FIAlab-3000 Operations Manual
Contents
1. compone A ringe Pump settings Quick Configure Syringe Pump Syringe Size uL 2500 FiAlab 3000 Fl Alab 3500 Pump Type wL FlAlab 3500 6 14 Instrument Mame Serial Port Custom syringe Pump ACCE pt Sub address RH5 485 address m A M Remove Cancel E Select component Define New from Existing Components to modify add Click Accept after modification Note Name must be unique Settings for the pumps and valves are described here Each pump or valve setting relating to communications includes the RS 232 serial port subaddress and for syringe pumps RS 485 address MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 7 B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology RS 232 Many of the main and peripheral components controlled by FIAlab are RS 232 Serial Port These include the syringe pumps usually though some may be RS 485 peristaltic pumps injection and selection valves autosamplers PMT FL and others The user must assign the correct serial port and sub address for each component which is dependent upon the specific configuration of the purchased system In nearly all cases the entire system will be plugged into a single serial port most likely serial port 1 Just set all serial port settings to 1 which 1s the default and Sub addre2. FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 28 B FIAlab Instruments Inc lab Leaders in Flow Injection Technology 6 0 Analyzing the Results After a run bring up the Analysis page and click Refresh Immediately click on Save Data and give the file a name It is imperative that you save the data before running any other programs The two tabs used for most applications are the Plots tab to visually see the results plotted and the Calibration tab to set the desired parameters prior to creating a report pa ls am ea Desi hides Pokereernal Caras Kanal a ooien A 37208E 04 Costco Cement hor Dit THAE E Ratnesh rT Mops daio Fleisch I thon od Mamm Shier Conceniedion litil Chick Hetresh to See Data Gick Metiech to See Dhaba i mm A E EI C o oem 08 02 Save Dats Load Data Add Report Rare Figure 6 1 Analysis Page The Calibration tab page allows the user to set the number of fitting coefficients i e 1 order straight line and 204 order fit Also the Correct for Drift option 1s selected here Correct for Drift 1s only applied if the run was made with Driftcor samples as described in section 5 0 The user can also select whether the report should be based on peak maximum or
3. button After the Defaults have been set then redo the Optimal Integration Time To include the spectrophotometer commands in your Dye Test protocol add the following instructions before the command to dispense the sample to the flowcell as shown in the example protocol provided in the Appendix Section 4 7 Spectrometer Reference Scan Spectrometer Absorbance Scanning At the end of the program after the sample has been completely delivered to the flowcell add the following command Spectrometer Stop Scan The following Dye Test program SIA dyetest2 fia also found in the training SIA folder on the included USB Flash Memory Stick uses the above spectrometer commands including automatic setting of the wavelengths It also includes a loop to repeat the command sequence 10 times which will demonstrate the repeatability of the measurements Run this script click on Start on the program page after you have loaded SIA dytest2 fia and observe the results from the spectrometer or analysis page You can observe the results occurring real time from the spectrometer page MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 20 B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology SIA dyetest2 fia this simple script primes the line washes flowcell aspirates sample and dispenses the sample to the flowcel
4. B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology Using template methods provided in the software There are several templates available that can be selected directly from the FIAlab program page It is recommended that you use these only after manually working with the system and trying to write a couple of your own programs with the help of the simpler programs like the Dye Test protocol so that you are familiar with how the system works The templates also include commands for the spectrometer and autosampler which will be covered in later sections Note the templates are typically a starting point for the user who ends up modifying them with regards to monitored wavelengths pump speeds volumes etc From the FIAlab program page select the desired method script to run as shown in the figure on the next page Under FIA Templates select SIA and then LOV 0 reagents since dye assays usually have zero reagents just the sample and carrier When using one of the methods provided it is assumed that all tubing is connected correctly and the reagents are all in place etc The selected script from the FIA Template library can be modified as needed If the script 1s modified save it with the Save button on the bottom of the Programming page and rename it Then to later open the saved script use the Open button at the bottom of the programming page Please review the FIAlab for Window
5. Jarda Ruzicka 2 B FIAlab Instruments Inc lab Leaders in Flow Injection Technology 2 0 Principles and Applications Sequential Injection Analysis SIA is the second generation approach to FIA compatible assays SIA usually consists of a single channel high precision bi directional pump a holding coil a multiposition valve and a flow through detector The system is initially filled with a carrier stream into which a zone of sample and a zone of reagent s are sequentially aspirated into a holding coil forming a linear stack These zones become overlapped due the parabolic profile induced by differences between flow velocities of adjacent streamlines Flow reversals and flow acceleration further promote mixing The multiposition BUFFER SAMPLE valve is then switched to the detector position and the flow direction is reversed propelling the sample reagent zones through the flowcell HOLDING COIL WASTE REAGENT 2 REAGENT 1 SIA in Lab On Valve LOV format is a groundbreaking technology that has won an enthusiastic acceptance in the research community for its versatility and in the routine laboratory for its reliability The LOV integrates manifold components including a fiber optic coupled flow through cell into a single unit mounted on a multiposition valve The SIA LOV is used as a platform for microSequential Injection as well as for Bead Injection and Sequential Injection Affinity Chromatography The L
6. 2 Flowcell Port 3 ReagentA won t be used for this dye exercise Port 4 ReagentB won t be used for this dye exercise Port 5 Sample Port 6 Carrier Filling the system with carrier solution and priming the ports Place the carrier inlet left side of syringe valve tube ideally I D 1 mm or more into the carrier solution bottle filled with 2100mL carrier Place the dye sample into a vial and insert the sampling tube from port 5 into the dye solution Using a 1 mL disposable syringe seated in the female Luer fitting on the opposite channel of the sample flow through port manually aspirate dye solution past port 5 and just into the plastic syringe the solution in the syringe will be waste Note for FIAlab 3200s use the top syringe pump for this exercise Figure 4 2 Photo of MicroSIA system configured with fiber optics and solution reservoirs attached MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 13 B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology FIAlab for Windows 5 0 ments Options About 5 31 2005 10 40 40 1 1010 md Program Analysis Exit Sequential Injection Analysis SEES Serial Port 1 Periskaltic Pump Syringe Pump Valve gt Syringe Settings E V alume uL U Flowrate Runtime sec Please Logo
7. Inc 2005 Graphics Copyright Jarda Ruzicka 10 B FIAlab Instruments Inc lab Leaders in Flow Injection Technology 4 0 Basic instrument functions and programmable flow With the exception of the fiber optic probes the FIAlab systems are shipped fully plumbed and ready to use for simple SIA assays e g dye concentration measurements enzymatic assay of glucose nitrite assays or any other one or two reagent based spectrophotometric assay It is critical that users first familiarize themselves with these types of assays prior to moving on to more advanced uses such as SIChromatography and Bioligand Interaction Assays The manifold should be configured as shown below Should you need to assemble or modify the system we suggest using 0 8mm 0 03 in LD teflon or PEEK tubing coded green for all manifold components Alternatively 0 5mm 0 02 in I D teflon or PEEK tubing coded orange can instead be used for ports 3 and 4 Block any unused ports with white Teflon filament 1 6mm O D supplied with the kit CARRIER BUFFER Figure 4 0 Manifold Configuration for SIA Typically port 1 waste port 2 flowcell port 3 reagent 1 port 4 reagent 2 port 5 sample flow through port port 6 carrier C connects to holding coil The relative position of the fiber optics suggested for training purposes are shown MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005
8. Settings L End Configure LOY configure LOY Lx Start NextSample command autosampler to first sample Message Next Sample TE Reset Data Wash and prime system and optimize spectrometer Camp Daan Peristaltic Pump Clockwise 50 Save Data I Wash LOY Stop Program Prime LOV Carrier Text Command Hardware Settings Optimize Integration Variable Peristaltic Pump Off Time sec Autosampler Wash 0 0 50 Number of Samples elo Open Save Pause Start Stop Emergency Check Exit MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 25 B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology 5 1 Sample Definition File FIAlab for Windows includes a very powerful approach to defining samples and standards located on sample racks using an autosampler The purpose of the Sample Definition File is to command the autosampler to the next desired sample at the desired time and have the software automatically know the type of sample whether it is an unknown or a standard and the standard concentration and how many repeat measurements to make The Sample Definition File page shown below is accessed from either the Autosampler page or by clicking on the Sample Definition File menu on the top of the Program page Select Edit Create File Please read this section and feel free to contact FIAlab
9. VALV E ee ette ones eee enuus 3l 7 1 MATERIALS REAGENTS AND SYSTEM CONFIGURATION nnns 32 LOADING THE COLUMN Te 33 7 3 RUNNING THE ASSAY AND INTERPRETING THE 33 7 4 REMOVING THE COLUMN Aeon uad P Iu anna as Ae Rae e dva uad 35 79 PROGRAM TEMPLATES 36 8 0 USING THE SYSTEM WITH OTHER TYPES OF DETECTORS FLOWCELLS esee 40 DAD VV dri P 41 VARRANT emm E 41 B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology 1 0 Introduction The FIAlab 3200 3500 and MicroSIA instruments are precise solution handling systems for introducing microliter to milliliter volumes of solutions in a reproducible and automated manner to nearly any instrument or device The systems are especially suited for Sequential Injection Analysis SIA Bead Injection Analysis BIA and Sequential Injection Chromatography SIC Up to four units can be slaved to one autosampler efficiently creating up to a four channel SIA system The advantage of SIA over the more traditional flow injection analysis FIA is that SIA consumes typically less than 1 10 the reagent and consequentially produces far less waste an important feature when deal
10. this simple script primes the line washes flowcell aspirates sample and reagent and dispenses the sample to the flowcell and collects data Repeats measurement 10 times Global Logon this line establishes communication Configure LOV this line tells software that the manifold is a LOV Set Wavelengths Hardware Settings Wavelength 1 nm 620 Hardware Settings Wavelength 2 nm 600 Hardware Settings Wavelength 3 nm 580 Hardware Settings Wavelength 4 nm 540 Hardware Settings Use Wavelength 4 as Reference Prime LOV ReagentA Prime LOV Sample Loop Start 10 prime sample line and wash flowcell Wash LOV Analyte New Sample aspirate 30 microliters of sample and 30 microliters of reagent Aspirate LOV Sample 30 Aspirate LOV ReagentA 30 Aspirate LOV Carrier 100 dispense to flowcell and collect data Multiposition Valve Flowcell syringe Pump Flowrate microliter sec 10 Syringe Pump Empty Spectrometer Reference Scan Spectrometer Absorbance Scanning Syringe Pump Delay Until Done Spectrometer Stop Scanning Loop End MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 23 B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology 4 6 Stop Flow Assays Choose either the two or three zone routine and include a stopped flow period in your assay cycle Do this by inserting Delay sec 45 into your a
11. through the port 5 and then stopped This sampling pump usually consists of a peristaltic pump MicroSIA FIAlab 3200 FIAlab 3500 solenoid pump or even secondary syringe pump e g using the FIAlab 3200 Example Run The FIA templates discussed in section 4 3 are setup to use the autosampler From the program page select FIA Templates SIA LOV 0 Reagents Then using blue dye in the autosampler vials and water for the carrier some surfactant in the carrier click Start Note to map out the autosampler racks e g which samples are standards which are unknowns please review the next subsection Programming Mew Font SaveEvents SafeLockMode Find 3 Sample Definition File FIA gt autosampler SIA LOV Method Number of Reage 2 127515 Co oe TTL DAC Setting One ml syringe assumed d eager Peristaltic Pump LOY 2 reagents Syringe Pump SIA Template For MicroSIA System with LO 50 Flow Assay Valve Global Logon logon to all components Sequential Injection Chromatography S Suas T S pectrome Sample Description load sample description Bioligand Interaction Assay Analyte Set Wavelengths Beep Hardware Settings Wavelength 1 nm 620 sec Hardware Settings Wavelength 2 nm 600 Ekal Hardware Settings Wavelength 3 nm 580 If Hardware Settings Wavelength 4 nm 540 Insert File Hardware Settings Use Wavelength 4 as Reference
12. within a 2 minute assay cycle HOLDING COIL Sequential Injection Ion Exchange Chromatography is based on ion exchange separation of biomolecules from their interaction with charged sites fixed on a stationary phase and their modification due to the pH and salt content of the mobile phase Their resolution is based on the gradual change in composition of the mobile phase which forms an elution gradient with a variable pH or salt content MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology 3 0 Software Hardware Installation The following software installation steps will only have to be performed once on a specific computer Note this section only describes how to install the software and establish communication between the software and the hardware Detailed instructions about their usage are described in later sections Computer Requirements MS Windows based PC running Windows XP or Windows 2000 Recommended 450 MHz or faster 256 MB of RAM at least 20 MB of free disk space an available serial port RS 232 and USB port There are low cost USB to RS 232 converters available from most computer stores for use on computers which do not have serial ports If using one of these converters please make note of which serial port number MS Windows assigns the converter to look
13. 00 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 24 B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology elution step see the note included with the Example Protocol for an Affinity microChromatography Assay in Section 7 5 7 4 Removing the Column With the system configured as described in Section 7 1 simply aspirating the beads back into the holding coil and sending them to waste via another port will remove the bead column To accomplish this the waste line extending from port 2 must be long enough and the waste reservoir must have enough solution in it so that when the beads are pulled back into the holding coil air is not aspirated into the flow cell and the lab on valve Washing steps included in the bead loading and assay programs prevent any waste solution from remaining in the flow cell and interfering with subsequent measurements A method template for removing the bead column Section 7 5 1s also included under Templates on the Program window In the first step carrier solution is pulled into the syringe from the reservoir to assist in flushing the beads from the system Then 50 microliters are aspirated from port 2 into the holding coil at 50 microliters second and the opaque column can be seen retreating into the holding coil Because solution being pulled around the fiber optics at that flow rate causes a change in pressure within the Lab
14. FIAlab Instruments F TA lab Leaders in Flow Injection Technology Operations Manual for the MicroSIA FIAlab 3200 and FIAlab 3500 Systems LO INTRODUCTION m 2 2 0 PRINCIPLES AND APPLICATIONS initis teuer S noA SEIS IEEE NS En XR ae YS venae Ro Er na SS UNO Sei IN Ue ENERO NER MEE YER 3 3 0 SOFTWARE HARDWARE INSTALLATION e eseessoreeus esa ra Yo SS Pa EPA ES Nba USER PS ER PEE EN USE PUR EVER PEE 5 3 1 FIALAB FOR S QVUNDOSYUS ne uda unt Eure RM Urbe Ku DID 6 3 2 HIALAB SYSTEM PUMPS AND VALVES 7 I SPECTROMETER 9 SO AUIOSAMPDEER e a denen E 10 4 0 BASIC INSTRUMENT FUNCTIONS AND PROGRAMMABLE FLOW ee eeee eee eene nette nnn tnus 11 4 1 FIBER OPTICS AND PERIPHERAL PORTS IN THE LAB ON VALVE eee 17 4 2 MANUAL CONTROL AND PRIMING OF THE SYSTEM 13 4 3 WRITING A DYE TEST ROUTINE AND RUNNING A nnns 16 4 4 SPECTROMETER SETUP AND RUNNING A ROUTINE WITH SPECTROPHOTOMETRIC DETECTION19 SU USEING AN AUTOSAMPLER E UIN SESS cU PISA Viae PES 25 6 0 ANALYZING THE RESUL S odes nean hao REM IS PERI DUE UR EO 29 7 0 AFFINITY MICROCHROMATOGRAPHY IN THE LAB ON
15. Graphics Copyright Jarda Ruzicka 11 FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology 4 1 Fiber Optics and Peripheral Ports in the Lab on valve Inserting the fiber optic probes The system has been plumbed with tubing and is almost ready for use The fiber optics must be inserted into the LOV since attaching them prior to shipment is not practical The relative placement of the fiber optic probes depends on the type of measurements you will be performing see Figure 2 1 For training purposes the fibers should be positioned for absorbance with a distance of 7 mm between the ends of the probes To attach the fibers first thread a 1 4 28 Upchurch nut onto each of the metal fiber casings followed by a ferrule and insert the fibers into the appropriate channels of the LOV see below Position the fibers and then tighten the fittings into the LOV Connect one of the fiber optics to the light source and the other to the spectrophotometer The operation of the spectrometer and will be discussed in Section 4 4 and detailed instructions can be found in the FIAlab for Windows Software Users Guide Note the ends of the optical fiber contained steel sheaths must be pushed in at least as far as the two exit ports port 2 and the unlabeled port above port 2 Typically the port labeled as 2 is plugged and the port above port 2 is connected to waste d _ To light source or detector Into LOV 7 mm Figu
16. H ampdorm Volume miceoliter Speed AutoDiuted percent cra ain Pe Humber of 5 amples Tere sec oo of S amples 500 mr IL z Wie Open Save Pause Ret Stop Energercy Check Ext The first simple program will simply automate aspirating sample from port 5 and dispensing the sample through the flowcell A simple SIA program script which simply aspirates sample and then dispenses it out through the flowcell would look like the following next page You can create this script your self by pointing and clicking as described below or you can simply load dyetest1 fia from the training SIA folder on the included USB Flash Memory Stick Click on the Open button on the bottom of the Program page to load a file To create or modify a program after changing to Edit Mode click on a component in the list on the right side of the Program window that you want to address e g Syringe Pump Once you do that a secondary list of MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 16 B FIAlab Instruments Inc F TA lab Leaders in Flow Injection Technology sub commands will appear Choose subcommand you want e g Unless you have to enter a number for flowrate volume or time delay push lt Enter gt on the keyboard to move to the next line to add the next comman
17. OV integrated flowcell can be utilized for absorbance adjustable path length and fluorescence measurements as shown below ABSORBANCE SHORT PATH CONFIGURATION Y ABSORBACE FLUORESCENCE ABSORBANCE amp LONG PATH FLUORESCENCE Figure 2 1 Configurations and relative positions of the fiber optic probes in the LOV flowcell For additional discussions please review Flow Injection Analysis Jarda Ruzicka 2 Ed self published 2000 with bibliography by E H Hansen on the included CD ROM MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 9 FIAlab Instruments Inc Leaders in Flow Injection Technology FIAlab Sequential Injection for the Assay of Biomolecules including Enzymatic Assays Bioligand Interaction Assays and Chromatography of Biomolecules are usually carried out on different instruments which necessitates mastering various types of hardware and software This fragmented approach supports a view of an apparent incompatibility of these techniques obscuring similarities of their underlying biochemical reactions and kinetics It is the versatility of programmable flow combined with a UV Vis or fluorescence detector that allows the gap between these techniques to be bridged by a single MicroSIA or FIAlab 3200 or 3500 system with FIAlab s unique Lab On Valve manifold For additional discussions please review Sequential Injection for Biom
18. are Drivers Follow Ocean Optics included step by step instructions to install their software and software drivers from the Ocean Optics included CD ROM Note You can alternatively download OOIBASE32 from ftp ftp oceanoptics com pub ooibase32 websetup exe Step 3 Verify that the OOIBASE32 software works with the spectrometer Reboot the computer Plug the spectrometer into the computer s USB port and wait 10 seconds Bring up the spectrometer page from FIAlab for Windows and click the Voltage button and then click single scan You should see data form in the top plot The data should change slightly each time you click single scan ra Spectrometer Typical results of voltage scan when there 1s no light source Master Master on click Single Scan while Voltage ujom button is selected 1 0 0 8 0 6 0 4 0 2 0 0 0 Disable Spectrum Plotting weleio d Dark Reference Volt Singdaissn Repeated Scan Scan to File MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 9 B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology 3 4 Autosampler Ignore this section if you are not using an autosampler Step 1 Power up the Autosampler Plug the provided power cables to Autosampler and turn Autosampler on Note the ASX260 520 sampler uses an external AC to DC con
19. at device manager from Windows control panel s system utilities The serial port number will be needed in configuring FIAlab for Windows software described later Please contact FIAlab if you have any questions MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 5 B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology 3 1 FIAlab for Windows Step 1 Install FIAlab for Windows Software Put included FIAlab CD ROM into CD ROM drive or flash memory stick into the USB port and an automatic startup program will give you the option to install FIAlab for Windows If the automatic program does not start locate the folder FIAlab 5 0 Software and from there start up fiasetup EXE or startup exe in the root folder of the flash memory stick Install FIAlab for Windows Step 2 Execute the fiareg reg file On the included Startup Floppy Disk or flash memory stick you will find the file fiareg reg Execute this file The startup exe on the flash memory stick has this as the second option This will install the specific configuration for your purchased system FIAlab for Windows should not be running when the registration file 1s executed THIS IS EXTREMELY CRITICAL YOUR SYSTEM WILL NOT RUN CORRECTY WITHOUT THIS STEP Step 3 Connect communication and power to FIAlab The system communicates to your computer via the serial co
20. c 2005 Graphics Copyright Jarda Ruzicka 33 B FIAlab Instruments Inc F TA lab Leaders in Flow Injection Technology Protein A the first set of peaks represent the varying amounts of non retained BSA flowing through the detector and the second set shows the elution of the captured IgG 4 alal Tine Gerke 0 t 00 Mam A ANC 2 40 BU eu Time see Au Heliesh Show Conceriraliori ShowDaaLebeli Maium Click Rehiech to Seem Data 9 Sawn fata Load fatal Add Data Refresh Fut Separation of Mouse IgG from bovine serum albumin on a renewable microcolumn of Protein A Sepharose 6B Sample mixture of 2 mg mL IgG and 4 6 mg mL BSA Mobile phase PBS pH 7 4 Eluant 5 uL 1M Column capacity 20 mg mL column volume 10 uL Flow cell volume 6 uL optical path 3mm The method template has been set up with the ability to monitor both peaks which is helpful when initially setting up an assay or troubleshooting For optimized assays where only the elution profile may be of interest the absorbance scanning need only run during the elution step When the template is loaded into the Program window the spectrometer commands during analyte delivery are written to collect data throughout the assay Once the assay has been optimized and only the elution profile 15 desired the program can be amended to collect data for only MicroSIA FIAlab amp 32
21. d Programming Editing Notes The underlying editing environment is similar to MS Wordpad so highlighted lines and sections can be cut Ctrl X copied Ctrl C and pasted Ctrl V To make the Program page full screen double click on the blue bar at the top of the page To return it back to normal size simply double click again on the top blue bar A couple of special commands should be inserted at the top of your program Global Logon and Configure LOV Global Logon automatically logs onto each component e g the FIAlab unit autosampler and spectrometer Configure LOV tells the software that the valve manifold mounted on the FIAlab is a LOV type manifold dyetest1 fia this simple script primes the line washes flowcell aspirates sample and dispenses the sample to the flowcell Global Logon this line establishes communication Configure LOV this line tells software that the manifold is a LOV prime sample line and wash flowcell Prime LOV Sample Wash LOV aspirate 100 microliters of sample Syringe Pump Valve Out Multiposition Valve Sample Syringe Pump Flowrate microliter sec 25 Syringe Pump Aspirate microliter 100 Syringe Pump Delay Until Done dispense to flowcell Multiposition Valve Flowcell Syringe Pump Empty Syringe Pump Delay Until Done MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 17
22. d change slightly each time you click single scan T praterie rebas EN iren mie es e meis e 1111 mus zT 2S TM Mam n5 2050 zu sema pan i 2 ee 2 Balnnos _ Figures 4 4 Good Results left Saturated Results right rude lipad Scan In figure 4 4 above plot on left demonstrates typical type of curve you want to see with voltage level above 1000 at the wavelengths of interest The flat portion of the curve on the right indicates that the detector is being saturated If your scans result in a saturated peak click on the Spectrometer Setup tab and adjust the integration time down by entering a new number in the box indicated in the figure below After adjusting the integration time switch back to the spectrometer graphing page and redo the scan Repeat these steps until a good reference scan is achieved Alternatively click on Optimize Integration Time on the spectrometer setup page see Figure 4 5 The optimization takes around 10 seconds If there is insufficient light throughput peak response is below 1000 counts then first check to make sure that there are no bubbles in the flowcell Increasing the amount of surfactant in the carrier will help prevent bubble formation Assuming that there are not any bubbles in the flowcell click on the Spectrome
23. dware Settings Use Wavelength 4 as Reference To run a script simply click on the Start button on the bottom of the program page However if you are using a spectrometer with your assay it is recommended that you read section 4 4 prior to your initial run Note For runs without an autosampler the software can be made to prompt the user automatically to insert the sample line into each vial For this option create a sample definition file as described in section 5 0 and then on the program page under the Sample Definition File menu select User Sample Prompt MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 18 B FIAlab Instruments Inc lab Leaders in Flow Injection Technology 4 4 Spectrometer Setup and Running a Routine with Spectrophotometric Detection Bring up the spectrometer window from FIAlab for Windows Be sure that you have connected the fiber optic cables to the lamp and light source as directed in Section 4 1 Also the following tests should be performed with water in the flowcell Before turning on the light source on the Spectrometer Graphing page click on Logon and then on Dark Scan Once the dark scan has been performed turn the lamp on click the Voltage button and then click single scan You should see data appear in the top plot which is a plot of response versus wavelength nm The data shoul
24. e complete packing an excess of beads 15 aspirated into holding coil in the second step of the template and step four sends any extra beads remaining in the groove or holding coil back to the bead reservoir Once the micro affinity column has been loaded it does not need to be removed unless an air bubble is introduced into the column elution is not achieved the beads will still be holding onto analyte or the set of assays 15 completed 7 3 Running the Assay and Interpreting the Results The main steps of the micro chromatography assay are washing of the microcolumn and flow cell perfusion of the sample through the microcolumn and elution of the retained analyte from the beads In the method template provided accessed via Templates on the Program window and shown in Section 7 5 the assay begins with a wash of the bead column followed by aspiration and disposal of a small amount of sample to prevent carryover and purging of the flow cell The flow cell is purged by sending carrier solution to port 6 through the tubing loop to an exit channel of port 2 where the solution 1s pushed past the fiber optics through the flow cell and out to waste Ata relatively high flow rate this step both rinses the flow cell and removes any bubbles that may have appeared Fifteen microliters of sample are then aspirated from port 5 into the holding coil and perfused through the microcolumn at port 2 followed by 45 microliters of carrier 3 tim
25. er string 2 Microcolumn Hardware Settings portname number string 3 Beads Hardware Settings portname number string 4 Eluant Hardware Settings portname number string 5 Sample Hardware Settings portname number string 6 Flowcell Bead conc 200 300 uL in bottom of Eppendorf tube 1 mL buffer Syringe Pump Valve In Syringe Pump Flowrate microliter sec 200 Syringe Pump Aspirate microliter 250 Syringe Pump Delay Until Done Syringe Pump Valve Out Multiposition Valve Beads Syringe Pump Flowrate microliter sec 20 Syringe Pump Aspirate microliter 125 Syringe Pump Delay Until Done Multiposition Valve Microcolumn Syringe Pump Flowrate microliter sec 50 Syringe Pump Dispense microliter 275 Syringe Pump Delay Until Done Multiposition Valve Beads Syringe Pump Dispense microliter 100 Syringe Pump Delay Until Done MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 37 FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology Example Protocol for an Affinity microChromatography Assay Global Logon logon to all components Sample Description load sample description file Set Wavelengths Hardware Settings Wavelength 1 nm 280 Hardware Settings Wavelength 2 nm 350 Hardware Settings Wavelength 3 nm 400 Hardware Settings Wavelength 4 nm 450 Configure LOV configure LOV Hardware Settings portna
26. es the sample volume The slow flow rate during perfusion 2 microliters second allows the analyte to interact with the immobilized ligand and be retained on the column Any non retained molecules continue through the column and flow cell and out to waste For the elution step 10 microliters of eluant are aspirated from port 4 into the holding coil and then perfused through the microcolumn followed by 70 microliters of carrier solution The eluant disrupts the molecular interaction releasing the analyte from the immobilized ligand and the analyte is carried through the flow cell where it 1s monitored in real time using fiber optic spectroscopy The height of the resulting peak corresponds to the amount of analyte retained on the beads In the top figure below copied from the Analysis window two peaks are observed as the assay 15 monitored by absorbance at 280 nm The first peak represents the non retained molecules as they pass through the flow cell and the second is the elution profile as eluant removes the analyte from the microcolumn and carries it through the flow cell Three replicate measurements are shown superimposed and demonstrate the reproducibility of the method In the lower figure a series of different volumes of sample a mixture of bovine serum albumin and mouse IgG are injected on a column of Protein A coated beads Since BSA does not bind to the MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments In
27. fialab flowinjection com for technical support for setting up a file with a specific application in mind Sample Definition File B File Edit Format Notepad Sample Definition File com Files WINFI4 5 0 Method Filessdefault csv Sample Definition T able View Rack SampleName Conc Statt Time hrs Period hrs ack 1 1 Blank 0 ack 1 std2 10 ack 1 std4 40 j Cu cu Cu J e e e Edit Selected File Refresh Total Number of Vials ees System Single Channel C Multi Channel Clear File Name Create New Sample DefinitionT emplate File Standards 4 Freq of OC Driftcor Sampling 20 v Create New File Pos of GC Driftcor Sample 3 Samples fso Initially the user will create a template click Create New File and edit the template to be consistent with their analysis requirements The options are as follows Standards Number of standards utilized for the assay These standards should be in the standards rack of the autosampler as defined by the autosampler page The typical setting is 5 Samples Number of unknowns to be analyzed during the assay These unknowns should be in the sample racks of the autosampler as defined by the autosampler page Freq of QC Driftcor Sampling How often to perform a quality control drift correction measurement Fo
28. ing with expensive chemicals and or hazardous by products Another beneficial characteristic of SIA is that for most assays the manifold configuration does not change only programming settings and the selected wavelengths are modified This manual will give simple instructions on how to configure and run the FIAlab unit This manual will only minimally cover the science of SIA the detailed operation and many additional options of the FIAlab 3200 3500 and MicroSIA systems usage of the FIAlab for Windows software and technical discussions of the specific chemistries However these areas are covered in detail within other manuals presentations included with your order For a detailed description of the fundamentals of SIA BIA and SIC as well as comprehensive information on other techniques compatible with the FIAlab systems please review the PowerPoint FIA SIA tutorials on the included CD For additional information about FIAlab for Windows please review the separate FIAlab for Window Software User s Manual During the installation process you will need the following 1 The Flash Memory Stick labeled FIAlab Software 2 The Ocean Optics CD if system includes the USB2000 spectrometer NOTE IS HIGHLY RECOMMENDED THAT THE USER CONTACT FIALAB 425 376 0450 TO ARRANGE A PHONE TUTORIAL TO WALK YOU THROUGH THE SYSTEM MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright
29. inge from the inlet and dispensing to waste from the outlet until all air has been expelled from the system leaving carrier in the holding coil of the Lab on valve At this time you should also prime the reagent ports purging them of air by aspirating perhaps 150 microliters from the reagent 1 reagent 2 and carrier ports ports 3 4 and 6 respectively pulling air from the tubing and a small amount of solution from the vials into the holding coil and dispensing those volumes to waste port 1 To fill the flowcell MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 14 B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology with carrier click on port 2 E and deliver 200 microliters through the flowcell using the appropriate steps described above NOTE Air bubbles lodged in the flowcell must be completely removed To dislodge any bubbles send a rapid burst of carrier solution e g 100 microliters 100 microliters sec through the flowcell Injecting a sample using manual controls With at least 200 microliters in the syringe turn the multiposition valve to port 5 E Change the volume to 25 microliters F and the flow rate to 25 microliters second C Click on lower bar H to aspirate the sample into the holding coil The blue colored dye zone should be observed moving from the channel of sample flow through p
30. integrated areas of the responses In most cases peak maximum is used Then click Report to see the tabulated results Please review the FIAlab for Windows software user s manual for details on the report generation The report is broken down into four channels one for each of the four monitored wavelengths If the user utilized the forth channel as the reference channel the responses for that channel will be zero MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 29 FIAlab Instruments Inc Leaders in Flow Injection Technology Example Partial Report of Channel 1 Analysis Details Report using calibration curve Set 1 Report using channel 1 FIAlab Response 3 7208E 04 3 555418E 02 concentration 2 771461E 04 concentration 2 R square of Fit 0 9998076 Time Drift Correction 0 1673019 0 1673019 3 813186E 05 time 4 825655E 08 time 2 name derived conc std1 0 119 std2 0 967 std3 2 908 std4driftcor 4 903 std5 10 118 std6 26 984 Unknownl 0 959 Unknown2 2 87 Unknown3 4 885 Unknown4 9 993 Unknown5 26 393 std4driftcor 4 904 Unknownlb 0 957 Unknown2b 2 811 Unknown3b 4 767 Unknown4b 10 08 Unknown5b 27 63 MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka true conc 0 1 3 5 10 NO NY NY NYO NYO NY NYY YY peak amplit
31. l and collects data Repeats measurement 10 times Global Logon this line establishes communication Configure LOV this line tells software that the manifold is a LOV Set Wavelengths Hardware Settings Wavelength 1 nm 620 Hardware Settings Wavelength 2 nm 600 Hardware Settings Wavelength 3 nm 580 Hardware Settings Wavelength 4 nm 540 Hardware Settings Use Wavelength 4 as Reference Prime LOV Sample prime sample line Loop Start 10 Wash LOV wash flowcell Analyte New Sample tell software to expect next sample aspirate 50 microliters of sample and 100 microliters of carrier Aspirate LOV Sample 50 Aspirate LOV Carrier 100 dispense to flowcell and collect data Multiposition Valve Flowcell syringe Pump Flowrate microliter sec 10 Syringe Pump Empty Spectrometer Reference Scan Spectrometer Absorbance Scanning Syringe Pump Delay Until Done Spectrometer Stop Scanning Loop End MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 21 B FIAlab Instruments Inc F TA lab Leaders in Flow Injection Technology 4 5 Stacking sample and reagent zones in the holding coil 1 Fill the vial for reagent 1 ReagentA with a red or yellow dye and prime the tubing to purge any air or previous solution Set Spectrophotometer Channel 1 to 620 nm and Channel Z2 to the wavelength of the selected dye 2 The program you write a
32. lso written for you as SIA dyetest3 fia shown on next page and on USB Memory Stick in the Training SIA folder should aspirate sample aspirate reagent and then reverse the flow flushing the combined zones and extra carrier through the flowcell Begin by setting the Aspirate Sample volume to 30 microliters and the Aspirate Reagent volume to 30 microliters Then dispense the combined zones plus 300 microliters of carrier solution through the flowcell at 5 microliters sec 3 Run the program From the spectrometer or analysis page observe the overlap of the zones compared to the one zone data observed in the previous section Repeat this experiment by sandwiching sample between two reagent zones Aspirate first 25 microliters of reagent then 50 microliters of sample and then another 25 microliters of reagent Compare the peak profiles between the two zone and three zone experiments Typical results are shown below However unlike these results unless you changed the sample concentration during each assay your peaks should all be nearly equal in height De Test JA E LL o i d ELO EL La dL Jd LET I IL4 EH SQOAAAAAAAAAA 0 4 Absorbance 100 200 Time Sec MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 20 B FIAlab Instruments Inc F TA lab Leaders in Flow Injection Technology SIA dyetest3 fia
33. me number string 1 Waste Hardware Settings portname number string 2 Microcolumn Hardware Settings portname number string 3 Beads Hardware Settings portname number string 4 Eluant Hardware Settings portname number string 5 Sample Hardware Settings portname number string 6 Flowcell Loop Start 5000 Next Sample command autosampler to first sample Analyte New Sample tell software to label next sample Peristaltic Pump Clockwise 50 Turn on peristaltic pump to bring sample to LOV Fill Syringe Syringe Pump Flowrate microliter sec 300 Syringe Pump Valve In Syringe Pump Fill Syringe Pump Delay Until Done Syringe Pump Valve Out Wash column Multiposition Valve Microcolumn Syringe Pump Flowrate microliter sec 10 Syringe Pump Dispense microliter 100 Syringe Pump Delay Until Done Peristaltic Pump Off Turn off pump assume sample made it to LOV Aspirate sample to remove carryover Aspirate LOV Sample 15 Purge Flow Cell Multiposition Valve Flowcell Syringe Pump Flowrate microliter sec 150 Syringe Pump Dispense microliter 190 Syringe Pump Delay Until Done Aspirate sample for Assay Aspirate LOV Sample 15 Send through column to flowcell Reset Time Multiposition Valve Microcolumn Syringe Pump Flowrate microliter sec 2 Syringe Pump Dispense microliter 60 NOTE The program as shown here will record data Spect ter Ref S poca omms nuaerenen vean for both the no
34. mm or RS 232 port Connect the included serial cable to the RS 232 IN port on the back of the FIAlab Connect the other end to your computer s serial port Connect the included 24 VDC power supply to the FIAlab Apply power plug the 24 VDC power converter into a 110 to 240 VAC supply You should hear a momentary swish swish noise from the syringe pump s Step 4 Test communication Start up FIAlab for Windows Click on the first button labeled FIAlab see below Then click on Logon You should hear the system s initialization followed by a Logon Successful statement at the bottom of the page Click on such commands as Fill and then Empty Also click on various valve port numbers In each case you should hear see the reaction of the system FIAlab for Windows 5 0 a Options na a j d FIAlab gt MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 6 B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology 3 2 FIAlab System Pumps and Valves This section describes how to add remove or modify a fluidic component pump or valve to the system Note provided the registry file was executed see step 2 of section 3 1 the software will automatically be fully configured for the specific system that was purchased Assuming no external pumps or valves are plugged into the back of the system this sec
35. n With all of your solutions ready to go call up the FIAlab software instrument control page shown above and initiate the instrument by clicking on the LOGON button point A The pump will activate and the valve will turn to the waste port port 1 Next click on the pump valve B to turn the position In Be sure that you hear the valve switch only once and that the label above the valve shows In If it switches twice or the wording says Out click on the valve again Set the flow rate to 200 microliters second by entering the number C and click on fill D to aspirate carrier into the syringe Note if the inlet tube has a large I D e g 1 mm no air bubbles will form in the syringe at this high intake flow rate Avoid using small I D tubing in the inlet syringe port for high aspiration flow rates After the pump has finished filling click on the pump valve again B to switch it Out in order to access the peripheral ports Change the flow rate to 25 microliters second Confirm that the multiposition valve is accessing the waste port by clicking on port 1 E and set the volume F to 500 microliters Activate the syringe pump to dispense the specified volume by clicking on the upper bar G which has an arrow pointing in the direction that the flow will go The carrier solution will pass through the holding coil the multiposition valve and out through the waste port Repeat the above steps filling syr
36. n retained peak as well as the elution Spectrometer Absorbance Scanning 4 Syringe Pump Delay Until Done profile If the assay has already been set up and Analyte elution by acid optimized or if troubleshooting is not the goal the Aspirate LOV Eluant 10 spectrometer commands in this section can be commented out By adding an apostrophe to the Send through column to flowcell SNP i beginning of each spectrometer command in blue Multiposition Valve Microcolumn Ms oe ee eee 2 the software is prevented from reading the line in the yringe Pump Dispense microliter l Spectrometer Reference Scan program This will result in the absorbance scanning Spectrometer Absorbance Scanning beginning at the next spectrometer command in Syringe Pump Delay Until Done green two sections below Spectrometer Stop Scanning Loop End MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 38 FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology Example Protocol for Removing the Bead Column Configure LOV configure LOV Waste Microcolumn Beads Hardware Settings portname number string Hardware Settings portname number string Hardware Settings portname number string Hardware Settings portname number string Hardware Settings portname number string Sample Flowcell 1 2 3 Hardware Settings po
37. ng the beads one for running the assay and one for removing the beads These three templates are listed below Programming Mew Font Save Events SafelockMode Find FIA Templates Sample Definition File FIA a autosampler LOW D reagents ds i Am etin Example Protocol for Affinity microChromatogr LOY 1 reagent BVOC Relay 12 ml syringe assumed LOW 2 reagents Relay Column in the conduit between port and opti Stop Flow Assay Sequential Injection Affinity Chromatography Load Beads Run Assay Remove Beads Delay sec End If Email IF Insert File Hardware Settings Add Data Loop End Loop Start t Message Next Sample Reset Data SIA Template For MicroSIA System with LOV je Global Logon logon to all components sample Descriptian load sample description file Time sec Wavelengths 0 0 ae aAA Number of Vials 3 34418B5E 08 Sample DEF Table 1 rack 1 Blank 07 0 eae BEADS ELUANT MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 36 B FIAlab Instruments Inc lab Leaders in Flow Injection Technology HOLDING COIL CARRIER BUFFER nETECTOR ES WASTE BEADS ELUANT Example Protocol for Loading the Bead Column Configure LOV configure LOV Hardware Settings portname number string 1 Waste Hardware Settings portname numb
38. ng the syringe pump to the central port of the Lab on Valve 1 for a system with a 500 microliter syringe should be made of 87 inches of 1 16 inch o d 0 03 inch 1 d Teflon tubing Upchurch 1522 The carrier for this assay is phosphate buffered saline Sigma P 3813 pH 7 4 containing 0 005 surfactant Brij 35 Sigma B4184 Surfactant is added to help prevent the formation of bubbles within the system Other detergents that do not absorb light at the desired detection wavelength such as Tween 20 can also be used The analyte is human IgG FITC conjugate Sigma F 9636 diluted to 1 ug uL with carrier solution The eluant is 0 1 M hydrochloric acid prepared from a 0 991 N stock solution Aldrich 31894 9 Once the tubing for the sample MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 32 B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology and eluant have been inserted into the respective reservoirs the solutions should be drawn into the tubing and small portions sent to waste so that the ports are primed and ready for the assay The beads are recombinant Protein A Sepharose 4B conjugate 10 1141 obtained from Zymed Laboratories Prior to use rinse an aliquot of the beads well by several rounds of centrifugation and solution exchange in order to remove the storage buffer which contains a preservative that could interfere
39. olecular Assays Jarda Ruzicka 1 Ed self published 2004 on the included CD ROM Additional literature can be found on the included Flash Memory Stick under Literature HOLDING COIL CARRIER BUFFER WASTE REAGENT 2 REAGENT 1 SIA based Enzymatic Assays are based on reaction rate measurements and carried out in a stopped flow mode where the reaction mixture is held for monitoring within the flow through cell Substrates assayed include glucose lactate urea glycerol and ethanol CARRIER BUFFER SIA based Bioligand Interaction Assays BIA are based on the monitoring of UV Vis spectra of translucent beads Sephadex Sepharose during their interaction with biomolecules in the assayed solution The protocol begins with the injection of microliter volumes of beads into the flowcell where the beads are captured and monitored while the analyte solution is passed through Native biomolecules typically absorb at 260 and 280 nm while labeled molecules absorb visible light and some also emit fluorescence Thus both nonlabeled and labeled biomolecules can be measured simultaneously HOLDING COIL CARRIER BUFFER UV vis DETECTOR BEADS ELUANT High Throughput microSequential Injection Affinity Chromatography is a redesigned variant of conventional affinity chromatography It uses a microcolumn integrated within the Lab On Valve manifold to carry out separation and assay of biomolecules on a Sephadex column
40. on 7 0 The fiber extending closest to port 3 needs to be positioned just past the immediate exit channel of port 2 A and the other fiber just at the far side of the downstream exit channel B allowing for bubbles to be pushed to waste The spectrometer setup should be configured with 100 ms integration time 5 detectors to average 1 sample to average and a sampling rate of 2 Hz Channel 4 is not used as a reference wavelength The recommended light source is an Ocean Optics deuterium UV lamp The Lab on valve needs to be plumbed with the following components the lengths given for tubing are approximate values A 10 inch piece of 1 8 inch o d 0 062 inch i d Teflon tubing Upchurch 1523 should be inserted between the syringe pump and carrier reservoir C Four inch pieces of 1 16 inch o d 0 03 inch i d Teflon tubing Upchurch 1522G should be inserted into each of ports 3 and 4 D a 9 inch piece needs to be attached to two outlets connecting port 6 to the exit channel extending directly from port 2 E and 12 inch pieces for waste lines should be inserted into both port 1 and the last open channel at port 2 F For the sample flow through port port 5 4 inches of 1 16 inch o d 0 02 inch 1 4 Teflon tubing Upchurch 1549OR should be inserted on the side closest to port 4 G Attach a 1 mL disposable syringe with Luer lock fitting to the other channel of port 5 H for use in priming the sample channel The holding coil HC connecti
41. on Valve a short delay of 3 seconds follows the aspiration step to allow the pressure to re equilibrate A second 50 microliter aspiration another 3 second delay and a third aspiration are followed by delivery of the holding coil contents to waste via port 1 Triplicate aspirations ensure that all of the beads have been removed from the Lab on Valve channel at port 2 The system 15 then ready for a new microcolumn to be loaded The volumes and flow rates used for removing the microcolumn were chosen because they easily transport the beads through the Lab on Valve to waste without clogging The system parameters and configuration allow for solution but not beads to flow past the fiber optics However the beads used in this work Sepharose 40 165 micrometer diameter can squeeze between the fibers and channel walls if a high enough flow rate and pressure are applied Because of the position of the fiber optics in the channels of port 2 rather than ending up in the waste reservoir trying to push the beads out and trying to squeeze them past the fiber optics clog the channel rather than achieving the goal of removing the beads MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 35 B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology 7 5 Program Templates There are three premade templates associated with affinity chromatography one for loadi
42. ort through the Lab on valve and into the holding coil Click on port 2 E of the multiposition valve set the volume at 200 microliters and the flow rate at 5 microliters sec Press the upper bar G to dispense the solutions and observe the passage of the dyed zone through the flowcell MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 15 B FIAlab Instruments Inc lab Leaders in Flow Injection Technology 4 3 Writing a Dye Test routine and running a method This section shows the user how to custom create a method script FIA program as well as how to use one of the already created templates provided with the program This first program will be a simple dye assay The included FIAlab for Windows Software manual includes many additional examples and detailed explanations of various commands For most assays only a very small subset of the available commands are actually required Using the main FIAlab for Windows window pull up the Program page by clicking on the button On the Safe Lock Mode drop down menu see the figure on the next page click on Locked and change to Edit Mode FIAlab for Windows 5 0 Instruments Options About 5 31 2005 10 40 34 FIAlab Edt Mode P Page she Command 7 pero Delsey Urti Done Irshskze Stop Vaive In Valve Out Cheech Status moun R
43. r example 12 means a QC Driftcor measurement will be made once after every 12 samples Set to 0 to turn off all QC Driftcor samplings recommended new users set this value to 0 More on this later in this section Pos of QC Driftcor Sample Location of QC Driftcor sample assumed to be in the standards rack More on this later in this section MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 26 B FIAlab Instruments Inc F TA lab Leaders in Flow Injection Technology The sample def file contains one line per sample or standard defining the sample name position in the autosampler concentration if it is a standard number of times to analyze and time delays between each analysis As an example see table below Rack Name Position Name Truth measure measurements Period B j 0 I 2 o i 0 10 20 Ix 2 o J 1 0 left rack 2 unknown2 0 1 0 left rack 4 unknown4 0 1 0 2 o 1 0 left rack unknown 2 0 1 0 2 1 o 0 left rack 7 unknown9 2 0 1 0 Field 1 is the Rack name The Autosampler contains several racks depending on brand model as defined in the configuration form from the Autosampler page Each Rack name in the sample definition file must exactly match a name defined in the Autosampler configuration form A a A a A a A a Field 2 is the Sample Po
44. re 4 1 Typical placements and mounting schematics of the fiber optics MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 12 B FIAlab Instruments Inc lab Leaders in Flow Injection Technology 4 2 Manual Control and Priming of the System The first exercise will involve manual operation of the system Though manual control is typically not required during daily routine assays it does help the user understand the system The next section 4 3 will discuss automated usage using the programming method scripting capabilities Also this initial exercise will not utilize an autosampler samples will be manually introduced For the purposes of instrument testing and the training session outlined below dye concentration measurements should be performed Use deionized water for the carrier adding one drop of detergent e g Joy dishwashing liquid per 50 mL water to help prevent bubble formation Initially also use water as reagents 1 and 2 For the sample use 0 01 bromothymol blue indicator solution prepared in 0 01 M sodium tetraborate to stabilize the pH of this acid base indicator Simple blue food coloring can be used instead diluted with water Typically 3 5 drops of food coloring per 50 ml of water is adequate For the purpose of this simple Dye exercise LOV ports will be utilized as follows Port 1 Waste Port
45. riftcor is a feature used to allow the software to automatically correct any response drift with time An example of this is with nitrate assays where the Cadmium column may degrade somewhat in the course of a run To correct for this add modify three or more lines in the sample definition file such that the name of the sample has the key phrase in it driftcor Each of these three or more lines must all point to the same vial with a concentration typical of your samples As a simple example the following uses the standard vial 4 both as part of the calibration curve shown in blue as well as the drift correction shown in red See the lines in red Obviously this correction is normally performed for much larger runs than the example below e g 200 sample vials Typically there are at least 8 samples between the drift correction vials and even up to 20 stndadrak 1 Blak at standardrak 4 Driftor_ ot l standard rack 1 std 0 0 NO unknown4 Driftcor unknown7 4 4 EN CNN 7 unknown 8 unknowns 9 EUN left rack standard rack standard rack 4 Driftcor 0 1 0 Note the drift correction is only applied when the Analysis page Calibration Tab Correct for Drift is checked This can be checked unchecked after a run to see the results both with without the correction MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual
46. rtname number string 4 Eluant 5 6 Syringe Pump Valve In Syringe Pump Flowrate microliter sec 200 Syringe Pump Aspirate microliter 200 Syringe Pump Delay Until Done Syringe Pump Valve Out Multiposition Valve Microcolumn Syringe Pump Flowrate microliter sec 50 Syringe Pump Aspirate microliter 50 Syringe Pump Delay Until Done Delay sec 3 Multiposition Valve Microcolumn Syringe Pump Aspirate microliter 50 Syringe Pump Delay Until Done Delay sec 3 Multiposition Valve Microcolumn Syringe Pump Aspirate microliter 50 Syringe Pump Delay Until Done Multiposition Valve Waste Syringe Pump Flowrate microliter sec 100 Syringe Pump Empty Syringe Pump Delay Until Done MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 39 B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology 8 0 Using the System with other types of Detectors Flowcells The FIAlab system is compatible with various types of detectors flowcells besides the built in flowcell in the LOV manifold and the USB2000 spectrometer For instance long path flowcells from 2 cm to 10 cm can be connected as well as the PMT FL Fluorometer To utilize a long path flowcell LPFC or PMT FL with the FIALab system LOV manifold the LPFC PMT FL input port is connected to the output waste port of the built in LOV flowcell So solution passes through
47. s software manual for additional details For many applications the scripts can be run without modification If the SIA scripts included in the FIA Templates do not satisfy your needs please keep in mind that FIAlab maintains a library of scripts for FIA SIA SIC BIA etc Please contact us with a description of your specific assay requirements We probably have a script in our extensive library that comes close to your needs We will then help you modify the script as needed i E DI M lampe Det mien Pis FLA Mothod Member of Fic BEER SE ws m saine asume a LOS ae i taya SIA Tomplats For bieron Sysipm weh F Hobs Logon legon io nil Comparrnis Sequens jamce Cort ae mike apre comple dereng moss Sol leery Teg terrier Tura Wali oh IT ri GE j re LU i i hist sample nash ond pom vatem mand aphmizm ope cients Facial Puno Claekweai 65 Select a template The most commonly required user modification to the template programs is to set the wavelengths as required by the specific assay to be run The following example lines show how to set the wavelengths in the script Four wavelengths will be collected and the 4 wavelength is used as the reference wavelength Set Wavelengths Hardware Settings Wavelength 1 nm 620 Hardware Settings Wavelength 2 nm 600 Hardware Settings Wavelength 3 nm 580 Hardware Settings Wavelength 4 nm 540 Har
48. sition Each rack has positions labeled one through num where num is the sample capacity of the rack named in field 1 Field 3 is the Sample name The name 15 arbitrary However having multiple samples with the same name will cause the software to group these samples and give such statistical information on the Analysis page as the mean and standard deviations Field 4 is the true concentration This is used if the specific defined vial is a standard If the vial contains an unknown this field should have a question mark or a for blank internally is treated the same as Use 0 to include a blank in the calibration curve Field 5 is the time to measure hours This is the time in hours after the start of the run that the first measurement will be made When multiple samples have the same start time the software will start at the first sample with this time and work its way down For most applications set to 0 Field 6 is the number of measurements The specified sample will be measured this number of times For most applications set to 1 Field 7 is the period between measurements in hours For most applications set to 0 MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 27 B FIAlab Instruments Inc F TA lab Leaders in Flow Injection Technology Sample Definition File Advanced Features Drift Correction D
49. spirate LOV Sample 50 Aspirate LOV ReagentA 50 dispense to FC and stop with sample partially in FC Dispense LOV 65 7715 quantity is critical collect data for 45 seconds Spectrometer Reference Scan Spectrometer Absorbance Scanning Delay sec 45 Spectrometer Stop Scanning Loop End MicroSIA FIAlab 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 24 B FIAlab Instruments Inc lab Leaders in Flow Injection Technology 5 0 Using an Autosampler Note Follow the instructions in Section 3 for connecting and configuring the autosampler before starting this section The Autosampler should be in very close proximity to the FIAlab so that the sample line the tubing between the Autosampler probe and port 5 of the Lab on valve is as short as possible Note however it is CRITICAL that the tubing between the Autosampler probe and the FIAlab system be long enough to allow free motion of the autosampler arm to each corner The tubing between the autosampler and the FIAlab should be made from PEEK or Teflon Tubing with 0 02 I D is recommended The autosampler probe line is connected to the upper port 5 on the Lab On Valve The lower port 5 is then connected to a sampling pump which pulls the sample through the flow through port 5 and on to waste see diagrams in section 2 0 Typically the sampling pump is only turned on momentarily to pull the sample up
50. ss values to comm the default In most cases the default settings will immediately control the MicroSIA FIAlab 3200 and FIAlab 3500 systems with no settings change Default Settings for internal components Component Syringe Pump Peristaltic Pump Valve Any peripherals e g extra syringe pump extra valve which are plugged into the RS 232 out of the FIAlab system will have the same serial port as the main system however will have a different sub address Correct Peripheral Settings Location Sub address Plugged into RS 232 A Out Same as RS 232A FIAlab usually 1 Plugged into RS 232 B Out Same as RS 232B FIAlab usually 1 RS 485 Settings of the Syringe Pump In nearly all cases the RS 485 address for syringe pumps are set to 1 One exception for the second syringe pump built into the FIAlab 3200 the RS 485 address 15 set to 2 Please inquire to FIAlab for additional information for more advanced configurations MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 8 B FIAlab Instruments Inc lab Leaders in Flow Injection Technology 3 3 Spectrometer Note if you purchased the computer from FIAlab ignore steps 1 through 3 Do not plug the USB2000 spectrometer into your computer s USB port until after the OOIBASE32 software has been installed Step 1 Close FIAlab for Windows Step 2 Install OOIBASE Softw
51. ssay protocol after the desired zones have been delivered to the flowcell An example two zone routine is written for you below as SIA dyetest4 fia and also will be found on USB Memory Stick in the Training SIA folder The position of the stopped flow period is CRITICAL to the success of stopped flow reaction rate assays During method development this quantity should be optimized by parameterization during several runs Your resulting script can used for any enzymatic assay by simply replacing the sample dye with a substrate e g glucose reagent 1 dye with an appropriate reagent e g glucose oxidase indicator and the carrier stream with an appropriate buffer and selection of the correct wavelengths SIA dyetest4 fia this simple script primes the line washes flowcell aspirates sample and dispenses the sample to the flowcell in stop flow mode and collects data Repeats measurement 10 times Global Logon this line establishes communication Configure LOV this line tells software that the manifold is a LOV Set Wavelengths Hardware Settings Wavelength 1 nm 620 Hardware Settings Wavelength 2 nm 600 Hardware Settings Wavelength 3 nm 580 Hardware Settings Wavelength 4 nm 540 Hardware Settings Use Wavelength 4 as Reference Prime LOV ReagentA Prime LOV Sample Loop Start 10 prime sample line and wash flowcell Wash LOV Analyte New Sample aspirate 50 microliters of sample and 50 microliters of reagent A
52. ter Setup tab and adjust the integration time up Note for certain UV applications below 250 nm and some bead assay applications it 1s not always feasible to achieve over 1000 counts This is usually ok MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 19 B FIAlab Instruments Inc lab Leaders in Flow Injection Technology r1 Spectrometer Spectrometer Graphing Spectrometer Setup Settings applied to all channels Integration Time msec Detectors to Average 1 3 5 Samples to Average Sample Rate Hz Plotting Threshold cnts Stream dala to file 67 set Absorbance i Use wavelength 4 as reference for Master Spec D Driver Turn Off Reference Warnings Lamp Hardware Settings for specific channels First Coefficient Second Coefficient Third Coefficient Intercept 0 ntegration i 3730185 i 398 05 nd Defaults Selected Spectrometer Master v Enabled Scale Voltage E 1 Use for fluorescence returns voltage when absorbance measurement is commanded S m ui Voltage Absorbance Single Scan Repeated Scan Scan to File Exit Figure 4 5 Spectrometer Setup Page If the user 1s uncertain as to the best overall settings on the spectrometer setup page with regards to the various options it is recommended they use the Default values obtained by clicking on Default
53. the built in flowcell and then into the LPFC PMT FL For the LPFC the fiber optics are hooked to the LPFC from the lamp and spectrometer in a similar fashion that one connects the fiber optics to the built in LOV flowcell Note to switch to a different spectrometer e g from USB2000 UV VIS to a USB2000 FL change the four spectrometer coefficients on the spectrometer setup page Then if you have more than one spectrometer plugged into the computer simultaneously select the desired spectrometer from the Hardware button on the spectrometer setup page MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 40 B FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology 9 0 Warnings The FIAlab system is a laboratory instrument which will not withstand extreme changes of humidity corrosion or prolonged vibration As with any laboratory instrument the system is NOT EXPLOSION PROOF and if small volumes of volatile or flammable liquids are pumped the system must be attended at all times Adequate ventilation must be provided to eliminate all fumes which might result from spillage caused by the operator or by rupture or disconnection of connecting tubes or syringes The same precautions must be observed when pumping corrosive liquids It is recommended that the operator follow routine laboratory precautions as required by law such as wearing safe
54. tion 3 2 can be ignored For each pump or valve included with the system whether internal or external the software must know a number of parameters For example for syringe pumps required information is the type of pump the syringe size it s serial port sub address and RS 485 address Each defined component must have a unique name and no name can be a subset of another name For instance syringe pump and second pump are ok together however syringe pump and pump are not ok since pump is a subset of syringe pump To modify a component s settings e g change the syringe size highlight click on the desired component under Existing Components modify the settings and click Accept Then click Logon after all the changes are made To delete a component highlight its name under Existing Components and click on Remove To add a component highlight Define New Highlight the type of component desired Syringe Pump Multiposition Valve or Peristaltic Pump type in a unique Instrument Name modify the settings and click Accept Sequential Injection Analysis SerialPort 1 Peristaltic Pump Syringe Pump Valve Update Existing Components Contact Closure Aspirate microliter Quantity TTL DAC Setting Command 7 Peristaltic Pump Dispense microliter Syringe Pump Delay Until Done Multiposition Valve Define New MN Flowrate micraliter sec
55. ty glasses and collecting generated waste If spillage of any liquid occurs 1 Immediately disconnect the system from the mains 2 Drain and neutralize the liquid using a damp cloth 3 Inspect the instrument for liquid penetration and corrosion REMEMBER DO NOT OPERATE THE INSTRUMENT WITH OPEN HOUSING OR WITH A DAMAGED ELECTRICAL CORD DO NOT OPERATE THE INSTRUMENT IN THE PRESENCE OF EXPLOSIVE FUMES DO NOT EXPOSE THE LAB ON VALVE MODULE TO ORGANIC SOLVENTS OR CONCENTRATED ACIDS OR BASES 10 0 Warranty FIAlab Instruments Inc warrants to the original purchaser that if the FIAlab 3200 is operated in a non corrosive atmosphere and under normal laboratory conditions it will repair or replace at its option any parts found to be defective in factory materials or workmanship within one year following the date of delivery This warranty is void if any part of the system is damaged by corrosive liquids or by improper use MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 41
56. ude 0 004 0 035 0 104 0 173 0 343 0 788 0 035 0 104 0 174 0 342 0 777 0 174 0 035 0 1 0 167 0 337 0 782 peak time sec 88 839 125 641 160 324 195 053 229 732 264 842 334 876 368 949 403 555 438 372 473 307 508 115 578 143 612 453 647 54 682 407 717 509 30 FIAlab Instruments Inc F 1A lab Leaders in Flow Injection Technology 7 0 Affinity microChromatography in the Lab on Valve Affinity chromatography relies on the selective interaction between molecules immobilized on a stationary phase and analyte molecules in solution With an appropriate pair of molecules selected analyte 1s selectively captured by the binding partner immobilized on the stationary phase while impurities and other molecules not of interest are not retained and pass through the column Changing the composition of the mobile phase 1 e pH or salt concentration effects the dissociation of the captured analyte from the stationary phase and for elution to a detector or recovery for secondary analysis in a separate instrument While columns typically used for traditional immunoaffinity chromatography applications are on the order of 1 5 milliliters the column size for microChromatography 15 in the range of 5 10 microliters Using Sequential Injection in the Lab on Valve format programmable flow 15 introduced to affinity chromatography providing an automatic means of renewing the stationary phase and the versatility and reliabilit
57. verter provided The AIM3200 3300 plugs direct to a 110 to 240 VAC supply The Autosampler will go into its initialization steps which takes around 10 seconds depending on the type of autosampler Step 2 Connect communication line Connect the Autosampler to an available serial port or the RS 232 A output on the back of the FIAlab The ASX autosamplers have COM 1 and COM 2 ports on their back panel You must use the COM 1 port If there are no available RS 232 Out ports on the back of the FIAlab they are being utilized by other components e g a PMT FL then the autosampler can be interfaced to a second computer serial port If your computer does not have a second serial port a low cost USB to serial port converter is available at most computer stores Step 3 Test Communication Bring up the Autosampler Page of FIAlab for Windows and set the serial port number consistent with the utilized serial port probably Serial Port 1 Click Logon Try sending a command such as send the sampler to wash as shown below highlight Wash and click Send button Autosampler Serial Port 1 Commands Down Goto Sample Up Get Position Get Error autosampler Set Name Celac ASX 260 520 C AIM3200 3300 Configure Cetac 4S 260 520 C 1250 C Samp3000 Sub address Create Select S ample Definition File Please Logon MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments
58. with the assay 7 2 Loading the Column The bead reservoir an Eppendorf tube with a hole punched in the lid allowing for insertion of the tubing should contain 200 300 uL of settled beads and 1 mL of carrier solution With the tubing at port 3 inserted into the reservoir and touching the bottom of the container and the system primed with carrier solution the system is ready to load the micro affinity column A method template for loading the beads and forming the micro affinity column Section 7 5 is included in the FIAlab software under Templates on the Program window The template consists of four steps 1 aspirate carrier 2 aspirate beads 3 deliver beads to load the column and 4 return extra beads remaining in the holding coil to the bead reservoir The volumes used for aspirating and delivering the beads can be changed if need be but have been optimized for the system setup outlined above While the color of the Lab on Valve makes it somewhat difficult to see small amounts of beads an opaque column can be seen building as the beads pack into the channel between port 2 and the fiber optic This is most easily seen with the magnifying glass To achieve reproducible data the entire channel between the fiber optic and the point where the groove meets port 2 on the multiposition valve must be completely packed with beads Incomplete packing allows the beads to shift and results in irreproducible capture and elution profiles To facilitat
59. y of sequencing the steps and data collection of an assay in variety of ways Affinity microChromatography can be applied to the purification of antibodies or other biomolecules as long as the species of interest are paired with selective capture ligands that can be immobilized on the stationary phase The following sections will outline the preparation for and running of affinity microchromatograpy assays in Lab on Valve systems Schematic of SI system configured for affinity microchromatography CARRIER BUFFER UV vis DETECTOR ae BEADS ELUANT Close up picture of chromatography microcolumn opaque channel between port 2 and fiber optic and downstream flowcell between the ends of the fiber optics for off column detection of non retained and eluted molecules MicroSIA FIAlab amp 3200 and FIAlab 3500 Operation Manual FIAlab Instruments Inc 2005 Graphics Copyright Jarda Ruzicka 31 B FIAlab Instruments Inc lab Leaders in Flow Injection Technology 7 1 Materials Reagents and System Configuration While the FIAlab system 1s shipped with the Lab on Valve already plumbed for affinity chromatography applications be sure that the system is set up with the following components and configuration For detection two 400 um fibers are inserted into the channels perpendicular to the exit channels for port 2 such that the distance between the fibers is 0 25 inches see second figure in secti
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