Technical Note11

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 Page

1 Nucleotide-related compoundsNucleotides AMP / ADP / ATP 1Sugar nucleotides UDP-galactose / UDP-glucose 1

2 Nucleic acid base-related compoundsPyrimidine / purine rings Nucleic acid bases 2Halogenated compounds Uracil derivatives 2

3 Other aromatics and heterocyclesCatechol rings Catecholamines 2Triazine rings Melamine derivatives 3

4 Peptide-related compounds Peptides of different chain lengths Peptides 35 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.

● 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.

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5C18-PAQ PBr

Column: Column size: 4.6mmI.D.-150mmMobile phase: Methanol/ 20mmol/l Phosphate buffer(pH7.0) = 10/90Flow rate: 1.0 ml/min Temperature: 30°CDetection: UV260nm

Sample: 1; Adenosine 5’-triphosphate (0.5mg/ml)2; Adenosine 5’-diphosphate (0.8mg/ml)3; Adenosine 5’-monophosphate (0.8mg/ml)

Inj.Vol.: 1.0µl

OH

N

NN

N

NH2

O

OH

OPO

OH

O

P

O

OH

OPHO

O

OHAdenosine 5'-triphosphate

k’(Peak.1)0.01

k’(Peak.1)0.52

NACALAI TESQUE, INC

5C18-PAQ PBr

Column: Column size: 4.6mmI.D.-250mmMobile phase: 100mmol/l Phosphate buffer(pH7.0)Flow rate: 1.0 ml/min Temperature: 30°CDetection: UV260nm

Sample: 1; Uridine-5'-diphosphate (0.8mg/ml)2; Uridine-5’-diphosphogalactose (0.8mg/ml)3; Uridine-5’-diphosphoglucose (0.8mg/ml)

Inj.Vol.: 1.0µl

O

OH

HOOH

OHNH

O

ON

O

OHOH

OP

OP

O

O OH O OH

O

OH

OHOH

OHNH

O

ON

O

OHOH

OP

OP

O

O OH O OH

2.Uridine-5’-diphosphogalactose 3.Uridine-5’-diphosphoglucose

Polar molecule separation by COSMOSIL PBr

2. Nucleic acid base-related compounds

● Pyrimidine / purine ringsCOSMOSIL PBr strongly retains analytes containing pyrimidine and purine rings, such as nucleobases. Compounds with more rings are retained more strongly.

● Halogenated compounds (uracil derivatives)COSMOSIL PBr offers stronger retention of halogenated analytes.

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.

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Column: Column size: 4.6mmI.D.-150mmMobile phase: Methanol/ 20mmol/l Phosphate

buffer(pH7.0) = 10/90Flow rate: 1.0 ml/min Temperature: 30°CDetection: UV260nm

Sample: 1; Cytosine (0.05mg/ml) 2; Uracil (0.05mg/ml) 3; Thymine (0.05mg/ml) 4; Guanine (0.05mg/ml) 5; Adenine (0.05mg/ml)

Inj.Vol.: 1.0µl

5C18-PAQ PBr

1. Cytosine

N

NH

NH2

O

2.Uracil

NH

NH

O

O

3.Thymine

NH

NH

O

O

4.Guanine 5.Adenine

NH

NNH

N

O

NH2

N

NNH

N

NH2

Pyrimidine Rings

Purine Rings

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Column: Column size: 4.6mmI.D.-150mmMobile phase: 20mmol/l Phosphate buffer(pH2.5)Flow rate: 1.0 ml/min Temperature: 30°CDetection: UV210nm

Sample: 1; 5,6-Dihydrouracil (0.005mg/ml)2; Uracil (0.005mg/ml)3; 5-Fluorouracil (0.005mg/ml)4; 5-Bromouracil (0.005mg/ml)

Inj.Vol.: 10µl

5C18-PAQ PBr

2.Uracil

NH

NH

O

O

3.5-Fluorouracil

NH

NH

O

O

F

4.5-Bromouracil

NH

NH

O

O

Br

1.5,6-Dihydrouracil

NH

NH

O

O

NACALAI TESQUE, INC

5C18-PAQ PBr

Column: Column size: 4.6mmI.D.-150mmMobile phase: 20mmol/l Phosphate buffer(pH2.5)Flow rate: 1.0 ml/min Temperature: 30°CDetection: UV280nm

Sample: 1; L-Noradrenaline (0.5mg/ml)2; L-Adrenaline (0.5mg/ml)3; Dopamine (0.5mg/ml)4; L-DOPA (0.5mg/ml)

Inj.Vol.: 1.0µl

k’(Peak.1)0.05

k’(Peak.1)0.81

1.L-NoradrenalineOH

OH

NH2HO

2.L-Adrenaline

OH

OH

NH

HO

3.DopamineOH

OH

NH2

4.L-DOPA

OH

OH

OHO

NH2

● 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.

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.

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.

NACALAI TESQUE, INC

5C18-PAQ PBr

Column: Column size: 4.6mmI.D.-250mmMobile phase: 20mmol/l Phosphate buffer(pH7.0)Flow rate: 1.0 ml/min Temperature: 30°CDetection: UV225nm

Sample: 1; Ammelide (0.1mg/ml)2; Cyanuric Acid (0.5mg/ml)3; Ammeline (0.1mg/ml)4; Melamine (0.1mg/ml)

Inj.Vol.: 1.0µl

t0: Retention time of the mobile phase

N

N

N

NH2HO

OHHN

NH

NH

O

OON

N

N

NH2HO

NH2

N

N

N

NH2H2N

NH2

1; Ammelide 2; Cyanuric Acid 3; Ammeline 4; Melamine

k’(Peak.1)0.12

k’(Peak.1)0.48

NACALAI TESQUE, INC

5C18-PAQ PBr

Column: Column size: 4.6mmI.D.-250mmMobile phase: 0.1%TFA-H2OFlow rate: 1.0 ml/min Temperature: 30°CDetection: UV220nm

Sample: 1; Glycine (25mg/ml)2; Glycylglycine (5mg/ml)3; Glycylglycylglycine (5mg/ml)4; Glycylglycylglycylglycine (5mg/ml)

Inj.Vol.: 1.0µl

H2N

HN

OH

O

O

H2NOH

O GlycylglycineGlycine

k’(Peak.1)0.00

k’(Peak.2)0.05

k’(Peak.2)0.20

k’(Peak.1)0.05

NACALAI TESQUE, INC

Column: PBrColumn size: 2.0mmI.D.-150mmMobile phase: 0.1% Pentafluoropropionic Acid

-Acetonitrile/ H2O = 10/90Flow rate: 0.2 ml/min Temperature: 40°CDetection: ESI-MS, Positive, SIM

Sample: Gentamicin (C1a, C2, C2a, C1)

Inj.Vol.: 1.0µl

OHHO

HNH2H

OH

O H

H2N H

OHH

O

H

HN H

NH2H

HO

HR2

R1H

R1 R2

C1 NHCH3 HC1a NH2 HC2 NH2 CH3

C2a CH3 NH2

1605

For research use only, not intended for diagnostic or drug use.

NACALAI TESQUE, INC.Nijo Karasuma, Nakagyo-ku, Kyoto 604-0855 JAPANTELFAXWebsiteE-mail

: +81-(0)75-251-1730: +81-(0)75-251-1763: www.nacalai.com: info.intl@nacalai.com

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 retention with PBr is insufficient, COSMOSIL HILIC is your next choice.

(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

Core-shell column “COSMOCORE PBr” is available.

0123456789

Cyt

osin

eU

raci

lTh

ymin

eG

uani

neA

deni

neL‐

Nor

adre

nalin

eL‐

Adr

enal

ine

L-D

OPA

Dop

amin

eTa

rtaric

Aci

dM

alic

Aci

dLa

ctic

Aci

dC

itric

Aci

dG

lyci

neVa

line

Lysi

neG

luta

mic

Aci

dP

heny

lala

nine

Am

mel

ide

Cya

nuric

Aci

dA

mm

elin

eM

elam

ine

Nic

otin

ic A

cid

Nic

otin

amid

eVi

tam

in B

1A

cryl

amid

eB

is-a

cryl

amid

eD

MS

OD

MF

C18-PAQ

PBr

Ret

entio

n ra

tio

of e

ach

sam

ple

(k’)

Nucleic acid bases

(1) Catecholamines

(2) Organic acids

(2) Amino acids

(2)Melamine

related materials

(3)Water soluble

vitamins

(1) Amides

(4) Solvents

(2)

Column SizeI.D. x Length (mm) Product Number

2.0 x 50 12943-612.0 x 100 13245-812.0 x 150 12392-812.0 x 250 13247-613.0 x 50 12592-613.0 x 100 13249-413.0 x 150 13250-013.0 x 250 13251-914.6 x 50 13252-814.6 x 150 12394-614.6 x 250 12395-51

Column SizeI.D. x Length (mm) Product Number

10 x 50 13253-7110 x 100 13254-6110 x 150 13255-5110 x 250 12397-3120 x 50 13257-3120 x 100 13258-2120 x 150 13259-1120 x 250 12398-2128 x 100 13260-7128 x 150 13261-6128 x 250 13262-51

Column SizeI.D. x Length (mm) Product Number

4.6 mm I.D. x 10 mm cartridge* 12444-14

10 x 20 12396-4120 x 20 13256-41

4.6 mm I.D. Cartridge Holder 38009-79

*Cartridge Holder is required.