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PAL System Setup Guide

1. SPE Filtration Manufacturers Phases Membranes Pore Sizes Biotage Polar Nylon 0 45um Jordi Reverse PES Orochem lon Exchange Polycarbonate UCT Mixed Mode Polypropylene Varian Special PTFE Polymeric PVDF Filters are available with a 1 0 um PES pre filter ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 9 Example of PAL Hardware Configuration for ITSP For illustration purposes let us assume an HTS PAL or MPS 3 is being configured to prepare samples for an LC application using ITSP It has the following accessories and modifications 1 N A LC Injection Valve 1 N A Fast Wash Station with 2 500mL Solvent Reservoirs 1 N A Syringe Adapter for 100uL Syringe 1 N A 100uL X Type Syringe 22S gauge point style3 1 N A Holder for 3 100mL Ink Well Solvent Reservoirs 2 N A Standard Tray Holder for 2 microplates holds sample trays elution trays and ITSP tray on PAL HW KIT ITSP Hardware Kit N A 350uL polypropylene shallow round well conical bottom 96 position microplate raw sample tray for filtration methods 2 N A 96 position deep well plate with 1 0mL clear conical inserts filtrate eluate tray and raw sample tray for SPE methods The ITSP tray holds 96 ITSP cartridges It is therefore convenient to operate with 96 position trays for samples and eluates in the demonstrations shown in this Set Up Guide although ITSP can work with any format of tr
2. These solvent reservoirs hold conditioning column wash and elution solvents used in the SPE method Depending on your method requirements you may need two sets In that case you can rename the second set SolvRes4 SolvRes5 and SolvRes6 f Positions ITSPdisp this is the location for ITSP cartridge disposal container usually located near the front of the Fast Wash Station The packing container in which the cartridges are shipped can be used for this purpose It is shipped with a biohazard sticker which can be attached to the container for proper disposal of used cartridges You don t need to add this object if you are planning to return spent cartridges to the ITSPprep tray after use Step 6 Backup the PAL configuration When you have finished configuring the PAL backup the configuration using the following steps a Click the Backup button in the main PAL Loader screen A dialog box entitled Backup Target Memory will be displayed b The path storage location and file name to be assigned to the new backup will be shown A customized path and or filename may be entered c Click the Backup button d Backup will proceed and information on the backed up components will be displayed in the Backup Log window The process will take several minutes The message Backup of Target Memory succeeded indicates a successful backup e Click the Close button to exit the Backup dialog box Click the Start PAL button to te
3. 2000 TRAY ITSPsmpl None GHI MT96ITSP 64500 135900 2000 TRAY ITSPprep None FGH ITSP96 64500 47200 2000 TRAY ITSPelut None GHI DW96ITSP 64500 135900 2000 ITSP Trays continued ooo ooo0o0 0 0 0 0 fo oO Oo O10 01 0 01 0 0 oO fo TRAY MT1 Frnt TRAY MT1 Rear TRAY MT2 Frnt TRAY MT3 Frnt TRAY MT3 Rear TRAY ITSPsmpl TRAY ITSPprep TRAY ITSPelut ITSP Injectors o p o oo oO oooooooo o a o e o e o e o o0 9 090 9 090 9 090 9 o oo00 0 0 0 0 0 0 oo00 0 0 0 0 0 0 oO D INJECTOR ITSPwast 23000 20000 None None NOTE If you cannot prepare samples with the cartridge in the cartridge tray e g because you wish to locate the cartridge tray in a stack then use INJECTOR ITSPwast to locate the cartridge over a waste port when applying solvents Contact ITSP Solutions for hardware modification options and required software changes CAUTION If TSPwast is located at the same location as the PAL default Waste then unexpected collisions may occur between the Injection Unit and Objects on the PAL deck especially during reset and error recovery routines In any case whether ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 33 ITSPwast is located at the same location as the PAL default Waste or any other waste location on the PAL deck the PAL may lose track of an ITSP cartridge that is left in this position during rese
4. Setting the Z offset 0 4mm above the septum allows 0 4mm of vertical overlap between the ITSP Lower Needle Guide Attachment nipple and the ITSP cartridge seal crown while still leaving 0 4mm tolerance in the Z axis The PAL can use this overlap to stabilize the ITSP cartridge over the elution well when it removes the needle from the cartridge to aspirate air for an air flush In the event this process appears to be unreliable for robust performance then simply return the cartridge temporarily to the ITSPprep tray between the elution step and the air flush step immediately following elution ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 28 For a more complete list of troubleshooting tasks please refer to the PAL Manuals from CTC PAL Cycle Composer User Manual PAL Macro Editor User Manual PAL Loader User Manual PAL Object Manager User Manual PAL Operation Recommendations Manual Tips and Hints for PAL Systems Manual HTS PAL User Manual GC PAL User Manual Combi PAL User Manual Updated versions of these manuals can be obtained from your instrument vendor or may be available from the CTC website www cte ch More information about ITSP Solutions products including ITSP can be found at www ITSP Solutions com ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 29 Appendix The following
5. time analysis time compared to a batch mode of operation ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 18 Programming the PAL for Sample Filtration using ITSP The PAL will follow these general steps as an example which can be customized to your specific analytical method to perform sample filtration on using ITSP by aspirating the sample through the ITSP filtration cartridge The ITSP filtration cartridge will be moved to the sample tray The sample will be aspirated through the ITSP cartridge into the PAL syringe The ITSP cartridge will be discarded The filtered sample will be dispensed into a filtrate tray The filtrate can then be injected using a standard PAL injection cycle These steps are repeated for each sample in the Sample List Typically the time required for the complete sample preparation injection process using ITSP is between 2 and 4 minutes Often the analysis method requires as long as or longer than this length of time Therefore there is no change to the throughput of the analytical instrument while there is a reduction in the overall time for the complete analysis sample preparation time analysis time compared to a batch mode of operation ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 19 Recommendations for Operating the PAL with ITSP ITSP Solutions suppor
6. PAL for you You will then need to add the appropriate hard ware and software modifications to use ITSP cartridges with your application With the example PAL hardware configuration as mentioned on the previous pages the remain der of this Guide will describe how to modify the PAL for using ITSP Step 2 Load the Objects into the PAL The ITSP Hardware Kit ships with preconfigured Objects that can be loaded into the PAL using Object Manager software from CTC version 2 1 0 or later If you are unsure how to use Object Manager the instrument vendor from whom the instrument was purchased or ITSP Solutions can help you through this process Load the following objects into the PAL TRAY HOLDERS MTHldri WASH STATIONS Washi MTHIdr2 Wash2 TRAY TYPES MT96 INJECTORS Waste DW96 LC Vivi ITSP96 MT96ITSP DW96ITSP TRAYS MT1Front VIALS SolvRes1 MT2Rear SolvRes2 ITSPsmpl SolvRes3 ITSPprep ITSPelut POSITIONS ITSPdisp The items marked with an asterisk are loaded from the ITSP Upgrade Object files The rest are loaded from the standard PAL Object files Position ITSPdisp can be added as a location for a disposal cup to throw away spent ITSP cartridges instead of returning them to ITSPprep ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 12 Step 3 Train the Tray Holder Reference Positions NOTE Remove the Syringe from the Injection Unit before performing
7. deep into the cartridge the needle will not have enough leverage against the bore of the cartridge to reliably maintain a vertical orientation during transport Penetrating too far of course can cause coring of the sorbent bed or filter membrane The ideal penetration depth is 20mm 6 Using the PAL objects provided by ITSP Solutions for tray types appropriate to the PAL deck configuration is important Tray type ITSP96 has the appropriate XYZ axis settings for the ITSPprep tray including the height of the seal crown above the septa as well as the ideal 20mm needle penetration depth The other ITSP tray type objects have the Z axis setting modified from their corresponding tray types to allow for the position of an ITSP cartridge above the tray as well as the ideal 20mm needle penetration depth But even when using the correct tray types you still have to train ITSP tray positions before operation 7 During programming and operation of an ITSP PAL it is vital that the ITSP wipe action unblocking the needle guide then retracting the needle then moving 6mm and 3mm in the Y and Z directions similar to magnetic vial transfer always be performed whenever the PAL leaves the ITSPprep tray without the ITSP cartridge Failure to include this step will predictably result in a crash or a bent needle usually in less than 100 cycle iterations ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing IT
8. including electronic storage and retrieval or translation into a foreign language without the prior agreement and written consent from the copyright owner 2013 ITSP Solutions Inc All rights reserved ITSP is protected under the following US Patents 6 859 615 amp 7 001 774 Canadian Patent 2 316 648 and European Block Patent EP 1174 701 Foreign Patents Pending ITSP is a trademark of ITSP Solutions Inc
9. might be helpful to write down the Offset X and Y parameters for MT1Rear You will use them for the ITSPsmpl tray position It is the same tray with ITSP cartridges positioned above the vials or inserts having the Offset Z position adjusted ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 15 for the height of the ITSP cartridge and the Needle Penetration adjusted for the depth of the ITSP cartridge An ITSP cartridge should be held in position above the tray when training Reference Positions for trays ITSPsmpl and ITSPelut ITSPsmpl of tray type MT96ITSP this 96 position shallow well microplate is the filtration sample tray located in the rear position of MTHIdr1 Its Reference Position is trained with an ITSP cartridge positioned above it MT1iFront of tray type DW96 this 96 position deep well microplate is the SPE sample tray located in the front position of MTHIdr1 MT2Rear of tray type DW96 this 96 position deep well microplate is the eluate or filtrate tray located in the rear position of MTHIdr2 It is also the injection sample tray for the LC Inject cycle NOTE It might be helpful to write down the Offset X and Y parameters for MT2Rear for later use You will use them for the TSPelut tray position It is the same tray with ITSP cartridges positioned above the vials or inserts having the Offset Z position adjusted for the height of the ITSP ca
10. pages contain reference materials for using ITSP on the PAL ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 30 Filename Conventions The following file extensions are used by Cycle Composer e pme PAL Method e pma PAL Macro e psl PAL Sample List e pol PAL Object List The Macro files SL Entry pma Method Entry pma Method Exit pma and e SLExit pma contain Macros which are automatically appended at the front and at the end of the Method and Sequence They are used for initialization before the Method and the Sequence and for cleanup after the Method and the Sequence The Macro files e Error Entry pma e Error Exit pma and e Reset pma contain Macros which are used for managing errors and interruptions The Object List file e latest_pal pol contains the most recent set of loaded Objects in the PAL e Traylnfo txt contains Tray Definitions The files e your sample list psl e your method pme and e your macro pma are the Cycle Composer Sample List Method and Macro files that pertain to your specific method All these files should be in each method folder directory Different Sample Lists Methods and Macros can reside in the same folder or in different folders at the end user s discretion but Cycle Composer will only operate from one folder at a time ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Se
11. to your applications In preparing this Guide it was necessary to make simplifying assumptions in the configuration in order to demonstrate a detailed hardware and software setup Some items used to perform the demonstrations in this Guide may not be practical for your application such as syringe size vial size types of trays etc Adaptation of the ITSP Methods in this Guide to your application may require different hardware If you are unsure which hardware options to order please contact your vendor or ITSP Solutions for assistance Adaptation of the ITSP Methods in this Guide to your application may require changes to the software Methods supplied with the ITSP Hardware Kit Please contact the vendor from whom the Kit was purchased or ITSP Solutions for assistance Please be aware that in some cases there may be additional charges associated with this service The ITSP Hardware Kit ships with hardware and software modifications necessary to convert a standard PAL system to be able to automate SPE and filtration sample preparation methods for LC and GC applications using ITSP cartridges The hardware modifications include an ITSP prep waste station see Figure 2 and an ITSP Lower Needle Guide replacement see Figure 3 for the Injection Unit Figure 2 ITSP Prep Waste Station Figure 3 ITSP Lower Needle Guide ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 8
12. 0000 None _TRAY_TYPE MW96ITSP G 31000 27000 None 4000 40000 None _TRAY_TYPE DW96ITSP_ H 44000 30000 None 4000 40000 None TRAY_TYPE VT54ITSP_ 22500 38500 None 4000 40000 None TRAY_TYPE _ITSP96 H 47000 17000 None 4000 40000 None _ TRAY_TYPE ITSP96v0 H 41000 17000 None 4000 40000 None ITSP Tray Types continued STRAYTYPE MT96 5000 10000 10500 osoo 15000 100 o TRAYTYPE mwas sooo 10000 28000 2700 1500 moo o TRAY TYPE Dwe sooo 10000 37000 3600 1sooo moo o TRAY TYPE vsa sooo 10000 32000 s1000 900 mso o TRAY TYPE DW96Mrer 5000 10000 37000 36000 1so0 moo o IRAYTYPE DMa6ITSP_ sooo 10000 23000 20000 1500 s200 o TRAY TYPE MT96ITSP Sooo 10000 23000 20000 1soo0 moo o STRAYTYPE MWS6ITSP 5o00 10000 23000 20000 15000 moo o TRAY_TYPE DWw9EITSP Sooo 10000 23000 20000 1500 moo o TRAYTYPE VTSaITsP_ sooo 10000 23000 2000 900 mso o Rav TYPE 1TSP96 sooo 10000 23000 20000 12000 10200 o TRAY TYPE ITSP96v0 sooo 10000 23000 20000 1500 moo o NOTE ITSP TRAY TYPE ITSP96v0 Legacy ITSP Cartridge Tray 12x8 All X Y and Z dimensions of the ITSP cartridge tray have changed as of June 1 2009 If you are using the original legacy version of the IT
13. First and foremost the needle needs to puncture the ITSP cartridge seal at the very center of the septum concentric with the z axis of the cartridge The center bore of the ITSP cartridge needle guide component is only slightly larger than the O D of a 22G syringe needle The target we are aiming for is the cartridge bore not the much larger diameter of the exposed septum on the seal 2 Secondly but equally important the needle needs to be inserted deep enough into the bore of the ITSP cartridge to provide a long enough moment arm for the needle to have sufficient leverage against the sides of the bore to reliably hold the cartridge in a vertical orientation during transport The 20mm needle penetration default setting of the ITSP PAL Tray Type objects achieves this objective 3 It is important to install the PAL ITSP Lower Needle Guide on the PAL and to check that the needle tip remains flush with the bottom of the PAL ITSP Lower Needle Guide when the PAL is at rest It has a nipple on the bottom that is designed to mate with the crown on the top of the ITSP cartridge seal to align the Z axes of the needle and cartridge If it is set too high the needle may catch on the inside edge of the ITSP Lower Needle Guide Attachment which can Z the needle If it is set too low the needle can stick in the septum or seal crown when moving toward or away from an ITSP cartridge causing an error 4 Those who use or set up a PAL for operation may
14. ITSP Hardware Kit Contents This Set Up Guide is for use with ITSP Solutions Product No HW KIT ITSP Hardware Kit Included in the kit are the following ITSP Solutions products e 1 HW 96P 96 position Aluminum Microplate see Figure 2 this is the tray that holds the ITSP cartridges on the PAL e 1 HW WR Teflon Waste Receptacle see Figure 2 this is a waste receptacle that is mounted underneath the ITSP cartridge tray It is used to carry away waste when conditioning loading and washing the ITSP cartridge in the ITSP tray e 1 HW LNG ITSP Lower Needle Guide see Figure 3 this replaces the lower needle guide on the PAL injection unit It ensures proper mating to the ITSP cartridge by centering the needle in the ITSP seal e 4 MicroLiter Analytical Supplies Inc 07 1200 96 position shallow well uLplate 350uL This plate is used to hold raw samples when performing ITSP filtration methods on the PAL e 1 USB drive with instructions and examples for using ITSP on the PAL e 2 MicroLiter Analytical Supplies Inc 07 8000 1000 96 position deep well uLplate with 1 0mL conical bottom clear glass inserts These plates are used to hold samples eluates and filtrates Please contact your instrument vendor or ITSP Solutions for sample ITSP SPE and filtration cartridges when adapting your application or method for ITSP on the PAL ITSP cartridges are available in a wide variety of specifications including
15. ITSP by MicroLiter iOO a E E E E A 855 395 8300 706 395 8300 www lTSPSolutions com 212 Northlake Drive Hartwell GA 30643 ITSP Set Up Guide Installing ITSP on the PAL For HW KIT ITSP Hardware Kit Set Up Guide Installing ITSP on the PAL ITSP SOLUTIONS INC Set Up Guide Installing ITSP on the PAL For ITSP Solutions Product No HW KIT ITSP Hardware Kit 4th Edition April 2013 The information in this document is subject to change without notice ITSP Solutions Inc makes no warranty of any kind with regard to this material including but not limited to the implied warranties or merchantability and fitness for a particular purpose ITSP Solutions Inc shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Contents of this publication may not be reproduced in any form or by any means including electronic storage and retrieval or translation into a foreign language without the prior agreement and written consent from the copyright owner Copyright 2013 ITSP Solutions Inc All rights reserved ITSP is protected under the following US Patents 6 859 615 amp 7 001 774 Canadian Patent 2 316 648 and European Block Patent EP 1174 701 Other US and Foreign Patents Pending ITSP is a trademark of ITSP Solutions Inc ITSP Solutions Inc Document ISDCooo1 Rev Da
16. ITSP cartridge is on the needle and some are not All positions paths and movements should be checked before use e When using Macros to operate the PAL with ITSP the PAL Macro Atom BLOCK NDLG Block NdlGuide On Check Released Off is used to grasp the ITSP cartridge in preparation for moving it around the deck and the PAL Macro Atom BLOCK NDLG Block NdlGuide Off Check Released Off is used to release the ITSP cartridge in preparation for leaving it in place at a position on the deck This means that the PAL Lower Needle Guide is not used to detect object positions in the Z axis when transporting an ITSP cartridge Therefore extra care is required in the setup and training of affected locations e During setup use the object setup function Check Pos on the PAL hand held terminal to verify the position of the PAL Injection Unit Lower Needle Guide in relation to the 8mm crimp seals on the ITSP cartridges It is important that the Needle Guide is well centered so that the syringe needle hits dead center on the ITSP cartridge This ensures that the ITSP cartridge will be transported accurately and reliably to different locations ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 20 e When locating trays on the PAL deck during setup it is important to position taller trays toward the front and shorter trays toward the rear Locat
17. J or later B or later for xt e Cycle Composer version 1 5 4 or later 1 6 0 or later for xt Check that the PAL Injection Unit Lower Needle Guide was correctly replaced with the ITSP Lower Needle Guide HW LNG Check that the needle tip is flush with the bottom surface of the ITSP Lower Needle Guide Attachment when the PAL is at rest If it is set too high the needle may catch on the inside edge of the ITSP Lower Needle Guide Attachment which can Z the needle If it is set too low the needle can stick in the septum or seal crown when moving toward or away from an ITSP cartridge causing an error During operation the set screw which holds the lower needle guide block may slip This is sometimes caused by the lower needle guide hitting the block during the release of the ITSP cartridge at ITSPdisp by the spring force of the bungee It is important that this set screw be securely fastened and periodically checked An alternative approach in the method is to return the ITSP cartridge to the ITSPprep tray after use instead of disposing of it at position ITSPdisp Check that the correct hardware revision of the ITSPprep tray is being used The latest version has larger sloped well walls requiring a wider X Y spacing of the wells to help guide an ITSP cartridge back in to place when returning it to the ITSPprep tray It also has a taller Teflon waste port underneath to improve fluid flow to the drain by increasing the slope This chan
18. SP cartridge tray shipped prior to June 1 2009 then use the ITSP96v0 tray type in the Legacy ITSP96 pol Object List file instead of the ITSP96 tray type in the ITSP96 pol Object List file The current version of the ITSP cartridge tray supports much more robust automation Contact ITSP Solutions if you wish to upgrade a legacy system to the current version x O O O x ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 32 NOTE Tray Type DW96Mrrr is the same as DW96 with the order of rows mirrored A1 is now H1 B1 is now G1 etc This tray type can be used where chain of custody is an issue and you do not wish to transport a cartridge over an unused well of the elution tray Its partner tray type is DM96ITSP which is the same as DW9GITSP with the rows mirrored ITSP Tray Holders TRAY_HOLDER MTHldri 0 138500 126500 None None Auto None None TRAY_HOLDER MTHldr2 0 138500 126500 None None Auto None None TRAY_HOLDER MTHldr3 0 138500 126500 None None Auto None None ITSP Trays TRAY MT1 Frnt None FGHI DW96 64500 47200 2000 TRAY MT1 Rear None GHI DW96 64500 135900 2000 TRAY MT2 Frnt None FGHI DW96 64500 47200 2000 fo lo fo Ko TRAY MT2 Rear None GHI DW96 64500 135900 2000 TRAY MT3 Frnt None FGHI DW96 64500 47200 2000 TRAY MT3 Rear None GHI DW96 64500 135900
19. SP on the PAL Page 24 Troubleshooting Below is a chart of some common troubleshooting tasks encountered when using ITSP for the first time Symptom or Error Message Possible Cause Recommended Action Object s not defined correctly Define Object s correctly Short Trays positioned in front Relocate short Trays behind of tall Trays in the same Tray Tall trays Collisions of the Injection Unit Holder Insufficient clearance set on Increase Z Retract value in some Object on the PAL the Z direction for the affected Object Incorrect Z value for the Redefine Z value for the Error Message 115 affected Object affected Object Object Not Detected Missing sample vial in tray Insert sample vial in tray Object is e g Tray1 LCVIv1 Z tolerance value set too low for Increase Z tolerance for the the affected Object affected Object Incorrect Z value for the Redefine Z value for the Error Message 147 affected Object affected Object Object Collision before Z Tolerance 7 tolerance value set too low for Increase Z tolerance for the _ Object is e g Tray1 LCVIv1 the affected Object affected Object Injection Unit dislodges ITSP Injection Unit does not clear the Insert an ITSP cartridge cartridge when moving about the ITSP cartridge in place properly wipe step at the appropriate PAL deck location in the method ITSP cartridge wipe step is used to wipe the cartridge off the Injection Unit Low
20. The Injection Unit will move to the HOME position 7 Verify the defined X Y Z Positions by pressing F1 Check Pos MTHIdr1 this is the left Tray Holder where you will place the sample trays MTHIdr2 this is the right Tray Holder where you will place the ITSP prep and eluate filtrate trays rT AgRP ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 13 Step 4 Install the ITSP Hardware Kit PAL Modifications Install the PAL modifications from the ITSP Hardware Kit before setting up the remaining Objects Figure 7 ITSP Lower Needle Guide Use a Torx T10 driver to replace the PAL Injection Unit Lower Needle with the ITSP Lower Needle Guide supplied in the kit Guide see Figure 7 Be careful not to let the short bungee cord slip off the right support rod or else you will need to disassemble the Injection Unit to re secure it An optional magnetic ITSP Lower Needle Guide is available if you need to transfer vials magnetically Install the ITSP Waste Receptacle see Figure 8 on the front position of the Tray Holder MTHIdr2 and screw the hose nipple screw into the Waste Receptacle through the calibration hole in the tray It is important for robust operation that you take extra care to squarely and securely mount the Waste Receptacle into position Then mount the ITSPprep tray on top and load it with cartridges before training this position on th
21. artridges on the PAL These files are installed in the appropriate application directory based on the specific instrument controlling the PAL If you are unsure where to put these files the instrument vendor from whom the instrument was purchased or ITSP Solutions can help you through this process ITSP Solutions can provide support with ITSP applications on the PAL up to the point of injection on the LC valve or GC inlet For assistance beyond the scope of applying ITSP on the PAL please contact the instrument vendor from whom the instrument was purchased Please be aware there may be charges assessed by the instrument vendor associated with these services ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 7 Supported Hardware ITSP Solutions supports the use of ITSP cartridges on any PAL system having sufficient accessories to prepare samples for LC and GC applications and having sufficient spare memory capacity to load the ITSP Objects Setup will require several pieces of hardware e g tray holders solvent reservoirs etc that may have shipped with the purchase of your PAL If you do not have these items ITSP Solutions can provide these at regular retail price You may also contact your local CTC dealer to acquire the necessary hardware The PAL is an extremely flexible platform with many options This flexibility allows a lab to customize a configuration tailored
22. ays vials and inserts configured on the PAL The ITSPprep tray mounts in the front tray position of a standard 2 position microplate tray holder over the positioning hole The PAL Injection Unit is modified by replacing the standard PAL Lower Needle Guide with the ITSP Lower Needle Guide included in the ITSP Hardware Kit The PAL system should be set up with locations for the following components e atray to hold the filtration raw samples if performing ITSP filtration which we will refer to as tray MT1 Rear or as ITSPsmpl in this guide a tray to hold the SPE raw samples if performing ITSP SPE which we will refer to as tray MT1 Frnt in this guide a tray to hold the eluted fraction or the filtrate before injecting which we will refer to as MT2 Rear or as ITSPelut in this guide a tray to hold the ITSP cartridges included in the Hardware Kit which we will refer to as ITSPprep in this guide solvent reservoirs for conditioning washing and eluting solvents a wash waste station and an LC injection valve for LC applications ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 10 The diagram in Figure 4 illustrates the relative locations of these objects on the PAL deck Tray Holder Tray Holder Solvent Fast Wash Injection Valve MTHidr1 MTHIdr2 Reservoir Holder Station for raw samples for SPE for solvents for cleaning for LC injection Filtration Samp
23. c is a consumable cartridge designed to automate filtration and solid phase extraction SPE on the Prep And Load PAL from CTC Analytics AG CTC or on the Gerstel MPS MultiPurpose Sampler The PAL or MPS can move the ITSP cartridge anywhere on the deck which allows significant flexibility in sample prep automation The ITSP cartridge requires a minimal amount of hardware modification to enable a standard PAL or MPS to prepare samples for both LC and GC applications Throughout this manual we will use the generic term PAL to refer to the CTC PAL or the Gerstel MPS Modifying a PAL for ITSP does not take away any prior functionality but simply adds the ability for the PAL to perform ITSP filtration and SPE methods in addition to any previous methods it was performing ITSP offers an excellent opportunity to improve the performance and throughput of the analytical lab Combining the sample preparation with the analysis of samples by the Mass Spectrometer can provide significant reductions some labs report 90 or more in solvents client sample requirements labor and hazardous waste for sample prep Explanation of the ITSP Cartridge The ITSP cartridge combines several features from the familiar manual SPE column and vacuum manifold into a proprietary cartridge designed to miniaturize and automate sample prep methods The cutaway drawing in Figure 1 illustrates the cartridge construction The needle of the syringe is inserted thro
24. cseciectasentpercavsaccicaenieasciantetaaateceianadeseaciaaeueias 3 Figure 2 ITSP Prep Waste Station sess sicsisiscsscssssriccrssserirssscsissosiseesescissrdesssisisosdsssssos s 7 Figure 3 ITSP Lower Needle Guide ccsscccsssccsssscecssseccssseccssseccesscceessecesssnsecseeeecsseeees 7 Figure 4 Example of Object Locations on an ITSP PAL ceeeesecseeceeeceeeeeeseeeeeeeeeees 10 Figure 5 Example ITSP Setup s sssesssesssosessesseessessseresesesstesseesoererereesseessesssesereseessesseeees 10 Figure 6 Reference Position for Tray Holders MTHIdr1 and MTHldr2 eee 12 Figure 7 ITSP Lower Needle Guide seesseessoeessseesseessssessseesseesssseesseessooessseesseessssessseesso 13 Figure 8 ITSP Waste Receptacle sisic sc cccscasaccasaaccaccsesssacevecesecesencadaeteancacaceasaccasiasaacesesesasets 13 Figure 9 Reference Position for ITSPelut wescesnssudecanstisursirnersnsnsnedairamamndanens 15 Please Note ITSP Installation Services Disclaimer If you purchase an ITSP Hardware Kit directly from ITSP Solutions to upgrade an existing PAL or MPS and decide that you would like assistance from your instrument vendor for hardware installation or software programming please be aware that they may charge for that service ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 3 Using ITSP on the PAL Instrument Top Sample Prep ITSP from ITSP Solutions In
25. e PAL Attach an appropriate length of tubing to the Waste Receptacle and to the Fast Wash Station waste drain fitting Figure 8 ITSP Waste Receptacle ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 14 Step 5 Train the remaining Object Reference Positions a Tray Types Make sure you have sufficient tray types loaded in the PAL for the trays you will need for your application You should not need to make any setup changes to the tray types simply confirm they exist in the PAL object list that the trays you are using are assigned the proper tray types and that you have trained at least the ITSP tray positions DW96 this is the tray type of the SPE sample tray and of the Eluate Filtrate tray used in this example It is a 96 position deep well microplate MT96 this is the tray type of the filtration method raw sample tray used in this example It is a 96 position shallow well microplate ITSP96 this is the tray type of the ITSP cartridge tray loaded with ITSP cartridges MT9GITSP this is the tray type of the filtration method raw sample tray used in this example It is a 96 position shallow well microplate with an ITSP cartridge positioned above it DW96ITSP this is the tray type of the elution tray used in this example It is a 96 position deep well microplate with an ITSP cartridge positioned above it b Trays Training some of the Tra
26. e preparation in LC and GC applications on any MPS PAL or PAL xt systems running e PAL or MPS Firmware version 2 5 2 or later and e PAL Object Set version J or later OR PAL xt Object Set version A or later depending on which PAL or MPS model is being upgraded for ITSP ITSP Solutions supports the following PAL programming software products as a programming environment in which to create or modify ITSP Methods for sample preparation on the PAL e PAL Cycle Composer with Macro Editor from CTC version 1 5 4 or later e Cycle Editor from CTC version 1 4 0 4 or later often embedded in other platforms such as MassHunter or ChemStation from Agilent Technologies e Maestro from Gerstel version 1 4 7 14 or later End users often control the PAL attached to their analytical instruments from that instrument s operating system instead of using Cycle Composer If your situation requires transferring to a different operating system then Instrument Control Components including Cycle Editor ICC CE conversion software from CTC may be required The ITSP Hardware Kit ships with preconfigured Objects that can be loaded into the PAL using Object Manager software from CTC version 2 1 0 or later If you are unsure how to use Object Manager the instrument vendor from whom the instrument was purchased or ITSP Solutions can help you through this process The ITSP Hardware Kit ships with software examples to demonstrate how to use ITSP c
27. er Needle Guide similar to a magnetic vial transport When using Macros to operate the PAL with ITSP before moving the syringe away from a location where it should be leaving an ITSP cartridge in place usually in the ITSP tray ITSPprep include the following PAL Macro Atoms MOVETO OBJECT Object Name ITSPprep Index SL index Penetration 0 MOVE_REL X 0 Y 6 z 3 to ensure the PAL injection unit does not dislodge the ITSP cartridge from the well as it moves away Or if moving back to the same location e g to perform an air flush include the PAL Macro Atoms MOVETO OBJECT Object Name ITSPprep Index SL index Penetration 0 MOVE_REL X 0 Y 6 Zz 3 MOVE REL X 0 Y 6 Z 0 NOTE In the above PAL Macro Atoms where y 6 and z 3 depending on observed versus desired behavior the values may need to be adjusted up or down to ensure the PAL injection unit clears the ITSP cartridge If y 6 is changed then adjust the associated y 6 accordingly ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 25 General Tips and Guidelines The following tips and guidelines may be helpful to reduce errors when using ITSP on the PAL 1 Check the software versions e PAL Firmware version 2 5 2 or later 4 1 3 or later for xt e PAL Object Set version
28. g the needle or introducing septa coring remnants from entering the LC injection valve Check that the ITSPprep tray has been squarely and securely fastened to the tray holder If it is not square then the X Y setting for the exact center of well 1 will not provide an appropriate setting for well 12 or 96 As the sample list progresses the object s offset settings for X and Y will gradually drift out of alignment Also if it is not secure then the tray may skew during operation also causing a misalignment Helpful hint It may sometimes be helpful to shim the edges of the tray with spare septa between the front or sides of the tray and the lip of the tray holder to ensure square and secure orientation The grip of the ITSP septa on the needle gradually decays with repeated needle penetrations of the septa Therefore design your method steps for a minimum number of penetrations per cycle Also discard rather than reuse each cartridge after one SPE or filtration cycle Check that the X Y and Z offsets are correctly set especially for the ITSPprep ITSPelut for SPE and ITSPsmpl for filtration trays The X and Y offsets should be set for the exact center of the septa in the ITSP cartridge The target we are aiming for is the center bore of the cartridge needle guide component not just anywhere in the septa The Z offset should be set somewhere between 0 2mm two clicks and 0 5mm five clicks above touching It is important
29. ge was made to the ITSP system in 2009 Earlier versions of the hardware will not match the current object set and will not perform as reliably on the PAL Contact ITSP Solutions if you wish to upgrade a legacy system to the current version or if you need help in determining which version you have Check that the Upper Needle Guide on the injection unit is adjusted to a height halfway between the Lower Needle Guide and the top of the needle where it connects to the syringe If it is set too high or too low then it does not provide the needle the appropriate support when penetrating septa which may cause a needle to flex or Z especially in the case of a 22 gauge needle which has a thinner wall than a 22S gauge needle Septa in the seal of the ITSP cartridge are thick in order to provide a strong seal against the needle for reliable transport and pressurization Check that the syringe needle is 22S gauge rather than 22 gauge point style 3 flat on the end The thicker barrel wall of the 22S gauge needle is much sturdier than that of the ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 26 10 11 22 gauge needle Some applications may require the use of a syringe that is only available with 22 gauge needles In these cases extra care should be taken in the remainder troubleshooting steps PAL setup and operating procedures to reduce the chance of either bendin
30. grade a legacy system to the current version Notice the raised nipple around the syringe needle hole on the ITSP needle guide s which mates with the crown on top of the seal on the ITSP cartridge to align the needle with the center of the cartridge HW NG HW LNG mounts onto the PAL replaces the PAL Lower close up of crown on seal Lower Needle Guide Needle Guide 10 UC8EC P ITSP cartridge The spacing of the wells in both the X axis and Y axis of the ITSPprep tray is wider than a standard 96 well microplate This is to provide room to slope the sides of each well to provide a larger target for the cartridge when the PAL returns it to the ITSPprep tray This feature builds in more forgiveness into the system to allow for things like manufacturing tolerances setup tolerances etc ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 22 Other Factors Besides the design features of ITSP there are other factors in the setup and operation of the PAL which contribute to robust ITSP PAL automation It is important that the cartridge Z axis remain parallel to the needle Z axis at all times in order for the PAL to reliably transport the ITSP cartridge around the deck of the PAL including the action of replacing a cartridge back into the ITSPprep tray without crashing or bending needles The factors which contribute most to proper vertical alignment of the cartridge are 1
31. ing tall trays behind short trays may cause the PAL Injection Unit to collide against the tall plates when accessing the first row on the short plates ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 21 Understanding Robust ITSP PAL Operation Design Features There are several design features of the ITSP cartridge and prep tray which contribute to robust ITSP PAL automation 1 Many standard 8mm vial crimp seal septa are 0 0040 We increased the septa thickness within the ITSP cartridge seal to 0 0060 This feature increases the strength of the septum s grasp on the needle during transport and also improves the hydraulic seal between the needle and the septum especially after repeated punctures The geometry of the top metal surface of the ITSP cartridge seal is similar to but different from a standard 8mm vial crimp seal It is modified from being flat to having a crowned upper surface that is designed to mate with the nipple on the bottom of the older PAL ITSP Needle Guide Attachment HW NG picture below and its replacement PAL modification the new PAL ITSP Lower Needle Guide HW LNG picture below The older modification was attached to the outside of the bottom of the PAL Lower Needle Guide requiring a height adjustment The newer modification replaces the PAL Lower Needle Guide with the ITSP version Contact ITSP Solutions if you wish to up
32. le Eluate Tray amp Tray Filtrate Tray SolvRes1 Washi LC Viv1 MT2 Rear MT2 Rear without ITSP cartridge without ITSP cartridge ITSPsmpl Elution Tray with ITSP cartridge ITSPelut with ITSP cartridge ITSP Cartridge Tray ITSPprep with ITSP cartridge SPE Sample Tray MT1 Frnt without ITSP cartridge Figure 4 Example of Object Locations on an ITSP PAL The photograph in Figure 5 shows an alternate configuration using a stack instead of a tray holder to hold raw samples ITSP is very flexible in supporting your choice of trays or vials for holding samples It even supports eluting to a closed vial if you use a Star cut septa snap cap on a 2mL vial Contact ITSP Solutions for more information on this option Figure 5 Example ITSP Setup ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 11 Steps to a Successful Installation Warning Prior to making any firmware changes for ITSP perform a PAL Firmware backup procedure using the PAL Loader software from CTC version 1 1 1 or later ITSP Solutions recommends that PAL users familiarize themselves with the guidelines in the following manuals from CTC e PAL User Manual e PAL Operation Recommendations Manual e Tips and Hints for PAL Systems Manual Step 1 Assemble the PAL This Guide assumes you are either modifying an existing PAL for ITSP or else your PAL vendor will assemble the
33. p and risking the needle tip coring either the sorbent bed or filter membrane inside the ITSP cartridge ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 27 12 Acommon step in an ITSP SPE method is to perform an air flush after applying solvent or sample to an ITSP SPE sorbent bed to thoroughly flush the liquid through the bed One way to accomplish this is to partially load the syringe with air before aspirating solvent or sample Another way is to aspirate and dispense the liquid then while still at the same location aspirating and dispensing air It is important to pull the needle out of the cartridge after dispensing liquid and before aspirating air to prevent back flushing the liquid through the cartridge a If performing this action over the ITSPprep tray be sure to program an ITSP wipe MOVE_REL 0 6 3 to make sure the needle comes all the way out of the septa before the air aspiration In this case you will need to compensate for the 6mm in the Y axis direction with a MOVE_REL 0 6 0 atom If performing this action over the ITSPelut tray after elution then be extra careful of the training of the ITSPelut X Y and Z offsets The X and Y should be in the exact center of the well with the Z offset set between 0 2mm and 0 5mm above the septum There is approximately 0 8mm between the top of the septum and the top of the seal crown
34. pushed off the needle until it is safe to release it The capacity of an ITSP cartridge is not limited by its small size because the analytical syringe of the PAL replaces the liquid reservoir of a manual SPE column ITSP cartridge capacity is determined by a wide variety of factors including e sorbent bed mass e affinity of the analytes of interest to the selected sorbent phase e affinity of the interference compounds in the sample matrix to the selected sorbent phase e size of the syringe times the number of loading cycles in each step of the method Manual SPE methods are typically scaled down by an order of magnitude or more when automated using ITSP Typical solvent and sample volumes are in the 100uL range using sorbent bed masses from 10mg to 25mg Explanation of the Hardware Set Up The Hardware Kit developed by ITSP Solutions is as technically significant as our cartridge and is a required purchase for robust performance The cartridges sit in a specially designed Prep Tray over a PTFE waste receptacle that has an attached drain hose so waste can be automatically diverted to an appropriate receptacle The Prep Tray is a 96 well array tray that we have redesigned to spread the wells apart so that we can reliably return the cartridge to the tray after moving it to another location on the platform Most of the SPE steps conditioning sample loading and the column wash after loading are performed in this position Once those s
35. ridge that is rounded ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 5 into a crown instead of flat The interaction of these two components allows the PAL to center up on the cartridge before piercing the septum This does two things First it helps prevent leakage by helping the PAL to pierce the same hole every time Second it helps the PAL maintain the cartridge in a vertical orientation during transport by helping the needle to enter the cartridge directly over the needle pathway in the cartridge ITSP cartridge transport is similar to the standard PAL needle vial transport in that it uses the grip of the septum on the needle to pick up the cartridge However the needle needs to enter the ITSP septa both deeply and in the very center for robust performance ITSP cartridge transport is similar to the standard PAL magnetic vial transport in that a wiping action must be performed when leaving the Prep Tray position to be certain that the cartridge stays in the tray when the tower moves to another position ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 6 Supported Software and Version Requirements Warning Prior to making any firmware changes for the ITSP installation perform a PAL Firmware backup procedure using the PAL Loader software ITSP Solutions supports the use of ITSP cartridges for sampl
36. rminate control of the PAL by the Loader program On the Keypad terminal the Job Queue screen is displayed ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 17 Programming the PAL for SPE using ITSP The PAL will follow these general steps as an example which can be customized to your specific analytical method to perform solid phase extraction using ITSP e The ITSP cartridge will be activated with organic conditioning solvent to prepare it for analyte adsorption e The ITSP cartridge will be equilibrated with aqueous conditioning solvent to prepare it for the sample matrix e The sample will be loaded onto the ITSP cartridge e The sample loaded ITSP cartridge will be washed with 1 or 2 wash solvents to remove interference compounds e The ITSP cartridge will be moved to the elution tray and the sample fraction eluted e The ITSP cartridge will be discarded e The eluted fraction can then be injected using a standard PAL injection cycle These steps are repeated for each sample in the Sample List Typically the time required for the complete sample preparation injection process using ITSP is between 3 and 7 minutes Often the analysis method requires as long as or longer than this length of time Therefore there is no change to the throughput of the analytical instrument while there is a reduction in the overall time for the complete analysis sample preparation
37. rs c However the ITSP crown nipple mating action can only compensate for one or two millimeters of error in the X or Y axes In addition for ITSP transport the PAL Lower Needle Guide is blocked so the PAL cannot use its Z axis seek object function when moving to a tray position while transporting a cartridge d Therefore it is critical that ITSP trays are squarely and securely installed and then carefully trained within 0 1mm to 0 2mm of the exact center of cartridge in the X axis and Y axis and from 0 2mm to 0 5mm high in the Z axis Failure to perform this process will predictably result in a crash or bent needle usually in much less than 100 cycle iterations 5 Horizontal presentation of the top of the cartridge seal to the bottom of the PAL ITSP Lower Needle Guide can contribute to proper orientation However a The ITSP crown nipple mating action ensures the needle enters the septum concentric with the Z axis of the cartridge in a properly installed ITSPprep tray b Ifthe needle punctures the septum off center and then is inserted into the cartridge the puncture in the septum will elongate and the elasticity of the septum rubber material will apply a strong sideways pressure tilting the cartridge to one side which will cause a crash or a bent needle Also an elongated puncture will dramatically reduce the hydraulic performance of the cartridge c Even if the needle punctures at dead center but is not then inserted
38. rtridge and the Needle Penetration adjusted for the depth of the ITSP cartridge e ITSPelut of tray type DW96ITSP ij this 96 position deep well microplate is the elution tray located in the rear position of MTHIdr2 It is the exact same tray as MT2Rear but its Reference Position is trained with ITSP cartridges positioned above the inserts see Figure 9 ITSPprep of tray type ITSP96 this 96 position tray supplied in the kit should be located in the front position of MTHIdr2 It should be trained with ITSP cartridges in place This tray is where the conditioning loading and washing steps occur when using SPE methods to prepare samples and so it sits on a Teflon base also supplied in the kit with a waste port and hose nipple screw for connection to waste tubing Figure 9 Reference Position for ITSPelut c Wash Stations Washi this is the rear position of the Fast Wash Station usually aqueous solvent Wash2 this is the middle position of the Fast Wash Station usually organic solvent d Injectors Waste this is the front position of the Fast Wash Station LC Vlv1 this is the LC Injection Valve ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 16 e Vials SolvRes1 this is the rear 100ml solvent reservoir SolvRes2 this is the middle 100ml solvent reservoir SolvRes3 this is the front 100ml solvent reservoir
39. sometimes skip the process of re training a PAL tray position after changing tray types on the deck They simply trust that changing the tray type object to the tray type nominally matching the tray physically installed on the deck will be sufficient a In many cases the PAL can compensate for minor Z axis errors by using the Lower Needle Guide seek object function Z Tolerance object setting and limit switch when moving to an object The PAL can compensate for minor X axis and Y axis errors because the relatively small diameter of the needle is aiming at a target that is comparatively large such as a vial opening Therefore minor errors in the X Y or Z settings for tray positions usually do not cause problems b With ITSP this is not the case It is very important to the successful performance of an ITSP PAL that tray objects especially ITSPprep be squarely and securely installed and then also very carefully trained after installation While this is an important point minor errors in setup and position training or minor slippage of trays during operation can be compensated for by the ITSP crown nipple mating action The mating of the crown on the top of the cartridge seal with the nipple on the bottom of ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 23 the ITSP PAL Lower Needle Guide as the PAL approaches ITSPprep compensates for minor misalignment facto
40. sssccnseaevnaseleaasedadecuuseyteteuataraeuachensetadtonata 17 Programming the PAL for Sample Filtration using ITSP eee eeeesseeeeeceeeeeeeeeeeeees 18 Recommendations for Operating the PAL with ITSP ee eeeseeseceeseeeeeeceeeeeeeeeeeeees 19 Understanding Robust ITSP PAL Operation csietsseesacevvescaccseevanacanaveacdeesisaceteennetvnavenners 21 Design Features saisasataivsiandescuaiesaneiei ineen isine a E E EEEE E EERE a 21 Other WCCO Ss oaiacocidinaecuensteszenacsiaunacaunseeanbenuurasoteatoneaWiaeencencecyaxsauaeseaneatansoteeacousanoniis 22 Pret les Iie sieisen e n Es EE e EEEE E EEE EEKE EEE EES EEEE RE 24 General Tips and Guidelines cece sss accaccenuscnansesesaucdoasaxasy eactansavacenaavnapeisactacskcseoeseenennnceaens 25 Appendiks E E eae 29 Filename C nventionS Seen Ree ete Re Clr eee Stee nit Mir a aia Ae eee r ar secre econ 30 Object Definitions in ITSP Upgrade PAL Object Lists eesseecceesceceeeceeeeeeeeeeeeees 31 IISP Dray Ty Des wsccoxciecavsecvastses cannesviciasvanceanteteave TEPEE TEEPEE KE N EREE Eaa aa 31 ITSP Tray Holders crcnrnccneiseeeoure ira en i E e a anaes ace Ei 32 i bed cal Ws peer eee Tree Cees E S rete S 32 ITSP Tia QCUOr S osshasvasaecudswesseecaudscenboecsaueinadeeied suosauadeadasduaekassneinadatanpannsaaasenasaesavaareeateenes 32 IFSP POSItIONS oeno a E ae E E E E EE 33 Set Up Guide Installing ITSP on the PAL Page 2 Table of Figures Figure 1 ITSP Cartridge Uta anycccctcas
41. t Up Guide Installing ITSP on the PAL Page 31 Object Definitions in ITSP Solutions ITSP Upgrade PAL Object Lists ITSP Tray Types TRAY_TYPE MmT96 99000 Oo O 63000 412 8 amp 8 Regular TRAY_TYPE Mw9e 99000 O O 63000 12 8 Regular TRAY_TYPE Dw96 99000 o O 63000 12 8 amp 8 Regular TRAY_TYPE vrs 4 111200 o 0 6o 9 Regular TRAY_TYPE DW96Mrrr 99000 O O 63000 12 8 amp 8 Regular _TRAY_TYPE DM96ITSP_ 99000 O O 63000 412 amp Regular TRAY_TYPE MT961TSP_ 99000 O O 63000 12 8 amp 8 Regular _TRAY_TYPE MwgeitsP 99000 O O 63000 412 amp Regular _TRAY_TYPE DwgeiTsp 99000 O O 63000 12 8 Regular TRAY_TYPE visaitsp 111200 o o so 9 Regular TRAY_TYPE iTsPp 105000 o O 66700 412 8 amp 8 Regular TRAY_TYPE ITsp96vo 99000 O O 63000 412 amp Regular ITSP Tray Types continued TRAY_TYPE MT96 G 1450 0O None 4000 40000 None TRAY_TYPE Mw96 H 31000 0O None 4000 40000 None TRAY_TYPE DW96 H 44000 3000 None 4000 40000 None TRAY_TYPE VT54 l 22500 14500 None 4000 40000 None TRAY_TYPE DW96Mrrr H 44000 4000 None 4000 40000 None _ _TRAY_TYPE DM96ITSP_ G 44000 30000 None 4000 40000 None TRAY_TYPE MT96ITSP_ G 14500 27000 None 4000 4
42. t and error recovery routines If this location instead of the recommended ITSP tray TSPprep is used for sample preparation then it is not advisable to leave the PAL unattended during ITSP sample preparation ITSP Positions POSITION _ ITSPdisp None 0 0 0 NOTE If you cannot or do not wish to return the cartridge to the ITSP tray when finished with sample preparation you may program the PAL to drop the spent cartridge into a disposal receptacle located at the Position TSPdisp The default location of ITSPdisp is 0 0 0 This position should be trained to the desired location for disposing of spent ITSP cartridges which can be the empty ITSP cartridge shipping container set on the bench near the PAL Fast Wash Station for example or the location set for changing syringes Document ISDCooo1 ITSP Solutions Inc Rev Date 130402 Set Up Guide Installing ITSP on the PAL ITSP SOLUTIONS INC The information in this document is subject to change without notice ITSP Solutions Inc makes no warranty of any kind with regard to this material including but not limited to the implied warranties or merchantability and fitness for a particular purpose ITSP Solutions Inc shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Contents of this publication may not be reproduced in any form or by any means
43. te 130402 Set Up Guide Installing ITSP on the PAL Table of Contents Using ITSP Ie eresie r E E E EEE ER 2 Explanation of the ITSP Cartridge oseeoeesseeeseeeeseesseesseseereessesstesseessesetessesseesseees 3 Explanation of the Hardware Set Up wciasecacveeslocevasedssrvensvadeoddeoreavuneinemeianranennaniass 4 Supported Software and Version Requirements cccssccsssseceesseceesseceesseeecsseeeceeseeeees 6 Supported FEAUC Wal Cce asa ancccacoceamsaeseaeancatacicantionssqsnevasetasyaenseunaeieunnyanraaaeantehaneareaeuctieennen 7 ITSP Hardware Kit Co tli Sc sacccsenseescdecaaniu sees eadaresceendtanteatadentestataniceas measeteneavemsaiaciecieiaie 8 Example of PAL Hardware Configuration for ITSP csccsssssssscsssseessccesesennececeees 9 Steps to a Ces still Tin seat Ooi kessneri eenieders rr Sesi 11 Step 1 Asse mble the PAD inninescinissnicst neneiia a ieia 11 Step 2 Load the Objects into the PAL sccsscsssssssscesseeseccescessecsserceseneeseccessceees 11 Step 3 Train the Tray Holder Reference PoSitions ccsscsscesssssseccesncesensessncess 12 Step 4 Install the ITSP Hardware Kit PAL Modifications ccccssecsssseceesteeeeees 13 Step 5 Train the remaining Object Reference Positions c cccesseecesneceesseeeeees 14 Step 6 Backup the PAL configuration sscssccssssssssccsssccsssseseccesseseneccesnecsensensneess 16 Programming the PAL for SPE using ITSP vecese
44. teps are complete the PAL aspirates a volume of elution solvent then moves to the cartridge Rather than dispensing again over the waste receptacle the PAL pierces the septum and inserts the needle to just above the sorbent With the needle still inserted into the cartridge the PAL retracts the cartridge into the tower moves to an open well in the elution tray usually right behind the Prep Tray location and dispenses the elution solvent through the sorbent to elute the compounds of interest into the open well Once the elution is complete the cartridge can be returned to the Prep Tray There is also the option of disposing of the cartridge into the original packaging to reduce human handling of a potential biohazard We provide biohazard labels with our packaging for quick visual reference The PAL can then perform a standard PAL LC Inject cycle on the eluate and start preparing the next sample while waiting on the Mass Spectrometer to perform its analysis cycle The standard operating procedure for SPE has multiple steps that require the needle to penetrate the septum during the method It is important that we minimize the number of holes we puncture in the septum in order to maintain a good seal around the needle To facilitate this we supply in the kit a replacement for the standard PAL lower needle guide with an ITSP lower needle guide which has a nipple on the bottom surrounding the needle hole We have a special crimp seal on the top of the cart
45. the following steps to prevent the possibility of bending the needle The objective is to define the Reference Positions for standard PAL Objects Make sure the Tray Holders Valve Solvent Reservoir Holders and Fast Wash Station are properly and securely mounted to the PAL X axis The following description is an example of how to teach the Reference Position for a Tray Holder The described procedure is also com mon to all other objects For Tray Holders the Refer M ence Position is a hole a slightly larger than the lower needle guide in the base plate near the front of the Tray Holder The lower needle guide should be centered in the hole with the bottom of the needle guide flush with the bottom of the base plate see Figure 6 On the PAL hand held ter Figure 6 Reference Position for Tray Holders MTHldr minal with the Job Queue and MTHIdr2 menu being displayed complete the following sequence 1 Menu Setup Objects Tray Holder MTHldr1 After selecting the object the X Y and Z positions for the selected object will be displayed 2 Highlight the item Position X with the cursor bars and press the inner knob to ENTER The Injection Unit will move to the previously defined X axis position 3 Rotate the outer knob to adjust the X axis position to the Tray Holder Reference Position Press the inner knob to ENTER the Position X value Repeat steps 2 4 for Positions Y and Z Press the F3 button Move to Zero
46. ts the following CTC Analytics guidelines when using ITSP cartridges on the PAL e SYRINGE SIZE a 100uL syringe is generally recommended although ITSP can accommodate any syringe size appropriate for your application X Type syringes have a much longer plunger life than C Line syringes and are more inert e FILL SPEED depending on your needle size and the viscosity of your solvents o Gauge 22S limits the fill speed to a maximum rate of 5 20uL s o Gauge 22 allows fill speeds up to 200uL s Warning Use of large ID needles may incur a risk of coring the ITSP cartridge septum ITSP Solutions therefore recommends using a 22S gauge needle e EJECT SPEED for 100uL Syringe 50 to 150uL s depending on your needle size and the viscosity of your solvents e The torque of the syringe plunger may need to be increased using motor setting MplgHigh to ensure reliable aspiration and dispensing speeds of solvents or samples through the ITSP cartridge sorbent bed or filter media e It is critical that syringes be primed before beginning sample preparation Prime the syringe by using the utility function Clean Syr on the PAL handheld terminal or include an adequate number of filling strokes in the Method or include the Macro Atom CLEAN SYR at the beginning of the cycle e g in SL Entry pma when using Cycle Composer e Some PAL Object positions are adjusted with respect to Z axis dimensions and needle penetration depths when the
47. ugh the septum The gripping action of the 8mm Crimp Seal septum allows the upper chamber of the Syringe Needle cartridge to be pressurized so that solvents can be forced through the retaining Adapter to Reduce membranes and the sorbent bed Instead of a a Headspace vacuum manifold ITSP uses the syringe Sorbent cup with plunger to provide hydraulic force in either direction across the sorbent bed or filter membrane The precision of the PAL is such Membrane Frit that you can control flow rates at micro volume levels so that you gain a very high degree of precision yet at the same time a Membrane Frit high degree of flexibility funnel Sorbent ITSP is bi directional so filtration of samples that could damage the syringe are also possible using ITSP The PAL dips the cartridge tip into the raw sample and Figure 1 ITSP Cartridge Cutaway aspirates only the filtrate into the syringe We use the precise movements of the PAL to move the cartridge around the platform To do so the needle stays inserted deep in the cartridge while the PAL Injection Unit moves ITSP Solutions Inc Document ISDCooo1 Rev Date 130402 Set Up Guide Installing ITSP on the PAL Page 4 to the location where the sample is eluted The cartridge grips the needle with the septum to keep the cartridge at a precise vertical orientation on the needle We program the needle guide to remain in its position so that the cartridge is not
48. when checking the Z offset parameter that you check the actual setting by actively selecting the Z offset parameter rather than simply using the check position function When using the check position function in the setup menu or go to position 1 in the utilities menu alone then you get a false reading of the actual Z parameter This is because the PAL Injection Unit seeks the object in this case the tray by feeling for it with a limit switch on the lower needle guide using the Z tolerance setting in the tray type object When you then while at position 1 of the tray actually select the Z offset parameter highlighting it on the remote which allows you to adjust the setting then the PAL Injection Unit moves up slightly to the real trained position setting This is the exact location the PAL will go to when transporting an ITSP cartridge ITSP transport utilizes the Block Needle Guide atom which forces a PAL to operate as if the Z tolerance is turned off set to o for all objects it moves to when in this mode The difference in these two Z heights can be several millimeters which is enough to cause a variety of errors including bent needles or object crashes during operation Check to make sure the needle penetration depths for all ITSP objects is set to 20mm This is deep enough into the cartridge to properly align the cartridge in a vertical orientation during transport without being too dee
49. y object Reference Positions will require holding an ITSP cartridge above a tray or insert Using the special ITSP tray types from the ITSP Hardware Kit that were loaded into the PAL during Step 2 using the Object Manager software from CTC will simplify those training steps NOTE When locating trays on the PAL deck during setup it is important to position taller trays toward the front and shorter trays toward the rear Locating tall trays behind short trays may cause the PAL Injection Unit to crash against the tall trays when accessing the first row on the short trays NOTE It is important to be careful when training ITSP tray types because the PAL does not sense objects when moving to positions with the Lower Needle Guide blocked as it is while transporting ITSP cartridges When training Reference Positions for the ITSP trays TSPsmpl ITSPelut and ITSPprep adjust the Z position such that the lower needle guide just touches the top of the ITSP cartridge and then back off up one half millimeter 0 50mm When setting up Tray Objects assign the proper Tray Type and then train the Reference Positions NOTE It is important to not skip this step for ITSP trays More information about critical factors affecting robust ITSP performance is covered in greater detail in later sections of this guide MT1Rear of TRAY TYPE MT96 this 96 position shallow well microplate is the tray located in the rear position of MTHIdr1 NOTE It

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