DESIGN FEATURES AND PERFORMANCE OF A DIRECT ......200 fmol enolase digest, analytical column: 1.7...
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TO DOWNLOAD A COPY OF THIS POSTER VISIT WWW.WATERS.COM/POSTERS ©2005 Waters Corporation DESIGN FEATURES AND PERFORMANCE OF A DIRECT-FLOW NANOSCALE HPLC SYSTEM COMBINED WITH COLUMNS PACKED WITH SUB 2 MICRON PARTICLES Jeffrey W. Finch, Martha D. Stapels, Keith Fadgen, Alistair Wallace , Hongji Liu, Geoff Gerhardt, James P. Murphy, Steve Ciavarini, Christopher C. Benevides, and John C. Gebler Waters Corporation, Milford, MA, USA OVERVIEW INTRODUCTION METHODS CONCLUSIONS Purpose • To design and demonstrate performance of a rug- ged, splitless nano– and capillary scale HPLC that yields excellent retention time reproducibility, can be used in trapping or direct-inject mode, and can with- stand high operating pressures to maximize chroma- tographic resolution. Methods • 75 μm ID nanocolumns packed with 1.7 μm C18 particles • Trapping and direct-inject mode • Mass spectrometry on Q-ToF instruments Results • Minimal loss in resolution with trapping mode • Peptide retention time standard deviations typically < 0.1 min • Analysis of complex biological digest samples with minimal carryover • UPLC for nanoscale chromatography (3,000 to 10,000 psi ) Due to higher MS sensitivity, nanoscale chromatography is often the method of choice for analysis of complex proteomic samples. Here we present results from the Waters nanoACQUITY UPLC™ sys- tem, a new nanoscale HPLC platform with the following features: • Direct flow without splitting • Based on ACQUITY UPLC™ platform • Accurate gradient delivery for excellent retention time repro- ducibility • Binary high-pressure mixing pump with wide dynamic flow range • Operation at elevated pressures for nanocolumns packed with particles < 2 microns • Heating Trapping Module (HTM) with simplified “forward flush” sample trapping scheme and integrated column heater • Consumables that deliver consistent performance • Nanocolumns packed with ACQUITY 1.7 μm bridged-ethyl hybrid (BEH) particles that provide higher separation effi- ciency • Variable flow chromatography (“peak parking”) capability Dedicated auxiliary pump for providing stable flow of “lockmass” solution to reference sprayer of Q-Tof NanoLock Spray source Figure 1. Columns packed with bridged-ethyl hybrid particles • Figure 2. nanoACQUITY UPLC system Components of the nanoACQUITY UPLC system (Figure 2A) are de- signed for operating at pressures up to 10,000 psi. The column heater (Figure 2B) of the HTM is mounted on a pivot which posi- tions the column outlet close to the MS source to minimize delay vol- ume. All fused-silica tubing in the sample path is preassembled with PEEK protective cladding, fittings, and pre-cut polished ends to minimize dead volume. The Sample manager is completely en- closed and can maintain samples at 4 ºC to 40 ºC. The needle and sample loop can be flushed with both a strong and weak sol- vent wash, eliminating sample carryover. The NanoFlow Sprayer (Figure 2C) minimizes postcolumn dead volume in an easy to use design. The wide dynamic flow range of the BSM makes it possible to con- figure the system for sample loading/trapping using the scheme shown in Figure 3. This greatly simplifies system fluidics, allows for robust operation in a “forward flush” mode, and eliminates the need for an extra loading pump. Lit Code Number 720001350EN Figure 3. Simplified sample loading/trapping scheme Figure 4. Direct inject vs. trapping performance Figure 4 compares LC/MS traces for enolase digest acquired us- ing the same analytical 1.7 μm BEH column for direct inject vs. trap modes. The data demonstrates that there is no significant loss in chromatographic resolution of the peptides with the addition of a trap column. The data also shows that sample losses are mini- mized. Figure 5. Performance with different gradient lengths 50 fmol MassPREP digest mixture (enolase, phosphorylase b, ADH, BSA), analytical column: 1.7 μm BEH C18 75 μm x 150 mm, flow rate: 400 nL/min, gradient: 2-60% B in 30, 60, and 90 min. Figure 5 shows the effect of different gradient lengths on perform- ance. The column pressure for these runs was approximately 5,200 PSI. Figure 6. LC/MS reproducibility at 300 nL/min, n = 6 MassPREP digest mixture (200 fmol each of enolase, phosphorylase b, hemoglobin, ADH, BSA) analytical column: 1.7 μm BEH C18 75 μm x 100 mm, 300 nL/min, gra- dient: 3-60% B for 60 min trap column: 5 μm Symmetry C18 180 μm x 20 mm, sam- ple trapping: 3% B at 4 μL/min for 3 min column backpressure ~3,300 psi Figure 6 shows traces from six consecutive injections of a 5 pro- tein digest, along with retention time std. dev. of four peptides. The data set demonstrates that the nanoACQUITY/Q-Tof yields good retention time reproducibility (std. dev. < 0.1 min) and re- producible MS response over an extended period of time. The 1.7 μm BEH column provides greater separation efficiency for peptides compared to columns packed with conventional particles (3 to 5 μm). Figure 7. Biological samples: in-gel and in-solution digests Figure 7 shows triplicate injections of in-gel and in-solution digests of biological tissue or fluid samples collected from rats. These chro- matograms were obtained after 48 (Fig. 7A) and 57 (Fig. 7B) prior injections of similar samples on the column and trap. Highly repro- ducible retention times and ion intensities enable calculation of ac- curate peptide ratios with Protein Expression informatics. Figure 8. UPLC on a nanoLC scale (9,000 psi) Figure 8 shows chromatograms from injections on a 30 cm BEH nanocolumn with system pressures exceeding 9,000 psi. Median peak widths (4σ) measured from SIC’s of peptides throughout the gradient were 23 sec (Fig. 8A) and 54 sec (Fig. 8B), respectively. Features and advantages of the nanoACQUITY UPLC include: • Direct nanoflow delivery without splitting • Highly reproducible retention times over extended analysis periods • Binary high pressure pump with wide dynamic flow range -supports analytical column flow rates from 100 to 5000 nL/min -simplified sample loading/trapping configuration • High pressure capabilities enabling the use of nano- columns packed with particles < 2 μm and lengths up to 300 mm • Columns packed with 1.7 μm bridged-ethyl hybrid particles-higher efficiency, higher resolution, greater selectivity • Reproducible ion signals for quantitative proteomics A very fine human hair, ~ 60 μm 1.7 μm BEH particles (~ 35 can fit across hair) 10 μm bridged-ethanes in silica matrix • Smaller, uniform particles=higher packing densities, higher efficiency • Stronger particles that can withstand high pressure • Chemically resistant (wide pH range) Solvent tray Heating and trapping module (HTM) Sample manager Binary solvent manager (BSM) Auxiliary solvent manager nano-Tee w/ magnetic mount to MS source trap outlet tubing to HTM valve analytical column B. Column heater detail inner compartment (swiveled open) A. System stack and modules C. Universal NanoFlow Sprayer RESULTS AND DISCUSSION 19.00 19.20 Time 0 100 % 0.07min 20.80 21.00 Time 0 100 % 0.08min Time 10.00 15.00 20.00 25.00 30.00 35.00 40.00 % 0 100 % 0 100 J091604HL_023 TOF MS ES+ TIC 1.29e5 J091604HL_002 TOF MS ES+ TIC 1.29e5 A. Direct Inject Mode B. Trap Mode Trap column: 5 μm Symmetry C18 180 μm x 20 mm. Sample trapping: 3% B at 5 μL/min for 3 min (from BSM) 200 fmol enolase digest, analytical column: 1.7 μm BEH C18 75 μm x 100 mm, flow rate: 250 nL/min, gradient: 3-60% B in 30 min 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 Time 0 100 % 0 100 % 0 100 % K050511jwf07 1: TOF MS ES+ BPI 1.38e4 20.07 17.46 14.75 21.69 K050512jwf14 1: TOF MS ES+ BPI 6.28e3 30.25 19.40 15.52 25.68 23.06 33.05 K050513jwf102 1: TOF MS ES+ BPI 7.40e3 39.77 23.94 16.08 22.09 19.85 33.05 31.67 25.54 36.03 42.45 45.31 30 min gradient 60 min gradient 90 min gradient Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 % 0 100 % 0 100 % 0 100 % 0 100 % 0 100 % 0 100 E041124mds23 TOF MS ES+ BPI 1.01e3 28.43 23.05 18.93 17.99 17.20 22.23 20.44 26.46 24.32 25.19 31.00 30.25 32.05 35.41 34.44 36.95 39.22 E041124mds24 TOF MS ES+ BPI 1.01e3 28.34 22.94 18.82 17.87 17.11 22.13 20.33 26.36 24.23 30.91 30.16 31.96 35.33 34.35 36.84 39.13 E041124mds25 TOF MS ES+ BPI 1.01e3 28.35 22.93 18.81 17.86 17.08 22.10 20.30 26.36 24.20 27.18 30.92 30.17 31.97 35.34 34.34 36.83 39.08 E041124mds26 TOF MS ES+ BPI 1.01e3 28.31 22.89 18.75 17.81 17.03 22.04 20.25 26.32 24.17 30.89 30.13 31.96 35.30 34.32 36.84 39.08 E041124mds27 TOF MS ES+ BPI 1.01e3 28.27 22.87 18.75 17.94 17.04 22.04 20.26 26.30 24.15 25.03 30.85 29.84 31.89 E041124mds28 TOF MS ES+ BPI 1.01e3 28.31 22.89 18.77 17.96 17.06 22.06 20.28 26.34 24.18 25.07 30.89 29.87 31.94 35.28 34.32 36.77 39.04 ♥ ♦ ♣ ♠ ♥ std. dev. = 0.07 min ♦ std. dev. = 0.06 min ♣ std. dev. = 0.05 min ♠ std. dev. = 0.05 min Inj #1 Inj #2 Inj #3 Inj #4 Inj #5 Inj #6 A. Analysis of in-gel digests of rat brain proteins with 60 min gradient to 60% B B. Analysis of in-solution digests of rat plasma proteins with 60 min gradient to 30% B 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 Time 0 100 % 0 100 % 0 100 % K050218mds61 1: TOF MS ES+ BPI 922 19.66 15.57 17.68 29.98 22.41 26.17 33.87 31.24 36.74 K050218mds62 1: TOF MS ES+ BPI 898 19.54 15.52 17.54 29.86 22.29 26.05 33.87 31.16 36.62 K050218mds63 1: TOF MS ES+ BPI 919 19.57 15.52 17.60 29.93 22.28 26.08 37.28 33.90 31.15 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Time 0 100 % 0 100 % 0 100 % K050505mds76 1: TOF MS ES+ BPI 694 16.09 33.43 19.99 25.52 29.71 68.13 48.79 41.88 36.77 57.85 53.51 64.66 K050505mds77 1: TOF MS ES+ BPI 745 33.28 16.07 20.05 25.44 29.61 68.13 48.84 41.78 37.05 57.73 53.49 64.68 K050505mds78 1: TOF MS ES+ BPI 688 33.20 16.07 25.29 29.47 68.15 49.10 41.80 36.61 58.14 53.82 65.04 MassPREP digest mixture (enolase, phosphorylase b, ADH, BSA), analytical column: 1.7 μm BEH C18 75 μm x 300 mm, 300 nL/min, gradient: 7-30% B for 90 min, trap column: 5 μm Symmetry C18 180 μm x 20 mm, sample trapping: 2% B at 4 μL/min for 3 min, column backpressure ~9,000 psi MassPREP E. coli digest standard, analytical column: 1.7 μm BEH C18 75 μm x 300 mm, 300 nL/min, gradient: 7-30% B for 5 hours, trap column: 5 μm Symmetry C18 180 μm x 20 mm, sample trapping: 2% B at 4 μL/min for 3 min, column backpressure ~9,000 psi A. 90 minute gradient B. 5 hour gradient 4000 3000 2000 1000 0 Counts 90 80 70 60 50 40 30 Retention Time (min) 6000 4000 2000 0 Counts 300 250 200 150 100 50 Retention Time (min)
Transcript of DESIGN FEATURES AND PERFORMANCE OF A DIRECT ......200 fmol enolase digest, analytical column: 1.7...
TO DOWNLOAD A COPY OF THIS POSTER VISIT WWW.WATERS.COM/POSTERS ©2005 Waters Corporation
DESIGN FEATURES AND PERFORMANCE OF A DIRECT-FLOW NANOSCALE HPLC SYSTEM COMBINED WITH COLUMNS
PACKED WITH SUB 2 MICRON PARTICLES
Jeffrey W. Finch, Martha D. Stapels, Keith Fadgen, Alistair Wallace, Hongji Liu, Geoff Gerhardt, James P. Murphy, Steve Ciavarini, Christopher C. Benevides, and John C. Gebler
Waters Corporation, Milford, MA, USA
OVERVIEW
INTRODUCTION
METHODS
CONCLUSIONS
Purpose • To design and demonstrate performance of a rug-
ged, splitless nano– and capillary scale HPLC that yields excellent retention time reproducibility, can be used in trapping or direct-inject mode, and can with-stand high operating pressures to maximize chroma-tographic resolution.
Methods • 75 µm ID nanocolumns packed with 1.7 µm C18
particles • Trapping and direct-inject mode • Mass spectrometry on Q-ToF instruments Results • Minimal loss in resolution with trapping mode • Peptide retention time standard deviations typically
< 0.1 min • Analysis of complex biological digest samples with
minimal carryover • UPLC for nanoscale chromatography (3,000 to
10,000 psi ) Due to higher MS sensitivity, nanoscale chromatography is often the method of choice for analysis of complex proteomic samples. Here we present results from the Waters nanoACQUITY UPLC™ sys-tem, a new nanoscale HPLC platform with the following features:
• Direct flow without splitting • Based on ACQUITY UPLC™ platform • Accurate gradient delivery for excellent retention time repro-
ducibility • Binary high-pressure mixing pump with wide dynamic flow
range • Operation at elevated pressures for nanocolumns packed with
particles < 2 microns • Heating Trapping Module (HTM) with simplified “forward
flush” sample trapping scheme and integrated column heater • Consumables that deliver consistent performance • Nanocolumns packed with ACQUITY 1.7 µm bridged-ethyl
hybrid (BEH) particles that provide higher separation effi-ciency
• Variable flow chromatography (“peak parking”) capability Dedicated auxiliary pump for providing stable flow of “lockmass” solution to reference sprayer of Q-Tof NanoLock Spray source Figure 1. Columns packed with bridged-ethyl hybrid particles
•
Figure 2. nanoACQUITY UPLC system
Components of the nanoACQUITY UPLC system (Figure 2A) are de-signed for operating at pressures up to 10,000 psi. The column heater (Figure 2B) of the HTM is mounted on a pivot which posi-tions the column outlet close to the MS source to minimize delay vol-ume. All fused-silica tubing in the sample path is preassembled with PEEK protective cladding, fittings, and pre-cut polished ends to minimize dead volume. The Sample manager is completely en-closed and can maintain samples at 4 ºC to 40 ºC. The needle and sample loop can be flushed with both a strong and weak sol-vent wash, eliminating sample carryover. The NanoFlow Sprayer (Figure 2C) minimizes postcolumn dead volume in an easy to use design. The wide dynamic flow range of the BSM makes it possible to con-figure the system for sample loading/trapping using the scheme shown in Figure 3. This greatly simplifies system fluidics, allows for robust operation in a “forward flush” mode, and eliminates the need for an extra loading pump.
Lit Code Number 720001350EN
Figure 3. Simplified sample loading/trapping scheme
Figure 4. Direct inject vs. trapping performance
Figure 4 compares LC/MS traces for enolase digest acquired us-ing the same analytical 1.7 µm BEH column for direct inject vs. trap modes. The data demonstrates that there is no significant loss in chromatographic resolution of the peptides with the addition of a trap column. The data also shows that sample losses are mini-mized. Figure 5. Performance with different gradient lengths 50 fmol MassPREP digest mixture (enolase, phosphorylase b, ADH, BSA), analytical column: 1.7 µm BEH C18 75 µm x 150 mm, flow rate: 400 nL/min, gradient: 2-60% B in 30, 60, and 90 min.
Figure 5 shows the effect of different gradient lengths on perform-ance. The column pressure for these runs was approximately 5,200 PSI. Figure 6. LC/MS reproducibility at 300 nL/min, n = 6 MassPREP digest mixture (200 fmol each of enolase, phosphorylase b, hemoglobin, ADH, BSA) analytical column: 1.7 µm BEH C18 75 µm x 100 mm, 300 nL/min, gra-dient: 3-60% B for 60 min trap column: 5 µm Symmetry C18 180 µm x 20 mm, sam-ple trapping: 3% B at 4 µL/min for 3 min column backpressure ~3,300 psi
Figure 6 shows traces from six consecutive injections of a 5 pro-tein digest, along with retention time std. dev. of four peptides. The data set demonstrates that the nanoACQUITY/Q-Tof yields good retention time reproducibility (std. dev. < 0.1 min) and re-producible MS response over an extended period of time. The 1.7 µm BEH column provides greater separation efficiency for peptides compared to columns packed with conventional particles (3 to 5 µm).
Figure 7. Biological samples: in-gel and in-solution digests
Figure 7 shows triplicate injections of in-gel and in-solution digests of biological tissue or fluid samples collected from rats. These chro-matograms were obtained after 48 (Fig. 7A) and 57 (Fig. 7B) prior injections of similar samples on the column and trap. Highly repro-ducible retention times and ion intensities enable calculation of ac-curate peptide ratios with Protein Expression informatics. Figure 8. UPLC on a nanoLC scale (9,000 psi)
Figure 8 shows chromatograms from injections on a 30 cm BEH nanocolumn with system pressures exceeding 9,000 psi. Median peak widths (4σ) measured from SIC’s of peptides throughout the gradient were 23 sec (Fig. 8A) and 54 sec (Fig. 8B), respectively. Features and advantages of the nanoACQUITY UPLC include: • Direct nanoflow delivery without splitting • Highly reproducible retention times over extended
analysis periods • Binary high pressure pump with wide dynamic flow
range -supports analytical column flow rates from 100 to
5000 nL/min -simplified sample loading/trapping configuration • High pressure capabilities enabling the use of nano-
columns packed with particles < 2 µm and lengths up to 300 mm
• Columns packed with 1.7 µm bridged-ethyl hybrid particles-higher efficiency, higher resolution, greater selectivity
• Reproducible ion signals for quantitative proteomics
A very fine human hair, ~ 60 µm
1.7 µm BEH particles (~ 35 can fit across hair)
10 µm
bridged-ethanes in silica matrix
• Smaller, uniform particles=higher packing densities, higher efficiency
• Stronger particles that can withstand high pressure
• Chemically resistant (wide pH range)
Solvent tray
Heating and trapping module (HTM)
Sample manager
Binary solvent manager (BSM)
Auxiliary solvent manager
nano-Tee w/ magnetic mount
to MS source
trap outlet tubing
to HTM valve
analytical column
B. Column heater detail
inner compartment (swiveled open)
A. System stack and modules
C. Universal NanoFlow Sprayer
RESULTS AND DISCUSSION
19.00 19.20Time0
100
%
0.07min
20.80 21.00Time0
100
%
0.08min
Time10.00 15.00 20.00 25.00 30.00 35.00 40.00
%
0
100
%
0
100J091604HL_023 TOF MS ES+
TIC1.29e5
J091604HL_002 TOF MS ES+ TIC
1.29e5
A. Direct Inject Mode
B. Trap Mode
Trap column: 5 µm Symmetry C18 180 µm x 20 mm. Sample trapping: 3% B at 5 µL/min for 3 min (from BSM)
200 fmol enolase digest, analytical column: 1.7 µm BEH C18 75 µm x 100 mm, flow rate: 250 nL/min, gradient: 3-60% B in 30 min
10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00Time0
100
%
0
100
%
0
100
%
K050511jwf07 1: TOF MS ES+ BPI
1.38e420.07
17.4614.75
21.69
K050512jwf14 1: TOF MS ES+ BPI
6.28e330.25
19.40
15.5225.68
23.06
33.05
K050513jwf102 1: TOF MS ES+ BPI
7.40e339.77
23.94
16.08 22.09
19.85
33.0531.6725.54
36.03
42.45
45.31
30 min gradient
60 min gradient
90 min gradient
Time5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
%
0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
0
100E041124mds23 TOF MS ES+
BPI1.01e3
28.4323.0518.93
17.9917.2022.2320.44
26.4624.3225.19
31.0030.25
32.0535.4134.44
36.95 39.22
E041124mds24 TOF MS ES+ BPI
1.01e328.3422.94
18.82
17.8717.1122.1320.33
26.3624.23 30.9130.1631.96
35.3334.3536.84 39.13
E041124mds25 TOF MS ES+ BPI
1.01e328.35
22.9318.81
17.8617.0822.1020.30
26.3624.2027.18
30.9230.1731.97 35.3434.34
36.83 39.08
E041124mds26 TOF MS ES+ BPI
1.01e328.31
22.8918.75
17.8117.03
22.0420.25
26.3224.17 30.8930.13
31.9635.3034.32
36.84 39.08
E041124mds27 TOF MS ES+ BPI
1.01e328.27
22.8718.75
17.9417.04
22.0420.26
26.3024.1525.03
30.8529.84 31.89
35.2334.2536.79 39.04
E041124mds28 TOF MS ES+ BPI
1.01e328.31
22.8918.77
17.9617.0622.0620.28
26.3424.1825.07 30.8929.87
31.9435.2834.32
36.77 39.04
♥ ♦ ♣ ♠
♥ std. dev. = 0.07 min ♦ std. dev. = 0.06 min ♣ std. dev. = 0.05 min ♠ std. dev. = 0.05 min
Inj #1
Inj #2
Inj #3
Inj #4
Inj #5
Inj #6
A. Analysis of in-gel digests of rat brain proteins with 60 min gradient to 60% B
B. Analysis of in-solution digests of rat plasma proteins with 60 min gradient to 30% B
10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00Time0
100%
0
100%
0
100%
K050218mds61 1: TOF MS ES+ BPI
92219.6615.57 17.68
29.9822.41 26.17
33.8731.24 36.74
K050218mds62 1: TOF MS ES+ BPI
89819.5415.5217.54
29.8622.29 26.0533.8731.16 36.62
K050218mds63 1: TOF MS ES+ BPI
91919.5715.52 17.60
29.9322.28 26.08 37.2833.9031.15
10.00 20.00 30.00 40.00 50.00 60.00 70.00Time0
100%
0
100%
0
100%
K050505mds76 1: TOF MS ES+ BPI
69416.09 33.4319.99 25.52 29.71
68.1348.7941.8836.77 57.8553.51 64.66
K050505mds77 1: TOF MS ES+ BPI
74533.2816.07
20.05 25.44 29.6168.1348.8441.7837.05 57.7353.49 64.68
K050505mds78 1: TOF MS ES+ BPI
68833.2016.07
25.29 29.4768.1549.1041.8036.61 58.1453.82 65.04
MassPREP digest mixture (enolase, phosphorylase b, ADH, BSA), analytical column: 1.7 µm BEH C18 75 µm x 300 mm, 300 nL/min, gradient: 7-30% B for 90 min, trap column: 5 µm Symmetry C18 180 µm x 20 mm, sample trapping: 2% B at 4 µL/min for 3 min, column backpressure ~9,000 psi
MassPREP E. coli digest standard, analytical column: 1.7 µm BEH C18 75 µm x 300 mm, 300 nL/min, gradient: 7-30% B for 5 hours, trap column: 5 µm Symmetry C18 180 µm x 20 mm, sample trapping: 2% B at 4 µL/min for 3 min, column backpressure ~9,000 psi
A. 90 minute gradient
B. 5 hour gradient
4000
3000
2000
1000
0
Cou
nts
90807060504030Retention Time (min)
6000
4000
2000
0
Cou
nts
30025020015010050Retention Time (min)
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