamicin
(C1a, C2, C2a, C1)
1.0µl
Inj.Vol.:
HN
H
HO
H
R2
R1
H
H
OH
H
O
H O
HO
NH2
H
H
O H
O
H
H H2N
H NH2
R1
R2
C1
NHCH3
C1a
NH2
H
C2
NH2
CH3
H
C2a
CH3
NH2
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Suitable Samples
Although hydrophilic compounds show weak retention by C18 columns, COSMOSIL PBr retains them using dispersion force. PBr
exhibits especially strong retention for compounds containing aromatic rings or heterocycles, amides and halogenated compounds. If
9
8
7
6
5
es
bas
acid
ic
le
Nuc
(1)
ines
lam
cho
Cate (2)
s
acid
anic
Org (2)
ids
o ac
Amin (2)
DMF
DMSO
Bis-acrylamide
Vitamin B1
Acrylamide
Nicotinamide
Nicotinic Acid
Melamine
Ammeline
Ammelide
Cyanuric Acid
Phenylalanine
Lysine
Glutamic Acid
Valine
Glycine
Citric Acid
Lactic Acid
Malic Acid
Tartaric Acid
L-DOPA
Dopamine
L‐Adrenaline
L‐Noradrenaline
Guanine
Adenine
Thymine
PBr
Uracil
4
3
2
1
0
C18-PAQ
Cytosine
Retention ratio
of each sample (k’)
retention with PBr is insufficient, COSMOSIL HILIC is your next choice.
ble
mine
r solu
Mela aterials Wate ins
ents
ides
vitam
ed m
Am ) Solv
relat (3)
(1)
(4
(2)
(Mobile phase)
(1) Methanol/ 20mmol/l Phosphate buffer(pH7.0) = 10/90
(2) 20mmol/l Phosphate buffer(pH2.5)
(3) 20mmol/l Phosphate buffer(pH7.0)
(4) Methanol/ H2O = 10/90
Ordering Informations
Column Size
I.D. x Length (mm)
2.0 x 50
2.0 x 100
2.0 x 150
2.0 x 250
3.0 x 50
3.0 x 100
3.0 x 150
3.0 x 250
4.6 x 50
4.6 x 150
4.6 x 250
Product Number
12943-61
13245-81
12392-81
13247-61
12592-61
13249-41
13250-01
13251-91
13252-81
12394-61
12395-51
Column Size
I.D. x Length (mm)
10 x 50
10 x 100
10 x 150
10 x 250
20 x 50
20 x 100
20 x 150
20 x 250
28 x 100
28 x 150
28 x 250
Product Number
13253-71
13254-61
13255-51
12397-31
13257-31
13258-21
13259-11
12398-21
13260-71
13261-61
13262-51
Column Size
I.D. x Length (mm)
4.6 mm I.D. x 10 mm
cartridge*
10 x 20
20 x 20
4.6 mm I.D. Cartridge
Holder
Product Number
12444-14
12396-41
13256-41
38009-79
*Cartridge Holder is required.
Core-shell column “COSMOCORE PBr” is available.
For research use only, not intended for diagnostic or drug use.
NACALAI TESQUE, INC.
Nijo Karasuma, Nakagyo-ku, Kyoto 604-0855 JAPAN
: +81-(0)75-251-1730
TEL
: +81-(0)75-251-1763
FAX
Website : www.nacalai.com
E-mail : [email protected]
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