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EfficiEnt REvERsEd-phasE MEthod dEvElopMEnt stRatEgy

acidic compounds

pH selectiv

ity

Basic and neut ral compounds

pH

0

5

10

15

20

25

30

35

40

0 2 4 6 8 10 12

Rete

ntio

n Fa

ctor

(k)

Acid

Base

Neutral

Note: Retention of neutral analytes not affected by pH

Increasedbase retention

Increased acid retention

Hybrid Particle pH Range

Silica pH Range

pH 10.0Acetonitrile

A1A2 A3

ACQUITY UPLC® BEH C18

A1 A2

A3pH 3.0Acetonitrile

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.000.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Minutes

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Minutes

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

pH 3.0Acetonitrile

pH 10.0Acetonitrile

B1

B1

B2

B2

N1

N1

N2

N2

ACQUITY UPLC® BEH Phenyl

Mobile phase pH is the most powerful method development selectivity tool for the separation of ionizable compounds. Large shifts in retention will be observed when changing analytes from their ionized to their unionized state. Screening methods at the extremes of pH (i.e. pH 3 and pH 10) quickly demonstrates the retention characteristics of the target analytes.

optim

iZa

tion

©2008 Waters Corporation. Waters, ACQUITY UltraPerformance LC, T he Science of What’s Possible, ACQUITY UPLC and ACQUITY are trademarks of Waters Corporation. 720001978EN SC-W P

Minutes

4.00 5.00 6.00 7.00 8.00 9.00 10.00

36 oC12,080 PSI

37 oC11,920 PSI

38 oC11,735 PSI

39 oC11,570 PSI

40 oC11,430 PSI

Minutes

4.00 5.00 6.00 7.00 8.00 9.00 10.00

41 oC11,295 PSI

42 oC11,160 PSI

43 oC11,010 PSI

44 oC10,875 PSI

45 oC10,700 PSI

Time10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00

%

1

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00

%

1

*

**

Rate of Change0.75%/ col . vol.

Rate of Change1.5%/ col. vol.

10 µm Particle1970’s

5 µm Particle1980’s

3.5 µm Particle1990’s

1.7 µm Particle2004

Optimal velocity range

ACQUITY UPLC® System

HPLC20

15

10

5

00 1 2 3 4 5 6 7

HET

P (µ

m)

Linear Velocity (µ, mm/sec)Flow Rate [mL/min]:

ID = 1.0 mm

ID = 2.1 mm

ID = 4.6 mm

0.04

0.15

0.7

0.07

0.03

1.4

0.10

0.45

2.1

0.13

0.6

2.8

0.17

0.75

3.5

0.20

0.9

4.2

0.24

1.05

4.9

Changes in temperature will influence the mass transfer of analytes into and out of the pores of the stationary phase and also the mobile phase viscosity. In general, an increase in temperature may resolve partially separated analytes. However, in some extreme cases, significant changes in selectivity can be observed.

Manipulation of the gradient slope can be a very effective way of optimizing a separation. However, as changes in gradient slope do not have a linear effect on resolution, care should be taken in monitoring possible changes in elution order. Changes in gradient slope will also impact sensitivity. Shallower gradient slopes will lead to a reduction in analyte sensitivity.

As can be seen from the van Deemter particle size comparison chart, conventional HPLC particles 3.5, 5 and 10 µm in size have limited optimal linear velocity operating ranges. The minimum point on each curve represents the highest efficiency. The curve for sub-2-µm particles is very flat, resulting in a larger optimal-linear-velocity range. Flow rate can now be utilized as an additional method optimization tool without sacrificing chromatographic efficiency.

smaller part icles enaBle p roduc t iv it yGradient slop einfluenc e of t emp erature

or or

acQuit y uplc® columns

BeH c18

Trifunctional C• 18, fully endcapped, bonded to Ethylene Bridged Hybrid (BEH) substrate

Particle Size 1.7 µm•

Pore Size:135Å; Surface Area: 185m• 2/g; Carbon Load: 18%

pH Range: 1–12; Low pH Temp Limit: 80 ˚C; High pH Temp Limit: 60 ˚C*•

Recommended Usage: General purpose column ideally suited to method development due to extreme pH •stability and applicability to the broadest range of compound classes.

BeH shield rp18

Monofunctional embedded polar C• 18, fully endcapped, bonded to Ethylene Bridged Hybrid (BEH) substrate

Particle Size: 1.7 µm•

Pore Size: 135Å; Surface Area: 185m• 2/g; Carbon Load: 17%

pH Range: 2–11; Low pH Temp Limit: 50 ˚C; High pH Temp Limit: 45 ˚C*•

Recommended Usage: Alternate selectivity to that of straight-chain C• 18, particularly with phenolic analytes. Compatible with 100% aqueous mobile phase composition.

BeH phenyl

Trifunctional C• 6-Phenyl, fully endcapped, bonded to Ethylene Bridged Hybrid (BEH) substrate

Particle Size: 1.7 µm•

Pore Size: 135Å; Surface Area: 185m• 2/g; Carbon Load: 15%

pH Range: 1–12; Low pH Temp Limit: 80 ˚C; High pH Temp Limit: 60 ˚C*•

Recommended Usage: Unique level of pH stability for a Phenyl bonded phase.•

Excellent method development column with alternate selectivity, particularly in regard for polyaromatic • compounds.

Hss c18

High coverage trifunctional C• 18, fully endcapped, bonded to high strength silica (HSS) substrate

Particle Size: 1.8 µm•

Pore Size: 100Å; Surface Area: 230 m• 2/g; Carbon Load: 15%

pH Range: 1–8; Low pH Temp Limit: 45 ˚C; High pH Temp Limit: 45 ˚C*•

Recommended Usage: High performance, general purpose C• 18 column for applications where a modern silica-based C18 selectivity is desired.

Provides superior peak shape for bases, excellent reproducibility and extremely long life times under • highly acidic conditions.

Hss c18 sB

Intermediate coverage trifunctional C• 18, not endcapped, bonded to high strength silica (HSS) substrate

Particle Size: 1.8 µm•

Pore Size: 100Å; Surface Area: 230 m• 2/g; Carbon Load: 8%

pH Range: 2–8; Low pH Temp Limit: 45 ˚C; High pH Temp Limit: 45 ˚C*•

Recommended Usage: Designed and optimized for method development where a C• 18 chemistry that provides alternate selectivity is desired.

Provides alternate Selectivity for Bases (SB) in low pH separations.•

Hss t3

Intermediate coverage trifunctional C• 18, fully endcapped, bonded to high strength silica (HSS) substrate

Particle Size: 1.8 µm•

Pore Size: 100Å; Surface Area: 230 m• 2/g; Carbon Load: 11%

pH Range: 2–8; Low pH Temp Limit: 45 ˚C; High pH Temp Limit: 45 ˚C*•

Recommended Usage: Specifically designed for enhanced retention of polar analytes.•

Extreme retentivity and full compatibility with 100% aqueous mobile phases.•

* Recommended pH and temperature limits for maximum column lifetime.

Minutes Minutes

ACQUITY UPLC® HSS T3 ACQUITY UPLC® HSS T3

A1 A2

A3

A1

A3

A2

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.000.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

ACQUITY UPLC® BEH Phenyl

B1 B2N1

N2

B1B2N1

N2ACQUITY UPLC® BEH Phenyl

Acetonitrile, pH 3.0 Methanol, pH 3.0

Acetonitrile, pH 10 Methanol, pH 10

Once the mobile phase pH has been selected, the next step is to evaluate the effects of different organic modifiers. Acetonitrile and methanol possess quite different chemical properties which can result in significantly different chromatographic elution profiles.

As methanol is significantly lower in elution strength, most analytes will be retained longer on the LC column when 100% methanol is used. For very challenging separations, a combination of acetonitrile and methanol as the organic modifier will provide additional selectivity.

2.1 x 50 mm ACN MeOH ACN MeOH

acQuity uplc® BeH c18 • • • •

acQuity uplc® BeH shield rp18 • • • •

acQuity uplc® BeH phenyl • • • •

acQuity uplc® Hss t3 • •

Chromatographic Conditions

Column Dimensions: 2.1 x 50 mm, 1.7 or 1.8 µmMobile Phase A1: 20 mM NH4COOH in H2O, pH 3.0Mobile Phase A2: 20 mM NH4HCO3 in H2O, pH 10.0Mobile Phase B1: AcetonitrileMobile Phase B2: MethanolFlow Rate: 0.5 mL/min Gradient: Time Profile Curve (min) %A %B 0.0 95 5 6 5.0 10 90 6 5.01 95 5 6 5.5 95 5 6 Injection Volume: 10.0 µLWeak Needle Wash: 3% methanolStrong Needle Wash: 90% acetonitrileTemperature: 30 ˚CDetection: UV @ 254 nmSampling Rate: 20 pts/secTime Constant: 0.1Instrument: Waters ACQUITY UPLC®, with Column Manager and ACQUITY™ PDA

O Si

O

O

O SiO

O

O SiO

O

O Si

CHPolar Group

3

CH3

O SiO

O

O Si

O

O

O SiO

O

O SiO

O

O Si

CHPolar Group

3

CH3

O SiO

O

O Si

O

O

O SiO

O

O SiO

O

O Si

CHPolar Group

3

CH3

O SiO

O

O Si

O

O

O SiO

O

O SiO

O

O Si

CHPolar Group

3

CH3

O SiO

O

O Si

O

O

O SiO

O

O SiO

O

O Si

CHPolar Group

3

CH3

O SiO

O

O Si

O

O

O SiO

O

O SiO

O

O Si

CHPolar Group

3

CH3

O SiO

O

colu

mn

selection

orG

an

ic mo

difier selectiv

ity

metHod dev elopment select iv it y scout inG protocol

st ep 1 rev ersed-pHase ret ent ion map: tHe importance of moBile pHase pH

st ep 2 orGanic modifier select ion

st ep 3 metHod opt imiZat ion

pH 10.0Acetonitrile

A1A2 A3

ACQUITY UPLC® BEH C18

A1 A2

A3pH 3.0Acetonitrile

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.000.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Minutes

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Minutes

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

pH 3.0Acetonitrile

pH 10.0Acetonitrile

B1

B1

B2

B2

N1

N1

N2

N2

ACQUITY UPLC® BEH Phenyl

ethylene Bridged Hybrid (BeH) phases

High strength silica (Hss) phases

pH 3 pH 10

Acetonitrile vs. Methanol

Aprotic [non-H-bonding] Protic [H-bonding]

Higher Elution Strength Lower Elution Strength

Lower Viscosity Higher Viscosity

[0.32 cP] [0.65 cP]