The Complete and Easy Guide to Configuring Your Specific Thermo NanoLC for Mass Spec Analysis
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1 The Complete and Easy Guide to Configuring Your Specific Thermo NanoLC for Mass Spec Analysis September 2013, Version 2 The world leader in serving science
2 Content Selection Tree EASY-nLC 1000 Slide 3 Selection Tree EASY-nLC II Slide 4 Selection Tree UltiMate 3000 RSLCnano Slide 5 How to Guideline Slide 37
3 Selection Tree EASY-nLC 1000 LC EASY-nLC 1000 Source Nanospray Flex EASY-Spray HV junction Liquid Junction Direct Junction setup Pre-Col EASY-Spray EASY-Spray Pre-Col Pre-Col Packed Packed Pre-Col Go to slide Slide 6 Slide 7 Slide 8 Slide 9 Slide 10 Slide 11 Slide 12 Slide 13 3
4 Selection Tree EASY-nLC II LC EASY-nLC II Source Nanospray Flex EASY-Spray HV junction Liquid Junction Direct Junction setup Pre-Col EASY-Spray EASY-Spray Pre-Col Pre-Col Packed Packed Pre-Col Go to slide Slide 14 Slide 15 Slide 16 Slide 17 Slide 18 Slide 19 Slide 20 Slide 21 4
5 Selection Tree UltiMate 3000 RSLCnano LC RSLCnano Source Nanospray Flex EASY-Spray HV junction Liquid Junction Direct Junction setup Pre-Col EASY- Spray EASY- Spray Pre-Col Pre-Col Packed Packed Pre-Col Go to slide Slide 22 Slide 23 Heated Room Temp Heated Room Temp Heated Room Temp Heated Room Temp Go to slide Slide 24 Slide 25 Slide 26 Slide 27 Slide 28 Slide 29 Slide 30 Slide 31 Slide 32 Slide 33 5
6 EASY-nLC 1000 with Precolumn and EASY-Spray Schematic Consumables Viper Union SC900 Acclaim Pepmap precolumn A/B mixing & venting Tee EASY-Spray column SC901 Various: ES801 ES802 ES803 6 Return to selection page EASY-nLC 1000
7 EASY-nLC 1000 with EASY-Spray Schematic Consumables A/B mixing & venting Tee EASY-Spray column SC901 Various: ES801 ES802 ES803 7 Return to selection page EASY-nLC 1000
8 EASY-nLC 1000 with Liquid Junction Precolumn Analytical UHPLC Schematic Preferred ES272 Consumables Out Tubing Waste In Tubing Viper on LC side only Viper on LC side only Union For 360 µm OD ES272 UPLC Liquid Junction Cross Kit ES269 HPLC Schematic 300 bar max HPLC Liquid Junction Cross Biege Sleeves ES257 Out Tubing Peek Union Pre- Waste In Tubing HV Liquid Junction Cross Analytical Peek Union Silica Red peek union for Sleeves for connecting OD 360 µm capillary to Beige (360µm x 1/32 ) unions Silica (IDEX) P-771 SC603 FS N-20 (New Objective) 8 Return to selection page EASY-nLC 1000
9 EASY-nLC 1000 with Liquid Junction Precolumn and Packed UHPLC Schematic Preferred ES272 Consumables out tubing Viper on LC Side Only Waste in tubing Viper on LC Side Only Union For 360 µm OD ES272 UPLC Liquid Junction Cross Kit ES269 HPLC Schematic 300 bar max Waste In Tubing Out Tubing Liquid Junction Cross Peek Union Pre- HV Packed HPLC Liquid Junction Cross Beige Sleeves ES257 Red peek union for (IDEX) P-771 Sleeves for connecting OD 360 µm capillary to Beige (360µm x 1/32 ) unions SC603 9 Return to selection page EASY-nLC 1000
10 EASY-nLC 1000 with Liquid Junction Analytical and UHPLC Schematic Preferred Consumables Out Tubing Waste In Tubing Viper on LC side only Viper on LC side only UPLC Liquid Junction Cross Kit ES269 HPLC Liquid Junction Cross Beige Sleeves ES257 HPLC Schematic 300 bar max Red peek union for (IDEX) P-771 Out Tubing Waste In Tubing HV Liquid Junction Cross Analytical Peek Union Silica Sleeves for connecting OD 360 µm capillary to Beige (360µm x 1/32 ) unions Silica SC603 FS N-20 (New Objective) 10 Return to selection page EASY-nLC 1000
11 EASY-nLC 1000 with Liquid Junction and Packed UHPLC Schematic Preferred Consumables out tubing Waste in tubing Viper on LC Side Only Viper on LC Side Only UPLC Liquid Junction Cross Kit ES269 HPLC Liquid Junction Cross Beige Sleeves ES257 HPLC Schematic 300 bar max Waste In Tubing Out Tubing Liquid Junction Cross Sleeves for connecting OD 360 µm capillary to unions Beige (360µm x 1/32 ) SC603 HV Packed 11 Return to selection page EASY-nLC 1000
12 EASY-nLC 1000 with Direct Junction Precolumn Analytical and Viper Schematic Viper Union Mixing/Venting Tee Black Sleeve Peak Union Consumables Viper Union SC900 Out Line to LC Pre- Analytical Metal Mixing/ Venting Tee SC901 Waste In Line from LC Non-Viper Schematic Red peek union for Sleeves for connecting OD 280 µm capillary to unions Black (280µm x 1/32 ) (IDEX) P-771 SC903 Out Tubing Union for 360 µm OD Pre- Waste In Tubing Venting Tee Analytical For non-viper Connections Beige Sleeve Peek Union Metal Metal Out Tubing Waste In Tubing Union for 360 µm OD Ultra High Pressure MicroTee for 360 µm OD Tubing Sleeves for connecting OD 360 µm capillary to unions Viper on LC side only Viper on LC side only Beige (360µm x 1/32 ) ES ES272 (IDEX) UH-750 SC Return to selection page EASY-nLC 1000
13 EASY-nLC 1000 with Direct Junction Analytical and Viper Schematic Mixing/Venting Tee Black Sleeve Consumables Out to LC Analytical Peek Union Metal Mixing/ Venting Tee Red peek union for SC901 (IDEX) P-771 Waste In Line from LC Non-Viper Schematic Out Tubing Waste In Tubing Venting Tee Analytical Beige Sleeve Metal Peek Union Sleeves for connecting OD 280 µm capillary to unions Metal Out Tubing Waste In Tubing Black (280µm x 1/32 ) Viper on LC side only Viper on LC side only Ultra High Pressure MicroTee for 360 µm OD Tubing Sleeves for connecting OD 360 µm capillary to Beige (360µm x 1/32 ) unions SC903 ES (IDEX) UH750 SC603 For non-viper Connections 13 Return to selection page EASY-nLC 1000
14 EASY-nLC II with Precolumn and EASY-Spray Schematic Consumables Adapter Mictrotight 1/16 to 1/32, peek (IDEX) P-881 Sleeve Acclaim Pepmap precolumn A/B mixing & venting Tee SC901 Acclaim PepMap 100 Pre EASY-Spray column Tubing Sleeve, 865 µm (0.034") ID x 1/16" OD, PEEK, Blue ES800 (IDEX) F Return to selection page EASY-nLC II
15 EASY-nLC II with EASY-Spray Schematic Consumables A/B mixing & venting Tee SC901 EASY-Spray column ES800 Sleeve Tubing Sleeve, 865 µm (0.034") ID x 1/16" OD, PEEK, Blue (IDEX) F Return to selection page EASY-nLC II
16 EASY-nLC II with Liquid Junction Precolumn Analytical and Schematic Out to LC Peek Union Pre- Waste In Line from LC Liquid Junction Cross HV Analytical Peek Union Silica Consumables HPLC Liquid Junction Cross Beige Sleeves Red peek union for Sleeves for connecting OD 360 µm capillary to Beige (360µm x 1/32 ) unions Silica ES257 (IDEX) P-771 SC603 FS N-20 (New Objective) 16 Return to selection page EASY-nLC II
17 EASY-nLC II with Liquid Junction Precolumn and Packed Schematic Out to LC Peek Union Pre- Waste In from LC Liquid Junction Cross HV Packed Consumables HPLC Liquid Junction Cross Beige Sleeves Red peek union for Sleeves for connecting OD 360 µm capillary to Beige (360µm x 1/32 ) unions ES257 (IDEX) P-771 SC Return to selection page EASY-nLC II
18 EASY-nLC II with Liquid Junction Analytical and Schematic Consumables Out to LC Waste In from LC Liquid Junction Cross Silica HPLC Liquid Junction Cross Beige Sleeves ES257 HV Analytical Peek Union Red peek union for Sleeves for connecting OD 360 µm capillary to Beige (360µm x 1/32 ) unions (IDEX) P-771 SC603 Silica FS N-20 (New Objective) 18 Return to selection page EASY-nLC II
19 EASY-nLC II with Liquid Junction and Packed HPLC Schematic Consumables Out to LC Waste In from LC HV Liquid Junction Cross Packed HPLC Liquid Junction Cross Beige Sleeves Sleeves for connecting OD 360 µm capillary to Beige (360µm x 1/32 ) unions ES257 SC Return to selection page EASY-nLC II
20 EASY-nLC II with Direct Junction Precolumn Analytical and Schematic Waste In from LC Out to LC Venting Tee Peek Union Pre- Analytical Peek Union Metal Consumables Red peek union for Sleeves for connecting OD 360 µm capillary to Beige (360µm x 1/32 ) unions Venting Tee Beige Sleeves (IDEX) P-771 SC603 SC601 Metal ES Return to selection page EASY-nLC II
21 EASY-nLC II with Direct Junction Analytical and Schematic Consumables Out to LC Waste In from LC Venting Tee Analytical Metal Peek Union Red peek union for Sleeves for connecting OD 360 µm capillary to Beige (360µm x 1/32 ) unions (IDEX) P-771 SC603 Venting Tee Beige Sleeves SC601 Metal ES Return to selection page EASY-nLC II
22 RSLCnano with Precolumn and EASY-Spray Schematic A B Consumables A B C Trap Ultimate 3000 RSLCnano EASY-Spray Connection Kit Viper Union Contents Slide SC900 WPS 3000 Autosampler Viper Union Ultimate 3000 RSLCnano EASY-Spray Connection Kit Acclaim Pepmap Pre- µ-precolumn holder EASY-Spray column Various: ES801 ES802 ES803 Shortest length necessary to attach valve to EASY-Spray 22 Return to selection page UltiMate 3000 RSLCnano
23 Schematic A B RSLCnano with EASY-Spray Consumables A B C Auto Sampler Valve WPS 3000 Autosampler Viper Union Ultimate 3000 RSLCnano EASY-Spray Connection Kit Viper Union EASY-Spray column Contents Slide SC900 Various: ES801 ES802 ES803 Ultimate 3000 RSLCnano EASY-Spray Connection Kit Shortest length necessary to attach valve to EASY-Spray 23 Return to selection page UltiMate 3000 RSLCnano
24 RSLCnano with Liquid Junction Precolumn Heated Analytical and UltiMate 3000 RSLCnano Pre-concentration nano LC kit µ-tight Union Sleeves for Connecting Capillary to µ-tight Union Contents Slide Black (280µm x 1/32 ) Beige (360µm x 1/32 ) (IDEX) P-720 SC903 SC603 HPLC Liquid Junction Tee Biege Sleeves Red peek union for Silica Only necessary if analytical column is not long enough to reach the liquid junction tee ŧ Note: This tubing has a 280 µm OD ES258 (IDEX) P-771 FS N-20-C12 (New Objective) 24 Return to selection page UltiMate 3000 RSLCnano
25 RSLCnano with Liquid Junction Precolumn Room Temperature Analytical and UltiMate 3000 RSLCnano Pre-concentration nano LC kit Contents Slide Out Tubing Viper on LC side only UHPLC Blind Plug (IDEX) P-116 Red peek union for Beige Sleeves 360 µm x 1/32 (IDEX) P-771 SC603 UPLC Liquid Junction Cross Kit ES269 Silica FS N-20 (New Objective) For non-viper Connections 25 Return to selection page UltiMate 3000 RSLCnano
26 RSLCnano with Liquid Junction Precolumn and Packed UltiMate 3000 RSLCnano Pre-concentration nano LC kit Contents Slide Out Tubing Viper on LC side only UHPLC Blind Plug (IDEX) P-116 Red peek union for (IDEX) P-771 UPLC Liquid Junction Cross Kit ES269 For non-viper Connections 26 Return to selection page UltiMate 3000 RSLCnano
27 RSLCnano with Liquid Junction Heated Analytical and UltiMate 3000 RSLCnano Direct Injection nano LC kit Contents Slide HPLC Liquid Junction Tee Biege Sleeves ES258 µ-tight Union (IDEX) P-720 Red peek union for (IDEX) P-771 Sleeves for Connecting Capillary to µ-tight Union Black (280µm x 1/32 ) Beige (360µm x 1/32 ) SC903 SC603 Silica Only necessary if analytical column is not long enough to reach the liquid junction tee ŧ Note: This tubing has a 280 µm OD FS N-20-C12 (New Objective) 27 Return to selection page UltiMate 3000 RSLCnano
28 RSLCnano with Liquid Junction Room Temperature Analytical and UltiMate 3000 RSLCnano Direct Injection nano LC kit Contents Slide Out Tubing Viper on LC side only UHPLC Blind Plug (IDEX) P-116 Red peek union for Beige Sleeves 360 µm x 1/32 (IDEX) P-771 SC603 UPLC Liquid Junction Cross Kit ES269 Silica FS N-20 (New Objective) For non-viper Connections 28 Return to selection page UltiMate 3000 RSLCnano
29 RSLCnano with Liquid Junction and Packed UltiMate 3000 RSLCnano Direct Injection nano LC kit Contents Slide UHPLC Blind Plug (IDEX) P-116 Out Tubing Viper on LC side only Red peek union for (IDEX) P-771 UPLC Liquid Junction Cross Kit ES Return to selection page UltiMate 3000 RSLCnano
30 RSLCnano with Direct Junction Precolumn Heated Analytical and UltiMate 3000 RSLCnano Pre-concentration nano LC kit µ-tight Union Contents Slide (IDEX) P-720 Red peek union for Metal (IDEX) P-771 E542 Sleeves for Connecting Capillary to unions Black (280µm x 1/32 ) Beige (360µm x 1/32 ) SC903 SC603 Only necessary if analytical column is not long enough to reach the liquid junction tee ŧ Note: This tubing has a 280 µm OD 30 Return to selection page UltiMate 3000 RSLCnano
31 RSLCnano with Direct Junction Precolumn Room Temperature Analytical and UltiMate 3000 RSLCnano Pre-concentration nano LC kit Contents Slide Red peek union for Beige Sleeves Beige (360µm x 1/32 ) (IDEX) P-771 SC603 Out Tubing Viper on LC side only Metal ES542 µ-tight Union (IDEX) P-720 For non-viper Connections Return to selection page UltiMate 3000 RSLCnano 31
32 RSLCnano with Direct Junction Heated Analytical and UltiMate 3000 RSLCnano Direct Injection nano LC kit Contents Slide Red peek union for (IDEX) P-771 µ-tight Union (IDEX) P-720 Sleeve for connecting to red peek union Beige (360µm x 1/32 ) SC603 Sleeves for Connecting Capillary to µ-tight Union Black (280µm x 1/32 ) Beige (360µm x 1/32 ) SC903 SC603 Metal ES542 Only necessary if analytical column is not long enough to reach the liquid junction tee ŧ Note: This tubing has a 280 µm OD 32 Return to selection page UltiMate 3000 RSLCnano
33 RSLCnano with Direct Junction Room Temperature Analytical and UltiMate 3000 RSLCnano Direct Injection nano LC kit Contents Slide Red peek union for (IDEX) P-771 Out Tubing Viper on LC side only Sleeves for Connecting to Unions Beige (360µm x 1/32 ) SC603 µ-tight Union (IDEX) P-720 Metal ES542 For non-viper Connections 33 Return to selection page UltiMate 3000 RSLCnano
34 UltiMate 3000 RSLCnano EASY-Spray Connection Kit UltiMate 3000 RSLCnano EASY-Spray connection kit Replacement P/N 300 µm I.D. x 5 mm, packed with Acclaim PepMap100 C18, 5 µm, 100Å (set of 5 cartridges) µ-precolumn holder, 5 mm, with 30 µm I.D. connecting tubing, nanoviper fittings nanoviper Capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 350 mm nanoviper Capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 550 mm nanoviper Capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 650 mm nanoviper Capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 750 mm nanoviper Capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 550 mm nanoviper Capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 650 mm nanoviper Sample Loop 20 µl, FS/PEEK sheathed Union Viper PTFE tubing, 500 µm I.D. x 100 cm, used as waste tubing /16" Universal Fingertight Fitting, one-piece design, extra long thread, 4 pieces Polypropylene vials for WPS with glass insert, 250 µl, 25 pieces Polypropylene caps for WPS vials, 25 pieces Cytochrome C digest, 1.6 nmol, Lyophilized For Pre-concentration Set Up Only 34 Return to selection page UltiMate 3000 RSLCnano
35 UltiMate 3000 RSLCnano Pre-concentration nano LC Kit UltiMate 3000 RSLCnano Pre-concentration nano LC Kit Replacement P/N 75 μm I.D. x 15 cm, packed with Acclaim PepMap RSLC C18, 2 μm, 100Å, nanoviper µm I.D. x 5 mm, packed with Acclaim PepMap100 C18, 5 µm, 100Å (set of 5 cartridges) μ-precolumn holder, 5 mm, with 30 μm I.D. connecting tubing, nanoviper fittings nanoviper capillary FS/PEEK sheathed 1/32" I.D. x L 20 μm x 350 mm nanoviper Capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 650 mm nanoviper Capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 550 mm nanoviper Capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 750 mm nanoviper Sample Loop 20 µl, FS/PEEK sheathed PTFE tubing, 500 µm I.D. x 100 cm, used as waste tubing /16" Universal Fingertight Fitting, one-piece design, extra long thread, 4 pieces Polypropylene vials for WPS with glass insert, 250 µl, 25 pieces Polypropylene caps for WPS vials, 25 pieces Cytochrome C digest, 1.6 nmol, Lyophilized Return to selection page UltiMate 3000 RSLCnano
36 UltiMate 3000 RSLCnano Direct Injection nano LC Kit UltiMate 3000 RSLCnano Direct Injection nano LC Kit Replacement P/N 75 μm I.D. x 15 cm, packed with Acclaim PepMap RSLC C18, 2 μm, 100Å, nanoviper nanoviper Capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 750 mm nanoviper Capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 550 mm nanoviper sample loop 1 μl, FS/PEEK sheathed I.D. x L 100 μm x 127 mm Polypropylene vials for WPS with glass insert, 250 µl, 25 pieces Polypropylene caps for WPS vials, 25 pieces Cytochrome C digest, 1.6 nmol, Lyophilized Return to selection page UltiMate 3000 RSLCnano
37 How to Guideline Making Viper Connections Slide 38 Making Connections with Sleeves Slide 39 Limit Pressure on EASY nlc Systems Slide 40 Limit Pressure on RSLCnano Slide 41 How Frequently Should I Replace My Unions and Sleeves? Slide 42 37
38 Making nanoviper and Viper Connections Carefully tighten the nut fingertight using the black knurled nut Do not overtighten Maximum of 1/8 turn after fingertight Remove the black knurled nut when tightened / after installation nanoviper fitting and capillary 38
39 Making Connections With Sleeves e.g. fused silica on a nano 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!). Push the sleeve all the way into the port. It is essential for a zero dead volume connection that the assembly seats firmly. Tighten the nut by two or three turns to make certain that the ferrule grabs the sleeve. Remove the pre-assembled fitting, and then slide the capillary (or column) into the sleeve. When re-installing the fitting together with the capillary (or column), make certain that the tip of the tubing does not extend the tip of the sleeve. Tighten the nut fingertight. Push the capillary (column) all the way into the sleeve (port) to minimize dead volume. Finally tighten the connection sufficiently to hold the pressure. It is essential for a zero dead volume connection that the assembly seats firmly, also under pressure. 39
40 Limiting Pressure on EASY nlc Systems To limit the EASY-nLC 1000 to 300 bars when running with a column setup rated to max 300 bar please change the Max pressure setting to 300 bar in: Methods: for Sample loading and Analytical and Precolumn equilibration Scripts: for Analytical and Precolumn equilibration Choose only flows for gradient, isocratic flow and idle flow that will create less than 300 bar on your column setup. 40
41 Limiting the Pressure on RSLCnano systems The RSLCnano has an upper and lower pressure limit The pressure limit can be set in each instrument method NC_Pump icon on the flow tab or directly on the control panel, using the sliders Upper limit Lower limit 41
42 How Frequently Should I Replace My Unions and Sleeves? Keep on hand When to replace Red peek union for (IDEX) P No more than two connections µ-tight Union (IDEX) P No more than two connections Sleeves for connecting OD 280 µm capillary to unions Sleeves for connecting OD 360 µm capillary to unions Black (280µm x 1/32 ) SC Every time you make a new connection Beige (360µm x 1/32 ) SC Every time you make a new connection Out Tubing Waste In Tubing Viper on LC side only Viper on LC side only Whenever viper is damage, silica is too short, or line is clogged Out Flow Line LC560 1 Waste In Flow Line LC562 1 Waste Viper Union SC900 2 When Viper is damaged or line is clogged Good to have on hand for EASY nanolc Leak Tests Ultimate 3000 RSLCnano EASY-Spray Connection Kit UltiMate 3000 RSLCnano Preconcentration nano LC kit UltiMate 3000 RSLCnano Direct Injection nano LC kit Contents Slide When tubing clogs or column fails Contents Slide When tubing clogs or column fails Contents Slide When tubing clogs or column fails 42
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