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User’s Manual Probot

D960R3

B-1

APPENDIX B

Peak Collection in Conjunction
with CHROMELEON® Software

B.1

Overview

This appendix describes how to setup the Probot

™

Micro Fraction Collector for

peak collection in conjunction with the CHROMELEON

software.

The peak detection capabilities of CHROMELEON can be used to control event
outputs (e.g. event outputs of the UltiMate

™

Micropump) so that a signal is

generated at the beginning of a peak and at the end of the peak. The conditions

that CHROMELEON uses to detect peaks can be programmed and adjusted to the

needs of the application.

The peak collection function of the Probot Micro Fraction Collector and the

Carrier

™

software involves the TTL inputs (Section 2.4.1 D).

The information provided in this appendix refers to CHROMELEON version 6.5
SP3 and to

Carrier version 2.0.

Summary of Contents for Probot 161403

Page 1: ...D960R3 Probot TM Micro Fraction Collector User s Manual P N 161403 www lcpackings com ...

Page 2: ...eserved No part of this manual may be reproduced or transmitted in any form or by any means without the written permission of LC Packings BV The following are Trademarks or Registered Trademarks of LC Packings A Dionex Company Probot µCarrier UltiMate UltiChrom FAMOS Thermos Switchos II Acurate UZ View UV Booster FluoBoost Pepmap µ Dumper µ Fluidics Fusica Nano Series Pico Series µ Guard µ Precolu...

Page 3: ...onents 2 3 2 3 1 Removing the Top Cover 2 4 2 3 2 Drive Table Safety Brackets 2 5 2 4 Installing the Probot with the UltiMate Capillary HPLC System 2 6 2 4 1 Electrical Connections 2 6 2 4 1 A RS 232 Connector 2 6 2 4 1 B START Connector 2 6 2 4 1 C REMOTE Connector 2 7 2 4 1 D TTL IN Connector 2 7 2 4 1 E INTERLOCK Connector 2 7 2 4 1 F RELAY Connector 2 7 2 4 1 G ANALOG IN Connector 2 8 2 4 1 H ...

Page 4: ...8 4 Create a New Application using the Wizard 2 34 2 8 5 Control the Table Position Manually 2 44 2 8 5 A Using the Manual Drive Panel 2 44 2 8 5 B Using the Manual Control Unit 2 44 2 8 6 Measure the Column X and Row Y Distance 2 45 2 8 7 Default Calibration and Acceleration Settings 2 47 CHAPTER 3 Maintenance and Troubleshooting 3 1 3 1 Overview 3 1 3 2 Maintenance 3 2 3 3 Replacing Major Compon...

Page 5: ...is 4 APPENDIX D The Vacuum Waste Removal Option 1 D 1 Overview 1 D 2 General Design 2 D 3 Setting up the Probot 3 D 3 1 Electrical Connections 3 D 3 2 Connecting the Vacuum Pump 3 D 3 3 Installing the Fused Silica Capillary 4 D 4 Setting up µCarrier and CHROMELEON 5 D 4 1 Enabling the Vacuum Waste Removal Option 5 D 4 2 Adjusting the Fused Silica Capillary and the Needle Height 5 D 4 3 Using the W...

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Page 7: ...literature the warranty indicated in the sales literature will prevail If the system includes equipment supplied by LC Packings but manufactured by a third party LC Packings makes no warranty of any kind express or implied including without limitation any warranty of merchantability or fitness for a particular purpose LC Packings will make available to you to the extent permitted the warranties of...

Page 8: ...negligence or otherwise for special incidental consequential or analogous damages including without limitation damages resulting from loss of use loss of profits loss of business or loss of goodwill even if LC Packings has been advised of the possibility of such damages This warranty comprises the entire warranty between LC Packings and the customer It overrides any warranty related language that ...

Page 9: ... SAFETY form if applicable is attached outside of the package so that we can properly track and account for your system 2 Please include the following a Company letterhead with the following information Your Name Complete Mailing Address Telephone Number fax number and e mail address Return Authorization Number A detailed description of the problem The name of the LC Packings personnel to whom you...

Page 10: ...Instructions for Returning Instruments x D960R3 User s Manual Probot This page intentionally left blank ...

Page 11: ...mage to equipment and or lead to invalid analytical results Note The information in a note statement relates to important information that should be read and understood before continuing Safety Precautions Note The following precautions should be followed to minimize the possibility of personal injury and or damage to property Note Make certain that you are familiar with the contents of this manua...

Page 12: ...t contained a potentially harmful substance 9 Wear protective eye goggles when handling mobile phases or operating the instrument An eye wash facility and a sink should be close to the unit If any mobile phase is splashed on the eyes or skin wash the affected area and seek medical attention 10 Dispose of all waste mobile phase in an environmentally safe manner that is consistent with all local reg...

Page 13: ...standard EN61010 1 Safety requirements for laboratory equipment Class I Installation cat II Pollution degree II WARNING LC Packings will not accept any liability for damages direct or indirect caused by connecting this instrument to devices which do not meet relevant safety standards EEC directives 89 336 and 92 31 EMC requirements applied with the following standards EN 50081 1 Generic emission s...

Page 14: ...CE Declaration xiv D960R3 User s Manual Probot This page intentionally left blank ...

Page 15: ...tion as well as a park position where the plate should be moved to during rest periods Various adapter tables are available for carrying the different targets When the Probot Micro Fraction Collector is employed with the µCarrier software the system provides the following features High precision X Y Z movement of the table any position within the working area can be programmed with a resolution do...

Page 16: ... LC CZE System Probot UltiMate UV Detector Vials Union Target FIGURE 1 1 General Configuration of the Probot Micro Fraction Collector The Probot Micro Fraction Collector is supplied with a µ Tee connection FIGURE 1 1 The lower end of the needle Tee holds a hollow needle in which a fused silica capillary is mounted the horizontal inlet is used to add reagent e g a matrix solution to the sample usin...

Page 17: ... directly in the Nano flow path default configuration Standard Needle Tee allows for adding the matrix solution through the hollow needle co axially at the needle tip supplied with the instrument Camera Option A camera kit P N 160590 is available which allows for easy monitoring of the needle and target s in conjunction with µCarrier 2 x or a standalone monitor Pipetting Option The pipetting optio...

Page 18: ... µ Tee with Needle Power Switch Dosage Module optional Target X Y Z Drive Table Adapter Camera optional FIGURE 1 2 Front View Probot Doors and Needle Cover Removed 1 4 The Manual Control Unit Mode LEDs X Axis Control Buttons Y Axis Control Buttons Z Axis Control Buttons Mode Buttons FIGURE 1 3 Manual Control Unit ...

Page 19: ...ication Designer software which is a Windows based program is used to control all functions of the Probot Micro Fraction Collector and to support time based fraction collection It can be used for peak collection in conjunction with any software package that allows for peak detection e g the LC Packings CHROMELEON Software and that can provide control signals e g contact closure signals Fractionati...

Page 20: ...ent Instructions discusses alignment procedures that are recommended whenever the instrument has been relocated or shipped APPENDIX D The Vacuum Waste Removal Option describes the features and usage of this option in conjunction with the CHROMELEON software APPENDIX E Installing and Using the Belkin USB Video Adapter describes the installation and usage of the Belkin VideoBusII Image Capture Devic...

Page 21: ...tem When the Probot Micro Fraction Collector is used in conjunction with the UltiMate system and the FAMOS Microautosampler please refer to the User s Manuals supplied with these units for additional information Chapter 2 provides the following installation information Removing Shipping Components Section 2 3 Installing the Probot with the UltiMate Capillary HPLC System Section 2 4 Electrical Conn...

Page 22: ...of the system The Probot Micro Fraction Collector requires approximately 480 mm 18 9 of linear bench space The lab bench should be capable of supporting the entire system for the LC Packings UltiMate FAMOS and Switchos II we recommend that the lab bench be capable of supporting at least 100kg 225 lb The power consumption of the Probot is 100 VA the power consumption of the UltiMate Capillary HPLC ...

Page 23: ...tems are missing this should be reported to the shipping agent and to LC Packings as soon as it is observed CAUTION Caution If there is any apparent damage to the instrument the user should investigate the nature of the damage before plugging the unit into the mains to ensure that powering up of the instrument will not create a hazardous condition or damage internal components If the damage appear...

Page 24: ...IGURE 2 1 Removing the Covers Left Side View c Remove the screw on the left side item 2 FIGURE 2 1 and the screw on the right side that hold the rear cover Remove the rear cover d Remove the two black screws on the lower left side item 3 4 FIGURE 2 1 and the two screws on the lower right side that hold the top cover 1 2 FIGURE 2 2 Removing the Rear Cover e Remove the two screws item 1 2 FIGURE 2 2...

Page 25: ...kets in place item 1 4 FIGURE 2 3 d Remove the brackets e Replace the covers 2 3 4 1 FIGURE 2 3 Removing the Safety Brackets Location of the Screws CAUTION Caution Do not keep these screws in the original holes inside Probot The movement of the Probot table may be seriously impaired by these screws Note Keep all screws and the two safety brackets in the accessory box in case it is necessary to mov...

Page 26: ...ty for damages directly or indirectly caused by connecting the Probot Micro Fraction Collector to instruments which do not meet relevant safety standards 2 4 1 A RS 232 Connector Connect the RS 232 cable to the RS 232 connector on the Probot and to the COM port of the personal computer TABLE 2 1 The Probot RS 232 Communication Cable Probot 9 pin male PC 9 pin female Description 2 3 TxD 3 2 RxD 5 5...

Page 27: ...e ground pin of the controlling device The output signal of the controlling device must be TTL level compatible Note If you are using a TTL output e g of the UltiMate Micropump the control logic will be inverted e g an inactive output of the Micropump will be recognized as an active input by the Probot 2 4 1 E INTERLOCK Connector The INTERLOCK connector is used if the Probot is to be installed in ...

Page 28: ...s input is reserved for future use 2 4 1 H VIDEO Connector If the camera option is installed in your Probot connect the BNC type connector of the VIDEO output to the monitor e g P N 160567 using the connecting cable provided with the camera 2 4 1 I POWER Connector Since the Probot Micro Fraction Collector is fitted with a universal power supply for input voltages from 90 to 260 V 47 62 Hz manual s...

Page 29: ...cell outlet of the UltiMate UV Detector a Depending on your application e g capillary or Nano LC use either a 30 µm I D 280 µm O D fused silica capillary P N 161473 for Nano LC or a 100 µm I D 280 µm O D capillary P N 160479 for capillary LC to extend the outlet capillary of the flow cell not included in the accessory kit b Install the fused silica capillary into the upper port of the µ Tee FIGURE...

Page 30: ...ing from the needle tee to port 1 PUMP b Connect port 2 FILL to one of the 3 vials Use the tubing supplied with the accessories kit Fill the vial with the matrix solution that fit the needs of your application c Connect port 3 PURGE to one of the 3 vials Use the tubing supplied with the accessories kit d To use the extra wash option connect port 4 AUX to one of the 3 vials Use the tubing supplied ...

Page 31: ...n of µCarrier To install the µCarrier software a Put the µCarrier CD ROM into the CD ROM drive If you have the Autostart function of Windows activated the setup program will start automatically If Autostart is not activated use the Windows Explorer or the Run command from the Windows Start menu to run SETUP EXE on the CD ROM All necessary files are decompressed and copied to your computer b Select...

Page 32: ...sample carriers trays and applications Easy file access via the new browser pane What You See Is What You Get operation WYSIWYG in both the editors and the wizards Basic fractionation task Only the most important settings need to be programmed what can be set by default is set by default Advanced fractionation task Advanced settings can be programmed to achieve for optimized applications New butto...

Page 33: ...rgets The application file holds the parameters for the sample carriers the tray and the tasks For each component a special wizard assisted Editor is available 2 6 3 The Editors and Wizards For the ease of use µCarrier provides various wizards and editors for the design of the sample carrier the tray and the application Sample Carrier Alignment Wizard Section 2 7 3 Sample Carrier Wizard Section 2 ...

Page 34: ...es have the extension rck the tray files have the extension try and the application files have the extension app Saves a new sample carrier a tray table or an application Activates the Sample Carrier Editor to create new or modify existing sample carriers Activates the Tray Editor to create new or modify existing trays Tray layout and spot grouping is available in this editor Activates the Applica...

Page 35: ...ctivated If a switch is activated a corresponding message is presented and the Probot will perform reference runs b In the next window that appears you will be asked whether or not a dosage module is present in your Probot Choose the Yes button if there is one present or choose the No button if not FIGURE 2 9 FIGURE 2 9 The Dosage Module Installed Window c After initializing of the Probot when run...

Page 36: ...ed so that it does not show some the previous dialogs on each start Select the Options menu in the main window to open the Options Window FIGURE 2 10 and adjust the Startup settings e g select Always start the software in the real device control mode to start up in real device control node FIGURE 2 11 The Settings Window Startup Section on the Main Tab ...

Page 37: ... the following sections will present in detail how to use and modify the predefined application for a time controlled fractionation application onto 6 Applied Biosystems OptiTof 24x24 targets While this example may not meet the specific needs of your application it is likely that it can be used with minor modification 2 7 2 A Loading a Predefined Application a Activate the Application Editor by se...

Page 38: ...tasks of this application are loaded in the Task List and the parameters of a selected task row are displayed in the right part of the Application Editor Selecting a different task row in the table will automatically load the corresponding parameters The tray with the six sample carriers and all groups are presented in the Group Mode section which is the lower part of the window ...

Page 39: ...e tray definition includes the exact positions of all sample carriers relative to each other e g multiple sampler carriers of the same type in predefined positions the µCarrier software needs only one spot as reference position to align all carriers in one step If the tray definition does not provide the exact relative X and Y positions the alignment has to be performed for each sample carrier ind...

Page 40: ... Aligning the Reference Position using the Camera g Finally adjust the table height Z axis so that the needle tip just touches the surface of the target This adjustment is the spotting height which will be used during spotting for all carrier positions selected in step b Note The optimal distance of the needle tip to the carrier surface depends on your application parameters e g the LC flow rate t...

Page 41: ...he tray one group per sample carrier and a meander spotting pattern FIGURE 2 17 6 Sample Carriers 1 Group on each Sample Carrier Check if this groups setup fulfills the requirements of the application you need to perform If not the group setup can be modified as follow a Select a group in the group list e g click on LC_Collect_1 in the table b To remove all spots from this group use the Clear Grou...

Page 42: ...task list FIGURE 2 18 e g to add delete or modify tasks Task List Tasks Task Description FIGURE 2 18 Available Tasks Task List and Task Description To delete a task in the task list select the task and press the Del key on the keyboard or click the right mouse key and then select Delete To add a task to the task list a Present Tasks section e g if the browser pane is currently visible by selecting...

Page 43: ...k The Basic Fractionation task is provided to simplify the setting up of such a task and it needs very few parameters to run it FIGURE 2 20 shows the parameters box The Basic Fractionation box is divided in three sections 1 How The column flow rate and the matrix flow rate is specified as multiples of the column flow rate via this box The Dispense 10 µl of matrix prior to fractionation option comp...

Page 44: ...ust ALWAYS be lower than the spotting height Section 2 7 3 If it s not lower you may scratch your MALDI target Note For faster spotting e g the spotting time needs to be decreased the Z coordinate for in task movements should be as close as possible to the spotting height Section 2 7 3 Check all tasks in the task list and save the application under any appropriate name µCarrier is now ready to fra...

Page 45: ...ionation of a NanoLC run The LC flow rate is 300 nL min the distance between columns and rows is 1 45 mm and the spot diameter is 1 mm The fraction collection time is 20 sec A MALDI matrix solution is added at a flow rate of 1 2 µL min 4 times the LC flow rate along with the fractionation of the mobile phase The start of the fractionation is synchronized with the injection of the UltiMate Capillar...

Page 46: ...ew button b The Confirm message box will be presented Choose Yes FIGURE 2 21 if you are willing to lose any unsaved data If you want to keep the existing tray and tasks setup quit with Cancel and start the wizard from the File menu New Sample Carrier FIGURE 2 21 Confirm Discarding the Tray and Tasks c The welcome screen of the Sample Carrier Wizard is presented FIGURE 2 22 Select the Create a new ...

Page 47: ...ion is enabled MALDI targets are usually planar surfaces height 0 making overfill control virtually impossible to use with MALDI targets Note The shape selected does not need to match exactly the shape of the actual spot s of the MALDI target Some manufacturers use triangular spot shapes or round and rectangular shapes on the same MALDI target Choose the shape that fits best with your target Circu...

Page 48: ... 24 by 24 in our example and the distance e g 1 45 mm between the rows and the columns The wizard automatically presents a preview of the target FIGURE 2 25 FIGURE 2 25 Sample Carrier Wizard Sample Carrier Dimensions g Choose Next to save the sample carrier You are prompted to define a name and a directory where you want µCarrier to save the sample carrier definition FIGURE 2 26 ...

Page 49: ... quit the wizard or continue to define or modify the next sample carrier The Sample Carrier Editor FIGURE 2 27 offers the same settings as the wizard but all at the same screen It is an ideal tool for experienced users and if only small changes of existing sample carriers are required FIGURE 2 27 Sample Carrier Editor ...

Page 50: ...ose Yes FIGURE 2 28 if you are willing to lose any unsaved data If you want to keep the existing tray and tasks setup quit with Cancel and start the wizard form the File menu New Tray FIGURE 2 28 Confirm Discarding the Tray and Tasks c The welcome screen of the Tray Setup Wizard is presented FIGURE 2 29 Select the Make a new tray option and confirm with the Next button FIGURE 2 29 Tray Setup Wizar...

Page 51: ...tiple carriers of the same type press the right mouse button and then select the Add multiple carriers option FIGURE 2 31 Enter the appropriate coordinates of your tray setup the Preview box will be updated according to the current settings Choose Next to add another carrier or finish the wizard FIGURE 2 31 Tray Setup Wizard 6 Sample Carriers added ...

Page 52: ...tray wizard option FIGURE 2 32 Alternatively you can start the Sample Carrier Alignment Wizard Section 2 7 3 or the Fractionation Application Wizard Section 2 8 4 2 8 3 Modify an Existing Tray Setup using the Tray Editor The Tray Editor offers more options than the Tray Setup Wizard It is an ideal tool for experienced users or if only small changes of existing tray setups are required You can add ...

Page 53: ...the reference point the red mark and keep the left mouse button press as long as you are moving the carrier This positioning is not very accurate use the Sample Carrier Alignment Wizard for exact positioning Section 2 7 3 e Groups can be defined in the Group Mode which can be accessed through the Group Edit button f Lock the carrier positions and save the setup Note The Application Wizard will aut...

Page 54: ...n presented in this section is based on the Advanced fractionation task If a Basic fractionation task is required please use the application editor to modify one of the predefined applications Note The figures screenshots provided below may vary depending on the choices made in the wizard For this example the most general choices were selected a Switch to the Application Editor by selecting the Ap...

Page 55: ...me Screen e Select the appropriate tray and choose Next FIGURE 2 36 FIGURE 2 36 The Fractionation Application Wizard Selecting a Tray f The fraction control mode will be specified in the next step FIGURE 2 37 The fraction control mode specifies if the collection is controlled by time or by a control signal or by the user ...

Page 56: ... when a peak starts or ends This requires that the operator monitors the entire chromatographic run and provides the right signal with each peak Note In time control mode the entire chromatogram is collected e g all peaks and waste With external and manual control the waste can be discarded Make certain that the peak detection parameters are properly set to avoid the possibility that peaks are tre...

Page 57: ...e option that is required for your application For this example select the Multiple injections collect each run option and choose Next to confirm the entry h In the next step the LC parameters are specified FIGURE 2 39 This includes the start signal the collection time automatically calculated if you enter start and end time the LC flow rate and the delay dead volume between the detector and the P...

Page 58: ...he Discrete volume to every fraction is selected Set an appropriate Dosage speed and then choose Next to confirm the entry Note The continuous addition of reagent makes certain that the matrix concentration is the same in all spots Use the Dispense 10 µl of matrix option to avoid any matrix dilution during a needle rest CAUTION Caution Exceeding the maximum flow rate limits may cause the rotary va...

Page 59: ...ct a group from the list and frame the spots while keeping the left mouse pressed Only if Multiple runs collect each run on a new group option was selected more than one group is available Each group represents then one LC run All groups are typically of the same size and one sample carrier is used for one LC run FIGURE 2 41 FIGURE 2 41 The Fractionation Application Wizard Selecting the Spots of M...

Page 60: ...ionation Application Wizard Standby Position l As the last step of the group definitions the Standby position where the needle stays before and after each collection needs to be defined Frame this position in the same way as described in the step above Choose Next to define a special Z coordinate for horizontal movements FIGURE 2 43 FIGURE 2 43 The Fractionation Application Wizard Z Coordinate for...

Page 61: ...ents It is defined as the height above the lowest table position 0 0 mm In addition to the Z movement XY movements can be optimized by choosing an appropriate spotting pattern e g meander vs saw tooth pattern To define the special Z coordinate for horizontal movements the table is moved to the desired travel height using the Manual Drive Panel Section 2 8 5 A m Carefully adjust the Probot table he...

Page 62: ...tial Start Run the application Select this option if the sample carriers were already aligned the application can run immediately Exit wizard allows you to review the application in the Application Editor and to make modifications if required o Select one of the options and choose Next to save the application and to finish the wizard Note It is recommended to review the application before running ...

Page 63: ...fy the application by changing task parameters deleting tasks or adding tasks From the Application Editor the run can be started by clicking the Run icon During the run the audit trail will be displayed on the top right section of the screen The needle position will be shown on the bottom FIGURE 2 46 FIGURE 2 46 The Run Application Window ...

Page 64: ...ar as presented in FIGURE 2 47 FIGURE 2 47 The Drive Panel b Move the table as follows Choose the left and right arrows to move the table to the left or right side X direction Choose the large up and down arrows to move the table to the back or to the front Y direction Choose the small up and down arrows located in the center to move the table up and down Z direction c To change the length of each...

Page 65: ...inates are displayed in mm e To change from step mode to continuous movement press the appropriate button of the manual control unit f To change the length of each step adjust the stepwidth parameter 2 8 6 Measure the Column X and Row Y Distance The dimensions of the target e g the exact distance between each column X1 X2 and the distance between each row Y1 Y2 are essential for the proper positio...

Page 66: ... center of the first vessel b Choose the Internal button and read the current coordinates c Choose the External button and then move the table along the X axis so that the needle is positioned exactly above to the center of the last vessel d Read the coordinates again and divide the distance between the first and last vessel by the number of vessels in that row column minus 1 Enter this value in t...

Page 67: ...obot Micro Fraction Collector FIGURE 2 49 In the case of a malfunction of the table drive or the syringe drive e g noise slow movement position errors check if all the settings are correct To check if the calibration and acceleration parameters are properly select the Settings option from the Tools menu Then select the Robot or Dosage Module tab FIGURE 2 49 FIGURE 2 49 The Robot and Dosage Module ...

Page 68: ...Installation and Getting Started 2 48 D960R3 User s Manual Probot This page intentionally left blank ...

Page 69: ...is to optimize the performance of the system and minimize down time Section 3 2 Replacing Components provides directions for replacing components due to wear or to re configure the system e g changing the syringe to meet the requirements of a different analytical procedure Section 3 3 Troubleshooting discusses a series of activities that should be used to determine the cause of a problem Section 3...

Page 70: ...esentative TABLE 3 1 Recommended Maintenance Schedule Frequency Operation Before operating check for any air bubbles in the fluidic lines and degas the wash solvent Check that there are no leaks of the fluidics connections Check that salts are not deposited by the fluidics joints or the needle Every Day When using buffer solutions flush the system thoroughly after use with a solvent that does not ...

Page 71: ...strument Disconnect the instrument from the electrical supplies before removing any panel or cover 3 3 1 Installing the Pipetting Option The pipetting option P N 161072 allows for pipetting from a source plate e g a well plate to a destination plate e g MALDI target It consists of a Teflon tube 45 cm I D 250 µm with fittings and a fused silica capillary 15 cm O D 280 µm I D 100 µm max 4 mm Flat Fe...

Page 72: ...Plug the connector into the socket on the back of the support bar item 3 FIGURE 3 2 d Connect the VIDEO output on the Probot back panel to the monitor using the connecting cable provided with the camera e Set the monitor to video mode f Adjust the camera position and tighten the knurled screws CAUTION Caution Make certain that the table and or the target does not touch the camera to avoid any dama...

Page 73: ... the tubing and check for any leakage Note Make sure that the plunger tip is placed correctly in the spring clip of the dosage module 3 3 4 Replacing the Dosage Unit The Probot Micro Fraction Collector is shipped with a dosage module option installed It is located in a small tray in the right side of the instrument To remove the dosage module a Remove the top cover Section 2 3 1 and disconnect the...

Page 74: ...e Probot If you attach the dosage module directly to the tray a malfunction of the dosage module may result 3 3 5 Replacing the Fractionation Needle of the µ Tee The fractionation needle is a fused silica tubing with an O D of 280 µm and an I D of 30 µm or 100 µm depending on the LC application Refer to TABLE 3 4 to select the appropriate needle for your application TABLE 3 2 I D of the Fractionat...

Page 75: ...eeve 330 µm I D µ Tee 1 2 3 4 2 mm 1 2 3 4 P N 162146 30 µm P N162144 100 µm P N162145 P N 162147 FIGURE 3 7 Fused Silica Fractionation Needle with Stainless Steel Supporting Needle e Connect the nut to the µ Tee to tighten the fused silica needle f Connect the fused silica column UV flow cell outlet and the connecting tubing from the rotary valve to the µ Tee g Mount the µ Tee on the Probot mount...

Page 76: ...Needle Tee d Unscrew the lower fingertight nut and remove the needle from the needle tee item 2 FIGURE 3 8 e When re installing the needle push the needle all the way into the needle tee while tighten the fingertight nut Make certain that the needle tip extends the stainless steel needle by approximately 1 mm f Re install the ground cable the needle cover and the fused silica capillary 3 3 7 Repla...

Page 77: ...instrument from the electrical supply before inspecting changing the fuse Replace the blown fuse by a fuse of identical type and rating To change the Fuse a Pull out the fuse holder FIGURE 3 9 The Fuse Compartment b Replace the blown fuse by a fuse of identical type and rating 1 6 A Slo Blo fuse c Re install the fuse holder ...

Page 78: ...e generated very shortly after each other Check the timing Sections B 6 and D 6 Wrong control parameter The Z stepper motor is missing steps Check the maximum speed and acceleration settings Section 2 8 7 The distance between needle and table increases during a run Wrong table alignment Check table alignment APPENDIX C Strange noise from the stepper motors table not moving The motor is losing step...

Page 79: ...2 160590 Built in camera system including the Belkin VideoBusII Image Capture Device 163973 BNC to Video Adapter 160563 Manual control unit for table positioning N A 160561 Microdosage unit for automatic addition of reagents N A 161718 µ Tee for Matrix Addition pre needle N A 161072 Propet fused silica Teflon tubing 100 µm 0 25 mm I D Pipetting Option 161080 100 µL syringe for Probot 162179 250 µL...

Page 80: ... and ferrule for Probot µ Tee needle assembly 162144 30 µm I D fused silica needles for Probot µ Tee needle assembly 2 each 162145 100 µm I D fused silica needles for Probot µ Tee needle assembly 2 each 162146 PEEK sleeves for Probot µ Tee needle assembly 330 µm I D 4x 30 mm 3x 10 mm 162148 Connecting tubing Probot rotary valve to µ Tee needle assembly Microtight Connection 161497 Microtight Union...

Page 81: ...Probot N A 161094 Door right acrylic without hinges for Probot N A 162104 Light holder and stand for Probot pen light 162105 Camera holder and stand for Probot camera 3 5 4 Adapter Tables P N Description Figure 163211 Table for MassTech MALDI Target 162340 a b Universal Table for AB4700 Proteomics Analyzer for 6 targets AB Q Star AB Voyager target Back view Top view 161717 Table for Ciphergen SELD...

Page 82: ...adjustment stripes 160575 Table Finnigan target circular dual collect 160574 Table Finnigan target linear dual collect 160573 Table Shimadzu target dual collect 160572 Table Shimadzu KRATOS Axima QIT for 2 targets 160570 Table Bruker target dual collect circular Notes a Replaces P N 160568 P N 161390 b Also compatible with the AB Opti TOF MALDI plate system with reusable holder and the AB Q Star o...

Page 83: ...han 20 µm Reference Position All axes are controlled by reference switches Reproducibility better than 1 motor step 2 5 µm Lifting Force Y axis 20 kp Dimensions WxDxH 480 mm 18 9 in x 500 mm 19 6 in x 300 mm 11 8 in Weight 23 kg 4 2 Electrical Power Requirements 90 260 V 47 62 Hz 100 VA Fuse 1 6 x Slo Blo 5 x 20 mm Stepper Motors X Y Z Axes 400 steps revolution for the X Y Z axis Stepper Motor Syr...

Page 84: ...mum Flow Rate a µ Tee installed 30 µL min 30 µm I D Fractionation Needle 300 µL min 100 µm I D Fractionation Needle Standard Needle Tee installed 300 µL min Maximum Pressure 20 bar 290 psi Note a Total flow rate of matrix solution and LC system 4 5 Safety Devices General All functions are stopped when a front door is opened an axis runs over its end switch the table exerts too much force on the ne...

Page 85: ... the dosage module refer to the µCarrier software user s manual Note All fluidics pathways of the dosage module e g the rotary valve the syringe and all tubing must be free of any air bubbles and must be properly purged A 1 1 The Step By Step Purge Routine FIGURE 2 5 Page 2 10 presents the dosage module with the syringe installed in the syringe drive FIGURE A 1 presents a schematic of this normal ...

Page 86: ...inge Drive Dosage Module PUMP FILL PURGE Rotary Valve Needle Tee µ Tee OR FIGURE A 2 Step 1 Purge the Tubing b Switch the rotary valve into the FILL position and withdraw the reagent solvent from the vial Fill the syringe completely and make sure that enough reagent solvent is in the vial FIGURE A 3 Waste Fill Aux Reagent Vials Syringe Drive Dosage Module PUMP FILL PURGE Rotary Valve Needle Tee µ ...

Page 87: ...lve back into the FILL position withdraw the reagent solvent and fill the syringe completely e Remove the plunger from the syringe Lower the syringe and allow the contents to drain via gravity then replace the plunger FIGURE A 5 Waste Fill Aux Reagent Vials Syringe Drive Dosage Module PUMP FILL PURGE Plunger removed Rotary Valve Needle Tee µ Tee FIGURE A 5 Step 4 Drain the Syringe f Reinstall the ...

Page 88: ...e valve into the PURGE position and purge again FIGURE A 6 Refill the syringe in the FILL position Waste Fill Spare Reagent Vials Syringe Pump Dosage Module PUMP FILL PURGE Pump Fill Rotary Valve Needle Tee µ Tee OR FIGURE A 6 Step 5 Reinstall the Syringe ...

Page 89: ...to control event outputs e g event outputs of the UltiMate Micropump so that a signal is generated at the beginning of a peak and at the end of the peak The conditions that CHROMELEON uses to detect peaks can be programmed and adjusted to the needs of the application The peak collection function of the Probot Micro Fraction Collector and the µCarrier software involves the TTL inputs Section 2 4 1 ...

Page 90: ... B 5 Trigger peakstart UV_VIS_1 Delta 0 1 true 2 00 Pump_Relay_1 State On Pump_Relay_1 Duration 1 00 EndTrigger Trigger peakend UV_VIS_1 1 0 and UV_VIS_1 Delta 0 03 true 2 00 Pump_Relay_2 State On Pump_Relay_2 Duration 1 00 EndTrigger The parameters UV_VIS_1 Delta and UV_VIS_1 can be used independently you can define either or both and they need to be modified according the requirements of your ap...

Page 91: ...arrier for peak collection using a waste group a Prepare a collection group with the amount of vessels required to collect all peaks and a waste group with the appropriate amount and size of vessels FIGURE B 2 FIGURE B 2 Defining the Collection Waste and Park Groups Note The number of vessels required to collect the peaks and the waste may differ and depends on the chromatography conditions e g th...

Page 92: ...olume between the detector flow cell and Probot needle outlet The delay time will be calculated automatically according to the flow rate of the LC system and the calculated volume FIGURE B 4 c Define the primary collect group used for sample collection select the Collect peaks only option and define the waste group used to collect the mobile phase in between the peaks on the Destination tab FIGURE...

Page 93: ...d flow rate e For the remote start of µCarrier select the Wait for contact start from the Relay Table tab FIGURE B 6 The µCarrier software will use the start delay from the General tab FIGURE B 4 FIGURE B 6 The Relay Table Tab f Start the program In the fraction action window you can see which fraction or waste has been put in what vessel FIGURE B 7 FIGURE B 7 Fraction Action Window To setup µCarr...

Page 94: ...1 to the Probot TTL 1 Peak start input as presented in TABLE B 1 c Connect the event output 8 relay 2 to the Probot TTL 2 Peak end input as presented in TABLE B 1 TABLE B 1 Connecting the Probot and the Micropump Probot Color Code UltiMate Micropump EVENTS Start Signal Start Lilac Event 3 pin 3 Ground Black Ground pin 4 TTL Inputs TT1 Peak Start TTL2 Peak End TTL1 pin 1 Red Event 7 pin 7 TTL2 pin ...

Page 95: ... PumpPressure Formula Formula Pump Masterpressure value PumpPressure Type Analog 0 000 UV autozero Flow 2 50 B 0 C 0 D 0 Wait UV Ready and Pump Ready and Sampler Ready Inject UV_VIS_1 AcqOn ColumnPressure AcqOn PumpPressure AcqOn Flow 2 50 B 0 C 0 D 0 Probot_Start On Duration 1 00 Trigger peakstart UV_VIS_1 Delta 0 1 true 2 00 Pump_Relay_1 State On Pump_Relay_1 Duration 1 00 EndTrigger Trigger pea...

Page 96: ...L 2 signals are generated immediately after each other the Probot moves the table to the next vial or stays in the waste vial if a waste position is selected Some older µCarrier version e g versions lower than 1 70 do not allow collection in only one waste vessel it requires that you define as many waste positions as collect positions Section B 3 To get only one virtual waste position with the pro...

Page 97: ...ent of the table of the Probot Micro Fraction Collector and how to align it Proper alignment is important to make certain that the distance of the fused silica needle remains constant at all X and Y positions Note It is recommended that you check the alignment whenever the instrument has been moved e g after shipping or relocation of the instrument ...

Page 98: ... If necessary adjust the four feet until the instrument is leveled properly Front View Right Side View FIGURE C 10 Leveling the Probot C 2 2 Test Needle and µCarrier Setup Note The following procedure discusses all steps using the standard needle If the µ Tee is installed all steps are identical except step a A piece of fused silica is used as test needle The table is then moved as close as possib...

Page 99: ...change from step mode to continuous movement press the appropriate button of the manual control unit To change the length of each step speed adjust the Stepwidth parameter C 2 3 Checking and Aligning of the X axis and Y axis To check align the X axis a Remove all targets from the table adapter and remove the adapter from the table b Lift the table and position the table so that one of the two smal...

Page 100: ...abeled 2 in FIGURE C 13 clockwise to lift the table on the left side turn the screws counterclockwise to lower the table on the left side Note Turn both screws labeled 2 in FIGURE C 13 by the same angle to keep the Y adjustment e g to lift the table on the left side by 1mm turn both screws approximately 30 degrees clockwise c Retighten the screws labeled 3 in FIGURE C 13 and check the X direction ...

Page 101: ... 6 Loosening all screws at the same time may lead to a misalignment of the limiter switches of the Y axis c Carefully rotate the table in the proper position d Carefully tighten the screw in the order 5 6 4 to prevent any movement of the table e Check the proper alignment of the Y axis and then check the X axis again f Repeat steps b e and repeat the X alignment if necessary g Replace the cover pl...

Page 102: ...Appendix C C 6 D960R3 User s Manual Probot This page intentionally left blank ...

Page 103: ...e solvent into a waste container The vacuum waste removal option includes the following items Lift magnet Vacuum pump Waste container Tubing and vacuum connectors To control the vacuum waste removal event outputs of the UltiMate capillary HPLC system are used in combination with the peak detection capability of CHROMELEON see also APPENDIX B The following sections provide information about how to ...

Page 104: ...less steel needle in the lower position e g the lift magnet is not powered The tip of the fused silica capillary is covered by the needle The vacuum will draw the solvent into the waste container Tubing from Vacuum Pump 1 mm Stainless Steel Needle Fused Silica Capillary FIGURE D 1 The Needle in lower Position FIGURE D 2 shows the needle tee in the upper position e g the lift magnet is powered The ...

Page 105: ...se the cables and connectors which are provided with the Probot and the UltiMate system TABLE D 2 Connecting the Probot and the Micropump Probot Color Code UltiMate Micropump EVENTS Start Signal Start Lilac Event 3 pin 3 Ground Black Ground pin 4 TTL Inputs TT1 Peak Start TTL2 Peak End TTL1 pin 1 Red Event 7 pin 7 TTL2 pin 3 Orange Event 8 pin 9 Ground pins 9 11 13 15 Blue Event 7 and 8 pins 8 10 ...

Page 106: ... capillary into the metal port above the needle tee using the installed nut a ferrule and a PEEK sleeve then guide the capillary through the upper port the needle tee and all the way through the needle FIGURE D 4 1 FIGURE D 4 Installing the Fused Silica Capillary c Turn the fitting until it is finger tight You should not be able to move the capillary laterally along the tubing axis item 1 FIGURE D...

Page 107: ...ge module cannot be used in conjunction with the waste removal option Select the Controls tab in the Settings menu then disable the Syringe pump module dosage module Enable the Vacuum waste removal option FIGURE D 5 FIGURE D 5 Disable the Dosage Module D 4 2 Adjusting the Fused Silica Capillary and the Needle Height The following steps are required to adjust the fused silica capillary properly in ...

Page 108: ...rtain that the distance tip of the fused silica capillary and the carrier is between 1 and 2 mm 0 004 0 008 inch D 4 3 Using the Waste Removal Option Example The following example shows how to use the waste removal option in conjunction with the peak detection capabilities of CHROMELEON To setup the µCarrier software a Prepare the tray in the standard way define as many groups as required for the ...

Page 109: ... the following lines have to be added to the program the underlined parameters are examples and chromatogram dependent This is an example in which the peak start is defined by the slope of the chromatogram and the peak end by the slope AND the absolute UV signal Depending on your chromatographic conditions other trigger rules may lead to better results Probot_Start On Duration 1 00 Trigger PeakSta...

Page 110: ... Normal SyringeSpeedFactor 0 2 SampleHeight 5 FlushVolume 5 0 WashVolume 50 RinseBetweenReinjections No UV_VIS_1 Wavelength 214 UV_VIS_1 Step 1 00 UV_VIS_1 Average Off Data_Collection_Rate 1 PumpPressure Formula Formula Pump Masterpressure PumpPressure Type Analog ColumnPressure Formula Formula Pump Columnpressure ColumnPressure Type Analog 0 000 Autozero Pump Flow 4 000 B 0 C 0 D 0 Wait UV Ready ...

Page 111: ...l wait for a start signal before the first peak if the Wait for contact start signal option is enabled on the Relay Table tab of the Fractionate task FIGURE D 7 In addition you need to connect the START input of the Probot to the start output of the starting device using the Start Cable see also Section B 3 If two Peak Start TTL 1 signals are generated immediately after each other the Probot moves...

Page 112: ...Appendix D D 10 D960R3 User s Manual Probot This page intentionally left blank ...

Page 113: ...tion Collector effective November 2004 The Belkin VideoBusII device serves to connect the video camera to your PC through a USB port and to display the camera image in a separate µCarrier window on your PC To use the Belkin video adapter and to display the camera image in µCarrier the following equipment is required Probot with built in camera µCarrier V2 0 Belkin F5U208 USB VideoBusII Image Captu...

Page 114: ... were not copied to the hard disk copy the files as discussed in Section 2 5 or download the appropriate drivers from www belkin com search for F5U208 Unpack the drivers by default the folder C F5U208 is created This is the path which you have to specify during the installation c Connect the Belkin video adapter to a free USB port The Windows Found New Hardware Wizard will start automatically d Fo...

Page 115: ...the video window FIGURE E 2 FIGURE E 2 The Video Camera Window of µCarrier b The video image should be visible If no image is visible refer to Section E 2 4 for troubleshooting information Refer to Section E 2 3 to optimize the video settings The Rate ms setting defines the frame rate of the image 100 ms is the default value that should be used To update the image at a higher frequency choose a lo...

Page 116: ...s a file that is larger than10 Mb To avoid any risk of a computer crash due to the large file size the maximum recording time is limited to 10 seconds E 2 3 Optimizing the Video Settings in µCarrier 2 0 To optimize the video image a Select the Settings button in the video camera window FIGURE E 2 and then choose the Device Settings tab FIGURE E 3 FIGURE E 3 Video Source Device Settings Tab b Set s...

Page 117: ...e µCarrier software and check if all settings are properly In order to check the installation of the Belkin VideoBusII device a Open the Control Panel via the Start button from the Windows Tool Bar b Open System and select the Hardware tab from the System Properties window and then choose the Device Manager c Click on the plus sign next to Imaging Devices Under this heading you should see Belkin U...

Page 118: ...n next to Sound video and game controllers Under this heading you should see a Belkin USB VideoBus II Audio f Right click on Belkin USB VideoBus II Audio and choose Uninstall g Close Device Manager and System Properties and unplug the adapter from the computer h Go to Start Run and type c windows inf if this fails you can try c winnt inf and click OK i Scroll through the list of files and locate t...

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