TR02AE

TR02AE-JF15

1

2

34

Unequalled durability against water elution

Imtakt Corp./JAPAN (info@imtakt.com)Imtakt USA (info@imtaktusa.com)North America Other Countries

0 5 10 min

1. fructose(2.5mg/mL)

2. glucose(3.75mg/mL)

3. sucrose(2.5mg/mL)

4. maltose(3.75mg/mL)

Aqueous durable silica-based aminopropyl columns have been used for a long time as a normal phase column for carbohydrate separation. However, these aminopropyl columns have a fatal flaw: "column bleeding" or the rapid deterioration in retention as a result of ligand desorption under aqueous elution.

Our newly-designed Unison UK-Amino offers a different approach from conventional columns: high durability against aqueous eluent. As the above chromatogram demonstrates, conventional columns experience a significant decline in retention when an aqueous mobile phase elutes through the column. UK-Amino, on the other hand, does not show any change in separation or retention. This is a significant development in the history of aminopropyl columns.

UK-Amino's design not only provides analytical power, but the 3um particle high-resolution column has other benefits including the minimization of LC-MS and LC-ELSD noise levels. UK-Amino can be applied to aqueous normal phases conditions, and separation optimization is possible while comparing to ODS columns using reversed-phase mode. One can expect significant results from this normal phase column of UK-Amino.

The times they are a-changin', from 5um to 3um ... Unison series

Aminopropyl type, Normal phase HPLC column

Pure spherical porous silica / 3um particle / Aminopropyl phase

0

20

40

60

80

100

120

0 100 200 300 400 500hours

% tR

sucrose

Unison UK-Amino, 250 x 2 mm

Unison UK-Amino, 250 x 2 mmacetonitrile /water = 75 /25

0.2mL/min (8MPa)37 deg.C, 4uL, ELSD

Conventional Aminopropyl Column

Initial

150 hr

220 hr

400 hr

500 hr

Analytical Conditions

water, 0.1mL/min, ambient

Revolutionary aqueous durability for aminopropyl phaseAqueous to non-aqueous Normal Phase Separation

3um particle high-speed and superior resolutionLC-MS applicable

304

Normal Phase and Anion Exchange Modes

Normal Phase Separation of Saccharides

50% ACN

20% ACN

0% ACN

1% AcOH

0.5% AcOH

75% ACN

3% AcOH

3% AcOH

3% AcOH

3% AcOH

0% ACN

0% ACN

Unison UK-Amino, 250 x 2 mm0.2 mL/min (9-14 MPa)37 deg.C, 260nm2.5mg/mL, 2uL

Aminopropyl stationary phases generally employ both a normal phase separation mode and an anion exchange mode derived from amino groups. There are two methods using Unison UK-Amino to separate ascorbic acid an its isomer isoascorbic acid (erythorbic acid).

Anion exchange3 chromatogramsfrom the bottom

Normal phase + anion exchange

4 chromatograms from the topn the normal phase mode, retention deteriorates as the polarity in the mobile phase rises. However, as the acetonitrile partition rises, retention increases and the two compounds completely separate at 75% acetonitrile. Moreover, the elution order is reversed from anion exchange mode due to the difference in interactions.

Unison UK-Amino provides excellent peak shape with 3um particle for hydrophilic monosaccharides and sugar alcohols separation.

Monosaccharides

1

2

3

4

5

0 5 10min

1

2

3

4

5

Unison UK-Amino, 250 x 2 mmacetonitrile /water = 80 /20, 0.2 mL/min, 37 deg.C, ELSD

glucose

rhamnose

xylose

mannose

fructose

O

OH

OH

OH

HOCH3

O

OH

OHOHHO

OOHOH

HOHO

CH2OH

O

OH

OHOHHO

CH2OH

Sugar Alcohols1

2

3

4

5

Unison UK-Amino, 250 x 2 mmacetonitrile /water = 80 /20, 0.2 mL/min37 deg.C, ELSD

0 5 10 15min

1

2

3 45

maltitol

OH

OHOH

OH

CH2OHO

OHO

OHHO

CH2OH

HOOH

OHOH

meso-erythritol

xylitol

myo-inositol

D-sorbitol

OH

OH

HO

CH2OH

CH2OH

H

HH

CH2OH

CH2OH

HOH

H

OH

OHH OH

H

OH

OH

OH

OH

HO

HO

In anion exchange mode, retention deteriorates as the ionic strength grows larger and pH-driven ionic interactions grow weaker (in this case, high acidic density = low pH). In this example, two compounds are completed separated with only a 0.5% acetic acid aqueous solution.

O

OH

HOCH2OH

HOH2C OH

0 10 20 min

anion exchange

normal phase

1 2

12

12

12

1 2

L-ascorbic acid(Vitamin C)

OHHO

CHOCH2

H OH

OO

isoascorbic acid(erythorbic acid)

OO

OHHO

CHOCH2

H OH

1 2

12

2

316

305 308

Normal Phase Separation of Saccharides

Arabinose, Galactose, Maltitol

Sorbitol and Mannitol

acetonitrile / water = 90 / 1050 deg.C, ELSD

1

D-sorbitol

CH2OH

CH2OH

HOH

H

OH

OHH OH

H

2

D-mannitol

CH2OH

CH2OH

HOH

H

HO

OHH OH

H

1

1

1

2

2

2

0 10 20 min

Unison UK-Amino, 250 x 2 mm0.2 mL/min (5MPa), 0.5 uL (2.5ug)

Unison UK-Amino, 250 x 4.6 mm1 mL/min (6MPa), 1.5 uL (7.5ug)

Brand-N (5um), 250 x 4.6mm1 mL/min (4MPa), 1.5 uL (7.5ug)

Sharp peak shape is possible with sugar alcohols by raising the column temperature settings.

Unison UK-Amino, 250 x 4.6 mmacetonitrile /water = 75 /25, 1 mL/min, 37 deg.C, ELSD

1 2 3

1

2

3

0 5 10 min

D-arabinose

OOH

OHHO

HO

D-galactose

O

OH

OHOHHO

CH2OH

maltitol

OH

OHOH

OH

CH2OHO

OHO

OHHO

CH2OH

Unison UK-Amino, 250 x 4.6 mmacetonitrile /water = 75 /25, 1 mL/min, 37 deg.C, ELSD

1

2

1 2

sucrose lactose

O

OH

OHOH

O

CH2OH

O

OH

OHHO

CH2OHO

OH

HOCH2OH

CH2OHO

OHO

OHHO

CH2OH

0 5 10 min

Sucrose and Lactose in Cafe au Lait

3

313

320 321

Normal Phase Separation of Saccharides

Reducing Sugar and its Reduced Alcohl

Unison UK-Amino, 250 x 4.6 mm,acetonitrile / water = 83 / 17, 1.0 mL/min, 50 deg.C, ELSD

1 2 3 41

2 3

4

0 5 10 15 min

D-glucose maltose

O

OH

OHOHHO

CH2OHO

OH

OHOH

CH2OHO

OHO

OHHO

CH2OH

D-sorbitol

CH2OH

CH2OH

HOH

H

OH

OHH OH

H

maltitol

OH

OHOH

OH

CH2OHO

OHO

OHHO

CH2OH

Cyclodextrins

Unison UK-Amino, 250 x 2 mmacetonitrile / water = 70 / 30 0.2 mL/min, 37 deg.C, ELSD

0 5 10 15 min

0 5 10 15 min

1

2

3 4

5

6 7

Unison UK-Amino, 250 x 2 mmA: acetonitrile, B: water, 25-40 %B (0-15min)0.2 mL/min, 37 deg.C, ELSD

2. laminaribiose (n=0)3. laminaritriose (n=1)4. laminaritetraose (n=2)5. laminaripentaose (n=3)6. laminarihexaose (n=4)7. laminariheptaose (n=5)

1

glucose

O

OH

OHOHHO

CH2OH

O

OH

OHHO

CH2OHO

OHHO

CH2OHO

OH

OHHO

CH2OH

O O

n

Laminariorigosaccharides

Unison UK-Amino provides exceptional separat ion ef f ic iency for ol igosaccharides and other chemical compounds with relatively higher molecular weights.

4

315

310

306

Aqueous Normal Phase Separation

Branched-Chain Amino Acids

Unison UK-Amino can conduct aqueous normal phase separatin even with chemical compounds other than carbohydrates. The column can optimally handle various compounds with its combination of electrostatic interactions and anion exchange mode. In that case, LC-UV/VIS, LC-ELSD and LC-MS is possible by optimizing the organic solvent strength and type, and by adjusting the buffer pH and ionic strength.

Unison UK-Amino, 150 x 2 mmacetonitrile /10mM ammonium acetate = 85 /15 0.2 mL/min, 37 deg.C, ELSD

0 5 10 min

1

2

3

3

L-valine

C C

H

OHOH2N

CH2H3C

H3C1

L-leucine

C C

H

OHOH2N

CH CH2H3C

H3C

2

L-isoleucine

CH2

CH3

H3C C C

H

OHOH2N

CH

In amino acid separation with both electrolytes, sharp peak shape is possible by controlling pH and ion strength with neutral acetate ammonium.

0 10 20 min

Unison UK-Amino, 150 x 2 mmacetonitrile /water /acetic acid = 80 /20 /20.2mL/min (5MPa), 37deg.C, 260nm, 1uL(1.3ug)

0 10 20 min

Unison UK-Amino, 250 x 2 mmwater / acetic acid = 100 / 0.50.2mL/min (14MPa), 37deg.C, 260nm, 0.2uL(0.5ug)

L-ascorbic acid(Vitamin C)

OHHO

CHOCH2

H OH

OO

isoascorbic acid(erythorbic acid)

OO

OHHO

CHOCH2

H OH

1

2

12

1 2anion exchange

normal phase

Ascorbic Acid and Erythorbic AcidAscorbic acid and its isomer erthyorbic acid can be separated in either normal phase or ion exchange modes. Unison UK-Amino can be used with acetic acid, a mild pH adjusting agent. Moreover, separation mode differences allow column users to select different elution orders and separation modes to suit their needs.

5

311

317

Aqueous Normal Phase Separation (Water-soluble vitamins)

Water-Soluble Vitamins

1

2

3

4

5

6

7

O O

OHHO

CHOCH2

H OH

L-ascorbic acid

nicotinamideN

C

O

NH2

nicotinic acidN

C

O

OH

thiamine

riboflavin

N

NH

N

N

O

OH3C

H3C

CH2CH CHCH2OH

OH

CH

OH OH

CH2CH2OHN

N

H3C

CH2 N

S

+ CH3

NH2

Co+NN

NN

CH3

CH3CH3

H3CH3C

H3C

CNCH2CH2CONH2

CH2CH2CONH2

NH2COCH2CH2NH2COCH2

NH2COCH2

CH2CONH2CH3 CH3

CH3

CH3

N

N

HO H

CH2OHOH

HH

C

H3C

O O

O-

O

PCH2CH2CONHCH2

H

cyanocobalamin

folic acid

HNCHC

ONH2N

CH2NHN

N

N C

C

O

CH2CH2OHOH

OH

O

1

2

34

5

6

7

1

2

3

4

5

6

0 10 20 min

Simple analysis is obtainable using acetic acid with water soluble vitamins. There is no need for ion-pair mode via reversed-phase separation. Moreover, gradient elution enables high speed analysis for a wide range of vitamins

Unison UK-Amin, 100 x 4.6 mmA: ACN / acetic acid = 100 / 5B: water / acetic acid = 100 / 52-70 %B (0-10min)1mL/min, 37 deg.C, 260 nm

Unison UK-Amino, 100 x 4.6 mmACN / water / acetic acid = 90 / 10 / 51mL/min, 37 deg.C, 260 nm

0 5 10 min

6

309

Non-Aqueous Normal Phase Separation

Tocopherols Steroids

Unison UK-Amino has a highly polar stationary phase enabling non-aqueous normal phase separation similar to silica columns. However, the presence of a dissociative group (amino group) and bound water in the stationary phase side means that highly reproducible analysis is possible by adding acetic acid and other pH modifiers.

1

2

3

4

Unison UK-Amino, 250 x 4.6 mmhexane / ethyl acetate / acetic acid = 80 / 20 / 0.11 mL/min, 37 deg.C, 295 nm

1

2

3 4

0 5 10 min

OCH2CH2CH2CH

CH3

CH3

CH3H3C

HO

CH3

CH3

3

OCH2CH2CH2CH

CH3

CH3

CH3HO

CH3

CH3

3

OCH2CH2CH2CH

CH3

CH3H3C

HO

CH3

CH3

3

OCH2CH2CH2CH

CH3

CH3HO

CH3

CH3

3

1 2 3

4 5 6

7 8 9

10 11 12

Unison UK-Amino, 250 x 4.6 mmA: hexane /acetic acid = 100 /0.1B: ethanol /acetic acid = 100 /110-30%B (0-30 min), 1 mL/min, 37 deg.C, 260 nm

1

2

3 4

5

67

8

9

10

11

12

0 10 20 30 min

progesteroneO

OC

CH3

corticosteroneO

OC

CH2OH

HO

deoxycorticosteroneacetate

hydrocortisone

cortisone

ethynylestradiol

O

OC

CH2OH

OHHO

O

OC

CH2OH

OHO

prednisolone

prednisone

O

OC

CH2OH

OHHO

6alpha-methylprednisolone

O

OC

CH2OH

OHHO

O

OC

CH2OH

OHO

O

OC

CH2O

O

CCH3

HO

CHC

OH

estriol

HO

OH

OH

estradiolHO

OH

estrone

HO

O

7

307 319

Imtakt Corp./JAPAN ( www.imtakt.com )Imtakt USA ( www.imtaktUSA.com )North America Other Countries

Batch-to-Batch Reproducibility

Ordering Information

Product Code / Price in JPYLength (mm)

1020305075

100150250500

UKA20 / 45,000UKA29 / 45,000UKA21 / 38,000UKA22 / 42,000UKA23 / 45,000UKA24 / 48,000UKA25 / 53,000UKA26 / 68,000

-

Particle Size: 3um, Stationary Phase: Aminopropyl

2 mm I.D.UKA30 / 45,000UKA39 / 45,000UKA31 / 38,000UKA32 / 42,000UKA33 / 45,000UKA34 / 48,000UKA35 / 53,000UKA36 / 68,000

-

3 mm I.D.--

UKA11 / 42,000UKA12 / 45,000UKA13 / 48,000UKA14 / 53,000UKA15 / 58,000UKA16 / 75,000

-

1 mm I.D.UKA00 / 45,000UKA09 / 45,000UKA01 / 38,000UKA02 / 42,000UKA03 / 45,000UKA04 / 48,000UKA05 / 53,000UKA06 / 68,000UKA07/110,000

4.6 mm I.D.--

UKA61 / 42,000UKA62 / 45,000UKA63 / 48,000UKA64 / 53,000UKA65 / 58,000UKA66 / 75,000

-

6 mm I.D.--

UKAP1 / 68,000UKAP2 / 80,000UKAP3 / 95,000UKAP4 /120,000UKAP5 /150,000UKAP6 /180,000

-

10 mm I.D.

23,00017,00023,000

Guard Cartridge UK-Amino

GCUKACGCUKASGCUKAM

5 x 1 mm, 3pcs5 x 2 mm, 3pcs

10 x 8 mm, 2pcs

For 1 mmFor 2 - 6 mm

For 10 mm

Prod.Code Price in JPY NoteSep. column I.D26,000

35,000

Guard Holder

GCH01S

GCH02M

For 1 - 6 mm

For 10 mm

(With a column coupler)

0 5 10min

UKAGD17E

UKAGD16E

UKAGC07D

Conventional aminopropyl stationary phases struggle to achieve solute retention and repeatable separations as the interactions are complicated due to the presence of both normal phase and anion exchange modes. Unison UK-Amino addresses this problem with a novel stationary phase design to provides excellent reproducibility.

Unison UK-Amino stationary phase has an excellent batch-to-batch reproducibility for normal phase + anion exchange mode.

Prod.Code Price in JPYSep. column I.D

Unison UK-Amino, 150 x 2 mmacetonitrile /water /acetic acid = 95 /5 /10.2 mL/min (3.5MPa) 37 deg.C, 270nm

1

2

3

4

5COOCH2CH2CH3

propylbenzoate

thymidinepKa = 9.8

HO

HOCH2 O

O

HN

O

CH3

N

COOH benzoic acidpKa = 4.2

uridinepKa = 9.2

O

HN

NO

HO

HOCH2 O

OH

NH2NC

O

H

HO

3-hydroxyphenylurea

1

2

3

4

5

318