synergi - phenomenex · 2008-10-16 · silanol acidity even under neutral mobile phases. with few...

237Phenomenex

HPLCS

ynergi

Ultra-Performance HPlc colUmnS

the concept Behind Synergi: Separate the entire Polarity SpectrumMuch like a carpenter needs a good set of tools, we envisioned analytical chemists would appreciate a unique set of chromatography columns that simplify method development. With the Synergi column line, we sought to eliminate the guesswork and simplify HPLC column selection, bearing in mind the diverse separation scenarios chromatographers might routinely encounter. We engineered column phases to provide improved performance over a set polarity range as well as provide solutions to often difficult to resolve compound mixtures. Whether you are looking to develop a new method or improve an existing one, try it first on a Synergi column.

material characteristics Calculated Packing Particle Pore Pore Surface Carbon Bonded Phase End Material Shape/Size Size Volume Area Load Coverage Capping (μm) (Å) (mL/g) (m2/g) % (μmole/m2) Synergi Max-RP Spher. 2.5 100 — 400 17 — TMSSynergi Hydro-RP Spher. 2.5 100 — 400 19 — HydrophilicSynergi Polar-RP Spher. 2.5 100 — 400 11 — HydrophilicSynergi Fusion-RP Spher. 2.5 100 — 400 12 — TMSSynergi Max-RP Spher. 4, 10 80 1.05 475 17 3.21 TMSSynergi Hydro-RP Spher. 4, 10 80 1.05 475 19 2.45 HydrophilicSynergi Polar-RP Spher. 4, 10 80 1.05 475 11 3.15 HydrophilicSynergi Fusion-RP Spher. 4, 10 80 1.05 475 12 N/A TMS

See p. 346 for information on the Synergi Column Selectors.

Mobile PhaseTypical Reversed Phase Organic Modified Conditions

High

Medium

Low

Sele

ctiv

ity

Non-Polar

Polar

Polar

FusionFusioFusionn

Hydro

Max

synergi™

Synergi is a trademark of Phenomenex, Inc.

If Synergi does not provide at least an equivalent separation as compared to a competing column of the similar particle size, similar phase and dimensions, send in your comparative data within 45 days and keep the Synergi column for FREE.

238 Phenomenex

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Hydro-phobic/philic reversed Phase retention

Polar and aromatic reversed Phase Selectivity

C18 with Polar EndcappingSynergi Hydro-RP

C12 with TMS EndcappingSynergi Max-RP

C12

C12 C12

C12

Improved Polar Selectivity with reduced run times

1.5-10.02.5 µm, 4 µm, and 10 µmPolar embedded C18For a balanced retention of polar, basic compounds and moderate retention of hydrophobics over a broad pH rangeAnalysis of polar, basic compounds with little or no MS phase bleed

pH stabilityParticle Size

PhaseApplication

Strength

Synergi fusion-rP

Low High

Hydrophobicity

Polarity

H-Bonding

Aromatic Selectivity

Silanol Activity (pH 2.5)LC/Ms Certified

1.5-7.52.5 µm, 4 µm, and 10 µmC18 with polar endcappingFor extreme retention of non-polar and extremely polar alkyl compoundsResolution of highly polar compounds under 100 % aqueous mobile phase conditions


PhaseApplication

Strength

Synergi Hydro-rP

Low High

Hydrophobicity

Polarity

H-Bonding


Silanol Activity (pH 2.5)UsP:L1

1.5-7.02.5 µm, 4 µm, and 10 µmEther-linked phenyl with polar endcappingFor extreme retention of polar and aromatic compoundsImproved peak shape for acidic and basic analytes and aromatic selectivity with methanol containing mobile phases


PhaseApplication

Strength

Synergi Polar-rP

Low High

Hydrophobicity

Polarity

H-Bonding


Silanol Activity (pH 2.5)

1.5-10.02.5 µm, 4 µm, and 10 µmReversed phase C12For hydrophobic, non-polar compounds over a wide pH range, with little or no MS phase bleedSharp peak shape for basic compoundsat neutral pH


PhaseApplication

Strength

Synergi max-rP

Low High

Hydrophobicity

Polarity

H-Bonding


Silanol Activity (pH 2.5)LC/Ms

Certified

maximum reversed Phase Performance for the Synergi line

see p. 379 for testing probes.



see p. 379 for testing probes.UsP:L11

UsP:L1

Ether-Linked Phenyl with Polar Endcapping Synergi Polar-RP

C18

C18 C18

C18

Polar Group

C18 with Polar EmbeddingSynergi Fusion-RP

TM TM

synergi™

239Phenomenex

HPLCS

ynergi

High efficiency 2.5 and 4 µm Silica

4 µm Silica – High efficiency at low Backpressures

Synergi utilizes a 4 µm 80 Å silica to provide column efficiencies closer to 3 µm columns but at low backpressures similar to 5 µm columns. Ultra-high purity silica (99.99 % metal-free) ensures minimal surface metal sites available for chelation and reduces silanol acidity even under neutral mobile phases. With few metal and silanol sites free to bind with sample compounds, peak shapes are sharp.

2.5 µm Silica – rapid results and complete resolution

With the increased emphasis on sample throughput; rapid, high-resolution techniques are very desirable. Thus we developed a 2.5 µm 100 Å ultra-pure silica (99.99 % metal-free) which is packed into the MercuryMS™ LC/MS cartridge system providing high efficiency, high-throughput screening. 2.5 µm Synergi Max-RP and Synergi Hydro-RP can reduce analysis time by up to 60 % without a loss of performance. Because Synergi 2.5 µm medias are slurry packed into the MercuryMS cartridges, resolution and peak shapes are equivalent to that typically found only in longer analytical HPLC columns

Columns: Synergi 4 µm Max-RP Waters 3.5 µm Symmetry C18 Zorbax 3.5 µm SB-C18Dimensions: 50 x 4.6 mmMobile Phase: A = Water with 0.1 % Formic acid B = Acetonitrile with 0.1 % Formic acidgradient: A/B (95:5) to A/B (5:95) in 4 minFlow rate: 4.0 mL/ minDetection: UV @ 254 nmTemperature: 30 °Cinjection: 2.5 μLsample: 1. Dexamethasone (2.5 μg) 2. Hydrocortisone (2.5 μg) 3. 11-α-Hydroxyprogesterone (2.5 μg) 4. 17-α-Hydroxyprogesterone (2.5 μg) 5. Progesterone (2.5 μg)

Zorbax®

3.5 µm SB-C18*Backpressure=216 Bar

Symmetry® 3.5 µm C18*

Backpressure=218 Bar

Synergi Silica characteristics

4 μm 80 ÅSILICA

Particle Particle Surface Pore Pore Metal Size Size Area Size Volume Content (μm) Distribution (m²/g) (Å) (mL/g) (ppm)

4.00 ± 0.10 1.80 475 ± 25 80 ± 10 1.00 ± 0.05 < 55

App

ID 1

2480

*See p. 248 for disclaimer information.

Columns: Synergi 4 µm Max-RP Luna® 5 µm C18 Luna® 3 µm C18Dimensions: 50 x 4.6 mmMobile Phase: Water / Acetonitrile (35:65)Detection: UV @ 254 nminjection: 1 µLTemperature: 30 °Csample: Naphthalene (1 µg)

low Backpressures

High efficiency

Lower Column Backpressure Compared to 3.5 µm Competitors

mercury mS™ cartridge SystemPacked with 2.5 µm Synergi Max-RP, Synergi Hydro-RP, Synergi Polar-RP and Synergi Fusion-RP

For more MercuryMS information, performance and technical specifications, see p. 274.

App

ID 1

2520

App

ID 1

2521

5 µm C18

4 µm Max-RP

3 µm C18

Low Backpressure!TM TM

synergi™

Synergi™ 4 µm Max-RPBackpressure=160 Bar

240 Phenomenex

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SynergI max-rP

a reversed phase c12 column

maximum reversed Phase Performance for the Synergi line

• Hydrophobic retention similar to a c18 – but it’s a c12

• 25 % more free silanol coverage than most c18 columns

• pH stable from 1.5 to 10 for sharp peaks for basic and tailing compounds

Who says you need a c18 column?

The bulky nature of C18 ligands results in relatively low coverage of surface silanols causing peak tailing. Nevertheless, C18 columns have the hydrophobic selectivity chromatographers rely on. To reduce peak tailing and still offer the preferred hydrophobic selectivity, we engineered Synergi Max-RP with a C12 bonded phase. A C12 ligand is sterically less hindered than a C18 and covers 25 % more of the silica surface, shielding more free silanols. When bonded to our extreme (475 m2/g) surface area silica, Synergi Max-RP gives the hydrophobic retention and methylene selectivity you would expect from a C18 column, but with sharper peaks, less tailing, and excellent reproducibility.

6.3706.740 6.930

8.2808.570

9.110

0.000

1.000

2.000

3.000

4.000

5.000

6.000

7.000

8.000

9.000

10.000

XTerra 5 µm MS C18 Zorbax 5 µm SB C18 Nucleosil 5 µm C18 Synergi 4 µm Max-RP Zorbax 5 µm XDB C18 Luna 5 µm C18(2)

k bu

tylb

enze

ne

Hydrophobic retention:synergi Max-rP (C12) Performs Like a C18

synergi Max-rP vs. Zorbax 3.5 µm XBD C18

C12 with TMS EndcappingSynergi Max-RP

C12

C12 C12

C12

Columns: XTerra 5 µm MS C18 Zorbax 5 µm SB C18 Nucleosil 5 µm C18 Synergi 4 µm Max-RP Zorbax 5 µm XDB C18 Luna 5 µm C18(2)Dimensions: 150 x 4.6 mm Mobile Phase: Acetonitrile/ Water (80:20)Flow rate: 1 mL/ min Detection: UV @ 254 nminjection: 1 µLTemperature: Ambientsample: Butylbenzene

Columns: Synergi 4 µm Max-RP Zorbax 3.5 µm Eclipse XDB C18Dimensions: 50 x 4.6 mm Mobile Phase: A = Water with 0.1 % Formic acid B = Acetonitrile with 0.1 % Formic acidgradient: A/B (95:5) to 100 % B in 5 minFlow rate: 1.5 mL/ minDetection: UV @ 254 nminjection: 5 µLTemperature: 30 °Csample: 1.Thiourea (0.25 µg) 2. Codeine (1 µg) 3. Chlorpheniramine (2 µg) 4. Propranolol (6 µg) 5. Desipramine (0.5 µg) 6. Ibuprofen (6 µg)

Sharper Peaks!

App

ID 1

2519

App

ID 1

2476

Zorbax® 3.5 µm XDB C18

1.5-10.02.5 µm, 4 µm, and 10 µmReversed phase C12For hydrophobic, non-polar compounds over a wide pH range, with little or no MS phase bleedSharp peak shape for basic compoundsat neutral pH


PhaseApplication

Strength

Synergi max-rP

Low High

Hydrophobicity

Polarity

H-Bonding


Silanol Activity (pH 2.5)LC/Ms

Certified see p. 379 for testing probes.

TM TM

Synergi™ 4 µm Max-RP


synergi™

241Phenomenex

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ynergi

Synergi max-rP (cont'd)

Sharper Peaks for Basic compounds

Ionic interactions created by free silanols also can contribute to peak tailing. Low pH mobile phases (~2.5) are often employed in order to protonate free silanols. Un-protonated free silanols can lead to poor peak shape for basic drugs. Utilizing a C12 bonded phase, a 25 % greater bonded phase density is achieved compared to typical C18 bonding, covering more free silanols. Benzylamine and phenol are used to probe for active silanol sites at pH 2.5; Synergi Max-RP shows lower silanol activity as compared to other C18 columns.

Columns: Synergi 4 µm Max-RP XTerra MS 5 µm C18Dimensions: 150 x 4.6 mm Mobile Phase: 20 mM Potassium phosphate pH 7 / Acetonitrile / Methanol (50:25:25)Flow rate: 1.5 mL/ minDetection: UV @ 210 nminjection: 1 µLTemperature: 30 °Csample: Methanol extract from Chlortrimeton Allergy Pills 1. Chlorpheniramine

reproducible Performance from pH 1.5 to 10

Our bonding and endcapping technology give Synergi 4 µm Max-RP stability from pH 1.5 (0.1 % TFA) to 10. This robust pH range ensures that there will be little bleed at low pH’s due to bonded phase hydrolysis and that a broad range of mobile phase modifiers can be used without damaging the column. It also allows analysts to use high pH’s to overcome basic ionization and to overcome sample solubility issues.

synergi Max-rP vs. Waters® XTerra® Ms

silanol Activity at Low pH: C12 vs. C18 Phases

Columns: XTerra 5 µm MS C18 Zorbax 5 µm SB C18 Nucleosil 5 µm C18 Synergi 4 µm Max-RP Zorbax 5 µm XDB C18 Luna 5 µm C18(2) Dimensions: 150 x 4.6 mmMobile Phase: Methanol/20 mM Potassium phosphate, pH 2.5 (30:70)Flow rate: 1 mL/ minDetection: UV @ 254 nminjection: 5 µLTemperature: Ambient sample: 1. Benzylamine 2. Phenol

App

ID 1

0896

Sharper Peaks!

Column: Synergi 4 µm Max-RP Dimensions: 30 x 2.0 mmPart no.: 00A-4337-B0 Mobile Phase: A = Water with 0.1 % TFA B = Acetonitrile with 0.1 % TFAgradient: A/B (95:5) to A/B (5:95) in 3 minFlow rate: 1.0 mL/ minDetection: UV @ 254 nminjection: 1 µLTemperature: 30 °Csample: Precipitated porcine serum (2:1 Acetonitrile:serum) containing: 1. Oxazepam (50 ng) 2. Temazepam (50 ng) 3. Nordiazepam (50 ng) 4. Diazepam (50 ng)

Column: Synergi 4 µm Max-RP Dimensions: 30 x 2.0 mmPart no.: 00A-4337-B0 Mobile Phase: Water with 0.1 % Ammonium hydroxide, pH 10 / Acetonitrile with 0.1 % Ammonium hydroxide, pH 10 (50:50)Flow rate: 0.5 mL/ minDetection: UV @ 254 nminjection: 5 µLTemperature: 30 °Csample: 1. Pyridine 2. Phenol 3. Toluene

stability @ pH 10.0

stability @ pH 1.5

0.048

0.059 0.060

0.079

0.088

0.095

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

Synergi 4 µm Max-RP Luna 5 µm C18(2) XTerra 5 µm MS C18 Zorbax 5 µm SB C18 Zorbax 5 µm XDB C18 Nucleosil 5 µm C18

alp

ha

of

ben

zyla

min

e/p

hen

ol

Over 18,000 ColumnVolumes ofMobile Phase!

XTerra® MS 5 µm C18

Over 18,000 ColumnVolumes ofMobile Phase!

App

ID 1

2481

App

ID 1

2482

TM TM

Peak Asymmetry = 1.25

Peak Asymmetry = 1.58

Synergi™ 4 µm Max-RP

synergi™

242 Phenomenex

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min2 4 6 8 10 12 14

min0 2 4 6 8 10 12 14

14840

12

3

4

1

2 34

SynergI fUSIon-rPa polar embedded c18 column

Balancing Polar, Basic and Hydrophobic compound retention• a polar embedded phase for improved separation of polar basic compounds

• a moderately hydrophobic column for applications requiring moderate retention

• low mS bleed with enhanced selectivity of a polar embedded phase

• 1.5 to 10 pH stability for method flexibility†

• Stable in 100 % aqueous conditions

Hydrophobic Basic Compounds

App

ID 1

4840

Typical C18

Balanced Retention! Antihistamines

Better Selectivity!

Columns: Synergi 4 µm Fusion-RP Typical C18 Dimensions: 150 x 4.6 mm Mobile Phase: 20 mm Potassium Phosphate, pH 2.5 / Acetonitrile (75:25)Flow rate: 1.0 mL/ minDetection: UV @ 210 nmsample: 1. Maleic acid 2. Chlorpheniramine 3. Triprolidine 4. Diphenhydramine

App

ID 1

4839

Columns: Synergi 4 µm Fusion-RP Waters 5 µm SymmetryShield™ C18 Supelco Discovery® 5 µm RP-AmideC16Dimensions: 150 x 4.6 mm Mobile Phase: 20 mm Potassium Phosphate, pH 2.5 / Methanol (70:30)Flow rate: 1.0 mL/ minDetection: UV @ 210 nmsample: 1. Phenylephrine 2. Phenylpropanolamine 3. Pseudoephedrine

Longer

Polar Retention

Less Hydrophobic Retention

†See p. 238 for pH Stability.

min0 1 2 3 4 5 6 7 8 9

min0 1 2 3 4 5 6 7 8 9

min0 1 2 3 4 5 6 7 8 9

14839

1 2 3

1 2 3

12 & 3

1.5-10.02.5 µm, 4 µm, and 10 µmPolar embedded C18For a balanced retention of polar, basic compounds and moderate retention of hydrophobics over a broad pH rangeAnalysis of polar, basic compounds with little or no MS phase bleed


PhaseApplication

Strength

Synergi fusion-rP

Low High

Hydrophobicity

Polarity

H-Bonding


Silanol Activity (pH 2.5)LC/Ms Certified

see p. 379 for testing probes.UsP:L1

Waters 5 µm SymmetryShield™ C18

Supelco Discovery® 5 µm RP-AmideC16

C18

C18 C18

C18

Polar Group

C18 with Polar EmbeddingSynergi Fusion-RP

Improved Polar Selectivity with reduced run times

Synergi Fusion-RP uses a polar embedded and a hydrophobic ligand to achieve improved selectivity. The C18 ligand gives Synergi Fusion-RP good hydrophobic retention and selectivity, while the polar embedded group provides enhanced polar retention. This dual-phase selectivity allows balanced polar, acidic, basic and hydrophobic compound retention and resolution. If you are working with mixtures of compounds with polar and non-polar characteristics, and having difficulties finding that perfect mix of selectivity, then you should try Synergi Fusion-RP.

no-nItrogen Polar Infused c18 column

By using a non-nitrogen containing polar embedded group, Synergi Fusion-RP is able to retain both acidic and basic compounds, with good peak shapes, in comparison to many competing polar embedded columns. The unique polar functionality, in addition to the C18 alkyl ligand, provides a balanced solution to numerous separation needs.

TM TM


synergi™

Synergi™ 4 µm Fusion-RP


243Phenomenex

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ynergi

Columns: Synergi 4 µm Fusion-RP Waters 5 µm SymmetryShield™C18Dimensions: 150 x 4.6 mm Mobile Phase: A: 20 mM Potassium Phosphate, pH 2.5 B: Methanolgradient: A/B (95:5) for 0.5 min A/B (20:80) in 15 minFlow rate: 1.0 mL/ minDetection: UV @ 254 nmsample: 1. Sulfanilamide 2. Sulfathiazole 3. Sulfamerazine 4. Sulfamethoxazole 5. Sulfaquinoxaline

sulfa Drugs

App

ID 1

4838


Enhanced Polar Retention!

extremely low lc/mS column Bleed

A careful control of the endcapping process combined with the chemical nature of the polar embedded group results in high phase stability with minimal ligand cleavage. The excellent bleed profile compared to a competitor's in the figure below shows Synergi Fusion-RP is well suited for LC/MS work.

100 % aqueous for added method flexibility

Use Synergi Fusion-RP for greater polar retention under 100 % aqueous conditions. The polar embedded group allows this phase to be run under 100 % aqueous conditions without loss of retentivity. Unlike typical C18 phases, the pores and the sorbent surface of Synergi Fusion-RP stay wet even after many hours of operation in 100 % aqueous mobile phase. This aqueous stability offers greater flexibility in application development.

Synergi fusion-rP (cont'd)1.5-10 pH Stable for rugged methods

The ability of Synergi Fusion-RP to operate in an extended pH range of 1.5-10 (under isocratic elution conditions) is the direct result of an exhaustive endcapping procedure, which is highly protective of the silica surface. pH stability is an indication of column ruggedness. pH tested at the extremes (1.5 and 10), for more than 4000 column volumes, the results below clearly show how rugged Synergi Fusion-RP is. Imagine how well this column will work for your application.

Column: Synergi 4 µm Fusion-RPDimensions: 150 x 4.6 mm Mobile Phase: 20 mM Potassium phosphate buffer pH 2.5Flow rate: 1.0 mL/ minDetection: UV @ 254 nminjection: 1 µLTemperature: 30 °Csample: 1. Thiourea 2. Adenine 3. Guanosine-5-monophosphate 4. Thymine

Aqueous stability

App

ID 1

4891

After 24 hours of 100 % Aqueous Conditions

At time=0

Dimensions: 150 x 4.6 mm Mobile Phase: A: 0.1 % CH3COOH in Water B: 0.1 % CH3COOH in Methanolgradient rate: 95:5 (A/B) linear to 5:95 over 8 min hold for 5 minFlow rate: 0.5 mL/ minDetection: Bruker-Daltonics Esquire 2000 IT Ion Source: ESI Scan Rate: 13000 m/z/s Scan Range: 50-1000

Bleed Profile

Low MS Background!

min0 2 4 6 8 10 12 14 16 18

min0 2 4 6 8 10 12 14 16 18

14838

12

3 4

5

12 3

4

5


14891

1

23 4

1

23 4

min0 2 4 6 8 1 0 1 2 1 4

min0 2 4 6 8 1 0 1 2 1 4

0 1 2 3 4 5 6 7 8 90 1 2 3 4 5 6 7 8 9 min

0.1x106

0.2

0.3

0.4

0.5

0.6

x107

pH stability Test

TIC:

Tot

al Io

n Cu

rren

t

pH testing was done under isocratic conditions with phosphate buffer. Formic acid and ammonium formate were also used as test buffers.

pH 10.0

pH 1.5

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

0

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2

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Column Volumes

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12000

00 1000 2000 3000 4000

Column Volumes

tR of toluene (min.)

K of toluene

symmetry of toluene

symmetry of pyridine

N of toluene (plates/column)

tR of toluene (min.)

K of toluene

symmetry of toluene

symmetry of pyridine

N of toluene (plates/column)

TM TM



synergi™

NucleosidesColumn: Synergi 4 µm Fusion-RPDimensions: 150 x 4.6 mmPartNo.: 00F-4424-E0MobilePhase: A: 20 mM Ammonium acetate, pH 4.5 B: AcetonitrileGradient: A/B (95:5) to (60:40) in 10 minFlowRate: 1 mL/minDetection: UV @ 260 nmSample: 1. Cytidine 2. Uridine 3. Guanosine 4. Thymine 5. Adenine

App

ID 1

5768Fluoropharmaceuticals

Column: Synergi 4 µm Fusion-RPDimensions: 150 x 4.6 mmPartNo.: 00F-4424-E0MobilePhase: 20 mm Potassium Phosphate, pH 2.5/ Acetonitrile (50:50)FlowRate: 1 mL/minTemperature: 30 °CDetection: UV @ 254 nmSample: 1. Flurbiprofen 2. Diflunisal

App

ID 1

4846

AdenineFamilyColumn: Synergi 4 µm Fusion-RPDimensions: 150 x 4.6 mmPartNo.: 00F-4424-E0MobilePhase: A: 20 mM Ammonium acetate, pH 4.5 B: AcetonitrileGradient: A/B (97:3) to (60:40) in 10 minFlowRate: 1 mL/minDetection: UV @ 260 nmSample: 1. ATP 2. ADP 3. AMP 4. Adenine 5. Adenosine

App

ID 1

5754Trichothecenes

Column: Synergi 4 µm Fusion-RPDimensions: 150 x 3.0 mmPartNo.: 00F-4424-Y0MobilePhase: A: 20 mM Ammonium Acetate, pH 4.5 B: AcetonitrileGradient: A/B (90:10) in 0.5 min to (20:80) in 15 minFlowRate: 0.5 mL/minDetection: Mass Spectrometer (MS)Sample: 1. T-2 5. 15-Acetyl-Deoxynivalenol 2. Nivalenol 6. 3-Acetyl-Deoxynivalenol 3. Deoxynivalenol 7. Diacetoxyscirpenol 4. Fusarenon-X 8. HT-2

App

ID 1

5541

DeoxynucleosidesColumn: Synergi 4 µm Fusion-RPDimensions: 150 x 4.6 mmPartNo.: 00F-4424-E0MobilePhase: A: 20 mM Ammonium acetate, pH 4.5 B: AcetonitrileGradient: A/B (95:5) to (60:40) in 10 minFlowRate: 1 mL/minDetection: UV @ 260 nmSample: 1. 2'-Deoxycytidine 2. 2'-Deoxyuridine 3. 2'-Deoxyguanosine 4. 2'-Deoxythymine 5. 2'-Deoxyadenine

App

ID 1

5766

0 2 4 6 8

14846

1

2

min0

15768

2

2

1

4

43

5

6 8 10

min0

15754

2

2

1

4

43

5

6 8 10 12

min0

15766

2

2

1

4

4

35

6 8 10

Excellentresolutionofnucleicacids,nucleotides,andnucleosides

244 Phenomenex

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Synergi fusion-rP (cont'd)

Applications

15541

3 4

5

6

7

8

0 2 4 6 8 10 12 14 16 18 min

2

1

Improve analyte sensitivity and reduce baseline noise with Strata SPE tubes and well plates. See p. 3 for more information.

synergi™

245Phenomenex

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ynergi

App

ID 1

4425

*See p. 248 for disclaimer information.

SynergI Hydro-rPa polar endcapped c18 column

Hydro-phobic/philic reversed Phase retention

• a c18 with enhanced retention of polar compounds

• Polar resolution enhanced in 100 % aqueous mobile phases

• extreme retention of hydrophobic compounds

a c18 with enhanced retention of Polar compounds

Extremely polar analytes do not retain well nor separate well on conventional C18 columns. Unlike conventional C18 columns, Synergi Hydro-RP’s C18 bonded phase is endcapped with a proprietary polar group to provide extreme retention of both hydrophobic as well as polar compounds via polar interactions, hydrogen bonding or electrostatic interactions. The high (475 m2/g) 4 µm silica surface area combined with a dense bonded phase coverage allows for substantial interaction between the sample analyte and the bonded phase yielding overall a very retentive C18 phase.

App

ID 1

4239

App

ID 1

4266

synergi Hydro-rP vs. Zorbax XDB

Columns: Synergi 4 µm Hydro-RP Zorbax Eclipse XDB 5 µm Dimensions: 150 x 4.6 mmMobile Phase: A=10 mM Triethylammonium formate pH 6 B=Acetonitrile with 10 mM Triethylammonium formate gradient: 15 % B to 65 % B in 15 minFlow rate: 1.5 mL/ minTemperature: AmbientDetection: UV @ 230 nminjection: 1 µL of beta-blockers mix each at 0.8 µg/ µLsample: 1. Atenolol 2. Pindolol 3. Nadolol 4. Acebutolol 5. Metoprolol 6. Labetalol

The polar endcapping of Synergi Hydro-RP provides unique selectivity in the separation of basic, polar drugs such as these beta-blockers.

Polar Retention!

Agilent TechnologiesZorbax® 5 µm Eclipse® XDB*Luna® 5 µm C18(2) 100 Å

App

ID 1

4424

Greater retention,polar selectivity!

C18 with Polar EndcappingSynergi Hydro-RP

1.5-7.52.5 µm, 4 µm, and 10 µmC18 with polar endcappingFor extreme retention of non-polar and extremely polar alkyl compoundsResolution of highly polar compounds under 100 % buffer mobile phase conditions


PhaseApplication

Strength

Synergi Hydro-rP

Low High

Hydrophobicity

Polarity

H-Bonding


Silanol Activity (pH 2.5)UsP:L1 see p. 379 for testing probes.

synergi Hydro-rP vs. Luna® C18(2)

Dimensions: 150 x 4.6 mmMobile Phase: 20 mM Potassium phosphate, pH 7.0 /Methanol (60:40)Flow rate: 1 mL/ minTemperature: 22 °CDetection: UV @ 210 nmsample: 1. Phenylephrine 2. Phenylpropanolamine 3. Pseudoephedrine 4. Methylparaben

Synergi™ 4 µm Hydro-RPSynergi™ 4 µm Hydro-RP 80 Å

synergi™

246 Phenomenex

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TM TM

Dimensions: 150 x 4.6 mmMobile Phase: Acetonitrile/20 mM Potassium phosphate pH 7.0 (65:35)Flow rate: 1.5 mL/ minTemperature: Ambientsample: 1. Butylbenzene 2. Amylbenzene

Hydrophobic retentivity Compared

Synergi Hydro-rP (cont'd)

extreme retention of Hydrophobic compounds

Synergi Hydro-RP shows increased hydrophobic retention and polar retention compared to other C18 phases. Increased hydrophobic retention allows for flexibility in fraction manipulation and modification of the organic component in the mobile phase. Enhanced hydrophobic retention allows for separation of compound that differ slightly in hydrophobicity, and results in compounds eluting in higher organic mobile phase, delivering a higher MS sensitivity versus more aqueous eluates.

Organic Acids

App

ID 1

4270

Column: Synergi 4 µm Hydro-RPDimensions: 250 x 4.6 mmPart no.: 00G-4375-E0Mobile Phase: 20 mM Potassium phosphate, pH 2.9Flow rate: 0.7 mL/ minTemperature: AmbientDetection: UV @ 220 nmsample: 1. Oxalic acid 2. Tartaric acid 3. Glycolic acid 4. Formic acid 5. Pyruvic acid 6. Malonic acid 7. Acetic acid 8. Maleic acid 9. Citric acid

explosives Mix: ePA Method 8330

App

ID 1

4768

Column: Synergi 4 µm Hydro-RPDimensions: 250 x 4.6 mmPart no.: 00G-4375-E0Mobile Phase: Water/Methanol/Acetonitrile (51:45:4)Flow rate: 0.8 mL/ minTemperature: 35 °CDetection: UV @ 254 nmsample: 1. HMX 2. RDX 3. 1,3,5-Trinitrobenzene 4. 1,2-Dinitrobenzene (Int Std) 5. Tetryl 6. 1,3-Dintrobenzene 7. Nitrobenzene 8. 2,4,6-Trinitrotoluene 9. 4-Amino-dinitrotoluene 10. 2-Amino-4,6-dinitrotoluene 11. 2,6-Dinitrotoluene 12. 2,4-Dinitrotoluene 13. 2-Nitrotoluene 14. 4-Nitrotoluene 15. 3-Nitrotoluene

0 10 20 min

12

3

4

5

6

78

9

10

11

12

1314

15

The chart was obtained by plotting hydrophob ic re ten t ion ( k fo r butylbenzene vs. methylene selectivity (log k for amylbenzene vs the number of methyl groups) under the stated conditions. A column with high hydrophobicity will better resolve two analytes which subtly differ in their overall hydrophobicity than a column with lower hydrophobic selectivity.

1

23

4

56

7

8

9

0 2 104 126 148 min

Polar retentionunder 100 %aqueous conditions!

synergi™

247Phenomenex

HPLCS

ynergi

Ether-Linked Phenyl with Polar Endcapping Synergi Polar-RP

App

ID 1

2527

1252

612

525

1248

5

Columns: Synergi 4 µm Polar-RP Waters 5 µm SymmetryShield C18 Waters 5 µm Symmetry C18 Waters XTerra 5 µm RP18Dimensions: 150 x 4.6 mmMobile Phase: 20 mM Potassium phosphate pH 3 / Methanol (50:50)Flow rate: 1.0 mL/ minDetection: UV @ 230 nmTemperature: Ambientinjection: 2 μLsample: 1. Metaproterenol (0.4 μg) 4. Alprenolol (0.3 μg) 2. Pindolol (0.6 μg) 5. Propranolol (0.04 μg) 3. Metoprolol (0.15 μg) 6. Ethylparaben (0.4 μg)

Polar resolution Compared

Waters 5 µm SymmetryShield™ C18*

Waters 5 µm XTerra® RP18*

Waters 5 µm Symmetry® C18*

SynergI Polar-rPether-linked phenyl column with polar endcapping

Polar and aromatic reversed Phase Selectivity• Increased retention of highly polar and aromatic compounds

• Polar resolution enhanced in 100 % buffer mobile phases

• Highly reproducible and stable phenyl phase

App

ID 1

0905

App

ID 1

0906

improved resolution and Peak shape

Waters 5 µm XTerra® RP18*

Columns: Synergi 4 µm Polar-RP Zorbax 5 µm SB-Phenyl Inertsil 5 µm Phenyl PH-3 Waters XTerra 5 µm RP18Dimensions: 150 x 4.6 mmMobile Phase: 20 mM Potassium phosphate pH 7.0/Acetonitrile (80:20)Flow rate: 1.0 mL/ minDetection: UV @ 254 nmTemperature: 25 °Csample: 1. Famotidine 2. Cimetidine 3. Ranitidine

Famotidine Peak

Enhanced Polar Selectivity!

Improved Selectivity!

1.5-7.02.5 µm, 4 µm, and 10 µmEther-linked phenyl with polar endcappingFor extreme retention of polar and aromatic compoundsImproved peak shape for acidic and basic analytes and aromatic selectivity with methanol containing mobile phases


PhaseApplication

Strength

Synergi Polar-rP

Low High

Hydrophobicity

Polarity

H-Bonding


Silanol Activity (pH 2.5)see p. 379 for testing probes.

GL Sciences®

Inertsil 5µm Phenyl PH-3* Zorbax® 5µm SB-Phenyl*

UsP:L11

Increased retention of Highly Polar and aromatic compounds

Synergi Polar-RP is an ether-linked phenyl phase with proprietary hydrophilic endcapping designed specifically to maximize retention and selectivity for polar and aromatic analytes. Aromatic selectivity can be further enhanced by the addition of methanol to the mobile phase. Methanol facilitates the π-π interactions between the aromatic rings of the analyte and the phenyl functional group of Synergi Polar-RP. This feature allows for improved polar retention that complements the more conventional C18 column chemistries, as well as provide improved peak shape and an alternative selectivity compared to other polar phases.

TM TM

synergi™

Synergi™ 4 µm Polar-RP




248 Phenomenex

HPLC

Syn

erg

i

TM

Aqueous stability

App

ID 1

0904

Synergi Polar-rP (cont'd)

Polar resolution enhanced in 100 % Buffer mobile Phases

The ether-linkage on the Synergi 4 µm Polar-RP phase contributes to not only sharp peak shape for acidic and basic analytes, but also ensures stability under highly aqueous mobile phase conditions. Very polar analytes, like formic acid, typically retain poorly on traditional alkyl-bonded phases. However, using a 100 % buffer mobile phase, the formic acid impurity is easily resolved from acetic acid. Other polar-embedded phases typically use a nitrogen-containing amide linkage or carbamate group; this can interfere with the resolution of highly acidic polar compounds. Since Synergi Polar-RP uses an ether-linkage as the polar embedded group, the result is improved peak shape and resolution of the highly acidic polar compound, formic acid.

Columns: Synergi 4 µm Polar-RP Waters XTerra RP18 5 µm Supelco Discovery 5 µm RP-Amide C16Dimensions: 150 x 4.6 mmMobile Phase: 20 mM Potassium phosphate pH 2.5/Methanol (97:3)Flow rate: 1.0 mL/ minDetection: UV @ 220 nmTemperature: 25 °Csample: 1. Formic acid 2. Acetic acid

Formic and Acetic Acids

Waters® XTerra®

RP18 5 µm*

Supelco Discovery RP-AmideC16 5 µm

®*

Column: Synergi 4 µm Polar-RP Dimensions: 30 x 2.0 mmPart no.: 00A-4336-B0 Mobile Phase: A = Water with 0.1 % TFA B = Acetonitrile with 0.1 % TFAgradient: A/B (95:5) to A/B (5:95) in 3 minFlow rate: 1.0 mL/ minDetection: UV @ 254 nmTemperature: 30 °Cinjection: 1 μLsample: Precipitated porcine serum (2:1 Acetonitrile:serum) containing: 1. Oxazepam (50 ng) 2. Temazepam (50 ng) 3. Nordiazepam (50 ng) 4. Diazepam (50 ng)

Better acidic compound resolution in 100 % buffer conditions!

Using a Phenyl PhasePhenyl phases can provide separations not achievable on alkyl phases (C18 or C12), such as increased retention for polar, aromatic compounds as well as reversals in analyte elution order. The unique selectivity is due to the interactions between the π electrons of the phenyl ring and π electrons from aromatic groups of the analytes.To fully express a phenyl phase’s unique aromatic selectivity, methanol should be used as the organic component rather than acetonitrile. It is presumed the π electrons of the “CN” bond in acetonitrile compete for the phenyl “binding sites” on the stationary phase, thus limiting the amount of π-π interaction between the stationary phase and the analyte molecules and the separation is based only on hydrophobic retention.

synergi™


*Comparative separations may not be representative of all applications. Synergi and Polar-RP are trademarks of Phenomenex, Inc., Luna and Phenomenex are registered trademarks of Phenomenex, Inc.

Discovery is a registered trademark of Sigma-Aldrich® Biotechnology. Phenomenex has no affiliation with Sigma-Aldrich®. Columns used for comparison studies were manufactured by and purchased from Sigma-Aldrich®.

Nucleosil is a registered trademark of Macherey Nagel. Phenomenex has no affiliation with Macherey Nagel. Columns used for comparison studies were manufactured by and purchased from Macherey Nagel.

Inertsil is a registered trademark of GL Sciences Inc.. Phenomenex has no affiliation with GL Sciences Inc.. Columns used for comparison studies were manufactured by and purchased from GL Sciences Inc.

SymmetryShield is a trademark of Waters Corp. XTerra and Symmetry are registered trademarks of Waters Corp. Phenomenex has no affiliation with Waters Corp. Columns used for comparison studies were manufactured by and purchased from Waters Corp.

Zorbax Bonus-RP is a trademark of Agilent Technologies. Zorbax SB and Zorbax Eclipse XDB are registered trademarks of Agilent Technologies. Columns used for comparison studies were manufactured by and purchased from Agilent Technologies.

249Phenomenex

HPLCS

ynergi

4 µm Analytical Columns (mm) SecurityGuard™Cartridges

30 x 4.6 50 x 4.6 75 x 4.6 150 x 4.6 250 x 4.6 4 x 3.0 mm Max-RP I 00A-4337-E0 I 00B-4337-E0 I 00C-4337-E0 I 00F-4337-E0 I 00G-4337-E0 AJ0-6074Hydro-RP 00A-4375-E0 00B-4375-E0 00C-4375-E0 00F-4375-E0 00G-4375-E0 AJ0-7511 Polar-RP 00A-4336-E0 00B-4336-E0 00C-4336-E0 00F-4336-E0 00G-4336-E0 AJ0-6076Fusion-RP 00A-4424-E0 00B-4424-E0 00C-4424-E0 00F-4424-E0 00G-4424-E0 AJ0-7557

4 µm Microbore and Minibore Columns (mm) SecurityGuard™Cartridges

50 x 1.0 150 x 1.0 250 x 1.0 30 x 2.0 50 x 2.0 75 x 2.0 150 x 2.0 250 x 2.0 4 x 2.0 mm Max-RP I 00B-4337-A0 I 00F-4337-A0 I 00G-4337-A0 I 00A-4337-B0 I 00B-4337-B0 I 00C-4337-B0 I 00F-4337-B0 I 00G-4337-B0 AJ0-6073Hydro-RP 00B-4375-A0 00F-4375-A0 00G-4375-A0 00A-4375-B0 00B-4375-B0 00C-4375-B0 00F-4375-B0 00G-4375-B0 AJ0-7510 Polar-RP 00B-4336-A0 00F-4336-A0 — 00A-4336-B0 00B-4336-B0 00C-4336-B0 00F-4336-B0 00G-4336-B0 AJ0-6075Fusion-RP 00B-4424-A0 00F-4424-A0 00G-4424-A0 00A-4424-B0 00B-4424-B0 00C-4424-B0 00F-4424-B0 00G-4424-B0 AJ0-7556

4 µm Narrow Bore Columns (mm) SecurityGuard™Cartridges

30 x 3.0 50 x 3.0 75 x 3.0 150 x 3.0 250 x 3.0 4 x 2.0 mm

Max-RP I 00A-4337-Y0 I 00B-4337-Y0 I 00C-4337-Y0 I 00F-4337-Y0 I 00G-4337-Y0 AJ0-6073Hydro-RP 00A-4375-Y0 00B-4375-Y0 00C-4375-Y0 00F-4375-Y0 00G-4375-Y0 AJ0-7510Polar-RP 00A-4336-Y0 00B-4336-Y0 00C-4336-Y0 00F-4336-Y0 00G-4336-Y0 AJ0-6075Fusion-RP 00A-4424-Y0 00B-4424-Y0 00C-4424-Y0 00F-4424-Y0 00G-4424-Y0 AJ0-7556

orderIng InformatIon

for ID: 2.0-3.0 mm

for ID: 3.2-8.0 mm

for ID: 9-16 mm 18-30 mm

for ID: 9-16 mm 18-30 mm

† Requires MercuryMS cartridge holder see p. 276

10 µm Bulk Packings

100g 1kg 5kg Max-RP I 04G-4350 I 04K-4350 I 04L-4350 Hydro-RP 04G-4376 04K-4376 04L-4376 Polar-RP 04G-4351 04K-4351 04L-4351Fusion-RP 04G-4425 04K-4425 04L-4425

Larger quantities of bulk media available upon request.

4 µm Capillary Columns (mm)

50 x 0.30 150 x 0.30 250 x 0.30 50 x 0.50 150 x 0.50 250 x 0.50 Max-RP I 00B-4337-AC I 00F-4337-AC I 00G-4337-AC I 00B-4337-AF I 00F-4337-AF I 00G-4337-AFHydro-RP 00B-4375-AC 00F-4375-AC 00G-4375-AC 00B-4375-AF 00F-4375-AF 00G-4375-AF

MercuryMS LC/MS Cartridges† (mm)

10 x 2.0 10 x 4.0 20 x 2.0 20 x 4.0Qty Pricing 2.5 µm Phase I I I ISynergi Hydro-RP 00N-4387-BO-CE 00N-4387-DO-CE 00M-4387-BO-CE 00M-4387-DO-CESynergi Max-RP 00N-4372-BO-CE 00N-4372-DO-CE 00M-4372-BO-CE 00M-4372-DO-CESynergi Fusion-RP 00N-4423-BO-CE 00N-4423-DO-CE 00M-4423-BO-CE 00M-4423-DO-CESynergi Polar-RP — 00N-4371-DO-CE 00M-4371-BO-CE 00M-4371-DO-CE

for ID: 2.0-3.0 mm

4 µm Semi-Prep and Preparative Columns (mm) SecurityGuard™Cartridges

250 x 10 250 x 15 150 x 21.2 250 x 21.2 250 x 30 10 x 10 mm 15 x 21.2 mm Max-RP I 00G-4337-N0 I 00G-4337-AK I 00F-4337-P0 I 00G-4337-P0 I 00G-4337-U0 AJ0-7275 I AJ0-7275 Hydro-RP 00G-4375-N0 00G-4375-AK 00F-4375-P0 00G-4375-P0 00G-4375-U0 AJ0-7512 AJ0-7275Polar-RP 00G-4336-N0 00G-4336-AK 00F-4336-P0 00G-4336-P0 00G-4336-U0 AJ0-7276 AJ0-7275 Fusion-RP 00G-4424-N0 00G-4424-AK 00F-4424-P0 00G-4424-P0 00G-4424-U0 AJ0-7558 AJ0-7275

10 µm Analytical, Semi-Prep, Preparative and Pilot Scale Columns (mm) SecurityGuard™Cartridges

250 x 4.6 250 x 10 250 x 15 250 x 21.2 250 x 30 250 x 50 10 x 10 mm 15 x 21.2 mm Max-RP I 00G-4350-E0 I 00G-4350-N0 I 00G-4350-AK I 00G-4350-P0 I 00G-4350-U0 I 00G-4350-V0 AJ0-7275 AJ0-7842 Hydro-RP 00G-4376-E0 00G-4376-N0 00G-4376-AK 00G-4376-P0 00G-4376-U0 00G-4376-V0 AJ0-7512 AJ0-7843 Polar-RP 00G-4351-E0 00G-4351-N0 00G-4351-AK 00G-4351-P0 00G-4351-U0 00G-4351-V0 AJ0-7276 AJ0-7845 Fusion-RP 00G-4425-E0 00G-4425-N0 00G-4425-AK 00G-4425-P0 00G-4425-U0 00G-4425-V0 AJ0-7558 AJ0-7844

Method Development and Method Validation Kits can be customized for your application. Please call your technical consultant at Phenomenex.

See p. 277 for HST Columns.See p. 282 for Fused Silica Capillary Adapter.See p. 274 for MercuryMS LC/MS Columns.See p. 284 for Axia Packed Columns.

**‡

Axia™ Packed Columns (mm)

50 x 21.2 100 x 21.2 50 x 30 75 x 30 100 x 30 4 µm I I I I I

Max-RP 00B-4337-PO-AX 00D-4337-PO-AX 00B-4337-UO-AX 00C-4337-UO-AX 00D-4337-UO-AX Hydro-RP 00B-4375-PO-AX 00D-4375-PO-AX 00B-4375-UO-AX 00C-4375-UO-AX 00D-4375-UO-AX Polar-RP 00B-4336-PO-AX 00D-4336-PO-AX 00B-4336-UO-AX 00C-4336-UO-AX 00D-4336-UO-AX 10 µm

Max-RP — 00D-4350-PO-AX 00B-4350-U0-AX — 00D-4350-UO-AX Hydro-RP — — 00B-4376-UO-AX — — Polar-RP — — 00B-4351-UO-AX — —

**‡

*

*

*

*SecurityGuard™ Analytical Cartridges require holder, Part No.: KJ0-4282‡Semi-prep SecurityGuard™ Cartridges require holder, Part No.: AJ0-7220

**PREP SecurityGuard™ Cartridges require holder, Part No.: AJ0-8223

synergi™


/10pk

/10pk

/10pk

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synergi - phenomenex · 2008-10-16 · silanol acidity even under neutral mobile phases. with few...

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