[ ]1

XBridge™ HPLC Columns

Unrivaled Versatility and Performance

The XBridge™ family of HPLC columns is designed for maximum flexibility in HPLC methods

development. Robust separations can be developed in less time and with greater confidence,

allowing chromatographers to fully utilize the power of pH. By combining cutting-edge bonding,

endcapping and particle synthesis with industry-leading manufacturing and quality, XBridge

columns are the trusted solution for your most challenging chromatographic separations.

■ Flexibility to work over an unparalleled range of mobile phase pH and temperature

■ Seamless method migration to UltraPerformance LC® technology

■ Unmatched column lifetime

BEH TEcHnology

[ ]4

First introduced in 1999 in the XTerra® family of HPLC columns, Waters patented organic/inorganic Hybrid Particle Technology (HPT)* surmounted significant limitations of silica-based reversed-phase packing materials: particularly hydrolytic instability at high pH. The second generation HPT, known as BEH Technology™, incorporated in both ACQUITY UPLC® BEH columns and XBridge HPLC columns, marks a new milestone in chromatography. Finally, the full power of pH can be used in routine LC methods development.

*U.S. Patents 6,686,035; 7,223,473; 7,250,214

In BEH particle synthesis the bis (triethoxysilyl) ethane monomer contains a preformed ethylene bridge. This ethylene bridge imparts the high pH dissolution resistance of polymers into the silica particle backbone without sacrificing any of the silica particle’s structural integrity or efficiency.

BEH T EcHnology ParT iclE SynT HESiS

ParT iclE DiSSoluT ion aT HigH pH

3OH-

Si

Si(OH)4

O

O O Si

O

O Si

O

O Si

O

O Si

O

O Si

O

OHOH

OSi

O

O O

O O Si

O

O O O O

O Si

O

O O O O O O O

OH OH OH

SiO O Si O Si O Si O Si O Si OSiO O Si O Si

Core

SiO O Si

O

O Si

O

O Si

O

O Si O Si

O

OHOH

OSi

O

CH2 O

O O Si

O

OH OH O

O Si

CH2

CH2

O O O O O O O

OH OH CH2 CH2

CH2

CH2

SiO O

O

Si O Si O Si O Si O Si OSi

Si

O

O

O

Si

Si

O

CH2

OH

Si O O

O

O

O

O Si O Si

Core

3OH-

Si

Si(OH)4

O

O O Si

O

O Si

O

O Si

O

O Si

O

O Si

O

OHOH

OSi

O

O O

O O Si

O

O O O O

O Si

O

O O O O O O O

OH OH OH

SiO O Si O Si O Si O Si O Si OSiO O Si O Si

Core

SiO O Si

O

O Si

O

O Si

O

O Si O Si

O

OHOH

OSi

O

CH2 O

O O Si

O

OH OH O

O Si

CH2

CH2

O O O O O O O

OH OH CH2 CH2

CH2

CH2

SiO O

O

Si O Si O Si O Si O Si OSi

Si

O

O

O

Si

Si

O

CH2

OH

Si O O

O

O

O

O Si O Si

Core

■ Only 4 bonds need to be hydrolyzed ■ Up to 6 bonds need to be hydrolyzed

■ Si(OH)4 has high solubility in water ■ Hydrophobic, lower reactivity than silica

■ Easy to dissolve silica at pH > 7 ■ Si-O-Si bonds form as others break

■ Loss of efficiency

Silica Particle XBridge Particle

Tetraethoxysilane(TEOS)

Polyethoxysilane(BPEOS)

Bis(triethoxysilyl)ethane(BTEE)

+4

Si

EtO

EtO OEtEtO

Si

E tOE tO

EtO

Si

OEt

OEt

OEt

Si

E tO

O

CH2 CH2

CH2

CH2

Si O Si

E tO

OE t

Si

O

O

OEt

Si

O

SiOO

Et

OOO

Et

E t Et n

Anal. Chem. 2003, 75, 6781-6788

BEH TEcHnology

[ ]5

Hybrid Particle attribute

Surface hybrid groups reduce surface silanol concentration

Internal bridging groups provide high interconnectivity

Internal and surface hybrid groups add hydrophobicity

BEnEfiTS of HyBriD T EcHnology

BEH Technology affords chromatographers the flexibility to scale between UltraPerformance LC, analytical and preparative scale chromatographic platforms. Combining BEH Technology with patented Optimum Bed Density (OBD™) preparative column design, XBridge Prep OBD columns provide high loadability, maximum efficiency and long column lifetime. Both XBridge HPLC particles and 1.7 µm ACQUITY UPLC BEH particles feature BEH Technology, thus enabling seamless migration of HPLC separations to UPLC Technology. Whether a chromatographer is interested in sub-2 µm UPLC separations, developing purification methods or analytical scale separations, BEH Technology provides precise and reliable solutions.

Benefit for rP-HPlc

Reduced USP peak tailing factors for bases

Increased chemical & mechanical stability

Increased high pH stability of column

Peptide Separation Technology

Oligonucleotide Separation Technology

ACQUITY UPLC BEH Columns

XBridge Columns

Bioseparations BEH Columns

XBridge OBD Prep Columns

BEH T EcHnology P roDucTS

xBridgE c18/c8

[ ]6

XBridge C18/C8

The two most popular HPLC phases, C18 and C8, are the workhorses of LC methods development. Useful for a wide variety of separations, these columns are familiar to all separation scientists. XBridge C18 and C8 columns offer methods development flexibility. By employing trifunctional bonding and advanced endcapping, excellent reproducibility, performance and column lifetime across the entire pH range (1-12) is observed.

* Nominal Values

O Si

O

O

O SiO

O

O SiO

O

O Si

CHPolar Group

3

CH3

O Si

O

O

O SiO

O

O SiO

O

O Si

CHPolar Group

3

CH3

Ligand Type Trifunctional C18 Trifunctional C8

Available Particle Sizes (µm) 2.5, 3.5, 5, 10 2.5, 3.5, 5, 10

Ligand density* 3.1 µmol/m2 3.2 µmol/m2

Carbon Load* 18% 13%

endcap Style Proprietary Proprietary

pH range 1-12 1-12

Low pH Temp. Limits 80 °C 60 °C

High pH Temp. Limits 45 °C 45 °C

USP L1 L7

Pore diameter* 135Å 135Å

Pore Volume* 0.7 mL/g 0.7 mL/g

Surface Area* 185 m2/g 185 m2/g

Bond

ed P

hase

BEH

Parti

cle

XBridge C18 and C8 Column Characteristics

low-pH STaBil iT y

Low-pH instability is due to siloxane bond cleavage via acid hydrolysis. In order to improve the low-pH stability of bonded phases, the rate of hydrolysis must be slowed. The XBridge C18, C8 and Phenyl chemistries utilize proprietary trifunctional bonding and endcapping to accomplish this. Under accelerated low-pH test conditions, XBridge C18 columns show little retention loss (greater hydrolytic stability) when compared to columns packed with phases prepared using monofunctional silanes.

HigH-pH STaBil iT y

The failure of silica-based chromatographic particles at elevated pH due to nucleophilic attack by hydroxide ions on its structural siloxane bonds. Chromatographically, this results in column voids, peak shape degradation and/or efficiency loss. XBridge particles incorporate ethylene bridges within the silica matrix which resist particle dissolution. Up to six siloxane bonds would need to be broken to free a single ethylene-bridged unit from a particle. Chemical stability is demonstrated in the accelerated high-pH lifetime shown below.

Zorbax® SB C18

XBridge C18

SunFire C18

Intersil® ODS-3

Ace® C18

Gemini™ C18

Luna® C18 (2)

0 20 40 60 80 100

Hours in 1% TFA at 80 ˚C to 20% Retention Loss

XBridge C18

XTerra MS C18

Gemini™ C18

Zorbax® Extend C18

Luna® C18 (2)

YMC™ Pro C18

0 50 100 150 200

Hours in 50 mM TEA at 50 ˚C to 50% Efficiency Loss

ManiPulaTing pH for oPTiMal METHoDS DEvEloPMEnT

In reversed-phase chromatography, mobile phase pH can impart selectivity and retentivity changes for ionizable compounds. Acidic compounds show increased retention at pH values below their pKa, and basic compounds exhibit longer retention at pH values above their pKa. Many pharma-cologically active compounds are ionizable; thus a column with a wider usable pH range enables greater flexibility for pharamaceutical methods development. XBridge columns have a wide usable pH range (pH 1-12), enabling unrivaled methods development flexibility.

Lincomycin hydrochloride is a well established antibiotic drug used in human and veterinary medicine, effective primarily against gram-positive pathogens. T he current USP method suffers from poor peak shapes and sensitivity. In developing an improved method, pH can be utilized to realize improved peak shape and resolution.

Once pH has been selected, gradient slope was optimized to improve resolution.

xBridgE c18/c8

[ ]7

AU

AU

AU

0

0.5

0

0.5

0

0.5

0 5 10 15 min

Column: XBridge C18, 3.0 x 100 mm, 3.5 µmMobile Phase A1: 100 mM Potassium phosphate, pH 2Mobile Phase A2: 100 mM Potassium phosphate, pH 7Mobile Phase A3: 100 mM Potassium phosphate, pH 12Mobile Phase B: ACNMobile Phase C: H2OFlow Rate: 0.6 mL/minGradient: Time Profile (min) %A %B %C 0.0 20 5 75 15.0 20 50 30 Temperature: 30 °CInjection Volume: 20 µL @ 30 mg/mL in waterDetection: UV @ 215 nm Instrument: Waters Alliance® 2695 with 2996 PDA

0 5 10 15 min

Column: XBridge C18, 3.0 x 100 mm, 3.5 µmMobile Phase A: 100 mM Potassium phosphate, pH 12Mobile Phase B: AcetonitrileMobile Phase C: WaterGradient: Time Profile (min) %A %B %C 0.0 20 15 65 15.0 20 35 45 Flow Rate: 0.6 mL/minInjection Volume: 20 µL @ 30 mg/mL in waterTemperature: 30 °CDetection: UV @ 215 nmInstrument: Waters Alliance 2695 with 2996 PDA

pH 2Poor peak shapeColumn overloadLow retention – ionized

pH 7Better peak shapeBetter retention – 50% ionizedCo-elution

pH 12Best peak shapeGood retention – 100% non-ionizedBest resolution

excellent loading

Optimized resolution

excellent peak shape

0.25%

1.1%

7.3%

xBridgE c18/c8

[ ]8

EliMinaT ing MS BlEED

0.0E+00

5.0E+06

1.0E+07

1.5E+07

2.0E+07

2.5E+07

3.0E+07

0 2 4 6 8 10 12 14 16 18 20 22 min

No column

XBridge C18

Ion

Cou

nts

Mass spectrometry in combination with HPLC provides analytical chemists with greater sensitivity and selectivity. In order to obtain the full benefit of LC/MS, the utilization of low bleed LC columns is essential. The BEH Technology incorporated into XBridge columns virtually eliminates LC/MS bleed by providing increased hydrolytic stability and particle strength. As seen below, when compared to a background MS scan, neglibible bleed is observed.

Taking aDvanTagE of PHoSPHaT E BuffErS

Phosphate buffers possess unique selectivity, excellent UV transparency and good buffering capacity at multiple pKa values. However, due to the aggressive nature of phosphate, traditional silica columns often exhibit significantly reduced column lifetime. XBridge columns demonstrate excellent performance using phosphate buffers across the entire pH range due to the improved chemical resistance of BEH Technology particles.

HigH/low PH SwiTcHing

The ability to switch between high and low pH on a single column can result in a dramatic reduction in instrument downtime as well as a significant cost savings when working at multiple pH levels. Traditionally, separate columns are allocated for each pH level in order to prevent premature column degradation. BEH Technology particles, when combined with novel and innovative bonding and end-capping, enables the use of XBridge HPLC columns at a higher pH than conventional columns, but enables “pH switching” without performance decrease. In other words, a single XBridge column can be used to run under both low and high pH conditions, thereby eliminating the time required to change columns, and the need to have multiple columns on-hand to run low and high pH separately.

In this evaluation, the effect of aggressively switching mobile phase pH for every injection was investigated using a mixture of acidic, basic and neutral compounds. Each column was first equilibrated at pH 3 followed by a single injection of the test mixture. The column was then equilibrated at pH 10 followed by a single injection of the test mixture. This sequence was repeated for several thousand injections without deterioration on the XBridge C18 column.

V0 BA

N

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min

Inj. #2

Inj . #4800

B = baseA = acidN = neutral

B = baseA = acidN = neutral

V0

BA N

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min

Inj. #2

Inj . #1724

V0 BA

N

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min

Inj. #2

Inj . #4800

B = baseA = acidN = neutral

B = baseA = acidN = neutral

V0

BA N

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min

Inj. #2

Inj . #1724

XBridge Column

gemini™ Column

0 1 2 3 4 5 6 7 8 min

0 1 2 3 4 5 6 7 8 min

0 1 2 3 4 5 6 7 8 min

1

6

6

6

5

5

5

4

4

4

3

3

3

2

2

2

1

1

pH 2

pH 7

pH 12

Waters Manufacturing Facility

Waters Corporation has been awarded the following registrations and certifications:

• USFoodandDrugAdministrationregistered:cGMP’s and Class 1 medical devices

• ISO9001:2000

• ISO13485:2003

xBridgE c18/c8

[ ]9

STaBil iT y for DEPEnDaBlE PErforManc E

BEH Technology produces particles designed for improved column performance and long column lifetime under harsh and demanding LC operating conditions. Using state-of-the-art synthesis techniques Waters has designed and developed LC columns capable of providing usable lifetimes that far exceed leading conventional HPLC column brands under both low and high pH conditions.

Waters state-of-the-art facilities manufacture HPLC and UPLC columns that maximize laboratory performance. We are a primary manufacturer of silica and hybrid particles and are able to continually monitor and control the complete manufacturing process over the lifetime of the product. This allows for unprecedented control and repeatability between batches of material.

rEP roDuciBil iT y for MET HoDS DEv EloPMEnT

Waters continues to set the industry benchmark for batch-to-batch reproducibility. Beginning with the Symmetry® brand of columns in 1994 and continuing with XTerra, Atlantis® and SunFire™ HPLC columns, Waters columns provide consistent results. XBridge columns are manufactured in the same cGMP, ISO9001 and ISO13485 certified facilities that produce these industry-leading HPLC column brands. Methods development scientists can be assured that the separations achieved today will be reproducible year after year.

0 10 20 30 min

The results above were obtained as part of an extensive HPLC column evaluation by a leading pharmaceutical company. A total of 18 columns were evaluated under demanding conditions for stability of efficiency and selectivity during extended use, as well as batch-to-batch reproducibility. This evaluation resulted in the selection of XBridge HPLC columns as the principle methods development column worldwide.

Data courtesy of Novartis.

0 5 10 15 20 25 30 min

Batch Run 1

Batch Run 13

Batch Run 15

Batch Run 34

Batch Run 36

Batch Run 3

XBridge C18, pH 2

XBridge C18, pH 11.3

1 batch run = 500 column volumes

5 µm

5 µm

10 µm

3.5 µm

3.5 µm

xBridgE pHEnyl

[ ]10

XBridge PHenyL

Phenyl columns are often used in separations where alternate selectivity is required, particularly when the analytes of interest contain an aromatic ring. Traditionally, phenyl columns have been under-utilized due to inadequate stability under low pH conditions. Methods development scientists are no longer pH-limited when utilizing a phenyl LC column. Due to the novel bonding and endcapping of BEH particle technology, XBridge Phenyl columns provide excellent low and high pH stability, resulting in the most stable and reproducible phenyl column on the market.

SuPErior PErforManc E

XBridge Phenyl columns combine trifunctional bonding of the phenyl hexyl-ligand with proprietary endcapping techniques to produce industry-leading stability at low pH – while still providing excellent peak shape.

excellent Peak Shape

10 20 30 40 50 60 70 80 90 min

Zorbax® SB Phenyl

XBridge PhenylAmitriptylineNUSP = 9300T USP = 1.20

AmitriptylineNUSP = 920T USP = 6.59

Probe: Ethylparaben

50

60

70

80

90

100

0 20 40 60 80 100

Hours in 1% TFA in H2O pH 1.0 at 80 °C

XBridge™ Phenyl

Agilent Zorbax® SB-Phenyl

Phenomemex® Luna® Phenyl-Hexyl

Varian Pursuit® Diphenyl

Agilent Zorbax® Eclipse XDB Phenyl

Restek Ultra Phenyl

% In

itial

Ret

entio

n

O Si

O

O

O SiO

O

O SiO

O

O Si

CHPolar Group

3

CH3

Ligand Type Trifunctional C6 Phenyl

Available Particle Sizes (µm) 2.5, 3.5, 5

Ligand density* 3.0 µmol/m2

Carbon Load* 15%

endcap Style Proprietary

pH range 1-12

Low pH Temp. Limits 80 °C

High pH Temp. Limits 45 °C

USP L11

Pore diameter* 135Å

Pore Volume* 0.7 mL/g

Surface Area* 185 m2/g

* Nominal Values

Bond

ed P

hase

BEH

Parti

cle

XBridge Phenyl Column Characteristics

industry-Leading Stability at Low-pH

xBridgE pHEnyl

[ ]11

uniquE SElEcT iv iT y

XBridge Phenyl columns provide alternate selectivity compared to straight-chain-alkyl columns as well as embedded-polar-group columns, particularly for compounds containing an aromatic moiety. This provides greater flexibility in developing orthogonal methods for challenging separations.

uT il izing low pH To MEET MET HoDS DEv EloPMEnT cHallEngES

The separation of aromatic acids has historically been difficult using traditional C18 columns. These compounds are derivatives of phenol and possess aromatic rings, therefore, making a phenyl column a logical solution. However, it has been demonstrated that low pH is critical to achieving a suc-cessful separation of this class of compounds, which is problematic for a majority of phenyl columns on the market with regards to stability. XBridge Phenyl columns provide the answer. Not only is the desired selectivity achieved (via pi-pi interactions), improved chemical stability enables column longevity and robust operation at low pH.

4 8 12 16 20 min

Acids

Bases

0 1 2 3 4 5 6 7 8 9 10 11 12 min

Aromatic acids are used in several common herbicides, including Silvex, Picloram and Weed-B-Gone. Many other organic acids are very polar acidic analytes that can be difficult to retain on traditional C18 columns, however are well retained using the XBridge Phenyl columns.

Column: XBridge Phenyl 4.6 × 100 mm, 3.5 µm

Part Number: 186003334

Mobile Phase A: H2O

Mobile Phase B: ACN

Mobile Phase C: 100mM NH4HCOOH pH 3

Flow Rate: 1 mL/min

Gradient: Time Profile

(min) %A %B %C

0.0 85 5 10

8.00 65 25 10

10.00 10 80 10

11.00 10 80 10

12.00 85 5 10

15.00 85 5 10

Injection Volume: 20 µL

Sample Diluent: 10 µg/mL of all analytes in H2O

Column Temperature: 35 °C

Sample Temperature: 20 °C

Detection: UV @ 280 nm

Sampling Rate: 5 points/sec

Filter Response: 1.0

Needle Wash: 5/95 MeOH/H2O

Instrument: Alliance 2695 with 2996 PDA

Compounds

1. Gallic acid

2. Protocatechuic acid

3. 3,4 Dihydroxyphenylacetic acid

4. 4-Hydroxybenzoic acid

5. Picrolam

6. Vanillic acid

7. Syringic acid

8. Salicylic acid

9. 2,4-Dichlorophenoxyacetic acid (Weed-B-Gone)

10. 2-(2,4,5-Trichlorophenoxy) propanoic acid (Silvex)

Changes in selectivity can be observed with different ligands. As shown here, the phenyl ligand provides enhanced retention and alternate selectivity for tricyclic antidepressants (peaks 6,7 relative to peak 8).

1

1

1

2

2

2

3

3

3

4,5

4

4

5

5

6

6

6

7

7

7

8

8

8

9

9,10

9

11

11

11

10

10

12

12

12

XBridge Phenyl

XBridge C18

XBridge Shield rP18

1

23 4

5

6

7

8 9 10

XBridge SHieLd rP18

Columns containing embedded polar groups are becoming more popular with methods development scientists due to their alternate selectivity when compared to traditional C18 chemistries. The XBridge Shield RP18 offers complementary selectivity as well as superior peak shape for bases. The patented Shield Technology* incorporates a carbamate group embedded into the bonded phase that “shields” surface silanols. Additionally, it is this func-tionality that enables compatibility with fully aqueous mobile phases without risk of pore dewetting, resulting in reliable and robust retention.

*U.S. Patent 5,374,755 [Neue et al.]

xBridgE SHiEld rp18

[ ]12

* Nominal Values

O Si

O

O

O SiO

O

O SiO

O

O Si

CHPolar Group

3

CH3

Ligand Type Monofunctional Embedded Polar Group

Available Particle Sizes (µm) 2.5, 3.5, 5, 10

Ligand density* 3.3 µmol/m2

Carbon Load* 17%

endcap Style TMS

pH range 2-11

Low pH Temp. Limits 30 °C

High pH Temp. Limits 45 °C

USP L1

Pore diameter* 135Å

Pore Volume* 0.7 mL/g

Surface Area* 185 m2/g

Bond

ed P

hase

BEH

Parti

cle

XBridge Shield rP18 Column Characteristics

wHaT iS SHiElD T EcHnology?

In the early 1990s, novel bonded phases were created in which polar functional groups were embedded into C18 and C8 alkyl chains. One of the key benefits was a reduction in silanol interactions with basic analytes resulting in improved peak shape. These first generation materials were prepared using a two-step surface modification in a mixture of derivatized and underivatized amine groups which varied from batch-to-batch. Thus, the analytes were retained via both reversed-phase and anion-exchange mechanisms.

Second generation materials are prepared using a one-step surface modification in which the functional group is built into a silane. A single surface reaction with the silane yields only one possible ligand structure with no possibility of anion exchange. This approach was shown to provide excellent batch-to-batch reproducibility. The elimination of anion-exchange mechanisms is also important as it prevents the pos-sibility of secondary interactions with negatively charged analytes.

Shield Technology benefits:

■ Reduced silanol interactions with basic analytes. This deactivation or “Shielding”, results in improved peak shape for basic analytes.

■ Unique selectivity is also observed for embedded polar group bonded phases, with polar and basic analyte retention factors generally reduced and non-polar analytes relatively unaffected.

■ Shield bonded phases give stable and reproducible analyte retention times in 100% aqueous mobile phases. Conventional alkyl phases suffer from dewetting under these conditions and retention times are greatly diminished.

A review of Waters’ Bonded Phase Shield Technology and its Use in High Performance Liquid Chromatography (HPLC) White Paper

Literature Reference 720000207EN

Literature Reference

oPT iMizing SElEcT iv iT y for MET HoDS DEv EloPMEnT

When developing chromatographic methods, some of the tools available to the separation scientist include pH, column chemistry and organic modifier. The XBridge Shield RP18 column provides an alternate selectivity while possessing the ability to successfully operate over a broad pH range with a variety of solvents. Utilizing these key features, the chromatographer is able to quickly optimize the test conditions necessary to achieve a successful separation.

Large selectivity changes when comparing orthogonal column chemistries in different organic modifiers.

SuPErior SEParaT ion of BaSES

Some C18 columns show secondary interactions with basic compounds due to weak cation exchange with residual surface silanols resulting in poor peak asymmetry. The XBridge Shield RP18 column, with patented Shield Technology, reduces silanol interaction with basic analytes, resulting in highly efficient, symmetrical peaks.

xBridgE SHiEld rp18

[ ]13

0.00

0.02

0.04

0.06

AU

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 min

Compounds1. Nordoxepin2. Trimethoprim3. Nortriptyline4. Doxepin5. Imipramine6. Amitriptyline7. Trimipramine

Column: XBridge Shield rP18, 4.6 X 50 mm, 3.5 µmPart Number: 186003042Mobile Phase A: H2OMobile Phase B: MeOHMobile Phase C: 100 mM NH4HCO3

Flow Rate: 1.0 mL/min

Gradient: Time Profile (min) %A %B %C 0 90 0 10 2 30 60 10 10 16 74 10 11 90 0 10 13 90 0 10

Injection Volume: 20 µLSample Concentration and Diluent: 10 µg/mL in H2OTemperature: 35 °CDetection: UV @ 254Sampling Rate: 5 points/secondTime Constant: 1.0Needle Wash: 5/95 MeOH/H2OInstrument: Alliance 2695 with 2996 PDA

Acids

Bases

20 4 6 8 10 12 14 16 18 min

20 4 6 8 10 12 14 16 18 min

20 4 6 8 10 12 14 16 18 min

1

2

2

2

1

1

3

3

3

4

4

4

5

5

5

6

6

6

7,12

7

7

8

8,9

8

9

9

10

10

10

11

11

11

12

12

1

2

3

4 5

67

pH 10XBridge™ C18

Methanol

XBridge™ Shield rP18

Methanol

XBridge™ Shield rP18

Acetonitrile

* Nominal Values

O Si

O

O

O SiO

O

O SiO

O

O Si

CHPolar Group

3

CH3

Ligand Type Unbonded BEH

Available Particle Sizes (µm) 2.5, 3.5, 5

pH range 1 - 8

Low pH Temp. Limits 45 °C

High pH Temp. Limits 45 °C

USP L3

Pore diameter* 130Å

Pore Volume* 0.7 mL/g

Surface Area* 185 m2/gBEH

Parti

cle

XBridge HiLiC Column Characteristics

xBridgE Hilic

[ ]14

XBridge HiLiC

Hydrophilic Interaction Chromatography (HILIC) is a chromatographic technique that can be used to improve retention of very polar species that are poorly retained by reversed-phase chromatography. XBridge HILIC columns provide alternative selectivity to reversed-phase columns as well as improvements in peak shape, retention and column lifetime compared to silica-based HILIC columns. Due to rugged and robust BEH Technology, XBridge HILIC columns set a new standard in performance for HILIC separations.

EnHanc ED rET EnT ion

HILIC retention mechanisms are a complex combination of partitioning, ion-exchange and hydrogen bonding, resulting in enhanced retention for polar analytes. Retention occurs with a polar stationary phase in combination with mobile phases composed of acetonitrile concentrations greater than 80%, offering dramatically more retention than reversed-phase.

V0 = 0.35 min

V0 = 0.33 min

Choline

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 min

HON+

XBridge C18 reversed-Phase

Retention Factor = 0

XBridge HiLiC

Retention Factor = 1.8 1 2 3 4 5 6 min0

XBridge HILIC provides retention when no retention is achieved by reversed-phase.

Compounds1. 6-Acetylmorphine2. Morphine3. Morphine-3-β-glucuronide

XBridge C18 reversed-Phase

XBridge HiLiC

3

2

1

12

3

XBridge HILIC provides alternative selectivity to reversed-phase. Polar metabolites of morphine are retained greater under HILIC conditions.

coMPlEMEnTary SElEcT iv iT y

XBridge HILIC columns provide complementary selectivity to reversed-phase columns. In some cases, a reversal in elution order is observed in addition to improved retentivity, which can be advantageous when developing orthogonal separations for polar analytes.

Bond

ed P

hase

cHEMical STaBil iT y

Intermediate mobile phase pH is often needed to alter the charge state of the analyte and/or substrate to promote retention. For silica-based materials, this often leads to particle dissolution, resulting in decreased column life. XBridge HILIC columns are built on BEH Technology resulting in exceptional column longevity, providing several thousand injections without performance decrease due to chemical degradation.

xBridgE Hilic

[ ]15

2 min10

1. Acetylcholine 2. Choline

3.0 x 107

2.0 x 107

1.0 x 107

Intensity

3.0 x 107

2.0 x 107

1.0 x 107

Intensity

0.2 1.0 2.0 min

ON+

O

HON+

Condition/Equilibrate:200 µL CH3OH/ 200 µL H2O

Load:300 µL spiked human plasma

with 2% H3PO4

Wash 1:200 µL 2% HCOOH in H2O

Wash 2:200 µL CH3OH

Wash 3:200 µL CH3CN

Elute:50 µL 5% NH4OH in CH3CN

Direct Injection of Eluate:Eliminates lengthy evaporation

and reconstitution steps

6-Acetylmorphine10 ng/mL spiked into human plasma

Morphine10 ng/mL spiked into human plasma

0 2 4 6 min

HO

O

O

NCH3

H

O

HO

O

HO

NCH3

H

XBridge C18 reversed-Phase

injection 10

injection 1000

injection 2000

XBridge HiLiC

1

2

3

Column: XBridge HiLiC, 2.1 x 50 mm, 3.5 μmMobile Phase A: 95:5 acetonitrile: water with 10 mM NH4+ CH3COO- pH 5.5Mobile Phase B: 50:50 acetonitrile: water with 10 mM NH4+ CH3COO- pH 5.5Flow Rate: 0.5 mL/minGradient: 1 – 99% B over 2 minutes, 1% B at 2.1 min, hold for 0.4 minInjection Volume: 2.0 μLTemperature: 30 ˚CDetection: UV @ 254 nm

XBridge HILIC exhibits exceptional chemical resistance resulting in long column lifetime.

2 1

1

2

10.5 x response 8.6 x response

XBridge HILIC provides enhanced mass spectrometry response resulting in lower limits of detection.

EnHanc ED MaSS SPEcT roMET ry SEnSiT iv iT y

Utilization of HILIC has grown in popularity, primarily due to the extensive adoption of mass spectrometry as a detector and the necessity of im-proving sensitivity for the quantitation of polar analytes. Unlike reversed –phase which utilizes high aqueous mobile phases to induce retention of polar compounds, HILIC employs an acetonitrile-rich mobile phase. This high organic mobile phase is easily desolvated, resulting in improved ionization efficiency and mass spectrometry response.

SHorT EnED SaMPlE HanDling ST EPS

When employing solid phase extraction (SPE) methods for sample clean-up, a high organic fraction is often used to selectively elute analytes of interest from the device. Before injecting this fraction onto a reversed-phase column, this organic fraction must first be evaporated then reconstituted with a portion of aqueous solvent. These two steps are often the lengthiest steps in an SPE procedure. With XBridge HILIC columns, the high organic fraction can be directly injected, thus eliminating the need for evaporation and increasing sample throughput.

XBridge HILIC provides direct injection of SPE eluents, thus increasing sample throughput.

Opiates in Human PlasmaOasis® MCX µElution Plate

Compounds1. Uracil2. 5-f luorocytosine3. Cytosine

Uplc TEcHnology

[ ]16

In 2004, Waters revolutionized LC separation science with the development of the ACQUITY UltraPerformance LC (UPLC) system. High resolution UPLC separations are realized in a system that is capable of fully utilizing LC columns that are efficiently packed with pressure tolerant sub-2 µm particles. BEH Technology is one of the key enablers behind this new separations platform and provides the efficiency, strength and pH range required for UPLC separations.

Since ACQUITY UPLC BEH columns and XBridge HPLC columns both incorporate BEH Technology, methods can be seamlessly transferred between these particle platforms. Chromatographic separations can be scaled between UPLC Technology, analytical and preparative scale effortlessly. Chromatographers can take advantage of a wide range of particle sizes (i.e., 1.7, 2.5, 3.5, 5 and 10 µm), while preserving method efficacy.

uPlc T EcHnology – SPEED anD rESoluT ion

For isocratic separations, resolution (Rs) is proportional to particle size. T hus, smaller particles provide higher resolution.

Abs

orba

nce

at 2

70 n

m

0.04

0.08

Abs

orba

nce

at 2

70 n

m

0.08

Abs

orba

nce

at 2

70 n

m

0.04

Abs

orba

nce

at 2

70 n

m

0.04

Abs

orba

nce

at 2

70 n

m

Rs (2,3) = 2.90

Rs (2,3) = 7.42

Rs (2,3) = 4.58

Rs (2,3) = 4.57

Rs (2,3) = 6.18

2.1 x 30 mm, 1.7 µm

2.1 x 50 mm, 1.7 µm

2.1 x 100 mm, 1.7 µm

2.1 x 150 mm, 5.0 µm

UPLC

HPLC

2.1 x 150 mm, 1.7 µm

0.00 0.50 1.00 1.50

0.00 1.00 2.00 3.00 4.00 5.00

0.00 2.00 4.00 6.00 7.00

0.00 0.50 1.50 2.001.00 2.50

0.00 5.00 15.0010.00 20.00

ULTRA

SPEED

ULTRA

RESOLUTION

SPEED WITH

RESOLUTION

RESOLUTION

WITH SPEED

UPLC Method development protocol

HPLC Method development protocol

dEvElop METHodS FaSTEr wiTH Uplc TEcHnology

26.8 HoursToTal ScrEEning TiME

pH 3 / Acetonitrile pH 3 / Acetonitrile

pH 3 / Methanol

pH 3 / Methanol

pH 10 / Acetonitrile

pH 10 / Acetonitrile

pH 10 / Methanol

pH 10 / Methanol

Screening Time: 6.7 Hours / Column x 4 Columns

8 HoursToTal ScrEEning TiME

Screening Time: 2 Hours / Column x 4 Columns

Developing methods utilizing a systematic screening approach is one of the fastest and most effective ways to create new HPLC separations. Evaluating combinations of pH, column chemistry and organic modifier, streamline the information necessary to enable a confident decision on how to proceed with a method. But what if that data could be obtained even faster? The answer lies in UPLC Technology.

Shown here is a typical methods development protocol utilizing HPLC and UPLC Technology. Both techniques provide the same amount of information. The difference is the amount of laboratory time necessary to acquire this information. In this example, UPLC Technology provided the answer in 1/3 of the time! This entire protocol can be completed in a single work day while maintaining the same quality of information. UPLC Technology greatly enhances the speed with which methods can be developed and efficiency with which a laboratory operates.

rEacHing a nEw lEv El of P rEParaT iv E coluMn DuraBil iT y

Poor lifetimes and inconsistent performance of preparative columns have been of significant concern to the purification chemist. Cost per purification must be reduced, and the loss of samples due to premature column failure minimized. After many years of research on column packing and design, Waters developed the Optimum Bed Density (OBD) preparative column design*, effectively addressing these concerns. This format is recognized in the industry as

delivering the most stable, efficient, and reproducible preparative columns.

The combination of rugged XBridge packings and OBD design takes preparative column performance to a new level, ensuring direct scalability, maximum efficiency, and long column lifetimes.

XBridge Prep Columns deliver the same high loading capacity and reliability expected of our XTerra Prep products, while generating lower column backpressure and higher efficiency.

Maximum efficiency/30% Lower Backpressure

0 2 4 6 8 10 min

0 2 4 6 8 10 min 0 2 4 6 8 10 min

0 2 4 6 8 10 minXTerra Prep MS C18, 19 x 50 mm, 5 µmInjection Volume: 660 µLTotal Load: 198 mgMaximum Backpressure: 1000 psi

XBridge Prep C18, 19 x 50 mm, 5 µmInjection Volume: 660 µLTotal Load: 198 mgMaximum Backpressure: 700 psi

Mobile Phase A: 0.1% DEA in waterMobile Phase B: 0.1% DEA in acetonitrileSample Concentration: 300 mg/mL in DMSOInstrument:: AutoPurification® SystemDetection: UV @ 260 nm

Compounds1. Labetolol (50 mg/mL)2. Quinine (50 mg/mL)3. Diltiazem (50 mg/mL)4. Verapamil (100 mg/mL)5. Amitriptyline (50 mg/mL)

Flow Rate: 23.8 mL/minGradient: Time Profile (min) %A %B 0.0 95 5 1.79 95 5 6.79 5 95 7.79 5 95

Flow Rate: 23.8 mL/minGradient: Time Profile (min) %A %B 0.0 95 5 1.79 95 5 6.79 5 95 7.79 5 95

0 2 4 6 8 10 12 14 16 18 20 min

0 2 4 6 8 10 12 14 16 18 20 min 0 2 4 6 8 10 12 14 16 18 20 min

0 2 4 6 8 10 12 14 16 18 20 min

The OBD preparative column design delivers the equivalent packed bed density to that of the analytical column and therefore ensures direct scalability.

XBridge C18, 4.6 x 100 mm, 5 µmInjection Volume: 30 µLTotal Load: 6 mg

Mobile Phase A: 10 mM ammonium acetate, pH 10Mobile Phase B: Acetonitrile/100 mM Ammonium bicarbonate (90/10)Flow Rate: 1.06 min/mL (ana); 18 min/mL (prep)Gradient: 10-min gradient from 5% A to 95% BDetection: UV @ 270 nm

Compounds1. Econazole (100 mg/mL)2. Miconazole (100 mg/mL)

XBridge Prep C18, 19 x 100 mm, 5 µmInjection Volume: 510 µLTotal Load: 102 mg

direct Scalability

prEparaTivE oBd TEcHnology

[ ]18

*UK Patent # GB2408469

1 2

3

45

12

3

4

5

12 1 2

0

100

100

100

0

0

0 1 2 3 4 5 6 7 min

%

%

%

BEnEfiTS of pH in iSolaT ion anD Purif icaT ion

-1

100

100

100

-8

00 2 4 6 8 10 min

%

%

%

Low pH for acidic analytes delivers the highest loadability, as well as the best retention and separation.

High pH for basic analytes delivers the best loadability, as well as the best retention and separation.

XBridge C18

XBridge C18

Loading of Bases

Loading of Acids

rEDucing coST By EXT EnDing coluMn lifET iME

injection 1

injection 7,000

Optimum Bed density (OBd) Preparative Columns Brochure

Literature Reference 720002336EN

Literature Reference

prEparaTivE oBd TEcHnology

[ ]19

The demand for rapid high-purity compound isolation places strong emphasis on the integrity and stability of the preparative column. Complex, sparingly-soluble starting materials are often dissolved in strong solvents, such as DMSO. This combination of poor solubility and pressure shocks associated with large injection volumes of pure organic solvent are the primary contributors to early column failure and chromatographic bed collapse.

The OBD design exhibits exceptional resistance to mechanical chromatographic bed failure and delivers consistent column-to-column performance, reducing cost through extended lifetimes.

1,2

1

1

1

1

1

2

2

2

2

2

3

3

3

3

3

3

Bases1. Nordoxepin2. Doxepin3. Amitriptyline

Acids1. Oxacillin2. Cloxacillin3. Dicloxacillin

x5 in y axis

x20 in y axis

0.1 mg

0.8 mg

0.1 mg

0.3 mg

0.5 mg

0.2 mg

pH 2.3

pH 2.3

pH 7

pH 7

pH 10

pH 10

pEpTidE SEparaTionS TEcHnology

[ ]20

Waters Peptide Separation Technology columns incorporate BEH Technology particles. They are specially tested with a peptide map in order to ensure the stability of peptide separation methods as well as predictable behavior with the variety of samples encountered in proteomics, protein characterization and peptide synthesis. Available in either 130Å or 300Å pore sizes and in particle sizes from 1.7 µm to 10 µm, they provide:

■ Narrow, symmetrical peaks for maximum resolution

■ Excellent separation of a wide variety of peptides

■ Superior peak shape and retention in formic acid and trifluoracetic acid for optimal chromatography

■ Seamless method migration from sub-2 µm UPLC Technology to 10 µm preparative HPLC separations.

BEH Technology columns can be used to increase the resolution of complex peptide digests. By utilizing the 1.7 µm BEH UPLC particles in longer column dimensions, the chromatographer can achieve ultra resolution. The resolving power of an LC column can be expressed by its length-to-particle-size ratio (l/dp). Columns with the highest l/dp ratios provide greater efficiencies and are used in the most difficult separations. An example would be the Peptide Separation Technology ACQUITY UPLC BEH columns which are available in 150 mm lengths. Their l/dp ratio is more than 88,000 and provide efficiencies of > 35,000 plates per separation.

HigHEr rESoluT ion PEPT iDE MaP Ping wiT H uPlc

Resolution of a complex peptide mixture from a phosphorylase b protein digest is shown above. Compared to using traditional HPLC technology, improved component resolution is obtained on a Waters ACQUITY UPLC system using Waters Peptide Separation Technology columns containing 1.7 µm BEH particles.

30 40 50 60 70 80 90 min

30 40 50 60 70 80 90 min

HPLC

Peak Capacity = 372

UPLC

Peak Capacity = 723

oligonUclEoTidE SEparaTionS TEcHnology

[ ]21

Waters Oligonucleotides Separations Technology (OST) columns utilize hybrid particle BEH Technology and are available in both 1.7 µm UPLC and 2.5 µm HPLC particle formats. This provides the flexibility to meet a wide variety of isolation and analytical needs while still delivering exceptional resolution and column lifetime.

The separation of detritylated synthetic oligonucleotide samples is based on the well established method of ion-pair, reversed-phase chromatography. ACQUITY UPLC OST C18 and XBridge OST C18 columns are well suited for this type of analysis and provide:

■ Separation efficiencies equivalent or better than PAGE-CGE or ion-exchange HPLC methods

■ The ability to resolve large oligonucleotide sequences due to the enhanced resolving power obtained using sub-3 µm particles

■ Scaleable column offerings for lab scale isolation needs

■ Exceptional column life for reduced cost per analysis.

ouTSTanDing coluMn lifET iME

Separation of 55 – 25 mer detritylated Oligode-oxythymidine Ladder

XBridge OST C18 columns are preferred for detritylated oligonucleotide purifications due to their availability in a variety of column sizes. The separation scientist has the ability to select the most appropriate size column based on the amount of oligonucleotide sample available. This results in maximum component resolution and recovery of the target product from non-desired failure sequences.

XBridge OST C18 Column Selection guide for detritylated Oligonucleotide Purification

Column: XBridge OST C18, 2.5 µm (2.1 x 50 mm)Mobile Phase: A: 10% MeOH / 90% (385mM HFIP + 14.3mM TEA) B: 25% meOH / 75% (385mM HFIP + 14.3mM) TEA Column Temp: 60 °C

Gradient: 0 – 100% B in 30 min (10-25 % MeOH).Flow Rate: 1.0 mL/minDetection: 260 nm, 5 scans per secondHPLC System: Waters Alliance Bio 2796 with PDA

Column (mm) Approx Mass Load (µmoles)** Flow Rate (mL/min)

2.1 x 50 0.04 0.2

4.6 x 50 0.20 1.0

10.0 x 50 1.00 4.5

19.0 x 50* 4.00 16.0

30.0 x 50* 9.00 40.0

50.0 x 50* 25.00 110.0

* OST Custom Column

** Values are only approximates and vary depending on oligonucleotide length, base composition, and “heart-cutting”

fraction collection method used.

injection #4 injection #1001

[ ]22

■ Analysis of Statin drugs

The statins (or HMG-CoA reductase inhibitors) form a class of hypolipidemic agents, used as pharmaceutical agents to lower cholesterol levels in people with or at risk for cardiovascular disease. They lower cholesterol by inhibiting the enzyme HMG-CoA reductase, which is a rate-limiting enzyme of the mevalonate pathway involved in cholesterol synthesis. Inhibition of this enzyme in the liver stimulates LDL receptors, resulting in an increased clearance of low-density lipoprotein (LDL) from the bloodstream and a decrease in blood cholesterol levels.

In addition to lipid-lowering activity, statins exhibit properties linked to the prevention of atherosclerosis. Researchers also speculate that statins help to prevent cardiovascular disease by inhibiting thrombus formation, stabilizing plaque levels, improving endothelial function and moderating inflammatory responses.

Compounds1. Pravastatin2. Atorvastatin3. Lovastatin4. Simvastatin

0 1 3 5 7 min

XBridge Shield RP18 columns effectively separate closely related compounds, allowing for rapid identification.

xBridgE SolUTionS - pHarMacEUTical

■ Analysis of Caffeine and Metabolites

Caffeine is a central nervous system and metabolic stimulant, and is used both recreationally and medically to reduce physical fatigue and restore mental alertness when unusual weakness or drowsiness occurs. Caffeine stimulates the central nervous system first at the higher levels, resulting in increased alertness and wakefulness, faster and clearer flow of thought, increased focus, and better general body coordination.

Column: XBridge Phenyl, 4.6 × 100 mm, 3.5 µm Part Number: 186003334 Mobile Phase A: H2OMobile Phase B: ACNMobile Phase C: 100 mM NH4HCO3 Flow Rate: 1.0 mL/min Gradient: Time Profile (min) %A %B %C 0.0 89 1 10 9.00 66 24 10 10.00 89 1 10 20.00 89 1 10Injection Volume: 10 µL Sample: Caffeine (10 µg/mL), Theobromine (10 µg/mL), 1-Methylxanthine, (10 µg/mL), 1,3-Dimethyluric Acid (10 µg/mL), 1,7-Dimethylxanthine, (10 µg/mL),1,7-Dimethyluric Acid (10 µg/mL) in H2O/NH4HCO3 (90/10) Column Temp.: 30 °CSample Temp.: 15 °C Detection: UV @ 280 nmSampling Rate: 5 points/secFilter Response: 0.2Instrument: Waters Alliance 2695 with 2998 PDA

XBridge Phenyl columns provide a rapid and robust separation for the analysis of caffeine and its metabolites.

Compounds1. 1,7-Dimethyluric acid 2. 1-Methylxanthine 3. 1,3-Dimethyluric acid4. 1,7-Dimethylxathine5. Theobromine6. Caffeine

0 2 4 6 8 min

Column: XBridge Shield rP18, 4.6 x 50 mm, 3.5 µm

Part Number: 186003042

Mobile Phase A: H2O

Mobile Phase B: ACN

Mobile Phase C: 100 mM NH4HCO3 , pH 10

Flow Rate: 1.2 mL/min

Injection Volume: 20 µL

Sample Concentration and Diluent: 10 µg/mL in H2O

Temperature: 30 °C

Detection: UV @ 248

Sampling Rate: 5 points/second

Filter Response: 1.0

Needle Wash: 5/95 MeOH/H2O

Instrument: Waters Alliance 2695 with 2996 PDA

1

2

3

4

5

6

1

2

34

xBridgE SolUTionS - Food SaFETy

[ ]23

For additional XBridge application notes, visit www.waters.com/xbridge

Literature Reference

■ Analysis of Food Additives and Preservatives

Saccharin is an artificial sweetener. 4-Hydroxybenzoic acid is primarily known as the basis for the preparation of its esters, known as parabens, which are used as preservatives. Dehydroacetic acid, methyl paraben and sorbic acid are preservatives used in cosmetics and foods.

T he XBridge Phenyl column provided a rapid quantification of the compounds of interest.

■ Analysis of Patulin in Apple Juice

Patulin is a mycotoxin produced by several species of molds which may appear on a variety of foods including grains, fruits and cheeses of the fungi capable of producing patulin. Penicillum expansum is the most common and is often isolated from decaying apples. Due to its toxicity, various governing bodies have established maximum daily intake of patulin, with a primary concern being apple juice consumption by children.

In this method, patulin can be completely resolved from hydroxymethylfurfural, a common interfering compound.

Data was provided courtesy of Dr. Vural Gökmen, Food Engineering Department, Hacettepe University, Ankara, Turkey

3 4 5 6 min

Column: XBridge C18, 4.6 x 100 mm, 3.5 µm

Part Number: 186003033

Mobile Phase: 0.1% HCOOH:ACN (95:5, v/v)

Flow Rate: 0.75 mL/min

Temperature: 25 °C

Detection: 276 nm

1 2 3 4 5 6 min

Compounds1. Saccharin2. p-Hydroxybenzoic acid 3. Sorbic acid4. Methylparaben 5. Dehydroacetic acid

Column: XBridge Phenyl, 4.6 × 100 mm, 3.5 µmPart Number: 186003334 Mobile Phase A: 20 mM KH2PO4, pH 2.5 Mobile Phase B: ACN Flow Rate: 1.0 mL/min Isocratic Mobile Phase Composition: 75%A; 25%B Injection Volume: 10 µL

Sample: Saccharin (100 µg/mL), p-Hydroxybenzoic Acid (10 µg/mL), Dehydroacetic Acid (100 µg/mL), Methylparaben (25 µg/mL), Sorbic Acid (10 µg/mL) in KH2PO4/ACN (75/25) Column Temperature: 30 °CSample Temperature: 15 °C Detection: UV @ 240 nmSampling Rate: 5 points/secFilter Response: 0.2Instrument: Waters Alliance 2695 with 2996 PDA

1

2

1

23

45

Compounds1. Hydroxymethylfurfural2. Patulin

xBridgE SolUTionS –EnvironMEnTal

[ ]24

■ Analysis of dnPH derivatives

Regulatory agencies around the world are interested in measuring formaldehyde and other aldehydes in the air. Many public health groups are interested in implications of these aldehyde causing respiratory irritation and potentially carcinogenic effects from prolonged exposure. Producers of products that can contribute aldehydes emissions to air and indoor pollutants are manufacturers of building materials and wood products, fabric and textiles, and automotive companies.

1 3 5 7 min

XBridge C18 columns rapidly separate the compounds of interest for quantification.

Column: XBridge Phenyl, 3.5 µm, 4.6 × 100 mm

Part Number: 186003334

Mobile Phase A: H2O

Mobile Phase B: ACN

Mobile Phase C: 0.2% HCOOH in H2O

Flow Rate: 1.2 mL/min

Gradient: Time Profile (min) %A %B %C 0.0 40 50 10 2.67 40 50 10 6.67 0 90 10 7.33 40 50 10 11.00 40 50 10

Injection Volume: 10 µL

Sample: Acetaldehyde-DNPH (10 µg/mL), Acetone- DNPH (10 µg/mL), Cyclohexanone-DNPH (10 µg/mL), Formaldehyde-DNPH (10 µg/mL), Crotonaldehyde-DNPH (10 µg/mL) in

H2O/ACN (60/40)

Column Temperature: 30 °C

Sample Temperature: 15 °C

Detection: UV @ 254 nm

Sampling Rate: 5 points/sec

Filter Response: 0.2

Instrument: Waters Alliance 2695 with 2996 PDA

Compounds

1. Formaldehyde-DNPH

2. Acetaldehyde-DNPH

3. Acetone-DNPH

4. Crotonaldehyde-DNPH

5. Cyclohexanone-DNPH

1 2

3

4

5

■ Analysis of explosives

EPA method 8330 describes the use of high performance liquid chromatography (HPLC) with ultraviolet (UV) detection for the analysis of samples containing or suspected of containing explosive compounds. Method 8330 provides for the detection in parts per billion (ppb) of explosive compounds in soil, water and sediments.

2 6 10 14 18 22 min

Column: XBridge Phenyl, 2.1 x 150 mm, 3.5 µm

Part Number: 186003324

Mobile Phase: 10 mM Ammonium formate/Isopropanol

Flow Rate: 0.25 mL/min

Injection: 10 µL

Temperature: 40 °C

Detection: UV @ 254 nm

System: Waters Alliance HPLC system with 2487 Dual λ Absorbance detector

Compounds 1. HMX 2. RDX 3. 1,3,5-Trinitrobenzene 4. 1,3 Dinitrobenzene 5. Nitrobenzene 6. TNT 7. Tetryl 8. 2 Amino-4,6 dinitrotoluene 9. 2,4 dinitrotoluene 10. 4 Amino-2,6 dinitrotoluene 11. 2,6 dinitrotoluene 12. 4-Nitrotoluene 13. 2-Nitrotoluene 14. 3-Nitrotoluene

XBridge Phenyl columns accurately separated 14 explosive compounds in samples of interest.

1

2

3

4

5

6

7

8

9

10 11

1213 14

xBridgE SolUTionS –EnvironMEnTal

[ ]25

For additional XBridge application notes, visit www.waters.com/xbridge

Literature Reference

■ Analysis of Aldehydes and Ketones as dnPH derivatives

EPA methods T011, 554, and 8315 describe the use of high performance liquid chromatography (HPLC) with ultraviolet (UV) detection for the identification of aldehydes and ketones as Dinitrophenylhydrazine (DNPH) derivatives in drinking water (554); soil, air, water, waste or stacks collected by method 0011 (8315 Option 1) ambient air (T011), and samples collected from indoor air by method 0100 (8315 Option 2). EPA methods 554 and 8315 Option 1 target the same 12 compounds. Likewise, methods T011 and 8315 Option 2 target the same 15 compounds. Several analytes are common to all four methods.

In combination with these EPA methods, XBridge Phenyl columns assisted in the successful identification of DNPH derivatives in environmental samples.

Column: XBridge Phenyl, 4.6 x 150 mm, 3.5 µm

Part Number: 186003335

Mobile Phase: Water/tetrahydrofuran/acetonitrile

Flow Rate: 1.5 mL/min

Injection: 20 µL each of AccuStandard mix (M- 8315-R1-DNPH and M-8315-R2-DNPH) diluted 1:5 in 40:60 water/acetonitrile

Temperature: 35 °C

Detection: UV @ 360 nm

System: Waters Alliance HPLC system with UV detection

1. Formaldehyde 2. Acetaldehyde 3. Propanal 4. Crotonaldehyde 5. Butanal 6. Cyclohexanone 7. Pentanal 8. Hexanal 9. Heptanal 10. Octanal 11. Nonanal 12. Decanal

1. Formaldehyde 2. Acetaldehyde 3. Acetone 4. Acrolein 5. Propanal 6. Crotonaldehyde 7. Butanal 8. Benzaldehyde 9. Isovaleraldehyde 10. Pentanal 11. o-Tolualdehyde 12. p-Tolulaldehyde 13. m-Tolulaldehyde 14. Hexanal 15. 2-5 Dimethylbenzaldehyde

DNPH Derivatives of

DNPH Derivatives of

4 86 1210 1614 18 20 22 min

4 86 1210 1614 18 20 min

1

1 2

3

4

5

6

78 9 10

11

1213

14

15

2

3

4

56

7

89

1011

12

ordEring inForMaTon

[ ]26

XBridge Analytical Columns

Dimensions Type Particle Size C18 C8 Shield RP18 Phenyl HILIC

1.0 x 50 mm Column 2.5 µm 186003118 186003164 186003136 186003306 —

2.1 x 10 mm Guard 2.5 µm 1860030561 1860030741 1860030651 1860033591 1860044552.1 x 20 mm IS™ Column 2.5 µm 186003201 186003167 186003139 186003307 —

2.1 x 30 mm Column 2.5 µm 186003084 186003099 186003091 186003308 1860044562.1 x 50 mm Column 2.5 µm 186003085 186003101 186003092 186003309 1860044573.0 x 20 mm IS Column 2.5 µm 186003087 186003168 186003140 186003310 —3.0 x 20 mm Guard 2.5 µm 1860030572 1860030752 1860030662 1860033602 —3.0 x 30 mm Column 2.5 µm 186003121 186003169 186003141 186003311 —3.0 x 50 mm Column 2.5 µm 186003122 186003170 186003142 186003312 1860044584.6 x 20 mm IS Column 2.5 µm 186003088 186003172 186003144 186003313 —4.6 x 20 mm Guard 2.5 µm 1860030582 1860030762 1860030672 1860033612 1860044594.6 x 30 mm Column 2.5 µm 186003089 186003173 186003145 186003314 —4.6 x 50 mm Column 2.5 µm 186003090 186003174 186003096 186003315 1860044604.6 x 75 mm Column 2.5 µm 186003124 186003175 186003146 186003316 186004461

1.0 x 50 mm Column 3.5 µm 186003126 186003177 186003148 186003317 1860044291.0 x 100 mm Column 3.5 µm 186003127 186003178 186003149 186003318 —1.0 x 150 mm Column 3.5 µm 186003128 186003179 186003150 186003319 —2.1 x 10 mm Guard 3.5 µm 1860030591 1860030771 1860030681 1860033621 1860044302.1 x 20 mm IS Column 3.5 µm 186003019 186003180 186003151 186003320 —2.1 x 30 mm Column 3.5 µm 186003020 186003046 186003035 186003321 1860044312.1 x 50 mm Column 3.5 µm 186003021 186003047 186003036 186003322 1860044322.1 x 100 mm Column 3.5 µm 186003022 186003048 186003037 186003323 1860044332.1 x 150 mm Column 3.5 µm 186003023 186003049 186003038 186003324 1860044343.0 x 20 mm IS Column 3.5 µm 186003024 186003181 186003152 186003325 —3.0 x 20 mm Guard 3.5 µm 1860030602 1860030782 1860030692 1860033632 —3.0 x 30 mm Column 3.5 µm 186003025 186003182 186003153 186003326 —3.0 x 50 mm Column 3.5 µm 186003026 186003050 186003039 186003327 1860044353.0 x 100 mm Column 3.5 µm 186003027 186003051 186003040 186003328 1860044363.0 x 150 mm Column 3.5 µm 186003028 186003052 186003041 186003329 —4.6 x 20 mm IS Column 3.5 µm 186003029 186003183 186003154 186003330 —4.6 x 20 mm Guard 3.5 µm 1860030612 1860030792 1860030702 1860033642 1860044374.6 x 30 mm Column 3.5 µm 186003030 186003184 186003155 186003331 1860044384.6 x 50 mm Column 3.5 µm 186003031 186003053 186003042 186003332 1860044394.6 x 75 mm Column 3.5 µm 186003032 186003185 186003043 186003333 —4.6 x 100 mm Column 3.5 µm 186003033 186003054 186003044 186003334 1860044404.6 x 150 mm Column 3.5 µm 186003034 186003055 186003045 186003335 1860044414.6 x 250 mm Column 3.5 µm 186003943 186003963 186003964 186003965 —

2.1 x 10 mm Guard 5 µm 1860030621 1860030801 1860030711 1860033661 1860044422.1 x 20 mm IS Column 5 µm 186003107 186003186 186003156 186003336 —2.1 x 30 mm Column 5 µm 186003129 186003187 186003157 186003337 1860044432.1 x 50 mm Column 5 µm 186003108 186003011 186002999 186003338 1860044442.1 x 100 mm Column 5 µm 186003109 186003012 186003002 186003339 1860044452.1 x 150 mm Column 5 µm 186003110 186003013 186003003 186003340 1860044463.0 x 20 mm IS Column 5 µm 186003130 186003188 186003158 186003341 —3.0 x 20 mm Guard 5 µm 1860030632 1860030812 1860030722 1860033672 —3.0 x 30 mm Column 5 µm 186003111 186003189 186003159 186003342 —3.0 x 50 mm Column 5 µm 186003131 186003190 186003160 186003343 1860044473.0 x 100 mm Column 5 µm 186003132 186003191 186003004 186003344 1860044483.0 x 150 mm Column 5 µm 186003112 186003014 186003005 186003345 —3.0 x 250 mm Column 5 µm 186003133 186003192 186003161 186003346 —4.6 x 20 mm IS Column 5 µm 186003134 186003193 186003162 186003347 —4.6 x 20 mm Guard 5 µm 1860030642 1860030822 1860030732 1860033682 1860044494.6 x 30 mm Column 5 µm 186003135 186003194 186003163 186003348 1860044504.6 x 50 mm Column 5 µm 186003113 186003015 186003006 186003349 1860044514.6 x 75 mm Column 5 µm 186003114 186003195 186003007 186003350 —4.6 x 100 mm Column 5 µm 186003115 186003016 186003008 186003351 1860044524.6 x 150 mm Column 5 µm 186003116 186003017 186003009 186003352 1860044534.6 x 250 mm Column 5 µm 186003117 186003018 186003010 186003353 186004454

Order online at www.waters.com

ordEring inForMaTon

[ ]27

XBridge Preparative Columns

Dimensions Type Particle Size C18 C8 Shield RP18 Phenyl

2.1 x 100 mm MVKit 3.5 µm 186003766 186003777 186003788 1860037993.0 x 100 mm MVKit 3.5 µm 186003767 186003778 186003789 1860038003.0 x 150 mm MVKit 3.5 µm 186003768 186003779 186003790 1860038014.6 x 100 mm MVKit 3.5 µm 186003769 186003780 186003791 1860038024.6 x 150 mm MVKit 3.5 µm 186003770 186003781 186003792 1860038032.1 x 150 mm MVKit 5 µm 186003771 186003782 186003793 1860038043.0 x 100 mm MVKit 5 µm 186003772 186003783 186003794 1860038053.0 x 150 mm MVKit 5 µm 186003773 186003784 186003795 1860038064.6 x 100 mm MVKit 5 µm 186003774 186003785 186003796 1860038074.6 x 150 mm MVKit 5 µm 186003775 186003786 186003797 1860038084.6 x 250 mm MVKit 5 µm 186003776 186003787 186003798 186003809

XBridge Column Method Validation Kits

1 Requires Universal Sentry Guard Holder - 2.1 x 10 mm WAT097958

2 Requires Universal Sentry Guard Holder - 3.0 x 20 mm/4.6 x 20 mm WAT046910

3 Requires 10 x 10 mm Prep Guard Holder 289000779

4 Requires 19 x 10 mm Prep Guard Holder 186000709

For ACQUITY UPLC Columns Ordering Information visit:

www.waters.com/acquitycolumns

For Bioseparations and Analyses Ordering Information visit:

www.waters.com/biosep

Dimensions Type Particle Size C18 C8 Shield RP18 Phenyl

10 x 10 mm Guard 5 µm 1860029721 1860029911 1860029831 1860033541

10 x 50 mm Column 5 µm 186002973 186003264 186003257 18600327110 x 100 mm Column 5 µm 186003255 186003265 186003258 18600327210 x 150 mm Column 5 µm 186002974 186003266 186003259 18600327310 x 250 mm Column 5 µm 186003256 186003267 186003260 18600327419 x 10 mm Guard 5 µm 1860029752 1860029922 1860029842 1860033552

OBD 19 x 30 mm Column 5 µm 186002976 186003268 186003261 186003275OBD 19 x 50 mm Column 5 µm 186002977 186002993 186002985 186003356OBD 19 x 100 mm Column 5 µm 186002978 186002994 186002986 186003357OBD 19 x 150 mm Column 5 µm 186002979 186002995 186002987 186003358OBD 19 x 250 mm Column 5 µm 186004021 186004023 186004022 186004024OBD 30 x 50 mm Column 5 µm 186002980 186002996 186002988 186003277OBD 30 x 75 mm Column 5 µm 186002981 186003269 186003262 186003278OBD 30 x 100 mm Column 5 µm 186002982 186002997 186002989 186003279OBD 30 x 150 mm Column 5 µm 186003284 186003083 186002990 186003276OBD 30 x 150 mm Column 5 µm 186004025 — — —OBD 50 x 50 mm Column 5 µm 186003933 186003934 186003935 186003936OBD 50 x 100 mm Column 5 µm 186003937 186003938 186003939 186003940OBD 50 x 150 mm Column 5 µm 186003929 — — —OBD 50 x 250 mm Column 5 µm 186004107 — — —

10 x 10 mm Guard 10 µm 1860038893 1860040033 1860039883 —10 x 150 mm Column 10 µm 186003890 186004004 186003989 —10 x 250 mm Column 10 µm 186003891 186004005 186003990 —OBD 19 x 10 mm Guard 10 µm 1860038924 1860040064 1860039914 —OBD 19 x 50 mm Column 10 µm 186003893 186004007 186003992 —OBD 19 x 100 mm Column 10 µm 186003901 186004008 186003993 —OBD 19 x 150 mm Column 10 µm 186003894 186004009 186003994 —OBD 19 x 250 mm Column 10 µm 186003895 186004010 186003995 —OBD 30 x 100 mm Column 10 µm 186003930 — — —OBD 30 x 150 mm Column 10 µm 186003896 186004011 186003996 —OBD 30 x 250 mm Column 10 µm 186003897 186004012 186003997 —OBD 50 x 50 mm Column 10 µm 186003898 186004013 186003998 —OBD 50 x 100 mm Column 10 µm 186003902 186004014 186003999 —OBD 50 x 150 mm Column 10 µm 186003899 186004015 186004001 —OBD 50 x 250 mm Column 10 µm 186003900 186004016 186004002 —

Austria and European Export (Central South Eastern Europe, CIS and Middle East) 43 1 877 18 07

Australia 61 2 9933 1777

Belgium 32 2 726 1000

Brazil 55 11 5094-3788

Canada 1 800 252 4752 x2205

China 86 10 8586 8899

CIS/Russia +7 495 3367000

Czech Republic 420 2 617 1 1384

Denmark 45 46 59 8080

Finland 09 5659 6288

France 33 1 30 48 72 00

Germany 49 6196 400600

Hong Kong 852 29 64 1800

Hungary 36 1 350 5086

India and India Subcontinent 91 80 2837 1900

Ireland 353 1 448 1500

Italy 39 02 27 421 1

Japan 81 3 3471 7191

Korea 82 2 820 2700

Mexico 52 55 5200 1860

The Netherlands 31 76 508 7200

Norway 47 6 384 60 50

Poland 48 22 833 4400

Puerto Rico 1 787 747 8445

Singapore 65 6273 1221

Spain 34 93 600 9300

Sweden 46 8 555 11 500

Switzerland 41 56 676 70 00

Taiwan 886 2 2543 1898

United Kingdom 44 208 238 6100

All other countries: Waters Corporation U.S.A. 1 508 478 2000

1 800 252 4752

www.waters.com

Sales offices

© 2008 Waters Corporation. Waters, The Science of What’s Possible, OBD, Oasis, XBridge, SunFire, XTerra, BEH Technology,

ACQUITY UPLC, Alliance, AflaTest, Atlantis, Symmetry, IS, ACQUITY, UPLC, UltraPerformance LC and AutoPurification are trademarks of Waters Corporation. All other trademarks are the

property of their respective owners.

720001255EN April 2008 SC-W P

The quality management system of Waters’ manufacturing facilitiesin Taunton, Massachusetts and Wexford, Ireland complies with the Interna-tional Standard ISO 9001:2000 Quality Management and Quality Assurance Standards. Waters’ quality management system is periodically audited by the registering body to ensure compliance.