Tips and Hints for PAL Systems including general technical HPLC
Contents
1. i Gauge 22 Needle gauge versus fill speed and Needle volume sNOSesgs 7 i 0 72 mm 0 41 mm Needle gauge Fill speed Time to fill a syringe Needle volume a Tt af Gauge 22S 5 to 10 wL s 0 90 uL g Gauge 22S Gauge 22 200 ul s 6 73 uL aah Example 100 uL syringe 0 72 mm ee F 0 15 mm P p syang O Tt T Point Style No 3 Square cut Needle Gauge 19 OD 1 04 mm Mandatory for prep valve with bore size 0 75 mm Needle with Gauge 22 fits into valve bore 1 Maximum fill speed before cavitations are observed Fill speed example with solvent water methanol 1 1 2 Needle volume for 51 mm standard needle 10 Microtiter Deepwell PI nd the Piercing of Foil Heat foil Well Alu foil Se a ee particles Foil cutter Heat foil Well Particle free Foil cutter Article No MSZ 02 01 Foil cutter can be used for all types of heat foils Do not use the adhesive seal foils Organic solvents such as DMSO can dissolve glue sof Ping Diag fr ee Transferring sample in uL range easier to handle than in nL range Transfer sample first to a trap to focus with a high flow rate Use partial loop filling to minimize loss of sample Adjust sample and wash solvent to chromatographic strength of mobile phase Use a backflush mode to optimize transfer from trap to next device Example Trap can be used for sample preparation Eliminate2. myw Tubing seats correctly at the bottom m Tubing doesn t reach the bottom introducing dead volume Tubing reaches the bottom before ferrule seats Valco Parker ail Rheodyne Swagelok b 1D Tubing Internal Diameter versus Tubing Volume Examples are calculated for a tube length of 100 mm The tubing internal diameter must be adjusted to flow rate valve type and application to avoid high backpressure or chromatographic irregularities Tubing ID Tubing Tubing Tubing Points to consider are inch iimm volume Delay volume of entire HPLC System ooo Time needed for gradient to go active at column inlet 0 040 1 00 78 55 uL Adjust tubing diameters and length 0 020 0 50 19 64 uL Solvent reservoir to pump Cavitation Pump head to mixing T Low backpressure 0 010 0 25 4 91 uL Mixing T to injection valve Low backpressure as short as possible 0 005 0 13 133 uL Valve to column ID as small as possible considering backpressure Column to detector ID as small as possible 0 0025 0 064 0 32 uL if possible smaller than 4 considering backpressure Valve Type Flow Rate Range Tubing ID Tubing Bore Size in mm from to inch mm from to see diagram below 0 75 mm 5 to 100 mL min 0 25 6 35 mm 1 Reservoir Pre
3. Wash solvent in Loop for next injection Avoid organic solvents with high elution potential Is the chromatographic strength adjusted remaining in loop to the Mobile Phase 2 Valve system 1 1 Is the valve intact Are any leaks observed Is the waste line open At loop fill observe movement and speed of s a solvent front Is the backpressure at normal level l Can the valve be activated l Menu Utilities Injectors LC Vlv F1 Is the Valve Type dimensions appropriate for the flow rate See Point 1 3 Needle seal 1 1 Does the seal fit in size Gauge 22 19 Is the seal tight See Point 8 4 Needle penetration Is the Injection Unit needle guide Is needle penetration in valve inlet port Z Axis correctly positioned adjusted See Point 8 5 Nuts and ferrules Are the tubings cut square and are Are only matched nuts and ferrules used e they open without any restriction Are all connections remade dead volume l See Point 6 6 Loop injection Full loop injection Is the loop overfilled 3 to 5 times See Point 3 Partial loop filling Consider also the valve volume as indicated in Is the rule of 20 to 60 of the loop content applied 1 Point 2 7 Syringe Is the plunger tight Is the syringe intact High throughput applications Is a change to the X Type syringe indicated High affinity of a compound for metal 8 Material replacement I Compare flow rate vs tubing ID Tubing sst to PEEK
4. High flow rate for fast sample transfer on trap Low flow rate optimized for LC MS application Mobile phase from first pump reconditions the trap Advantage Clean design with ease of troubleshooting Two dedicated flow ranges pumps Disadvantage Two pumps required PAL Syringe Low flow rate mb Backflush Equilibration of trap m Load sample from loop to trap high flow rate Pump 1 EE Backflush with low flow rate on column detector LC M Pump 2 E Equilibrate trap with high flow rate Pump 1 Low flow rate supports analytical column and LC MS Pump 2 12 Carry over Troubleshootin Carry over and repeatability 2 What is the composition of the analytical sample solvent Wash solvent Sample vial r m What is the composition of the mobile s phase One Wash steps for biological samples e 1st Wash cycle Aqueous solvent e 2nd Wash cycle Organic solvent e 1st Wash cyle before next sample Pre wash with aqueous solvent Eliminate organic solvents in syringe and valve Does the composition of the washing solvents match No Important points to check Influence on Influenceon Remarks carry over repeatability Wash solvents See Point 12 above Does the wash solvent match i
5. buffer salt or high molecular weight compounds Solid Phase Extraction SPE Restricted Access Material RAM First wash step for biological samples must be with aqueous medium followed by organic solvent Last wash step must be with aqueous medium achieved with a pre wash directly before next injection Molecular Imprint Polymer MIP PAL System used as a Syringe Pump Loop is replaced with a cartridge or trap PAL System used as a Syringe Pump to load the cartridge or trap Slow deposition of the sample solution 1 mL s Advantage Simple setup Only one valve and one HPLC pump required lt i Stepper mode PAL lt _ Syringe 1uL s flow rate Waste Analytical column or detector t Pump nano flow Load m gt Backflush Valve System Single Pump with Splitter Sample transfer with high flow rate Control of the analytical flow rate with the splitter device after valve switches Place splitter as close as possible to column Advantage One pump system required Disadvantage 3 valve setup Splitter must work reliably PAL Syringe Waste 1 Sample loop E High flow rate loading trap E Low flow rate for LC MS cam 4 High flow rate loading m gt Low flow rate backflush Valve System and Two pump Setup Two pumps are operated independently of each other
6. full loop 2 ports and 1 engraving Loop overfill full loop Content of 2 ports and 1 engraving Partial loop filling 1 port and 1 engraving Partial loop filling Content of 1 port engraving is injected Valve Bore Size Volume for Valve Volume for Valve Volume for and Valve Type Port Engraving Partial Loop Loop Overfill 0 75 mm Port 1 2725 nL Other ports 2725 nL 6623 nL 3898 nL Cheminert sst Engraving 1173 nL 0 40 mm Port 1 155nL Other ports 155 nL 545 nL 700 nL W Type sst Engraving 390 nL 0 40 mm Port 1 615 nL Other ports 220 nL 1005 nL 785 nL Cheminert sst Engraving 170 nl 0 40 mm Port 1 650nL Other ports 345 nL 1165 nL 820 nL Cheminert PAEK Engraving 170 nL 0 25 mm Port 1 320 nL Other ports 75 nL 465 nL 390 nL Cheminert sst Engraving 70 nL 0 25 mm Port 1 290 nL Other ports 130 nL 490 nL 360 nL Cheminert PAEK Engraving 70 nL 0 15 mm Micro Port 1 273 nL Other ports 30 nL 343 nL 313 nL Cheminert sst Engraving 40 nL 0 15 mm Micro Port 1 514nL Other ports 40 nL 594 nL 554 nL Cheminiert PAEK Engraving 40 nL 0 10 mm Nano Port 1 8 nL Other ports 8 nL 29 nL 21 nb Cheminert PAEK Engraving 13 nL All values are theoretical values Values may change without notice and are provided by VICI Valco Rheodyne PD 7991 Port to port volume 195 nL 294 nL for vertical port Port 1 3 General Rules for Loop Filling Lb sample in Graphic A Mobile phase Flow gt Full loop
7. injection Partial loop filling Overfill Loop 3 to 5 times Reliable working range 20 to 60 of loop content Example 1 Small loop size Loop 20 uL Volume needed to fill loop Example Small loop size Loop 20 wL 4 to 12 uL sample volume 60 to 100 pl Loops with larger volume can be filled within a range of 20 to max 80 of Example 2 Large loop size Loop 200 uL Use at least 300 uL to fill the loop content 200 uL or larger loop Disregarding the rules will result in poor repeatability The reason for these rules is the principle of hydrodynamic flow patterns in the solvent front reaching the loop inlet and outlet See graphic A above Injection speed Example for a 20 uL loop Injection speed is 5 to 10 u s 20 60 Higher speed will cause turbulence in the loop resulting in poor repeatability Reliable working range Injection speed is a PAL method parameter and must be adjusted for the type of solvent viscosity and boiling point loop size or rather loop internal diameter and the valve bore size See the recommended method parameters listed in the PAL firmware software overview If a limited amount of sample solution is available use of low volume pick up mode is recommended The sample solution is embedded in a solvent sandwich separated by small air gaps The sample plug is positioned mid loop Important iv nt Use a solvent with a lower elution power than the starting compos
8. or fused silica See No 7 directly above Valve rotor I Is a change from Valcon H to Valcon E indicated See Point 1 Valve body Observe material advantages and limitations Is a change from sst to PEEK indicated Product information is available from supplier PAL method parameters 7 9 Syringe fill speed Cavitation Gauge 22S or 22 See Point 9 Pullup delay Is the time long enough For recommendations see the PAL Injection speed Is the loop fill speed too high firmware overview or the PAL User Manual Eject speed Is the syringe eject speed Eject speed is a syringe parameter high enough Air bubbles are ejected during fill strokes 0 HPLC column Is the inlet frit clean replaced HPLC System backpressure 1 1 Era al General rule is to flush the column trap Is the selected equilibration time long enough 5 to 10 times with the starting condition of the Is column equilibration timed correctly gradient 12 Detection and integration Is the peak detection and integration verified Is the signal within the dynamic linear range of the detector Peak tailing Baseline assignment S N ratio Area rejected Peak slope detection Linearity CTC Analytics Where design meets performance CTC Analytics AG reserves the right to make improvements and or changes to the products described in this poster at any time without notice In no case shall CTC Ana
9. psi 340 bar C2V 1346D CTC K 6 0 25 1 16 C2V 1C46 C2 13R6 10 to 500 L min Biocompatible Valve C PAEK Valcon E for semi micro columns ID 1 and 2 mm Vertical port Max pressure 5000 psi 340 bar C2V 0006D CTC K 6 0 15 1 16 C2V 0C06 C2 00R6 100 nL min to 100 L min Injection Valve for micro columns C sst Valcon H Vertical port bore 0 25 mm reduced to 0 15 mm at bottom Vertical port Max pressure 5000 psi 340 bar C2V 0346D CTC K 6 0 15 1 16 C2V 0C46 C2 03R6 100 nL min to 100 L min Biocompatible Injection Valve for micro C PAEK Valcon E columns Vertical port bore 0 25 mm reduced to 0 15 mm at bottom Vertical port Max pressure 5000 psi 340 bar CN2 4346D CTC 6 0 10 1 32 CN2 4C461 CN2 43R6 10 nL min to 10 L min Biocompatible Valve for column C PAEK Valcon E switching only Stator sst wetted parts No vertical port PAEK C72VX 1696D CTC K 6 0 25 1 16 C72V 1C96 C72 16R6 10 to 500 L min Valve for ultra high pressure use N60C Valcon E3 Max pressure 15 000 psi 1030 bar Vertical port C72VX 6696D CTC K 6 0 15 1 16 C72V 6C96 C72 66R6 100 nL minto 100 L min Valve for ultra high pressure use C N60C Valcon E3 Max pressure 15 000 psi 1030 bar Vertical port PD7991 6 0 20 1 16 7991 005 7991 999 10 to 500 L min Valve for ultra high pressure use R Ti plated PEEK blend Vertical port bore 0 25 mm Vertical port with SPC 1 RPC 10 reduced to 0 20 mm at bottom Max pressure 15 000 psi 1030 bar Listing is not complete Only most common valve types listed Other co
10. Asisten Prep and Load Platform Tips and Hints for PAL Systems including general technical HPLC information 1 Valve Type and Characteristics 2 Valve Type Bore Size versus Valve Volume 3 General Rules for Loop Filling 4 Low Volume Pick up 5 Plumbing Diagram for the W and Cheminert Valve Types 7 Tubing Internal Diameter versus Flow Rate 8 Needle Guide and Needle Seals 9 Syringes and Needles 10 Microtiter Deepwell Plates and the Piercing of Foils 11 Examples of Plumbing Diagrams for Micro Nano Flow Applications 12 Carry over Troubleshooting 1 Valve T nd Characteristi Valve P N No of Ports Valve Tube Stator Rotor Flow Rate Range Remarks Valve Type Bore Size Connect P N P N Inlet Port inmm ininch Material Material C2V 3006 CTC K 6 0 75 1 16 C2V 3C06 C2 30R6 5 to 100 mL min Preparative application L sst Valcon H Bore size requires needle Gauge 19 Vertical port Max pressure 5000 psi 340 bar DC6WK K 6 0 40 1 16 na SSAC6W 0 5 to 5 0 mL min Valve for standard HPLC application W sst Valcon H Column ID 4 mm flow 1 mL min Vertical port Max pressure 5000 psi 340 bar C2V 2346 CTC K 6 0 40 1 16 C2V 2C46 C2 23R6 0 5 to 5 0 mL min Biocompatible Valve for standard C PAEK Valcon E HPLC flow rates Vertical port Max pressure 5000 psi 340 bar Q2V 1006D CTC K 6 0 25 1 16 C2V 1C06 C2 10R6 10 to 500 L min Valve for semi micro columns C sst Valcon H ID 1 and 2 mm Vertical port Max pressure 5000
11. ctly Loss of steps from Z stepper motor PAL will recover steps at point of axis referencing Possibility that next object in cycle will not be detected if next step follows directly without axis referencing Example Wash Station Needle Guide and Seals replacement information Needle Seal and Needle Guide Change the Needle Seal at regular intervals Do not use steel ferrules for a PAEK valve stator Select the appropriate Needle Seal Guide as listed in the table below o inn EE Effect if Needle Seal is not tight Loss of sample or no sample transfer to valve Filling injection port Port 1 with sample solution Carry over Needle Penetration in Valve Inlet Port Path Menu Utilities Injectors F3 move to injector e Activate Needle Penetration e Move needle down slowly until a clearly audible click occurs e Motor pressure release 2 steps up e Check position again Correct position shown in graphic Needle Seal H anonnndnann ED Needle Penetration in wrong position If needle is too high No seal possible Carry over If needle is too low Needle tip hits top of valve body Possible needle distortion P N Description Remarks PAL NdlSeal Needle Seal Gauge 22 Transparent FEP tubing wit
12. e brine Syringe CIC Hamilton Syringe description max vol ml Article No P N Description GlassOD mm Gauge Point style PST Remarks X Type syringes SyrX for high throughput and biocompatible applications 25 SyrX G25 22S 3 1702 CTC 225 3 inert 77 22S 3 Metal flange red color 50 SyrX G50 22S 3 1702 CTC 22S 3 inert 77 22S 3 Metal flange red color 100 SyrX G100 225 3 1710 CTC 225 3 inert 6 6 22S 3 Metal flange red color 100 SyrX G100 22 3 1710 CTC 22 3 inert 6 6 22 3 Metal flange blue color C Type syringes SyrC standard HPLC syringes 10 SyrC G10 225 3 203194 1701 CTC 225 3 6 6 225 3 Metal flange 25 SyrC G25 22S 3 203075 1702 CTC 22S 3 77 228 3 Metal flange 100 SyrC G100 22S 3 203077 1710 CTC 225 3 6 6 225 3 Metal flange 100 SyrC G100 22 3 203235 1710 CTC 22 3 6 6 22 3 Metal flange 250 SyrC G250 22 3 203079 1725 CTC 22 3 Til 22 3 Metal flange 500 SyrC G500 22 3 203349 1750 CTC 22 3 77 22 3 Metal flange 1000 SyrC G1000 22 3 203081 1001 CTC 22 3 Hil 22 5 Glass flange 2500 SyrC G2500 22 3 203083 1002 CTC 22 3 9 5 22 3 Glass flange 5000 SyrC G5000 22 3 203085 1005 CTC 22 3 155 22 3 Glass flange G Gastight Plunger Teflon Plunger Tip for X Line Teflon Polymer mix Replacement plungers are available at CTC X Type Syringes are sold exclusively by CTC Flange color indicates needle internal diameter Example Gauge 22 blue or Gauge 22S red Syringe Needles Standard Needle for HPLC Technique
13. h sst ferrule PAL NdlSealP Needle Seal for PAEK Valve PEEK ferrule to protect PAEK valve stator Gauge 22 Transparent FEP tubing PAL NdlSealR Needle Seal 22 Rheodyne Transparent FEP tubing with sst Rheodyne ferrule PAL NdlSeal 19 Needle Seal Gauge 19 Blue colored Teflon tubing to differentiate from Gauge 22 sst ferrule MV 30 12 Needle Guide for Gauge 22 Needles sst material MV 30 30 Needle Guide for Gauge 22 Needles PEEK material for PAEK valve MV 30 52 Needle Guide for Gauge 19 Needles Groove in rim to differentiate from Gauge 22 type sst material 2 Syringes and Need es CTC Analytics X Type Syringe developed for e High throughput applications Biological samples drug discovery proteomics Practically zero carry over EXtra long life and EXtra low carry over No metal contact Inert smooth surface I Adjustable plunger Color coded flange Plunger stem Glass barrel inner New inert plunger Needle fixation to Deactivated needle with _ protects plunger indicating needle sealed with surface polished tip polymer with barrel No sample extremely smooth inorganic tip from being internal diameter temperature and sealed for significantly longer contact with glue or glass layer prevents metal squeezed and solvent inertness Stable lifetime cement contact Stable to organic stable material to organic solvents solvents acids and bases acids and bases or or brin
14. ition of the PONEN mobile phase gradient alr sample A sample macro for the Cycle Composer software is available from any CTC air Analytics representative The macro is written with built in flexibility to adapt to solvent syringe loop and sample volumes Sample in Sample in Vent waste 7 ent waste To column Sample loo Sample loo Carrier mobile p P P phase Carrier mobile phase Carrier mobile phase VICI Valco W Type Valve VICI Valco Cheminert Type Valve Rheodyne PD7991 K Conical rotary valves Flat plate rotary valve 15 000 psi 1030 bar Flat plate rotary valve No biocompatible version available Biocompatible version available Rheodyne Make Before Break MBB technology features reduced pressure transients during valve actuation Do not use a nut or a ferrule from a vendor other than specified for the product While tightening the nut keep tubing tightly positioned to ensure correct pilot distance Do not over tighten the nut ferrule Do not reuse an installed nut ferrule for any other connection Eliminate trapped air by installing nut ferrule in wetted ports only Disregarding these basic rules will lead to Dead volume Peak deformation or peak splitting Carry over effect Male nut internal Ferrule Tubing Pilot
15. lytics AG be held liable for any coincidental or consequential damage in connection with or arising from the use of this information 2008 CTC Analytics AG All rights reserved CTC acknowledges all tradenames and trademarks used as the property of their respective owners PAL is a registered trademark of CTC Analytics AG Switzerland CTC Analytics AG Tel 41 61 7658100 Industriestrasse 20 Fax 41 61 7658199 CH 4222 Zwingen E mail support ctc ch Switzerland Web www palsystem com P N PAL HintsLC 08
16. nfigurations such as 10 port or internal loop valves are available Key and Remarks to the Table Valve Type and Characteristics Rotor material e Valcon H Reinforced carbon fiber composite PTFE lubricated inert P N engineered polymer Standard material from VICI Valco e CTC Specific modifications for CTC Analytics AG e Valcon E Polyarylether ketone Teflon PAEK Teflon material if biocompatibility is required e Valcon T Polyimide Vespel PTFE carbon composite A choice when high temperature is required e Valcon E3 Wear resistant coating for UHPLC proprietary VICI Valco K indicates a kit containing nuts ferrules Needle Guide Needle Seals and Teflon waste tubing Valve type e ee ae rotary valve VICI Valco For material specific information and limitations consult VICI Valco product e C Cheminert ype Flat plate rotary valve VICI Valco information bulletin Plumbing for two different valve types is shown below R Rheodyne flat plate rotary valve Bore Size Valve connection port inlet outlet path diameter See graphic at right Stator sst Stainless steel N60 corresponds to S316 N60C Stator coated for UHPLC use PAEK polymer related to well known PEEK material Rotor engraving Bore size diameter 2 Valve Type Bore Size versus Valve Volume Valve volume specification Sample loss at injection Valve contribution not overfill volume Loop overfill
17. parative application 0 040 1 0 mm 2 5 Same ID for entire HPLC sytem plumbing to avoid backpressure 0 40 mm 0 5 to 5 mL min 0 125 3 18 mm 1 Reservoir Standard HPLC 0 020 0 50 mm 2 3 Pump to Valve Column ID 4mm 0 010 0 25 mm 4 5 Valve to Detector 0 25 mm 10 to 500 pL min 0 040 1 0 mm 1 Reservoir Standard HPLC 0 010 0 25 mm 2 3 Pump to Valve Column ID 1 to 2 mm 0 005 0 13 mm 4 5 Valve to Detector 0 15 mm 100 nL to 100 wL min 0 020 0 50 mm 1 Reservoir Micro flow application 0 020 0 50 mm 2 Pump Head to Mixing T 0 005 0 13 mm 3 Mixing T to Valve 0 005 0 13 mm 4 5 Valve to Detector 0 10 mm 10 nL to 10 L min 0 020 0 50 mm 1 Reservoir Nano flow application Fused silica 50 ym Fused silica 50 wm Ome Fused silica 25 um 2 Pump Head to Mixing T Mixing T to Detector flow rates gt 1 wL min 3 5 Mixing T to Detector flow rates lt 1 wL min Data for Tubing IDs are recommendations only Variations depend on the application mobile phase flow rate column ID and sample load Column Tubing from to Solvent reservoir to pump Pump head to mixing T Mixing T to injection valve Injection valve to column Column to detector aAePWN 8 Needle Guide and Needle Seals Positioning of Injection Unit on Valve Needle Guide Position Injection Unit approx 2 mm above bottom of Valve Needle Guide Lap 2 mm gap Effect if Injection Unit is not positioned corre
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