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Increasing the Throughput of UHPLC
Increasing the Throughput of UHPLC
Increasing the Throughput of UHPLC
Increasing the Throughput of UHPLC
Increasing the Throughput of UHPLC
Increasing the Throughput of UHPLC
Increasing the Throughput of UHPLC
Increasing the Throughput of UHPLC
Increasing the Throughput of UHPLC
Increasing the Throughput of UHPLC
Increasing the Throughput of UHPLC
Increasing the Throughput of UHPLC
Increasing the Throughput of UHPLC
Increasing the Throughput of UHPLC
Increasing the Throughput of UHPLC
Increasing the Throughput of UHPLC
Increasing the Throughput of UHPLC
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Increasing the Throughput of UHPLC

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UHPLC has proven to be an effective way to reduce analysis times without losing separation efficiency through the use of small particle and core-shell column technologies. The use of higher column …

UHPLC has proven to be an effective way to reduce analysis times without losing separation efficiency through the use of small particle and core-shell column technologies. The use of higher column temperatures and shorter column lengths has allowed the analysis speed of UHPLC to be further increased. A number of high-speed UHPLC applications and conditions will be presented which now allow up to four analytical runs to be completed in only a one-minute timeframe.

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  • Here is the separation of the phenones on a ZirChrom 3 micron column performed at different temperatures. As temperature in increased from 40C to 120C, the run times decrease from 11.4 minutes to only 3.25 minutes, an improvement of about 3.5 times. However, at 120C, the back pressure is so low that it is possible to increase the linear velocity also and cut the run time from 3.25 minutes down to less than 1 minutes. So overall, a combination of temperature and flow can increase the analysis speed by more than a factor of 10.
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    • 1. Increasing the Throughput of UHPLCWilliam Hedgepeth, Rachel Lieberman,Shimadzu Scientific Instruments, Columbia, MD, USA,800-477-1227, www.ssi.shimadzu.com
    • 2. IntroductionUHPLC has proven to be an effective way to reduceanalysis times without losing separation efficiency throughthe use of small particle and core-shell columntechnologies. The use of higher column temperatures andshorter column lengths has allowed the analysis speed ofUHPLC to be further increased. A number of high-speedUHPLC applications and conditions will be presented whichnow allow up to four analytical runs to be completed in onlya one-minute timeframe.2
    • 3. BackgroundUHPLC has been gaining momentum as a way to shorten HPLC analytical runtimes. Initially, small totally porous particle (sub-2 um) columns were used toachieve these results; however, there is a growing popularity for the use ofsuperficially porous particles that can deliver similar results with lower systempressures. An evaluation was conducted with each type of column to see howmuch throughput could be obtained at or above their maximum operatingtemperatures.Elevated and high-temperature LC has also been gaining attraction as a way tospeed analytical runtimes. Two columns (polymer based and a polybidentate)designed specifically for higher temperature HPLC analysis (> 100°C) were alsoanalyzed to see the effect increased temperature could have.Recently, a new high-throughput autosampler has been introduced that provides aninjection time of only seven seconds, with a total injection cycle time of 14 seconds.The use of this autosampler allows the completion of four analytical runs within aone-minute timeframe without any load ahead or pre-injection techniques.3
    • 4. Methodology/ProcedureAnalytical runs were conducted under isocratic conditions using Water/Acetonitrilemixtures. High-throughput runs were evaluated with a 250 ppm paraben (Methyl,Propyl, Butyl) mix on a Phenomenex Kinetex XB-C18 (30 x 3mm, 1.7 um) “core-shell” type column and a Shim-pack XR-ODSIII (50 x 2.0 mm, 1.6 um) column.Column temperature was set at or slightly above the recommended maximumtemperature and the flow rate was increased as much as possible. High-temperature analytical runs were evaluated with a 100 ppm phenone(Acetophenone, Butyrophenone, Hexaphenone, Octaphenone) mix on a ZirchromPBD (50 x 3 mm, 3 um) column and a Shodex ET-RP1 4D (150 x 4.6mm, 4 um)column. Column temperatures were increased from 40oC to 150oC to determine theeffect on runtime and peak efficiency.High-temperature data was obtained from a Shimadzu Nexera system and high-throughput data was obtained on a Shimadzu Nexera system with the new SIL-30ACMP autosampler.4
    • 5. Kinetex Core-shell Column Figure 1: Kinetex XB-C18, 60% ACN, 75oC, 4 mL/min, Paraben mix,5 runtime: 4.2 seconds, 7,940 psi, 2-minute timescale
    • 6. Kinetex Core-shell ColumnFigure 2: 1 minute timescale of Figure 16
    • 7. Shimadzu XR-ODSIII 1.6 um Column Figure 3: Shim-pack XR-ODSIII, 60% ACN, 80oC, 2.25 ml/min, Paraben mix, runtime: 6.6 seconds, 17,300 psi7
    • 8. Shimadzu XR-ODSIII 1.6 um ColumnFigure 4: 30 second timescale of Figure 38
    • 9. High temperature polymer columnFigure 5: Shodex ET-RP1, 1 mL/min, 60% ACN, from bottom 40 oC,60oC, 80oC, 100oC, 120oC, 140oC, 150oC9
    • 10. High Temperature Bidentate ColumnFigure 6: Zirchrom PBD, 0.6 mL/min, 35% ACN, from bottom 60 oC,80oC, 100oC, 120oC, 140oC, 150oC10
    • 11. Elevated Temperatures Column: ZirChrom-PBD, MP: 40% acetonitrile, Analytes: valerophenone, hexaphenone, heptaphenone, octaphenone RT=11.4min 40℃ RT=7.83min 60℃0.2mL/min RT=5.87min 80℃ RT=3.26min , RS=4.99 RT decreased 3.5x by increasing temp. from 40° C to 120°C. 120℃ Increased flow rate further RT=0.94min , RS=4.45 reduced the RT to less than 1 minute (<1/10).0.7mL/min 120℃ 0.0 2.5 5.0 7.5 10.0 12.5
    • 12. High Throughput UHPLC/MS/MSRequirements:High-speed scanning rates to obtain enough data points to reducepeak distortion (15,000 u/sec)Fast polarity switching speeds (15 msec) to combine ionization modesShort pause time when switching measurements between compounds(1 msec)Technology to keep ion momentum in collision cell12
    • 13. High-throughput UHPLC/MS/MS Analysis 14 sec cycle time analysis  ⇒ Ultra fast analysis by combination of SIL-30AC MP and LCMS-8030  ⇒ Ultra fast 14 sec analysis without compromise of performance LC/MS 1:235.40>86.10(+) 2:256.10>167.10(+) Event   125000 3:281.10>86.10(+) Compound Q1 m/z Q3 m/z # 1 Lidocaine 235.4 86.1 100000 2 Diphenhydramine 256.1 167.1 3 Imipramine 281.1 86.1 75000 Column : Shim-pack XR-ODSⅡ 1.5 mmID×30 mm, 2.2 µm MP : acetonitrile / water =25/75 50000 containing 0.1 % formic acid Flow rate : 1.2 mL/min 25000 Ionization : ESI(+) 0 0.0 0.25 0.5 0.75 1.0 min
    • 14. Carryover DiscussionThere are two ways to reduce carryover: 1) Remove it by rinsing or 2) Prevent it in the firstplace. Rinsing can be effective; however, with the need for increased throughput, taking thetime needed for rinsing may not be the best option. Careful choice of materials used in thedesign and construction of an autosampler will go a long way to prevent carryover. A lowcarryover autosampler design is necessary for successful high-throughput conditions.Carryover for ionic compounds can be reduced by:1) Removing adsorbed sample from the system with rinsing solution – time penalty.2) Controlling element adsorption by changing sample needle composition or by coating theneedle with chemically inert materials.Carryover for hydrophobic compounds can be reduced by:1) Removing adsorbed sample from the rotor seal groove by rinsing or flushing the systemwith organic solvents – time penalty.2) Controlling sample adsorption by changing the rotor seal material and geometry.14
    • 15. Minimized Carryover to Support LC/MS/MS  Carryover level of chlorohexidine    ⇒ Very low carryover of a stubborn compound, chlorhexidine ⇒ Chlorhexidine 500 ug/mL →carryover 0.0001%! (x10,000) 2.0 Chlorhexidine 500 ng/µL 1.5 LCMS-8030 m/z 253.2 > 170.1 1.0 Blank (0.0001 %) 0.5 0.0 0.00 0.25 0.50 0.75 1.00 min Carryover of chlorhexidine in LCMS-8030 analysis
    • 16. ResultsRSD data for 1 uL injection, 4.2 second run (n = 9): Methyl paraben0.17%, Propyl paraben 0.28%, Butyl paraben 0.37%. A total of fouranalytical runs could be completed in less than one minute.Pressure for ET-RP1 column was decreased from 1730 to 1185 psi(40-150oC), retention time of octaphenone was decreased from 7.85min to 1.79 min.Pressure for PBD column was decreased from 1620 to 950 psi (40-150oC), retention time of octaphenone was decreased from 6.86 minto 1.30 min. An additional analysis showed the runtime could beshortened tenfold with increasing column temperature.16
    • 17. ConclusionsThe use of sub-2 micron and core-shell columns at elevatedtemperatures and flow rates in conjunction with a new high-throughput autosampler allowed up to four analytical runs to beobtained within a one-minute timeframe. High-quality data could stillbe achieved with a 1 uL injection and a 4.2 second runtime.Increasing the temperature on columns designed for temperaturesabove 100oC not only reduced analysis times, but could greatlyimprove the peak shape of late eluting compounds.High-throughput UHPLC/MS/MS is practical with a low carryoverautosampler design that reduces rinsing requirements and a massspectrometer designed for high-speed analysis.17

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