HPLC Columns Cosmosil

1
NACALAl TESQUE, lnc. is commiIIed Io Ihe well-bein of all humaniIy Ihrouh enhancin Ihe research capabiliIies of Ihe
worldwide scienIifc communiIy. We sIrive Io connecI counIries Ihrouh lon-Ierm business relaIionships based on IrusI and
friendship.
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when KyoIo was Ihe capiIal of Japan, Ihe ancesIors of Nakarai TakaIoshi, our presenI-day presidenI supplied IradiIional
Japanese and Chinese medicines Io Ihe lmperial Family. The documenIed hisIory of our company bean in 1846 when Ihe
company`s founder opened Nakarai Mansuke ShoIen, LId. Our more Ihan 160 year-lon presence in KyoIo, in walkin disIance
from Ihe preserved lmperial Household rounds is a place of honor and presIie in Japan. ln 1968 Ihe company`s reaenI
deparImenI spun of and became Nakarai Chemicals, lnc. ln a move Io propel Ihe company Io Ihe fuIure Ihe name was chaned
Io Nacalai Tesque, lnc. in 1988.
CenIerin around research chemicals, Ihe acIive felds of Nacalai Tesque include fne chemicals, dianosIics, consumables and
kiIs for bioIechnoloy, and relaIed laboraIory equipmenIs and supplies. We imporI and disIribuIe Io Japanese research
laboraIories, universiIies, food and beverae indusIry and pharmaceuIical companies commodiIies from Ihe Iop bioIechnoloy
manufacIurers of Ihe world and exporI our own producIs.
Nacalai Tesque esIablished iIs own Nacalai Research lnsIiIuIe as parI of our lon-Ierm sIraIey Io keep us aI Ihe fronIier of
science. WiIh Ihe Iechnoloy and experIise IhaI developed TANAKA PloI`known as a column evaluaIion meIhod, our hihly
qualified researchers are conIinuously invenIin new producIs and sIeadily improvin our convenIional commodiIies. These
consIanI eforIs and reaI achievemenIs are peaked in our HPLC packin maIerials and columns. We are proud of Ihe excellence
of our HPLC columns in accuracy, endurance, reasonable price and sIron Iechnical supporI IhrouhouI. We packed in our
HPLC columns all of our experIise, Ihe besI available maIerials and innovaIions, culminaIin in our specialIy columns.
Nacalai Tesque has realized IhaI Ihe company`s fuIure depends on Ihe compleIe saIisfacIion of our precious cusIomers. We are
keen Io provide you wiIh Ihe besI service and supporI and hope IhaI you kindly review Ihe conIenI of Ihis caIaloue Io eI
acquainIed wiIh our producIs. Please conIacI us wiIh any inquiries. Your commenIs will be hihly appreciaIed.
CORPORATE PROF|LE
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OORPORATE PROFILE

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OONTENTS

2-8
GENERAL INFORMATION

4-6
OOSMOSIL HPLO OOLUMN
1. COSMOSlL/COSMOGEL

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General descripIion of Ihe COSMOSlL/COSMOGEL packin maIerials

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Column selecIion uide

9
COSMOSlL chromaIoram lndex/COSMOSlL applicaIion daIa

9
COSMOSlL silica packin maIerial

10-11
Performance uaranIee

12
ConvenIional columns versus hih performance columns

18
USP specifcaIions

18
2. Reversed Phase ChromaIoraphy-OcIadecyl Iypes

14
Cs-MS-

16-17
Cs-AR-

18-19
Cs-PAQ

20-21
8. Reversed Phase ChromaIoraphy-SpecialIy columns

22
CholesIer

28-26
)NAP

26-27
PYE

28-29
NPE

80
PBB-R

81
4. Reversed Phase ChromaIoraphy-Alkyl chains columns

82
Caa-AR-Cs-MSC+-MSTMS-MS

82-88
6. Reversed Phase ChromaIoraphy-Phenyl IypeCyano Iype

84
6PE-MS6CN-MS

84
6. Silica Based PreparaIive Columns

86
7. Normal Phase ChromaIoraphy

86
SL-

86-87
8. Hydrophilic lnIeracIion ChromaIoraphy

88
HlLlC

88-89
9. Saccharide Analysis

40
Suar-D

40-48
NHa-MS

48
10. ProIein SeparaIion Wide Pore Columns

44
Reversed phase chromaIoraphy

44
ProIein-R

44-46
Cs-AR-800Cs-AR-800C+-AR-800Ph-AR-800

46-47
Gel flIraIion chromaIoraphy

48
Diol-120-Diol-800-

48-49
lon-exchane chromaIoraphy

60
DEAEQACMSP

60-62
Hydrophobic inIeracIion chromaIoraphy

68
HlC

68
11. SpecialIy Columns for Fullerene

64
Buckyprep

66
Buckyprep-M

67
PBB

68
PYENPE

69
CONTENTS
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LIQUID OHROMATOGRAPHY RELATED PRODUOTS
1. Packin MaIerials for Column ChromaIoraphy
Packin MaIerials for Reversed Phase PreparaIive Liquid ChromaIoraphy

62
Cs-OPN

68-64
Cs-PREP

66-66
Packin MaIerials for Normal Phase PreparaIive Liquid ChromaIoraphy

67
SL-ll-PREP

67-68
Silica Gel (sphericalneuIral)

69
2. Liquid ChromaIoraphy RelaIed ProducIs

71
lon-pair reaenIs

71-72
Labelin reaenIs

72
8. PreflIraIion Tools for Liquid ChromaIoraphy

78
Cosmonice FilIer

78-74
Cosmospin FilIer

78-74
Guard carIride holder

76
Column preflIer

76
Column spare flIer for preflIer

76
Column connecIin Iube

76
T TEOHNIOAL NOTE
1. SelecIiviIy of packin maIerials in reversed phase liquid chromaIoraphy

78
2.

PreparaIion of mobile phase for HPLC

86
8. Sample preIreaImenI for HPLC

88
4.

Baseline noise in radienI eluIion

92
6.

TroubleshooIin for increased pressure

98
6.

EfecI of uard column

96
7.

TroubleshooIin for normal phase chromaIoraphy

97
8.

lnner diameIer of column (scale down and scale up)

99
9.

Packin insIrucIion

101
N APPLIOATION DATA
1. ChromaIoram index/ApplicaIion daIa

104
2. Reference lisI

106
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The Structure of the Screw-in Type Oo|umns
Genera| Ordering Information
When placin an order wiIh us or makin an enquiry, please conIacI our lnIernaIional Business DevelopmenI Group or your local
disIribuIor. Please clearly idenIify Ihe producI in quesIion when submiIIin your enquiry.
The speed of innovaIion is acceleraIin. We always have brand new or improved columns noI lisIed here. There are also many
oIher producIs Nacalai Tesque can supply. Therefore we ure you Io make enquiries.
GeneraI |nformation
End-ftting Types
Columns in Ihe recenI COSMOSlL line-up are shipped wiIh WaIers Iype end fIIin. Upon requesI diferenI end fIIin Iypes may
be available. Please enquire!
Oo|umn Identifcation
AI Ihe end of each secIion, Ihe COSMOSlL and COSMOGEL packed columns are lisIed in a way IhaI Ihe parIicle size, sIaIionary
phase, column size of Ihe packin maIerial can be easily deIermined.
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7918-81 C
OSMOSlL 6
s-MS- 4.6 mm l.D.

'
`160 mm W
!
aIers

When placin an order, please clearly indicaIe Ihe producI number (1), producI name (2), parIicle size (8), Iype of sIaIionary
phase (4), column size (6) and Ihe end fIIin Iype (6).
Column lenIhmm)
lnside diameIer
mm l.D.)
Micropore diameIer 7)
ParIicle size,m)
Silica el
Q
QScrew pipe FiIIin Piece
Column End Plu
FilIer
Product Description and Avai|abi|ity
Please noIe IhaI Ihe producI specifcaIions are sub|ecIed Io chanes and Ihe manufacIurin of some producI may be sIopped.
Please consulI Ihe Iable on pae 18 for cross-reference informaIion on old producIs and Iheir newer and beIIer equivalenIs.
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Warranty O|aims
The manufacIurer will replace defecIive columns if noIifed wiIhin 2 weeks of receipI of Ihe producI by Ihe cusIomer under Ihe
followin condiIions:
1) Column abnormaliIies are due Io accidenIs in shippin or rouh handlin.
2) The number of efecIive plaIes of Ihe column is considerably lower Ihan Ihe minimum uaranIeed IheoreIical plaIe number
documenIed in Ihe inspecIion reporI IhaI accompanies each column.
Please conIacI Ihe lnIernaIional Business DevelopmenI Group of Nacalai Tesque or your local disIribuIor for addiIional
informaIion.
Terms and Oonditions of Sa|e
Terms are sub|ecI Io condiIions seI forIh by Ihe auIhorized Nacalai Tesque dealers in each counIry.
Not for O|inica| Use
Nacalai Tesque producIs are noI inIended for clinical use. While clinical applicaIions may be shown, Ihese producIs are noI
validaIed for clinical use.
COEMOEIL MFLC COLOMN
J. COEMOEIL / COEMOGEL
E. HCVC|SCd FhaSC Ch|CmatCQ|aphy
OCtadCCyl typCS
U. HCVC|SCd FhaSC Ch|CmatCQ|aphy
EpCClalty CClumnS
4. HCVC|SCd FhaSC Ch|CmatCQ|aphy
Alkyl ChalnS CClumnS
U. HCVC|SCd FhaSC Ch|CmatCQ|aphy
FhCnyl typCSCyanC typCS
U. ElllCa HaSCd F|Cpa|atlVC CClumnS
7. NC|mal FhaSC Ch|CmatCQ|aphy
H. Myd|CphlllC IntC|aCtlCn Ch|CmatCQ|aphy
D. EaCCha|ldC AnalySlS
JO. F|CtCln ECpa|atlCn WldC FC|C CClumnS
JJ. EpCClalty CClumnS !C| FullC|CnC
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1. COSMOS|L / COSMOGEL
Sample
SeparaIion
mode
Packin
maIerial
SIaIionary phase Special feaIures and applicaIions Pae
Oranic
compounds
(low M.W.)
Reversed
phase
Cs-MS-
OcIadecyl roup
MulIi-purpose Cs column. MonofuncIional
silylaIion on ulIra-pure silica el for
separaIion of Ihe widesI rane of
compounds
16
Cs-AR-
MulIi-purpose Cs column usin ulIra-
pure silica el. FeaIures sIron acid
resisIance and suiIable for a wide rane
of separaIion
18
Cs-PAQ
Reversed phase column, compaIible wiIh
100% waIer based mobile phases
20
CholesIer CholesIeryl roup
Usable under Ihe same condiIion as Cs
Unique riid cholesIeryl sIrucIure improves
separaIion
28
)NAP NaphIhyleIhyl roup
SIroner )-) inIeracIion Ihan Phenyl
column
26
PYE PyrenyleIhyl roup MosI powerful )-)inIeracIion 28
NPE NiIrophenyleIhyl roup
SeparaIion uIilizin )-) inIeracIion and
Dipole-dipole inIeracIion
80
PBB-R
PenIabromobenzyl
roup
SeparaIion uIilizin dispersion force 81
Caa-AR- Docosyl roup
Alkyl chain columns excepI Cs column 82
Cs-MS OcIyl roup
C+-MS BuIyl roup
TMS-MS TrimeIhyl roup
PE-MS PhenyleIhyl roup
)-) inIeracIion 84
CN-MS Cyanopropyl roup
Normal phase SL-
Normal Phase chromaIoraphy wiIh non-
polar oranic solvenIs
86
Hydrophilic
inIeracIion
HlLlC Triazole
ReIains hihly polar compounds IhaI
would noI be reIained in Cs column
88
Monosaccharide
Oliosaccharide
Hydrophilic
inIeracIion
Suar-D
Secondary/TerIiary
amine
A novel sIaIionary phase for saccharide
separaIion. ExIended column life
and increased sIabiliIy. AlIernaIive Io
aminopropyl Iype
40
NHa-MS Aminopropyl roup Primary amine bonded column 48
ProIeins
Reversed
phase
ProIein-R OcIadecyl roup
The mosI suiIable reverse phased column
for proIeins
44
Cs-AR-800 OcIadecyl roup Wide pore Iype reversed phase columns
wiIh hih acid resisIance recommended
for Ihe separaIion of proIeins,
polypepIides, nucleic acids and oIher
lare molecules
46
Cs-AR-800 OcIyl roup
C+-AR-800 BuIyl roup
Ph-AR-800 Phenyl roup
Gel
permeaIion
Diol-120-
Diol roup
Silica-based el flIraIion column for hih
speed separaIion of proIeins and waIer
soluble polymer
48
Diol-800-
lon-exchane
DEAE DieIhylaminoeIhyl Iype Weak anion-exchane
60
QA
QuarIernary ammonium
Iype
SIron anion-exchane
CM CarboxymeIhyl Iype Weak caIion-exchane
SP Sulfopropyl Iype SIron caIion-exchane
Hydrophobic
inIeracIion
HlC
Hydrophobic inIeracIion charomaIoraphy
column for proIein separaIion
68
Fullerenes
Buckyprep Pyrenylpropyl roup SIandard column for fullerenes separaIion 66
Buckyprep-M PhenoIhiazinyl roup Desined Io separaIe meIallofullerenes 67
PBB
PenIabromobenzyl
roup
Desined for Ihe preparaIive separaIion
of fullerenes usin carbon disulphide,
o-dichlorobenzene and Ioluene
68
PYE PyrenyleIhyl roup
SeparaIion of Fullerene and sIrucIural
isomer 69
NPE NiIrophenyleIhyl roup SeparaIion of derivaIived fullerene
Genera| description of the OOSMOSIL / OOSMOGEL packing materia|s
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AOranic compounds (Low M.W.)
OcIadecyl roup bonded column (Cs,ODS) are recommended as frsI-choice columns for separaIions of oranic compounds
(Low M.W.). lf Ihere is noI enouh separaIion or no reIenIion usin COSMOSlL Cs columns, COSMOSlL series ofer many
kinds of specialIy columns.
For Cs-series selecIion uide, please refer Io pae 16.
ASaccharides
COSMOSlL Suar-D is recommended for Ihe separaIion of monosaccharides and oliosaccharides as a frsI-choice column.
For Ihe separaIion of suar derivaIives, COSMOSlL Cs-PAQ is suiIable as well.
AProIeins
Please selecI based on Ihe separaIion mode.
AFullerenes
COSMOSlL Buckyprep is mosI suiIable for Ihe separaIion of fullerenes.
Oo|umn Se|ection Guide
NoI enouh
No reIenIion
ReIains hihly polar compounds
IhaI would noI be reIained in C18
columns (pae 88)
For Ihe separaIion of monosaccharides
and oliosaccharides (pae 40)
Usable under Ihe same
condiIion as ODS
Hih planariIy selecIiviIy
(pae 28)
NoI enouh
NoI enouh
MosI powerful
)-) inIeracIion
pae 28)
SelecIion uide
C
18
(ODS)
HlLlC
Suar-D
CholesIer
)NAP
PYE
NPE
Phenyl
PBB-R
SIroner )-)inIeracIion
Ihan Phenyl column
pae 26)
COSMOSlL ChromaIoram lndex and COSMOSlL ApplicaIion DaIa are now available on our websiIe (websiIe : hIIp://www.
nacalai.com). The online version includes more Ihan 6,700 chromaIorams and more Ihan 1,000 applicaIion daIa usin
COSMOSlL columns. These indexes are useful for opIimizin analyIical condiIions. lf you have any quesIions reardin Ihe
applicaIion daIa or separaIions of compounds noI lisIed here, please feel free Io e-mail us aI info.inIl@nacalai.co.|p.
COSMOSlL ChromaIoram lndex/ApplicaIion DaIa
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Introduction
Superior HPLC columns can be produced only wiIh excellenI packin
maIerials and superb packin Iechnique. COSMOSlL columns are well known
for Iheir hih efciency and hih-resoluIion separaIions. Based on spherical,
IoIally porous silica, COSMOSlL columns provide enhanced chemical and
mechanical sIabiliIy as well as very hih surface coverae.
The selecIion of Ihe Cs chemisIries available enables Ihe chromaIorapher Io
Iailor separaIion Io special applicaIions. The ulIra pure silica based MS-
series wiIh widely exIended pH rane are developed for improved separaIion
of basic compounds. The Cs AR- phase provides increased acid resisIance.
Four unique bonded chemisIries are available for Cosmosil specialIy columns:
CholesIer, )NAP, PYE and HlLlC. These specialIy columns may improve Ihe separaIion compared wiIh convenIional columns.
Five hihly effecIive phases for fullerene separaIion are also available: Buckyprep, Buckyprep-M, PBB, PYE and NPE.
COSMOGEL packin maIerials are non-silica based and provide superior performance in ion exchane columns.
Fiure. Microscopic phoIoraph of Ihe
silica el
Raw materia| si|ica ge|
COMSOSlL is based on ulIra pure porous spherical silica el (puriIy: 99.99% or hiher). Low-puriIy silica el conIains meIal
impuriIy which may cause inIerference in Ihe separaIion, especially for meIal coordinaIion compounds.
Column size 4.6 mm l.D. `160 mm
Mobile phase aceIoniIrile : waIer - 60 : 60
Flow raIe 1.0 ml/min
TemperaIure 80T
DeIecIion UV 264 nm
Sample Paeonol
6Cs-MS-
UlIra-pure silica el
ConvenIional Silica el
MeIal coordinaIion compounds
The compounds, which have 2 or more hydroxyl roups, carboxyl
roups, carbonyl roups and/or amino roups in Ihe ad|acenI posiIions,
can complex wiIh meIal impuriIy, which resulIs in peak Iailin.
Table. MeIal conIenI percenIae (ppm)
Packin maIerial Al Ca Fe M Na Ti ToIal
UlIra pure silica el 4 10 11 8 12 <1 41
Normal silica el 82 460 26 88 66 184 786
Stationary phase construction
While Cs columns are mosI widely used in reversed phase HPLC, iI is imporIanI Io disIinuish beIween Iwo very differenI
bonded phase formaIs.
Monomeric Iype Cs formaI incorporaIes Ihe bondin of Ihe Cs alkyl chain Io a sinle silica aIom on Ihe silica el backbone.
Monomeric Iype columns such as Ihe COSMOSlL Cs-MS- and Ihe MS series have excellenI synIhesis reproducibiliIy, very
ood loI-Io-loI reproducibiliIy and shorI mobile phase equilibraIion Iimes.
On Ihe oIher hand, Ihe polymeric Cs formaI incorporaIes a Iri-funcIional silylaIion procedure whereby Ihe ocIadecyl roup is
bonded Io 2 or 8 silica aIoms on Ihe silica el backbone. This increases silylaIion resulIs in far reaIer column sIabiliIy
parIicularly in acidic mobile phase condiIions. SIereo reconiIion capabiliIy is also reaIer Ihan IhaI of Ihe monofuncIional
silylaIion Iype Cs columns. The polymeric formaI is ofered in Ihe AR- and Ihe enIire AR-800 series of COSMOSlL columns.
Please refer Io producI descripIions and applicaIion chromaIorams for selecIion uidance.
Fiure. Diarams of diferenI sIaIionary phase consIrucIions (before end-cappin IreaImenI)
Monomeric Iype Cs Polymeric Iype Cs Silica el surface
Cs-MS- Cs-AR-,Cs-PAQ SL-
OOSMOSIL Si|ica Packing Materia|
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6Cs-MS- Cs column wiIh much
residual silanol roups
End-capping treatment
The silanols (Si-OH roups) on Ihe silica surface provided bondin siIe for sIaIionary phases. However, parI of Ihe silanol roups
remain un-capped as residual silanol roups even afIer Ihe end-cappin IreaImenI, Ihey cause peak Iailin for basic compounds.
COSMOSlL packin maIerials for reversed phase chromaIoraphy are of near-perfecIly end-capped residual silanol roups.
R-NH
+
(Basic compounds)
Si-O
(Free silanol roup)

Basic compound can form ionic bonds wiIh
residual free silanols. The ionic bondin causes
peak Iailin of basic compounds if a silica-
based column is noI perfecIly end-capped.
Mobile phase meIhanol20 mmol/l phosphaIe bufer (pH7) - 20 : 80
TemperaIure 80T
DeIecIion UV264 nm (0.16AUFS)
Sample 1.Uracil
2.N-MeIhylbenzylamine
8.N,N-DimeIhylbenzylamine
4.Benzylalcohol
2. N-MeIhyl Benzylamine 8. N,N-DimeIhyl Benzylamine
+C/P-k`(Cafeine)/k`(Phenol) in 80% meIhanol.
Synthesis Reproducibi|ity
By usin sIricIly selecIed silica el and consIanI synIhesis condiIions, Ihe chemically bonded Iype column reIains a variance of
Ihe capaciIy facIor (k`) beIween synIheIic loIs of wiIhin 10% and a variance of Ihe separaIion facIor (+) of wiIhin 6%.
The fures below show in raphic form Ihe loI inspecIion resulIs of synIhesized packin maIerial (COSMOSlL 6Cs-MS- ).
Fiure 1 shows Ihe variance of sIaIionary phase (ocIadecyl roup) inIroduced volume which is Ihe basic indicaIor of Ihe qualiIy
of Ihe packin maIerial. Fiure 2 shows Ihe end-cappin efciency of Ihe packin maIerial. The variance amon Ihe loIs is
reduced Io Ihe minimum in Ihe COSMOSlL packed columns.
k`Nap-k`value of NaphIhalene in Ihe 70%
meIhanol.
k`(NaphIhalene, 70% MeIhanol) + (Cafeine/Phenol, 80% MeIhanol)
Fiure 1. Variance of Ihe combinin volume
beIween silica el and Cs.
Fiure 2. Variance of end-cappin efciency of
Ihe packin maIerial.
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ValidaIed columns
ProducI number ProducI name Column size
88019-81 COSMOSlL 6Cs-MS- 4.6 mm l.D. `160 mm
88020-41 COSMOSlL 6Cs-MS- 4.6 mm l.D. `260 mm
88144-81 COSMOSlL 6Cs-AR- 4.6 mm l.D. `160 mm
88146-21 COSMOSlL 6Cs-AR- 4.6 mm l.D. `260 mm
Qua|ity guarantee of packing materia| (Va|idation)
The sIricI qualiIy conIrol sysIem of Nacalai Tesque supporIs Ihe cusIomers wiIh an individual lnspecIion ReporI`which
accompanies each and every COSMOSlL and COSMOGEL Packed Column (excepI uard columns) and an addiIional CerIifcaIe
of Analysis`for Ihe COSMOSlL 6Cs-MS- and 6Cs-AR- (4.6 mm l.D. `160 mm and 4.6 mm l.D. `260 mm).
COSMOSlL cerIifcaIe of analysis
ValidaIe Ierms of Ihe physical properIies of Ihe silica el, Ihe carbon conIenI, polar selecIiviIy, hydrophobiciIy, silanol capaciIy,
sIeric selecIiviIy, inacIive deree Io basic and chelaIin compounds.
[DescripIion of ValidaIed Ierms]
1) Physical properIy of basic silica el maIerial
parIicle size, specifc surface area, pore capaciIy, pore size
2) Carbon conIenI
The bondin raIe of Cs roups is shown. The hiher iI is, Ihe hiher Ihe carbon conIenI is.
8) Polar selecIiviIy
The selecIiviIy of polar roup is shown.
4) HydrophobiciIy
The hydrophobiciIy of packin maIerial is shown. The hiher Ihe separaIion facIor is.
6) Silanol capaciIy
The raIe of residual silanol on Ihe silica surface is shown. The lower iI is, Ihe ealier Ihe eluIion of cafeine.
6) SIeric selecIiviIy
SIeric selecIiviIy of Ihe silica el is characIerized by Ihis facIor, as Ihe bulkier is reIained loner.
7) lnacIive deree Io basic compounds and 8) lnacIive deree Io chelaIin compounds
The lower Ihe separaIion facIor is and Ihe hiher Ihe efecIive number raIio is, Ihe beIIer Ihe basic compounds and chelaIin compounds are separaIed.
ValidaIed Ierms 6Cs-MS- 6Cs-AR-
1) Basic silica el maIerial Yes Yes
2) Carbon conIenI Yes Yes
8) Polar selecIiviIy
SeparaIion facIor of MeIhyl benzoaIe Io Benzene
Yes Yes
4) HydrophobiciIy
SeparaIion facIor of Toluene Io Benzene
Yes Yes
6) Silanol capaciIy
SeparaIion facIor of Caffeine Io Phenol
Yes Yes
6) SIeric selecIiviIy
SeparaIion facIor of Triphenylene Io o-Terphenyl
No Yes
7) lnacIive deree Io basic compounds
SeparaIion facIor and effecIive plaIe number raIio of N-AceIylprocainamide Io Benzylalcohol
Yes No
8) lnacIive deree Io chelaIin compounds
SeparaIion facIor and effecIive plaIe number raIio of 8-Quinolinol Io Propylbenzene
Yes Yes
Performance Guarantee
C
O
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M
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l
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H
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L
C

C
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L
U
M
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18
Qua|ity guarantee of OOSMOSIL packed co|umn
lnspecIion reporI conIains daIa of number of IheoreIical plaIes (N), peak
asymmeIry (s), capaciIy facIor (k`), separaIion facIor (+) and pressure.
A period of more Ihan 27 years has passed since Ihe firsI COSMOSlL 6Cs columns were developed and offered for sale.
ConIinuous Iechnical improvemenI has made many of Ihese columns obsoleIe and noI of Ihe hihesI qualiIy and performance
available any more. However, many lon-Ierm users conIinue Io employ Ihese older convenIional columns for rouIine analysis
and qualiIy conIrol. NeverIheless, Ihe manufacIure of Ihese older columns will evenIually cease and we sIronly ure cusIomers
Io replace Ihe convenIional columns wiIh Iheir hiher performance equivalenIs ouIlined in Ihe Iable below. For addiIional
informaIion, conIacI Ihe manufacIurer or your local disIribuIor direcIly.
lnspecIion reporI
ConvenIional Columns versus Hih Performance Columns
ConvenIional columns (old) Hih performance columns (new)
6Cs-AR 6Cs-AR-
6Cs 6Cs-MS-
6Cs-MS 6Cs-MS-
6Cs-P 6Cs-PAQ
6Cs-P-MS 6Cs-PAQ
6Cs 6Cs-MS
6TMS 6TMS-MS
6PE 6PE-MS
6CN-R 6CN-MS
6NHa 6NHa-MS
6Cs-800 6Cs-AR-800
6Cs-800 6Cs-AR-800
6C+-800 6C+-AR-800
6SL 6SL-
USP Code DescripIion Recommended columns
L1
OcIadecyl silane chemically bonded Io porous silica or ceramic micro-parIicles, 1.7
Io 10 ,m in diameIer, or a monoliIhic rod
COSMOSlL Cs-MS-
COSMOSlL Cs-AR-
COSMOSlL Cs-PAQ
COSMOSlL Cs-AR-800
L8 Porous silica parIicles, 6 Io 10 ,m in diameIer COSMOSlL SL-
L7
OcIylsilane chemically bonded Io IoIally porous silica parIicles, 1.7 Io 10 ,m in
diameIer
COSMOSlL Cs-MS
COSMOSlL Cs-AR-800
L10 NiIrile roups chemically bonded Io porous silica parIicles, 8 Io 10 ,m in diameIer COSMOSlL CN-MS
L11 Phenyl roups chemically bonded Io porous silica parIicles, 1.7 Io 10 ,m in diameIer
COSMOSlL PE-MS
COSMOSlL Ph-AR-800
L18 TrimeIhylsilane chemically bonded Io porous silica parIicles, 8 Io 10 ,m in diameIer COSMOSlL TMS-MS
L20
Dihydroxypropane roups chemically bonded Io porous silica parIicles, 6 Io 10 ,m in
diameIer
COSMOSlL Diol-120-
COSMOSlL Diol-800-
L28
An anion-exchane resin made of porous polymeIhacrylaIe or polyacrylaIe el wiIh
quarIenary ammonium roups, abouI 10 ,m in size
COSMOGEL QA
L26
BuIyl silane chemically bonded Io IoIally porous silica parIicles, 8 Io 10 ,m in
diameIer
COSMOSlL C+-MS
COSMOSlL C+-AR-800
The USP (U.S. Pharmacopeia) specifcaIions are lisIed below wiIh Ihe recommended COSMOSlL HPLC columns.
USP specifcaIions
C
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Introduction
The reversed phase HPLC column is mosI commonly used because of Ihe hih IheoreIical plaIe number, excellenI separaIion
characIerisIics, reproducibiliIy, afordable cosI and ease of use. Columns packed wiIh Ihe ocIadecyl roup bonded Iype silica el
(Cs, ODS) are Ihe mosI widely employed. We ofer Ihree Iypes of ocIadecyl roup bonded columns: COSMOSlL Cs-MS-, Cs-
AR- and Cs-PAQ, each of which has a diferenI separaIion properIy.
2. Reversed Phase Chromatoyraphy - OctadecyI types
Materia| characteristics
Packin maIerial Cs-MS- Cs-AR- Cs-PAQ
Silica el hih puriIy porous spherical silica
Averae parIicle size 8616 ,m
Averae pore size approx. 120
Specifc surface area approx. 800 m/
SIaIionary phase
ocIadecyl roup
Bondin Iype monomeric polymeric
Main inIeracIion hydrophobic inIeracIion
End-cappin IreaImenI near-perfecI IreaImenI
Carbon conIenI approx. 16 approx. 17 approx. 11
pH rane 2 10
z
1.6 7.6
z
2 7.6
FeaIure
This phase is recommended for
mosI applicaIions buI parIicularly
efecIive for basic compounds.
This phase is recommended
for separaIions requirin acidic
mobile phase condiIions. lI also
shows superior molecular shape
selecIiviIy Io monomeric Iype
Cs columns.
This phase is desined Io
ofer superior reIenIion of polar
compounds and excellenI
reproducibiliIy in hihly aqueous
mobile phases, even in 100%
aqueous.
zOpIimum pH rane of columns based on silica el is beIween 2 and 7.6.
Di#erence of separation property
Mobile phase meIhanol : waIer - 80 : 20
TemperaIure 80T
DeIecIion UV 264 nm
6Cs-MS- 6Cs-AR- 6Cs-PAQ
Sample 1. Valerophenone (0.17 ,)
2. BuIyl BenzoaIe (0.17 ,)
8. BuIylbenzene (8.0 ,)
4. o-Terphenyl (0.17 ,)
6. Amylbenzene (8.0 ,)
6. Triphenylene (0.021 ,)
C
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M
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H
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C

C
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L
U
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16
Oo|umn se|ection based on app|ication data
We prepare followin applicaIion daIa Io help you selecI separaIion condiIions.
ACOSMOSlL ChromaIoram lndex
More Ihan 6,700 sinle compound eluIion profles wiIh full chromaIoraphic condiIion descripIion are available. They are noI
only an incredible help for chromaIoraphers, buI also can be used as references in choosin condiIions for similar compounds.
These daIa are available aI our web siIe: hIIp://www.nacalai.co.|p/cosmosil/
AApplicaIion daIa of subsIances in Japanese Pharmacopoeia, 16
Ih
version (222 daIa)
We prepare daIa of drus usin Ihree kinds of Cs columns IhaI are specified in HPLC analysis in ApplicaIion DaIa of
SubsIances in Japanese Pharmacopoeia, 16
Ih
version. The daIa are available aI our web siIe.
hIIp://www.nacalai.co.|p/en/cosmosil/TheJP16.hIm, or Iype Cosmosil Japanese Pharmacopoeia`aI a search siIe.
ACOSMOSlL ApplicaIion DaIa
COSMOSlL ApplicaIion DaIa is now available on our websiIe. The online version includes more Ihan 1,000 applicaIion daIa
usin COSMOSlL columns. The online daIa are searchable by name of sample and column. lf you have any quesIions reardin
Ihe applicaIion daIa or separaIions of compounds noI lisIed here, please feel free Io e-mail us aI info.inIl@nacalai.co.|p.
Oo|umns se|ection by mobi|e phase
lf a mobile phase is deIermined, use Ihe followin charI Io selecI an appropriaIe COSMOSlL column.
Refer Io applicaIion daIa above for choosin a mobile phase of new analysis.
Ad|usImenI of pH is required for dissociaIive compounds.
Generally acidic mobile phase is suiIable for acidic compounds, and neuIral mobile phase is suiIable for basic compounds.
lf you are noI sure abouI Ihe mobile phase, Iry Cs-MS- frsI.
6C18-MS-
6C18-AR-
6C18-PAQ MeIhanol or AceIoniIrile concenIraIion of 10% or less
Acidic (pH8 or less)
No
No
Yes
Yes
lf Ihe mobile phase is unknown, frsI choice is 6C18-MS-.
C
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16
The COSMOSlL 6Cs-MS- is a well-balanced column wiIh beIIer basic performance such as sharper peaks of basic compounds
and chelaIin compounds, and hih IheoreIical plaIe number. lI is Ihe mosI popular HPLC column because we produce very
consisIenI producIs and minimize variaIion from loI Io loI. FurIhermore we provide abundanI applicaIion daIa wiIh Ihe column, so
iI will help you Io choose an analysis condiIion of your sample.
Mobile phase meIhanol20 mmol/l phosphaIe
bufer (pH7) - 20 : 80
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. Procainamide (0.88 ,)
2. N-AceIylprocainamide (0.26 ,)
8. Benzyl Alcohol (6.68 ,)
Procainamide Benzylamine Pyridine
Bufer (pH7) - 20 : 80
TemperaIure 80T
DeIecIion UV 264 nm0.16AUFS
Sample 1. Uracil
2. Benzylamine
8. N-MeIhylbenzylamine
4. N,N-DimeIhylbenzylamine
6. Benzyl Alcohol
Mobile phase aceIoniIrile waIer - 80 : 70
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. Pyridine (0.4 ,)
2. Phenol (1.7 ,)
App|ication Data
Column size 4.6 mm l.D. 160 mm
bufer (pH7) - 80 : 70
TemperaIure 80T
Sample 1. AsIropine SulfaIe
2. Scopolamine Hydrobromide
bufer (pH7) - 80 : 70
TemperaIure 80T
Sample 1. AceIoaminophen
2. AnIipyrine
8. 4-AminoanIipurine
4. PhenaceIin
Ana|ysis of basic compounds
The COSMOSlL 6Cs-MS- column, Iakin advanIae of a new end-cappin IreaImenI, can replace Ihe oriinal COSMOSlL Cs
(ODS) column. A new end-cappin IreaImenI wiIh polar roups for shield efecI`has sinifcanIly improved peak shape for basic
compounds.
Ot-MS-
Cholineric blocker AnIipyreIic analesic
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Column size
l.D. `lenIh (mm)
ProducI number
2.0 ` 60 06614-01
4.6 ` 10 88066-71
4.6 ` 60 88066-61
4.6 `100 88067-61
Column size
l.D. `lenIh (mm)
ProducI number
28 `260 84626-61
60 `260 06886-41
60 `600 84681-71
COSMOSlL 16Cs-MS- Guard Column
Column size
l.D. `lenIh (mm)
ProducI number
28 `60 06886-61
60 `60 84627-41
COSMOSlL 6Cs-MS- Guard Column
Column size
l.D. `lenIh (mm)
ProducI number
1.0 `60 02824-81
1.0 `160 02896-01
2.0 `80 06876-71
2.0 `60 04866-21
2.0 `100 06697-81
2.0 `160 88026-91
2.0 `260 06761-61
8.0 `100 06468-61
8.0 `160 84246-81
8.0 `260 84264-11
4.6 `80 84841-61
4.6 `60 88017-01
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `100 88018-91
4.6 `160 88019-81
4.6 `260 88020-41
6.0 `160 88021-81
6.0 `260 88022-21
10 `60 06789-21
10 `160 84866-91
10 `260 88028-11
20 `160 06091-41
20 `260 88024-01
28 `260 06760-71
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 88014-81
4.6 `10 CarIride 88016-89
10 `20 88016-11
20 `20 06790-81
20 `60 84871-71
28 `60 84847-01
Ordering information
Stabi|ity under a|ka|ine condition
The durabiliIy under exIremely hih pH is reaIly improved in Ihe new COSMOSlL 6Cs-MS- compared wiIh old COSMOSlL
6Cs-MS. AlIhouh iI is possible Io use COSMOSlL under alkaline condiIion for a limiIed Iime, some deIerioraIion may occur
afIer lon exposure. We recommend pH 7.6 and below for lon-Ierm usae. BeIween Ihe measuremenIs, meIhanol : 20 mM/l
Tris bufer (pH 10.0) - 60 : 60 mobile phase was run aI 1.0 ml/min fow raIe, no sinifcanI deIerioraIion could be observed.
For more informaIion on 16Cs-MS-, please see pae 86.
For fow raIe and device of semi-micro columns, or preparaIive columns, please see pae 99.
Va|idation
of Analysis`for Ihe COSMOSlL 6Cs-MS- (4.6 mm l.D. `160 mm and 4.6 mm l.D. `260 mm). For more informaIion, please
refer Io pae 12.
APreparaIive column (ParIicle size : 16 ,m)
COSMOSlL 16Cs-MS- Packed Column
AFasI LC column (ParIicle size : 8 ,m)
Decrease of k`in % Decrease of N in %
T
r
(
%
)

(
0

m
i
n

I
a
k
e
n

a
s

1
0
0
)
Time (hr)
T
r
(
%
)

(
0

m
i
n

I
a
k
e
n

a
s

1
0
0
)
Time (hr)
TemperaIure 80T
DeIecIion UV 264 nm
Sample BaphIhalene
Fiure 6/A, ReIenIion facIor for NaphIhalene Fiure 6/B, TheoreIical plaIe numbers for Pyridine
TemperaIure 80T
DeIecIion UV 264 nm
Sample Pyridine
AAnalyIical column (ParIicle size : 6 ,m)
C
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M
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H
P
L
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C
O
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18
Acidic resistance
The acidic resisIance of COSMOSlL 6Cs-AR- is much improved compared wiIh commercially available monomeric Iype
ocIadecyl sIaIionary phases. lI reIains hih performance even in case of acidic mobile phases commonly used Io separaIe acidic
compounds and pepIides.
Ot-AR-
The COSMOSlL 6Cs-AR- packed column feaIures a polymeric Iype of Cs reversed phase maIerial. The column employs an
ulIra-pure silica el low in meIal impuriIies and has near-perfecI end-cappin. ln addiIion, iI has sIroner acid resisIance Ihan Ihe
COSMOSlL 6Cs-AR. The COMSOSlL 6Cs-AR- column is especially efecIive for Ihe separaIion of chelaIin compounds as
well as boIh acidic and basic compounds.
Fiure. DecomposiIion IesI in 0.1% TrifuoroaceIic Acid soluIion aI 60T.
CapaciIy facIor (k`) - NaphIhalene, Mobile phase: 70% meIhanol
Paraben Salicylic Acid Oranic Acid
Column size 4.6 mm l.D. x 160 mm
Mobile phase meIhanol : 20 mmol/l
phosphoric acid - 20 : 80
TemperaIure 80T
DeIecIion UV 264 nm 0.6AUFS
Sample 1. Gallic acid (0.68 ,)
2. ProIocaIechuic Acid (0.68 ,)
8. GenIisic Acid (0.68 ,)
4. PhIhalic Acid (0.68 ,)
Mobile phase aceIoniIrilewaIer - 60 : 60
TemperaIure 80T
DeIecIion UV 264nm
Sample 1. MeIhyl o-HydroxybenzoaIe (0.26 ,)
2. EIhyl o-HydroxybenzoaIe (0.026 ,)
8. n-Propyl o-HydroxybenzoaIe (0.026 ,)
4. n-BuIyl o-HydroxybenzoaIe (0.026 ,)
Mobile phase aceIoniIrilewaIer - 70 : 80
TemperaIure 80T
DeIecIion UV 264 nm0.2 AUFS
Sample 1. MeIhyl SalicylaIe (2.8 ,)
2. EIhyl SalicylaIe (2.6 ,)
8. Propyl SalicylaIe (2.8 ,)
4. BuIyl SalicylaIe (2.6 ,)
Mobile phase meIhanol : 1 mmol/l EDTA 20mmol/l
phosphoric acid - 70 : 80
TemperaIure 80T
DeIecIion UV 264 nm0.6 AUFS
Sample HonokiIiol (1 ,)
Mobile phase aceIoniIrile : 20mmol phosphoric asid - 6 : 96
TemperaIure 80T
DeIecIion UV 240nm0.2AUFS
Sample Oxine-copper
Mobile phase aceIoniIrile20 mmol/l
phosphaIe bufer (pH7) - 60 : 40
TemperaIure 80T
Sample 1. AceIylaceIone (0.76 ,)
2. 8-Quinolinol (0.70 ,)
8. Benzene (14.66 ,)
AceIylaceIone Oxine-copper HinokiIiol
App|ication Data
Time (hr)
ConvenIional monomeric Cs
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LO-MS App|ication data
ldenIifcaIion of herbal medicine consIiIuenIs by LC-MS
Va|idation
of Analysis`for Ihe COSMOSlL 6Cs-MS- (4.6 mm l.D. `160 mm and 4.6 mm l.D. `260 mm). For more informaIion, please
refer Io pae 12.
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `100 88148-41
4.6 `160 88144-81
4.6 `260 88146-21
6.0 `160 88146-11
6.0 `260 88147-01
10 ` 60 06869-21
10 `160 84860-41
10 `260 88149-81
20 `160 84816-01
20 `260 88160-41
28 `260 84862-91
Column size
l.D. `lenIh (mm)
ProducI number
1.0 ` 60 02966-21
1.0 `160 02961-61
2.0 ` 80 06098-71
2.0 ` 60 84400-81
2.0 `100 84469-11
2.0 `160 87992-61
2.0 `260 06272-71
8.0 `100 06791-71
8.0 `160 88028-61
8.0 `260 88029-61
4.6 ` 80 06877-61
4.6 ` 60 88142-61
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 88141-61
4.6`10 CarIride 88008-89
10 `20 88148-91
20 `20 84468-61
20 `60 84479-81
28 `60 84868-81
Column size
l.D. `lenIh (mm)
ProducI number
28 `260 87978-61
60 `260 88068-71
60 `600 06884-61
Column size
l.D. `lenIh (mm)
ProducI number
28 `60 88080-11
60 `60 88067-81
Column size
l.D. `lenIh (mm)
ProducI number
2.0 ` 60 06478-91
4.6 ` 10 88068-41
4.6 ` 60 88069-81
4.6 `100 88070-91
For more informaIion on 16Cs-AR-, please see pae 86.
COSMOSlL 16Cs-AR- Guard Column
COSMOSlL 6Cs-AR- Guard Column COSMOSlL 6Cs-AR- Packed Column
COSMOSlL 16Cs-AR- Packed Column
COSMOSlL 8Cs-AR- Packed Column
AconiIIine (M.W. 646.76)
HPLC
6Cs-AR-
2.0mml.D. `160mm
UV
MS MS
lmpuriIy (M.W. 686) AconiIIine (M.W. 646)
TareI subsIance
C
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20
The COSMOSlL 6Cs-PAQ is a new member of our Cs column family which already includes Ihe monomeric Iype MS- and Ihe
polymeric Iype AR- . This new column mainIains sIable reIenIion Iime even in 100% aqueous mobile phases. The new
polymeric linkin sIyle ives Ihis column a sIron acidic resisIance so IhaI iI is compaIible wiIh mobile phases of acidic pH IhaI
can permanenIly damae convenIional ocIadecyl sIaIionary phases. This Iype is especially ood for separaIion of hydrophilic
compounds.
Ot-PAQ
Stab|e Performance under 100% aqueous conditions
The fure shows Ihe chane of reIenIion Iime for Ihymine wiIh
100% aqueous mobile phase (20 mmol/l phosphaIe bufer pH7).
The sample was analyzed 4 Iimes. Flow of mobile phase was
Ihen sIopped for 1 hour. The sample was analyzed under Ihe
same condiIion aain afIer 1 hour. The convenIional Cs column
showed chane of reIenIion Iime, buI COSMOSlL 6Cs-PAQ
mainIained sIable reIenIion Iime.
Oranic acid
Mobile phase 20 mmol/l phosphoric acid
TemperaIure 80T
Sample 1. Glycolic Acid (6.8 ,) 4. CiIric Acid (6.8 ,)
2. Malic Acid (6.8 ,) 6. Maleic Acid (0.068 ,)
8. LacIic Acid (18 ,) 6. Fumaric Acid (0.081 ,)
Mobile phase 20 mmol/l phosphoric acid
TemperaIure 80T
Sample 1. TarIaric Acid (4.0 ,)
2. AceIic Acid (18 ,)
8. Succinic Acid (18 ,)
4. Propionic Acid (18 ,)
Mobile phase 20 mmol/l phosphaIe bufer (pH7)
TemperaIure 80T
Sample 1. CyIosine (0.1 ,)
2. Uracil (0.1 ,)
8. Guanine (0.1 ,)
4. Thymine (0.2 ,)
6. Adenine (0.06 ,)
Nucleic-acid base dNTP
Mobile phase 20 mmol/l phosphaIe bufer (pH7)
TemperaIure 80T
Sample 1. dCTP
2. dTTP
8. dGTP
4. dATP
1 mmol/ each
ln|ecIion vol. 0.6 ,l
Ascorbic acid
Mobile phase 20 mmol/l Phosphoric Acid
TemperaIure 80T
Sample Ascorbic Acid (ViIamin C) (0.1 ,)
App|ication data
ConvenIional Cs
mobile phase fow sIopped analysis resumed
I
r
(
%
)

(
0
m
i
n

I
a
k
e
n

a
s

1
0
0
)
120
0 60 100 160
min
100
98
96
94
92
90
C
O
S
M
O
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l
L

H
P
L
C

C
O
L
U
M
N
21
Mobile phase aceIoniIrile : meIhanol : waIer - 8 : 4 : 88
TemperaIure 80T
Pressure 10.6 MPa
Datacou|tesyofD|.B.S|imizu,D|.K.Sa|ata,lnstitutefo|C|emicalPesea|c|,KyotoUnive|sity
2-PhenyleIhyl lycoside
2-PhenyleIhyl-}-melibioside
1
2-PhenyleIhyl--}-enIiobioside
2
2-PhenyleIhyl-}-lacIoside
8
2-PhenyleIhyl--}-cellobioside
4
2-PhenyleIhyl--}-malIoside
6
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `100 06799-91
4.6 `160 02486-71
4.6 `260 02486-81
6.0 `160 84419-61
6.0 `260 06800-41
10 ` 60 06801-81
10 `160 84466-41
10 `260 84876-21
20 `160 84476-11
20 `260 84878-61
28 `260 84466-71
Column size
l.D. `lenIh (mm)
ProducI number
1.0 ` 60 06792-61
1.0 `160 06798-61
2.0 ` 80 06878-61
2.0 ` 60 06794-41
2.0 `100 06470-71
2.0 `160 84449-71
2.0 `260 06796-81
8.0 `100 06796-21
8.0 `160 06797-11
8.0 `260 06798-01
4.6 ` 80 06879-41
4.6 ` 60 84461-21
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 02484-91
10 `20 84467-61
20 `20 06808-11
20 `60 06804-01
28 `60 84466-81
Column size
l.D. `lenIh (mm)
ProducI number
28 `260 06888-21
60 `260 06890-71
60 `600 06891-61
Column size
l.D. `lenIh (mm)
ProducI number
28 `60 06887-81
60 `60 06889-11
For more informaIion on 16Cs-PAQ, please see pae 86.
COSMOSlL 16Cs-PAQ Guard Column
COSMOSlL 6Cs-PAQ Guard Column
COSMOSlL 6Cs-PAQ Packed Column
COSMOSlL 16Cs-PAQ Packed Column
1
2
8
4
6
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
22
3. Reversed Phase Chromatoyraphy - SpeciaIty coIumns
Introduction
Reversed phase HPLC columns have been widely used because of Iheir superior resoluIion, hih IheoreIical plaIe number and
ease of use. Since hydrophobic inIeracIion is Ihe dominanI separaIion mechanism in reversed phase chromaIoraphy,
convenIional sIaIionary phases such as Cs and Cs do noI ofer opIimum selecIiviIy for compounds wiIh similar hydrophobiciIy.
COSMOSlL ofers a broad selecIion of columns wiIh unique sIaIionary phases for separaIion of Ihese difculI analyIes. These
columns ofer improved separaIion of sIrucIurally similar compounds IhaI are difculI Io analyze wiIh a Cs Iype column.
Packin maIerial CholesIer )NAP PYE NPE PBB-R
SIaIionary phase

CholesIeryl roup NaphIyleIhyl proup PyrenyleIhyl roup NiIrophenyleIhyl
roup
PenIabromobezyl
roup
Bonded Iype monomeric Iype
Main inIeracIion
hydrohobic
inIeracIion
molecular shape
selecIiviIy
hydrohobic
inIeracIion
)-)inIeracIion
hydrohobic
inIeracIion
)-)inIeracIion
dispersion force
chare-Iansfer
inIeracIion
hydrohobic
inIeracIion
)-)inIeracIion
dipole-Dipole
inIeracIion
hydrohobic
inIeracIion
dispersion force
End-cappin IreaImenI near-perfecI IreaImenI
Carbon conIenI approx 20 approx 11 approx 18 approx 9 approx 8
FeaIure
Usable under
condiIion Ihe same
as Cs
Hih molecular
sharp selecIiviIy
SIroner )-)
inIeracIion Ihan
Phenyl column
SIronesI )-)
inIeracIion
Dipole-Dipole
inIeracIion
Dispersion force
inIeracIion
NoI enouh
No reIenIion
Usable under Ihe same condiIion as ODS
Hih planariIy selecIiviIy
(pae 28)
C
18
(ODS) CholesIer
)NAP
HlLlC
Phenyl
Suar-D
ReIains hihly polar compounds IhaI would noI
be reIained in C18 columns (pae 88)
For Ihe separaIion of monosaccharides
and oliosaccharides (pae 40)
PYE
NPE PBB-R
NoI enouh
NoI enouh
MosI powerful )-)inIeracIion (pae 28)
SIroner )-)inIeracIions Ihan Phenyl column
(pae 26)
Oo|umn se|ection guide
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
28
Oho|ester
COSMOSlL CholesIer is a reversed phase HPLC column wiIh cholesIeryl roups bonded silica packin maIerial, which provides
equivalenI hydrophobiciIy like IradiIional alkyl roup bonded silica packin maIerials (Cs, Caa). However, CholesIer ofers sIron
molecular shape selecIiviIy for hydrophobic compounds Io yield unique and reproducible separaIion paIIerns followin Ihe same
analyIical condiIions used wiIh oIher ODS columns.
Hydrophobic interaction
Fiure 1 shows Ihe comparison of hydrophobic inIeracIions wiIh
compeIi Ii ve Cs col umns. Chol esIer provi des Ihe same
hydrophobiciIy as alkyl roup bonded Iypes (Cs, Caa). lI is noI
necessary Io chane Ihe analyIical condiIions when replacin Cs
or Caa columns wiIh CholesIer.
Mo|ecu|ar shape se|ectivity
The sIaIionary phase of CholesIer has very riid sIrucIures and
can disIinuish differenI molecular shapes. CholesIer offers
improved separaIion for sIrucIurally similar compounds IhaI are
difficulI Io analyze wiIh alkyl roup bonded maIerials (Cs and
Caa). As in Ihe followin example CholesIer reIains planar
Triphenylene loner Ihan sIereoscopic o-Terphenyl.
6Cs-MS-
+-1.64
CholesIer
+-8.66
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. o-Terphenyl (0.10 ,)
2. Triphenylene (0.01 ,)
o-Terphenyl Triphenylene
Fiure1. Comparison of hydrophobic inIeracIions
Fiure 2. Comparison of molecular sharp selecIiviIy
1.80 1.86 1.40 1.46 1.60 1.66
CholesIer
6C18-MS-
6C18-AR-
A company C18
B company C18
D company C18
C company C18
E company C18
F company C80
zParIicle size : 6,m (excepI E company ; 8,m)
(CH2), 80% meIhanol
0 1 2 8 4
CholesIer
6C18-MS-
6C18-AR-
A company C18
B company C18
D company C18
C company C18
E company C18
F company C80
(T/O),90% meIhano
zParIicle size : 6,m (excepI E company ; 8,m)
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
24
Improvement in separation
COSMOSlL CholesIer provides enhanced selecIiviIy over IradiIional Cs columns and ofers reaIer performance in separaIin
isomers or oIher closely relaIed compounds. COSMOSlL CholesIer is ideal for meIhod developmenI and serves as an excellenI
alIernaIive Io IradiIional Cs columns. The fure below shows analyIical daIa of chloroaceIophenone isomers. These isomers are
difculI Io separaIe wiIh Cs and Caa, buI Ihey are well resolved by COSMOSlL CholesIer.
ENciency of preparative separation
The fure below shows Ihe comparison of efciency of preparaIive separaIion wiIh a Cs column. BoIh columns show ood
separaIion. However, sample loadin capaciIy for preparaIive separaIions can be afecIed by a slihI diference in separaIion
abiliIy. COSMOSlL CholesIer can load 4 Iimes of sample volume compared wiIh Cs columns.
DeIecIion UV 264 nm
Sample 1. Bibenzyl
2. AnIhracene
Mobile phase 100% meIhanol
TemperaIure 80T
Mobile phase: 60% meIhanol, Sample : ChloroaceIophenone zParIicle size: 6,m (excepI E company; 8,m)
A company C18
B company C18
C company C18
D company C18
E company C18
F company C80
1
2
2 1
1
1 2
2
ln|ecIion volume : 10,l
ln|ecIion volume : 40,l
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
26
App|ication Data
CaIechin
Saikosaponin
Mobile phase A : aceIoniIrile : 20 mmol/l phosphaIe bufer (pH2.6) - 10 : 90
BaceIoniIrile : 20 mmol/l phosphaIe bufer (pH2.6) - 80 : 70
Bconc. 0 100% 20 min linear radienI
TemperaIure 80T
DeIecIion UV 280 nm
Flow raIe aceIoniIrile : waIer - 46 : 66
Mobile phase 1.0 ml/min
TemperaIure 80T
DeIecIion ELSD (Gain - 6, AIIen - 8)
Sample 1. Saikosaponin c (1.6 ,)
2. Saikosaponin a (1.6 ,)
8. Saikosaponin ba (1.6 ,)
4. Saikosaponin b (1.6 ,)
6. Saikosaponin d (1.6 ,)
Sample 1. Cafeine (0.12 ,)
2. EpiallocaIechin (EGC) (1.20 ,)
8. CaIechin (C) (0.60 ,)
4. EpicaIechin (EC) (0.60 ,)
6. EpiallocaIechin allaIe (EGCG) (0.24 ,)
6. EpicaIechin allaIe (ECG) (0.80 ,)
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `160 06976-61
4.6 `260 06977-61
10 `160 08011-91
10 `260 06979-81
20 `160 06088-71
20 `260 06982-71
28 `260 06986-41
Column size
l.D. `lenIh (mm)
ProducI number
1.0 `160 06968-71
1.0 `260 06969-61
2.0 ` 80 08666-61
2.0 `160 06971-11
2.0 `260 06972-01
8.0 `160 06978-91
8.0 `260 06974-81
4.6 ` 60 06869-21
4.6 `100 06691-61
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 06976-71
10 `20 06978-41
20 `20 06980-91
20 `60 06981-81
28 `60 06988-61
6Cs-MS- CholesIer
6Cs-MS- CholesIer
COSMOSlL CholesIer Guard Column
COSMOSlL CholesIer Packed Column
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
26
COSMOSlL )NAP is a reversed phase HPLC column wiIh naphIyleIhyl roup bonded silica packin maIerial. The naphIhyleIhyl
roup is composed of Iwo fused aromaIic rins and forms sIron )-) inIeracIions wiIh unsaIuraIed compounds. This column
offers improved separaIion of compounds such as posiIional isomers IhaI are difficulI Io analyze wiIh alkyl roup bonded
maIerials.
1NAP
Oomparison of 1-1 interactions
COSMOSlL )NAP shows sIroner )-) inIeracIions Ihan
phenyl columns.
Fiure. Comparison of )-) inIeracIions
Ana|ytica| data
ToluniIrile
Adrenal corIical hormone
0.4 0.6 1.0 1.2 1.4
CholesIer
C18-MS-
PE-MS
)NAP
NiIrobenzene/Benzene), 60%MeIhanol
1.6 0.8
)NAP
(60% meIhanol)
6Cs-MS-
(40% meIhanol)
6PE-MS
(40% meIhanol)
TemperaIure 80T
DeIecIion UV 264 nm
Sample o-ToluniIrile (2.0 ,)
m-ToluniIrile (2.0 ,)
o-ToluniIrile (1.0 ,)
)NAP
(60% meIhanol)
6Cs-MS-
(60% meIhanol)
6PE-MS
(60% meIhanol)
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. Prednisolone (0.88 ,)
2. HydrocorIisone (0.88 ,)
8. CorIisone (0.88 ,)
O
HO
O
OH
HO
2
OH
O
O
HO
O
8
1
O
OH
HO
HO
O
CH8
CH8
CH8
CN
CN
CN

C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
27
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `160 08086-41
4.6 `260 08086-81
10 `160 08088-11
10 `260 08089-01
20 `160 08092-41
20 `260 08098-81
28 `260 08096-11
Column size
l.D. `lenIh (mm)
ProducI number
1.0 `160 08076-61
1.0 `260 08077-61
2.0 ` 80 08666-41
2.0 ` 60 08667-81
2.0 `160 08078-41
2.0 `260 08079-81
8.0 `160 08080-91
8.0 `260 08081-81
4.6 ` 60 08088-61
4.6 `100 08084-61
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 08082-71
10 `20 08087-21
20 `20 08090-61
20 `60 08091-61
28 `60 08094-21
Berberine
)NAP 6Cs-MS- 6PE-MS
Mobile phase meIhanol : 20 mmol/l phosphaIe bufer (pH2.6) -
6Cs-MS- 40 : 60
6PE-MS 60 : 80
)NAP 80 : 20
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. PalmaIine choride (0.2 ,)
2. Berberine Hydrochoride (0.2 ,)
N+
CHO
O
OCH
OCH
C
-
1
O
O
N+
CHO
OCH
C
-
2
CH
lsofavone
)NAP 6Cs-MS- 6PE-MS
Mobile phase A10% aceIoniIrile / 20mmol/l phosphaIe bufer (pH 2.6)
B60% aceIoniIrile / 20mmol/l phosphaIe bufer (pH 2.6)
BConc. 0 100% 20min linear radienI
TemperaIure 80T
DeIecIion UV 260 nm
Sample 1. Daidzin (0.06 ,)
2. GlyciIin (0.06 ,)
8. GenisIin (0.06 ,)
4. Daidzein (0.06 ,)
6. GlyciIein (0.076 ,)
6. GenisIein (0.026 ,)
COSMOSlL )NAP Guard Column
COSMOSlL )NAP Packed Column
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
28
COSMOSlL PYE column is a reversed phase column wiIh 2-(1-pyrenyl) eIhyl roups bonded silica packin maIerial. This column
uIilizes )-) inIeracIions oriinaIin from Ihe planar pyrene rin sIrucIure Io separaIe sIrucIural isomers.
PYE
Oomparison of 1-1 interactions with 1NAP
COSMOSlL PYE provides much sIroner )-) inIeracIions
Ihan )NAP.
Fiure. Comparison of )-) inIeracIions
0.0 1.0 2.0 2.6
NiIrobenzene/Benzene), 60% meIhanol
8.0 1.6
CholesIer
C18-MS-
PE-MS
)NAP
PYE
0.6
Ana|ytica| data
SIerol
PhIhaloniIrile
PYE
(96% meIhanol)
6Cs-MS-
(98% meIhanol)
)NAP
(90% meIhanol)
TemperaIure 80T
DeIecIion UV 210 nm
SampleSample 1. CholesIerol
2. SIimasIeroll
8. CampesIerol
4. SiIosIerol
HO
R
2:
R -
1: 8:
4:
PYE
(80% meIhanol)
6Cs-MS-
(80% meIhanol)
)NAP
(60% meIhanol)
TemperaIure 80T
DeIecIion UV 264 nm
Sample o-PhIhaloniIrile (PhIhaloniIrile) (0.8 ,)
m-PhIhaloniIrile (lsophIhaloniIrile) (8.0 ,)
o-PhIhaloniIrile (TerephIhaloniIrile) (0.16 ,)
o-,p-
m-
p-
o-
m- p-
m-
o-
CN CN
CN
CN
CN

CN
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
29
Column size
l.D. `lenIh (mm)
ProducI number
1.0 `160 02861-71
2.0 `160 88042-61
2.0 `260 84460-81
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 87908-11
10 `20 88041-71
20 `20 06867-91
20 `60 84476-21
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `160 87887-91
4.6 `260 87989-11
10 `260 87996-11
20 `260 88044-41
Tocopherol
TemperaIure 80T
DeIecIion UV 296 nm
Sample +-Tocopherol (1.0 ,)
}-Tocopherol (1.0 ,)
\-Tocopherol (1.0 ,)
?-Tocopherol (1.0 ,)
O
HO
R1
R2
R8
PYE (90% meIhanol)
6Cs-MS- (96% meIhanol) )NAP (90% meIhanol)
R
1
R
2
M
M
M
H
H
M
H
H
R
8 M M M M
COSMOSlL 6PYE Guard Column
COSMOSlL 6PYE Packed Column
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
80
COSMOSlL NPE column is a reversed phase column wiIh niIrophenyleIhyl roups bonded silica packin maIerial. This column
provides unique reIenIion characIerisIics, slihIly diferenI from Ihe COSMOSlL PYE column, uIilizin boIh dipole-dipole and
)-) inIeracIions.
NPE
Se|ectivity for dipo|e
SelecIiviIy for dipole is evaluaIed based on Ihe separaIion of 1,5-diniIronaphIhalene and 1,8-diniIronaphIhalene.
DiniIronaphIhalenes (peak 1 and 2) were sIronly reIained on PYE and NPE because of )-) inIeracIion compared wiIh
dimeIhylnaphIhalenes. However, Ihere is a slihI diference beIween Ihese Iwo columns. While 1,5-diniIronaphIhalene (peak 2)
was preferenIially reIained on PYE, 1,8-diniIronaphIhalene (peak 1) was reIained loner on NPE. The resulIs wiIh NPE indicaIe
Ihe presence of sIron dipole-dipole inIeracIion. The Iwo niIro roup dipoles in 1,8-diniIronaphIhalene are alined for a much
reaIer dipolar couplin wiIh Ihe bonded niIrophenyl roup in NPE Ihan 1,5-diniIronaphIhalene.
Mobile phase 6NPE meIhanol : waIer - 70 : 80
6PYE meIhanol : waIer - 90 : 10
6Cs-MS- meIhanol : waIer - 80 : 20
TemperaIure 80T
DeIecIion UV 264 nm
1 2 8 4 6
6NPE 6PYE 6Cs-MS-
Sample 1. 1,8-DiniIronaphIhalene (0.21 ,)
2. 1,5-DiniIronaphIhalene (0.11 ,)
8. NaphIhalene (0.26 ,)
4. 1-MeIhylnaphIhalene (0.86 ,)
6. 1,5-DimeIhylnaphIhalene (0.42 ,)
Attention
1. MeIhanol is recommended as a mobile phase for COSMOSlL NPE column. AceIoniIrile is noI recommended because iI has
many )elecIrons and inIerferes )-) inIeracIions beIween a sample and Ihe sIaIionary phase.
2. The sIaIionary phase of COSMOSlL NPE, niIrophenyl roup, has a lare UV absorpIion. When Ihe sIaIionary phase deIaches
from silica el and eluIes, even a slihI quanIiIy can be deIecIed and causes baseline noise. ln such a case, wash Ihe column
wiIh IeIrahydrofuran. DeIachmenI of a small amounI of Ihe sIaIionary phase does noI deIerioraIe a column`s separaIion abiliIy.
8. COSMOSlL NPE column is noI suiIable for radienI analysis.
Column size
l.D. `lenIh (mm)
ProducI number
1.0 `160 06897-01
2.0 `160 84828-61
2.0 `260 84879-91
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 87904-01
10 `20 88046-81
20 `20 06868-81
20 `60 06869-71
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `160 87902-21
4.6 `260 87990-71
10 `260 06469-11
20 `260 88046-21
COSMOSlL 6NPE Guard Column
COSMOSlL 6NPE Packed Column
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
81
App|ication data
Haloen exchane reacIion producIs
COSMOSlL PBB-R sIronly reIains iodine aIom which has a lare dispersion force, Ihan bromine aIom. So iI can separaIe
haloen exchane reacIion producIs IhaI are difculI Io analyze wiIh Cs column.
COSMOSlL PBB is a reversed phase column wiIh penIabromobenzyl roups bonded silica packin maIerial. This column
provides unique selecIiviIy for sIrucIurally similar compounds uIilizin Ihe dispersion force inIeracIion. The dispersion force
inIeracIion of COSMOSlL PBB makes iI useful for separaIion of sIrucIural isomers diferin only by a double bond.
PBB-R
6PBB-R 6Cs-MS-
SurfacIanI aenI
Cs column can noI separaIe TriIon X-100 mixIurem, because (-OCHaCHa-) roup has liIIle hydrophobiciIy. However, COSMOSlL
PBB-R can separaIe Ihem because iI disIinuishes diference in Ihe dispersion force, which depends on iIs molecular weihI.
6PBB-R 6Cs-MS-
Mobile phase 6PBB-R meIhanol : waIer - 100 : 0
TemperaIure 80T
DeIecIion UV 264 nm
Sample TriIon X-100 (60 ,)
Column size
l.D. `lenIh (mm)
ProducI number
1.0 `160 06899-81
2.0 `160 06900-81
2.0 `260 06904-91
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 06704-11
10 `20 06721-81
20 `20 06911-91
20 `60 06722-71
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `160 06697-21
4.6 `260 06698-11
10 `260 06699-01
20 `260 06700-61
Mobile phase 6PBB-R meIhanol : waIer - 100 : 0
TemperaIure 80T
DeIecIion UV 264 nm
Sample Haloen exchane reacIion producIs
Samolecou|tesyofD|.H.Yamamoto,PlKEN
R-l or Br
COSMOSlL 6PBB-R Guard Column
COSMOSlL 6PBB-R Packed Column
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
82
COSMOSlL alkyl Iype columns also include sIaIionary phases wiIh packin maIerials of Caa (Dococyl roup), Cs (OcIadecyl
roup), C+ (BuIyl roup) and TMS (TrimeIhyl roup). The order of reIenIion force by hydrophobiciIy of each packin maIerial is
Caa-Cs`Cs`C+`TMS. The columns havin lower hydrophobiciIy Ihan Cs or Caa are efecIive for separaIion of hih hydrophobic
compounds and compounds wiIh bi difference in hydrophobic. HydrophobiciIy of Caa is abouI Ihe same as Cs. However,
sIereoselecIiviIy of Caa is hiher Ihan Cs, and so Caa may provide beIIer separaIion.
4. Reversed Phase Chromatoyraphy - AIkyI chains coIumns
E#ect of a|ky| chain |ength on reversed phase
The shorIer alkyl chain sIaIionary phase shows shorIer reIenIion Iime for non-polar compounds such as benzene and Ioluene and
loner reIenIion for polar compounds such as aceIophenone and benzoaIe.
6Caa-AR- 6Cs-MS- 6Cs-MS 6C+-MS
TemperaIure 80T
DeIecIion UV 264 nm
6TMS-MS
Packin maIerial Caa-AR- Cs-MS- Cs-MS C+-MS TMS-MS
SIaIionary phase
dococyl roup ocIadecyl roup ocIyl roup buIyl roup IrimeIhyl roup
Bondin Iype polymeric monomeric
End cappin IreaImenI near-perfecI IreaImenI
Carbon conIenI approx. 19 approx. 16 approx. 10 approx. 7 approx. 6
Sample 1. AceIophenone (0.06 ,)
2. MeIhyl BenzoaIe (0.6 ,)
8. Benzene (2.0 ,)
4. Toluene (2.0 ,)
Ozz-AR-Ot-MSO+-MSTMS-MS
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
88
Column size
l.D. `lenIh (mm)
ProducI number
6.0 `160 88167-41
6.0 `260 88168-81
10 `260 88169-21
20 `260 88170-81
Column size
l.D. `lenIh (mm)
ProducI number
4.6 ` 60 88168-81
4.6 `100 88164-71
4.6 `160 88166-61
4.6 `260 88166-61
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 88161-01
10 `20 88162-91
Column size
l.D. `lenIh (mm)
ProducI number
6.0 `160 88167-71
6.0 `260 88168-61
10 `260 88169-61
20 `260 88160-11
Column size
l.D. `lenIh (mm)
ProducI number
4.6 ` 60 88168-11
4.6 `100 88164-01
4.6 `160 88166-91
4.6 `260 88166-81
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 88161-81
10 `20 88162-21
Column size
l.D. `lenIh (mm)
ProducI number
6.0 `160 06860-91
6.0 `260 06861-81
10 `260 04969-91
20 `260 06188-41
Column size
l.D. `lenIh (mm)
ProducI number
4.6 ` 60 06848-41
4.6 `100 06849-81
4.6 `160 04698-61
4.6 `260 04699-41
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 04881-21
10 `20 06664-81
Column size
l.D. `lenIh (mm)
ProducI number
6.0 `160 88177-11
6.0 `260 88178-01
10 `260 88179-91
20 `260 88180-61
Column size
l.D. `lenIh (mm)
ProducI number
4.6 ` 60 88178-61
4.6 `100 88174-41
4.6 `160 88176-81
4.6 `260 88176-21
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 88171-71
10 `20 88172-61
COSMOSlL 6Caa-AR- Guard Column
COSMOSlL 6Caa-AR- Packed Column
COSMOSlL 6Cs-MS Packed Column COSMOSlL 6Cs-MS Guard Column
COSMOSlL 6C+-MS Packed Column COSMOSlL 6C+-MS Guard Column
COSMOSlL 6TMS-MS Packed Column COSMOSlL 6TMS-MS Guard Column
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
84
The COSMOSlL PE-MS columns (phenyleIhyl roup) and Ihe COSMOSlL CN-MS columns (cyanopropyl roup) provide a
secondary separaIion mode ()-) inIeracIion). These columns are recommended when Ihe oIher alkyl chain sIaIionary phases
do noI ofer opIimum selecIiviIy for sIrucIurally similar compounds.
5. Reversed Phase Chromatoyraphy - PhenyI type, Cyano type
Packin maIerial PE-MS CN-MS
Averae parIicle size approx. 6 ,m
SIaIionary phase
phenyleIhyl roup cyanopropyl roup
Bondin Iype monomeric
Main inIeracIion
hydrophobic inIeracIion
)-)inIeracIion
Carbon conIenI approx. 10% approx. 7%
Column size
l.D. `lenIh (mm)
ProducI number
6.0 `160 88187-81
6.0 `260 88188-71
10 `260 88189-61
20 `260 88190-21
Column size
l.D. `lenIh (mm)
ProducI number
4.6 ` 60 88188-21
4.6 `100 88184-11
4.6 `160 88186-01
4.6 `260 88186-91
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 88181-41
10 `20 88182-81
Column size
l.D. `lenIh (mm)
ProducI number
6.0 `160 88287-21
6.0 `260 88288-11
10 `260 88289-01
20 `260 88240-61
Column size
l.D. `lenIh (mm)
ProducI number
4.6 ` 60 88288-61
4.6 `100 88284-61
4.6 `160 88286-41
4.6 `260 88286-81
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 88281-81
10 `20 88282-71
5PE-MS5ON-MS
COSMOSlL 6PE-MS Guard Column
COSMOSlL 6PE-MS Packed Column
COSMOSlL 6CN-MS Packed Column COSMOSlL 6CN-MS Guard Column
86
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
.
Please refer Io pae 17 for 16Cs-MS-, pae 19 for 16Cs-AR- and pae 21 for 16Cs-PAQ.
Packin maIerial 16Cs-MS- 16Cs-AR- 16Cs-PAQ
SIaIionary phase ocIadecyl roup (please refer Io pae 14)
Bondin Iype monomeric polymeric
Carbon conIenI approx. 16% approx. 17% approx. 11%
pH rane 2 10
z
1.6 7.6
z
2 7.6
FeaIure
This phase is recommended
for mosI of applicaIions buI
parIicularly efecIive for basic
oranic compounds.
This phase is recommended for
Ihe separaIions requirin acidic
mobile phase condiIions. lI also
shows superior molecular shape
selecIiviIy Io monomeric Iype
Cs columns.
This phase is desined Io
ofer superior reIenIion of polar
compounds and excellenI
reproducibiliIy in hihly aqueous
mobile phases, even in 100%
aqueous.
6. SiIica Based Preparative CoIumns
COSMOSlL series is available in 10 mm l.D. and 20 mm l.D. for semi-preparaIive applicaIions and 28 mm l.D. and 60 mm l.D.
for preparaIive scales.
For column sizes and packin maIerials noI lisIed below, conIacI eiIher your local disIribuIor or Ihe manufacIurer direcIly.
zOpIimum pH rane of columns based on silica el is beIween 2 and 7.6.
Column COSMOSlL 16Cs-AR-
60 mm l.D. `260 mm
Flow raIe 60 ml/min
TemperaIure room IemperaIure
DeIecIion UV 264 nm
Preparative separation using 50 mm I.D. co|umn
Carbazol is exIracIed from anIhracene oil (coal Iar) and required hih puriIy because iI is ofIen used for analyIical applicaIions.
Followin is Ihe preparaIive separaIion of carbazol usin a 60 mm l.D. COSMOSlL 16Cs-AR-.
Column COSMOSlL 6Cs-AR-
4.6 mm l.D. `160 mm
DeIecIion UV 264 nm
ln|ecIion volume : 0.6 ,l (26 ,) ln|ecIion volume : 8000 ,l
(160,000 ,)
Please refer Io TECHNlCAL NOTE 8, lnner diameIer of column (scale down and scale up) aI pae 99.
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
86
UlIra-pure silica el of more Ihan 99.99% puriIy is used for Ihe COSMOSlL SL- packed column series. This column provides
improved separaIion and reproducibiliIy for compounds wiIh carbonyl or phenol hydroxyl roups, which are ofIen problemaIic Io
separaIe usin convenIional silica el columns.
7. NormaI Phase Chromatoyraphy coIumn
Packin maIerial SL-
FeaIure
Hih puriIy Silica Gel (`99.99%) wiIh specially IreaImenI
SuiIable for preparaIive separaIion
Ana|ysis of acids and amide without ionic additives
Oomparison with medium-pressure co|umns
COSMOSlL SL- ofers sharper peak compared wiIh packin maIerials for medium-pressure liquid chromaIoraphy and open
chromaIoraphy.
Column 6SL- A company carIride (80 ,m silica el) B company carIride (60 ,m silica el)
Column size 20 mm l.D. `260 mm 26 mm l.D. `104 mm 26 mm l.D. `104 mm
Pressure 1.8MPa 0.26MPa 0.16MPa
ChromaIoram

6SL- (PuriIy: 99.99% or more)
Mobile phase eIhanol : hexane - 10 : 90
Flow raIe 1 ml/min
TemperaIure 80T
DeIecIion UV 264 nm, 0.6AUFS
Sample 1. Benzoic Acid (4.0 ,)
2. Salicylic Acid (6.0 ,)
8. Saliculamide (4.0 ,)
6SL
Mobile phase eIhanol : hexane - 6 : 96
Flow raIe 10 ml/min
DeIecIion UV 264 nm
Sample 1. o-Xylene (8.8 m)
2. Cinnamyl Alcohol (1.7 m)
8. o-NiIrobenzyl Alcohol (4.2 m)
SL-
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
87
Mobile phase eIhyl aceIaIe : hexane - 10 : 90
Flow raIe 4.6 mm l.D. : 1 ml/min
20 mm l.D. : 18.9 ml/min
DeIecIion UV 264 nm 1 2
Sca|ing up from ana|ytica| to preparative separation
SIep 1 SIep 2 SIep 8
Mobile phase is opIimized by usin 4.6
mm l.D. `260 mm column
Maximum load capaciIy is opIimized by
usin 4.6 mm l.D. `260 mm column
PreparaIive separaIion by usin 20 mm
l.D. `260 mm column
As mobile phase, hihly soluble solvenI
for sample is suiIable.
Maximum in|ecIion volume 66 ,l (28 m)Maximum in|ecIion volume 1,060 ,l (626
m) (19 Iimes hiher Ihan 4.6 mm l.D.)
1
2
1 2
1 2
Column size
l.D. `lenIh (mm)
ProducI number
6.0 `160 88008-71
6.0 `260 88004-61
10 `260 88006-61
20 `260 88006-41
28 `260 84868-61
Column size
l.D. `lenIh (mm)
ProducI number
4.6 ` 60 87999-81
4.6 `100 88000-01
4.6 `160 88001-91
4.6 `260 88002-81
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 87997-01
10 `20 87998-91
20 `20 06874-91
20 `60 06876-81
28 `60 84869-61
Column size
l.D. `lenIh (mm)
ProducI number
4.6 ` 10 88069-61
4.6 ` 60 88060-21
4.6 `100 88061-11
Column size
l.D. `lenIh (mm)
ProducI number
28 `260 06898-41
60 `260 06896-21
60 `600 06896-11
Column size
l.D. `lenIh (mm)
ProducI number
28 `60 06892-61
60 `60 06894-81
COSMOSlL 6SL- Guard Column
COSMOSlL 6SL- Packed Column
Please refer Io TECHNlCAL NOTE 8, lnner diameIer of column (scale down and scale up) aI pae 99.
COSMOSlL 16SL- Guard Column
Sample 1. 1-NaphIhol
2. 2-NaphIhol
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
88
COSMOSlL 6C
18
-PAQ
COSMOSlL HlLlC
Mobile phase :
aceIoniIrile : 10 mmol/l CH
8
COONH
4
=60 : 40
Mobile phase :
20 mmol/l phosphaIe bufer (pH2.6)
-6 mmol/l sodium 1-ocIanesulfonaIe (lon pair reaenI)
wiIhouI ion pair reaenIs
wiIh ion pair reaenIs
wiIhouI
ion pair
reaenIs
Mobile phase :
20 mmol/l phosphaIe bufer (pH2.6)
COSMOSlL HlLlC is a new column for hydrophilic inIeracIion chromaIoraphy wiIh Triazole bonded silica packin maIerial. The
hydrophilic inIeracIion chromaIoraphy is a variaIion of normal phase chromaIoraphy where a polar sIaIionary phase is used
wiIh a mobile phase which conIains a hih concenIraIion of waIer miscible oranic solvenI and a low concenIraIion of aqueous
eluenI. The main reIenIion mechanism is Ihe parIiIionin of Ihe polar analyIes beIween Ihe polar sIaIionary and Ihe non-polar
mobile phase. As iI is also called aqueous normal phase`, Ihe eluIion order is similar Io IhaI of normal phase and Ihe sample
eluIion is in Ihe order of increasin hydrophiliciIy. WiIhouI usin ion-pair reaenI COSMOSlL HlLlC reIains hihly polar analyIes
IhaI would noI be reIained in reversed phase chromaIoraphy. lI also shows a weak anion-exchane mechanism wiIh Ihe
posiIively chared sIaIionary phase, Ihus acidic compound is sIronly reIained.
Oomparison with Ot
COSMOSlL HlLlC can separaIe lycine and lycyllycine wiIhouI ion-pair reaenI. AlIhouh Cs column can separaIe Ihem wiIh
ion-pair reaenIs, Ihere are some disadvanIaes such as column equilibraIion, preparaIion of mobile phase and column
deIerioraIion.
Packin maIerial HlLlC
SIaIionary phase
Triazol
lnIeracIions hydrophilic inIeracIion, anion exchane
Ob|ecI subsIance hydrophilic compound, acidic compound
FeaIure SuiIable for non-reIainin by Cs
8. HydrophiIic |nteraction Chromatoyraphy
HILIO
Column size 4.6 mm l.D. ` 260 mm
TemperaIure 80T
DeIecIion UV 210 nm
Sample 1. Glycine (6.0 m/ml)
2. Glycyllycine (0.126 m/ml)
ln|ecIion volume 2.0 ,l
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
89
Column COSMOSlL HlLlC
Mobile phase aceIoniIrile : 10 mmol/l CHaCOONH+ - 86 : 16
TemperaIure 80T
DeIecIion ELSD(Gain - 6, AIIen - 8)
Sample 1. Leucine (1.0 m/ml)
2. lsoleucine (1.0 m/ml)
8. Valine (1.0 m/ml)
Ascorbic Acid BCAA(amino acid branched-chain)
Column COSMOSlL HlLlC
Mobile phase aceIoniIrile : 100 mmol/l CHaCOONH+ - 80 : 20
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. Sorbic Acid (0.06 m/ml)
2. lsoascorbic Acid (0.8 m/ml)
8. Ascorbic Acid (0.8 m/ml)
Column size
l.D. `lenIh (mm)
ProducI number
8.0 `160 07871-61
8.0 `260 07872-61
4.6 `160 07066-61
4.6 `260 07067-41
10 `260 07069-21
20 `260 07060-81
28 `260 07876-21
Column size
l.D. `lenIh (mm)
ProducI number
1.0 `160 07869-11
1.0 `260 07870-71
2.0 ` 80 08668-21
2.0 ` 60 07062-91
2.0 `100 08669-11
2.0 `160 07064-71
2.0 `260 07489-91
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 07066-61
10 `20 07068-81
20 `20 07864-91
20 `60 07878-41
28 `60 07874-81
Optimizing ana|ytica| conditions
COMSOSlL HlLlC ChromaIoram lndex, which includes 164 chromaIorams usin COSMOSlL HlLlC, is now available online aI
hIIp://www.nacalai.co.|p/en/cosmosil/. This index is useful for opIimizin analyIical condiIions for hydrophilic inIeracIion
chromaIoraphy.
COSMOSlL HlLlC Guard Column
COSMOSlL HlLlC Packed Column
App|ication data
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
40
Saccharides are noI reIained on sIandard Cs columns because of Ihe low hydrophobiciIy of compounds. COSMOSlL Suar-D
and NHa-MS are specifically desined for separaIion of saccharides. COSMOSlL Cs-PAQ is recommended for hydrophobic
lycosides or saccharide derivaIives.
9. Saccharide AnaIysis
1`||]v;||]
Packin maIerial Suar-D NHa-MS
SIaIionary phase
secondary/IerIiary amine aminopropyl
Bondin Iype polymeric
Ob|ecI subsIance monosaccharide, oliosaccharide
Carbon conIenI approx. 4
FeaIure
Hih durabiliIy
Good quanIiIaIive analysis
DifferenI selecIiviIy from Suar-D
ConvenIionally aminopropyl bonded sIaIionary phases are used for liquid chromaIoraphic analysis of mono- and
oliosaccharides. General shorIcomins of Ihe convenIional aminopropyl bonded phases are Iailin and adsorpIion of cerIain
saccharides and Ihe eneral low durabiliIy (shorI acIive life) of Ihese columns. These problems are addressed and solved by Ihe
novel COSMOSlL Suar-D, resulIin in beIIer (sharper) separaIion and much improved durabiliIy.
Sugar-D
Durabi|ity
DecomposiIion condiIion
SoluIion waIer
Column 4.6 mm l.D. `260 mm
TemperaIure 80T
DeIecIion Rl
Sample MalIose
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
41
Adsorption characteristics
CerIain Iypes of saccharides such as arabinose or alacIose are parIially or Iemporarily adsorbed on convenIional aminopropyl
sIaIionary phases causin Iailin or no eluIion aI all. COSMOSlL Suar-D provides superior separaIion and hih recovery for
Ihese saccharides.
OliomalIoses
App|ication data
Mono- and Oliosaccharides
TemperaIure 80T
DeIecIion Rl
Sample 1. Glucose 10 ,)
2. MalIose 10 ,)
8. MalIoIriose 10 ,)
4. MalIoIeIraose 10 ,)
6. MalIopenIaose 10 ,)
6. MalIohexaose 10 ,)
7. MalIohepIaose 10 ,)
TemperaIure 80T
DeIecIion Rl
Sample 1. Rhamnose 10 ,)
2. FrucIose 10 ,)
8. Glucose 10 ,)
4. Sucrose 10 ,)
6. MalIose 10 ,)
6. Rafnose 10 ,)
TemperaIure 80T
DeIecIion Rl
Sample 1. Glycerol 10 ,)
2. meso-EryIhriIol 10 ,)
8. XyliIol 10 ,)
4. GluciIol 10 ,)
6. MalIiIol 10 ,)
6. lnosiIol 10 ,)
Polyols
COSMOSlL Suar-D Aminopropyl Iype
,
0
600
1000
1600
2000
2600
8000
8600
4000
0 2 8 10
P
e
a
k

a
r
e
a
4 6
Arabinose
GalacIose
MalIiIol
0
600
1000
1600
2000
2600
8000
8600
4000
,
P
e
a
k

a
r
e
a
0 2 4 6 8 10
Arabinose
GalacIose
MalIiIol
C
h
r
o
m
a
I
o
r
a
m
A
n
a
l
i
I
i
c
a
l

c
u
r
v
e
TemperaIure 80T
DeIecIion Rl
Sample 1. Arabinose
2. GalacIose
8. MalIiIol
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
42
COSMOSlL Suar-D
80% aceIoniIrile
COSMOSlL 6NHa-MS
76% aceIoniIrile
A company (aminopropyl Iype)
76% aceIoniIrile
B company (aminopropyl Iype)
76% aceIoniIrile
C company (aminopropyl Iype)
76% aceIoniIrile
C company (Polyamine Iype)
76% aceIoniIrile
D company (Carbamoyl Iype)
76% aceIoniIrile
E company (PenIaeIhylene imine Iype)
76% aceIoniIrile
F company (amino Iype)
76% aceIoniIrile
Oomparison
The separaIion and Ihe adsorpIion of monosaccharides were compared usin COSMOSlL Suar-D and oIher companies`
columns. SeparaIion of aldoses, conIainin aldehyde roup per molecule, is usually problemaIic wiIh undesirable adsorpIion.
COSMOSlL Suar-D provides excellenI separaIions for Ihese saccharides.
Mobile phase aceIoniIrile : waIer
TemperaIure 80T
DeIecIion Rl
Sample 1. Rhamnose (10 ,)
2. Xylose (10 ,)
8. FrucIose (10 ,)
4. Mannose (10 ,)
6. Glucose (10 ,)
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
48
Retention
The convenIional aminopropyl column is slihIly more reIenIive Ihan Suar-D. The reIenIion Iime of Suar-D can be ad|usIed by
increasin Ihe concenIraIion of aceIoniIrile in Ihe mobile phase by 6%-10%.
TemperaIure 80T
COSMOSlL Suar-D
80% aceIoniIrile
aminopropyl Iype
76% aceIoniIrile
DeIecIion Rl
Sample 1. Glucose
2. MalIose
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `160 06896-71
4.6 `260 06897-61
10 `260 06692-71
20 `260 06698-61
Column size
l.D. `lenIh (mm)
ProducI number
2.0 `260 06689-81
8.0 `160 06690-91
8.0 `260 06691-81
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 06894-81
10 `20 06696-81
20 `60 06694-61
The COSMOSlL 6NHa-MS is a column wiIh aminopropyl roup bonded silica packin maIerial. lI is recommended for separaIion
of monosaccharides and oliosaccharides. While Suar-D is our frsI choice for separaIion of saccharide, 6NHa-MS can ive
beIIer separaIion Ihan Suar-D in some cases dependin on samples.
NHz-MS
TemperaIure 80T
DeIecIion Rl
6NHa-MS
Mobile phase; aceIoniIorile : waIer - 76 : 26
Suar-D
Mobile phase; aceIoniIorile : waIer - 80 : 20
Sample 1. Sucrose 10 ,)
2. MalIose 10 ,)
8. LacIose 10 ,)
4. Trehalose 10 ,)
Column size
l.D. `lenIh (mm)
ProducI number
10 `260 88249-71
20 `260 88260-81
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `160 88246-11
4.6 `260 88246-01
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 88241-61
10 `20 88242-41
20 `60 06098-91
COSMOSlL Suar-D Guard Column
COSMOSlL Suar-D Packed Column
COSMOSlL 6NHa-MS Guard Column
COSMOSlL 6NHa-MS Packed Column
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
44
10. Protein Separation Wide Pore CoIumns
Oomparison of separation
ProIein-R shows sharper peaks for proIeins Ihan convenIional Cs wide pore columns.
Packin maIerial ProIein-R
Averae parIicle size 6,m
Averae pore size approx. 800 7
SIaIionary phase ocIadecyl roup
Main lnIeracIions hydrophobic inIeracIion
FeaIure
Hih recovery raIe
Acid-resisIanI
Protein-R
COSMOSlL ProIein-R is a reversed phased HPLC column desined specifcally for pepIide and proIein separaIions. This column
provides improved separaIion, hih recovery raIe and acid-resisIanI, which are ofIen problemaIic Io separaIe usin convenIional
Cs-8007(wide pore) and C+-8007(wide pore).
ProIein-R Cs-800 7
Mobile phase A : 0.06%TFA - 20% aceIoniIrile
B : 0.06%TFA - 60% aceIoniIrile
B conc. 0 100% 20 min linear radienI
TemperaIure 80T
DeIecIion UV 220 nm
Sample 1. Ribonuclease A (1.0 ,)
2. CyIochrome C (1.0 ,)
8. Lysozyme (1.0 ,)
4. BSA (1.0 ,)
6. Myolobin (1.0 ,)
6. Ovalbumin (1.0 ,)
Recovery rate
The fure below shows recovery raIes for proIeins usin diferenI columns. ProIein-R shows a hiher recovery raIe Ihan C+-800
and a much hiher recovery raIe Ihan Cs-800.
0% 20% 40% 60% 80% 100%
ProIein-R
Recovery raIe
Myolobin (MW:17000)
BSA (MW:66000)
CyIochrome C (MW:12400)
Ribonuclease A (MW:18700)
COSMOSlL
6C
18
-AR-800
C
18
-8007
A company
COSMOSlL
6C
4
-AR-800
C
4
-8007
B company
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
46
Durabi|ity
The fure below shows durabiliIy aainsI acidic mobile phase of various columns. ProIein-R shows a hiher acid durabiliIy Ihan
C+-800.
DeradaIion IesI wiIh 0.1%-TrifuoroaceIic Acid aI 60T
(k`) : NapIhalene in Ihe mobile phase (meIhanol : waIer - 60 : 60)
App|ication of peptide separation
6Cs-MS- (pore size 120 ) shows beIIer separaIion of low molecular weihI proIeins, buI ProIein-R shows beIIer separaIion of
hih-molecular weihI proIeins.
Column size
l.D. `lenIh (mm)
ProducI number
10 `160 06629-91
10 `260 06680-61
20 `160 06681-41
20 `260 06682-81
Column size
l.D. `lenIh (mm)
ProducI number
2.0 `160 06614-71
4.6 ` 60 06626-81
4.6 `160 06626-21
4.6 `260 06627-11
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 06618-81
10 `20 06628-01
COSMOSlL ProIein-R Guard Column
COSMOSlL ProIein-R Packed Column
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
46
Ot-AR-300Ot-AR-300O+-AR-300Ph-AR-300
COSMOSlL ofers a varieIy of sIaIionary phases wiIh wide-pore silica el maIerial for separaIions of polypepIides and proIeins.
Packin maIerial 6Cs-AR-800 6Cs-AR-800 6C+-AR-800 6Ph-AR-800
SIaIionary phase
ocIadecyl roup ocIyl roup buIyl roup phenyl roup
Main lnIeracIion hydrophobic inIeracIion
hydrophobic inIeracIion
)-) inIeracIion
Carbon conIenI approx. 12 approx. 7 approx. 6 approx. 7
Oomparison of separation
6Cs-AR-800 6Cs-AR-800 6C+-AR-800 6Ph-AR-800
Mobile phase A : 0.06%TFA - 20% aceIoniIrile
B : 0.06%TFA - 60% aceIoniIrile
B conc. 0 100% 20 min linear radienI
Flow raIe 1 ml/min
TemperaIure 80T
DeIecIion UV 220 nm
Sample Hemolobine, Bovine (10 ,)
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
47
Column size
l.D. `lenIh (mm)
ProducI number
10 `160 84249-91
10 `260 88047-11
20 `160 84477-01
20 `260 88048-01
Column size
l.D. `lenIh (mm)
ProducI number
4.6 ` 60 87966-81
4.6 `160 87968-11
4.6 `260 87969-01
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 87966-41
10 `20 06862-41
Column size
l.D. `lenIh (mm)
ProducI number
10 `160 84846-21
10 `260 84247-11
20 `160 06861-61
20 `260 84864-71
Column size
l.D. `lenIh (mm)
ProducI number
4.6 ` 60 87961-81
4.6 `160 87968-61
4.6 `260 87964-61
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 87960-91
10 `20 84464-61
Column size
l.D. `lenIh (mm)
ProducI number
10 `160 87917-41
10 `260 87918-81
20 `160 87919-21
20 `260 87920-81
Column size
l.D. `lenIh (mm)
ProducI number
4.6 ` 60 87911-01
4.6 `160 87918-81
4.6 `260 87914-71
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 87910-11
10 `20 87966-11
Column size
l.D. `lenIh (mm)
ProducI number
10 `160 06866-11
10 `260 84267-61
20 `160 06866-01
20 `260 84468-21
Column size
l.D. `lenIh (mm)
ProducI number
4.6 ` 60 87961-61
4.6 `160 87968-81
4.6 `260 87964-21
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 87960-61
10 `20 84268-41
COSMOSlL 6Cs-AR-800 Guard Column
COSMOSlL 6Cs-AR-800 Packed Column
COSMOSlL 6Cs-AR-800 Guard Column COSMOSlL 6Cs-AR-800 Packed Column
COSMOSlL 6C+-AR-800 Guard Column COSMOSlL 6C+-AR-800 Packed Column
COSMOSlL 6Ph-AR-800 Guard Column COSMOSlL 6Ph-AR-800 Packed Column
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
48
App|ication data
Oa|ibration curve of proteins
COSMOSlL Diol-120- and Diol-800- el flIraIion columns are ideal for Ihe size-based separaIion of proIeins and oIher waIer
soluble polymers. The separaIion MW rane is 6,000 700,000 dalIons for proIeins and 800 800,000 dalIons for waIer
soluble polymers when Diol-120- and Diol-800- are used in series.
Column COSMOSlL 6Diol-7.6 mm l.D. `600 mm
Mobile phase 20 mmol/l phosphaIe bufer (pH 7.0) + 100 mmol/l NaaSO+
TemperaIure 80T
Sample M. W. Sample M. W.
Thyrolobulin 660,000 AlbuminOvalbumin) 46,000
CaIalase 260,000 Peroxidase 40,000
Glucose Oxidase 160,000 Carbonic Anhydrase 80,000
Uricase 128,000 +-ChymoIrypsinoen A 26,700
Choline Oxidase 96,000 +-ChymoIrypsin 26,200
Transferrin 86,000 Trypsinoen 24,000
Conalbumin 77,600 Trypsinbovine) 28,800
MalaIe Dehydroenase 70,000 Myolobin 17,000
+-Glucosidase 68,600 Lysozyme 14,800
AlbuminBSA) 66,000 Ribonuclease A 18,700
+-Amylase 62,600 CyIochrome C 12,400
FeIuin 48,000 AproIinin 6,600
Gly-Gly 182
Column COSMOSlL 6Diol
Mobile phase 20 mmol/l phosphaIe bufer (pH 7.0)
+100 mmol/l NaaSO+
DeIecIion UV 220 nm
Sample 1. Thyrolobulin
2. Glucose Oxidase
8. Conalbumin
4. Peroxidase
6. Myolobin
6. AproIinin
Ge| f|tration chromatography - Dio|-120-Dio|-300-
6Diol-800-
7.6 mm l.D. `600 mm
6Diol-120-
7.6 mm l.D. `600 mm
Packin maIerial 6Diol-120- 6Diol-800-
Averae pore size approx. 120 approx. 800
Specifc surface area diol roup
Ob|ecI subsIance proIein, waIer soluble polymer
Flow raIe 0.6-1.0ml/min)
SelecIion of pore size
ProIein 6,000-100,000 Da 10,000-700,000 Da
WaIer soluble polymer 800-80,000 Da 600-800,000 Da
EluIion volume (ml)
L
o

M
W
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
49
WaIer soluble polysIyrene
Column COSMOSlL 6Diol-800-
7.6 mm l.D. `800 mm
Mobile phase 20 mmol/l phosphaIe buferpH 6.7)
TemperaIure 80T
DeIecIion UV 264 nm
Sample Sodium PolysIyrene SulfonaIe
1. MW780,000
2. MW100,000
8. MW86,000
4. MW 8,000
7.6 mm l.D. `800 mm
TemperaIure 80T
DeIecIion Rl
Sample PolyeIhylene Glycol
1. PEG20,000
2. PEG6,000
8. PEG4,000
4. PEG1,000
6. PEG800
PolyeIhylene lycol
Pullulan
7.6 mm l.D. `800 mm
Mobile phase waIer
TemperaIure 80T
DeIecIion UV 264 nm
Sample Pullulan
1. P-800
2. P-100
8. P-60
4. P-20
6. P-6
Column size
l.D. `lenIh (mm)
ProducI number
7.6 `800 88060-61
7.6 `600 88061-41
Column size
l.D. `lenIh (mm)
ProducI number
7.6 `60 88049-91
Column size
l.D. `lenIh (mm)
ProducI number
7.6 `800 88068-21
7.6 `600 88064-11
Column size
l.D. `lenIh (mm)
ProducI number
7.6 `60 88062-81
COSMOSlL 6Diol-120- Guard Column
COSMOSlL 6Diol-120- Packed Column
COSMOSlL 6Diol-800- Packed Column COSMOSlL 6Diol-800- Guard Column
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
60
The packin maIerials for COSMOGEL ion-exchane lass columns are based on hydrophilic polymeIhacrylaIe 10 ,m parIicles
wiIh a 1000 pore size. COSMOGEL packed columns are available wiIh DEAE, a weak anion exchaner; QA, a sIron anion
exchaner ; CM, a weak caIion exchaner; and SP, a sIron caIion exchaner. The availabiliIy of four diferenI ion exchaners
provides chromaIoraphers wiIh Ihe fexibiliIy of column selecIion based on chare diferences of samples.
Ion-exchange Ohromatography - DEAEQAOMSP
Packin maIerial DEAE QA CM SP
Type
DieIhylaminoeIhyl Iype
weak anion exchane
QuaIernary ammonium Iype
sIron anion exchane
CarboxymeIhyl Iype
weak caIion exchane
Sulfopropyl Iype
sIron caIion exchane
Gel IoIally porous spherical hydrophilic polymer
FuncIional roup N
+
H(CaHs)a N
+
(CHa)a COO
SOa
CounIer ion Cl
Cl
Na
+
Na
+
CapaciIymeq/) 0.6 0.4 0.8 0.4
60% lonizaIion pH 10.8 11.0 6.7 2.6
pH rane <11 whole area `4 whole area
Flow raIe (appropriaIe) 7.6 mm l.D. / 8.0 mm l.D. ; 0.6 1.0 ml/min20 mm l.D. ; 4.0 6.0 ml/min
Flow raIe (maximum) 7.6 mm l.D. / 8.0 mm l.D. ; 1.6 ml/min20 mm l.D. ; 8.0 ml/min
Pressure (maximum) 1.6 MPa
TemperaIure 10 60T
Se|ection of the mobi|e phase
Generally, anion-exchane columns are operaIed wiIh Ihe mobile phase pH aI leasI one poinI hiher Ihan Ihe isoelecIric poinI (pl)
of samples, while caIion-exchane columns are operaIed wiIh Ihe mobile phase pH aI leasI one poinI lower Ihan Ihe pl. The
eluIion force of bivalenI ions such as Bis-Iris HCl is sIroner Ihan univalenI ions such as Tris HCl.
Anion exchane (DEAE, QA) pH CaIion exchane (CM, SP)
4.0 Formic acid bufer
Piperazine bufer 6.0 AceIic acid bufer
Bis-Tris bufer 6.6 Phosphoric acid bufer
Tris bufer 8.0 HEPES bufer
MonoeIhanolamine bufer 9.6
The iniIial mobile Phase (A) is 20-60 mmol/l of one of Ihe above menIioned bufer soluIions and Ihe fnal
mobile phase (B) is Ihe mobile phase (A) wiIh an addiIion of 20-600 mmol/l of salI.
Table. Bufer Iype and pH
C
O
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M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
61
App|ication data
SeparaIion of proIeins on anion exchane columns
The hiher Ihe neaIive chare, Ihe loner Ihe sample is reIained on an anion exchane column. As shown below, wiIh a weak
alkaline mobile phase, Ihe lower Ihe isoelecIric poinI, Ihe loner Ihe sample is reIained.
Se|ection of organic so|vents
WaIer miscible solvenIs can be used when Ihe eluIion is noI sIron enouh. COSMOGEL columns can be used wiIh up Io 20%
of waIer miscible solvenIs such as aceIoniIrile and 2-propanol.
Se|ection of sa|ts
Hih concenIraIion of salIs, enerally NaCl, is used in eluIion bufers. When sIroner eluIion bufer is needed, CaCla or MCla
can be used for DEAE and QA columns, and NaaSO+ can be used for CM and SP columns.
Column COSMOGEL DEAE7.6 mm l.D. `76 mm
Mobile phase A: 20 mmol/l Tris-HCl bufer (pH 8.2)
B: 600 mmol/l NaCl in bufer A
B 0100 /80min linear radienI
TemperaIure 80T
Sample 1. Myolobin (20 ,)
2. Transferrin (60 ,)
8. Trypsin lnhibiIor (70 ,)
SeparaIion of proIeins on caIion exchane columns
The hiher Ihe posiIive chare, Ihe loner Ihe sample is reIained on caIion-exchane column. As shown below, wiIh a weak
acidic mobile phase, Ihe hiher Ihe isoelecIric poinI, Ihe loner Ihe sample is reIained.
Column COSMOGEL CM7.6 mm l.D. `76 mm
Mobile phase A: 20 mmol/l phosphaIe bufer (pH 6.6)
B: 600 mmol/l NaCl in bufer A
B 0 100 /80 min linear radienI
TemperaIure 80T
Sample 1. Myolobin (60 ,)
2. Ribonuclease A (60 ,)
8. CyIochrome C (60 ,)
4. Lysozyme (60 ,)
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
62
Samp|e |oading capacity and reso|ution
Up Io 1 m of sample can be well separaIed on an 8 mm l.D. column.
lf Ihe resoluIion is hih enouh, 10 m of sample can be separaIed.
R
e
s
o
l
u
I
i
o
n

f
a
c
I
o
r

(
R
s
)
RTransferrin / Trypsin lnhibiIor)
RMyolobin / Transferrin)
Sample size (m)
CondiIion
Column COSMOGEL DEAE GLASS Packed Column, 8.0 mm l.D. `76 mm
Mobile phase A: 20 mmol phosphaIe bufer (pH 8.2)
B: 600 mmol NaCl in bufer A
B 0 100/80 min liner radienI
TemperaIure 80T
DeIecIion UV 280 nm
Sample 1. Myolobin
2. Transferin
8. Trypsin lnhibiIor,Soybean
ToIal in|ecIion volume: 0.2 m ToIal in|ecIion volume: 12.6 m
ProducI name
Column size
l.D. `lenIh (mm)
ProducI number
COSMOGEL DEAE Glass Packed Column
8.0 `76
87846-81
COSMOGEL QA Glass Packed Column 87846-71
COSMOGEL CM Glass Packed Column 87844-91
COSMOGEL SP Glass Packed Column 87847-61
ProducI name
Column size
l.D. `lenIh (mm)
ProducI number
COSMOGEL DEAE SIainless Packed Column
7.6 `76
48871-91
COSMOGEL QA SIainless Packed Column 48878-71
COSMOGEL CM SIainless Packed Column 48876-61
COSMOGEL SP SIainless Packed Column 48877-81
COSMOGEL Glass Packed Column
COSMOGEL SIainless Packed Column
C
O
S
M
O
S
l
L

H
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L
C

C
O
L
U
M
N
68
COSMOSlL 6HlC is desined for one sIep desalIin and separaIion of proIeins. Hydrophobic inIeracIion chromaIoraphy (HlC) is
an effecIive meIhod for purificaIion and separaIion of proIeins (especially enzymes) based on differences in Iheir surface
hydrophobiciIy. Since Ihis meIhod does noI use oranic solvenIs like reversed phase chromaIoraphy, Ihere is only a liIIle loss in
enzyme acIiviIy and Ihe IerIiary sIrucIure of proIeins.
App|ication data
SeparaIion of proIein sIandards
A bufer wiIh hih salI concenIraIion, usually 1-2 mol/l of (NH+)aSO+, is used as an iniIial mobile phase for adsorpIion of
samples Io a weakly hydrophobic sIaIionary phase. The eluIion is done wiIh a decreasin salI radienI.
Mobile phase A: 20 mmol/l phosphaIe bufer + 100 mmol/l NaaSO+ + 1.6 mol/l (NH+)aSO+ (pH6.0)
B: 20 mmol/l phosphaIe bufer + 100 mmol/l NaaSO+ (pH6.7)
B 0 100% /10 min linear radienI
TemperaIure 80T
DeIecIion UV 220 nm
Sample 1. Myolobin (0.6 m/ml)
2. }-LacIolobulin (1.0 m/ml)
8. Hemolobin (2.6 m/ml)
4. BSA (1.0 m/ml)
ln|ecIion vol. mixIure 2.0,l
Packin maIerial HlC
Hydrophobic Interaction Ohromatography - HIO
SeparaIion of crude beIa-Glucosidase
Mobile phase A: 20 mmol/l phosphaIe bufer + 100 mmol/l NaaSO+ + 2.0 mol/l (NH+)aSO+ (pH6.0)
B: 20 mmol/l phosphaIe bufer + 100 mmol/l NaaSO+ (pH6.7)
B 0 100% /10 min linear radienI
TemperaIure 80T
DeIecIion UV 220 nm
ConcenIraIion 10.0 m/ml
ln|ecIion vol. 1.6,l
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `60 04268-21
COSMOSlL 6HlC Packed Column
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
64
SeparaIion of fullerenes, especially preparaIive-scale separaIion, on convenIional HPLC columns is always problemaIic due Io
Ihe low solubiliIy and small recovery of fullerenes. COSMOSlL offers a varieIy of columns desined for preparaIive-scale
separaIion of fullerenes includin hiher fullerenes, meIallofullerenes and fullerene derivaIives.
11. SpeciaI CoIumn for FuIIerenes
Packin maIerial Buckyprep Buckyprep-M PBB PYE NPE
SIaIionary phase
pyrenylpropyl phenoIhiazinyl roup penIabromobenzyl roup pyrenyleIhyl roup niIrophenyleIhyl roup
Bondin Iype monomeric
End cappin IreaImenI near-perfecI none near-perfecI near-perfecI near-perfecI
Carbon conIenI approx. 17 approx. 18% approx. 8% approx. 18% approx. 9%
FeaIure
SIandard column
for fullerenes
separaIion.
Desined
Io separaIe
meIallofullerenes.
Desined for
preparaIive
separaIion of Cea,
C:a.
SeparaIion of
fullerene and
sIrucIural isomers.
SeparaIion
of fullerene
derivaIives
Oomparison of retention in to|uene
Column 4.6 mm l.D `260 mm
Mobile phase Ioluene
TemperaIure 80T
DeIecIion UV 812 nm
Sample 1. Cea
2. C:a
6Cs-MS- Buckyprep Buckyprep-M PYE NPE PBB
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
66
Toluene is Ihe mosI commonly used solvenI for fullerene separaIion.
Suggested so|vents
Chlorobenzene SIroner eluenI Ihan Ioluene. Recommended for hiher fullerenes.
o-Dichlorobenzene
SIroner eluenI Ihan chlorobenzene.
1,2,4-Trichlorobenzene
SIronesI eluenI.
lI can be used as a washin solvenI for hiher fullerenes. To wash a
column, in|ecI 8 ml of 1,2,4-Irichlorobenzene Io a 4.6 mm l.D. `260
mm column and 60 ml Io a 20 mm l.D. `260 mm column afIer every
operaIion.
n-Hexane Weak eluenI. Recommended for weakly reIained fullerenes.
AceIoniIrile Weak eluenI. Recommended for weakly reIained fullerenes.
NoIe : Use Ihem afIer flIraIion or disIillaIion, if Ihey are noI for HPLC.
Column 4.6 mm l.D `260 mm
Mobile phase 6Cs-MS- : Ioluene : aceIoniIrile - 66 : 46
Buckyprep : Ioluene
TemperaIure 80T
DeIecIion UV 286 nm
Sample Fullerene Ioluene exIracI2.6 m/ml)
6Cs-MS-
4.6 ,l 70 ,l
2,600,l
Buckyprep
4.6 ,l 2,600 ,l
So|ubi|ity and boi|ing point of each so|vent for Osa
Comparison wiIh Cs
SolvenI m/ml b.p. (T)
MeIhanol 0.001 64.6
AceIoniIrile 0.018 81.8
n-Hexane 0.046 68.7
Toluene 8.2 111
Chlorobenzene
7.0 182
Carbon disulfde 12 46.8
o-Dichlorobenzene
27 180
1,2,4-Trichlorobenzene 21.8 218
: R.S.Ruof, eI al., J.Phy.Chem.,97,8879 (1998)
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
66
COSMOSlL Buckyprep is a pyrenylpropyl roup bonded silica based column specifcally desined for fullerene separaIion. The
unparalleled separaIion capabiliIies have enabled COSMOSlL Buckyprep Io become Ihe world benchmark of HPLC column for
fullerene separaIion. COSMOSlL Buckyprep reIains fullerenes very sIronly wiIh a mobile phase of 100% Ioluene and exceeds
Ihe in|ecIion volume of a sIandard Cs column by a facIor of 86. Therefore, preparaIive-scale separaIion can be obIained wiIh a
260 mm `4.6 mm l.D. analyIical column.
Buckyprep
TemperaIure 80T
DeIecIion UV 812 nm
Sample Cea, CeaO
Mobile phase: chlorobenzene Mobile phase: Ioluene
Hiher fullerene
TemperaIure 80T
DeIecIion UV 812 nm
Sample Cea, C:a, C:e, Cs+
SeparaIion of hiher fullerenes on Buckyprep
TemperaIure 80T
DeIecIion UV 286 nm
DeIivaIized fullerene
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `260 87977-61
10 `260 87981-91
20 `260 87982-81
28 `260 84846-11
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 87988-71
10 `20 87984-61
20 `60 84874-41
28 `60 06871-21
COSMOSlL Buckyprep Guard Column
COSMOSlL Buckyprep Packed Column
App|ication data
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
67
COSMOSlL Buckyprep-M is a phenoIhiazinyl roup bonded silica based column specifically desined for meIallofullerene
separaIion. MeIallofullerenes are reIained more sIronly Ihan oIher fullerenes on Ihis column. COSMOSlL Buckyprep-M is also
efecIive for Ihe separaIion of hiher fullerenes and fullerene derivaIives.
Buckyprep-M
Buckyprep-M Buckyprep
Column size 4.6 mm l.D `160 mm
TemperaIure 80T
DeIecIion UV 812 nm Samolecou|tesyofD|.H.S|ino|a|a,Deoa|tmentofC|emist|y,NagoyaUnive|sity.
Buckyprep Buckyprep-M
Buckyprep Buckyprep-M
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `260 04188-71
10 `260 04141-11
20 `260 04142-01
28 `260 06878-01
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 04189-61
10 `20 04140-21
20 `60 84474-81
28 `60 06872-11
COSMOSlL Buckyprep-M Guard Column
COSMOSlL Buckyprep-M Packed Column
App|ication data
C
O
S
M
O
S
l
L

H
P
L
C

C
O
L
U
M
N
68
App|ication data
SeparaIion of hiher fullerenes
COSMOSlL PBB is a penIabromobenzyl roup bonded silica based column useful for preparaIive-scale separaIion of fullerenes.
lI can be used wiIh o-Dichlorobenzene, which has reaIer solubiliIy for fullerenes Ihan Ioluene. The loadin capaciIy of
COSMOSlL PBB for Cea and C:a can be Ihree Iimes reaIer Ihan COSMOSlL Buckyprep.
PBB
Preparative separation of fu||erenes
The loadin capaciIy of COSMOSlL PBB for Cea and C:a can be Ihree Iimes reaIer Ihan COSMOSlL Buckyprep.
PBB Buckyprep
Column Size 20 mm l.D. `260 mm
Flow raIe 18 ml/min
DeIecIion UV 286 nm
Sample Crude fullerenes8.6 m/ml)
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `260 87980-01
10 `260 87986-61
20 `260 87986-41
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 87987-81
10 `20 87988-21
20 `60 84876-81
Mobile phase o-Dichlorobenzene
DeIecIion UV 810 nm
Column size 4.6 mm l.D. `260 nm
TemperaIure 80T
Sample Cea,C:a,C:e,Cs+
Mobile phase 1,2,4-Trichlorobenzene
DeIecIion UV 810 nm
Mobile phase Carbon Disulfde
DeIecIion UV 880 nm
COSMOSlL 6PBB Guard Column
COSMOSlL 6PBB Packed Column
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PYENPE
Fu||erene Ohromatogram Index
Fullerene ChromaIoram lndex includes more Ihan 100 chromaIorams. lf you are inIeresIed in Ihis index, please feel free Io
e-mail us aI info.inIl@nacalai.co.|p.
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `260 87989-11
10 `260 87996-11
20 `260 88044-41
28 `260 84800-91
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 87908-11
10 `20 88041-71
20 `60 84476-21
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `160 87902-21
4.6 `260 87990-71
10 `260 06469-11
20 `260 88046-21
Column size
l.D. `lenIh (mm)
ProducI number
4.6 `10 87904-01
10 `20 88046-81
20 `60 06869-71
COSMOSlL 6PYE Guard Column
COSMOSlL 6PYE Packed Column
COSMOSlL 6NPE Packed Column COSMOSlL 6NPE Guard Column
LIOOIO CMHOMA1OGHAFMY
HELA1EO FHOOOC1E
J. FaCklnQ MatC|lalS !C| CClumn Ch|CmatCQ|aphy
E. Llquld Ch|CmatCQ|aphy HClatCd F|CduCtS
U. F|Clt|atlCn 1CClS !C| Llquld Ch|CmatCQ|aphy
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1. Packiny MateriaIs for CoIumn Chromatoyraphy
Introduction
Open column chromaIoraphy is an excellenI and easy Iechnique for lare-scale preparaIion and purificaIion aI low cosI.
COSMOSlL ofers boIh normal and reversed phase packin maIerials based on IoIally porous spherical silica, which provides
hiher separaIion, less pressure and hiher reproducibiliIy Ihan irreular silica.
Packing materia|s for reversed phase preparative Iiquid chromatography
Cs-OPN Cs-PREP
Averae parIicle size 76, 140 ,m 40, 76, 140 ,m
Averae pore size approx. 120 7
SIaIionary phase ocIadecyl roup
Carbon conIenI approx. 19
End cappin IreaImenI IreaIed
Materia| characteristic
+++ : very suiIable,++ ; suiIable,+, applicable,- : noI applicable
Open column
chromaIoraphy
concenIraIion of
oranic solvenI
76Cs-OPN
140Cs-OPN
40Cs-PREP
76Cs-PREP
140Cs-PREP
70% or less +++ - -
70% or more ++ + +++
Middle pressure column chromaIoraphy + +++ ++
Usefu| range of Ot-OPN and Ot-PREP
Fiure. Packin maIerial in waIer
LefI Cs-OPN provides ood resoluIion
RihICs-PREP foaI up
ConcenIraIion of solvenIs SuiIable packin maIerials
Open column chromaIoraphy
Flash column chromaIoraphy
Hih (70% or more)
Midium pressure
column chromaIoraphy
Low (70% or less)
76C18-PREP
140C18-PREP
76C18-OPN
140C18-OPN
40C18-PREP
Se|ection guide
How Io selecI Ct preparaIive packin maIerials.
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Ot-OPN
ConvenIional reversed phase Cs packin maIerials are resIricIed Io abouI 80-60% waIer in Ihe mobile phase. The COSMOSlL
Cs-OPN is a new WaIer-WeI`Cs packin maIerial developed for reversed phase open column chromaIoraphy. The Cs-OPN
maIerial can be used in 100% aqueous efuenIs.
Oharacteristic
The exIernal surface of Ihe Cs-OPN el is coaIed wiIh hydrophilic roup Io increase weIIabiliIy of Ihe el, and ocIadecyl roup
is bonded in Ihe pore of Ihe el. This physical characIerisIic of Ihe el makes Ihe reversed phase open column chromaIoraphy
possible wiIh 100 % waIer.
Packin maIerial COSMOSlL 76Cs-OPN
Column size 20 mm l.D. `260 mm bed heihI
DeIecIion UV 264 nm
Separation of Theobromine and Theophy||ine
Fiure shows Ihe samples are clearly separaIed by reversed open column chromaIoraphy wiIh 70% of waIer.
Inuence of partic|e size
Column size 10 mm l.D. `260
mm bed heihI
Mobile phase meIhanol : waIer -
20 : 80
DeIecIion UV 264 nm
Sample 1. Theobromide (TB)
2. Theophylline (TP)
8. Cafeine (CF)
76Cs-OPN
1
2
8
0 80 60 90 120 min) 240 210 180 160
140Cs-OPN
1
2
8
min) 0 80 60 90 120
0 80 60 90 120 160min)
1
2
HN
N
N
N
O
O
CH
8
CH
8
N
N
N
N
O
O
CH
8
H
H
8
C
1 2
Sample 1. Theobromide (100 m)
2. Theophylline (100 m)
Fiure. ChromaIoram of Theobromide and Theophylline separaIed on Cs-OPN
Table. Comparison beIween 76 ,m and 140,m parIicle size silica
ParIicle Flow raIe TheoreIical plaIe number Rs
SeparaIion
Iime(min)
SolvenI
consumpIion(ml)
,m) ml/min) TB TP CF TB/TP TP/CF
76 0.26 400 890 840 1 1.74 240 60
140 0.6 800 280 260 0.9 1.4 100 60
Hydrophobic roup
Hydrophilic roup
Silica el
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F|ow rate
Since reversed phase chromaIoraphy enerally employs hih viscosiIy solvenIs
such as waIer and meIhanol, Ihe flow raIe is lower Ihan IhaI of normal phase
chromaIoraphy. The flow raIe of reversed phase depends on Ihe mobile phase
composiIion. Fiure indicaIes IhaI Ihe fow raIe of Ihe COSMOSlL 140Cs-OPN
(140 ,m in parIicle size) is abouI 2.6 Iimes hiher Ihan IhaI of Ihe COSMOSlL
76Cs-OPN.
Fiure.
ConcenIraIion of meIhanol aainsI fow raIe
Column 10 mm l.D. `180 mm bed heihI
(raviIaIional liquid fow)
Separation of p-Oreso| and p-Ethy|pheno| by norma| and reversed phase mode
Since Ihe sIrucIural diference beIween o-Cresol and o-EIhylphenol is only one meIhylene roup, iI is difculI Io separaIe such
samples under normal phase condiIion. On Ihe oIher hand, Ihe samples are clearly separaIed under reversed phase condiIion
wiIh COSMOSlL Cs-OPN packin maIerial.
Packin maIerial COSMOSlL 76Cs-OPN
Column size 10 mm l.D. `180 mm bed hihI
DeIecIion UV 264 nm
Sample 1. o-Cresol
2. o-EIhylphenol
Packin maIerial Silica Gel 120 (160-826 mesh)
Mobile phase n-Hexane : eIhyl aceIaIe - 90 : 10
DeIecIion UV 264 nm
Sample 1. o-Cresol
2. o-EIhylphenol
1
2
Silica Gel 120
1,2
0 80 60 min)
76Cs-OPN
0 80 60 90 min)
1
2
Reproducibi|ity and washing methods
Wash Ihe COSMOSlL Cs-OPN packin maIerial wiIh IeIrahydrofuran, chloroform or oIher solvenIs Io remove Ihe impuriIies. This
packin maIerial has excellenI reproducibiliIy and can be used repeaIedly.
"CAUTlON`
Do noI wash wiIh basic solvenIs of pH 7 or more which will dissolve Ihe silica el or pH 2 or less which will cleave Ihe Cs sIaIionary phase. Dry Ihe
packin maIerial aI 60C or less. See end of Ihis chapIer for packin meIhod.
Fiure. Column size and required amounI of Cs-OPN packin maIerial
Column l.D.(mm) Bed heihI(mm) AmounI of Cs-OPN()
10
160 4
260 7
20
160 17
260 28
80
160 88
260 68
Oo|umn size and required amount of packing materia|
ProducI name ProducI number PKG size
COSMOSlL 76Cs-OPN
87842-66 100
87842-96 600
87842-11 1 k
COSMOSlL 140Cs-OPN
87878-16 100
87878-46 600
87878-61 1 k
COSMOSlL Cs-OPN
0 20
meIhanol)
1.6
40 60 80 100
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0.8
0.6
0.4
0.2
0
140Cs-OPN
76Cs-OPN
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Ot-PREP
Partic|e size, ow rate and theoretica| p|ate number
Because reversed phase chromaIoraphy employs efuenIs of hih viscosiIy such as meIhanol and waIer, Ihe fow raIe is lower
Ihan IhaI of normal phase chromaIoraphy, which uses efuenIs of low viscosiIy such as hexane and eIhyl aceIaIe.
The lare parIicle size Cs bulk maIerials are widely used for lab Io process scale purifcaIions. COSMOSlL ofers Ihree diferenI
parIicle sizes of Cs packin maIerials.
Hiher IheoreIical plaIe number can be obIained wiIh lower fow raIe.
Fiure 1. ConcenIraIion of meIhanol aainsI fow raIe
Column : 10 mm l.D. `180 mm bed heihI (raviIaIional liquid fow)
Fiure 2. Flow raIe aainsI IheoreIical plaIe number
Column : 20 mm l.D. `800 mm
140Cs-PREP
76Cs-PREP
40Cs-PREP
MeIhanol (%)
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60 60 70 80 90 100
1.0
0.8
0.6
0.4
0.2
0
140Cs-PREP
76Cs-PREP
40Cs-PREP
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Flow raIe (ml/min)
0
0
1000
2000
8000
4000
2 4 6 8 10
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App|ication Data
SeparaIion of ViIamin E
SeparaIion of crude drus
Crude drus IhaI are sIronly hydrophobic can be separaIed by Ihe medium pressure chromaIoraphy (closed columns).
Packin maIerial COSMOSlL 40Cs-PREP
Mobile phase meIhanol
DeIecIion UV 280 nm 0.2ALFS
Sample 1. Dl-+-Tocopherol (6 m)
2. Dl-+-Tocopherol AceIaIe (6 m)
Packin maIerial COSMOSlL 40Cs-PREP
Mobile phase meIhanol : 0.06% TFA - 70 : 80
DeIecIion UV 264 nm, 0.06ALFS
Sample 1. Baicalin (40 m)
2. Baicalein (120 m)
8. Woonin (40 ,)
60min)
1
2
0 10 20 80 40
min)
1
2
0 10 20 80
8
Oo|umn size and required amount of Ot-PREP packing materia|
Column l.D.(mm) Bed heihI(mm) Column volume(ml) AmounI of Cs-PREP()
8
800 16 9
600 26 16
10
800 26 16
600 40 26
20
800 96 66
600 160 96
80
800 210 126
600 860 220
60
800 660 860
600 980 600
COSMOSlL 40Cs-PREP
87982-86 100
87982-16 600
87982-81 1 k
COSMOSlL 76Cs-PREP
87988-76 100
87988-06 600
87988-21 1 k
COSMOSlL 140Cs-PREP
87984-66 100
87984-96 600
87984-11 1 k
Packing method
Please refer Io TECHNlCAL NOTE 9 ; Packin insIrucIion aI pae 101.
COSMOSlL Cs-PREP
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SL--PREP Silica el 60 neuIral
Averae parIicle size 76 ,m 140 ,m 76 ,m 140 ,m
ParIicle size rane
42 106 ,m
160 826 mesh)
74 210 ,m
70 280 mesh)
42 106 ,m
160 826 mesh)
74 210 ,m
70 280 mesh)
Averae pore size approx. 120 7 approx. 60 7
Specifc surface area approx. 800 m/ approx. 600 m/
ApplicaIion Open column chromaIoraphy/Flash column chromaIoraphy
Packing materia|s for norma| phase preparative |iquid chromatography
DiferenI Iype of silica el is also available, please see pae 69.
SL--PREP
Column size 10 mm l.D. `260 mm
Mobile phase hexane : eIhanol - 96 : 6
TemperaIure 80T
(6 ,m 0.82AUFS)
Sample 1. Chrysazin
2. o-Cresol
COSMOSlL SL--PREP is ulIra pure silica el packin maIerial more Ihan 99.99% puriIy. COSMOSlL SL--PREP provides
improved separaIion and reproducibiliIy for compounds wiIh carbonyl or phenol hydroxyl roups, which are ofIen problemaIic on
convenIional silica el maIerials.
*All chromaIorams shown below are obIained wiIh silica el packed inIo sIainless sIeel columns.
Inuence of the partic|e size on peak shape
The SL--PREP is available in fve sizes : 8, 6, 16, 76 and 140 ,m (parIicle diameIer). The peak shapes depend on Ihe parIicle
size, however, Ihe eluIion order remains Ihe same.
Materia| Oharacteristic
Se|ection guide
SelecIion fow of packin maIerials for normal phase preparaIive chromaIoraphy.
Acid resisIance of compounds
NoI decompose in low acid
Decomposed in low acid
SL--PREP
Silica el 60 (neuIral)
Unknown
6 ,m(for HPLC) 76 ,m 140 ,m
1
2
1
2
1
2
0 6 10
0 6 10 0 6 10
min)
min) min)
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Performance for chelaIin compounds
Hihly purifed silica el of Cosmosil SL--PREP enables separaIion of meIal coordinaIion compounds wiIhouI adsorpIion.
TemperaIure 80T
Sample 1. Quinizarin
2. o-NiIrobenzyl Alcohol
TemperaIure 80T
Sample 1. Salicylic Acid
2. Salicylamide
COSMOSlL 76SL--PREP
88012-64 100
88012-86 600
88012-61 1 k
COSMOSlL 140SL--PREP
88018-64 100
88018-26 600
88018-41 1 k
Packing method
Our convenIional producI
(76 ,m)
Silica el -A company
(64 210 ,m)
0 10 20
1
2
0 10 20
2
76SL--PREP
(76 ,m)
0 10 20
1
2
Silica el-A company
2
0 10 20
Our convenIional producI
(76 ,m)
0 10 20
76SL-PREP
(76 ,m)
1
2
0 10 20
COSMOSlL SL--PREP
Performance for oranic acid and amide
min) min) min)
min) min) min)
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Decompose
0 80 60 (min)
Si|ica ge| (spherica|neutra|)
TemperaIure 80T
Sample Sample A (100 m/ml)
lnIernal sIandard MeIhyl BenzoaIe (10 m/ml)
ln|ecIion volume 8 ,l
Oomparison with conventiona| si|ica ge|
PurifcaIion of AceIal
Silica el 60 (sphericalneuIral)
lnIernal sIandard
TemperaIure 80T
Sample Sample B (200 m/ml)
lnIernal sIandard MeIhyl BenzoaIe (10 m/ml)
ln|ecIion volume : 8 ,l
Silica el (sIandard)
lnIernal sIandard
Silica el 60 (sphericalneuIral)
lnIernal sIandard
Since convenIional silica els are weakly acidic, some pH sensiIive compounds may be decomposed durin Ihe purifcaIion by
column chromaIoraphy wiIh Ihe acidic silica els. The pH of Silica el 60 (sphericalneuIral) is ad|usIed Io nearly neuIral for
Ihe separaIion of noI only pH sensiIive compounds buI also new compounds IhaI Ihe physical properIies are sIill unknown.
*All chromaIorams shown below are obIained wiIh silica el packed inIo sIainless and sIeel columns.
Recovery up
0 80 60 (min)
Silica el (SIandard)
lnIernal sIandard
Decomposed
EluIed
0 80 60 (min)
0 80 60 (min)
Sample A
Sample B
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Silica el 60, spherical, neuIral
76 ,m
80611-64 100
80611-86 600
80611-61 1 k
80611-06 6 k
80611-22 26 k
Silica el 60, spherical, neuIral
140 ,m
80618-94 100
80618-66 600
80618-81 1 k
80618-62 26 k
Si|ica ge|for co|umn chromatograph)
Silica el 60 (spherical, neuIral)
ProducI name ParIicle size ProducI number PKG size
Silica el (irreular)
Silica el 60
approx. 70-280 mesh
80724-66 600
80724-71 1 k
80724-84 6 k
80724-42 26 k
approx. 280-400 mesh
80721-86 600
80721-01 1 k
80721-14 6 k
80721-72 26 k
approx. 2-20 ,m 80787-24 6 k
Silica el (spherical)
Silica el 60, spherical
approx. 70-280 mesh
80781-71 1 k
80781-42 26 k
approx. 160-826 mesh
80788-61 1 k
80788-22 26 k
Silica el 120, spherical
approx. 70-280 mesh 80784-41 1 k
approx. 160-826 mesh 80786-81 1 k
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The use of ion pair reaenIs as mobile phase addiIives exIends Ihe applicabiliIy of reversed phase HPLC. lonic or hihly polar
compounds are difficulI Io analyze by reversed phase usin only oranic solvenI and buffer soluIion because of Ihe shorI
reIenIion Iime. lon pair reaenIs are sIron hydrophobic ions which form neuIral ion pairs wiIh opposiIely chared samples
molecules, makin Ihe efcienI ODS columns amenable Io separaIe ionic or hihly polar samples.
Nacalai ofers a broad rane of ion pair reaenIs for pharmaceuIical compounds and oIher hihly polar maIerials.
When usin ion pair reenIs, ample Iime should be allowed for esIablishin equilibrium and for cleanin Ihe column.
When usin ion pair reenIs wiIh an alkyl chain of Ca or shorIer, iI Iypically Iakes 20 minuIes for esIablishin equilibrium and 80
minuIes for cleanin. lI may Iake more Ihan 1 hour Io clean Ihe column when usin ion pair reaenIs wiIh an alkyl chain loner
Ihan Ca. Therefore, iI is hihly recommended Io prepare a column for exclusive use wiIh ion pair reaenIs.
Ion pair reagents
General use of ion pair reaenIs in Ihe mobile phase.
A: Ba ic compound
Add oranic olvenI and ad|u I Io:
aceIoniIrile : bufer - 10 : 90 + ion pair reaenI
or
meIhanol : bufer - 20 : 80 + ion pair reaenI
Prepare Sodium 1-heprane ulfonaIe
and 20 mmol/l KHaPO+ oluIion.
Prepare TeIra-n-buIyl ammonium
pho pahIe and 20 mmol/l
pho pahIe bufer oluIion.
Add ion pair reaenI Io each mixIure unIil Ihe concenIraIion of ion pair reaenI
become 6 mmol/l. FilIer Ihe oluIion by u in flIer uch a MlLLlCUP-HV.
Replace olvenI in Ihe column by waIer and Ihen e Iabli h
equilibrium wiIh Ihe mobile pha e for aI lea I 20 min.
B: Acidic compound
SIarI Io analy i
Table 1. ReIenIion Iime and separaIion ad|usImenI
Troubles SoluIions
1. Good peak separaIions buI reIenIion Iime is Ioo lon or Ioo
shorI.
1. Too lon : increase oranic solvenI concenIraIion by 10%.
Too shorI : decrease oranic solvenI concenIraIion by 10%.
2. Good reIenIion Iime bur peaks are noI sharply separaIed.
2. Use ion pair of loner alkyl chain or Ihe same ion pair buI
double Ihe concenIraIion.
8. No diference in separaIions beIween usin ion pair and noI
usin, or no chane appears Iryin soluIion No.2.
8. For basic compounds analysis, decrease pH value.
For acidic compounds analysis, increase pH value.
4. ReIenIion Iime of Ihe inIeresIed compound is Ioo lon.
4. Use ion pair of shorIer alkyl chain or same ion pair of half
concenIraIion.
2. Liquid Chromatoyraphy ReIated Products
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for Basic samples
ProducI name Grade SIorae ProducI number PKG size
Sodium 1-BuIanesulfonaIe SP RT 81881-94 6
Sodium 1-BuIanesulfonaIe
(0.6M soluIion)
SP RT 81882-84 6`10 ml
Sodium 1-PenIanesulfonaIe SP RT
81780-64 6
81780-22 26
Sodium 1-HexanesulfonaIe SP RT
81629-24 6
81629-82 26
Sodium 1-HexanesulfonaIe
(0.6M soluIion)
SP RT
81682-64 10 ml
81682-06 6`10 ml
Sodium 1-HepIanesulfonaIe SP RT
81628-84 6
81628-92 26
Sodium 1-OcIanesulfonaIe SP RT
81729-04 6
81729-62 26
Sodium 1-OcIanesulfonaIe
(0.6M soluIion)
SP RT
81788-84 10 ml
81788-76 6`10 ml
Sodium 1-NonanesulfonaIe SP RT 81626-44 6
Sodium 1-DecanesulfonaIe SP RT 81429-84 6
Sodium 1-UndecanesulfonaIe SP RT 82080-04 6
Sodium 1-DodecanesulfonaIe SP RT 81426-64 6
Sodium Lauryl SulfaIe SP RT 81628-82 26
TeIra-n-buIylammonium Bromide SP R 82824-72 26
TeIra-n-buIylammonium Chloride EP R
82986-64 6
82986-22 26
TeIra-n-buIylammonium HydroensulfaIe GR RT 82924-62 26
TeIra-n-buIylammonium lodide SP R
82906-64 6
82906-12 26
TeIra-n-buIylammonium PerchloraIe SP R
82906-44 6
82906-02 26
TeIra-n-buIylammonium PhosphaIe SP R 82929-64 6
TeIra-n-buIylammonium PhosphaIe
(0.6M soluIion)
SP RT
82926-26 10 ml
82926-84 6`10 ml
Labe|ing reagents
Dansyl Chloride SP RT 10427-91 1
p-Bromophenacyl Bromide (PBPB) GR R 06802-92 26
8,6-DiniIrobenzoyl Chloride (DNBC) SP A 18680-44 6
N- 9-Acridinyl)maleimide (NAM) SP R 00842-64 60 m
NBD Chloride SP R 24118-61 1
o-PhIhalaldehyde (OPA) SP R
27824-61 1
27824-74 6
27824-82 26
for Acid samples
(sIorae) A : Cool and dark, RT : Room IempraIure, R : RefrieraIor
(sIorae) A : Cool and dark, RT : Room IempraIure, R : RefrieraIor
78
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3. PrehItration TooI for Liquid Chromatoyraphy
W series (Aqueous so|ution)
W series are insIalled wiIh low adsorpIion hydrophilic durapore flIer (polyvinylidenedifuoride,
PVDF). W series can be used wiIh boIh aqueous and oranic solvenIs. They are besI suiIed
for preflIraIion of proIein and oIher bioloical samples.
ln|ecIion of samples conIainin parIiculaIes (microparIicles, precipiIaIes, colloid subsIances)
will clo HPLC columns, shorIen in|ecIor life, and resulI in exIensive mainIenance on pumps.
Cosmonice flIers are used Io remove parIiculaIes from samples and prolon Ihe life of HPLC
sysIem componenIs. There are Iwo Iypes of Cosmonice flIers as sIaIed below.
Oosmonice f|ter
S series (Organic so|vents)
S series are insIalled Tefon flIer (polyIeIrafuoroeIhylene, PTFE) wiIh sIron resisIance Io
oranic solvenIs, acids and alkalis. They are besI suiIed for prefilIraIion of samples wiIh
aressive oranic solvenIs such as chloroform and IeIrahydrofuran.
ProducI name Pore size Process volume Hold-up volume ConnecIion ProducI number PKG size
Cosmonice FilIer
W (Aqueous) 4 mm
0.46 ,m less Ihan 1 ml <10 ,l
lnleI : luer-lock
OuIleI : luer-slip
ConnecIable
needles
06648-04 100 pk
Cosmonice FilIer
W (Aqueous) 18 mm
0.46 ,m 0.6-10 ml < 80 ,l 06644-94 100 pk
Cosmonice FilIer
W (Aqueous) 26 mm
0.46 ,m 8-60 ml < 100 ,l 06646-84 60 pk
Cosmonice FilIer
W (SolvenI) 4 mm
0.46 ,m less Ihan 1 ml < 10 ,l 06641-24 100 pk
Cosmonice FilIer
W (SolvenI)) 18 mm
0.46 ,m 0.6-10 ml < 80 ,l 06642-14 100 pk
Cosmospin filIers are used Io remove fine parIicles and precipiIaIes from samples by cenIrifuaIion. They uIilize Omnipore
hydrophilic PTFE membrane flIer, which has a wide rane of chemical resisIance. Cosmospin flIers are Ihe besI choice for
HPLC sample flIraIion. Two pore sizes, G (0.2 ,m) and H (0.46 ,m), are available.
Oosmospin f|ter
ProducI name Pore size
Maximum
sample volume
Hold-up
volume
Maximum
cenIrifual force
RoIor size
(fxed-anle)
FilIraIion
area
Color
ProducI
number
PKG size
Cosmospin
FilIer G
0.2 ,m 0.4 ml 6 ,l 6,000` 1.6 ml 0.2 cm brown 06649-44 100 pk
Cosmospin
FilIer H
0.46 ,m 0.4 ml 6 ,l 6,000` 1.6 ml 0.2 cm whiIe 06640-84 100 pk
Dimension : DiameIer 10.6 mm `LenIh 46 mmMembrane : Omnipore Hydrophilic PTFESample reservoir and collecIion Iube : Polypropylene
- Add Ihe sample inIo Sample reservoir
- CenIrifue afIer cappin
- Recover Ihe flIraIe from Sample collecIion Iube
Sample
reservoir
parIicle
sample
Sample
vial
74
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Chemical compaIibiliIy
SolvenI
Cosmonice
W series
Cosmonice
S series
Cosmospin SolvenI
Cosmonice
W series
Cosmonice
S series
Cosmospin
AceIic acid, lacial + + + Hydroen peroxide (8%) + +
AceIic acid, 6% + + + Hypo (phoIo) + + +
AceIone - + + lsobuIyl alcohol + + +
AceIoniIrile + + + lsopropyl aceIaIe + + +
Ammonia soluIion (conc.) + + lsopropyl alcohol + + +
Ammounium hydroxide (6N) + + + Kerosene + + +
Amyl alcohol + + + MeIhyl alcohol + + +
Benzene + + - MeIhyl eIhyl keIone - + +
Benzyl alcohol (1%) + + - MeIhyl isobuIyl keIone + + -
Boric acid + + NiIric acid (6N) + +
BuIyl aceIaIe + NiIrobenzene + + -
Carbon IeIrachloride + + + Ozone (10ppm in waIer) - + -
CelloSolve (eIhyl) solvenI + + + Paraldehyde +
Chloroform + + + PeI base oils + + +
Cyclohexanone - + - PenIane + + -
DichloromeIhane + + - PeIroleum eIher + +
DimeIhylaceIamide - + + Phenol (6.0%) + + -
DimeIhylformamide + + + 2-Propanol + + +
Dioxane + + + PhosphaIe bufer soluIion + +
DMSO - + - SeawaIer + + +
EhIyl alcohol + + + Silicone oils + + +
EIhers + + + Sodium hydroxide (conc.) + + +
EIhyl aceIaIe + + + Sulfuric acid (6N) +
EIhylene lycol + + + Toluene + + -
Formaldehyde + + + THF - +
Freon, TF or PCA solvenI + + + TrichloroaceIic acid + + +
Gasoline + + + TrichloroeIhane + + -
Glycerine (Glycerol) + + + TrichloroeIhylene + + -
Hexane + + - TFA + + -
Hydrochloride (6N) + + + Xylene + + +
Hydrofuoric acid - + -
+ : Recommended,- : NoI recommended,(blank) : No daIa
76
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Others
COSMOSlL Guard CarIride Holder
COSMOSlL Guard CarIride Holder 88009-79 1PKG
COSMOSlL Column PreflIer
COSMOSlL Column PreflIer 89861-19 1PKG
COSMOSlL Column Spare FilIer for PreflIer
COSMOSlL Column Spare FilIer for PreflIer 89689-09 2PKG
COSMOSlL Column ConnecIin Tube
COSMOSlL Column ConnecIin Tube 87848-69 1PKG
1ECMNICAL NO1E
J. EClCCtlVlty C! paCklnQ matC|lalS ln |CVC|SCd phaSC
llquld Ch|CmatCQ|aphy
E. F|Cpa|atlCn C! mCbllC phaSC !C| MFLC
U. EamplC p|Ct|CatmCnt !C| MFLC
4. HaSCllnC nClSC ln Q|adlCnt ClutlCn
U. 1|CublCShCCtlnQ !C| lnC|CaSCd p|CSSu|C
U. ECCt C! Qua|d CClumn
7. 1|CublCShCCtlnQ !C| nC|mal phaSC Ch|CmatCQ|aphy
H. InnC| dlamCtC| C! CClumn {SCalC dCwn and SCalC up)
D. FaCklnQ lnSt|uCtlCn
78
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Reversed phase chromaIoraphy is Ihe mosI commonly used meIhod of HPLC, because of Ihe hih IheoreIical plaIe
number, excellenI separaIion characIerisIics, reproducibiliIy, and ease of use. Columns packed wiIh ocIadecyl roup
bonded Iype silica el (Cs, ODS) are Ihe mosI widely used reversed phase chromaIoraphy. However, Cs columns
provide insufcienI separaIion for compounds similar in hydrophobiciIy because Ihe main separaIion mechanism of Cs
column is based on hydrophobic inIeracIion. lI may improve separaIion of compounds wiIh similar hydrophobiciIy by
usin loner columns, chanin mobile phases or chanin IemperaIure. However, in many cases, iI is probably mosI
effecIive Io use differenI packin maIerials which reIain compounds base on a secondary inIeracIion in addiIion Io
hydrophobic inIeracIion.
AI Nacalai, we ofer a varieIy of COSMOSlL reversed phase packin maIerials. Summary of Ihese packin maIerials and
Iheir respecIive reIenIion mechanism are in Table 1. ReIenIion of compounds in each sIaIionary phase depends on
summaIion of Ihe inIeracIions. Therefore, comprehension of each inIeracIion leads Io selecIion of an appropriaIe
column.
Table1. SIaIionary phase and inIeracIion of packin maIerials
Cs-MS- Cs-AR- Cs-MS PE-MS )NAP PYE NPE PBB-R CholesIer
Averae
parIicle size
8616 ,m 6 ,m
Averae pore
size
approx. 120 7
Specifc
surface area
approx. 800m/
SIaIionary
phase
Si
CH8
H8C
Si
OH
Si
CH8
H8C
Si
CH8
H8C
Si
CH8
H8C
Si
CH8
H8C
Si
CH8
H8C
NO2
Si
CH8
H8C
O
B
B
B
B
B
H8C
H8C
O
Si
CH8
H8C
ocIadecyl ocIadecyl ocIyl phenyleIhyl naphIhyleIhyl pyrenyleIhyl niIrophenyleIhyl penIabromobenzyl cholesIeryl
Types monomeric polymeric monomeric monomeric monomeric monomeric monomeric monomeric monomeric
lnIeraciIon
hydrophobic
inIeracIion
hydrophobic
inIeracIion
hydrophobic
inIeracIion
hydrophobic
inIeracIion
)-)inIeracIion
hydrophobic
inIeracIion
)-)inIeracIion
hydrophobic
lnIeracIion
)-)inIeracIion
dispersion
lnIeracIion
shape selecIiviIy
hydrophobic
lnIeracIion
)-)inIeracIion
dipole-dipole
inIeracIion
hydrophobic
inIeracIion
dispersion
inIeracIion
hydrophobic
inIeracIion
shape selecIiviIy
End cappin near-perfecI IreaImenI
Carbon conIenI approx. 16% approx. 17% approx. 10% approx. 10% approx. 11% approx. 18% approx. 9% approx. 8% approx. 20%
1. Se|ectivity of packing materia|s in reversed phase |iquid chromatography
TEOHNIOAL NOTE
79
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Se|ectivity
SelecIiviIy for polar funcIional roup is evaluaIed based on Ihe separaIion of benzene, niIrobenzene, which has a niIro
roup, and anisole, which has a meIhoxy roup. The chromaIorams below show separaIion of Ihe Ihree compounds on
four COSMOSlL columns : Cs-MS-, PE-MS, )NAP and PYE. EluIion order on Ihe Cs column is as followin :
niIrobenzene, anisole and benzene. EluIion orders on Ihe aromaIic columns are reversed. SeparaIion on Ihe Cs column
is based on hydrophobic inIeracIion only. On Ihe oIher hand, Ihe packin maIerials on Ihe oIher Ihree columns have
aromaIic rins and reverse Ihe eluIion order by )-) inIeracIion.
The raph of selecIiviIy for polar funcIional roup is shown below. Amon nine COSMOSlL columns, PYE and NPE
columns have Ihe hihesI selecIiviIy facIors for polar roups. As Io mobile phases, meIhanol is more efecIive Ihan
aceIoniIrile for separaIion usin )-) inIeracIion.
1)SelecIiviIy for polar funcIional roup
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. NiIrobenzene (0.18 ,)
2. Anisole (1.6 ,)
8. Benzene (4.0 ,)
0.6 1.0 1.6 2.0 2.6 8.0
Cs-MS
PBB-R
NPE
PE-MS
)NAP
PYE
CholesIer
Cs-AR-
Cs-MS-
+(NiIrobenzene/Benzene)
60% meIhanol
40% aceIoniIrile
App|ication
SeparaIion of IoluniIrile posiIion isomers
ToluniIriles have Ihree posiIion isomers. lI is difculI Io separaIe orIho and para isomers by Cs or PE-MS column
because of lack of poor )-) inIeracIion. On Ihe oIher hand, Ihe isomers are well separaIed on PYE or NPE column
which has sIron )-) inIeracIion.
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. o-ToluniIrile (2.0 ,)
2. m-ToluniIrile (2.0 ,)
8. o-ToluniIrile (1.0 ,)
CH8
CN
CH8
CN
CH8
CN
m- o- o-
Cs-MS-
40% meIhanol)
PYE
60% meIhanol)
PE-MS
40% meIhanol)
NPE
40% meIhanol)
Cs-MS-
60% meIhanol)
)NAP
60% meIhanol)
PYE
60% meIhanol)
NO
2
OCH
8
1 2
8
PE-MS
60% meIhanol)
80
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N
O
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Se|ectivity
SelecIiviIy for dipole is evaluaIed based on Ihe separaIion of 1,5-diniIronaphIhalene and 1,8-diniIronaphIhalene.
DiniIronaphIhalenes (peak 1 and 2) were sIronly reIained on PYE and NPE because of )-) inIeracIion compared wiIh
dimeIhylnaphIhalenes. However, Ihere is a slihI diference beIween Ihese Iwo columns. While 1,5-diniIronaphIhalene
(peak 2) was preferenIially reIained on PYE, 1,8-diniIronaphIhalene (peak 1) was reIained loner on NPE. The resulIs
wiIh NPE indicaIe Ihe presence of sIron dipole-dipole inIeracIion. The Iwo niIro roup dipoles in 1,8-diniIronaphIhalene
are alined for a much reaIer dipolar couplin wiIh Ihe bonded niIrophenyl roup in NPE Ihan 1,5-diniIronaphIhalene.
2)SelecIiviIy for dipole
Cs-MS-
80% meIhanol)
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. 1,8-DiniIronaphIhalene (1,8-DNN) (0.21 ,)
2. 1,5-DiniIronaphIhalene (1,5-DNN) (0.11 ,)
4. 1-MeIhylnaphIhalene (0.86 ,)
6. 1,5-DimeIhylnaphIhalene (0.42 ,)
NPE
70% meIhanol)
PYE
90% meIhanol)
0.0 0.6 1.0 1.6
Cs-MS
PBB-R
NPE
PE-MS
)NAP
PYE
CholesIer
Cs-AR-
Cs-MS-
+(1,8-DNN/1,5-DNN)
meIhanol sysIem
aceIoniIrile sysIem
App|ication
SeparaIion of phIhaloniIrile posiIion isomers
PhIhaloniIriles have Ihree posiIion isomers. NPE or PYE compleIely separaIes Ihese compounds due Io )-) inIeracIion.
FurIhermore, NPE sIronly reIains o-phIhaloniIrile due Io dipole-dipole inIeracIion.
1 2 4 8
CH8
CH8
NO2 NO2 NO2
NO2
CH8
6
Cs-MS-
80% meIhanol)
NPE
40% meIhanol)
PYE
80% meIhanol)
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. o-PhIhaloniIrile (0.8 ,)
2. m-PhIhaloniIrile (8.0 ,)
8. o-PhIhaloniIrile (0.16 ,)
-
CN
CN
CN
CN
CN
CN
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Se|ectivity
SelecIiviIy for polyaromaIic compounds is evaluaIed based on Ihe separaIion of benzene, naphIhalene and anIhracene.
The eluIion orders in all columns are Ihe same : benzene, naphIhalene and anIhracene. ReIenIion increases in all
columns wiIh increasin number of aromaIic rins. ln addiIion, hihly dispersive packin maIerials such as PBB and
PYE show much sIroner reIenIion for polyaromaIic compounds due Io dispersion inIeracIion.
8)SelecIiviIy for polyaromaIic compounds
Cs-MS-
90% meIhanol)
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. Benzene (1.67 ,)
8. AnIhracene (0.0068 ,)
PBB-R
90% meIhanol)
PYE
80% meIhanol)
1 2 8 4 6
Cs-MS
PBB-R
NPE
PE-MS
)NAP
PYE
CholesIer
Cs-AR-
Cs-MS-
+(NaphIhalene/Benzene)
80% meIhanol
70% aceIoniIrile
App|ication
SeparaIion of dibenzosuberone and dibenzosuberenone
Cs reIains dibenzosuberone (peak 1) loner Ihan dibenzosuberenone (peak 2). On Ihe oIher hand, PBB-R and PYE
reIain dibenzosuberenone (peak 2), which has a )-elecIron con|uaIed sysIem, loner Ihan dibenzosuberone (peak 1).

Cs-MS-
80% meIhanol)
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. Dibenzosuberone (0.1 ,)
2. Dibenzosuberenone (0.026 ,)

PBB-R
100% meIhanol)
PYE
90% meIhanol)
82
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Se|ectivity
SelecIiviIy for molecular shape is evaluaIed based on Ihe separaIion of chrysene and benzaanIhracene. The isomers
of Iwo polyaromaIic hydrocarbons, which consisI of four benzene rins, are difculI Io separaIe because of Ihe similar
hydrophobiciIy or aromaIiciIy. However, PYE and CholesIer columns, which reconize molecular shape, enable Ihem Io
separaIe chrysene and benzaanIhracene.
4)SelecIiviIy for molecular shape
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. Chrysene (0.04 ,)
2. BenzaanIhracene (0.04 ,)
0.96 1.00 1.06 1.10 1.16
Cs-MS
PBB-R
NPE
PE-MS
)NAP
PYE
CholesIer
Cs-AR-
Cs-MS-
meIhanol sysIem
aceIoniIrile sysIem
+(Chrysene/Benz[a]anIhracene)
ApplicaIion
SeparaIion of diasIereomers (Ihreo- and eryIhro-)
Cs cannoI separaIe Ihe Ihreo and eryIhro forms. On Ihe oIher hand, PYE reIains Ihe planar eryIhro form loner Ihan
Ihe Ihreo form.

Cs-MS-
90% meIhanol)
PYE
90% meIhanol)
CholesIer
100% meIhanol)
PE-MS
80% meIhanol)
Cs
80% meIhanol)
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. t||eo form
2. e|yt||o form
PYE
80% meIhanol)
88
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Se|ectivity
SelecIiviIy for halide is evaluaIed based on Ihe separaIion of chlorobenzene and bromobenzene. PBB-R shows Ihe
hihesI selecIiviIy facIor due Io dispersion inIeracIion of Ihe fve bromine aIoms.
6)SelecIiviIy for halide
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. Toluene (8.8 ,)
2. Chlorobenzene (8.8 ,)
8. Bromobenzene (8.8 ,)
1.0 1.1 1.2 1.8 1.4
Cs-MS
PBB-R
NPE
PE-MS
)NAP
PYE
CholesIer
Cs-AR-
Cs-MS-
+(Bromobenzene/Chlorobenzene)
60% meIhanol
60% aceIoniIrile
App|ication
SeparaIion of haloen exchane reacIion producIs
PYE and PBB-R reIain dispersed iodine aIom loner Ihan bromine aIom. As a resulI, PYE and PBB-R can separaIe
Ihe complicaIed bromine and iodine compounds IhaI Cs cannoI separaIe.
1
CH8 Cl
8 2
Br
Cs-MS-
60% meIhanol)
PBB-R
60% meIhanol)
PYE
60% meIhanol)
Cs-MS-
90% meIhanol)
PBB-R
100% meIhanol)
PYE
100% meIhanol)
TemperaIure 80T
DeIecIion UV 264 nm
Sample haloen exchane reacIion producI
R
R
R
R
R-l or Br
Samolecou|tesyofD|.H.Yamamoto,
PlKEN,CondensedMolecula|Mate|ialslaoo|ato|y
84
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L

N
O
T
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Se|ectivity
SelecIiviIy for hydrophobiciIy is evaluaIed based on Ihe separaIion of alkylbenzenes. Two Cs and CholesIer show
similar hih selecIiviIy for hydrophobiciIy. OIher columns show less hydrophobic selecIiviIy Ihan Cs.
6)SelecIiviIy for hydrophobiciIy
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. Benzene (1.67 ,)
2. Toluene (1.67 ,)
8. EIhylbenzene (1.67 ,)
4. Propylbenzene (1.67 ,)
6. BuIylbenzene (1.67 ,)
6. Amylbenzene (1.67 ,)
0 2 4 6 8 10
Cs-MS
PBB-R
NPE
PE-MS
)NAP
PYE
CholesIer
Cs-AR-
Cs-MS-
k'(Amylbenzene)
80% meIhanol
70% aceIoniIrile
Lower concenIraIion of oranic solvenI in mobile phase leads Io much reIenIion in reversed phase chromaIoraphy. ln
case of NPE, when meIhanol concenIraIion is reduced Io 60%, Ihe reIenIion Iimes increase Io Ihose similar Io Cs wiIh
80% meIhanol.
(CH
2
)
n
-H
n-0-6
Cs-MS- CholesIer PBB-R PYE NPE
NPE
80% meIhanol) 60% meIhanol)
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2. Preparation of mobi|e phase for HPLO
ComposiIion Iable for mobile phase 1L usin meIhanol and waIer
MeIhanol / WaIer MeIhanol () DisIilled waIer ()
90/10v/v) 711.9 99.8
80/20v/v) 682.8 199.6
70/80v/v) 668.7 299.6
60/40v/v) 474.6 899.8
60/60v/v) 896.6 499.1
40/60v/v) 816.4 698.9
80/70v/v) 287.8 698.7
20/80v/v) 168.2 798.6
10/90v/v) 79.1 898.4
ComposiIion Iable for mobile phase 1L usin aceIoniIrile and waIer
AceIoniIrile / WaIer AceIoniIrile () DisIilled waIer ()
90/10v/v) 707.4 99.8
80/20v/v) 628.8 199.6
70/80v/v) 660.2 299.6
60/40v/v) 471.6 899.8
60/60v/v) 898.0 499.1
40/60v/v) 814.4 698.9
80/70v/v) 286.8 698.7
20/80v/v) 167.2 798.6
10/90v/v) 78.6 898.4
CauIion : MeIhanol and aceIoniIrile are hazardous subsIances, do noI use for medical purpose. Always process in a laboraIory hood and wear an eye
proIecIion and a mask.
Column COSMOSlL 6Cs-MS-
DeIecIion 264 nm 0.16 AUFS
TemperaIure 80T
Sample 1 uracil
2. naphIhalene
1) Oranic solvenI/ aqueous mixed mobile phase
1)-1. Preparation of methano| : water = 70 : 30 (v/v) 1L
QMeasure 700 ml of meIhanol in a measurin cylinder.
Measure 800 ml of disIilled waIer in a measurin cylinder.
Mix Q and Ihorouhly and deas.
The beIIer approach is Io prepare Ihe mobile phase ravimeIrically raIher Ihan volumeIrically. Followin is example of
preparaIion.
TEOHNIOAL NOTE
Inuence of organic so|vent composition in mobi|e phase on the retention time.
1% di#erence in the composition signifcant|y changes the retention.
Special aIIenIion should be paid Io measure correcI
amounI of oranic solvenI as Ihe reIenIion Iime is
sinifcanIly chaned by 1% diferenI composiIion.
Di#erences between acetonitri|e and methano| in reversed phase |iquid chromatography
AceIoniIrile (for HPLC) MeIhanol (for HPLC)
Pressure
%
Me hanl
Cncen a in f anic len
P
e

(
M
P
a
)
Ace ni ile
0 20 40 60 80 I 00
0
2
4
6
8
I 0
Back pressure differs dependin on species of oranic
solvenIs and mixin raIio. Back pressure of aceIoniIrile is
lower Ihan IhaI of meIhanol aI Ihe same concenIraIion.
TemperaIure 80T
86
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AceIoniIrile (for HPLC) MeIhanol (for HPLC)
EluIion sIrenIh
EluIion sIrenIh of aceIoniIrile/waIer is sIroner Ihan meIhanol/ waIer aI Ihe same
concenIraIion. WiIhin Ihe rane of abouI 80%-80% in Ihe concenIraIion of Ihe oranic
solvenI, eluIion sIrenIh of aceIoniIrile/waIer is almosI equivalenI Io IhaI of meIhanol/waIer
which was raised meIhanol concenIraIion abouI 10% (ex : aceIoniIrile/waIer - 60/40
meIhanol/waIer - 70/80)
Absorbance
AceIoniIrile has a lower UV absorbance in far
UV reion (less Ihan 260 nm) .
MeIhanol has a hiher UV absorbance Ihan
aceIoniIrile in far UV reion (less Ihan 260 nm).
Deas of mobile
phase
When aceIoniIrile is mixed wiIh waIer, iI is
endoIhermic. lI is difculI Io deas.
When meIhanol i s mi xed wi Ih waIer, iI i s
exoIhermic. lI is easy Io deas.
0
2
4
6
8
10
12
14
0 10 20 80 40 60 60 70 80 90 100 %
AceIoniIrile (sample: Ioluene)
MeIhanol (sample: Ioluene)
MeIhanol (sample: phenol)
AceIoniIrile (sample: phenol)

C
a
p
a
c
i
I
y

f
a
c
I
o
r

)
approx. 10%
ConcenIraIion of oranic solvenI
TemperaIure 80T
2) Oranic solvenI/ bufer mixed mobile phase
2)-1.Preparation of methano| : 20 mmo|/| phosphate bu#er (pH2.5) = 80 : 20 (v/v) 1L
PreparaIion of 20 mmol/l phosphaIe bufer (pH2.6)
QPrepare 20 mmol/l sodium dihydroenphosphaIe aqueous soluIion.
Prepare 20 mmol/l phosphoric acid aqueous soluIion.
Ad|usI Ihe pH Io 2.6 by mixin Q wiIh .
FilIer under reduced pressure Io remove insoluble subsIance IhaI may deIerioraIe pump-seal and clo columns
(0.46 ,m or smaller pore size is recommended).
(Easy meIhod)
Q Dissolve 1.81 of sodium dihydroenphosphaIe and 1.06 of phosphoric acid in disIilled waIer Io make 1 L
soluIion.
FilIer Ihe soluIion under reduced pressure Io remove insoluble subsIance (0.46 ,m or smaller pore size is
recommended).
Confrm IhaI Ihe soluIion is pH2.6.
PreparaIion of meIhanol : 20 mmol/l phosphaIe bufer (pH2.6) - 80 : 20 1L
Measure 200 ml of 20 mmol/l phosphaIe bufer (pH2.6) in a measurin cylinder.
2)-2. Preparation of methano| : 20 mmo|/| phosphate bu#er (pH7.0) = 80 : 20 (v/v) 1L
PreparaIion of 20 mmol/l phosphaIe bufer (pH7.0)
QPrepare 20 mmol/l sodium dihydroenphosphaIe aqueous soluIion.
Prepare 20 mmol/l di-sodium hydroenphosphaIe aqueous soluIion.
Ad|usI Ihe pH7.0 by mixin Q wiIh .
87
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(Easy meIhod)
Q Dissolve 1.14 of sodium dihydroenphosphaIe and 1.49 of di-sodium hydroenphosphaIe in disIilled waIer Io
make 1L soluIion.
recommended).
PreparaIion of MeIhanol : 20 mmol/l phosphaIe bufer (pH7.0) - 80 : 20 1 L
Measure 200 ml of 20 mmol/l phosphaIe bufer (pH7.0) in a measurin cylinder.
The beIIer approach is Io prepare Ihe mobile phase ravimeIrically raIher Ihan volumeIrically. Followin is example of
preparaIion.
meIhanol : 20 mmol/l
phosphaIe bufer
meIhanol ()
20 mmol/l phosphaIe
bufer (pH2.6) ()
20 mmol/l phosphaIe
bufer (pH7.0) ()
90/10v/v) 711.9 99.8 99.9
80/20v/v) 682.8 199.6 199.8
70/80v/v) 668.7 299.4 299.7
60/40v/v) 474.6 899.2 899.6
60/60v/v) 896.6 499.0 499.6
40/60v/v) 816.4 698.8 699.4
80/70v/v) 287.8 698.6 699.8
20/80v/v) 168.2 798.4 799.2
10/90v/v) 79.1 898.2 899.1
CauIion : MeIhanol and aceIoniIrile are hazardous subsIances, do noI use for medical purpose. Always process in a laboraIory hood and
wear an eye proIecIion and a mask.
8) PreparaIion of ion pair reaenI conIainin mobile phase
3)-1.Preparation of 5 mmo|/| sodium J-butanesu|fonate containing 20 mmo|/| phosphate bu#er (pH2.5)
QPrepare 6 mmol/l sodium 1-buIanesulfonaIe conIainin 20 mmol/l sodium dihydroenphosphaIe aqueous soluIion.
Prepare 6 mmol/l sodium 1-buIanesulfonaIe conIainin 20 mmol/l phosphoric acid aqueous soluIion
Ad|usI Ihe pH Io 2.6 by mixin Q wiIh .
(Easy meIhod)
Q Dissolve 1.81 of sodium dihydroenphosphaIe, 1.06 of phosphoric acid and 0.80 of sodium 1-buIanesulfonaIe
in disIilled waIer Io make 1L soluIion.
recommended).
z0.6M sodium 1-buIanesulfonaIe aqueous soluIion is also available from Nacalai Tesque.
88
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1) FilIraIion
FilIraIion is a common meIhod used for separaIin solids from liquids. lI exIends a column`s life by minimizin column
damaes from solid conIaminanIs such as parIicles, sedimenIs and colloid subsIances. lI also improves reproducibiliIy
of analyIical daIa. We ofer boIh syrine-Iype and spin-Iype flIers for sample flIraIion.
PreIreaImenI before HPLC analysis is ofIen required for samples of low concenIraIion or samples conIainin analyIical
conIaminanIs. lI improves reproducibiliIy and sensiIiviIy in analysis, and proIecIs HPLC columns. The preparaIion
meIhods are diferenI accordin Io Ihe each sample. The followins are examples of diferenI preIreaImenIs.
QSyrine flIer QCenIrifual flIer
Cosmonice flIer Cosmospin flIer
Easy Io use. JusI aIIach a flIer on Iop of a syrine. Easy Io use by cenIrifuaIion.
W (aqueous sysIem)
S (solvenI sysIem)
pore diameIer : 0.2 ,m
pore diamaIer : 0.46 ,m
QRequired equipmenI
SyrineSample boIIle CenIrifue
QProducI informaIion
Please see pae 78 Please see pae 78
3. Samp|e pretreatment for HPLO
TEOHNIOAL NOTE
Oosmonice f|ter
How Io use :
QFill a syrine wiIh Ihe sample you wanI Io flIer.
AIIach a Cosmonice flIer Io Ihe syrine.
Push Ihe syrine pluner Io flIer Ihe sample.
Analyze Ihe flIered sample by HPLC.
Oosmospin f|ter
ComponenIs : Sample reservoir
Sample collecIion Iube
How Io use :
Q lnserI a Cosmospin sample reservoir inIo a Cosmospin
sample collecIion Iube.
Add a sample inIo Ihe Cosmopsin sample reservoir.
Close Ihe sample collecIion Iube cap and cenIrifue.
Remove Ihe sample reservoir and collecI Ihe flIered sample
in Ihe sample collecIion Iube.
Analyze Ihe flIered sample by HPLC.
Sample collecIion Iube

Sample reservoir
lmpuriIies
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ProIein precipiIaIion is commonly used Io
remove proIeins in samples for downsIream
analysis. For example, when analyzin dru
concenIraIion in blood samples, proIeins have
Io be removed frsI. OIherwise, proIeins may be
adsorbed in columns and inIerfere wiIh Ihe
anal ysi s. Common meI hods f or pr oI ei n
precipiIaIion include salIin ouI, isoelecIric
poinI precipiIaIion and precipiIaIion wiIh oranic
sol venIs. The fol l owi n shows a eneral
procedure for proIein precipiIaIion wiIh oranic
solvenIs.
Procedure for proIein precipiIaIion :
2) ProIein precipiIaIion
Q Sample of blood.
ProIeins
Add precipiIanI, for insIance, eIhanol or meIhanol, 0.2-4 Iimes Ihe amounI of Ihe sample.
Sample Io blood
Mix well Ihe sample soluIion.
Keep aI a cool place for 16 min.
ProIeins will be separaIed.
CenIrifue aI 1700 G for 16 min.
Remove Ihe oranic solvenI layer.
ProIeins will be precipiIaIed aI Ihe boIIom.
HPLC analysis
*
1
SelecIion of precipiIanI
Choose precipiIanIs of hih solubiliIy and ad|usI Io a suiIable pH.
Followins are examples of precipiIanIs.
Oranic solvenI aceIone, aceIoniIrile
Acid IrichloroaceIic acid, perchloric acid
8) UlIraflIraIion
UlIraflIraIion is a meIhod Io concenIraIe proIeins
or oI her macr omol ecul es I hr ouh a semi -
per meabl e membr ane wi I h def i ned por es.
UlIrafilIraIion is applicable for sample desalIin,
concenIraIin proIeins from diluIe soluIion such
as urine samples, or deproIeinizin samples wiIh
hih proIein concenIraIion (i.e. blood serum or
plasma). Followin is a eneral procedure for
ulIraflIraIion.
Procedure for ulIraflIraIion :
QChoose an appropriaIe ulIraflIraIion Iube (flIer device). Add sample
Io Ihe sample reservoir. Close Ihe cap of Ihe flIer uniI.
CenIrifue accordin Io flIer device manufacIurers recommendaIion.
ProIeins will remain aI Ihe flIraIion membrane. ConcenIraIed proIein
soluIion can be recovered from Ihe sample reservoir. FilIered soluIion
is in Ihe collecIion Iube.
Analyze Ihe flIered soluIion and/or Ihe concenIraIed proIein soluIion
by HPLC.
90
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SolvenI exIracIion is a meIhod Io separaIe compounds due Io Iheir unequal solubiliIy in Iwo immiscible liquid phases,
usually waIer and an oranic solvenI. The meIhod is used Io concenIraIe hihly hydrophobic compounds, and
consequenIly increase analyIical sensiIiviIy. A buffer soluIion is added Io sample Io opIimize Ihe pH and IareI
subsIance is Ihen exIracIed by an oranic solvenI such as eIher and chloroform. However, when IareI subsIance is
combined wiIh proIeins, solvenI exIracIion may noI work well.
Procedure for solvenI exIracIion meIhod :
4) SolvenI exIracIion meIhod
QSample of blood
Solve IareI subsIance
Sample
AiIaIe Ihe soluIion
AiIaIe Ihe soluIion
7AiIaIe Ihe soluIion
SaIuraIe Ihe soluIion wiIh oranic solvenI (such as Chloroform, eIher or aceIic eIher, several
Iimes Ihe amounI of Ihe samples) and ammonium carbonaIe (neuIral). (SalIin ouI*
1
)
Basic compounds Acid compounds NeuIral compounds
NeuIral compounds in basic or acid
soluIion remain in waIer layer, and neuIral
compounds soluble in oranic solvenI
parIiIioned boIh in oranic solvenI layer
and waIer layer. Therefore, solvenI exIrac-
Iion is noI suiIable meIhod for neuIral
compound.
Acidize by
addin HCl
Alkalize by addin
ammonia or NaOH
Sample waIer soluIion
Ammonium carbonaIe
Oranic solvenI layer
Oranic solvenI layer
Sodium sulfaIe anhydrous
SeparaIe Ihe oranic solvenI layer inIo oIher Iube*
2
and dehydraIe Ihe soluIion by addin
sodium sulfaIe, (anhydrous)
WaIer in oranic solvenI will be crysIallized as sodium
sulfaIe hydraIe
*
1
SalIin ouI required for parIiIionin Ihe IareI
subsIance inIo oranic layer.
*
2
Causion : SupernaIanI is noI always oranic solvenI.
lI depends on Ihe specifc raviIy of Iwo layers.
Take ouI IareI subsIance in oranic solvenI layer wiIh syrine.
AIIach a Cosmonice flIer Io Ihe syrine, and flIer Ihe soluIion.
Refer Io pae 88, FilIraIion for Ihe proIocol of
Cosmonice flIer.
]Analyze Ihe soluIion by HPLC.
91
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PreIreaImenI by ion-exchane resin may be efecIive for samples IhaI Ihe solvenI exIracIion meIhod cannoI be adapIed
due Io iIs emulsifcaIion. A preliminary experimenI may be required for Ihe selecIion of resin and experimenIal condiIions.
For example, a neaIively chared compound is sIronly adsorbed on an anion-exchane resin such as DEAE cellulose
resin. Therefore, Ihe IareI compound is collecIed by increasin salI concenIraIion of bufer soluIion or ad|usIin pH of
eluIion bufer afIer washin of oIher weakly adsorbed undesired subsIances.
Procedure for ion exchane :
6) lon exchane
SIu lass wool in 1ml size of micropipeIIe Iip, and lock iI verIically as a column.
Make sure IhaI Ihe lass wool does noI o Ihrouh by addin pressure wiIh a sIick.
SIu lass wool wiIh pasIeur pipeIIe
Lock Ihe column verIically wiIh clamp
CauIion : The volume of resin will be sinifcanIly
increased afIer swellin.
QMix and swell appropriaIe amounI of resin wiIh waIer or TE bu er soluIion.
Basic compounds Acidic compounds
CaIion-exchane resin Anion-exchane resin Applicable resin
Add new TE bu er immediaIely afIer Ihe frsI bu er
oes Ihrouh so IhaI Ihe resin does noI dry.
Pour approximaIely 100 l of suspended resin in Ihe column, and
wash Ihe resin wiIh 800 l of TE bu er soluIion Ihree Iimes.
NeuIralize Ihe soluIion
Analyze neuIralized soluIion by HPLC.
Basic compounds Acidic compounds
Add alkaline soluIion in eluIion bu er
Apply sample in Ihe column.
CollecI Ihe flIraIe and apply in Ihe column
aain. RepeaI Iwice.
Add acidic soluIion in eluIion bu er
Wash Ihe resin wiIh 800 l of TE bu er soluIion Ihree Iimes.
92
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N
O
T
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4. Base|ine noise in gradient e|ution
ln radienI analysis, incompleIe mixin of mobile phases or impuriIies in waIer of mobile phase can cause baseline
noise. ln Ihe former case, iI can be improved by usin a proper mixer before in|ecIor (Baseline 12). ln Ihe laIIer case,
iI can be improved by usin a pre-column. lmpuriIies in waIer are adsorbed on Ihe pre-column (Baseline 28).
COSMOSlL 6Cs-AR- 4.6 mm l.D. x 10 mm or 10 mm l.D. x 20 mm as a pre-column.
Baseline 1
lncompleIe mixin
of mobile phase
Fiure 1
Mixer
Column COSMOSlL 6Cs-AR-800 4.6 mm l.D. `160 mm
Precolumn COSMOSlL 6Cs-AR- 4.6 mm l.D. `10 mm
Mobile phase A : 0.1% TFA conIainin waIer
B : 0.1% TFA conIainin 96% aceIoniIrile
B 0% 100%/80 min linear radienI
TemperaIure 80T
DeIecIion UV 220 nm
Baseline 2
lmpuriIies in waIer
Fiure 2
Pre-column
Baseline 8
Fiure 8
Drain
EluenI A
EluenI B
Pump A
Pump B
ln|ecIor
Column DeIecIor
EluenI A
EluenI B
Pump A
Pump B
Mi er
ln|ecIor
Column DeIecIor
Drain
EluenI A
EluenI B
Pump A
Pump B
Mi er
ln|ecIor
Column DeIecIor
Drain
Prex
column
TEOHNIOAL NOTE
98
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N
O
T
E
5. Troub|eshooting for increased pressure
RepeaIed analysis may increase back pressure. ConIinuous use of HPLC columns under hih pressure can cause
deIerioraIion and overload of Ihe equipmenI. Therefore, iI is imporIanI Io moniIor column back pressure reularly and
solve Ihe problem Iimely.
The back pressure increase can be due Io cloin of a column or cloin of Ihe equipmenI. FirsI of all, idenIify Ihe
cloin siIe.
Q
Recorder
QSucIion flIer Pump Line flIer Pre-column ln|ecIor Guard column AnalyIical column DeIecIor
wasIe soluIion
mobile phase
SchemaIic diaram of HPLC sysIem
Remove analyIical column firsI and connecI Ihe plumbin from Ihe back of HPLC sysIem direcIly Io Ihe deIecIor.
Measure Ihe pressure of fowin mobile phase wiIhouI an HPLC column. Generally equipmenI should hardly eneraIe
any pressure. lf Ihere is sinifcanI fow pressure, disconnecI Ihe sysIem componenIs one by one Io idenIify Ihe cloed
componenI(s). Possible causes and soluIions of cloed equipmenI are discussed in secIion ll below.
lf Ihe fow pressure wiIhouI a column is normal, Ihen pressure increase is due Io cloin of a column. ln Ihis case, one
needs Io deIermine Ihe causes and wheIher iI is Iime Io replace Ihe column. Possible causes and soluIions of cloed
column are discussed in reaIer deIails in secIions l and lll.
Pressure increase radually in
lon-Ierm use
Yes
DeIerioraIion of column due Io lon-Ierm use
Refer Io secIion 1
Pressure increase rapidly in
shorI-Ierm use
Yes
Flow pressure wiIhouI a column is 0-0.8 MPa
Cloin of column
Refer Io secIion
Flow pressure wiIh a column is 0.8 MPa or
hiher

Cloin of equipmenI
Refer Io secIion
.SoluIion in case an HPLC column is cloed in shorI-Ierm use.
SelecI Ihe possible cause of cloin accordin Io Ihe followin fow charI.
SIep1
SalI deposiIion
Use mobile phase of hih concenIraIion oranic solvenI rihI afIer usin bufer
Yes
Cause 1
NO
SIep2
ForeI Io flIer mobile phase
Sample is noI dissolved enouh
Yes
Cause 2
NO
SIep8
Analyzin samples which Iend Io absorb Io a column (i.e. proIein samples)
Sample deposiIion in column
Yes
Cause 8
TEOHNIOAL NOTE
SympIom Possible Cause
94
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Cause 1 SalI is deposiIed on a column.
SoluIion : Wash columns for 80 minuIes aI half of Ihe analyIical flow raIe wiIh 10% oranic solvenI
(meIhanol or aceIoniIrile) in waIer Io dissolve deposiIed salI. lf Ihe siIuaIion is noI improved, wash
wiIh 100% waIer under Ihe same condiIion.
PrevenIion : To swiIch Io hih concenIraIion oranic solvenI afIer usin a bufer, frsI wash a column wiIh a
mobile phase noI conIainin salI (wiIh Ihe same concenIraIion of oranic solvenI as Ihe bufer),
Ihen swiIch Io Ihe mobile phase of hiher oranic solvenI concenIraIion. Example : To chane
mobile phase from 10/90 (v/v) aceIoniIrile/20mmol/l phosphaIe bufer (pH2.6) Io 90/10 (v/v)
aceIoniIrile/waIer, frsI wash for 16 minuIes wiIh 10/90 (v/v) aceIoniIrile/waIer, and Ihen swiIch
Io 90/10 (v/v) aceIoniIrile/waIer.
Cause 2 Column flIer is cloed by sample or impuriIies.

SoluIion : ConnecI Ihe column in reverse direcIion, and Ihen wash Ihe column for 80 minuIes aI half of Ihe
usual analyIical fow raIe wiIh Ihe mobile phase used for analysis. lf Ihe siIuaIion is noI improved,
chane Ihe end fIIin in Ihe fronI of column. (We can replace end fIIins wiIh a paid service fee.)
PrevenIion : We recommend flIerin sample and/or mobile phase. For more informaIion, please see pae 88
TECHNlCAL NOTE 8. Sample preIreaImenI for HPLC 1) flIraIion.
Cause 8 Sample may be adsorbed Io packin maIerial or deposiIed in a column.

SoluIion : Wash for 80 minuIes aI half of analyIical fow raIes wiIh a solvenI which adsorbed subsIances are
dissolved in. The followins are how Io wash each Iype of columns.
[Reversed phase columns]
a) When an absorbed subsIance is noI proIein, wash wiIh meIhanol or IeIrahydrofuran.
b) When an absorbed subsIance is proIein, wash wiIh 60-70% of aceIoniIrile/waIer (conIainin
0.1% of IrifluoroaceIic acid). However proIeins may be deposiIed in hih concenIraIion of
oranic solvenI dependin on varieIies.
[COSMOSlL Suar-D/NHa/HlLlC columns]
Wash wiIh 60/60 (v/v) aceIoniIrile/waIer for NHa-MS and 100% waIer for Suar-D and HlLlC
columns.
[COSMOSlL SL-]
Wash wiIh meIhanol, IeIrahydrofuran or eIhanol.
PrevenIion : Choose appropriaIe preIreaImenI for each sample. For more informaIion, please see pae 88
TECHNlCAL NOTE 8. Sample preIreaImenI for HPLC 1) filIraIion. We also recommend usin
uard column. For more informaIion for uard columns, please see pae 96 TECHNlCAL NOTE 6.
EfecI of uard columns.
CauIion : When wash columns, do noI connecI column exiI and leI Ihe soluIion Ihrouh.
Lon Ierm of washin may deIerioraIe Ihe performance of columns.
Do noI use sIronly alkaline soluIion (more Ihan pH 7.6) or sIronly acidic soluIion (less Ihan
pH 1.6) for silica el base packin maIerial.
SIore columns wiIh manufacIurer recommended sIorae solvenI afIer washin,
When Ihe siIuaIion is noI improved, replace Ihe column.
.SoluIions in case pressure is Ioo hih because of cloed equipmenI.
FirsI of, idenIify Ihe specifc cloin siIe by disconnecIin Ihe componenIs in Ihe sysIem one by one and
checkin Ihe fow pressure. The followins are possible common causes.
Cause 1 SalI is deposiIed in plumbin.
SoluIion : Flow waIer Io Ihe plumbin wiIhouI connecIin a column and any oIher equipmenI. Washin ouI
Ihe plumbin in a reversin connecIion is also an efecIive way. lf Ihe siIuaIion is noI improved,
replace iI wiIh a new one.
Cause 2 Check-valve of pump is cloed by sIain

SoluIion : Wash Ihe check-valve wiIh a sIain dissolvin solvenI. Take aparI Ihe washable parI, soak iI in Ihe
solvenI, Ihen clean in an ulIrasonic cleaner.
Cause 8 Manual in|ecIor is cloed wiIh sIain

SoluIion : Wash wiIh a sIain dissolvin solvenI. Soak roIor seal and line flIer in waIer and clean Ihem in an
ulIrasonic cleaner. lf Ihe siIuaIion is noI improved, replace Ihe in|ecIor.
PrevenIion : lI exIends Ihe life Iime of an HPLC sysIem Io mainIain reular wash of Ihe sysIem. Wash Ihe
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sysIem Ihe same as wash an HPLC column. When Ihe mobile phase conIains salI, wash for
10-16 minuIes wiIh a mobile phase which has Ihe same composiIion buI noI conIainin salI. For
example, when usin 60/60 (v/v) meIhanol/20mmol/l phosphaIe bufer, wash wiIh 60/60 (v/v)
meIhanol/waIer. When Ihe mobile phase conIains haloen, acid and/or base, wash for 10-16
minuIes wiIh mobile phase which has Ihe same composiIion buI noI conIainin haloen, acid and/
or base.
1.SoluIions in case a column is damaed from lon Ierm use
Every column will have Io be replaced evenIually. Performance of a column is expecIed Io deIerioraIe slowly
afIer lon Ierm use. One has Io decide wheIher iI is Iime Io replace Ihe column.
Cause 1 Column deIerioraIion resulI from lon Ierm use.
SoluIion : Wash accordin Io SoluIion Io SecIion , Cause 8.
PrevenIion : Same as PrevenIion in SecIion , Cause 8. When Ihe column condiIion is noI improved, you
could conIinue Io use Ihe column if peak shapes do noI chane and Ihe maximum pressure is less
Ihan 20 MPa. However, we recommend replacin Ihe column because iI place exIra burden on
Ihe equipmenI.
Cause 2 Silica el in Ihe column may be cracked because of lon Ierm use.
SoluIion : Replace Ihe columns.
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6. E#ect of guard co|umn
Inspection of co|umns
We recommend IhaI Ihe performance of a column be examined reularly. DeIerioraIed columns shall be replaced Iimely.
lf a deIerioraIed uard column conIinues Io be used, Ihe packed column will also deIerioraIe.
Examp|e of using guard co|umns
The followin chromaIorams show analysis examples usin a COSMOSlL 6Cs-MS- analyIical column (4.6 mm l.D. `
160 mm) and Ihe same column connecIed wiIh iIs uard column (4.6 mm l.D. ` 10 mm). There is no chane in
separaIion characIerisIics since Ihe packin maIerial of Ihe uard column is idenIical Io IhaI used in Ihe main column.
The use of uard columns Io proIecI boIh analyIical and
preparaIive columns is hihly recommended.
COSMOSlL uard columns are packed wiIh packin
maIerials idenIical Io IhaI used in analyIical and preparaIive
columns. As a resulI, Ihe COSMOSlL uard columns do noI
conIribuIe Io any decrease in Ihe performance of Ihe main
column.
Please see respecIive paes of each column.
Please see pae 76 for Ihe parIs necessary Io use uard columns or uard column carIrides.
WiIhouI uard column WiIh uard column
WiIh a deIerioraIed uard column AfIer chanin Ihe uard column
Guard columns Guard carIrides
TEOHNIOAL NOTE
Column COSMOSlL 6Cs-MS- 4.6 mm l.D. `160 mm
COSMOSlL 6Cs-MS- 4.6 mm l.D. `10 mm
Mobile phase meIhanol : waIer - 70:80
TemperaIure 80T
DeIecIion UV 264 nm
Sample 1. BeIameIhasone 17-ValeraIe (0.26,)
2. lsoamyl BenzoaIe (2.6,)
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7.Troub|eshooting for norma| phase chromatography
Q How can l converI from reversed phase mode Io normal phase mode or vice versus usin Ihe same HPLC
equipmenI?
7 To converI from reversed phase mode Io normal phase mode, or vice versus, fush Ihe equipmenI wiIh a
solvenI IhaI is miscible wiIh boIh Ihe currenI mobile phase and Ihe inIended mobile phase. ConnecI Ihe
HPLC pump direcIly wiIh Ihe deIecIor, and replace Ihe solvenIs accordin Io followin insIrucIions.
SolvenI conversion from reversed phase Io normal phase
Q To converI a mobile phase wiIhouI bufer soluIion in reversed phase Io normal phase, replace solvenIs
accordin Io Ihe followin sIeps :
1)Flush Ihe equipmenI wiIh a solvenI for reversed phase. For example, meIhanol/HaO (v/v-60/60).
2) Flush Ihe equipmenI wiIh a solvenI miscible Io boIh mobile phases. For example, IeIrahydrofuran,
eIhanol.
8)Flush Ihe equipmenI wiIh a solvenI for normal phase. For example, hexane/eIhyl aceIaIe
To converI a mobile phase wiIh buffer soluIion in reversed phase Io normal phase, replace solvenIs
accordin Io Ihe followin sIeps :
1) Flush Ihe equipmenI wiIh a solvenI wiIh bufer soluIion for reversed phase. For example, meIhanol/
phosphaIe bufer (v/v-60/60).
2) Flush Ihe equipmenI wiIh a solvenI wiIh Ihe composiIion same as 1) and wiIhouI salI, for example
meIhanol/HaO (v/v-60/60).
8) Flush Ihe equipmenI wiIh a solvenI miscible Io boIh mobile phases. For example, IeIrahydrofuran,
eIhanol.
4)Flush Ihe equipmenI wiIh a solvenI for normal phase. For example, hexane/eIhyl aceIaIe.
SolvenI conversion from normal phase Io reversed phase
Q To converI from normal phase Io a mobile phase wiIhouI buffer soluIion in reversed phase, replace
solvenIs accordin Io followin sIeps :
1) Flush Ihe equipmenI wiIh a solvenI for normal phase. For example, hexane/eIhyl aceIaIe.
2) Flush Ihe equipmenI wiIh a solvenI miscible Io boIh mobile phases. For example IeIrahydrofuran,
eIhanol.
8) Flush Ihe equipmenI wiIh a solvenI for reversed phase. For example, meIhanol/HaO (v/v-60/60).
To converI from normal phase Io a mobile phase wiIh bufer soluIion in reversed phase, replace solvenIs
accordin Io followin sIeps :
1) Flush Ihe equipmenI wiIh a solvenI for normal phase. For example, hexane/eIhyl aceIaIe.
2) Flush Ihe equipmenI wiIh a solvenI miscible Io boIh mobile phases. For example IeIrahydrofuran,
eIhanol.
8) Flush Ihe equipmenI wiIh a solvenI wiIh Ihe composiIion same as 4) and wiIhouI salI, for example
meIhanol/HaO (v/v-60/60).
4) Flush Ihe equipmenI wiIh a solvenI for reversed phase. For example, meIhanol/phosphaIe bufer (v/v-
60/60).
Q.My fow raIe is noI sIable. How can l IroubleshooI?
7. Possible causes for unsIable fow raIe can be a malfuncIionin check valve or air in a mobile phase. Wash
Ihe check valve Ihorouhly by ulIrasonic cleaner. SolvenIs wiIh a low boilin poinI such as n-Hexane and
n-HepIane eneraIe air easily. To prevenI air eneraIion, deas Ihe mobile phase sufcienIly.
Qln spiIe of usin Ihe same condiIion, Ihe reIenIion Iime is diferenI. How can l solve Ihe problem?
7 One possible cause is unsIable fow raIe. Please refer Io Q2 secIion.
AnoIher possible cause is variaIion of polar componenI in mobile phases. ln normal phase chromaIoraphy,
Ihe reIenIion Iime depends on Ihe concenIraIion of small amounIs of very polar consIiIuenIs in Ihe mobile
phase. This is especially Irue for waIer conIenI in a mobile phase. ln Ihis case, always use fresh solvenIs in
a mobile phase. lf sample solvenI includes waIer, chane Io a solvenI wiIhouI waIer or decrease in|ecIion
volume. lf a column conIains waIer, remove waIer from Ihe column by washin iI wiIh eIhanol.
TEOHNIOAL NOTE
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QHow can l wash Ihe COSMOSlL SL- column?
7The SL- column can be washed wiIh IeIrahydrofuran, meIhanol, eIhanol, meIhylene chloride, n-Hexane or
n-HepIane.
QHow can l sIore Ihe COSMOSlL SL- column?
7 SIore Ihe SL- column wiIh Ihe shippin screw IihIen in order Io prevenI Ihe solvenI in Ihe column from
volaIilizaIion. ln case where a solvenI conIainin haloens is used, replace Ihe solvenI in Ihe column wiIh a
solvenI wiIhouI haloens such as n-HepIane before sIorin.
QTCould l choose a mobile phase based on TLC daIa?
QTl eI no peaks. How can l IroubleshooI?
7TFirsI of, make sure IhaI Ihere is no problem wiIh Ihe sysIem. lf Ihe problems are wiIh Ihe sample, sample
solvenI or mobile phase, Iry followin check lisI.
1. The analyIe may noI be eluIed from a column because Ihe reIenIion of Ihe analyIe is Ioo sIron. ln Ihis
case, use a sIroner eluenI (mobile phase).
2. The sample conIains chelaIin compounds or basic compounds. They may be adsorbed Io Ihe packin
maIerials. ln Ihis case, add 0.1% - 1% acid (IrifuoroaceIic acid or aceIic acid) Io Ihe mobile phase.
EA/Hex : eIhyl aceIaIe/hexane sysIem
EIOH/Hex : eIhanol/hexane sysIem
DlE/Hex : diisopropyl eIher/hexane sysIem
0 0.1 0.2 0.8 0.4 0.6 0.6 0.7 0.8
Rf
0
1
2
8
4
6
6
k'
EA/Hex
DlE/Hex
EIOH/Hex
Ql eI peak Iailin in my run. WhaI can l do abouI iI?
7Q ln case where Ihe sample conIains acidic compounds, add approx. 0.6 of aceIic acid Io Ihe mobile
phase.
ln case where Ihe sample conIains basic compounds, add approx. 0.6 of IrieIhylamine Io Ihe mobile
phase.
7T The reIenIion Iimes of HPLC can be deduced from Ihe Rf values of TLC. Refer Io Ihe fure below.
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8.Inner diameter of co|umn (sca|e down and sca|e up)
The fure below shows eneral parameIers for 1.0 mm Io 60 mm l.D. COSMOSlL columns : fow raIe, equipmenI, inner
diameIer of pipe, applicaIion, surface raIio (compared wiIh 4.6 mm l.D.) and parIicle size. lI may help Io scale up or
down from Ihe mosI commonly used 4.6 mm l.D. column.
lnner diameIer
(mm l.D.)
1.0 2.0 8.0 4.6 10 20 28 60
Flow raIe
(ml/min)
0.06 0.2 0.4 1.0 6.0 18 87 70
DeIecIor cell
ln|ecIor
for semi-micro for analyIical for preparaIive
lnner diameIer of
pipe (mm)
0.06 0.1 0.2-0.8 1.0
ApplicaIion
LC-MS
solvenI savin
solvenI savin
wiIh sIandard
sysIem
sIandard
preparaIive
(small scale)
preparaIive
(medium
scale)
preparaIive
(lare scale)
preparaIive
(super lare
scale)
Surface raIio wiIh
4.6 mm l.D.
0.06 0.19 0.48 1.00 4.78 18.90 87.06 118.16
ParIicle size (,m) 8 or 6 6 16 or more
Sca|e down
When scalin down from Ihe mosI commonly used analyIical column (4.6 mm l.D.) Io a semi-micro or 8.0 mm l.D.
analyIical HPLC column (of Ihe same column lenIh), sample loadin dose is proporIionaIe Io Ihe cross secIion of
column. The 8.0 mm l.D. columns provide hih sensiIiviIy and solvenI savin wiIhouI Ihe need Io chane Ihe exisIin
equipmenI seIIins. Semi-micro columns (2.0 mm l.D. and 1.0 mm l.D.) provide hiher sensiIiviIy and enable analysis of
minor componenIs, buI one needs Io chane Ihe pipin of HPLC equipmenI, Ihe in|ecIor and Ihe deIecIor cell for semi-
micro columns.
Column size 4.6 mm l.D. `160 mm 8.0 mm l.D. `160 mm 2.0 mm l.D. `160 mm 1.0 mm l.D. `160 mm
ChromaIoram
Flow raIe (ml/min) 1.0 0.4 0.2 0.06
Pressure (MPa) 8.4 8.6 8.8 8.6
ln|ecIion volume(,l) 1.0 0.4 0.2 0.06
DeIecIor Cellln|ecIor for analyIical for semi-micro
DeIecIor sensiIiviIy(AUFS) 0.08 0.04
lnner diameIer of pipe (mm) 0.26 0.10 0.06
Column COSMOSlL 6Cs-MS- Sample 1. Benzene
Mobile phase aceIoniIrile : waIer - 70 : 80 2. Toluene
Flow raIe 1.0 ml/min 8. EIhylbenzene
TemperaIure 80T 4. Propylbenzene
DeIecIion UV 264 nm 6. BuIylbenzene
6. Amylbenzene
TEOHNIOAL NOTE
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Sca|e up
When scalin up from analyIical column (4.6 mm l.D.) Io preparaIive HPLC (of Ihe same packin maIerial (parIicle size)
and lenIh), sample loadin capaciIy is proporIionaIe Io Ihe cross secIion of column.
Column size 4.6 mm l.D. `260 mm 10 mm l.D. `260 mm 20 mm l.D. `260 mm
ChromaIoram
Flow raIe (ml/min) 1.0 6.0 19.8
Pressure (MPa) 6.6 6.9 6.8
ln|ecIion volume(,) 126 626 2,600
DeIecIor Cellln|ecIor for analyIical for preparaIive
lnner diameIer of pipe (mm) 0.26 1.0
Column COSMOSlL 6SL- TemperaIure 80T
Mobile phase aceIic eIher : eIhanol - 4 : 1 DeIecIion UV 264 nm
Flow raIe 1.0 ml/min Sample TriIon X-100
Oomparison of partic|e size
When chane parIicle size of packin maIerial from 6 ,m Io 16 ,m, Ihe number of IheoreIical plaIe (N) is reduced by
one-Ihird, and Ihe pressure is reduced by one-ninIh. As shown in Ihe fures below, when a small amounI of sample is
in|ecIed, Ihere is a bi diference in Ihe number of IheoreIical plaIes beIween 6 ,m and 16 ,m. However, when a lare
amounI of sample is in|ecIed, Ihere is noI much diference beIween Ihe Iwo. Therefore Ihe low pressure packin maIerial
(parIicle size 16 ,m) is recommended for preparaIive columns (28 mm l.D. or more).
TemperaIure 80T
DeIecIion UV 264 nm
Pressure 6Cs-AR- : 10.9 Mpa
16Cs-AR- : 1.8 MPa
Sample 1. Uracil
2. MeIhyl BenzoaIe
8. Toluene
4. NaphIhalene
6Cs-AR- 16Cs-AR-
The number of IheoreIical plaIe of
N-4. NaphIhalene peak.
ln|ecIion volume - 1 ,l
ln|ecIion volume - 100 ,l
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9.Packing instruction
S|urry packing
1. Use a sIandard open lass column, close Ihe sIopcock, pack a small amounI of absorbenI coIIon in
Ihe boIIom of Ihe column and add solvenI Io approximaIely 1/8 of Ihe column lenIh.
2. Add a Ihin layer (6 mm) of sea sand Io Ihe surface of Ihe absorbenI coIIon.
8. Prepare a slurry soluIion of Ihe packin maIerial (80% w/v) wiIh solvenI rihI before packin. (Make
sure Io prepare enouh slurry soluIion Io form a column bed sufcienI Io separaIe Ihe compounds of
inIeresI.)
4. SimulIaneously open Ihe sIopcock and add Ihe slurry soluIion Io Ihe column Io form Ihe column
bed.
6. AfIer packin Ihe column, wash Ihe newly packed column bed wiIh 6-10 column volumes of solvenI.
Allow Ihe bed Io sIabilize overnihI in solvenI.
6. Add a Ihin layer (6 mm) of sea sand Io Ihe Iop of Ihe bed in order Io prevenI disIurbance of Ihe Iop
of Ihe column bed durin sample or solvenI addiIion.
Dry packing
1. Wash Ihe column in meIhanol and mounI Ihe end fIIin on Ihe boIIom of Ihe dried lass column as
shown in Ihe diaram Io Ihe lefI.
2. Add dry packin maIerial while enIly Iappin Ihe column frequenIly up and down Io insure even
disIribuIion.
8. ConIinue Io add packin maIerial while Iappin unIil Ihe lasI 6 cm of Ihe column lenIh.
4. For Ihe lasI 6 cm, add 1 cm of dry maIerial aI a Iime while Iappin conIinuously.
6. AfIer havin flled Ihe column, weI Ihe surface of Ihe bed wiIh solvenI Io form a faI surface.
6. Remove all maIerial adherin Io Ihe lass rim. MounI and IihIen Ihe end fIIin.
7. Pump 6-10 column volumes of solvenI Ihrouh Ihe column Io remove all air and Io sIabilize Ihe bed.
Oonnecting method
lnserI (1), (2), (8) inIo a 1/16 inches lon Tefon Iube and Ihen IihIen Ihe ferrule properly.
Max. Pressure
Column l.D. (mm) Pressure (MPa)
8 6.0
20 8.0
80 2.0
60 2.0
Oo|umn size and proper ow rate for medium pressure co|umn chromatography
Column l.D. (mm) Column lenIh (mm) Flow raIe (ml/min)
8 - 10 160 - 600 1 - 2
20 160 - 600 4 - 10
80 160 - 600 10 - 26
60 160 - 600 26 - 60
NoIe : To avoid hih pressure aI hih fow raIe, use Iubin
wiIh an l.D. of 0.6 mm or more. lf Ihe flow raIe
becomes very hih, keep Ihe pressure under 2.0
MPa.
The structure of co|umns
Glass FilIer
Glass Iube
NuI
EndfIIin
End plu
8)O-rin Tube
1)Male nuI 2)Ferrule
TEOHNIOAL NOTE
sea sand
packin
maIerial
sea sand
absorbenI
coIIon
sIopcook
Iowel
Iable
packin
maIerial
AFFLICA1ION OA1A
J. Ch|CmatCQ|am lndCX/AppllCatlCn data
E. HC!C|CnCC llSt
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1. Chromatoyram |ndex/AppIication Data
AOOSMOSIL Ohromatogram Index
More Ihan 6,700 sinle compound eluIion profles wiIh full chromaIoraphic condiIion descripIion are available. They are noI
only an incredible help for chromaIoraphers, buI also can be used for reference in choosin condiIions for similar compounds.
AOOSMOSIL App|ication Data
COSMOSlL ApplicaIion daIa is now available on our websiIe. The online version includes more Ihan 1,000 applicaIion daIa
usin COSMOSlL columns. The online daIa is searchable by name of sample and column. lf you have any quesIions reardin
Ihe applicaIion daIa or separaIions of compounds noI lisIed here, please feel free Io e-mail us aI info.inIl@nacalai.co.|p.
These daIa are available aI our web siIe : hIIp://www.nacalai.co.|p/en/cosmosil/index.hIml
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2. Reference List
No. Column TiIle AuIhor Journal, Vol. (lssue), Year, Pae
1 6Cs-MS-
AvailabiliIy of ParIially Milled Rice as a Daily Source of
\-AminobuIyric Acid
K. lwaki and Y. KiIada
Food Science and Technoloy Research, 18(1),
2007, 41-44
2 6Cs-MS-
Molecularly imprinIed polymers for Iriazine herbicides prepared by
mulIi-sIep swellin and polymerizaIion meIhod Their applicaIion Io
Ihe deIerminaIion of meIhylIhioIriazine herbicides in river waIer
H. Sambe, K. Hoshina and J. Hainaka
Journal of ChromaIoraphy A, 1162(1-2), 2007,
180-187
8 6Cs-MS-
Screenin of ACE-inhibiIory pepIides from a random pepIide-
displayed phae library usin ACE-coupled liposomes
Y. Kumada, N. HashimoIo, F. Hasan, M.
Terashima, K. Nakanishi, A. Junbauer and S.
KaIoh
Journal of BioIechnoloy, 181(2), 2007,
144-149
4 6Cs-MS-
CharacIerizaIion of Ihe TriIium-Labeled Analo of L-Ihreo-}-
BenzyloxyasparIaIe Bindin Io GluIamaIe TransporIers
K. ShimamoIo, Y. OIsubo, Y. Shieri, Y.
Yasuda-KamaIani, M. SaIoh, S. Kaneko, and T.
Nakaawa
Molecular Pharmacoloy, 71, 2007, 294-802
6 6Cs-MS- New Cas SIeroidal Glycosides from Tubocapsicum anomalum
N. KiyoIa, K. Shinu, K. Yamauchi, Y.
YoshiIake, K. Harano, H. YoshimiIsu, T. lkeda
and T. Nohara
Chemical & PharmaceuIical BulleIin, 66(1),
2007, 84
6 6Cs-MS-
Cu-caIalyzed arylaIion of phosphinic amide faciliIaIed by ()-Irans-
cyclohexane-1,2-diamine
J. Li, S. Zhan, C. Tao, Y. Fu and Q. Guo
Chinese Chemical LeIIers, 18(9), 2007,
1088-1086
7 6Cs-MS-
DeIerminaIion of Caffeine and Caffeine-RelaIed MeIaboliIes in
Ephedra-ConIainin SIandard Reference MaIerials Usin Liquid
ChromaIoraphy wiIh Absorbance DeIecIion and Tandem Mass
SpecIromeIry
J. B. Thomas, K. E. Sharpless, S. MiIvalsky, M.
Roman, J. Yen, M. B. SaIIerfield
Journal of AOAC lNTERNATlONAL, 90(4),
2007, 984-940
8 6Cs-MS-
Medicinal Flowers. XVl. New Dammarane-Type TriIerpene
TeIralycosides and GasIroproIecIive Principles from Flower Buds of
Panax insen
M. Yoshikawa, S. SuimoIo, S. Nakamura, H.
Sakumae and H. MaIsuda
2007, 1084
9 6Cs-MS-
BioacIive ConsIiIuenIs from Chinese NaIural Medicines. XXlll.
AbsoluIe SIrucIures of New MeasIimane Glycosides, Sedumosides
A+, As, Ae, H, and l, and HepaIoproIecIive MeasIimanes from
Sedum sarmenIosum
K. Ninomiya, T. Morikawa, Y. Zhan, S.
Nakamura, H. MaIsuda, O. Muraoka and M.
Yoshikawa
2007, 1186
10 6Cs-MS-
BioacIive ConsIiIuenIs from Chinese NaIural Medicines. XXlV.
Hypolycemic EffecIs of Sinocrassula indica in Suar-Loaded
RaIs and GeneIically DiabeIic KK-Ay Mice and SIrucIures of New
AcylaIed Flavonol Glycosides, Sinocrassosides A, Aa, B, and Ba
M. Yoshikawa, T. Wan, T. Morikawa, H. Xie and
H. MaIsuda
2007, 1808
11 6Cs-AR-
SeparaIion and ldenIificaIion of Ihe FifIeen ConsIiIuenIs in
ForsyIhiae FrucIus
W. Huan and S. Sheu
Journal of Food and Dru Analysis, 16(1),
2007, 88-89
12 6Cs-AR- New isoflavones from Belamcandae Rhizoma
M. Moriyasu, Y. li, M. lchimaru, K. lwasa, J.
Kobayakawa, F. SaIo-Nishimori, Y. MaIsukawa
and C. Naase
Journal of NaIural Medicines, 61(8), 2007,
829-888
18 6Cs-AR-
SimulIaneous deIerminaIion of anIhraquinones in rhubarb by hih-
performance liquid chromaIoraphy and capillary elecIrophoresis
J. Koyama, l. MoriIa and N. Kobayashi
188-189
14 6Cs-AR- Two New MeasIimanes from Ihe Leaves of Cucumis saIivus H. Kai, M. Baba and T. Okuyama
2007, 188
16 6Cs-AR- Three New MonoIerpenoids from Ihe FruiI of Genipa americana
M. Ono, N. lshimaIsu, C. Masuoka, H.
YoshimiIsu, R. Tsuchihashi, M. Okawa, J. Kin|o, T.
lkeda and T. Nohara
Chemical & PharmaceuIical BulleIin, 66 (4),
2007, 682
16 6Cs-AR- A Novel Oxazolidine Linker for Ihe SynIhesis of PepIide Aldehydes M. Tanaka, S. Oishi, H. Ohno and N. Fu|ii
lnIernaIional Journal of PepIide Research and
TherapeuIics, 18(1-2), 2007, 271-279
17 6Cs-AR-
AcIive SiIe Mappin of Amylo-+-1,6-lucosidase in Porcine Liver
Glycoen Debranchin Enzyme Usin Fluoroenic 6-O-+-Glucosyl-
malIooliosaccharides
E. YamamoIo, Y. Makino and K. Omichi
Journal of BiochemisIry, 141(6), 2007,
627-684
18 6Cs-AR-
AnIi-plaIeleI AreaIion TriIerpene Saponins from Ihe Galls of
Sapindus mukorossi
H. Huan, W. Tsai, C. Liaw, S. Wu, Y. Wu and Y.
Kuo
2007, 1412-1416
19 6Cs-AR-
OxidanIs in Ihe Gas Phase of CiareIIe Smoke Pass Throuh Ihe
Lun Alveolar Wall and Raise SysIemic OxidaIive SIress
Y. Yamauchi, F. Nasu, A. Harada and M.
KuniIomo
Journal of Pharmacoloical Sciences, 108(8),
2007, 276-282
20 6Cs-AR-
OxidaIive couplin of Ihe pyroallol B-rin wiIh a alloyl roup durin
enzymaIic oxidaIion of epiallocaIechin 8-O-allaIe
Y. Li, T. Tanaka and l. Kouno PhyIochemisIry, 68(7), 2007, 1081-1088
21 6Cs-AR-
GomadalacIones A, B, and C: novel 8-oxabicyclo[8.8.0]ocIane
compounds in Ihe conIacI sex pheromone of Ihe whiIe-spoIIed
lonicorn beeIle, Anoplophora malasiaca
H. Yasui, T. Akino, T. Yasuda, M. Fukaya, S.
Wakamura and H. Ono
TeIrahedron LeIIers, 48(18), 2007, 2896-2400
22 6Cs-AR-
ValidaIion of a Novel HPLC MeIhod for Ihe DeIerminaIion of
Raloxifene and lIs PharmacokineIics in RaI Plasma
Zh. Y. Yan, Zh. F. Zhan, X. B. He, G. Y. Zhao
and Y. Q. Zhan
ChromaIoraphia, 66(8-4), 2007, 197-201
28 6Cs-AR- AnIimalarial Flavonol Glycosides from Euphorbia hirIa
Y. Liu, N. Murakami, H. Ji, Pedro Abreu, S.
Zhan
PharmaceuIical Bioloy, 46(4), 2007, 278-281
24 6Cs-AR- HPLC DeIerminaIion of LovasIaIin in RaI Tissue Z. Zhan and Z. Yan ChromaIoraphia, 66(7-8), 2007, 487-491
26 6Cs-AR-
SensiIive deIerminaIion of ko|ic acid in foodsIuffs usin PVP
(polyvinylpyrrolidone) modified aceIylene black pasIe elecIrode
X. Yan, and H. Zhan Food ChemisIry, 102(4), 2007, 1228-1227
26 6Cs-AR-
Rubusuaviins AF, Monomeric and Oliomeric EllaiIannins from
Chinese SweeI Tea and Their + -Amylase lnhibiIory AcIiviIy
H. Li, T. Tanaka, Y.Zhan, C.Yan and l. Kouno
2007, 1826
27 6Cs-AR-
AnalyIical meIhod for deIerminaIion of diniIropyrenes usin as
chromaIoraphyhih-resoluIion mass specIromeIry
T. Nakao, O. Aozasa, S. OhIa and H. MiyaIa
862-867
28 6Cs-AR-
EnzymaIic SynIhesis of Hydroxycinnamic Acid Glycerol EsIers Usin
Type A Feruloyl EsIerase from Asperillus nier
M. Tsuchiyama, T. SakamoIo, S. Tanimori, S.
MuraIa and H. Kawasaki
Bioscience, BioIechnoloy, and BiochemisIry,
71(10), 2007, 2606-2609
1. Reversed phase chromaIoraphy OcIadecyl Iypes (6Cs-MS-6Cs-AR-6Cs-PAQ)
106
I
A
P
P
L
l
C
A
T
l
O
N

D
A
T
A
1 CholesIer
MeIachromins LQ, new sesquiIerpenoid quinones wiIh an amino
acid residue from spone Sponia sp.
Y. Takahashi, T. KuboIa, J. FromonI and J.
Kobayashi
TeIrahedron, 68(86), 2007, 8770-8778
2 CholesIer
PepIide ThioesIer SynIhesis via an Auxiliary-MediaIed N S Acyl
ShifI ReacIion in SoluIion
K. Nakamura, H. Mori, T. Kawakami, H. Ho|o, Y.
Nakahara and S. AimoIo
lnIernaIional Journal of PepIide Research and
TherapeuIics, 18(1-2), 2007, 191-202
8 6PYE
ComparaIive AssessmenI of Oranochlorine ConcenIraIions in
Porpoise Melon and Blubber
S.C. Gardner, G.M. YliIalo, U. Varanasi
Marine Mammal Science, 28(2), 2007,
484-444
4 6PYE
ApplicaIion of ln-Line Liquid ChromaIoraphy-AccuraIe RadioisoIope
CounIin-Mass SpecIromeIry (LC-ARC-MS) Io EvaluaIe MeIabolic
Profile of [8H]-Mefenamic Acid in RaI Plasma
W. Lam, C. Loi, J. AIherIon, W. SIolle, J. EasIer, A.
MuIlib
Dru MeIabolism LeIIers, 1(8), 2007, 179-188
6 6PBB-R
An assessmenI of Ihe reIenIion behaviour of polycyclic aromaIic
hydrocarbons on reversed phase sIaIionary phasesThermodynamic
behaviour on Cs and phenyl-Iype surfaces
S. Kayillo, G. R. Dennis and R. A. Shalliker
188-140
2. Reversed phase chromaIoraphy Special columns
29 6Cs-AR-
EfficienI lncorporaIion of UnsaIuraIed FaIIy Acids inIo VoliciIin-
RelaIed Compounds in SpodopIera liIura (LepidopIera: NocIuidae)
T. Abishi, N. Yoshinaa, K. Noe, R. Nishida and
N. Mori
71(2), 2007, 607-610
80 6Cs-AR-
Clonin and characIerizaIion of a new heIero-ene clusIer of
nonribosomal pepIide synIheIase and polykeIide synIhase from Ihe
cyanobacIerium MicrocysIis aeruinosa K-189
A. Nishizawa, A. B. Arshad, T. Nishizawa, M.
Asayama, K. Fu|ii, T. Nakano, K. Harada and M.
Shirai
The Journal of General and Applied
Microbioloy, 68(1), 2007, 17-27
81 6Cs-AR-
FaIIy Acid Amides, Previously ldenIified in CaIerpillars, Found
in Ihe CrickeI Teleoryllus Iaiwanemma and FruiI Fly Drosophila
melanoasIer Larvae
N. Yoshinaa, T. Aboshi, C. lshikawa, M.
Fukui, M. Shimoda, R. Nishida, C. G. LaiI, J. H.
Tumlinson and N. Mori
Journal of Chemical Ecoloy, 88(7), 2007,
1876-1881
82 6Cs-AR-
Chanes in pimenI conIenI and surface micro-morpholoy of in cuI
carnaIion flower peIals under hih-IemperaIure condiIions
Fukai S., Manabe Y., Yankhamman P, Takamura
T.
The Journal of HorIiculIural Science and
BioIechnoloy, 82(6), 2007, 769-776
88 6Cs-AR-
ProIecIive effecI of Ihe Chinese prescripIion Kanen-karyu aainsI
hih lucose-induced oxidaIive sIress in LLC-PK cells
T. Yokozawa, Y. A. Kim, H. Y. Kim, T. OkamoIo
and Y. Sei
Journal of EIhnopharmacoloy, 109(1), 2007,
118-120
84 6Cs-AR-
Precursor-feedin sIraIey for Ihe producIion of solanine, solanidine
and solasodine by a cell culIure of Solanum lyraIum
M. Lee, J. Chen, C. Lin, Y. Chen and M. Lu Process BiochemisIry, 42(6), 2007, 899-908
86 6Cs-AR-
AnIi-inflammaIory EffecI of Oyaksunisan in Peripheral Blood
Mononuclear Cells from Cerebral lnfarcIion PaIienIs
Y. Kim, H. So, J. Kim, C. Park, J. Lee, W. Woo, K.
Cho, B. Moon and R. Park
Bioloical & PharmaceuIical BulleIin, 80(6),
2007, 1087
86 6Cs-AR-
2-Geranyl-1,4-naphIhoquinone, a Possible lnIermediaIe of
AnIhraquinones in a Sesamum indicum Hairy RooI CulIure
T. FurumoIo, T. Ohara, T. Kubo, Y. Kawanami
and H. Fukui
71(10), 2007, 2600-2602
87 6Cs-AR-
Facile DiscriminaIion of Aldose EnanIiomers by Reversed-Phase
HPLC
T. Tanaka, T. Nakashima, T. Ueda, K. Tomii and l.
Kouno
2007, 899
88 6Cs-AR-
Fenycin anIibioIics isolaIed from B-FS01 culIure inhibiI Ihe rowIh
of Fusarium moniliforme Sheldon ATCC 88982
Hu, Lian Bin, Shi, Zhi Qi, Zhan, Tin, Yan,
Zhi Min
FEMS Microbioloy LeIIers, 272(1), 2007,
91-98
89 6Cs-AR-
DeIecIion of Aminophenylnorharman, a Possible Endoenous
MuIaenic and Carcinoenic Compound, in Human Urine Samples
R. Nishiaki, Y. ToIsuka, H. KaIaoka, H.
Ushiyama, S. GoIo, T. Akasu, T. WaIanabe, T.
Suimura and K. Wakabayashi
Cancer Epidemioloy Biomarkers & PrevenIion,
16, 2007, 161-166
40 6Cs-AR-
UIilizaIion of hydrophobic bacIerium Rhodococcus opacus B-4 as
whole-cell caIalysI in anhydrous oranic solvenIs
S. YamashiIa, M. SaIoi, Y. lwasa, K. Honda, Y.
Sameshima, T. Omasa, J. KaIo and H. OhIake
Applied Microbioloy and BioIechnoloy, 74(4),
2007, 761-767
41 6Cs-AR-
Mechanism of Ihe ProIecIive EffecI of lnIraperiIoneally AdminisIered
AonisIs for Formyl PepIide RecepIors aainsI ChemoIherapy-
lnduced Alopecia
T. Tsuruki, K. TakahaIa and M. Yoshikawa
71(6), 2007, 1198-1202
42 6Cs-AR-
DifferenIial lnIracellular Sinalin Ihrouh PAC1 lsoforms as a
ResulI of AlIernaIive Splicin in Ihe FirsI ExIracellular Domain and
Ihe Third lnIracellular Loop
M. Ushiyama, R. lkeda, H. Suawara, M. Yoshida,
K. Mori, K. Kanawa, K. lnoue, K. Yamada, and A.
MiyaIa
Mol Pharmacol 72, 2007, 108-111
48 6Cs-AR-
SIrucIure-acIiviIy relaIionships of 1'-aceIoxychavicol aceIaIe
homoloues as new nuclear exporI sinal inhibiIors
Y.Liu, N.Murakami, S.Zhan, T.Xu Pharmazie, 62(9), 2007, 669-662
44 6Cs-AR-
MeIhoxy- and Fluorine-SubsIiIuIed Analos of O-1802: SynIhesis
and in ViIro Bindin AffiniIy for Ihe CB1 Cannabinoid RecepIor
S. Tobiishi, T. Sasada, Y. No|iri, F. YamamoIo, T.
Mukai, K. lshiwaIa and M. Maeda
2007, 1218
46 6Cs-AR-
ReacIive Oxyen Scavenin AcIiviIy of MaIured Whiskey and lIs
AcIive Polyphenols
K. Koa, A. Tauchi, S. Koshimizu, Y. Suwa, Y.
Yamada, N. Shirasaka and H. Yoshizumi
Journal of Food Science, 72(8), S212-S217
46 6Cs-AR-
ToIal synIhesis of miraziridine A and idenIificaIion of iIs ma|or
reacIion siIe for caIhepsin B
H. Konno, K. Kubo, H. Makabe, E. Toshiro, N.
Hinoda, K. Nosaka and K. Aka|i
TeIrahedron, 68(88), 2007, 9602-9618
47 6Cs-AR-
Analysis of Polyamines, 1-Aminocyclopropane-1-carboxylic Acid and
Iheir Con|uaIed Forms in Floral Orans of Hibiscus syriacus L.
S. Seo, l. Shim, K. Usui and S. Fu|ihara
Journal of Ihe Japanese SocieIy for HorIiculIural
Science, 76(2), 2007, 149-166
48 6Cs-AR-
Chemical Profilin and Gene Expression Profilin durin Ihe
ManufacIurin Process of Taiwan Oolon Tea OrienIal BeauIy`
J. Cho, M. MizuIani, B. Shimizu, T. KinoshiIa, M.
Oura, K. Tokoro, M. Lin and K. SakaIa
Bioscience, BioIechnoloy, and
BiochemisIry,71(6), 2007, 1476-1486
49 6Cs-AR- BacIerial c-di-GMP ls an lmmunosIimulaIory Molecule
D. K. R. Karaolis, T. K. Means, D. Yan, M.
Takahashi, T. Yoshimura, E. Muraille, D. PhilpoII,
J. T. Schroeder, M. Hyodo, Y. Hayakawa, B. G.
TalboI, E. BrouilleIIe and F. Malouin
The Journal of lmmunoloy, 178, 2007,
2171-2181
60 6Cs-AR-
N-lycans of recombinanI human acid -lucosidase expressed in Ihe
milk of Iransenic rabbiIs
S. P Jonen, G. J Gerwi, B. R Leeflan, K.
Koles, M. LM Mannesse, P. HC van Berkel, F.
R Pieper, M. A Kroos, A. JJ Reuser, Q. Zhou, X.
Jin, K. Zhan, T. Edmunds and J. P Kamerlin
Glycobioloy, 17, 2007, 600 - 619
61 6Cs-PAQ
Medicinal Flowers. Xll. New SpirosIane-Type SIeroid Saponins wiIh
AnIidiabeIoenic AcIiviIy from Borassus flabellifer
M. Yoshikawa, F. Xu, T. Morikawa, Y.
Ponpiriyadacha, S. Nakamura, Y. Asao, A.
Kumahara and H. MaIsuda
2007, 808
62 6Cs-PAQ
ProducIion of 2-PhenyleIhanol in Roses as Ihe DominanI Floral
ScenI Compound from L-Phenylalanine by Two Key Enzymes,
a PLP-DependenI Decarboxylase and a PhenylaceIaldehyde
ReducIase
M. Sakai, H. HiraIa, H. Sayama, K. SekiuIi,
H. lIano, T. Asai, H. Dohra, M. Hara and N.
WaIanabe
71(10), 2007, 2408-2419
107
I
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P
L
l
C
A
T
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O
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D
A
T
A
8. Reversed phase chromaIoraphy OIhers
1 6Cs-MS
EnanIioselecIive synIhesis of d- and l- + -meIhylcysIeine wiIh
hydanIoinase
T. Ohishi, H. Nanba, M. Suawara, M. lzumida,
T. Honda, K. Mori, S. Yanaisawa, M. Ueda, N.
Naashima and K. lnoue
TeIrahedron LeIIers, 48(19), 2007, 8487-8440
2 6Cs-MS
Decreased lnIesIinal CYP8A and P-lycoproIein AcIiviIies in RaIs
wiIh Ad|uvanI ArIhriIis
S. Uno, A. Kawase, A. Tsu|i, T. Tanino and M.
lwaki
Dru MeIabolism and PharmacokineIics, 22(4),
2007, 818-821
8 6Cs-MS
PacliIaxel-2 -EIhylcarbonaIe Prodru Can CircumvenI
P-lycoproIein-mediaIed Cellular Efflux Io lncrease Dru CyIoIoxiciIy
T. Tanino, A. Nawa, E. Kondo, F. Kikkawa, T.
Daikoku, T. Tsurumi, C. Luo, Y. Nishiyama, Y.
Takayanai, K. Nishimori, S. lchida, T. Wada, Y.
Miki and M. lwaki
PharmaceuIical Research, 24(8), 2007,
666-666
4 6TMS-MS
BioderadaIion of nonylphenol polyeIhoxylaIes under Fe(lll)-reducin
condiIions
J. Lu, Q. Jin, Y. He, and J. Wu Chemosphere, 69(7), 2007, 1047-1064
4. Normal phase chromaIoraphy
1 6SL-
PrenylIerphenyllin and lIs Dehydroxyl Analos, New CyIoIoxic
SubsIances from a Marine-derived Funus Asperillus candidus lF10
H. Wei, H. lnada, A. Hayashi, K. HiashimoIo, P.
Pruksakorn, S. Kamada, M. Arai, S. lshida and M.
Kobayashi
The Journal of AnIibioIics, 60(9), 2007,
686-690
2
6SL-
6Cs-AR-
Two New SIeroid Glycosides and a New SesquiIerpenoid Glycoside
from Ihe Underround ParIs of Trillium kamIschaIicum
M. Ono, C. Takamura, F. SuiIa, C. Masuoka, H.
YoshimiIsu, T. lkeda and T. Nohara
2007, 661
6. Saccharide analysis columns
1 Suar-D
OxidaIive couplin reacIion of arbuIin and enIisaIe caIalyzed by
horseradish peroxidase
T. Kiso, M. Shizuma, H. Murakami, T. Kiryu, K.
Hozono, T. Terai and H. Nakano
Journal of Molecular CaIalysis B: EnzymaIic,
46(1-2), 2007, 60-66
2 Suar-D
SIrucIural Basis for ReconiIion of Hih Mannose Type
GlycoproIeins by Mammalian TransporI LecIin VlP86
T. SaIoh, N. P. Cowieson, W. HakamaIa, H.
ldeo, K. Fukushima, M. Kurihara, R. KaIo, K.
YamashiIa, and S. WakaIsuki
J. Biol. Chem., 282(88), 2007, 28246-28266
8 Suar-D
lmprovemenI in lacIic acid producIion from sIarch usin + -amylase-
secreIin LacIococcus lacIis cells adapIed Io malIose or sIarch
K. Okano, S. Kimura, J. NariIa, H. Fukuda and A.
Kondo
Applied Microbioloy and BioIechnoloy,
76(64), 2007, 1007-1018
4 Suar-D CucurbiIane Glycosides from Unripe FruiIs of SiraiIia rosvenori
D. Li, T. lkeda, T. Nohara, J. Liu, Y. Wen, T.
SakamoIo and G. Nonaka
2007, 1082
6 6NHa-MS
Blood LysophosphaIidylcholine (LPC) Levels and CharacIerisIic
Molecular Species in NeonaIes: Proloned Low Blood LPC Levels in
Very Low BirIh WeihI lnfanIs.
TakaIera A., Takeuchi A., Saiki K., Morioka l.,
Yokoyama N., MaIsuo M.
PediaIric Research., 62(4), 2007, 477-482
1 6Cs-AR-800
Porcine erminal anioIensin l-converIin enzyme: lsolaIion,
characIerizaIion and molecular clonin
K. Takeuchi, H. Araki, T. Sakaue, Y. YamamoIo,
M. Fu|iwara, K. Nishi and l. Ohkubo
ComparaIive BiochemisIry and Physioloy ParI
B: BiochemisIry and Molecular Bioloy, 146(2),
2007, 216-226
2 6Cs-AR-800
A Novel Arinine Kinase wiIh SubsIraIe SpecificiIy Towards d
-arinine
K. Uda and T. Suzuki The ProIein Journal, 26(6), 2007, 281-291
8 6Cs-AR-800
TherapeuIic EffecIs of a Re-ComplexCon|uaIed BisphosphonaIe
for Ihe PalliaIion of MeIasIaIic Bone Pain in an Animal Model
K. Oawa, T. Mukai, D. Asano, H. Kawashima, S.
Kinuya, K. Shiba, K. HashimoIo, H. Mori and H.
Sa|i
Journal of Nuclear Medicine, 48(1), 2007,
122-127
4 6Cs-AR-800
Chemical desin of a radiolabeled elaIinase inhibiIor pepIide for Ihe
imain of elaIinase acIiviIy in Iumors.
H. Hanaoka, T. Mukai, S. HabashiIa, D. Asano, K.
Oawa, Y. Kuroda, H. Akizawa, Y. lida, K. Endo,
T. Saa
Nuclear Medicine and Bioloy, 84(6), 608-610
6 6Cs-AR-800
RecombinanI human anIiIhrombin expressed in Chinese hamsIer
ovary cells shows in vivo efficacy on raI DlC model similarly Io
plasma-derived anIiIhrombin reardless of differenI N-lycosylaIion
M. Hirose, M. Tsukada, F. Hirayama, Y. Kubo, M.
Ka|ii, S. Mochizuki, N. and H. Ohi
Thrombosis Research, 119(6), 2007, 681-641
6 6Cs-AR-800
AssessmenI of Re chelaIe-con|uaIed bisphosphonaIe for Ihe
developmenI of new radiopharmaceuIicals for bones.
T. Uehara, Z. Jin, K. Oawa, H. Akizawa, K.
HashimoIo, M. Nakayama, Y. Arano
Nuclear Medicine and Bioloy, 84(1), 2007,
79-87
7 6Cs-AR-800
Assembly of Iwo disIincI dimers and hiher-order oliomers from full-
lenIh Iau
Sahara N., Maeda S., Murayama M., Suzuki T.,
Dohmae N., Yen S., Takashima A.
European Journal of Neuroscience, 26(10),
2007, 8020-8029
8 6Cs-AR-800
Molecular Clonin of cDNA for Trehalase from Ihe European
Honeybee, Apis mellifera L., and lIs HeIeroloous Expression in
Pichia pasIoris
J. Lee, S. SaiIo, H. Mori, M. NishimoIo, M.
Okuyama, D. Kim, J. WonchawaliI, A. Kimura
and S. Chiba
71(9), 2007, 2266-2266
9 6C+-AR-800
Geranyleranyl reducIase involved in Ihe biosynIhesis of archaeal
membrane lipids in Ihe hyperIhermophilic archaeon Archaeolobus
fulidus
M. Murakami, K. Shibuya, T. Nakayama, T.
Nishino, T. Yoshimura and H. Hemmi
FEBS Journal, 274(8), 2007, 806-814
10 6Diol-800-
PolypepIide linkers suiIable for Ihe efficienI producIion of dimeric
scFv in Escherichia coli
Y. Kumada, T. Kawasaki, Y. Kikuchi and S.
KaIoh
Biochemical Enineerin Journal, 86(2), 2007,
168-166
6. ProIein separaIion wide pore columns
7. SpecialIy columns for Fullerene
1 Buckyprep
ProducIion and CharacIerizaIion of HeIeroaIom-encapsulaIed
MeIallofullerene, CaHo@Csa
K. Sakauchi, R. Fu|ii, T. Kodama, H. Nishikawa,
l. lkemoIo, Y. Achiba and K. Kikuchi
ChemisIry LeIIers, 86(7), 2007, 882
2 Buckyprep
SynIhesis and characIerizaIion of difluoromeIhylene-
homo[60]fullerene, Cea(CFa)
A. S. Pimenova, A. A. Kozlov, A. A. Goryunkov, V.
Yu. Markov, P. A. Khavrel, S. M. Avdoshenko, l.
N. loffe, S. G. Sakharov, S. l. Troyanov and L. N.
Sidorov
Chem. Commun, 2007, 874 - 876
108
I
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A
T
l
O
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D
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1 76Cs-OPN
ComposiIions of Royal Jelly ll. Oranic Acid Glycosides and SIerols
of Ihe Royal Jelly of Honeybees (Apis mellifera)
T. Kodai, K. Umebayashi, T. NakaIani, K.
lshiyama and N. Noda
2007, 1628
2 76Cs-OPN
ldenIificaIion and CharacIerizaIion of Cannabinoids ThaI lnduce Cell
DeaIh Ihrouh MiIochondrial PermeabiliIy TransiIion in Cannabis
Leaf Cells
S. MorimoIo, Y. Tanaka, K. Sasaki, H. Tanaka,
T. Fukamizu, Y. Shoyama, Y. Shoyama, and F.
Taura
J. Biol. Chem., 282(28), 2007, 20789-20761
8 76Cs-OPN Microbial meIabolism of sIeviol and sIeviol-16+,17-epoxide
L. Yan, F. Hsu, S. Chan, J. Chen, J. Hsu, C.
Hsu, P. Liu and S. Lin
PhyIochemisIry, 68(4), 2007, 662-670
4 76Cs-OPN + -Glucosidase inhibiIors from Devil Iree (AlsIonia scholaris) N. J. Anurakkun, M. R. Bhandari and J. KawabaIa Food ChemisIry, 108(4), 2007, 1819-1828
6 76Cs-OPN
A GalacIolipid Possesses Novel Cancer ChemoprevenIive EffecIs by
Suppressin lnflammaIory MediaIors and Mouse B16 Melanoma
C.Hou, Y. Chen, J. Wu, C. Huan, S. Wan, N.
Yan and L Shyur
Cancer Research, 67, 2007, 6907-6916
6 76Cs-OPN
EpicaIechin con|uaIed wiIh faIIy acid is a poIenI inhibiIor of DNA
polymerase and anioenesis
K. MaIsubara, A. SaiIo, A. Tanaka, N. Naka|ima,
R. Akai, M. Mori and Y. Mizushina
Life Sciences, 80(17), 2007, 1678-1686
7 76Cs-OPN CharacIerizaIion of a naIural inducer of coral larval meIamorphosis
M. KiIamura, T. Koyama, Y. Nakano and D.
Uemura
Journal of ExperimenIal Marine Bioloy and
Ecoloy, 840(1), 2007, 96-102
8 76Cs-OPN
Tricalysiolide G and Iricalysiols A and B: rearraned enI-kaurane-
Iype and enI-kaurane-Iype diIerpenoids from Ihe leaves of Tricalysia
dubia (Lindl.) Ohwi
D. He, K. MaIsunami, H. OIsuka , T. ShinzaIo, M.
AramoIo, M. Bando and Y. Takeda
46-60
9 76Cs-OPN
A novel noninvasive meIhod for assessin luIaIhione-con|uaIe
efflux sysIems in Ihe brain
T. Okamura, T. Kikuchi, K. Fukushi, Y. Arano and
T. lrie
Biooranic & Medicinal ChemisIry, 16(9), 2007,
8127-8188
10 76Cs-OPN
New prenylaIed flavones from ArIocarpus champeden, and Iheir
anIimalarial acIiviIy in viIro
A. WidyawaruyanIi, Subehan, S. K. Kalauni, S.
Awale, M. NindaIu, N. C. Zaini, D. Syafruddin, P.
B. S. Asih, Y. Tezuka and S. KadoIa
410-418
11 76Cs-OPN
Two New SesquiIerpenoid Glucosides from Ihe Aerial ParIs of
Saussurea involucraIe
X. Wan, S. Gesan, W. Jiao, X. Liao and L.
Din
Journal of lnIeraIive PlanI Bioloy, 49(6),
2007, 609-614
12 76Cs-OPN lnhibiIory EffecIs of 6, 6, 7-Trihydroxyflavones on Tyrosinase H Gao, J Nishida, S SaiIo, J KawabaIa Molecules, 12, 2007, 86-97
18 76Cs-OPN
Molecular Clonin, Expression and ProperIies of an +/
}-GalacIoside + 2,8-SialylIransferase from Vibrio sp. JT-FAJ-16
Y. Takakura, H. TsukamoIo and T. YamamoIo
Journal of BiochemisIry, 142(8), 2007,
408-412
14 76Cs-OPN
PurificaIion, Clonin, and Expression of an +/}-GalacIoside +-2,8-
SialylIransferase from a Luminous Marine BacIerium, PhoIobacIerium
phosphoreum
H. TsukamoIo, Y. Takakura, and T. YamamoIo J. Biol. Chem., 282(41), 29794-29802
16 140Cs-OPN
TLC Analysis of a Corrinoid Compound from Dark Muscle of Ihe
Yellowfin Tuna (Thunnus albacares)
M. Nishioka, Y. Tanioka, E. MiyamoIo, T.
EnomoIo, F. WaIanabe
Journal of Liquid ChromaIoraphy & RelaIed
Technoloies, 80(16), 2007, 2246-2262
16 40Cs-PREP
CP6484, a novel quaIernary carbapenem wiIh poIenI anIi-MRSA
acIiviIy and reduced IoxiciIy
T. Maruyama, Y. YamamoIo, Y. Kano, M.
Kurazono, E. MaIsuhisa, H. TakaIa, T. TakaIa, K.
AIsumi, K. lwamaIsu and E. ShiIara
Biooranic & Medicinal ChemisIry, 16(19),
2007, 6879-6887
8 Buckyprep Bioloical EffecIs of Cea Fullerenes in viIro and in a Model SysIem
S. V. PryluIska, O. P. MaIyshevska, l. l. Grynyuk,
Yu. l. PryluIskyy, U. RiIIer, P. Scharff
Molecular CrysIals and Liquid CrysIals, 468(1),
2007, 266-274
4 Buckyprep
The enIhalpy of formaIion of fullerene fluoride CeaFs and Ihe C-F
bond enery
T. S. Papina, V. A. Lukyanova, A. A.
Goryunkov, l. N. loffe, l. V. GoldI, A. G.
Buyanovskaya, N. M. Kabaeva and L. N. Sidorov
Russian Journal of Physical ChemisIry A, Focus
on ChemisIry, 81(10), 2007, 1660-1664
6 Buckyprep
SynIhesis and molecular sIrucIure of 1,6,11,16,18,
24,27,86-Cea(CFa)s
A. A. Goryunkov, E. l. Dorozhkin, N. B. Tamm, D.
V. lnaIeva, S. M. Avdoshenko, L. N. Sidorov
and S. l. Troyanov
Mendeleev CommunicaIions, 17(2), 2007,
110-112
6 Buckyprep ProducIion of endohedral Xe-hiher fullerenes by ion implanIaIion
S. WaIanabe, T. KaIabuchi, N. S. lshioka, S.
MaIsuhashi, H. MuramaIsu
Journal of RadioanalyIical and Nuclear
ChemisIry, 272(8), 2007, 467-469
7 Buckyprep The former CeaFe is acIually a double-caed adducI: (CeaFe)(Cea)
A. A. Goryunkov, l. N. loffe, P. A. Khavrel, S.
M. Avdoshenko, V. Yu. Markov, Z. Maze|, L. N.
Sidorov and S. l. Troyanov
Chem. Commun., 2007, 704 - 706
8 Buckyprep
FormaIion of sinle-wall carbon nanoIubes in Ar and niIroen as
aImosphere by usin laser furnace Iechnique
S. Suzuki, N. Asai, H. KaIaura and Y. Achiba
The European Physical Journal D - AIomic,
Molecular, OpIical and Plasma Physics,
48(1-8), 2007, 148-146
9 Buckyprep
Two-dimensional hoppin moIion of encapsulaIed La aIoms in
silylaIed Laa@Csa
T. Wakahara, M. Yamada, S. Takahashi, T.
Nakahodo, T. Tsuchiya, Y. Maeda, T. Akasaka,
M. Kako, K. Yoza, E. Horn, N. Mizoroi and S.
Naase
Chem. Commun., 2007, 2680 - 2682
10 Buckyprep
Technoloy for manufacIure of pure fullerenes Cea, C:a and a
concenIraIe of hiher fullerenes
Yu. S. Grushko, V. P. Sedov and V. A. Shilin
Russian Journal of Applied ChemisIry, 80(8),
2007, 448-466
11 Buckyprep
SynIhesis and CaIalyIic AcIiviIy of 1-Allyl and 8-Allyl, EIhyl, and
Hydrido Complexes of RuIhenium-PenIameIhyl[60]fullerene
Y. MaIsuo, T. UemaIsu, E. Nakamura
European Journal of lnoranic ChemisIry,
2007(18), 2007, 2729 - 2788
12 Buckyprep GeneraIion of Cea nanoparIicle aerosol in hih mass concenIraIions
A. GupIa, W. C. ForsyIhe, M. L. Clark, J. A. Dill
and G. L. Baker
Journal of Aerosol Science, 88(6), 2007,
692-608
18 Buckyprep
X-ray sIrucIure and DFT sIudy of C-Cea(CFa)a.
A hih-enery, kineIically-sIable isomer prepared aI 600 C
l. E. Kareev, N. B. ShusIova, D. V. Peryshkov, S.
F. Lebedkin, S. M. Miller, O. P. Anderson, A. A.
Popov, O. V. BolIalina and S. H. SIrauss
Chem. Commun., 2007, 1660 - 1662
14 Buckyprep
Hiher IrifluoromeIhylaIed derivaIives of Cea, Cea(CFa)e and
Cea(CFa)s SynIhesis, sIrucIure, and IheoreIical sIudy
S. l. Troyanov, A.A. Goryunkov, E. l. Dorozhkin, D.
V. lnaIeva, N. B. Tamm, S. M. Avdoshenko, l. N.
loffe, V. Yu. Markov, L. N. Sidorov, K. Scheurel
and E. KemniIz
Journal of Fluorine ChemisIry, 128(6), 2007,
646-661
16
Buckyprep
6PBB
FormaIion of novel Si-fullerene compound maIerials usin a hih-
densiIy silicon-ion plasma
T. HiraIa, R. Suzuki and R. HaIakeyama Thin Solid Films, 616(9), 4177-4181
8. Packin maIerials for reversed phase preparaIive liquid chromaIoraphy

HPLC Columns Cosmosil

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1

s-MS- 4.6 mm l.D.

(Free silanol roup)

Sample collecIion Iube

Cause 2 Column flIer is cloed by sample or impuriIies.

Cause 8 Sample may be adsorbed Io packin maIerial or deposiIed in a column.

Cause 2 Check-valve of pump is cloed by sIain

Cause 8 Manual in|ecIor is cloed wiIh sIain

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