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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, Hongji Liu, Geoff Gerhardt, James P. Murphy, Steve Ciavarini, Christopher C. Benevides, and John C. Gebler Waters Corporation, Milford, MA, USA

OVERVIEW

INTRODUCTION

METHODS RESULTS AND DISCUSSION

CONCLUSIONS

Purpose: To design and demonstrate performance of a rugged, splitless nano– and capillary scale HPLC that yields excellent retention time reproduci- bility, can be used in trapping or direct-inject mode, and can withstand high operating pressures to maximize chromatographic 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 )

ASMS 2005

Due to higher MS sensitivity, nanoscale chromatography is often the method of choice for analysis of complex proteomic samples. Here we pre-sent results from the Waters nanoACQUITY UPLC™ system, a new nano-scale HPLC platform with the following features:

• Direct flow without splitting • Based on ACQUITY UPLC™ platform • Accurate gradient delivery for excellent retention time reproducibility • 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 efficiency

• Variable flow chromatography (“peak parking”) capability • Dedicated auxiliary pump for providing stable flow of “lockmass” so-lution to reference sprayer of Q-Tof NanoLockSpray source

Figure 1. Columns packed with bridged-ethyl hybrid particles

A very fine human hair, ~ 60 µm

5 µm particles (~12 can fit across hair)

1.7 µm BEH particles (~ 35 can fit across hair)

bridged-ethanes in silica matrix

10 µm

Solvent tray

Heating and trapping module (HTM)

Sample manager

Binary solvent manager (BSM)

Auxiliary solvent manager (ASM)

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

Components of the nanoACQUITY UPLC system (Figure 2A) are designed for operating at pressures up to 10,000 psi. The column heater (Figure 2B) of the HTM is mounted on a pivot which positions the column outlet close to the MS source to minimize delay volume. All fused-silica tubing in the sample path is preassembled with PEEK protective cladding, fittings, and pre-cut pol-ished 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 solvent wash, eliminating sample carryover. The NanoFlow Sprayer (Figure 2C) minimizes postcolumn dead volume in an easy to use design.

Figure 3. Simplified sample loading/trapping scheme

4. Sample Loop injected 5. Sample loads onto trap

1. Open HTM valve 2. Ramp BSM to trapping flow rate 3. Load sample in loop

6. Reduce BSM flow rate 7. Close HTM valve 8. Take loop offline 9. Gradient flows through trap and analytical column

6

A. B.

C. D.

The wide dynamic flow range of the BSM makes it possible to configure 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.

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 nanocolumns 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

Figure 4. Direct inject vs. trapping performance

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

Figure 4 compares LC/MS traces for enolase digest acquired using the same analytical 1.7 µm BEH column for direct inject vs. trap modes. The data dem-onstrates 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 minimized.

19.00 19.20Time0

100

%

0.07min

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%

0.08min

Time10.00 15.00 20.00 25.00 30.00 35.00 40.00

%

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100

%

0

100J091604HL_023 TOF MS ES+

TIC1.29e5

J091604HL_002 TOF MS ES+ TIC

1.29e5

A. Direct Inject Mode

B. Trap Mode

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, gradient: 3-60% B for 60 min trap column: 5 µm Symmetry C18 180 µm x 20 mm, sample trapping: 3% B at 4 µL/min for 3 min column backpressure ~3,300 psi

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

100

E041124mds23 TOF MS ES+ BPI

1.01e3

28.4323.0518.93

17.9917.20

22.2320.4426.4624.32

25.1931.00

30.2532.05

35.4134.4436.95 39.22

E041124mds24 TOF MS ES+ BPI

1.01e3

28.3422.94

18.82

17.8717.11

22.1320.3326.3624.23 30.91

30.16

31.9635.3334.35

36.84 39.13

E041124mds25 TOF MS ES+ BPI

1.01e3

28.3522.9318.81

17.8617.08

22.1020.3026.3624.20

27.1830.92

30.17

31.9735.3434.34

36.83 39.08

E041124mds26 TOF MS ES+ BPI

1.01e3

28.31

22.8918.75

17.8117.03

22.0420.25

26.3224.17 30.89

30.1331.96

35.3034.3236.84 39.08

E041124mds27 TOF MS ES+ BPI

1.01e3

28.2722.8718.75

17.9417.04

22.0420.26

26.3024.15

25.0330.85

29.8431.89

35.2334.2536.79 39.04

E041124mds28 TOF MS ES+ BPI

1.01e3

28.3122.89

18.77

17.9617.06

22.0620.2826.3424.18

25.0730.89

29.8731.94

35.2834.3236.77 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

Figure 6 shows traces from six consecutive injections of a 5 protein 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 reproducible MS response over an extended period of time. The 1.7 µm BEH column provides greater separation efficiency for pep-tides compared to columns packed with conventional particles (3 to 5 µm).

Figure 2. nanoACQUITY UPLC system

Figure 5. Performance with different gradient lengths

• Smaller, uniform particles=higher pack-ing densities, higher efficiency

• Stronger particles that can withstand high pressure

• Chemically resistant (wide pH range)

C. Universal NanoFlow Sprayer

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

30 min gradient

60 min gradient

90 min gradient

Figure 5 shows the effect of different gradient lengths on performance. The column pressure for these runs was approximately 5,200 PSI.

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 chromatograms were obtained

A. Analysis of in-gel digests of rat brain proteins with 60 min gradient to 60% B

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%

K050511jwf07 1: TOF MS ES+

BPI

1.38e4

20.07

17.4614.75

21.69

K050512jwf14 1: TOF MS ES+

BPI

6.28e3

30.2519.40

15.52

25.6823.06

33.05

K050513jwf102 1: TOF MS ES+

BPI

7.40e3

39.77

23.94

16.08 22.09

19.85

33.05

31.6725.54

36.03

42.45

45.31

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

Figure 8. UPLC on a nanoLC scale (9,000 psi)

after 48 (Fig. 7A) and 57 (Fig. 7B) prior injections of similar samples on the column and trap. Highly reproducible retention times and ion intensi-ties enable calculation of accurate peptide ratios with Protein Expression informatics.

B. Analysis of in-solution digests of rat plasma proteins with 60 min gradient to 30% B

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100

%

0

100

%

K050218mds61 1: TOF MS ES+

BPI

922

19.6615.5717.68

29.9822.41 26.17

33.8731.24 36.74

K050218mds62 1: TOF MS ES+

BPI

898

19.5415.5217.54

29.8622.29 26.05

33.8731.16 36.62

K050218mds63 1: TOF MS ES+

BPI

919

19.5715.52 17.60

29.9322.28 26.08

37.2833.9031.15

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K050505mds76 1: TOF MS ES+

BPI

694

16.09 33.4319.99 25.52 29.71

68.1348.7941.8836.77 57.8553.51 64.66

K050505mds77 1: TOF MS ES+

BPI

745

33.2816.0720.05 25.44 29.61

68.1348.8441.7837.05 57.7353.49 64.68

K050505mds78 1: TOF MS ES+

BPI

688

33.2016.0725.29

29.4768.1549.1041.80

36.61 58.1453.82 65.04

Figure 8 shows chromatograms from injections on a 30 cm BEH nanocolumn with system pressures exceeding 9,000 psi. Median peak widths (4σ) meas-ured from SIC’s of peptides throughout the gradient were 23 sec (Fig. 8A) and 54 sec (Fig. 8B), respectively.

A. 90 minute gradient

B. 5 hour gradient

4000

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Counts

90807060504030

Retention Time (min)

6000

4000

2000

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Counts

30025020015010050

Retention Time (min)

trap column: 5 µm Symmetry C18 180 µm x 20 mm sample trapping: 3% B at 5 µL/min for 3 min (from BSM)