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Dionex UltiMate 3000 Applications

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UltiMate 3000 RSLCnano
Standard Applications
Revision:
Date:
Doc.no.
©
2012 Thermo Fisher Scientific Inc.
V1.0 R3
January 2012
4820.4103

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  • Page 1 UltiMate 3000 RSLCnano Standard Applications Revision: V1.0 R3 Date: January 2012 Doc.no. 4820.4103 © 2012 Thermo Fisher Scientific Inc.
  • Page 2 UltiMate 3000 RSLCnano – Standard Applications...

  • Page 3: Table Of Contents

    2.1.1 Connections ......................6 2.1.2 Sample Preparation ..................... 7 2.1.3 Mobile Phases ..................... 8 Installing the UltiMate 3000 RSLCnano system ............9 Application Overview ..................... 12 Direct Injection onto a Nano Column ..............13 2.4.1 Hardware Layout ....................13 2.4.2 Fluidic Setup .....................
  • Page 4 UltiMate 3000 RSLCnano – Standard Applications 2.11.3 Installation tips ....................44 2.11.4 Testing the application ..................44 2.12 Tandem nano LC ..................... 46 2.12.1 Hardware Layout ....................46 2.12.2 Fluidic Setup ..................... 47 2.12.3 Installation Tips ....................48 2.12.4 Testing the Application ..................48 FAQ ...........................

  • Page 5: Introduction

    UltiMate 3000 RSLCnano – Standard Applications 1 Introduction This document describes the standard setup, recommended experimental conditions and testing procedures to run standard applications on the Thermo Scientific Dionex UltiMate 3000 RSLCnano system. Notes: This document is intended for Thermo Fisher Scientific (or authorized) service personnel as well as customers to assist in the installation and system test of UltiMate...

  • Page 6: Additional Documents

    In addition to the information provided in this manual, the following documents are available: • Operating Instructions of the individual modules • Installation Qualification manual for installation of the UltiMate 3000 RSLCnano • Micro Fraction Collection Option for the Thermo Scientific Dionex UltiMate WPS- 3000PL Nano/Cap Autosampler •...

  • Page 7: Application Setup

    2.1.1 Connections All high-pressure fittings used in the applications on the UltiMate 3000 RSLCnano system are made with nanoViper. nanoViper is a fingertight high-pressure fitting that is dead volume free by design and back pressure resistant up to 1000 bar. The fittings are assembled in the factory to ensure consistent fittings and prevent experimental failure due to bad connections.

  • Page 8: Sample Preparation

    UltiMate 3000 RSLCnano – Standard Applications Nano connector sleeve (~1.1 cm) PTFE (1.8 cm) Figure 3: nano connector (top) and PTFE (bottom) sleeve comparison 1. Slide the golden nut and silver union onto one of the fused silica outlets; slide the second golden union on the other fused silica end.

  • Page 9: Mobile Phases

    UltiMate 3000 RSLCnano – Standard Applications 2.1.3 Mobile Phases • Always use fresh solvents. • When running online multidimensional approaches, make sure that the mobile phase of the first dimension will allow for trapping the sample on the (trap) column used on the second dimension.

  • Page 10: Installing The Ultimate 3000 Rslcnano System

    UltiMate 3000 RSLCnano – Standard Applications Installing the UltiMate 3000 RSLCnano System The general UltiMate 3000 RSLCnano system overview is shown in Figure 5. The modular nature of the Thermo Scientific Dionex UltiMate 3000 platform does allow modifications to this layout.
  • Page 11 UltiMate 3000 RSLCnano – Standard Applications NC_Pump Loading pump Rear seal wash system Flow meter Purge screws Inline filter Purge screw Tubing guides Snap-in valves Column compartment Figure 6: Detailed overview of the NCS-3500RS interior Figure 7: Detailed overview of the NCP-3200RS interior, components are identical to the NC_Pump in the NCS-...
  • Page 12 UltiMate 3000 RSLCnano – Standard Applications Before the parts of the application kit are installed, the UltiMate 3000 RSLCnano system has to be prepared and primed. To prepare the system, the following steps are required. For more details, see the operating instructions for the respective module.

  • Page 13: Application Overview

    UltiMate 3000 RSLCnano – Standard Applications Application Overview Table 1: Property overview of different flow selectors Flow Selector I.D. Total Flow Rate Type (Sum of channel A and B) Nominal Minimum Maximum Nano 6041.0002 500 nL/min 50 nL/min* 1000 nL/min Capillary 6041.0003...

  • Page 14: Direct Injection Onto A Nano Column

    UltiMate 3000 RSLCnano – Standard Applications Direct Injection onto a Nano Column 2.4.1 Hardware Layout The preferred setup is presented in Figure 8 and consists of: SR-3000 5035.9200 NCP-3200RS 5041.0030 VWD-3400RS 5074.0010 3 nL flow cell 6074.0270 WPS-3000TPL RS 5826.0020 Application kit: 6720.0300...
  • Page 15 UltiMate 3000 RSLCnano – Standard Applications The alternative setup is presented in Figure 9 and consists of: SRD-3400 5035.9245 NCS-3500RS 5041.0010 VWD-3400RS 5074.0010 3 nL flow cell 6074.0270 WPS-3000TPL RS 5826.0020 Application kit: 6720.0300 Figure 9: Setup for a Direct Injection experiment onto a...

  • Page 16: Fluidic Setup

    (P/N 6040.2304) Figure 10: Fluidic connections for a Direct Injection experiment onto a nano column Table 2: UltiMate 3000 RSLCnano Direct Injection nano LC kit (P/N 6720.0300) contents Item Replacement P/N 75 µm I.D. x 15 cm, packed with Acclaim PepMap RSLC C18, 2 µm, 164534 100Å, nanoViper...

  • Page 17: Testing The Application

    UltiMate 3000 RSLCnano – Standard Applications 2.4.4 Testing the Application The direct injection can be tested using the following conditions: Property Setting Mobile phase A 100% water + 0.05% TFA Mobile phase B 20%/80% (v/v) water/ACN + 0.04% TFA Sample Cytochrome C digest 1 pmol/μL, prepared according to the instruction sheet...
  • Page 18 UltiMate 3000 RSLCnano – Standard Applications The advantage of such a setup is the ease of use and a minimum loss of peptides, especially hydrophilic ones. The prerequisites of this setup are desalted samples, since all that is injected will enter the MS, and an investment of extra analysis time to accommodate the loading of...

  • Page 19: Direct Injection Onto A Capillary Column

    UltiMate 3000 RSLCnano – Standard Applications Direct Injection onto a Capillary Column 2.5.1 Hardware Layout The recommended setup is presented in Figure 12 and consists SRD-3400 5035.9245 NCS-3500RS 5041.0020 VWD-3400RS 5074.0010 45 nL flow cell 6074.0280 WPS-3000TPL RS 5826.0020 Application kit: 6720.0305...

  • Page 20: Fluidic Setup

    (P/N 6040.2304) Figure 13: Fluidic connections for a Direct Injection experiment onto a capillary column Table 3: UltiMate 3000 RSLCnano Direct Injection capillary LC Kit (P/N 6720.0305) contents Item Replacement P/N 300 µm I.D. x 15 cm, packed with Acclaim PepMap RSLC C18, 2 µm, 164537 100Å, nanoViper...

  • Page 21: Installation Tips

    UltiMate 3000 RSLCnano – Standard Applications 2.5.3 Installation Tips Follow the General Recommendations for Applications (→ page 6). 2.5.4 Testing the Application The direct injection can be tested using the following conditions: Property Setting Mobile phase A 100% water + 0.05% TFA Mobile phase B 20%/80% (v/v) water/ACN + 0.04% TFA...

  • Page 22: Pre-Concentration Onto A Nano Column

    UltiMate 3000 RSLCnano – Standard Applications Pre-concentration onto a Nano Column 2.6.1 Hardware Layout The recommended setup is presented in Figure 15 and consists SRD-3400 5035.9245 NCS-3500RS 5041.0010 10-port sw. valve 6041.0001 VWD-3400RS 5074.0010 3 nL flow cell 6074.0270 WPS-3000TPL RS 5826.0020...

  • Page 23: Fluidic Setup

    Chromeleon templates if necessary. Figure 16: Fluidic connections for a Pre-concentration experiment onto a nano column Table 4: UltiMate 3000 RSLCnano Pre-concentration nano LC kit (P/N 6720.0310) contents Item Replacement P/N 75 µm I.D.

  • Page 24: Installation Tips

    UltiMate 3000 RSLCnano – Standard Applications 2.6.3 Installation Tips • Follow the General Recommendations for Applications (→ page 6). • The design of the nano trap column provides the easiest connections, but must only be used in forward flush operation. Please check the indicated flow direction when installing a nano trap column.
  • Page 25 UltiMate 3000 RSLCnano – Standard Applications To evaluate the result of the experiment, the chromatogram should look very similar to the one obtained with direct injection. For details on interpretation and troubleshooting of the Cytochrome C nano LC separation, see the FAQ section on page 50.

  • Page 26: Pre-Concentration Onto A 200 Μm Monolithic Column

    UltiMate 3000 RSLCnano – Standard Applications Pre-concentration onto a 200 µm Monolithic Column 2.7.1 Hardware Layout The recommended setup is presented in Figure 18 and consists SRD-3400 5035.9245 NCS-3500RS 5041.0020 10-port sw. valve 6041.0001 VWD-3400RS 5074.0010 3 nL flow cell 6074.0270...

  • Page 27: Fluidic Setup

    Chromeleon templates if necessary. Figure 19: Fluidic connections for a Pre-concentration experiment onto a monolithic column Table 5: UltiMate 3000 RSLCnano Pre-concentration monolithic LC kit (P/N 6720.0320) contents Item Replacement P/N PepSwift Monolithic Capillary Column, 200 µm I.D.

  • Page 28: Installation Tips

    UltiMate 3000 RSLCnano – Standard Applications 2.7.3 Installation Tips • Follow the General Recommendations for Applications (→ page 6). • Using a stainless steel loop (on a standard system) instead of a PEEK loop is recommended in case of injecting highly diluted samples.
  • Page 29 UltiMate 3000 RSLCnano – Standard Applications Figure 20: Typical chromatogram for Pre-concentration of 0.5 pmol Cyt C onto a monolithic column Tip: When the trap column is switched in line with the analytical column, a large positive peak is detected at 214 nm. This is due to the different UV absorbance of...

  • Page 30: Pre-Concentration On A Capillary Column

    UltiMate 3000 RSLCnano – Standard Applications Pre-concentration on a Capillary Column 2.8.1 Hardware Layout The recommended setup is presented in Figure 21 and consists SRD-3400 5035.9245 NCS-3500RS 5041.0020 10-port sw. valve 6041.0001 VWD-3400RS 5074.0010 45 nL flow cell 6074.0280 WPS-3000TPL RS 5826.0020...

  • Page 31: Fluidic Setup

    Chromeleon templates if necessary. Figure 22: Fluidic connections for a Pre-concentration experiment onto a capillary column Table 6: UltiMate 3000 RSLCnano Pre-concentration capillary LC kit (P/N 6720.0315) contents Item Replacement P/N 300 µm I.D.

  • Page 32: Installation Tips

    UltiMate 3000 RSLCnano – Standard Applications 2.8.3 Installation Tips • Follow the General Recommendations for Applications (→ page 6). • The design of the nano trap column provides the easiest connections, but must only be used in forward flush operation. Please check the indicated flow direction when installing a nano trap column.

  • Page 33: Salt Steps With Nano Column

    UltiMate 3000 RSLCnano – Standard Applications 2D Salt Steps with Nano Column 2.9.1 Hardware Layout The recommended setup is presented in Figure 23 and consists SRD-3400 5035.9245 NCS-3500RS 5041.0010 2x 10-port sw.valve 6041.0001 VWD-3400RS 5074.0010 3 nL flow cell 6074.0270 WPS-3000TPL RS 5826.0020...

  • Page 34: Fluidic Setup

    Chromeleon templates if necessary. Figure 24: Fluidic connections for a 2D LC Salt Plugs experiment Table 7: UltiMate 3000 RSLCnano 2D salt plugs kit (P/N 6720.0325) contents Item Replacement P/N 300 µm I.D. x 10 cm, packed with Poros 10 S with connections, 130 µm I.D.

  • Page 35: Installation Tips

    UltiMate 3000 RSLCnano – Standard Applications 2.9.3 Installation Tips • Follow the General Recommendations for Applications (→ page 6). • The design of the nano trap column provides the easiest connections, but must only be used in forward flush operation. Please check the indicated flow direction when installing a nano trap column.

  • Page 36: Testing The Application

    UltiMate 3000 RSLCnano – Standard Applications 2.9.4 Testing the Application The 2D salt plug setup can be tested using the following conditions: Property Setting Mobile phase A 100% water + 0.05% TFA Mobile phase B 20%/80% (v/v) water/ACN + 0.04% TFA Loading solvent 95%/5% (v/v) water/ACN + 0.025% TFA...

  • Page 37: Salt Solutions Preparation

    UltiMate 3000 RSLCnano – Standard Applications 2.9.5 Salt Solutions Preparation The following protocol can be used to prepare the salt plugs: • Prepare two stock solutions using the loading solvent: 1) 2000 mM NaCl (for example, 467.5 mg of NaCl in 4 ml of loading solvent)

  • Page 38: Automated Off-Line 2D Lc Of Peptides, Micro Scx X Nano Rp

    UltiMate 3000 RSLCnano – Standard Applications 2.10 Automated off-line 2D LC of Peptides, micro SCX x nano RP 2.10.1 Hardware Layout The recommended setup with one detector is presented in Figure 25, when using two detectors a dual stack, as shown in Figure 26, is recommended.
  • Page 39 UltiMate 3000 RSLCnano – Standard Applications Figure 26: Setup for an Automated Off-line 2D Mic Nan experiment using two detectors The recommended setup using 2 detectors is Tip: The µFC option limits presented in Figure 26 and consists of: the upper pressure of the SRD-3400 5035.9245...

  • Page 40: Fluidic Setup

    Chromeleon templates if necessary. Figure 27: Fluidic connections for an Automated Off-line 2D experiment Table 8: UltiMate 3000 RSLCnano Automated Off-line SCX-RP peptides kit (P/N 6720.0330) contents Item Replacement P/N 1.0 mm I.D. x 15 cm, packed with Polysulfoethyl ASP, 5 µm, 300Å, nanoViper 164566 75 µm I.D.

  • Page 41: Installation Tips

    UltiMate 3000 RSLCnano – Standard Applications Table 8: UltiMate 3000 RSLCnano Automated Off-line SCX-RP peptides kit (P/N 6720.0330) contents - Continued Item Replacement P/N nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 750 mm 6041.5280 nanoViper sample loop 50 µL, FS/PEEK sheathed I.D. x L 250 µm x 408 mm 6826.2450...

  • Page 42: Testing The Application

    UltiMate 3000 RSLCnano – Standard Applications 2.10.4 Testing the Application The Mic Nan SCX – RP application setup can be tested using the following conditions: Property Setting Mobile phase A 100% water + 0.05% TFA Mobile phase B 20%/80% (v/v) water/ACN + 0.04% TFA...

  • Page 43: Automated Off-Line 2D Lc, Cap Rp (Basic) X Nan Rp (Acidic)

    UltiMate 3000 RSLCnano – Standard Applications 2.11 Automated off-line 2D LC, Cap RP (basic) x nan RP (acidic) 2.11.1 Hardware Layout Figure 28: Setup for an Automated Off-line 2D Cap Nan experiment The recommend setup is presented in Tip: The µFC option limits...

  • Page 44: Fluidic Setup

    Chromeleon templates if necessary. Figure 29: Fluidic connections for an Automated Off-line 2D experiment Table 9: UltiMate 3000 RSLCnano Automated Off-line RP-RP peptides kit (P/N 6720.340) contents Item Replacement P/N 75 µm I.D. x 15 cm, packed with Acclaim PepMap RSLC C18, 2 µm, 100Å,...

  • Page 45: Installation Tips

    UltiMate 3000 RSLCnano – Standard Applications Table 9: UltiMate 3000 RSLCnano Automated Off-line RP-RP peptides kit (P/N 6720.340) contents - Continued Item Replacement P/N PTFE tubing, 500 µm I.D. 100 cm, used as waste tubing 6720.0077 1/16" Universal Fingertight Fitting, one-piece design, extra long thread, 4 pcs.
  • Page 46 UltiMate 3000 RSLCnano – Standard Applications Property Setting Sample Protein mix digest, prepared according to the included instruction sheet Injection volume Sample: 10 μL Fractions: 20 μL Gradient RP basic 4-60% B in 15 min, 90% B for 5 min,...

  • Page 47: Tandem Nano Lc

    UltiMate 3000 RSLCnano – Standard Applications 2.12 Tandem nano LC 2.12.1 Hardware Layout Figure 30: Setup for a Tandem nano LC experiment Tip: The components to The recommended setup is presented in Figure 30 convert the WPS- and consists of: 3000FC are included in the application kit.

  • Page 48: Fluidic Setup

    (P/N 6825.0020). All necessary parts are included in the kit. For further information, refer to the operating instructions for the module. Table 10: UltiMate 3000 RSLCnano Tandem nano LC kit (P/N 6720.0335) contents Item Replacement P/N 75 µm I.D.

  • Page 49: Installation Tips

    UltiMate 3000 RSLCnano – Standard Applications Table 10: UltiMate 3000 RSLCnano Tandem nano LC kit (P/N 6720.0335) contents - Continued Item Replacement P/N Cleaving stone 160483 Upgrade kit nano/cap WPS-3000TFC 6824.0030 1/32” 2 pos 6 port nano switching valve 6825.0020 Fittings for nano valve 6720.0080...
  • Page 50 UltiMate 3000 RSLCnano – Standard Applications Property Setting Gradient RP 4% to 55% B in 30 min 90% B for 5 min 15 min equilibration WPS temperature 5°C Loading flow 5 μL/min Flow rate 300 nL/min (nano flow selector)

  • Page 51: Faq

    UltiMate 3000 RSLCnano – Standard Applications 3 FAQ Interpreting a Chromatogram A typical Cytochrome C separation is shown in Figure 32. The different areas of a chromatographic separation are marked inside the figure. Figure 32: Example Cytochrome C separation with different parts of the run identified.
  • Page 52 UltiMate 3000 RSLCnano – Standard Applications...

  • Page 53: The Use Of Tfa And Fa

    UltiMate 3000 RSLCnano – Standard Applications The use of TFA and FA The separation of peptides by reversed phase is typically done in the presence of an ion pairing agent. The typical ion pairing agents serve a double function. First, these weak acids bring the pH of the solvents down to pH 2-3, causing almost all peptides to have an overall positive charge.

  • Page 54: Unstable Baseline

    3.4.2 Unstable Baseline Unstable baselines can have various causes. The UltiMate 3000 RSLCnano pumps are designed to provide the best gradient precision, but solvent miscibility can present a problem. Therefore, Thermo Fisher Scientific recommends using a minimum of 5% water in the organic mobile phase.

  • Page 55: Appendix - Traditional Capillary Connections

    UltiMate 3000 RSLCnano – Standard Applications 4 Appendix – Traditional Capillary Connections • When installing fused silica tubing (for example, the capillary column): Insert the PEEK sleeve with the stainless steel nut (standard) or PEEK nut (biocompatible system) and the appropriate ferrule in a port (or the preassembly tool, when working on a biocompatible system!).
  • Page 56 UltiMate 3000 RSLCnano – Standard Applications • When using a micro tight union, always use the gauge plug provided with the union to make sure that the connection is well centered. Gauge Plug     M icrot ight...

  • Page 57: Appendix - Common Spare Parts In Nano Lc

    5 Appendix – Common Spare Parts in nano LC The three tables below list the most common parts to be used with the UltiMate 3000 RSLCnano system. They are divided by columns (Table 12), tubing (Table 13, Table 14), and hardware (Table 15).
  • Page 58 UltiMate 3000 RSLCnano – Standard Applications Table 13: Matrix for connection tubing for the UltiMate 3000 RSLCnano system [Color code] Length (mm) 20 µm 50 µm 75 µm 100 µm 150 µm [Orange] [Brown] [Black] [Red] [Purple] 6041.5120 6041.5123 6041.5126 6041.5810...
  • Page 59 UltiMate 3000 RSLCnano – Standard Applications Table 15: List of hardware components for the UltiMate 3000 RSLCnano system Item Low dispersion 2 pos 10 port valve high pressure for NCS-3x00 6041.0001 Low dispersion 2 pos 6 port valve high pressure for NCS-3x00 6041.0004...

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