NEW AND EXPANDED! ZORBAX HPLC Columns for LC/MS and High Throughput HPLC 31 Order online at: www.agilent.com/chem/store ZORBAX HPLC Columns LC/MS Columns and Cartridges ZORBAX LC/MS columns and cartridge columns Column choices for LC/MS are wide ranging, depending on the sample to be studied. For standard analytical samples, column choices focus on finding short columns with high resolution to reduce analysis 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 internal diameter columns allow good sensitivity and fast flow rates that are compatible with standard electrospray, APCI (atmospheric pressure chemical ionization) and APPI (atmospheric pressure photoionization) 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. These columns are available in popular ZORBAX Eclipse XDB and StableBond bonded phases. Additional bonded phases can be packed upon request. These bonded phases are compatible with the typical volatile mobile phase additives used for LC/MS – including TFA, formic acid, and acetic acid. • Fast analysis for high speed LC/MS with Rapid Resolution and Rapid Resolution HT columns • Many column choices for LC/MS compatibility — from nano to analytical • Narrow bore columns for improved sensitivity • Transfer methods from longer columns with 5 μm particles with no loss in resolution • Capillary and nano columns are most often used to analyze complex protein and peptides samples by LC/MS. More information on these columns can be found on page 42 of this guide. Reduce Analysis Time by 70% Using Short LC/MS Cartridge Columns Column: ZORBAX StableBond SB-C18 Mobile Phase: 85% Water with 0.1% TFA 15% Acetonitrile Flow Rate: 1.0 mL/min Temperature: 35°C Sample: 1. Phenylalanine 2. 5-Benzyl-3,6-dioxo-2-piperazine Acetic Acid 3. Asp-phe 4. 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 min A: Water with 0.2% Formic Acid B: Methanol with 0.2% Formic Acid Temperature: 35°C Sample: 2 μL Organic Acids 1. Gallic 2. Protocatechuic 3. Hydrocaffeic 4. Gentisic 5. Vanillic 6. Syringic 4.6 x 30 mm, 3.5 μm P/N 833975-902 2.1 x 30 mm, 3.5 μm P/N 873700-902 Time (min.) 4.6 x 50 mm, 3.5 um P/N 835975-902 4.6 x 30 mm, 3.5 um P/N 833975-902 4.6 x 15 mm, 3.5 um P/N 831975-902
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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
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
Flow Rate: 0.5 mL/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
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
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Rapid Resolution 3.5 µm columns are available in narrowbore configurations for great compatibility with LC/MS.
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.
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
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
1
23 4
56 7
1 23 4
56 7
1
2 3 45
6 7
12 3 4
56 7
12
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6 74.6 x 250 mm, 5 µmP/N 990967-902
Rapid Resolution 4.6 x 150 mm, 3.5 µmP/N 963967-902
Rapid Resolution 4.6 x 75 mm, 3.5 µmP/N 966967-902
Rapid Resolution 4.6 x 50 mm, 3.5 µmP/N 935967-902
Rapid Resolution 4.6 x 50 mm, 1.8 µmP/N 925975-902
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
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
Temperature: Ambient
Detection: UV 254 nm
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
• 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
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
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.
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
Detection: UV 215 nm
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
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mA
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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
ZORBAX Capillary and Nano Columns
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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.
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
Injection Volume: 0.1 µL
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
ZORBAX Capillary and Nano Columns
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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
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
ZORBAX Capillary and Nano Columns
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