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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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.
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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.
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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
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10. High Temperature Bidentate Column
Figure 6: Zirchrom PBD, 0.6 mL/min, 35% ACN, from bottom 60 oC,
80oC, 100oC, 120oC, 140oC, 150oC
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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
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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.
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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.
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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.
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Hinweis der Redaktion
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.