advantages of polymer- based hplc columns – an … denko europe gmbh 1 advantages of polymer-based...

Showa Denko Europe GmbH 1

Advantages of polymer-based HPLC columns

an alternative for ODS

Yukiko Higai

Shodex, Showa Denko Europe GmbH

Showa Denko Europe GmbH

Content

(1) Comparison between Silica and Polymer-based

RP columns

(2) Characteristics of Polymer RP columns

- an example: a new column type

(3) Characteristics of Polymer HILIC columns

2


HPLC

60-70% of samples

can be applied

Reversed Phase (RP)

(mainly ODS)

Size ExclusionIon Exchange

(Ion Chromatography)Normal PhaseLigand Exchangeetc.

Use of HPLC in general

3


Silica-based exODS Polymer-based exODP

Higher resolution Relatively lower resolution

Reasonable price Relatively higher price

Narrow pH range pH2-7 Wide pH range pH2-12

Impossible to use underthe low salt concentration

Possible to use underthe low salt concentration

Short life time Long life time

Impossible to injectlarge concentration sample

Possible to inject large concentration sample

Not enough separation of hydrophilic substances

Good separation ofhydrophilic substances

Need ion-pair reagent to separate basic substances

Possible to separate basic substances without any reagent

(1) Comparison between Silica and Polymer-based RP


Problems of Silica-based RP columns

O

O

O

Si OH

Si OH

O

Si OH

O

O

OHSi

O

OHSi

Si O Si

Si O Si

OSi Si

Residual silanol capping

difficult to remove ionic adsorption

Lot-to-lot differences

effect of metal coodination, difficult to control residual silanols

Low durability against solvents

Si

O

Si

OHSi

OH

Si

HO

O Si C18

OH

OH

5


Sample suitable for ODS analysis

Suitable for ODS Unsuitable for ODS

Molecularsize

LowHigh also sample including

high MW impurities

PolarityCompounds

with different polarityCompounds

with similar polarity

Low to Moderate High or Ionic

Solution Acidic or Neutral Basic

6

other choice Polymer


(2) Characteristics of polymer-based RP columns

a) Separation Almost the same

b) Analysis of basic sample Good

c) pH range in eluent Wide

d) Effect of salt concentration in eluent Low

e) Reproducibility Good

X=OR, COOR, etc

Si

O

Si

O

O

O

H

SiC

CH

X


a) Separation performance (1)

Polymer-based Silica-based

Eluent : CH3CN / H2O = 50 / 50

Flow rate : 1.0mL/min

Detector : UV (262nm)

Column temp. : 40oC

Almost the same separation

Sample : 5L1. 0.2% Methylethyl ketone

2. 0.2% Diethyl ketone

3. 0.4% Di-n-propyl ketone

4. 0.6% Di-n-butyl ketone


a) Separation performance (2)

Column : Shodex RSpak DE-413

ODS column from other manufacturer

Eluent : 10mM H3PO4 aq.


Detector : RI

Column temp. : 50 oC

Sample : 0.2% each,

5L

1. Lactitol

2. Erythritol

Better separation

of hydrophilic samples

9


Amine

compounds

R-N+

-O-SiResidual

Silanol

adsorption

Polymer-based column Silica-based column

Tends to cause tailing( ion-pair reagent needed)

Less Tailing

b) Analysis of basic compounds (1)

10


b) Analysis of basic compounds (2)

(ODS)

Eluent : 0.1% 1-Pentanesulfonic acid

sodium salt aq./CH3CN=80/20


Column temp. : 60oC

ODP-50no need

ion-pair reagent

(ODP-50 4D)

Eluent : 50mM Phosphate buffer (pH10)

/CH3CN=80/20


Column temp. : 30

ODSwith

ion-pair reagent

no need of ion-pair reagent

11


c) Better resolution in high pH (1)

Durability in Alkaline condition

better selectivity

higher TPN

for example

ODS

1 7 14

ODP

2 13

Available pH

Good for the separation

of basic sample under

alkaline conditions

12


pH 11pH 7

Basic sample (local anesthetic drug)

Column: Shodex Asahipak ODP-50 4DEluent: 25mM Phosphate buffer/CH3CN (60/40)

Flow Rate: 0.6mL/min

Detector: UV(254nm)

Column temp.: 30oC

pH 3

Working condition of

Polymer column!

13

c) Better resolution in high pH (2)


Sample : 1mL/mL Scopolamine50LODS-A ODS-C

Low concentration for ionic drugs

excellent for LC/MS

ODP-50

25m

M10m

M1m

M

Column : Shodex Asahipak ODP-50 4D

ODS Columns from other manufacturer

Eluent : Phosphate buffer(pH7.0)/CH3CN=60/40


Detector : UV (254nm)

Column temp. : 30 oC

14

d) Effect of salt concentration in analysis of ionic drugs


e) Reproducibility

Rt

(min)

Lot A Lot B Lot C CV (%)

1 2.504 2.505 2.482 0.425

2 3.148 3.152 3.123 0.409

3 4.878 4.894 4.847 0.400

4 7.961 8.001 7.920 0.415

Peak

area

(UV x

sec)


1 36616 37614 36414 1.422

2 32168 33166 31934 1.648

3 48560 50090 48075 1.756

4 64814 66686 64276 1.583

Peak

area

(%)


1 20.102 20.055 20.151 0.195

2 17.659 17.683 17.673 0.056

3 26.658 26.706 26.605 0.155

4 35.581 35.555 35.571 0.030

Control of batch differences

15


When ODS is not suitable

Problem Solution

ODP2 HPHigh MW impurityHigh polarity (ionic)

16

- an example: a new column type

ex) direct analysis of drugs in biological fluids


Characteristics of polymer-based RP ODP2 column

proteindrug with low Mw

ODSODP2 HP

Polyhydroxymethacrylate gel Silica gel

ca. 100-120ca. 40

More adsorption of proteinsLess adsorption of proteins

Advantages:Hydrophilic surface of packing

material

Small pore size

Proteins will be removed without sample pre-treatment

less time-consuming

17


a) Analysis of high polarity substances

-1.00

-0.50

0.00

0.50

1.00

-1 0 1 2

log P

log

k'

ODP2 HP

ODP-50

ODS(B)ODS(A)

High

Lo

wH

igh

Ret

enti

on

Polarity Low

Column : Shodex ODP2 HP-4D (4.6mmID x 150mm)

Eluent : H2O/CH3CN=75/25


Column temp. : 40oC

Strong retention of high polarity substances


ODP2 HP-4D Polymer-based RP column (Shodex Existing column)

3123

4

15

45

6

N: 13,200

7

5

6N: 5,700

7

1 2

0 5 10 0 5 1015 min min

Sample : 5L

1. Uracil 30mg/L2. Theobromine 75mg/L

3. Caffeine 130mg/L

4. Phenol 300mg/L

5. Methyl benzoate 350mg/L

6. Toluene 1000mg/L

7. Naphthalene 150mg/L

Column : Shodex ODP2 HP-4D(4.6mmID x 150mm)

Eluent : H2O/CH3CN=55/45Flow rate : 0.6mL/min

Detector : UV(254nm)Column temp. : 40oC

Column : Shodex Asahipak ODP-50 4D(4.6mmID x 150mm)

Eluent : H2O/CH3CN=35/65Flow rate : 0.6mL/minDetector : UV(254nm)Column temp. : 40oC

hydrophilic

Better separation for hydrophilic materials

b) Analysis of hydrophilic compound


90909090

100100100100

110110110110

120120120120

130130130130

140140140140

150150150150

160160160160

0000 20202020 40404040 60606060 80808080 100100100100 120120120120 140140140140

Injection number

Pre

ssu

re c

han

ge

rati

o [

%]

Column : ODP2 HP-2B (2.0mmID x 50mm), ODS(A) (2.0mmID x 50mm)Eluent : 1mM CH3COONH4 aq./CH3CN=90/10Flow rate : 0.2mL/minDetector : UV (220nm)Column temp. : 30oC

c) Removing proteins

ODP2 HP-2B

ODP2 HP-2BODS(A)

Sample : 5L

BSA 7.0mg/mL

Proteins are not adsorbed in ODP2 HP and the pressure did not increase.

No adsorption

no C18 end-capping

Hydrophilic surface

minminminmin0000 0.50.50.50.5 1.01.01.01.0 1.51.51.51.5 2.02.02.02.0

Longer column life


d) LC/MS

Column : ODP2 HP-2B (2.0mmID x 50mm) Eluent : 10mM CH3COONH4 aq./CH3CN=70/30Flow rate : 0.2mL/minDetector : UV (220nm), ESI-MS Column temp. : 30oC

Sample :

1. Barbital 500g/L

2. BSA 7.0mg/L

There is no ion suppression by protein.

SIMSIMSIMSIMSIM

m/zm/zm/zm/zm/z ====183.2183.2183.2183.2=183.2

SIMSIMSIMSIMSIM

m/zm/zm/zm/zm/z ====183.2183.2183.2183.2=183.2

BarbitalBarbitalBarbitalBarbitalBarbital

Peak Area : Peak Area : Peak Area : Peak Area : 100100100100%%%%Peak Area : 100%

Peak Peak Peak Peak Peak HightHightHightHightHight : : : : 100100100100%%%%: 100%

BarbitalBarbitalBarbitalBarbitalBarbital

Peak Area : Peak Area : Peak Area : Peak Area : 107107107107%%%%Peak Area : 107%

Peak Peak Peak Peak Peak HightHightHightHightHight : : : : 102102102102%%%%: 102%

BarbitalBarbital

UV (220nm)

0 1.0 2.0 3.0 4.0

Time (min)

100000

200000

300000

Inte

nsi

ty

0 1.0 2.0 3.0 4.0

Time (min)

0

100000

200000

300000

Inte

nsi

ty

0 1.0 2.0 3.0 4.0

Time (min)

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

uA

U Inject to MS

UV (220nm)

0 1.0 2.0 3.0 4.0

Time (min)

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

uA

U

0.120.120.120.12

Inject to MS

Blank with BSA

BSA

Sample pre-treatment

Pore size: 40SEC effect to

remove protein

Bar

bit

al r

eco

very

rat

io [

%]

ODP2 HP Blank

ODP2 HP with BSA

BarbitalPeak Area :100%

0.0 1.0 2.0 3.0 4.0

Time (min)

Inte

nsi

tyIn

ten

sity

0.0 1.0 2.0 3.0 4.0

Time (min)

0

20000

40000

60000

80000

100000

Inte

nsi

ty

0

20000

40000

60000

80000

100000

Inte

nsi

ty

0

20000

40000

60000

80000

100000

0

20000

40000

60000

80000

100000

ODS(A)Blank

ODS(A) with BSA




TIC

m/Z 182.5-183.5

TIC

m/Z 182.5-183.5


(3) Characteristics of Polymer-based amino column (HILIC)

a) Enables sugar analysis at room temp.

(alkali atmosphere)

b) Long life time/stability.

Regeneration possible with alkali solvents

c) Reduced noise level because of less bleeding

22


Comparison with an amide column at low temp.

Column : Shodex Asahipak NH2P-50 4E (4.6mmID x 250mm), Amide column from another manufacturer (4.6mmID x 250mm)

Eluent : CH3CN / H2O = 75 / 25

Flow rate : 1.0mL/min Detector : Shodex RI Column temp. :30deg-C

a) Sugar analysis at room temp.

Amino columnAlkali atmosphere due to amino groups

No anomers even at room temp.

Amide column

Not alkali atmosphere

(acryl amide groups)

Need high temp. to avoid anomers


Silica

-based

Polymer-based

Comparison with a silica-column in Life timeb) Long life time/stability


c) Reduced noise level less bleeding

Silica Amino column

Polymer Amino ColumnAbsolute amount 500 ng 100 times better

Noise level: 30pA

Lower limit (S/N=3)

Absolute amount 100 ng

Noise level: 0.8pA

Lower limit (S/N=3)


Reduced noise level because of less bleeding

Source: LMS CO., LTD. Japan

25



Column : Shodex Asahipak NH2P-50 4E (250mmLx4.6mmID) Eluent : (A) CH3CN / H2O = 50 / 50

(B) 0 to 7min CH3CN=57/43 to 50/50 (Linear gradient) 7 to 16min CH3CN=50/50 Flow rate : 1.0mL/min

Detector : ELSD Column temp. : 30oC Sample : 0.5%, 20 L

Sample : 0.5% Hydrolyzed dextran, 20 L

Poly- and oligosaccharides

Gradient mobile phase enables a better separation of hydrolyzed dextran in Mw. ELSD detectionis effective.


Summary: Polymer vs ODS

1. Separation performance similar

Good separation for hydrophilic substances

2. Better separation of basic substances

Possible to analyze trace level of basic substances

No need of ion-pair reagent

3. Alkaline solvent can be used

Wide selection of solvent for analyses

4. Low influence of salt

Better peak shape under low concentration of salt

lower ion suppression

5. Good Reproducibility

27


for details >>>

www.shodex.de

www.bioanalytics.co.il

Thank you for your attention!

advantages of polymer- based hplc columns – an … denko europe gmbh 1 advantages of polymer-based...

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