Increasing the Throughput of UHPLC

William Hedgepeth, Rachel Lieberman,Shimadzu Scientific Instruments, Columbia, MD, USA, 800-477-1227, www.ssi.shimadzu.com

Introduction

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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Background

UHPLC has been gaining momentum as a way to shorten HPLC analytical run times. Initially, small totally porous particle (sub-2 um) columns were used to achieve these results; however, there is a growing popularity for the use of superficially porous particles that can deliver similar results with lower system pressures. An evaluation was conducted with each type of column to see how much throughput could be obtained at or above their maximum operating temperatures.

Elevated and high-temperature LC has also been gaining attraction as a way to speed analytical runtimes. Two columns (polymer based and a polybidentate) designed specifically for higher temperature HPLC analysis (> 100°C) were also analyzed to see the effect increased temperature could have.

Recently, a new high-throughput autosampler has been introduced that provides an injection 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 a one-minute timeframe without any load ahead or pre-injection techniques.3

Methodology/Procedure

Analytical runs were conducted under isocratic conditions using Water/Acetonitrile mixtures. 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 maximum temperature 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 Zirchrom PBD (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 the effect 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.

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Kinetex Core-shell Column

Figure 1: Kinetex XB-C18, 60% ACN, 75oC, 4 mL/min, Paraben mix, runtime: 4.2 seconds, 7,940 psi, 2-minute timescale

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Kinetex Core-shell Column

Figure 2: 1 minute timescale of Figure 1

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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 psi

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Shimadzu XR-ODSIII 1.6 um Column

Figure 4: 30 second timescale of Figure 3

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High temperature polymer column

Figure 5: Shodex ET-RP1, 1 mL/min, 60% ACN, from bottom 40oC, 60oC, 80oC, 100oC, 120oC, 140oC, 150oC

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High Temperature Bidentate Column

Figure 6: Zirchrom PBD, 0.6 mL/min, 35% ACN, from bottom 60oC, 80oC, 100oC, 120oC, 140oC, 150oC

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Elevated Temperatures

0.0 2.5 5.0 7.5 10.0 12.5

40℃

60℃

80℃

120℃

0.2mL/min

RT=11.4min

RT=7.83min

RT=5.87min

RT=3.26min , RS=4.99

Column: ZirChrom-PBD, MP: 40% acetonitrile, Analytes: valerophenone, hexaphenone, heptaphenone, octaphenone

RT decreased 3.5x by increasing temp. from 40C to 120C. Increased flow rate further reduced the RT to less than 1 minute (<1/10).

RT decreased 3.5x by increasing temp. from 40C to 120C. Increased flow rate further reduced the RT to less than 1 minute (<1/10).

120℃0.7mL/min

RT=0.94min , RS=4.45

High Throughput UHPLC/MS/MS

Requirements:

High-speed scanning rates to obtain enough data points to reduce peak distortion (15,000 u/sec)

Fast polarity switching speeds (15 msec) to combine ionization modes

Short pause time when switching measurements between compounds (1 msec)

Technology to keep ion momentum in collision cell

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14 sec cycle time analysis14 sec cycle time analysis

　　⇒ Ultra fast analysis by combination of SIL-30ACMP and LCMS-8030

　　⇒ Ultra fast LC/MS analysis without compromise of performance

High-throughput UHPLC/MS/MS Analysis

min

0

25000

50000

75000

100000

125000 3:281.10>86.10(+)2:256.10>167.10(+)1:235.40>86.10(+)

0.0 0.25 0.5 0.75 1.0 min

0

25000

50000

75000

100000

125000 3:281.10>86.10(+)2:256.10>167.10(+)1:235.40>86.10(+)

0.0 0.25 0.5 0.75 1.0

14 sec14 sec

Event #

Compound Q1 m/z Q3 m/z

1 Lidocaine 235.4 86.1

2 Diphenhydramine 256.1 167.1

3 Imipramine 281.1 86.1

Column : Shim-pack XR-ODS 1.5 mmID×30 mm, 2.2 µmⅡ

MP : acetonitrile / water =25/75

containing 0.1 % formic acid

Flow rate : 1.2 mL/min

Ionization : ESI(+)

Carryover Discussion

There are two ways to reduce carryover: 1) Remove it by rinsing or 2) Prevent it in the first place. Rinsing can be effective; however, with the need for increased throughput, taking the time needed for rinsing may not be the best option. Careful choice of materials used in the design and construction of an autosampler will go a long way to prevent carryover. A low carryover 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 the needle 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 system with organic solvents – time penalty.

2) Controlling sample adsorption by changing the rotor seal material and geometry.

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Carryover level of chlorohexidineCarryover level of chlorohexidine

　　 ⇒ Very low carryover of a stubborn compound, chlorhexidine

⇒ Chlorhexidine 500 ug/mL →carryover 0.0001%!

Minimized Carryover to Support LC/MS/MS

Carryover of chlorhexidine in LCMS-8030 analysis

m/z 253.2 > 170.1

LCMS-8030LCMS-8030

0.00 0.25 0.50 0.75 1.00 min

0.0

0.5

1.0

1.5

2.0(x10,000)Chlorhexidine 500 ng/µL

Blank (0.0001 %)

Results

RSD data for 1 uL injection, 4.2 second run (n = 9): Methyl paraben 0.17%, Propyl paraben 0.28%, Butyl paraben 0.37%. A total of four analytical 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 7.85 min 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 min to 1.30 min. An additional analysis showed the runtime could be shortened tenfold with increasing column temperature.

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Conclusions

The use of sub-2 micron and core-shell columns at elevated temperatures and flow rates in conjunction with a new high-throughput autosampler allowed up to four analytical runs to be obtained within a one-minute timeframe. High-quality data could still be achieved with a 1 uL injection and a 4.2 second runtime.

Increasing the temperature on columns designed for temperatures above 100oC not only reduced analysis times, but could greatly improve the peak shape of late eluting compounds.

High-throughput UHPLC/MS/MS is practical with a low carryover autosampler design that reduces rinsing requirements and a mass spectrometer designed for high-speed analysis.

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