Polar molecule separation - ナカライテスク株式会社...Polar molecule separation by...
Transcript of Polar molecule separation - ナカライテスク株式会社...Polar molecule separation by...
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.
NACALAI TESQUE, INC
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.
NACALAI TESQUE, INC
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
NACALAI TESQUE, INC
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: [email protected]
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.