zorbax hplc columns for lc/ms and high throughput...

NEW ANDEXPANDED!

ZORBAX HPLC Columns for LC/MS and High Throughput HPLC

31Order online at: www.agilent.com/chem/store

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ZORBAX LC/MS columns and cartridge columnsColumn choices for LC/MS are wide ranging, depending on the sample to be studied. For standardanalytical samples, column choices focus on finding short columns with high resolution to reduceanalysis time for high-throughput LC/MS. Column choice is also guided by the desired flow rate. LC/MS systems operate at flow rates from 1µL/min to 1 mL/min and therefore smaller internaldiameter columns allow good sensitivity and fast flow rates that are compatible with standardelectrospray, APCI (atmospheric pressure chemical ionization) and APPI (atmospheric pressurephotoionization) interfaces. ZORBAX Rapid Resolution (3.5 µm) and Rapid Resolution HT (1.8 µm)columns in 15, 30, and 50 mm lengths provide excellent resolution and fast analysis times. Thesecolumns are available in popular ZORBAX Eclipse XDB and StableBond bonded phases. Additionalbonded phases can be packed upon request. These bonded phases are compatible with the typicalvolatile mobile phase additives used for LC/MS – including TFA, formic acid, and acetic acid.

• Fast analysis for high speed LC/MS withRapid Resolution and Rapid ResolutionHT columns

• Many column choices for LC/MS compatibility — from nano to analytical

• Narrow bore columns for improved sensitivity

• Transfer methods from longer columnswith 5 µm particles with no loss inresolution

• Capillary and nano columns are mostoften used to analyze complex proteinand peptides samples by LC/MS. Moreinformation on these columns can befound on page 42 of this guide.

Reduce Analysis Time by 70% Using ShortLC/MS Cartridge Columns

Column: ZORBAX StableBond SB-C18

Mobile Phase: 85% Water with 0.1% TFA15% Acetonitrile

Flow Rate: 1.0 mL/min

Temperature: 35°C

Sample: 1. Phenylalanine2. 5-Benzyl-3,6-dioxo-2-piperazine

Acetic Acid3. Asp-phe4. Aspartame

Maximize Sensitivity with Narrow-Bore LC/MS Cartridges

Column: Rapid Resolution SB-C18, 3.5 µm

Mobile Phase: Gradient: 5 - 35% B in 6 minA: Water with 0.2% Formic AcidB: Methanol with 0.2% Formic Acid

Temperature: 35°C

Sample: 2 µL Organic Acids1. Gallic2. Protocatechuic3. Hydrocaffeic4. Gentisic5. Vanillic6. Syringic

4.6 x 30 mm, 3.5 µmP/N 833975-902

2.1 x 30 mm, 3.5 µmP/N 873700-902

Time (min.)

4.6 x 50 mm, 3.5 umP/N 835975-902

4.6 x 30 mm, 3.5 umP/N 833975-902

4.6 x 15 mm, 3.5 umP/N 831975-902

32 Order online at: www.agilent.com/chem/store

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sZORBAX High Throughput and LC/MS HPLC Columns and Cartridge Columns

ZORBAX Rapid Resolution and Rapid Resolution HT LC/MS Cartridge Columns Ordering Information (requires Hardware Kit 820555-901)

Column Size Particle SB-C18 SB-C8 Eclipse XDB-C18 Eclipse XDB-C8 Description (mm) Size (µm) USP L1 USP L7 USP L1 USP L7 Cartridge Columns

Rapid Resolution Cartridge 4.6 x 30 3.5 833975-902 833975-906 933975-902 933975-906

Rapid Resolution Cartridge, 3/pk 4.6 x 30 3.5 833975-932 833975-936 933975-932 933975-936







Rapid Resolution HT Cartridge 4.6 x 50 1.8 825975-902 925975-902

Rapid Resolution HT Cartridge, 3/pk 4.6 x 50 1.8 825975-932 925975-932











Column Hardware Kit 820555-901 820555-901 820555-901 820555-901

Standard Columns

Rapid Resolution Column 4.6 x 50 3.5 835975-902 835975-906 935967-902 935967-906

Rapid Resolution HT Column 4.6 x 50 1.8 822975-902 922975-902

Rapid Resolution HT Column, 3/pk 4.6 x 50 1.8 822975-932 922975-932

Rapid Resolution Column 2.1 x 50 3.5 871700-902 871700-906 971700-902 971700-906



Recommend 01090-68702, low volume in-line filter, in place of a guard column with both Rapid Resolution and Rapid Resolution HT columns.

Column: ZORBAX Rapid Resolution HT SB-C18 2.1 x 30 mm, 1.8 µmP/N 823700-902

Mobile Phase: Gradient: 15-50% B in 1 min. hold for 1.5 min.A: 0.2% Formic Acid pH,B: ACN + 0.2% Formic Acid

Post Time: 1.5 min


Injection Volume: 1 µL

Temperature: Ambient

HPLC: Agilent 1100 with WPS and ADVR on

Detection: APCI, Positive ion

MS Conditions: Peakwidth: 0.10 min

Scan: 150-600 Da, step 0.1

Fragmentor: 70

Gas Temp: 350° C Vaporizer:

Sample: 1. Lincomycin2. Clindamycin

Use Rapid Resolution HT (High Throughput) Columns for Fast LC/MS

This figure shows an LC/MS analysis with APCI using a Rapid Resolution HT column. The rapid gradient takes2.5 minutes and re-equilibration is done in another 1.5 minutes for a total analysis time of only 4 minutes onthis 2.1 x 30 mm, 1.8 µm column.

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Services and Compliance Products

ZORBAX High Throughput and LC/MS HPLC Columns and Cartridge Columns


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Additional columns suitable for LC/MS are listed in the Rapid Resolution and LC/MS columns ordering guide on page 35.Configurations not shown are available upon request.For more information on these and other columns, consult the Agilent Web site at www.agilent.com/chem

Additional ZORBAX LC/MS Columns Ordering Information Column Size Particle SB-C18 SB-C8 SB-CN SB-PhenylDescription (mm) Size (µm) USP L1 USP L7 USP L1 0 SB-C3 USP L11 SB-Aq

ZORBAX StableBond Columns

Narrow Bore 2.1 x 150 5 883700-922 883700-906 883700-905 883700-909 883700-912

Narrow Bore 2.1 x 50 5 860975-902 860975-906 860975-905 860975-909 860975-912 860975-914

Narrow Bore 2.1 x 150 3.5 830990-902 830990-906 830990-914

Narrow Bore 2.1 x 50 3.5 871700-902 871700-906 871700-914

Column Size Particle Eclipse XDB-C18 Eclipse XDB-C8 Eclipse XDB-PhenylDescription (mm) Size (µm) USP L1 USP L7 USP L11 Rx-C18 Rx-C8 Bonus-RP Extend-C18

ZORBAX Rx, Eclipse XDB, Bonus-RP and Extend-C18 Columns

Narrow Bore 2.1 x 150 5 993700-902 993700-906 993700-912 883700-902 883700-906 883725-901 773700-902

Narrow Bore 2.1 x 50 5 960967-902 960967-906 960967-912 861971-901 760450-902

Narrow Bore 2.1 x 150 3.5 930990-902 930990-906

Narrow Bore 2.1 x 50 3.5 971700-902 971700-906 861700-902

Rapid Resolution HT Columns

Mass-Selective Detector

Valves

Well-Plate LC System

Sample Capacity Extension

Integrated Controller

Agilent 1100 Series HT LC/MS High-Throughput System for Fastest AnalysisAgilent Technologies provides a total system solution for high-throughput analyses. Although severalAgilent 1100 Series HPLC configurations can be upgraded/optimized for use with Rapid ResolutionHT columns, Agilent now offers a completely optimized 1100 system, designed specifically for high-throughput HPLC analysis (with or without LC/MS detection). The system includes a fast autosamplerwith a cycle time as low as 30 seconds, and a sample capacity extension module that can handle up to80 96/384 shallow well plates (7,680/30,720 total samples) or 24 54-vial plates (1,296 total samples).

ZORBAX Rapid Resolution 3.5 µm HPLC Columns

Agilent ZORBAX Rapid Resolution columns, with a 3.5 µm particle size, reduce analysis time andincrease sample throughput for any application when compared to 5 µm columns. Rapid Resolutioncolumns are available from 15-150 mm, in both 4.6 and 2.1 mm internal diameters, so the bestconfigurations are available for high-throughput, LC/MS, combinatorial chemistry and rapid analyticalapplications. Rapid Resolution 3.5 µm particles have superior mechanical strength, so every column hasan extremely stable packed bed and provides a comparable lifetime to 5 µm columns. These are availablein all of the modern ZORBAX bonded phases — StableBond, Eclipse XDB, Bonus-RP, and Extend-C18.

• Reduce analysis time and solvent usageand increase sample throughput

• High efficiency in short and ultra-short column lengths

• Available in analytical (4.6 mm) and narrow-bore (2.1 mm) i.d.

• Comparable lifetime to 5 µm columns

Order online at: www.agilent.com/chem/store

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Rapid Resolution 3.5 µm columns are available in narrowbore configurations for great compatibility with LC/MS.

Column: ZORBAX Bonus-RP

Mobile Phase: 80% 25 mM NH4Ac, pH 5.520% ACN

Temperature: 40°C

Detection: 254 nm

Sample: 1. Caffeine2. Doxylamine3. Chlorpheniramine4. Triprolidine

Rapid Resolution Columns Reduce Analysis Time While Maintaining Resolution

Bonus-RP4.6 x 150 mm, 5 µmP/N 883668-901


Rapid Resolution Bonus-RP4.6 x 75 mm, 3.5 µmP/N 866668-901


Narrow BoreRapid Resolution Bonus-RP2.1 x 50 mm, 3.5 µmP/N 861700-901


Column: ZORBAX StableBond SB-C184.6 x 75 mm, 3.5 µmP/N 866953-902

Mobile Phase: A: 25 mM NaH2PO4, pH 3.0B: MeOH


Temperature: 35°C

Detection: UV 254 nm

Sample: 1. Diltiazem2. Dipyridamole3. Nifedipine4. Lidoflazine5. Flunarizine

Run Method Development Chromatograms on Rapid Resolution Columns

A : B 20 : 80 30 : 70 40 : 60

Time (min.)

Time (min.)

Time (min.)

Time (min.) Time (min.) Time (min.)

Rapid Resolution 4.6 x 75 mm, 3.5 µm columns are a good choice for initial method development because they allowyou to quickly determine if a given column and mobile phase combination can produce the desired separation.

ZORBAX Rapid Resolution 3.5 µm HPLC Columns


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ZORBAX Rapid Resolution 3.5 µm Columns Ordering Information Column Size Particle SB-C18 SB-C8 SB-CN SB-PhenylDescription (mm) Size (µm) USP L1 USP L7 USP L1 0 SB-C3 USP L11 SB-Aq

ZORBAX StableBond Columns

Rapid Resolution 4.6 x 150 3.5 863953-902 863953-906 863953-905 863953-912 863953-914




Solvent Saver Plus 3.0 x 150 3.5 863954-302 863954-306 863954-305 863954-312 863954-314

Solvent Saver Plus 3.0 x 100 3.5 861954-302 861954-306 861954-305 861954-309 861954-312 861954-314

Narrow Bore RR* 2.1 x 150 3.5 830990-902 830990-906 830990-914

Narrow Bore RR 2.1 x 100 3.5 861753-902 861753-906 861753-905 861753-912 861753-914

Narrow Bore RR 2.1 x 50 3.5 871700-902 871700-906 871700-914

MicroBore RR 1.0 x 150 3.5 863600-902 863600-906

MicroBore RR 1.0 x 50 3.5 865600-902 865600-906

MicroBore RR 1.0 x 30 3.5 861600-902 861600-906

Column Size Particle Eclipse XDB-C18 Eclipse XDB-C8 Eclipse XDB-Phenyl Rx-C18 Rx-C8Description (mm) Size (µm) USP L1 USP L7 USP L11 USP L1 USP L7 Bonus-RP Extend-C18

ZORBAX Rx, Eclipse XDB, Bonus-RP, and Extend-C18 Columns

Rapid Resolution 4.6 x 150 3.5 963967-902 963967-906 963967-912 863967-902 863953-906 863668-901 763953-902

Rapid Resolution 4.6 x 100 3.5 961967-902 961967-906 861967-902 861953-906 861953-914 764953-902

Rapid Resolution 4.6 x 75 3.5 966967-902 966967-906 966967-912 866967-902 866953-906 866668-901 766953-902

Rapid Resolution 4.6 x 50 3.5 935967-902 935967-906 935967-912 735953-902

Solvent Saver Plus 3.0 x 150 3.5 963954-302 963954-306 963954-312 863967-302 863954-306

Solvent Saver Plus 3.0 x 100 3.5 961967-302 961967-306 961967-312 861967-302 861954-306 864668-301 764953-302

Narrow Bore RR* 2.1 x 150 3.5 930990-902 930990-906

Narrow Bore RR 2.1 x 100 3.5 961753-902 961753-906 861767-902 861753-906 861753-914 761753-902

Narrow Bore RR 2.1 x 50 3.5 971700-902 971700-906 861700-901 735700-902

MicroBore RR 1.0 x 150 3.5 963600-902 963600-906 863608-901 763600-902

MicroBore RR 1.0 x 50 3.5 965600-902 965600-906 865608-901 765600-902

MicroBore RR 1.0 x 30 3.5 961600-902 961600-906 861608-901 761600-902

*RR: Rapid Resolution 3.5 µm columns.Configurations not shown are available upon request.For more information on these and other columns, consult the Agilent Web site atwww.agilent.com/chem

Get the most from your RapidResolution HT columns

ZORBAX Rapid Resolution HT columns provide very highefficiency in short column lengths. To take full advantage ofthis efficiency, it is important to optimize your 1100 SeriesHPLC with low-volume tubing and a low-volume detectorflow cell. For a quick list of the simple modifications neededfor ultimate performance, click on the link at this Web site:www.agilent.com/lccolumntips

ZORBAX Rapid Resolution High Throughput 1.8 µmColumns and Cartridges

Agilent now introduces the latest addition to its family of High Throughput (HT) HPLC columns. TheZORBAX Rapid Resolution HT series of columns and cartridges raise the industry performance standardby using a totally porous, 1.8 µm particle as the packing material for short (50 mm) and very short (15 and 30 mm) columns and cartridges. These short column lengths enable ultra high-speed analyseswhile maintaining high resolution. It is possible to reduce analysis time by up to 95% in comparison to 250 mm length columns. The small particle size provides double the efficiency of a 3.5 µm column in the same column length providing the highest efficiency and resolution possible in the shortest column lengths. This efficiency surpasses that of 2 and 2.5 µm particle size columns. The 1.8 µm Rapid Resolution HT columns take high-speed, high-resolution HPLC to a new level with either fasteranalyses while maintaining resolution or higher resolution while maintaining analysis speed.

• Ultra high speed, high resolution analyseswith short Rapid Resolution HT columnsand cartridges packed with totally porous,1.8 µm packings

• High Throughput labs — double yourlaboratory productivity with columns of half the length but with no loss inresolution!

• LC/MS labs — improve your spectralclarity by increasing your sampleresolution (vs. a 3.5 µm or 5 µmpacking) without increasing columnlength or analysis time

• Packed with popular Eclipse XDB-C18and StableBond-C18 phases

• Long column lifetimes — thousands of injections possible


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Reduce Analysis Time Dramatically with Rapid Resolution HT Columns

Rapid Resolution HT Columns Provide Double the Efficiency of Rapid Resolution Columns

This figure shows that Rapid Resolution HT columns can provide double the efficiency of a 3.5 µm column in thesame column length. This high efficiency can be used for very high-resolution, high throughput analyses.

Columns: ZORBAX Eclipse XDB-C18

Mobile Phase: 73% MeOH: 27% 20 mM Phosphate Buffer, pH 7.0

Flow Rate: 1 mL/min


Detection: UV 254 µm

Sample: 1. Uracil2. Naproxen3. Mefanamic Acid4. Butyl Paraben5. Propranolol6. Napthalene7. Dipropyl Phthalate

Columns: ZORBAX SB-C184.6 x 50 mm

Mobile Phase: 25% Water, 75% MeOH



Detection: UV 254 µm

Sample: QC: 1. Uracil2. Phenol3. 4-CI-Nitrobenzene4. Toluene

Rapid Resolution4.6 x 50 mm, 3.5 µmP/N 835975-902

Rapid Resolution HT4.6 x 50 mm, 1.8 µmP/N 822925-902

Plates (N)1. 34762. 45853. 56734. 6180

Plates (N)1. 65602. 89583. 115084. 12266

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Rapid Resolution 4.6 x 150 mm, 3.5 µmP/N 963967-902




This figure shows the dramatic reduction in analysis time possible by using Rapid Resolution HT columns. Chromatogram A shows a separation that takes 11.5 minutes on a 25 cm, 5 µm column. Rapid Resolution (3.5 µm) columns, shown in chromatogram B and C, reduce analysis time substantially, but with a slight compromise in resolution. The Rapid Resolution HTcolumn reduces analysis time to 2.2 minutes, an 80% reduction, while still maintaining baseline resolution.


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Higher resolution providesimproved results for LC/MSwith more accurateidentification and spectralclarity. The Rapid ResolutionHT columns are packed withboth Eclipse XDB-C18 andStableBond-C18 bondedphases for use from pH 1-9.And these columns can beused reliably for thousandsof injections.

ZORBAX Rapid Resolution High Throughput 1.8 µm Columns and Cartridges

Higher Resolution with Rapid Resolution HT Columns

If the same length 1.8 µm Rapid Resolution HT column is substituted for a 3.5 µm Rapid Resolution column, the significant increase in column efficiency results in higher peak resolution as shown above. This increased resolution will improve quantitation and enhance LC/MS spectral clarity while maintaining high analysis speed.

ZORBAX Rapid Resolution HT Columns Provide the Highest Efficiency Possible

This comparison clearly shows the 1.8 µm ZORBAX Rapid Resolution HT column produces 30% higher column efficiencythan the 2.5 µm columns, correcting for differences in column length.

Column: Eclipse XDB-C184.6 x 50 mm

Mobile Phase: 73% Methanol, 23% 20mM Phosphate Buffer, pH 7

Flow Rate: 1 mL/min.


Injection Volume: 1.0 µL


Sample: 1. Uracil 2. Naproxen3. Mefenamic Acid4. Butyl Paraben 5. Propranolol6. Dipropyl Phthalate7. Napthalene

Column: See Left

Mobile Phase: 60% Methanol 40% Water

Flow Rate: 1 mL/min.




Sample: QC Test1. Uracil 2. Phenol 3. 4-CI-Nitrobenzene4. Toluene

Rapid ResolutionEclipse XDB-C18, 3.5 µmP/N 935967-902

N7 = 6,838Rs4,5 = 1.2

Rapid Resolution HTEclipse XDB-C18, 1.8 µmP/N 922975-902

N7 = 11,985Rs4,5 = 1.6

N = 3366N = 3190

N = 6613N = 6138

Time (min.) Time (min.)

Time (min.)

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Competitive-C184.6 x 20 mm, 2.5 µm

Avg = 164,000 plates/m

Rapid Resolution HT SB-C184.6 x 30 mm, 1.8 µmP/N 823975-902

Avg = 213,000 plates/m


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Column: ZORBAX SB-C184.6 x 30 mm, 1.8 µmP/N 823975-902

Mobile Phase: 60% Methanol: 40% Water

Flow Rate: 1 ml/min



Sample: QC Test:

1. Uracil2. Phenol3. 4-CI-Nitrobenzene4. Toluene

Injection Number

ZORBAX Rapid Resolution High Throughput (HT) HPLC Columns and Cartridges

Long Lifetime with High Efficiency Using Rapid Resolution HT Columns

More than 2500 injections were made on this 1.8 µm, 4.6 x 30 mm ZORBAX StableBond-C18 RapidResolution HT cartridge column with no compromise in performance. Efficiency remained high, peak retentiontimes (RT) were consistent, and peak tailing (TF) remained low. This indicates a stable and long-lived column.

Efficiency of:

Injection No. 4-CI-Nitrobenzene Toluene

1 6635 6138

100 6857 6372

200 6882 6381

300 6895 6381

400 6883 6294

500 6815 6312

600 6687 6236

700 6653 6115

800 6695 6221

900 6781 6300

1050 6810 6302

1300 6858 6528

2034 6823 6343

Configurations not shown are available upon request.For more information on these and other columns, consult the Agilent Web site atwww.agilent.com/chem

Rapid Resolution HT Columns and Cartridges Ordering InformationColumn Size Particle StableBond Eclipse

Description (mm) Size (µm) SB-C18 XDB-C18Rapid Resolution HT Columns





Rapid Resolution HT Cartridges (require Hardware Kit 820555-901)













Rapid Resolution HT Cartridge Hardware Kit 820555-901 820555-901

ZORBAX Solvent Saver Columns


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• Provide 60% reduction in mobile phaseusage and waste generation comparedto a 4.6 mm i.d. column at theequivalent flow rate (linear velocity)

• Provide 2- to 3-fold signal-to-noise (S/N ratio) improvement

• Deliver optimal LC/MS performance atintermediate flow rates — ideal for bothESI and APCI/APPI modes of LC/MS

• Can be used with most conventional LC instrument configurations withoutmodification

Solvent Saver Columns Provide up to 60% Reduction in Solvent Use and Waste

This separation of antibacterials on a 4.6 and3.0 mm i.d. columns, shows that solvent use isreduced by 50% just by changing to theSolvent Saver column with no change in thechromatography. This reduces the cost ofanalyses dramatically.

Solvent Saver Columns Increase Sensitivity

This figure shows sensitivity is increased 2-3 timeswith Solvent Saver columns compared to 4.6 mm i.d.columns when the same mass sample is injected. Nochange in the HPLC instrumentation is required to seethe sensitivity improvements.

Solvent Waste 31 mL4.6 x 150 mm, 5 µmP/N 883975-902

Solvent Waste 15 mL3.0 x 150 mm, 5 µmP/N 883975-302 Less solvent

consumption,less waste

ZORBAX Solvent Saver Columns have a 3.0 mm internal diameter. This is ideal for reducingsolvent usage by 50% over 4.6 mm i.d. columns. Solvent Saver columns are also ideal forLC/MS. With a typical flow rate of 0.5 mL/min, these columns are compatible with electrospray,atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization(APPI) MS interfaces. These columns also improve sensitivity 2 to 3 times over 4.6 mm i.d.columns. Solvent Saver columns can be used with conventional HPLC instruments and are agood choice for cost effective analyses.

Column: ZORBAX SB-C18

Mobile Phase: 20% ACN: 80% 0.2 M Na2HPO4+ 0.1 M Citric Acid, pH 2.6


Sample: Peak Identification:

1. Sulfamerazine2. Furazolidone3. Oxolinic Acid4. Sulfadimethoxine5. Sulfaquinoxaline6. Nalidixic Acid

Column: ZORBAX SB-C18150 mm, 3.5 µm

Mobile Phase: 25% Methanol: 75% 0.4% Formic Acid

Detection: UV, 254 nm

Time (min.) Time (min.)

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Increase insensitivity

4.6 mm i.d.P/N 863953-902

3.0 mm i.dP/N 863954-302

4’5,7 - trihydroxyflavanone

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Quercetin

ZORBAX Solvent Saver Columns


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Solvent Saver Columns are Ideal for LC/MS

Solvent Saver columns are ideal for LC/MSbecause the typical 0.5 mL/min flow rate allowssamples to be evaluated and analyzed withoutchanging columns when the MS interface ischanged from electrospray to APCI.

Agilent 3.0 mm ZORBAX Solvent Saver and Solvent Saver Plus Columns Ordering InformationZORBAX StableBond (80Å) Solvent Saver

Column Size Particle SB-C18 SB-C8 SB-CN SB-PhenylDescription (mm) Size (µm) USP L1 USP L7 USP L10 SB-C3 USP L11 SB-Aq

Solvent Saver 3.0 x 250 5 880975-302 880975-306 880975-305 880975-309 880975-312 880975-314

Solvent Saver 3.0 x 150 5 883975-302 883975-306 883975-305 883975-309 883975-312 883975-314



ZORBAX StableBond (300Å) Solvent Saver

Column Size Particle Size 300SB-C18 300SB-C8 300SB-CN Description (mm) Size (µm) USP L1 USP L7 USP L10 300SB-C3

Solvent Saver Plus 3.0 x 150 3.5 863974-302 863974-306 863974-309

Solvent Saver Plus 3.0 x 100 3.5 861973-306

ZORBAX Eclipse XDB (80Å), Bonus-RP (80Å), and Rx (80Å) Solvent Saver

Column Size Particle XDB-C18 XDB-C8 XDB-Phenyl Rx-C18 Rx-C8Description (mm) Size (µm) USP L1 USP L7 USP L11 Bonus-RP USP L1 USP L7

Solvent Saver 3.0 x 250 5 990967-302 990967-306 990967-312 880668-301 880967-302 880975-306

Solvent Saver 3.0 x 150 5 993967-302 993967-306 993967-312 883668-301 883967-302 883975-306




ZORBAX Extend-C18 Solvent Saver

Column Size Particle Pore SizeDescription (mm) Size (µm) 80Å

Solvent Saver 3.0 x 250 5 770450-302




Configurations not shown are available upon request.

Time (min.)

Column: ZORBAX SB-C18Solvent Saver Plus3.0 x 100 mm, 3.5 µmP/N 861954-302

Mobile Phase: A: 70% Methanol+0.4% Formic AcidB: 30% Water+0.4% Formic Acid


Detection: A: UV 254 nmB: Positive Ion APCIC: Positive Ion Electrospray

Sample Steroids:1. Triamcinolone2. Hydrocortisone3. Cortisone acetate4. Deoxycorticosterone5. Hydroxyprogesterone6. Progesterone

ZORBAX MicroBore (1.0 mm i.d.) HPLC Columns


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MicroBore (1 mm i.d.) columns are often a good choice when sample sizes are limited. They willimprove detection limits 5 times over 2.1 mm i.d. columns when the same sample mass is used.This increase in sensitivity can be critical. MicroBore columns use low flow rates (typically ~50 µL/min). Therefore, these columns are ideal for use with detectors requiring low flow ratessuch as some mass spectrometers.

MicroBore columns perform optimally with HPLC systems purchased or modified for microboreuse. A wide variety of bonded phases are available including: StableBond SB-C18, SB-C8, and300SB-C18; Eclipse XDB-C18 and XDB-C8; Bonus RP, Extend-C18; and most recently Poroshell300SB-C18, 300SB-C8, and 300SB-C3. Guard columns are also now available with anadjustable tube stop depth to provide a perfect zero dead volume connection every time.

• High sensitivity for small sample sizes

• Compatible with LC/MS interfaces

• Wide variety of bonded phases

Separation of a Tryptic Digest on a ZORBAX MicroBore 300SB-C18

This example of a tryptic digest separated on a MicroBore column demonstrates the high sensitivity and resolutionpossible with 1.0 mm i.d. columns.

ZORBAX MicroBore (1.0 mm i.d.) HPLC Column Ordering InformationColumn Size Particle Description (mm) Size (µm) SB-C18 SB-C8 300SB-C18 300SB-C8

MicroBore 1.0 x 250 5 861630-902

MicroBore RR* 1.0 x 150 3.5 863600-902 863600-906 863630-902 863830-906

MicroBore RR 1.0 x 50 3.5 865600-902 865600-906 865630-902 865630-906

MicroBore RR 1.0 x 30 3.5 861600-902 861600-906

MicroBore Guard 1.0 x 17 5 5185-5920 5185-5920 5185-5920 5185-5920

Column Eclipse EclipseDescription XDB-C18 XDB-C8 Bonus-RP

MicroBore RR 1.0 x 150 3.5 963600.902 963600-906 863608-901

MicroBore RR 1.0 x 50 3.5 965699-902 965699-906 865608-901

MicroBore RR 1.0 x 30 3.5 961600-902 961600-906 861608-901

MicroBore Guard 1.0 x 17 5 5185-5921 5185-5921 5185-5922

Column Poroshell Poroshell PoroshellDescription Extend-C18 300SB-C18 300SB-C8 300SB-C3

MicroBore 1.0 x 75 5 661750-902 661750-906 661750-909

MicroBore RR 1.0 x 150 3.5 763600-902

MicroBore RR 1.0 x 50 3.5 765600-902

MicroBore RR 1.0 x 30 3.5 761600-902

MicroBore Guard 1.0 x 17 5 5185-5926 5185-5968 5185-5968 5185-5968

* RR: Rapid Resolution 3.5 µm columnsConfigurations not shown are available upon request.

Column: ZORBAX 300SB-C181.0 x 150 mmP/N 863630-902

Mobile Phase Gradient: 2-60% B in 60 Min.A: 0.1% TFAB: 0.075% TFA/80% ACN


Flow Rate: 50 µL/min.

Temperature: 50°C

Sample: Tryptic Digest of rhGH, 2 µL

Time (min.)

ZORBAX Capillary and Nano Columns


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Agilent ZORBAX capillary (0.5 mm, 0.3 mm i.d.) and nano (0.1 mm, 0.075 mm i.d.) columns are nowavailable in a wide variety of phases, pore sizes, and dimensions. These columns are ideal for verysample-limited applications because they provide enhanced sensitivity by reducing on-column sampledilution. This high sensitivity can be provided with exceptional reproducibility using Agilent columns andlow dispersion HPLC instruments. Reproducibility is a critical issue when working with small sample sizes.

The fastest growing application for capillary and nano columns is 2D LC/MS for complex proteomicssamples. This technique typically employs cation exchange, reversed-phase enrichment, and reversed-phase analysis capillary columns. Samples are adsorbed to the cation exchange column under low saltconditions. Successively more concentrated salt eluents are used to elute fractions of peptides/proteins,which are then preconcentrated on a reversed-phase enrichment column and then backflushed ontothe reversed-phase analysis column to be separated with a reversed-phase gradient analysis. Agilentprovides all the columns needed for the 2-D separation — the SCX column for the first dimension, thereversed-phase trapping column, and the reversed-phase column for the second dimension.

• Highest sensitivity for your smallest sample sizes

• Compatible with all LC/MS interfaces

• Internal diameters of 0.5 mm, 0.3 mm, 0.1 mm, and 0.075 mm

• Packings/phases for both small and large molecules (80Å and 300Å poresizes, respectively)

• Ideal for 1D and 2D (proteomics) applications

Separation of Peptides on Capillary Columns

This example shows a peptide standard mixture separated on a variety of the ZORBAXcapillary columns. These chromatograms demonstrate the wide range of selectivities available, which can be used to optimize your specific separation.

Columns: 0.3 x 150 mm

Column Flow: 5.5 µl/min

Primary Flow: Low Solvent Consumption:

200-500 µl/min

Mobile Phase: Water + 0.05% TFA, pH = 2.2 = A

Acetronitrile + 0.045% TFA = B

Gradient 0.5%B/min: at 0 min = 1%B,

at 60 min = 31%B, at 70 min = 50%B, at 75 min = 85%B,

at 80 min = 85%B, at 81 min = 1%B, at 110 min = 1%B

DAD UV Detector: 206/10 nm, ref 450/80 nm

Temperature: 30°C

Injection Volume: 0.1 µl, automatic delay volume reduction was activated

Time (min.)

Abu

ndan

ce (

mA

U)

0 20 40 60

0

100

200

300

400

12 3 4 5

12 3 4 5

12 3 4 5

12 3 4 5

1 2 3 4 5

1 2 3 4 5

12 3 4

5

1= Gly-Tyr, 5 ng/100 nl

2= Val-Tyr-Val, 20 ng/100 nl

3= Met Enkephalin, 28 ng/100 nl

4= Low Enkephalin, 20 ng/100 nl

5= Angiotensin ΙΙ, 20 ng/100 nl

300SB-C8, 3.5 µmP/N 5065-4460

Eclipse XDB-C18, 5 µmP/N 5064-8291

300SB-C18, 5 µmP/N 5064-8291

SB-C18, 5 µmP/N 5064-8255

300SB-C18, 3.5 µmP/N 5064-8267

300Extend-C18, 3.5 µmP/N 5065-4464



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High Sensitivity with Capillary Columns

Sample limited applications require capillary column dimensions to minimizeon-column sample dilution and to enhance sensitivity. The 0.3 mm capillary inthis example provides 100 times more sensitivity than the standard 4.6 mmcolumn. Nano-bore (0.1 mm-0.075 mm i.d.) columns can provide up to 2000times more sensitivity for your most limited sample applications.

Column: ZORBAX SB C180.3 mm: P/N 5064-82550.5 mm: P/N 5064-82561.0 mm: P/N 863600-9024.6 mm: P/N 883975-902

Sample: 200 ng Biphenyl

Excellent Column-to-Column Reproducibility with Agilent Capillary Columns

Excellent reproducibility is seen for a separation of polar organic acids on three differentStableBond-C18, 0.5x150 mm, 5 µm columns. Retention (k) varied less than 0.8% RSDand selectivity (α) varied less than 0.4% RSD.

Time (min.)

Time (min.)

Column: ZORBAX SB-C18

0.5 x 150 mm, 5 µm

P/N 5064-8256

Mobile Phase: A: 75% H2O with 0.4% formic acid

B: 25% MeOH with 0.4% formic acid

Flow Rate: 20 µL/min

Temperature: 25°C


Sample: 1. Protocatechuic acid

2. Chlorogenic acid

3. Caffeic acid

4. Syringic acid

5. p-coumaric acid

6. Ferulic acid

SN DE43A00906

SN DE43A00908

SN DE43A00927



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A: Instrument Configuration for 2-D HPLC for Proteomics Applications

Instrumental set up of the Agilent Nanoflow Proteomic Solution system.

Micro degasser

Micro degasser

2nd pump

Nanopump

Therm. well-plate autosampler

Thermostat LC/MSD Trap

Therm. column comp. & micro valve

Nano interface and nano column

B: Typical Column Configuration for 2-D HPLC

Flow path of the Agilent 1100 Series Nanoflow Proteomics Solution system. 1: Sample loading, elution from SCX and trapping on enrichment column: 2: Valve switch in column compartment, elution from enrichment column, separation on RP and MS-analysis.

Nanopump

Nanopump

Detection(1100 SeriesLC/MSD Trap)

Strong cationexchangecolumn

Strong cationexchangecolumn

Detection(1100 SeriesLC/MSD Trap)

Waste

Waste

1100 Series columncompartment

Reversed-phase column

Reversed-phase column

Reversed-phase enrichment column

1100 Series columncompartment

1100 Series microwell plate autosampler

1100 Series microwell plate autosampler

Waste

Waste

1100 Seriesquaternary pump

1100 Seriesquaternary pump

Reversed-phase enrichment column

1.

2.

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Figures A and B show a typical instrumentand column configuration for 2D LC/MSanalyses and Figure C shows the reversed-phase results from the 2-D separation for aselection of salt fractions. The sample is atryptic digest of bovine serum albumin (BSA)

.

Tryptic digest of bovine serum albumin (BSA). The base peak chromatograms show a electionof fractions from a 2-dimensional HPLC separation. Single chromatograms represent peptidesfrom BSA eluting at a given salt concentration followed by enrichment and reversed phasechromatography.

C: 2-D HPLC with Nano HPLC Columns Provided Clear Identification of Proteins in a Complex Sample

Time (min.)

Column: 2D HPLC

Columns: Strong Cation Exchange PolySulfoethyl Aspartamide:0.3 x 35 mm, 5 µm

Enrichment Column: ZORBAX 300SB-C18, 0.3 x 5 mm, 5 µmP/N 5065-9913

Reversed-Phase: ZORBAX 300SB-C180.075 mm x 150 mm, 3.5 µmP/N 5065-9911

Flow Rate: Quaternary Pump: 30 µL/min Nanopump: 300 nL/min

Gradient: Separation of Tryptic Digest of 10 Proteins

Quaternary Pump: Isocratic 3% Acetonitrile/0.1% Formic Acid

Nanopump: A = Water, 0.1% Formic Acid, B = ACN, 0.1% Formic Acid 6 min = 3%B, 120 min = 60%B, 125 min = 80%B, 130 min = 80% B, 131 min = 3%B, 140 min = 3% B

Post Time: 10 min

Injection Volume: 1 to 8 µL

Salt Step Elution: 8ml of 10 mM-100 mM KCI (10 mM increments), 125 mM, 150 mM, 200 mM, 300 mM, 500 mM, 1M.

Source: Nano ESI, drying gas flow: 5L/min, drying gas temp.: 225°C.

Ion Trap: Skim: 1:35 V, cap exit offset: 115 V, octopole 1:12 V, octopole 2:3.5 V, trap drive: 80 V. ICC: on, averages: 4, max accutime: 150 ms; target 60.000, ion mode positive, MS/MS mode.


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Material No. of Identified Peptides No. of Identified Proteins

Bio-SCX Series II 626 232

Competitor SCX 479 185


The ZORBAX Bio-SCX Series II column can be used as the first dimension in either an on-line or off-line 2-D HPLC separation of peptides. The column can retain and isolate many peptide fragmentsso the subsequent reversed-phase separation ultimately identifies more proteins present.

The 2-D separation, shown on the previous page, is done with 3 different capillary and nanocolumns. An SCX column for thefirst dimension, a reversed-phasecapillary trapping column, and areversed-phase nano separationcolumn for the second dimension.ZORBAX columns are available forthe complete 2-D separation.

Sample: E-Coli Digest

LC/MS: Agilent 1100 Nanoflow Proteomics Solution System, Ion trap SL

Columns: SCX: Agilent SCX, 0.3 x 35 mm (or 0.75 x 150 mm), 3.5 µm particlesP/N 5065-9912

RP: ZORBAX 300SB-C18, 0.075 x 150 mm, 3.5 µm particlesP/N 5065-9911

Enrichment: ZORBAX 300SB-C18, 0.3 x 5 mm, 5 µm particlesP/N 5065-9913

Chromatography:

1st Dimension (Cap Pump): Solvent A: 5% ACN + 0.1% FASolvent B: 5% ACN, 500 mM NaCl + 0.1% FA; flow 5 (or 20) µL/min

Injection Volume: 5 µL

Nano Pump: Solvent: A = Water + 0.1% FA; B = ACN + 0.1% FAGradient: 0 min = 5%B, 5 min = 5%B, 8 min = 15%B, 68 min = 45%B,

70 min = 75%B, 71 min = 75%B, 71.01 min = 0%B, 80 min = 5%B

Flow: 450 nL/min

ZORBAX HPLC Capillary Column Ordering Information (glass-lined stainless steel)Column Size Particle Eclipse Poroshell 300Extend Bio-SCXDescription (mm) Size (µm) SB-C18 XDB-C18 300SB-C18 300SB-C8 300SB-C8 C18 Series II

Capillary 0.8 x 50 5065-9942

Capillary 0.5 x 250 5 5064-8258 5064-8286 5064-8266

Capillary 0.5 x 150 5 5064-8256 5064-8287 5064-8264

Capillary 0.5 x 75 5 5065-4468

Capillary 0.5 x 35 5 5064-8254 5064-8296 5064-8294

Capillary RR* 0.5 x 150 3.5 5064-8262 5064-8288 5064-8268

Capillary RR 0.5 x 35 3.5 5064-8260 5064-8298 5065-4459

Capillary 0.3 x 250 5 5064-8257 5064-8269 5064-8265

Capillary 0.3 x 150 5 5064-8255 5064-8291 5064-8263

Capillary 0.3 x 35 5 5064-8253 5064-8297 5064-8295 5065-9912

Capillary RR 0.3 x 150 3.5 5064-8261 5064-8271 5064-8267 5065-4460 5065-4464

Capillary RR 0.3 x 100 3.5 5064-8259 5065-4461 5065-4465

Capillary RR 0.3 x 75 3.5 5064-8270 5065-4462 5065-4466

Capillary RR 0.3 x 50 3.5 5064-8300 5065-4463 5065-4467

Replacement Screens, 10/pk 5065-4427 5065-4427 5065-4427 5065-4427 5065-4427 5065-4427

Agilent Nano HPLC Column Ordering Information (PEEK)Column Size ParticleDescription (mm) Size (µm) 300SB-C18 300SB-C8

Nano RR 0.1 x 150 3.5 5065-9910

Nano RR 0.075 x 150 3.5 5065-9911

Nano RR 0.075 x 50 3.5 5065-9924 5065-9923

Trap/Guard, 5/pk 0.3 x 5 5 5065-9913 5065-9914

Trap/Guard Hardware Kit 5065-9915 5065-9915

* RR: Rapid Resolution 3.5 µm columnsConfigurations not shown are available upon request.

ZORBAX Bio SCX Series II Column Helps Resolve More Peptides by 2-D LC/MS

zorbax hplc columns for lc/ms and high throughput...

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