Increasing the Throughput of UHPLC
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The document discusses advancements in ultra-high-performance liquid chromatography (UHPLC) that enhance throughput and reduce analytical run times. It highlights the effectiveness of using small particle and core-shell columns at elevated temperatures, along with a high-speed autosampler, allowing up to four analytical runs to be completed in under one minute. Additionally, the study addresses techniques to minimize carryover and maintain high-quality data in rapid analyses.
Increasing the Throughput of UHPLC
- 1. Increasing the Throughput of UHPLC William Hedgepeth, Rachel Lieberman, Shimadzu Scientific Instruments, Columbia, MD, USA, 800-477-1227, www.ssi.shimadzu.com
- 2. 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. 2
- 3. 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
- 4. 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. 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 Column Figure 2: 1 minute timescale of Figure 1 6
- 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 psi 7
- 8. Shimadzu XR-ODSIII 1.6 um Column Figure 4: 30 second timescale of Figure 3 8
- 9. High temperature polymer column Figure 5: Shodex ET-RP1, 1 mL/min, 60% ACN, from bottom 40 oC, 60oC, 80oC, 100oC, 120oC, 140oC, 150oC 9
- 10. High Temperature Bidentate Column Figure 6: Zirchrom PBD, 0.6 mL/min, 35% ACN, from bottom 60 oC, 80oC, 100oC, 120oC, 140oC, 150oC 10
- 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/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 12
- 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 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. 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. 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. 16
- 17. 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. 17
Editor's Notes
- #12: 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.