Optimizing Agilent 1100 HPLC Systems to achieve UHPLC

TECHNICAL REPORTMODifying AgileNT ® 1100 HPLC SySTeMS TO ACHieve “UHPLC-like”perFOrMANCe wiTH HALO ® Fused-Core ® COLuMNSAgilent 1100 HPLC systemWith a few modifications, an Agilent 1100HPLC can produce UHPLC-like performanceusing HALO Fused-Core columns.IntroductionThe Agilent 1100 HPLC systems are foundin many labs, and their performance intheir as-shipped configuration is excellentwhen used with conventional HPLCcolumns. However, the recent introductionof columns packed with Fused-Core particles offers Agilent 1100 usersthe opportunity to obtain even higherperformance from their existing HPLCequipment. HALO columns packed withFused-Core particles deliver resolutionand speed similar to sub-2-µm UHPLCcolumns, but generate only 40% to 50%of the back pressure produced by sub-2-µmcolumns. (See Figure 1.) The high efficiencyand lower back pressure of HALO columnsmake it possible to obtain “UHPLC-like”performance from conventional 400-barequipment, like the Agilent 1100.HALO columns, like their sub-2-µmcolumn counterparts, produce smaller peakvolumes (50% to 75% smaller) than conventionalcolumns of the same dimensions.Because of the smaller peak volumes,it is important to keep extra columnband broadening appropriate for theparticular column geometry used. Howyour Agilent 1100 instrument is configured,and the method settings youchoose will determine the amount of extracolumn band broadening, and, ultimatelythe efficiency and resolution you canobtain with a given HALO columngeometry. Many HALO columns can beused quite effectively with the Agilent1100 in its standard configuration, butmuch more performance can be obtainedby modifying the equipment and settingsfor optimum results. For example,a 4.6 x 100 mm HALO column canreduce analysis time by 67% compared to a4.6 x 250 mm, 5 µm conventional columnusing a standard Agilent 1100 quaternarysystem under identical conditions.However, by making a few modificationsto the Agilent 1100, the same HALOcolumn will yield 46% more plates. Themoderate back pressure and high flowvelocity characteristics of HALO columnsfacilitate a 85% reduction in analysis timecompared to a conventional column, whilemaintaining resolution. (see Figure 2).Figure 1: HALO columns exhibit UHPLC-like performance at conventionalHPLC pressure1.7 µm1.8 µm2.2 µm2.7 µm Fused-Core 3.43 µm3.5 µm5 µm 122.85.27.78.70 2 4 6 8 10RELATIVE BACK PRESSUREIn HPLC, peak volume decreases ascolumn ID or length decreases. Smallpeak volumes are more vulnerable to extracolumn band broadening, thus preventingmaximum column efficiency from beingachieved. While very good results canbe obtained with many of the largervolume HALO columns using the Agilent1100 in its standard configuration, youwill likely find it worthwhile to reducethe amount of extra column volumeand adjust detector settings to achievemuch better results with some of thesmaller volume HALO columns.In this technical report we first discussextra column band broadening and theinstrument parameters that contribute toit. Next, we recommend how an Agilent1100 can be configured to get the mostperformance from specific HALO columns.Finally, we provide step-by-step instructionsfor making those recommendedmodifications to the Agilent 1100.5 µm3.5 µm3 µm2.2 µmSub-2 µmHALO0 50k 100k 150k 200k 250kEFFICIENCY: N/METERHALO columns packed with Fused-Core particles provide over 80% of the efficiency (theoreticalplates, N) and 90% of the resolving power of sub-2-µm columns, but require less than half the backpressure. This lower pressure permits HALO columns to be used with conventional 400 bar-limitHPLC equipment and achieve speed and resolution very similar to UHPLC.

Figure 2: An Agilent 1100 modified to reduce extra column band broadeningpermits significantly better performance to be obtained from HALO columns.Standard configured Agilent 1100Conventional column: 4.6 x 250 mm, 5 μmFlow rate: 1.0 ml/minPressure: 91 barR s = 6.50 minutes 12Standard configured Agilent 1100HALO column: 4.6 x 100 mmFlow rate: 1.0 ml/minPressure: 124 barR s = 5.2R s = 6.3The HALO column delivers a much faster separation compared to a conventional columnwhen using a standard configured Agilent 1100, but the resolution is less than expected.When the Agilent 1100 is modified to reduce extra column band broadening, 46% moreplates and higher resolution are obtained from the same HALO column. The Fused-Coreparticles packed in the HALO column permit a higher flow velocity to be used withoutsacrificing resolution or exceeding the pressure limit of the Agilent 1100. The result is areduction in analysis time of 85% while maintaining similar resolution compared to theconventional column.Extra Column Band BroadeningN = 25,900N = 16,7000 minutes4Agilent 1100 modified to reduce extra column band broadeningHALO column: 4.6 x 100 mmFlow rate: 2.0 ml/minPressure: 247 barN = 24,5000 minutes2In liquid chromatography the observed peak width (w obs) is comprised of thedispersion (or band broadening) from the chromatographic process itself (w col)and from dispersion elsewhere in the HPLC system (w ec). The relationship can bedescribed by this equation:w 2 obs =w2 col +w2 ecThe observed, column-related, and extra columnrelatedvariances are related similarly,σ 2 = σ 2 +σ 2 obs col ecwhere w = 4σ, σ is the standard deviation of the peakand σ 2 is the peak variance.The total extra column variance (σ 2 ec) is equal tothe sum of the variances from the various contributorsto band broadening (assuming all variablesare independent):σ 2 ec= σ 2 injection+ σ 2 tubing + σ 2 flow cell+ variance fromdetector response time and data rateThe importance of extra column band broadeningcan be assessed relative to the peak width or volumeat the retention time or volume for each analyte.In general, the contributions to band broadeningand peak volume are inversely proportional to theretention time or volume of the analyte, so that extracolumn band broadening is most important for theleast retained analytes in a separation.For a given HPLC system configuration, the extracolumn volume, or ECV, is the total volume outsideof the column that contributes to the observed peakvolume. For an Agilent 1100 the ECV includes thevolumes associated with the following items:• the injection volume,• the flow path in the autosampler and valve,• the tubing from the autosampler to the precolumnheat exchanger,• the precolumn heat exchanger,• the tubing from precolumn heat exchanger tothe column,• the tubing from the column to the detectorflow cell,• the detector flow cell, and• any volume added by in-line filters, unions, guardcolumns, etc.NOTE: For simplicity, any contributions to bandbroadening due to fittings and connections areconsidered to be negligible with proper choice ofhardware and good practices, and will be ignored.The total extra column band broadening also includesthe dispersion due to electronic signal filteringfrom the detector. With Agilent UV-VIS detectorsthe ChemStation® software Peak Width setting isdirectly related to detector response time, andinversely related to data acquisition rate for aparticular detector type and model. Typically, you canfind the relationships among Peak Width setting,response time, and data rate summarized in atable in the appropriate detector manual. The largerthe Peak Width setting is, the slower the data rate,and the greater the amount of signal filtering. Conversely,smaller Peak Width settings are associatedwith faster data rates, less signal filtering, and moreshort-term noise. A general recommendation is to2

Figure 3: Extra Column Volume, ECVPumpSampleInjectionFlow CellColumnDetectorTABLE A: Standard configured Agilent 1100 (as typically shippedfrom Agilent)Acceptable configuration for the following HALO columns:4.6 x 150 mm, 4.6 x 100 mm, and 4.6 x 75 mm.Description Part No. Volume (μL)ConnectingTubing and FittingsWasteNeedle seat capillary for standardautosampler 0.17 x 100 mm, GreenOr, needle seat capillary for well-plateautosampler 0.17 x 100 mm, GreenG1313-87101 2.5G1367-87302 2.5SolventReservoirData CollectionIn this schematic of an HPLC system, the gold color indicates where extracolumn volume, ECV, is located.Stainless steel tubing from autosamplerto column compartment heat exchanger0.17 x 180 mm, GreenColumn compartment heat exchanger 3 uLStainless steel tubing from columncompartment heat exchanger to column0.17 x 90 mm, GreenStainless steel tubing from column todetector flow cell 0.17 x 380 mm, GreenG1313-87305 4.53.0G1316-87300 2.2G1315-87311 9.4set the detector Peak Width setting to be equal to or slightly lessthan the half-height width of the narrowest peak of interest in thechromatogram. In practice, it is wise to compare performance (peakwidths, tailing factors, signal-to-noise ratios) from chromatogramsacquired with several detector Peak Width settings above and belowthe value for the narrowest peak, in order to choose the best settingfor your needs. If the peak width setting is too small, the observedpeak widths will be minimized and peak heights maximized, but thesignal-to-noise ratio will suffer due to increased baseline noise. If thepeak width setting is too large, baseline noise will be reduced, but thepeaks will be shorter and broader and may begin to tail and merge.Several report styles (Performance, Performance and Noise, andExtended Performance) within the Data Analysis View of theChemStation software can help you find an appropriate or optimumsetting for your column and conditions. Fortunately, the datarate is automatically set when you choose the Peak Width settingas just described. Nevertheless, a good rule of thumb is toensure that the data rate is sufficiently fast to acquire atleast 20 points across the narrowest peak. As an example, for apeak width at half height of 0.05 min., the 4σ baseline width(1.7 X PW ½) is 5.1 sec., and the data rate should be at least20 points per 5.1 seconds, or ~ 4 Hz.The detector flow cell (volume and design), the detector responsetime and the data rate are typically the largest contributors to bandbroadening. These are the parameters you should optimize firstwhen trying to get the most performance from HALO columns.If you are using HALO columns with ID ≤ 3.0 mm, you mayalso have to minimize connecting tubing volume, heat exchangervolume, and injection volume.Recommended Instrument Configurations to Use withSpecific HALO ColumnsThe smaller the peak volume produced by a column, the moreimportant it is to make sure that extra column volume (ECV) doesnot rob you of the resolving power that the column can deliver.Depending on the dimensions of the HALO column you wish touse, three different configurations of the Agilent 1100 will berecommended; the standard configuration (as shipped from theStandard flow cell for VWD10 mm, 14 μL, 400 barStandard flow cell for DAD/MWD10 mm, 13 µL, 120 barTABLE B: Low ECV configured Agilent 1100G1314-60082 14.0G1315-60022 13.0Total ECV 34.6/35.6Acceptable configuration for the following HALO columns:4.6 x 150 mm, 4.6 x 100 mm, 4.6 x 75 mm, 4.6 x 50 mm, 4.6 x 30mm, 3.0 x 150 mm, 3.0 x 100 mm, and 3.0 x 75 mm.Description Part No. Volume (μL)Needle seat capillary for standardautosampler 0.12 x 100 mm, RedOr, needle seat capillary for well-plateautosampler 0.12 x 150 mm, RedStainless steel tubing from autosamplerto column compartment heat exchanger0.12 x 180 mm, RedColumn compartment heat exchanger3 uLStainless steel tubing from columncompartment heat exchanger to column0.12 x 70 mm, RedStainless steel tubing from column todetector flow cell 0.12 x 180 mm, RedAlternate longer length stainless steeltubing from column to detector flow cell0.12 x 280 mm, RedSemi-micro flow cell for VWD6 mm, 5 μL, 120 barSemi-micro flow cell for DAD/MWD6 mm, 5 μL, 120 barG1313-87103 1.3G1367-87303 1.9G1313-87304 2.33.0G1316-87303 0.9G1313-87304 2.301090-87610 3.6G1314-60083 5.0G1315-60011 5.0Total ECV 14.8/16.73

TABLE C: Ultra-low ECV configured Agilent 1100Acceptable configuration for the following HALO columns:4.6 x 150 mm, 4.6 x 100 mm, 4.6 x 75 mm, 4.6 x 50 mm, 4.6 x 30 mm,3.0 x 150 mm, 3.0 x 100 mm, and 3.0 x 75 mm, 3.0 x 50 mm, 3.0 x 30 mm,2.1 x 150 mm, 2.1 x 100 mm, 2.1 x 75 mm, 2.1 x 50 mm, 2.1 x 30 mm.Description Part No. Volume (μL)Needle seat capillary for standardautosampler 0.12 x 100 mm, RedOr, needle seat capillary for well-plateautosampler 0.12 x 150 mm, RedStainless steel tubing from autosamplerto low volume heat exchanger0.12 x 180 mm, RedLow volume heat exchanger 1.6 uL(includes connecting tubing to column)Stainless steel tubing from column todetector flow cell 0.12 x 180 mm, RedAlternate longer length stainless steeltubing from column to detector flow cell0.12 x 280 mm, RedMicro flow cell for VWD 1 μL, 6 mm 40 barMicro flow cell for DAD/MWD2 μL, 3 mm 40 barTABLE D: Recommended Agilent 1100 configurations to use withspecific HALO columnsHALO Column Dimensions4.6 x 150 mm Standard, Low-ECV, Ultra-low ECV4.6 x 100 mm Standard, Low-ECV, Ultra-low ECV4.6 x 75 mm Standard, Low-ECV, Ultra-low ECV4.6 x 50 mm Low-ECV, Ultra-low ECV4.6 x 30 mm Low-ECV, Ultra-low ECV3.0 x 150 mm Low-ECV, Ultra-low ECV3.0 x 100 mm Low-ECV, Ultra-low ECV3.0 x 75 mm Low-ECV, Ultra-low ECV3.0 x 50 mm Ultra-low ECV3.0 x 30 mm Ultra-low ECV2.1 x 150 mm Ultra-low ECV2.1 x 100 mm Ultra-low ECV2.1 x 75 mm Ultra-low ECV2.1 x 50 mm Ultra-low ECV2.1 x 30 mm Ultra-low ECVG1313-87103 1.3G1367-87303 1.9G1313-87304 2.3G1316-80002 1.6G1313-87304 2.301090-87610 3.6G1314-60081 1.0G1315-60024 2.0Total ECV 8.5/11.4Notes:• The ChemStation peak width setting is optimized best by carrying out several runsunder method conditions using different settings. One can choose the setting thatgives the best signal to noise for all analytes, consistent with observed peak widthand USP tailing factors using Performance and Noise and Extended Performancereports from the Data Analysis view of the Agilent ChemStation software• In the autosampler, replacing the 0.17 x 100 mm needle seat capillary (Agilentpart number G1313-87101, 2.5 µL) with the 0.12 x 100 mm seat capillary (Agilentpart number G1313-87103, 1.1 µL) will reduce ECV and improve systemefficiency slightly.Agilent 1100 Configurationfactory), a low ECV configuration, and an ultra-low ECV configuration.See Tables A, B and C for the three different Agilent 1100 configurationswe recommend for specific HALO column dimensions.Modifying Your Agilent 1100 SystemModifying your Agilent 1100 system from the standard configurationto either the low ECV or ultra-low ECV configuration is quite simple,once you have the parts on hand. The Agilent part numbers needed tomake the modifications are listed in Tables A, B and C. Substitutionof similar sized capillary tubing from a source other than Agilent isgenerally okay, but we recommend that the replacement low volumeheat exchanger and detector flow cells come from Agilent to ensurecompatibility.Step 1: Replace the standard capillary tubing (0.17 x 180 mm, green,G1313-87305) connecting the autosampler valve to the heat exchangerin the column compartment with the low volume capillary tubing(0.12 x 180 mm, red, G1313-87304). For ultra-low ECV configurationconnect the outlet of the capillary tubing to the inlet union of thelow volume heat exchanger (G1316-80002). Position the low volumeheat exchanger in one of the wells of the column compartment heatexchanger block, and secure it using one of the column clips as needed.See Figure 4.Step 2: Replace the standard capillary tubing (0.17 x 90 mm, green,G1316-87300) connecting the heat exchanger to the column withthe low volume capillary tubing (0.12 x 70 mm, red, G1316-87303).For the ultra-low ECV configuration, connect the outlet tubing ofthe low volume heat exchanger (G1316-80002) directly to the columninlet using an appropriate stainless steel, PEEK or polyketone fitting.See Figure 4.Step 3: Replace the standard capillary tubing (0.17 x 380 mm, green,G1315-87311) connecting the column to the detector flow cell withthe low volume capillary tubing (0.12 x 180 mm, red, G1313-87304)or the alternate longer capillary tubing (0.12 x 280 mm, red,01090-87610). See Figure 4. Note: Do not connect the tubing to thedetector until the column and tubing have been flushed.Step 4: Prime the instrument and flush the tubing and columnwith either 100% methanol or acetonitrile. Some people find theyachieve faster equilibration by first flushing the capillary tubing beforeconnecting the column and flushing it.Step 5: Replace the standard detector flow cell with either a semimicroflow cell (low ECV) or a micro flow cell (ultra-low ECV).See Figure 5.Step 6: Connect the capillary tubing coming from the outlet ofthe HALO column to the detector flow cell. For diode-array andmultiwavelength detectors the capillary tubing is normally connectedto a zero dead volume union, which then is connected to thecapillary tubing connected into the flow cell body. For 2.1 mm HALOcolumns in shorter lengths (30, 50, 75 mm) it is beneficial to connectthe column directly to the capillary inlet tubing of the flow cell to minimizeextracolumn volume and extra connections. It may be necessaryto stack the column compartment directly above the DAD/MWDdetector in such situations.Step 7: Pump 100% methanol or acetonitrile through the tubing,column and the detector flow cell for ~ 5 minutes or until you observestable pressure and baseline.You should observe a flat baseline at 25-50 mAUFS with a UVdetector, and can now proceed to equilibrate your column with yourmobile phase and begin running samples.4

Figure 4: Replace standard capillary tubing (0.17 mm, green) withlow volume capillary tubing (0.12 mm, red).Step 1: Replacecapillary tubing fromsample injector valveto heat exchanger(3 μL) in the columncompartment.Step 2: Replacecapillary tubingconnecting the heatexchanger to thecolumn.Step 3: Replacecapillary tubingconnecting thecolumn to thedetector flow cell.band after injection and before the column is usually minimized oreliminated, if the injection volume is not too large and the startingmobile phase is not too strong. However, it continues to be importantto minimize extra column band broadening after the column(tubing and flow cell volume, and Chemstation Peak Width setting)for gradient separations when using low-volume, high efficiencyHALO columns.If you plan to only run gradient separations with your Agilent1100, you may be able to skip Steps 1 and 2 when modifyingthe equipment.For gradient separations the delay volume is also important becauseit imposes an isocratic hold to the start of the gradient program,such that the desired mobile phase composition reaches the columnat a time later than programmed by a factor of V d/F, where V dis thedelay volume and F is the volumetric flow rate. For a discussion ofthe translation of gradient methods please see our Quick Tips Guidehttp://www.mac-mod.com/QTpdf.ConclusionFigure 5: Replace standard detector flow cell with either asemi-micro flow cell (low ECV configuration) or a micro flow cell(ultra-low ECV configuration).Step 5: Removestandard detectorflow cell and replacewith a semi-microflow cell or a microflow cell.Figure 6 illustrates the benefits of modifying an Agilent 1100 toan ultra-low ECV configuration. Compared to a conventionalcolumn, analysis time is reduced by over 90% while resolutionis maintained. This performance compares favorably with UHPLC,but is accomplished with a conventional Agilent 1100 quaternarysystem by making a few simple, and inexpensive, modifications tothe equipment.Figure 6: An ultra-low ECV configured Agilent 1100 using aHALO column provides performance that compares favorably toUHPLC systems.Gradient SeparationsAll of the considerations and recommendations about reducingextra column band broadening which have been made thus far inthis technical report have dealt with isocratic separations. If you aredeveloping a new gradient method or translating an existing gradientmethod to a HALO column, the most important parameters arethe same ones that have been discussed for isocratic separations.However, there are two major differences.1. The extra column volume before the column is less importantthan the extra column volume after the column when using agradient, and,2. the delay volume (system volume from the point where solvents Aand B are mixed up to the column itself ) is important because it is amajor contributor to the “hold” time at the beginning of a gradient.Extra column volume before the column is less important for gradientseparations, because there will be a refocusing of the analytes atthe head of the column (unless pre-elution of polar analytes occurs)in a gradient separation. Consequently, any dispersion of the sampleConditionsColumn: HALO C18, 3.0 x 50 mmMobile Phase: 35% acetonitrile to90% acetonitrile in 1 minuteFlow Rate: 2.0 mL/min.Column Temperature: 40º CPressure: 304 bar —> 167 barResponse Time: < 0.125 sec1234Sample:1. Acetophenone2. Propiophenone3. Butyrophenone4. Benzophenone5. Valerophenone6. Hexanophenone7. Heptanophenone8. Octanophenone1.5 minutes7 80 minutes2An ultra-low ECV configured Agilent 1100 quarternary system using a highefficiency HALO column (Fused-Core particles) separates these 8 alkylphenonesin 90 seconds with ample resolution between all peaks. This speedand resolution compare favorably to the performance reported bychromatographers using UHPLC equipment and sub-2 μm columns. Thepressure generated by the HALO column is well within the acceptableoperating limits of the Agilent 1100.565

www.advanced-materials-tech.comLC501®HALO and Fused-Core are registered trademarks of Advanced Materials Technology, Inc.®Agilent and ChemStation are registered trademarks of Agilent Technologies, Inc.