Thermo Scientific UltiMate 3000 Standard Applications Manual
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Thermo Scientific UltiMate 3000 Standard Applications Manual

Rslcnano
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thermoscientific
UltiMate™3000
RSLCnano
Standard Applications Guide
Revision:
3.1
Date:
April 2019
Document No.:
4820.4103
Copyright © 2019 Thermo Fisher Scientific Inc. All rights reserved.

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Summary of Contents for Thermo Scientific UltiMate 3000

  • Page 1 thermoscientific UltiMate™3000 RSLCnano Standard Applications Guide Revision: Date: April 2019 Document No.: 4820.4103 Copyright © 2019 Thermo Fisher Scientific Inc. All rights reserved.
  • Page 2 Trademarks PEEK is a trademark of Victrex PLC. Valco is a trademark of Valco Instruments Co. All other trademarks are property of Thermo Fisher Scientific and its subsidiaries. Thermo Fisher Scientific Inc. provides this document to its customers with a product purchase to use in the product operation.
  • Page 3 Contact Us on http://www.thermofisher.com. Technical Assistance For technical support for HPLC products, contact your local Thermo Fisher Scientific support organization. For contact information, go to Contact Us on http://www.thermofisher.com. UltiMate 3000 RSLCnano Standard Applications Guide Page 3...
  • Page 4 Contacting Us Page 4 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 5: Table Of Contents

    Making Connections using the Nano Connector ........16 2.1.3 Installing and Configuring the Application Fluidics ........17 2.1.4 Interfacing the UltiMate 3000 RSLCnano with the Nanospray Flex™ Ion Source 17 2.1.5 Sample Preparation for Reversed Phase LC Separation ......18 2.1.6...
  • Page 6 2.12 Tandem Nano LC ....................71 2.12.1 Hardware Layout ..................71 2.12.2 Fluidic Setup .................... 72 2.12.3 Installation Tips ..................73 2.12.4 Testing the Application ................74 2.12.5 Tandem NanoLC-MS ................75 Page 6 UltiMate 3000 RSLCnano Standard Applications Guide...
  • Page 7 Common Application Related Consumables ............97 4.2.1 Columns ....................97 4.2.2 nanoViper Capillaries, Sample Loops and Connectors......99 UltiMate 3000 RSLCnano convenience bundles, list of contents ....... 101 Hardware Accessories ..................102 UltiMate 3000 RSLCnano Standard Applications Guide Page 7...
  • Page 8 Contents Page 8 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 9: Using This Manual

    Using this Manual 1 Using this Manual This chapter provides information about this manual, the conventions used throughout the manual, and the reference documentation that is available in addition to this manual. UltiMate 3000 RSLCnano Standard Applications Guide Page 9...

  • Page 10: About This Manual

    UV radiation and exposure to pressurized solvents. This manual contains important information about the correct care and use of the UltiMate 3000 RSLCnano. Please read this manual carefully before installing or running any of the applications described. Keep this manual close to the UltiMate 3000 RSLCnano for future reference and pass it on to any subsequent user.

  • Page 11: Conventions

    NOTICE Highlights information necessary to prevent damage to the instrument or invalid test results. TIP Highlights information of general interest or helpful information that can make a task easier or optimize the performance of the instrument UltiMate 3000 RSLCnano Standard Applications Guide Page 11...

  • Page 12: Typographical Conventions

    These include:  Table of contents entries  Index entries  Cross-references (in blue text), for example, to sections, figures or online reference materials Page 12 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 13: Reference Documentation

     Using this Manual Reference Documentation Further information relating to the UltiMate 3000 RSLCnano systems and associated applications is available as follows:  The UltiMate 3000 RSLCnano system webpage: https://www.thermofisher.com/order/catalog/product/ULTIM3000 RSLCNANO  Module operating instructions NCS-3500RS / NCP-3200RS pump modules...

  • Page 14: Application Setup

     Application Setup 2 Application Setup This chapter provides details on each of the application kits available for the UltiMate 3000 RSLCnano system. Page 14 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 15: General Recommendations For Applications

    1) is a fingertight high-pressure fitting that is virtually dead volume free by design and rated for backpressures up to 1200 bar. All high-pressure fittings used in the applications on the UltiMate 3000 RSLCnano system use nanoViper. The fittings are factory assembled to ensure quality and prevent experimental failure due to bad connections.

  • Page 16: Making Connections Using The Nano Connector

    Slide the other end of the fused silica into the connector sleeve. Make sure that the connection is dead volume free (i.e. that the ends meet in the middle Figure 4b). Page 16 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 17: Installing And Configuring The Application Fluidics

    This will ensure that all air is removed from the capillaries and connections and that no air is passed through the column. 2.1.4 Interfacing the UltiMate 3000 RSLCnano with the Nanospray Flex™ Ion Source LC−MS based applications using linear columns commonly use the Nanospray Flex Ion source (see...

  • Page 18: Sample Preparation For Reversed Phase Lc Separation

    C18 75 cm PepMap RSLC, P/N 164939) a beige sleeve (P/N SC603) should be used (both types are included with the ion source). For more details on connecting the UltiMate 3000 RSLCnano with a Thermo Scientific mass spectrometer, please refer to “The Complete and Easy...
  • Page 19 TIP: To limit the risk of peptide or protein adsorption on the walls of the vials, Thermo Fisher Scientific recommends using vials containing glass inserts (Polypropylene vials for WPS with glass insert, 250 µL, set of 100, P/N 6820.0027). UltiMate 3000 RSLCnano Standard Applications Guide Page 19...

  • Page 20: Mobile Phases

    IMPORTANT: When installing fresh mobile phase on the LC system, replace the mobile phase solvent in the bottle completely. DO NOT “top up” mobile phases to avoid solvent composition changes or unwanted components building up in the mobile phase bottles. Page 20 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 21: Available Trapping Columns

    Backpressure is lower compared to nano trap columns and therefore µ- precolumns can accommodate higher loading flows and are often preferred when large sample volumes need to be injected. UltiMate 3000 RSLCnano Standard Applications Guide Page 21...

  • Page 22: The Difference Between Forward Flush And Back Flush

    Figure 7 shows the different fluidic setups for a forward- and a back-flush fluidic pathway. Figure 7: The different fluidic configurations for forward-flush (left) and back-flush (right) Page 22 UltiMate 3000 RSLCnano Standard Applications Guide...
  • Page 23 NOTICE For pre-concentration applications, better chromatographic resolution (narrower chromatographic peaks) are produced when the µ-precolumn is installed in back-flush mode. UltiMate 3000 RSLCnano Standard Applications Guide Page 23...

  • Page 24: Installing The Ultimate 3000 Rslcnano System

     Application Setup Installing the UltiMate 3000 RSLCnano System 2.3.1 UltiMate 3000 RSLCnano System Components SRD-3400, (Optional): SRD-3200 with degassing or SR-3000 without degassing. NCS-3500RS module featuring - NC pump, up to 900 bar - Loading pump, micro Titanium up to 620 bar...

  • Page 25: Nc Pump Configurations

    Nano (Nan) 500 nL/min 50 nL/min 1000 nL/min Capillary (Cap) 5 µL/min 500 nL/min 10 µL/min Micro (Mic) 25 µL/min 2.5 µL/min 50 µL/min Table 1: Properties of the different flow selectors UltiMate 3000 RSLCnano Standard Applications Guide Page 25...
  • Page 26 Tubing guides Snap-in valves Column compartment Figure 9: NCS-3500RS with ProFlow flow meter NOTICE The maximum pump pressure available for the column is 900 bar if a ProFlow flow meter is installed. Page 26 UltiMate 3000 RSLCnano Standard Applications Guide...
  • Page 27 Purge screw Tubing guides Snap-in valves Column compartment Figure 10: NCS-3500RS with classic flow meter NOTICE The maximum pump pressure available for the column is 800 bar with a classic flow meter installed. UltiMate 3000 RSLCnano Standard Applications Guide Page 27...
  • Page 28  Application Setup NCP-3200RS 2.3.2.5 NCP-3200RS module featuring - NC pump Figure 11: NCP 3200RS pump Figure 12: NCP 3200RS pump with ProFlow flow meter installed Page 28 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 29: Software Compatibility For Ncx-3X00Rs Operation With Proflow And Classic Flow Meters

    For LC control using Chromeleon, ProFlow technology requires: ≥ SR 15b for Chromeleon 6.80 ≥ SR4 for Chromeleon 7.2 NOTICE The Chromeleon and SII software versions show in Figure 14 the minimum requirements. Later versions are fully compatible. UltiMate 3000 RSLCnano Standard Applications Guide Page 29...
  • Page 30  Application Setup Figure 14: UltiMate 3000 RSLCnano with ProFlow flow meter – compatibility matrix Page 30 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 31: Preparing The Rslcnano For Use

    2.3.4 Preparing the RSLCnano for Use The UltiMate 3000 RSLCnano system must be prepared and primed prior to use. A brief description of each step is given below. For detailed information on configuring the system, please refer to the respective operating instructions for each module.
  • Page 32 NC Pump solvent B - 80/20 (v/v) Acetonitrile / Water with 0.1% FA P/N LS122-500 Alternative for NC Pump solvent B (ProFlow only) - Acetonitrile with 0.1% FA P/N LS120-500 Auxiliary Solvents: 2.3.4.5 The following solvents are recommendations to ensure robust operation Page 32 UltiMate 3000 RSLCnano Standard Applications Guide...
  • Page 33 Flow Meter Type Refresh Solvent Change Solvent ProFlow 5 min 15 min Classic 10 min 30 min Table 2: NC Pump block purge times UltiMate 3000 RSLCnano Standard Applications Guide Page 33...
  • Page 34 TIP The descriptions above refer to the test locations in SII / CM 7.2. For Link information on where to find the test in CM 6.8 / DCMS please refer to the NCx-3x00 operating instructions. Page 34 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 35: Flow Meter Calibration

    They are also valid when the flow meter is transferred from one UltiMate 3000 RSLCnano system to another. NOTICE The solvent types are valid for a variation in composition ≤ 2% i.e.
  • Page 36 Once the test is finished, the measured viscosity values can be stored by selecting the ‘apply’ button for the respective channel. The results should be examined for plausibility (see Table 4 for reference values). Page 36 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 37: Application Overview

     Application Setup Application Overview Figure 15: Overview of the Applications available for the RSLCnano UltiMate 3000 RSLCnano Standard Applications Guide Page 37...

  • Page 38: Direct Injection Onto A Nano Column

    Figure 16: Setup for a Direct Injection experiment onto a nano column including the optional UV detector Page 38 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 39: Fluidic Setup

    Polypropylene vials for WPS with glass insert, 250 µL, 25 pcs 6820.0027 Polypropylene caps for WPS vials, 25 pcs 6820.0028 Cytochrome C digest, 1.6 nmol, Lyophilized 161089 Table 5: RSLCnano Direct Injection nano LC kit (P/N 6720.0300) contents UltiMate 3000 RSLCnano Standard Applications Guide Page 39...

  • Page 40: Installation Tips

    Table 6: Test conditions for direct injection of 500 fmol Cytochrome C onto a nano column Figure 18: Typical MS TIC for a direct injection of 500fmol Cytochrome C onto a nano column Page 40 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 41: Large Volume Injections

    MS, and ii) an investment of extra analysis time to accommodate the complete loading of sample at low flow rates. UltiMate 3000 RSLCnano Standard Applications Guide Page 41...

  • Page 42: Direct Injection Onto A Capillary Column

    Figure 19: Setup for a Direct Injection experiment onto a capillary column including the optional UV detector Page 42 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 43: Fluidic Setup

    Figure 20: Fluidic connections for a direct injection onto a capillary column TIP If no valve is available, a Viper union (P/N 6040.2304, ordered separately) can be used to connect the capillary (2) directly to the column (a). UltiMate 3000 RSLCnano Standard Applications Guide Page 43...
  • Page 44 Polypropylene vials for WPS with glass insert, 250 µL, 25 pcs 6820.0027 Polypropylene caps for WPS vials, 25 pcs 6820.0028 Cytochrome C digest, 1.6 nmol, Lyophilized 161089 Table 7: RSLCnano Direct Injection capillary LC kit (P/N 6720.0305) contents Page 44 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 45: Installation Tips

    Flow Rate 5 µL / min during gradient, 10 µL / min during wash and equilibration Table 8: Test conditions for direct injection of 1 pmol Cytochrome C onto a capillary column UltiMate 3000 RSLCnano Standard Applications Guide Page 45...
  • Page 46  Application Setup Figure 21: Typical chromatogram for a direct injection of 1pmol Cytochrome C onto a capillary column Page 46 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 47: Pre-Concentration Onto A Nano Column

    1x 10-port sw. valve 6041.0001A VWD-3400RS (optional) 5074.0010 3 nL flow cell 6074.0270 WPS-3000TPL RS 5826.0020 Application kit: 6720.0310 Figure 22: Setup for a pre-concentration experiment onto a nano column including the optional UV detector UltiMate 3000 RSLCnano Standard Applications Guide Page 47...

  • Page 48: Fluidic Setup

    FS/PEEK sheathed 1/32" I.D. x L 75 µm x 650 mm 6041.5775 nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 550 mm 6041.5760 nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 750 mm 6041.5280 Page 48 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 49: Installation Tips

    (see capillary 1 in Figure 17). One further capillary is required (P/N 6041.5260, capillary 2 in Figure 17) which must be ordered separately. UltiMate 3000 RSLCnano Standard Applications Guide Page 49...

  • Page 50: Testing The Application

    Table 10 Test conditions for pre-concentration injection of 1 pmol Cytochrome C onto a nano column Figure 24: Typical chromatogram for a pre-concentration of 500 fmol Cytochrome C onto a nano column Page 50 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 51: Pre-Concentration Onto A 200 Μm Monolithic Column

    1x.10 port sw valve 6041.0001A VWD-3400RS (optional) 5074.0010 3 nL flow cell 6074.0270 WPS-3000TPL RS 5826.0020 Application kit: 6720.0320 Figure 25: Setup for a Pre-concentration experiment onto a monolithic column including the optional UV detector UltiMate 3000 RSLCnano Standard Applications Guide Page 51...

  • Page 52: Fluidic Setup

    Figure Figure 26: Fluidic connections for a pre-concentration onto a monolithic column TIP The schematic shows a 10-port switching valve. This application can also be performed using a 6-port valve. Page 52 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 53: Installation Tips

    Test the pre-concentration setup using the following conditions: Property Setting Mobile Phase A 100% Water + 0.05% TFA Mobile Phase B 50 %/ 50% (v/v) Acetonitrile / Water + 0.04% TFA Loading Solvent Water + 0.05% HFBA UltiMate 3000 RSLCnano Standard Applications Guide Page 53...
  • Page 54 (injection) peak is detected at 214 nm. This is due to the different UV absorbance of the ion-pairing agents (e.g. HFBA vs. TFA) in the loading and analytical solvent. Page 54 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 55: Pre-Concentration Onto A Capillary Column

    1x.10 port sw. valve 6041.0001A VWD-3400RS 5074.0010 45 nL flow cell 6074.0280 WPS-3000TPL RS 5826.0020 Application kit: 6720.0315 Figure 28: Setup for a pre-concentration experiment onto a capillary column including the optional UV detector UltiMate 3000 RSLCnano Standard Applications Guide Page 55...

  • Page 56: Fluidic Setup

    FS/PEEK sheathed 1/32" I.D. x L 75 µm x 650 mm 6041.5775 nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 550 mm 6041.5760 nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 50 µm x 750 mm 6041.5580 Page 56 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 57: Installation Tips

    (see capillary 1 in Figure 20). One further capillary is required (P/N 6041.5560, capillary 2 in Figure 20) which must be ordered separately. UltiMate 3000 RSLCnano Standard Applications Guide Page 57...

  • Page 58: Testing The Application

    Table 14: Test conditions for pre-concentration injection of 500 fmol Cytochrome C onto a capillary column Figure 30: Typical chromatogram for a pre-concentration of 1 pmol Cytochrome C onto a capillary column Page 58 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 59: Easy-Spray Columns With The Rslcnano

    LC outlet using a viper union. In-built column temperature control ensures optimal retention stability and consistent chromatographic performance. Figure 32 shows the EASY-Spray source with an EASY-Spray column installed. UltiMate 3000 RSLCnano Standard Applications Guide Page 59...
  • Page 60 EASY Spray NG (ES082) TSQ Series Orbitrap Fusion™ Series Endura MD™ EASY-Spray (ES081) Exactive™ Series Orbitrap™ Series LTQ™ Series LCQ™ Dec XP Max Table 15: EASY-Spray ion sources and compatible mass spectrometers Page 60 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 61: Example Separation Performance Of An Easy-Spray Column

    33 A and PWHH 2.5 sec 12.00 12.10 12.20 Time (min) PWHH 3.0 sec 14.20 14.30 14.40 Time (min) Time (min) Figure 33: Base Peak Chromatogram of an EASY-Spray column in pre- concentration mode UltiMate 3000 RSLCnano Standard Applications Guide Page 61...

  • Page 62: Hardware Layout Direct Injection

    The instrument setup for pre- concentration with EASY-Spray presented in Figure 35 consists of: SRD-3400 5035.9245 NCS-3500RS 5041.0010A 1x 10-port sw.valve 6041.0001A WPS-3000TPL RS 5826.0020 Application kit: 6720.0395 Figure 35: Setup for pre-concentration with EASY- Spray Page 62 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 63: Fluidic Setup Using Easy-Spray Columns

     Application Setup 2.10.4 Fluidic Setup using EASY-Spray Columns The fluidic setups for EASY-Spray with an UltiMate 3000 RSLCnano are essentially identical to those used for the direct injection and pre- concentration applications already described. The only difference is that instead of connecting the separation column...
  • Page 64 6820.0027 Polypropylene caps for WPS vials, 25 pieces 6820.0028 Cytochrome C digest, 1.6 nmol, Lyophilized 161089 Table 16: UltiMate 3000 RSLCnano EASY-Spray connection kit (P/N 6720.0395) contents For a list of available EASY-Spray columns, see – Table 28: EASY-Spray columns in the Appendix.

  • Page 65: Easy-Spray Transfer Lines

    EASY-Spray source, attached to a nanoViper fitting via a fused silica capillary. Figure 36: EASY-Spray Transfer Lines EASY-Spray transfer lines are available in both nano fIow (P/N ES791A) and capillary /micro flow (P/N ES792A) compatible formats. UltiMate 3000 RSLCnano Standard Applications Guide Page 65...

  • Page 66: Salt Plugs With Nano Column

    5041.0010A 2x 10-port sw.valve 6041.0001A VWD-3400RS 5074.0010 3 nL flow cell 6074.0270 WPS-3000TPL RS 5826.0020 Application kit: 6720.0325 Figure 37: Setup for a 2D Salt Plugs experiment including the optional UV detector Page 66 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 67: Fluidic Setup

    300 µm I.D. x 5 mm, packed with Acclaim PepMap100 C18, 5 µm, 100Å (set of 5 160454 cartridges) µ-Precolumn holder, 5 mm, with 30 µm I.D. connecting tubing, nanoViper fittings 164649 nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 350 mm 6041.5240 UltiMate 3000 RSLCnano Standard Applications Guide Page 67...

  • Page 68: Installation Tips

    The salt plugs listed here have been chosen for the separation of the protein mix digest. The best sequence of plugs will highly depend on the affinity of the peptides present in the sample with the IEX column. Page 68 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 69: Testing The Application

    5 °C Loading flow rate 10 µL / min NC Flow Rate 300 nL / min (capillary flow selector) Table 18: Test conditions for a protein digest separation using 2D Salt Plugs UltiMate 3000 RSLCnano Standard Applications Guide Page 69...

  • Page 70: Salt Solution Preparation

    1000 µL 2 mM 20 µL 980 µL 1000 µL 1 mM 10 µL 990 µL 1000 µL Table 20: Guide for the preparation of the Salt Plugs (dilutions from 100mM NaCl) Page 70 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 71: Tandem Nano Lc

    3 nL flow cell 6074.0270 supply solvent from the bottles placed on WPS-3000FC 5824.0020 the solvent rack to the NCP at the bottom of Injection valve 6826.0011A the stack. Application kit: 6720.0335 UltiMate 3000 RSLCnano Standard Applications Guide Page 71...

  • Page 72: Fluidic Setup

    3000FC replaced with the 1/32” nano switching valve (P/N 6820.6232). All necessary parts are included in the kit. A detailed description of how to set-up, configure and control both hardware variants is given in TN72899. Page 72 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 73: Installation Tips

    If a loss in hydrophilic peptides is observed, the acetonitrile in the loading solvent can be removed and / or the 0.1 % FA can be replaced with 0.05% TFA or 0.1% TFA as required. UltiMate 3000 RSLCnano Standard Applications Guide Page 73...

  • Page 74: Testing The Application

    Oven Temperature 35 °C WPS temperature 5 °C Loading flow Rate 5 µL / min NC Pump flow rate 300 nL / min Table 22 Test conditions for running the tandem nanoLC application Page 74 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 75: Tandem Nanolc-Ms

    LC-MS systems. Complete LC method parameters for this set-up are also available for download from the AppsLab library. UltiMate 3000 RSLCnano Standard Applications Guide Page 75...

  • Page 76: High Throughput Tandem Capillary-Flow Lc-Ms

    Tandem capillary-flow LC workflows combine high sensitivity with high throughput, permitting the analysis of up to 200 samples per day whilst yielding 100% MS utilization. Details on how to connect and configure the UltiMate 3000 RSLCnano in order to run this application are provided in TN72827...

  • Page 77: Micro Lc Applications

    50 µm I.D. nanoViper capillaries. A list of the available nanoViper capillaries is available in Table 31 page If UV detection is required, the cap flow cell (45 nL; P/N 6074.0280) is recommended for all micro LC applications. UltiMate 3000 RSLCnano Standard Applications Guide Page 77...

  • Page 78: Ms Connection Kit

     Application Setup 2.15 MS Connection Kit The UltiMate 3000 RSLCnano MS connection kit (P/N 6720.0345; Table 23) contains a variety of common tubings, unions and connectors necessary to facilitate coupling of the LC column to a mass spectrometer interface.
  • Page 79 Keep in mind that internal diameter has a much bigger effect on dispersion then the length of the connecting tubing. Ensure that tubing with the correct diameter is used at all times. UltiMate 3000 RSLCnano Standard Applications Guide Page 79...

  • Page 80: Mass Spectrometry Interfaces For Linear Columns And / Or Interfacing The Uv Detector With The Ms

    MS instrument. The common interfaces applicable to linear columns and / or the VWD 3400RS UV detector available for Thermo Scientific mass spectrometers are shown below. For details on the connections, also see section 2.1.4.
  • Page 81 Legacy TSQ™ series (Quantum Access Max, Vantage, Ultra etc.) H-ESI Spray Insert for Ion Max NG Source P/N 80000-60321 Figure 42: Ionization probes for the Ion Max and Ion Max NG sources. UltiMate 3000 RSLCnano Standard Applications Guide Page 81...
  • Page 82 A compatibility matrix for the different low-flow options is shown in Figure Figure 43: Ionization probes for the Ion Max and Ion Max NG sources Page 82 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 83: Faqs

     FAQs 3 FAQs UltiMate 3000 RSLCnano Standard Applications Guide Page 83...

  • Page 84: Nc_Pump Solvent Recalibration - Best Practice

    Every time solvents are refreshed or when a retention time drift is observed. Viscosity Measurement Test 3.1.2.2 Every time solvents are exchanged or refreshed. TIP The test also acts as a diagnostic tool. Page 84 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 85: Interpreting A Chromatogram

    Since there are usually one (direct injection) or two (pre-concentration) valve switches involved in the application, introducing additional capillaries and connections, the dwell volume and gradient delay are not the same volume in direct injection and pre- concentration setups. UltiMate 3000 RSLCnano Standard Applications Guide Page 85...

  • Page 86: Troubleshooting Nano Lc Peptide Applications

    Cytochrome C digest separation; when working with a different standard use a trusted reference chromatogram for the expected values for number of peaks, intensity and elution window. Page 86 UltiMate 3000 RSLCnano Standard Applications Guide...
  • Page 87  FAQs Figure 45: Decision Tree for troubleshooting a low-flow UltiMate 3000 RSLCnano Standard Applications Guide Page 87...

  • Page 88: The Use Of Tfa And Fa

    TFA instead of FA, use the amounts given in Table 24. Figure 46 demonstrates the effect of ion pairing agent choice on the chromatographic separation of Cytochrome C. Page 88 UltiMate 3000 RSLCnano Standard Applications Guide...
  • Page 89  FAQs Figure 46: Comparison of Cytochrome C separation with different ion pairing agents. Top uses 0.05% TFA and bottom 0.1% FA UltiMate 3000 RSLCnano Standard Applications Guide Page 89...

  • Page 90: Minimizing Baseline Effects

    ‘burn in’ period. Allowing sufficient equilibration time for the lamp is necessary for obtaining a stable baseline. Lamps should be replaced after approximately 2000 hours. Older flow cells can be cleaned by flushing overnight with organic solvent or for a Page 90 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 91: Unstable Baseline

    Detector’s Operating Instructions for more details. 3.5.2 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 92: Typical Wps-3000Tpl Rs Autosampler Settings For Standard Injection Routines

    Flush volume used in full loop and partial loop 3 µL injections from the same vial. Loop Overfill Loop overfill factor used in full loop only Table 25: Typical WPS-3000TPL RS autosampler settings Page 92 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 93: Appendix

     Appendix 4 Appendix UltiMate 3000 RSLCnano Standard Applications Guide Page 93...

  • Page 94: Customized Sample Injection Routines

    4. Injecting the sample and starting the acquisition run 5. Washing the syringe and preparing for the next injection Step ‘1’ is often omitted, as samples are usually ready to inject when placed in the autosampler. Page 94 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 95: Example Μl Pickup Udp For Maximum Sample Pickup

    In the following example, 10 µL of sample is injected using a 20 µL loop using transport liquid placed in vial R1. Figure 47: Example UDP injection routine for maximizing µL pickup volume UltiMate 3000 RSLCnano Standard Applications Guide Page 95...

  • Page 96: Variable Injection Volumes

    Step 3 – Delete the text ‘Volume= 10 [µL]’ Step 4 - Replace with ‘Volume=system.Injection._Volume’ (make to place a comma at the end of the line) Step 5 – Save the method with an appropriate name Page 96 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 97: Common Application Related Consumables

    The tables below list consumables associated with the applications outlined in this handbook. 4.2.1 Columns The most common analytical columns used with the UltiMate 3000 RSLCnano system are listed below. For a comprehensive guide of all available column formats please refer to the Thermo Scientific Chromatography columns and consumables catalogue.
  • Page 98 PepSwift Monolithic Capillary Column, 200 μm I.D. x 25 cm, nanoViper 164542 Table 29: PepSwift Monolithic linear analytical columns Item PepSwift Monolithic Trap Column, 200 μm x 5 mm, set of 2, nanoViper 164558 Table 30: Pepswift Monolithic trap columns Page 98 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 99: Nanoviper Capillaries, Sample Loops And Connectors

    6041.5122 6041.5125 6041.5128 1100 6041.5711 6041.5828 Table 31: Matrix for connection tubing for the UltiMate 3000 RSLCnano system Description nanoViper, ID x L, 20 μm x custom length 6041.5299 nanoViper, ID x L, 50 μm x custom length 6041.5599A nanoViper, ID x L, 75 μm x custom length 6041.5799...
  • Page 100 Sample loop 20 μL with nanoViper fittings connections 6826.2420 Sample loop 50 μL with nanoViper fittings connections 6826.2450 Sample loop 125 μL with nanoViper fittings connections 6826.2412 Table 33: Sample loops with nanoViper fittings Page 100 UltiMate 3000 RSLCnano Standard Applications Guide...

  • Page 101: Ultimate 3000 Rslcnano Convenience Bundles, List Of Contents

     Appendix UltiMate 3000 RSLCnano convenience bundles, list of contents Description U3000 Nano RSLCnano with VWD U3000 Nano RSLCnano, no detector U3000 Cap flow RSLCnano, no detector U3000 Nano RSLCnano with EASY-Spray Item P/N: 5200.0350 P/N: 5200.0355 P/N: 5200.0356 P/N: 5200.0357...

  • Page 102: Hardware Accessories

    Classic flow meter with micro flow selector 6041.7903A Flow selector, Nano LC (recommended: 50–1,000 nL/min) 6041.0002 Flow selector, Capillary LC (recommended: 0.5–10 µL/min) 6041.0003 Flow selector, Micro LC (recommended: 5–50 µL/min) 6041.0014 Table 36: NC Pump Flow Control Page 102 UltiMate 3000 RSLCnano Standard Applications Guide...
  • Page 103 www.thermofisher.com Thermo Fisher Scientific Inc. 168 Third Avenue Waltham Massachusetts 02451...

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