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DAWN HELEOS Hardware Manual

1. cccccceseeeeeeeeseeeeeeeeeeeeens H 20 H 7 Trouble shooting And GiaQnOStiCs ccccceeceeeeeseeeeeeeeeeeeeeeeeeeeeens H 21 Please read over Section H 1 to gain an understanding of the components to be used Then read over either Section H 2 H 3 or H 4 depending on your configuration Finally read over Section H 5 for instrument settings Please read Section H 6 for instructions on accessing instruments via ASTRA V Finally if you experienced problems connecting to your instrument please read Section H 7 for diagnostics and trouble shooting MWH Rev A H 1 Appendix H Connecting to Network or PC H 1 Components H 1 1 Instrument connections Figure H 1 is a detail of the instrument back panel The Ethernet port designated with a yellow arrow is to be used for all connections in these instructions Please see Section H 3 for instructions on establishing a USB connection Alarm Auto Inject In In Analog Recycle Alarm Auto Inject Ethemet Out Out Out Out Heated Line Figure H 1 Detail of the back panel of the HELEOS The yellow arrow designates the Ethernet port H 2 MWH Rev A Components H 1 2 LAN connection MWH Rev A Figure H 2 shows a typical wall socket connection to a Local Area Network LAN If you are going to connect the instrument to a LAN you will need access to this type of socket Figure H 2 Wall socket LAN connection indicated by yellow arrow Appendix H
2. 0 H 17 Multiple instruments to computer not on LAN using an Ethernet switch H 18 Instrument Network Settings ccccccsseecceseececeeceeseueceeseueeecseeesseeseesseeeeeseaes H 19 Accessing instruments with ASTRA V u c sccccccsescecceeeeecceeeeeseeseeseaueeesseeeeesaaes H 20 Trouble ShoOoting and diagnostics ccccccseeececeeeeeeeeeeeeeeeeesseueeeseeeeeseeeeeseees H 21 Verifying instrument CONNECTIONS ccceccceeeeeeeeeeeeceeeeeceeeeeseeeeesaeeeeeaeeeesaeeeesseneenas H 21 INGEN scssi yes newantvednecnccnsetcertunetcaawmsveneceses Index 1 Contents vi MWH Rev A Introduction When you can measure what you are speaking about and express it in numbers you know something about it but when you cannot measure it when you cannot express it in numbers your knowledge is of a meager and unsatisfactory kind it may be the beginning of knowledge but you have scarcely in your thoughts advanced to the stage of science Lord Kelvin William Thomson Kelvin the 19th century physicist and mathematician who wrote that paragraph would have been very comfortable with the DAWN HELEOS Enhanced Optical System laser photometer and software The DAWN systems measuring scattered light at 18 different angles simultaneously can determine the molar masses of polymers and biopolymers from a thousand to hundreds of millions of daltons Certain options permit temperature control of the flow cell the us
3. Figure B 1 Ultra High Temperature read head and laser assemblies B 2 MWH Rev A Heating the Cell B 2 Heating the Cell B 2 1 B 2 2 Note MWH Rev A The HELEOS flow cell is designed to operate at temperatures up to 210 C with the Ultra High Temperature option The high temperature cell is designed around two cartridge heaters Temperature regulation is digitally controlled by the front panel computer The resolution of the controllers is 0 01 C and the accuracy is 1 C About the Thermocontrollers There are independent controllers for the read head and for the heated line Gf installed One controller controls the read head temperature If you purchased the optional heated lines the second controller controls the temperature of these heated lines Typically the heated line controller is slaved to the cell controller That is when the temperature of the cell is changed the temperature of the heated line 1s changed in sync On the System panel it is possible to break this slave relationship by unchecking the Sync button Then you can set the temperature of the heated line independently of the cell These controllers are designed to give the best possible temperature regulation They use a Proportional Integral Derivative PID control loop which measures the difference between the setpoint the temperature you desire and the process the temperature of your system Setting the Operating Temperature You
4. Bottom retainer O ring cord S5002 2006 M2 screw 2 5 3 3 Step 3 Cleaning the Flow Cell and Windows From here on you must be fastidious in your handling and cleaning of the MWH Rev A flow cell parts The smallest particle on the flow cell window or inside the bore can introduce stray light and distort your measurements 1 Clean your hands thoroughly or wear lint free gloves When you disassemble the cell be careful not to handle the glass cell s curved optical surfaces the sides 2 Clean the cell through bore a Cut a strip of lens tissue and roll it into a thin wick Or you may use Oral B SuperF loss which is available in most pharma cies The floss is a better tool as it cannot leave any fibers behind Insert the wick all the way through the cell bore then moisten it with a small amount of filtered alcohol While the wick is in the cell bore untwist it slightly move it back and forth to clean the cell then pull it out Immediately flush the bore with a stream of alcohol for 10 15 seconds The alcohol stream flushes out any fibers that may have been left behind by the tissue wick Blow out the alcohol for 10 15 seconds with inert dusting gas or let the glass bore drain in a vertical position Examine the bore with a magnifying loupe Look through the bore focusing on the bore exit Repeat from the opposite side See the Note at the end of this section 3 Clean the outside
5. 4 5 Chapter 4 Using the Display Window 4 3 3 4 3 4 4 3 5 4 3 6 Set Scale Milan 26 22606436 MAIN 2522606435 Hint ou may also adjust display range by navigating to the Change r axis button and press any ofthe four arrow keys Figure 4 4 Setting the scale numerically To toggle positive and negative tab to the button and press Enter 4 Tochange values tab to the Max field and enter a value Tab to the Min field and enter a value Press Enter Autoscale Changes the scaling so the display fills the window Setting Cell Temperature You set the cell temperature by using the numeric keypad to enter the value The up arrow key and 4 down arrow key toggle between positive and negative The HELEOS adjusts to within one tenth of a degree of the set temperature Laser Sets the laser to On or Off Comet Comet is an internally installed option that applies a radio frequency ultrasonic field which loosens particles that may have adhered to the cell walls and removes them on a daily basis When used on a regular basis the need to remove the flow cell for cleaning may be postponed indefinitely In addition periodically activating the device prevents new particles from adhering Recycle On the back panel is a connector for driving a 12V solenoid valve that can be plumbed to divert the flow from recycle to waste You can turn it on or off A timer setting on the Systems Panel allows it to
6. The software uses the rear laser monitor signal to normalize the scattering signals relative to incident laser beam power The method involves splitting the beam at its source and dividing background corrected values by the split signal The normalization factor the incident intensity 1s proportional to the beam emitted from the front of the laser and is obtained from the beam splitter on the laser assembly 3 8 Rear Laser Monitor is adjusted to about 5 volts before the instru ment is shipped If over time the rear laser monitor value begins to drop below 4 9 volts contact Wyatt Technical Support The laser may have reached the end of its useful lifetime You can view them graphi cally on the Main panel or remotely through the Diagnostic Manager s Data numeric page Forward Laser Monitor enables the HELEOS to measure transmit ted light through the flow cell and sample It is useful for detecting flow path obstructions such as air bubbles or large particles which reduce the transmitted signal intensity to near zero volts It can also be used to correct of the attenuation of the laser by absorbing samples Laser Current signal is used to gauge the lifetime of the laser As the laser ages the current required to provide a constant intensity slowly increases The initial laser current 1s recorded on the quality control worksheet provided with each instrument When the current reaches a value of 30 higher than the initial
7. Time Constant specifies the samples per second The default is 0 5 seconds Set Time Set Time sets the time displayed in the X axis of the graph Language You can set the language of the user interface to English or other supported foreign languages MWH Rev A System Panel 4 7 8 Restart ISI Restart ISI is used to restart or reset the instrument communication in the event that a remote client such as Astra V or the Diagnostic manager crashes 4 7 9 Load Factory Defaults Load Factory Defaults is used to reset the instrument to the settings installed when the instrument was shipped 4 7 10 Restart Instrument Restart Instrument turns off the HELEOS and turns it back on This is typically used only when installing a firmware update MWH Rev A 4 15 Chapter 4 Using the Display Window 4 8 Comm Panel The Comm panel allows you to connect to a computer network Main Batch GELS Alarm System Comm Ethernet Communication DHCP Instrument Mame IF Address et Be Wyatt 100 HELEOS subnet Mask Apply Figure 4 10 Comm DHCP Dynamic Host Configuration Protocol once the instrument is connected to a computer or LA the IP address and subnet mask will be assigned automatically When using DHCP it may take several minutes for the IP address to be assigned During this time the IP address and subnet mask on the front panel will read 0 0 0 0 Once the IP address and subnet mask have been assi
8. 4 75 psi Figure 4 8 Alarm panel 4 6 1 Alarm Signal State Select whether alarm input and output signals are active low Alarm IN If you select Active Low the instrument considers an Alarm In event to occur when the signal on this line transitions from 5 V to 0 V When an Alarm In event occurs the Alarm signal flashes on the LCD display and an Alarm Out signal is transmitted see Alarm Out If you don t select Active Low the instrument considers a transition on this line from 0 V to 5 V to be an Alarm In event Alarm OUT If Active Low is selected the instrument keeps this signal at 5 V for no alarm state and brings the signal to 0 V in the event of an alarm state In this context an alarm state occurs if the internal liquid leak sensor detects liquid or the internal vapor alarm detects organic solvent vapors or the rear panel connector Alarm In signal is active see Alarm In If Active Low is not selected the instrument keeps this signal at 0 V for no alarm state and brings the signal to 5 V in the event of an alarm state MWH Rev A 4 11 Chapter 4 Using the Display Window Note 4 12 4 6 2 Audio Alarm To turn off enable or disable the audio alarm display the Alarm Panel Tab to the Audio Alarm field then Enter to toggle the option Even when the audible alarm is turned off the back panel alarm output will remain active Here is a list of the alarms and their meanings Overheat not in Ambient v
9. Count Rate A 1 2 Correlation Function The Correlation Function displays the intensity correlation curve for a single slice of QELS data which is the raw dynamic light scattering data from which the hydrodynamics properties are derived The QELS measures the correlation function which is a statistical measurement of how the scattered intensity fluctuates It is a function of t which is a time difference For large values of t the correlation function approaches 1 0 indicating that the light intensity at time t 1s uncorrelated to the intensity at time t t For smaller values of t the correlation function increases indicating that the scattered intensity is correlated The time difference at which the correlation function transitions from being correlated to being uncorrelated is related to the molecular diffusion coefficient Small particles diffuse rapidly giving to rapid fluctuations of MWH Rev A A 1 3 A 1 4 MWH Rev A QELS Panel the scattered light which will have a short correlation time Correspondingly large particles diffuse slowly and have a long correlation time See the ASTRA V for Windows User s Guide for a more detailed explanation of the physics of QELS Integration Time Integration Time is the QELS sample rate in seconds of each QELS measurement The collection rate depends on the sample concentration the flow rate and molecule size In general the value chosen should be proportional to expe
10. Note 5 18 If you are not careful the cell could be reversed Make sure that the INlet manifold is in the rear position and the OUTlet manifold is in the forward position 1 Replace the cell assembly in the read head insert the two M3 screws and tighten with the 2 5 mm Ball driver Loosely tighten both screws and then alternately tighten them evenly until they are secure Reconnect the blue exit tubing with two 1 4 crescent wrenches Connect the white inlet tubing to the adapter union inside the heat exchanger See Figure 5 3 above Then slide either the COMET assembly or the spacer between the heat exchanger and the read head and bolt it into place with 2 M8x20 screws using the 2 5mm hex driver Connect to the HPLC system and check for leaks Bolt the heat exchanger onto the top of the COMET making sure that all of the tubing is contained within the heat exchanger cavity and it is not pinched between the heat exchanger and the COMET Replace the insulating cover with two M38x8 screws Replace the instrument cover MWH Rev A Flow to Batch Conversion 5 4 Flow to Batch Conversion You can easily alter the HELEOS read head to take measurements from a 20 mL scintillation vial The conversion procedure takes a minute or two The batch configuration permits a variety of uses that would not be possible with the flow configuration Among these are the ability to store and analyze sealed samples and to perform
11. and its interfaces clean of precipitates MWH Rev A Flow Cell 3 6 3 Cell Windows The cell s windows protrude into the flowing stream at the entrance and exit manifolds These miniature rods of glass are designed to minimize debris buildup on their flat ends and for the same reason have no recessed rims or edges Note The large surface facing out from the cell has been coated to minimize reflections shown in Figure Be careful not to scratch the coating during cleaning and do not use acids Cas oem Inlet manifold 4 C Window O ring Flow cell O rings N _ 4 Backing ring Ar Flow cell Ne indies Flow cell Sr S See warning A below for heated pa p instruments Z TL gt Bottom retainer i L O wN O ring cord V retainer Bottom cell erang retainer Figure 3 11 Exploded view of the flow cell assembly 30 C to 80 configuration MWH Rev A 3 13 Chapter 3 HELEOS Components Caution If you intend to operate your instrument above 80 C the flow cell must use the 9 mm O rings instead of the 6 mm O ring and backing ring combination used at lower temperature With the high temperature O ring configuration you may run the instrument over the entire temperature range however the dead volume at low temperatures will be increased If the instrument temperature is set above 80 C a warning message will appear on the front panel that the correct O rings must be
12. low temperature setting the same precaution must be obeyed e When heating the cell above 80 C double check the fittings for leaks as thermal expansion can open fittings that were otherwise sealed at room temperature MWH Rev A Removing the Cell Assembly B 3 Removing the Cell Assembly MWH Rev A The Ultra High Temperature cell assembly is the same as the ambient cell assembly but with several added components for insulation These instructions are for those instances when you need to remove the cell assembly typically to clean the flow cell or to convert to batch mode What you will need to remove the cell assembly e Two Crescent wrenches e 2 0 mm Ball driver e 2 5 mm Ball driver To remove the cell assembly do the following 1 Set the temperature of the cell and heated lines to 25 C and wait for the system to stabilize 2 Remove the bib from the cover of the instrument 3 Using the 2 0 mm Ball driver remove the read head cover plate by removing the four M8x10 screws holding it in place Me ie _ __l Figure B 2 Heated Lines Bib 4 Remove the heat exchanger by unscrewing I the two M8x12 screws P ENN Read head BED 5 Disconnect the short cover plate pieces of 1 6mm OD stainless steel tubing from the in line unions shown Heat a in figure B 4 using the exchanger two 14 Crescent wrenches COMET The adapter union or spacer connecting the brass heat exchanger to th
13. the optical fiber and collet will detach from the mount see Figure A 5 A 6 MWH Rev A Removing and Installing the Optical Fiber Receiver 5 The fiber will usually not need to be removed from the collet to clean or inspect it However if desired you may gently remove it by twisting it and drawing it out Collet fingers W _ ii lt p Figure A 5 Optical fiber in collet The end of the fiber should protrude about 1mm from the end of the collet 6 Check that the fiber slides freely into the fiber collet If it does not slide freely gently bend the collet fingers outward until it does 7 Screw the collet back into the position and thread into position until it begins to grab Do not over tighten or the collet fingers will bend inward and the fiber will not fit 8 Install the fiber into the collet and press it all the way until it reaches its stop If it does not go in all the way remove the collet and repeat 9 Gently tighten the collet until it clamps onto the fiber Again do not over tighten 10 When the collet is tight gently pull on the fiber It should not come loose A 3 2 To move the HELEOS QELS fiber positioner to a different detector location 1 Turn off the HELEOS system power 2 Remove the QELS fiber receiver as described above 3 Remove the photodetector from the new detector location a The detector is held into the read head with a rubber O ring and is connected on the back side to a s
14. 5 mm Ball driver Then insert the long M3 screw into the inlet manifold and tighten View the O rings through the bottom glass surface make sure the surface is clean and confirm that the bore is centered in each O ring Also examine the alignment pins to make sure they touch the cell glass on each side 5 15 Chapter 5 HELEOS Maintenance 5 16 Inspect the sides of the cell and apply a burst of air if you see any particles Replace the bottom cell retainer cord and O ring and attach the bottom cell retainer to the manifolds using the 1 5 mm Ball driver Clean the fittings that will connect the inlet and outlet tubes to the unions Use the same method for cleaning as you did for the window O rings a couple of drops of alcohol on lens tissue then dry with a burst of air Reinstall the blue and white coated tubing in the correct holes The inlet tubing has white insulation and an interior diameter of 0 005 The outlet tubing has blue insulation and an interior diameter of 0 010 Do not reverse the inlet and outlet tubing MWH Rev A Cleaning the Flow Cell and Windows 5 3 5 Step 5 Reinstalling the Flow Cell Ambient Version Important The following steps apply to the HELEOS Ambient version only See Step 5 Reinstalling the Flow Cell Heated Cooled Version on page 5 18 for the procedure for the HELEOS Heated Cooled version Note If you are not careful the cell could be reversed Make sure that the INlet
15. B 4 2 DANGER Place the HELEOS in line between the columns and RI detector The HELEOS has to be placed in line between the columns and the RI detector in the oven Therefore it is necessary to take the output line from the columns out of the oven through the HELEOS and back into the RI detector Use the heated lines provided with the HELEOS and additional stainless steel tubing inside the oven if needed The HELEOS can be placed either on the right or left hand side of most ovens Newer Waters 150C instruments have a pre drilled hole in the left side If you have an older Waters 150C you can drill a hole yourself on either side of the 150C at the level of the columns Make sure there are no cold spots where the heated lines connect to the oven the point of connection should be inside the injector or column compartments to ensure this The RI and autoinject cables should be attached to the RI detector integrator output and the autoinject terminals On the Waters 150C these are on the left side and are clearly marked Connecting the Heated Lines and Heating the HELEOS The HEATED LINE electrical connector on the side of the HELEOS contains live 48V DC pins Keep the dust cap on this connector whenever the temperature controller is on and the heated lines are not connected If the oven is at operating temperature such as 135 C leave it connected to the oven even when servicing the read cell assembly What you will need to con
16. Interference Filters ccccceccccsseeecseeeeceeseeseeseeeneeeeees E 2 MWH Rev A E 1 Appendix E Interference Filter Option E 1 Installing Interference Filters What you will need to install interference filters Anti static wrist strap Tweezers Nine interference filters with O rings Wooden or plastic spatula or similar tool To install interference filters do the following 1 2 10 Put on the anti static wrist strap Switch off the power to the instrument and laser then remove the instrument cover Ground yourself to the chassis and gently remove a photodiode from the read head using a pair of tweezers Be careful not to stress the solder connection of the lead to the PCB Also make sure the leads do not touch one another to cause a short circuit Carefully insert an interference filter into the diode hole using a wooden or plastic spatula Touch the outer edge of the interference filter only The mirrored side of the filter should face out towards the photodiode the colored side of the filter should face in towards the cell Insert the small O ring and push it firmly against the filter This holds the filter in place Remove the black O ring from the photodiode and push it into the shoulder of the hole Moisten the O ring then push the photodiode through the O ring into its hole Moistening the O ring ensures that the photodiode slides easily into place Repeat steps 3 to 7 for the
17. Panel Wiring Continued ee O C o fs ow p S E S S S Standard RJ45 wiring of White 10Base T 100Base TX 4 Orange Transmit E White 3 Green eo Zo f e e e O White 7 Brown on ne You may need to connect the wires to a connector provided with your device or to the device directly The following list contains additional information for various other connectors e Auto Inject Some injectors require programming in order for the closure to happen Make sure that an injection closes the circuit e Alarm In TTL input on red signal and green signal ground On the instrument display Alarm panel you may select the active state of this input see Alarm Panel on page 4 11 e Alarm Out TTL output on white signal and black signal ground On the instrument display Alarm panel you may select the active state of this output see Alarm Panel on page 4 11 e Recycle In TTL input on red signal and green signal ground When the signal on this line transitions from 0 V to 5 V the instru ment actuates an external solenoid valve by supplying power to the Recycle Out connector When the signal transitions from 5V to OV the Recycle valve is de actuated e Recycle Out The solenoid valve drives current on the white and black wires the current direction is irrelevant for the solenoid This signal may be connected to a user supplied solenoid valve or a Wyatt Technology Recycle unit which conta
18. a 4 6 COMC aero cession cece dase cutetetinns at tetgentetide a ccug 4 6 FIOCV CIC ona E E E A N nus ueueneen 4 6 BALE INN sni a a a a ee 4 7 Setting the Baseline and Normalization Coefficients s neeneneennennennernerneeeernnenn 4 8 Adjusting the Display Rang sscsrss sashes hace cons a a a te eeies 4 9 QELS ae anian aa 4 10 APANG a a a a REBT ef ee a 4 11 Alarm Signal State ccccccsssccccsssesececeeeeeseeeeeeeseeesceesseseeeseeeeessseaseeseageeeesaneeseneas 4 11 PIC IOC AR Nea ae a N a a 4 12 Syste Pane iaa a E 4 13 Read Head Temperature and Heated Line Temperature ccccceseeeeeeeeeeeeeees 4 13 PROC ClO siiri a O a OEE SE 4 14 COMET anisa N eluded a a Gece 4 14 PNAC OUUOU somesa A a a 4 14 PME CONSTANT caii aaa aa a a a 4 14 Se TIME i A 4 14 Lang gge toten a a E a Ea a 4 14 REStar ISI apii a E EA EA E E EA 4 15 Load Factory Delawlts sisisi a aa 4 15 Restart INSUUMENI soricei a aA EE aaa 4 15 COMM AME pesn E ES 4 16 Chapter 5 HELEOS Maintenance ccccccseeseeeeeneeeeeneeeeeaes 5 1 General MamntenanCe sicceuseigentoscecteanicotsseteuey E a a a 5 2 FIOW Gell Mantenan e stich evaresscsicrmsdensaiedsatcedvarinncscanactcdcnredaateeeseeesuaccacedar tens 5 3 Onne Cleaning sitesin a Ea aE Fea E 5 3 Parnicles jin ine Cell uir a E E 5 5 Contents iv MWH Rev A Contents Cleaning the Flow Cell and Windows ccccccecccccceeeeeeeeeeeeeeeeeeeeeeeeseaeeeeeesaaaes 5 6 step 1 Rem
19. a pure filtered 0 02 um solvent to flush the cell We recommend filtered ethanol or isopropanol be left in the cell e Do not flush the cell from Outlet to Inlet Backflushing the cell can cause particles to become lodged in the inlet tube which has a smaller inside diameter than the outlet tube e A mild detergent solution may also help clean the flow cell and may be kept in it overnight when the instrument is not in use then purged in the morning e There are two extra sets of inlet and outlet tubes in your hardware kit One set consists of 4 pieces of color code tubing white for inlet and blue for outlet This set of tubes is for use with the unions to make it easy to remove the flow cell for cleaning without breaking the seal at the manifolds The second set of color code tubes is for use without the unions MWH Rev A 5 3 Chapter 5 HELEOS Maintenance Note With either set you will need to bend the tubes in order to install them in the instrument The bend radius should not be less than the bend radius of tubing that comes installed in your HELEOS To avoid introducing particles into the flow cell flush the tubes after bending them and before installation COMET Option We also recommend the use of the Wyatt COMET option The COMET is a permanently installed ultrasonic flow cell cleaning system which operates on a different principle than traditional immersion bath based cleaners Traditional ultrasonic cleaners opera
20. below 80 C and you decide to operate above 80 C you must remove the backing rings and install the 9 mm flow cell O rings instead of the 6 mm O rings Above 80 C the O rings expand enough to crack the flow cell glass if the backing ring 1s installed If your instrument is configured to operate above 80 C and you decide to operate below 80 C install the backing rings and the 6 mm flow cell O rings This minimizes dead volume If dead volume is not an issue you may choose to use the high temperature O ring set over the entire temperature range When operating below ambient temperature be sure to connect a dry air or nitrogen source to the HELEOS Nitrogen Purge connector Light scattered from condensed water ruins your measurements It is a good idea to use dry air or nitrogen even at ambient or higher tem peratures to minimize the amount of dust within the instrument The temperature controller for the flow cell will not allow you to set a temperature below 20 5 C unless it detects at least 20psi of gas pressure on the nitrogen port This 1s prevent accidental condensation on the flow cell and read head If for some reason you need to open the flow cell heat it to at least 20 5C before opening the flow cell C 3 Appendix C Peltier Heated Cooled Option C 4 MWH Rev A Polarization Option The polarization option consists of a special grooved cell retainer which holds two strips of Polaroid film around the sides of the
21. can set the temperature of the cell on the Main display panel Alternatively you can navigate to the System panel and set the temperature of the cell and heated line separately 1 Navigate to the associated field and type in the new temperature Press Enter to select The system will ramp the temperature at 1 C per minute This is to ensure that differential thermal expansion of the cell materials do not cause damage 2 Allow the read head temperature to ramp to the setpoint temperature For example if you wish to operate your system at 150 C and your system is initially at 25 C it will take about two hours for the temperature to reach 150 C since the ramp rate is limited to 1 C per minute If you want to perform temperature ramping experiments contact Wyatt Technology for instructions on how to reprogram the ramp rate It can be changed programatically to as slow a rate as required B 3 Appendix B Ultra High Temperature Option B 2 3 Heated Lines B 2 4 B 4 The optional HELEOS heated lines can maintain temperatures up to 210 C in the inlet and outlet lines if the HELEOS is connected to other high temperature instruments and detectors The heated lines consist of two pieces of steel tubing that are insulated and contain a temperature sensor and a heater One of them is marked at both ends with a piece of metal tape so that you can distinguish the tubings as they protrude from the insulation Typically the hea
22. cell window 5 9 changing a fuse 3 4 channels 3 8 3 10 values 3 8 chromatography system 2 5 cleaning 5 2 flow cell and windows 5 6 5 11 flow cell on line 5 3 Comet 4 6 run time 4 14 start time 4 14 Comm panel 4 16 DCHP 4 16 communications Internet 1 6 concentration detector 2 7 connecting to LAN H 3 connecting to network H 1 connecting to PC H 1 connecting via ethernet H 16 connecting via USB H 13 connectors 90 Degree Detector 2 7 Auto Inject 2 7 AUX 1 2 3 4 2 7 Index 1 Index auxiliary 2 7 back panel 3 3 Heated Line B 11 Nitrogen Purge 2 3 Vapor Interlock 2 7 cooled HELEOS C 2 correlation function QELS A 2 cover 3 5 removing 3 6 crossover cable H 5 customer service 1 6 D dark offsets 2 4 depolarizing samples D 1 Detector board 3 17 detectors accessible 3 15 angles 3 10 viewing voltages 2 4 DHCP 4 16 H 19 diagnostics www wyatt com H 21 Display autoscale 4 6 setting the scale 4 6 display alarm panel 4 11 batch panel 4 7 Comm panel 4 16 heated line temperature 4 13 read head temperature 4 13 system panel 4 13 display panels button description 4 2 main 4 2 navigating 4 2 display range adjusting 4 5 display See LCD display dust detecting 3 8 5 13 dusting gas 5 6 E electronic fuse 3 3 environment dust 5 2 location 2 3 EOS Instrument Controller Board 3 17 equipment list 2 2 ethernet cable H 6 ethernet connection multiple instruments H 18 Index 2 one inst
23. flow cell The vertically polarized strip has vertical notches which can still be seen when installed in the flow cell assembly These strips detect the presence of depolarizing molecules or particles CONTENTS PAGE eM ASTANA O esera O E D 2 D 2 Normalization and Calibration with Polarization Strips Installed D 3 MWH Rev A D 1 Appendix D Polarization Option D 1 Installation What you need to install the polarization filters e 2 0 mm Ball driver e 2 5 mm Ball driver e Two Crescent wrenches e 1 5 mm Hex driver To install polarization filters do the following 1 Remove the bib from the cover of the instrument 2 Remove the cell assembly from the read head See Removing the Cell Assembly on page B 5 3 Use the 1 5 mm Hex driver to unscrew the two M2 screws holding the bottom cell retainer in place then remove the cell retainer 4 Install a vertically polarized strip facing the odd detectors left side including detector 11 at 90 and a horizontally polarized strip facing the even detectors right side The small notches at the edges of the Polaroid film indicate its horizontal or vertical orientation Reinstall the special polarizer bottom cell retainer Reinstall the flow cell assembly using two M3 screws Reconnect the tubing to the in line unions Reinstall the flow cell cover plate So a et Replace the instrument cover bib D 2 MWH Rev A Normalization and Calibration with Pola
24. lt 1 K lt 1 lt 1 lt 1 lt 1 lt 1 41 lt 1 41 41 lt 1 41 41 4141 KI nn Figure 4 6 Raw data 4 8 MWH Rev A Batch Panel Main Batch QELS Alarm 5 stem Comm Raw Data Set Baseline Normalize Normalization Auto Scale Set Scale Coefficients 1 000 1 000 1 000 1 000 1 000 1 000 1 000 1 000 1 000 1 000 1 1 1 1 1 1 5 a 4 B F T a 000 000 000 000 000 000 000 000 m M M Iw Iw Iw Iw Iw Iw Iw F M M Iw Iw Iw Iw Iw Figure 4 7 Normalized data 4 4 2 Adjusting the Display Range Adjusting the display range is the same as on the main page See Adjusting the Display Range on page 4 5 for more details MWH Rev A 4 9 Chapter 4 Using the Display Window 4 5 QELS panel For information on the QELS option see Appendix A Using QELS 4 10 MWH Rev A Alarm Panel 4 6 Alarm Panel The Alarm panel displays sensor information and lets you adjust alarm settings An alarm history is shown of the last few alarms and the time at which they occurred Some alarms are not visible for all instruments Main Batch QELS Alarm System Alarm IN M Active Low Alarm QUT Active Low i Audio Alarm Alarm history text box I Recycle Open DD Eerma comer I nz Pressure jes Flow Cell Temperature Lock ia Heated Line Temperature Lock Flow Cell Temperature 25 00 C Heated Line Temperature MA Me Pressure
25. manifold is in the rear position and the OUTlet manifold is in the forward position 1 Replace the cell assembly in j the read head insert the 7 two M3X20 screws and tighten with the 2 5 mm How call E Ball driver Loosely tighten cover plate both screws and then E alternately tighten them evenly until they are secure m3x20 a fol a a Manli O d 2 Reconnect the short pieces nee 4 of stainless steel tubing to the in line unions using the Rear N two 14 Crescent wrenches 3 Plugin the power cord and turn on the HELEOS 4 Connect the cell to your HPLC system and make sure the cell does not leak Make certain the fittings are tight and leak free Whenever you pump solvent through the cell check the Figure 5 10 Ambient Flow Cell Installation fittings at least twice during the first hour Use a piece of tissue and touch the top of the fitting where the tubing emerges no solvent should be visible on the tissue 5 Replace both sections of the flow cell cover plate Tighten the two Allen head screws with the 2 5 mm Ball driver 6 Replace the instrument cover bib MWH Rev A 5 17 Chapter 5 HELEOS Maintenance 5 3 6 Step 5 Reinstalling the Flow Cell Heated Cooled Version Important The following steps apply to the HELEOS heated cooled version only See Step 5 Reinstalling the Flow Cell Ambient Version on page 5 17 for the procedure for the HELEOS ambient version
26. naked eye after alkaline exposure The alkaline resistance class indicates the time in minutes required to decompose a 0 1 micrometer layer of glass in an alkaline solution at 90 C sodium hydroxide pH 10 Table G 1 Flow cell alkaline resistance classes Table 8 MWH Rev A G 5 Chapter G Flow Cell Properties Table G 1 Flow cell alkaline resistance visible surface changes Table 8 Visible Surface Description Changes ee Scarred surface but no visible coatings color change 90 2 Interference colors colors e mr staining White coating thick layers G 6 MWH Rev A Scattering Angles G 5 Scattering Angles The table below shows the scattering angles for two different flow cells in four different solvents at a wavelength of 690 nm Note that for a K5 cell in water the first two detectors are not available for an F2 cell in water the first three detectors are not available The mathematics behind these changes in scattering angles are discussed in the Flow Cell section of Chapter 3 Table G 1 Flow cell scattering angles part A ene ene mo psr 1 51876 1 51876 1 51876 1 61311 1 61311 1 6131 1 61311 fn 1 830 1 401 1 488 11 500 1330 1 401 1 488 1 500 Table G 2 Flow cell scattering angles part B 22 500 batch batch batch batch 28 000 N A 26 621 N A 18 281 32 000 14 440 30 834 N A 24 217 38 000 25 862 37 073 17 108 32 067 44 000 34 77
27. timed out Ping statistics for 172 280 1243 Packets Sent 4 Received Lost 4 108H loss Figure H 20 Failure to connect to instrument using ping MWH Rev A H 21 Chapter H Connecting to Network or PC H 22 MWH Rev A A air filter 5 2 alarm comet failed 4 12 enabling 3 17 4 3 external 4 12 leak 4 12 N2 pressure 4 12 overheat 4 12 recycle open 4 12 turn off 4 3 turning off 3 16 vapor 4 12 alarm history 4 11 alarm panel 4 11 alarm signal state 4 11 analog output 4 14 angles of scattering G 7 APD A 3 over illumination A 3 protection system A 4 aperture installation tool 5 9 ASTRA V F 1 ASTRA V for Windows 1 6 audible alarm 4 3 audio alarm disable 4 12 enable 4 12 turn off 4 12 Auto Inject connector 2 7 2 9 autotuning temperature control B 3 AUX 1 2 3 4 connectors 2 7 auxiliary connectors 2 7 devices 2 7 viewing signals 2 4 avalanche photodiode APD A 3 B back panel 3 3 Backing ring ambient and low temperatures 5 15 C 3 not used for high temperature 5 15 bare wire 2 7 MWH Rev A baseline noise sources B 9 batch conversion 5 19 temperature controlled B 12 batch cover 3 5 batch panel 4 7 bib on cover 8 5 black wire 2 7 blue wire 2 7 Board EOS instrument controller 3 17 nitrogen sensor 3 18 utility 3 17 vapor sensor 3 18 boards circuit 3 17 bubbles detecting 3 8 C calibration 2 5 after setup with oven B 7 polarization option D 3 temperature control B 10
28. to Network or PC H 4 Connecting via Ethernet when not on a LAN If the computer is not on the LAN it is possible to use the Ethernet port directly to connect to the instruments H 4 1 One instrument to computer not on LAN using crossover cable Connect the yellow crossover cable from the instrument directly to the Ethernet port on the computer Pag Figure H 15 Connecting one instrument directly to a computer that is not on the LAN using the yellow crossover cable H 16 MWH Rev A Connecting via Ethernet when not on a LAN H 4 2 One instrument to computer not on LAN using an Ethernet switch Connect the instrument to the Ethernet switch using a standard Ethernet cable Then connect the switch to the computer Ethernet port using a standard Ethernet cable Figure H 16 Connecting one instrument to the computer using an Ethernet Switch MWH Rev A H 17 Chapter H Connecting to Network or PC H 4 3 Multiple instruments to computer not on LAN using an Ethernet switch Connect each instrument to the Ethernet switch using a standard Ethernet cable Then connect the switch to the computer Ethernet port using a standard Ethernet cable Figure H 17 Connecting multiple instruments to a computer not on the LAN using an Ethernet switch H 18 MWH Rev A Instrument Network Settings H 5 Instrument Network Settings MWH Rev A Figure H 18 shows the standard settings on the instrument front panel that will w
29. wish clean the cell themselves Contact Wyatt customer service for details MWH Rev A Cleaning the Flow Cell and Windows 5 3 1 Step 1 Removing the Flow Cell Assembly In this first step you will remove the cell assembly from the read head 1 Put on the anti static wrist strap This is an important step The strap keeps the flow cell glass and windows from building up a static charge and attracting particles while being handled 2 Remove the two hex head screws with the 2 5 mm Ball driver provided then lift the dust cover off The flow cell assembly is now visible Figure 5 1 Flow cell assembly after insulating cover has been removed 3 Remove two M3 screws F 4 1 N j J Outlet tubing i TA fitting M3x20 screws Figure 5 2 Removing the heat exchanger MWH Rev A oT Chapter 5 HELEOS Maintenance 4 Remove the inlet tubing and outlet tubing at the fittings shown It does not need to be unplumbed at this point if you are simply removing the COMET to inspect the cell for dirt 5 Next unbolt the COMET by removing the two M3x20 screws Figure 5 3 Removing the COMET assembly Gently tilt the heat exchanger so that it can be placed aside or if unplumbed in the previous step remove the heat exchanger completely Slide the COMET away to the right Inlet tubing white insulation gt lt Outlet tubing blue insulation Disconnect in line un
30. 0 MWH Rev A 3 11 Chapter 3 HELEOS Components 3 6 Flow Cell 3 12 3 6 1 Flow Cell Design The patented flow cell is at the heart of the HELEOS and is critical to the instrument s unique measuring capabilities In many applications such as chromatography the ability to measure small samples is crucial so cell volumes must be minimal The total volume of the cell from the manifold inlet to the manifold outlet is about 70 uL The actual scattering volume the illuminated part of the sample that is viewed by the detectors is less than 1 uL In other light scattering instruments the cell walls are so close to the detected sample that the light scattered from the cell walls often overwhelms the small scattering signals from the sample The HELEOS flow cell design resolves this dilemma Because the windows are recessed in the manifolds away from the scattering volume any stray light from the air glass solvent interfaces is not seen by the detectors As a result the detectors measure scattering only from the sample and not from the cell Inlet manifold Outlet manifold Flow cell Bottom cell retainer Figure 3 10 Flow cell assembly 3 6 2 Laser Beam Orientation Another critical element of the HELEOS flow cell is the laser beam s orientation The laser passes in the same direction as the flowing stream This helps to minimize beam cell interface problems by keeping the cell
31. 2 43 253 29 254 39 323 50 000 42 776 49 396 38 775 46 270 57 000 51 542 56 533 48 656 54 147 64 000 59 961 63 650 57 881 61 873 72 000 69 337 71 767 67 988 70 590 81 000 79 710 80 886 79 063 80 315 90 000 90 000 90 000 90 000 90 000 99 000 100 29 99 114 100 94 99 685 108 00 110 66 108 23 112 01 109 41 117 00 121 23 117 37 123 41 119 22 126 00 132 16 126 52 135 47 129 21 ene ene O MN oh l o1 amp W MWH Rev A G 7 Chapter G Flow Cell Properties 16 134 00 142 49 138 86 135 16 134 70 147 41 143 11 138 86 138 33 17 141 00 152 55 147 40 142 49 141 89 160 49 153 48 147 40 146 69 18 147 00 163 28 155 39 148 87 148 12 180 00 164 94 155 39 154 41 A C 81 000 79 710 80 236 80 812 80 886 79 063 79 623 80 236 80 315 G 8 MWH Rev A Connecting to Network or PC These instructions contain a pictorial overview for connecting your HELEOS to a computer for data collection The instructions are divided into seven sections CONTENTS PAGE F COMPONE E H 2 H 2 Connecting to a LAN sciis naiiai H 9 H 3 Connecting via USB ssceseccuveendadcindcatinesssnsestammentemmiyuadautdetiacbeaniels H 13 H 4 Connecting via Ethernet when not on a LAN c sceeeeeeeeeeeeees H 16 H 5 Instrument Network Settings ccccccccsessseeeeeeeeeeeeeseeeeeeesaeeeeeeens H 19 H 6 Accessing instruments with ASTRA V
32. 432 C 0 557 Transformation Specific Heat Temperature c J gx K G 2 MWH Rev A Refractive Indices G 2 Refractive Indices Table 1 Glass Classification Refractive Index A 633nm MWH Rev A G 3 Appendix G Flow Cell Properties G3 Chemical Properties To interpret the CR FR SR and AR values see Definition of Terms Table 2 G 4 MWH Rev A Definition of Terms G 4 Definition of Terms Transformation Temperature Temperature at which deformation of precision finished surfaces anda change in the refractive index can occur Climate Resistance CR 1 4 Class CR 1 after 180 hours of exposure the glasses exhibit no or only slight signs of deterioration due to changing climatic conditions Under normal humidity conditions that prevail during the processing and storage of optical glasses no surface deterioration of class CR1 glasses is to be expected Resistance to Staining FR 0 5 Class FR 0 after exposure to a standard acetate solution pH 4 6 for over 100 hours no interference color staining is observed Resistance to Acids SR 1 4 Class SR 1 after a 100 hour exposure to an aggressive solution of 0 3n nitric acid pH 0 3 the smallest visible detectable thickness 0 1 micrometer is not dissolved Resistance to Alkalis AR 1 4 A two digit figure is used to express resistance to alkalis The digit after the decimal point indicates what surface changes are visible to the
33. 5 3 3 Step 3 Cleaning the Flow Cell and Windows 00000 5 11 5 3 4 Step 4 Reassembling the Flow Cell ccccccsessseeeeeeeeeeees 5 14 5 3 5 Step 5 Reinstalling the Flow Cell Ambient Version 5 17 5 4 Flow to Batch Conversion cccccccccccseseceeeeessececeeseceeeaeneeeessaeees 5 19 MWH Rev A 5 1 Chapter 5 HELEOS Maintenance 5 1 General Maintenance Note For general maintenance we suggest you do the following Keep the HELEOS on a flat clean surface with space behind and standing on its feet to allow proper air ventilation Keep the case clean Use a cloth dampened with water to clean it Allow the instrument to warm up for 30 minutes before taking measurements Keep the instrument cover on at all times with the bib installed Keep the cell inlet and outlet sealed when not in use to prevent solvent evaporation or introduction of particles Check the air filter every month or so When the air filter gets dusty pull the air filter cover off and remove the filter Then gently clean it with warm soapy water dry and replace You can also order replace ment filters from www wyatt com If you are in a dusty environment clean the filter more often than monthly Failure to keep the air filter clean will cause the instrument to heat up and will decrease the ability of the fan to blow dust particles out of the instrument In addition you will need to follow certain pro
34. Connecting to Network or PC H 1 3 Computer connections Computer connections can be made via either the Ethernet or USB port Figure H 3 shows these ports on a standard laptop computer Sections H 2 and H 4 describe instrument connections made via the Ethernet port Section H 3 describes connections made via the USB port Figure H 3 Ethernet and USB ports on the computer The USB ports are designated by a green arrow and the Ethernet port is designated by a yellow arrow H 4 MWH Rev A Components H 1 4 Crossover cable MWH Rev A A crossover cable can be used to make a direct connection from the instrument to an Ethernet port on a computer or to an Ethernet to USB adapter Please note that the crossover cable shipped with Wyatt Technology instruments is yellow to distinguish it from a standard Ethernet cable Please be careful to only use the yellow crossover cable where indicated Figure H 4 The Ethernet crossover cable shipped by Wyatt Technology is yellow Chapter H Connecting to Network or PC H 1 5 Ethernet cable A standard Ethernet cable is sometimes referred to as a patch cable or a straight through cable to distinguish it from the crossover cable in Section H 1 4 Ethernet cables provided by Wyatt Technology are black blue white or gray but never yellow yellow is reserved for the crossover cable For these instructions the Ethernet cable will always be black Figure H 5 Standard Ethernet cabl
35. HELEOS has no AUX gain settings The AUX input signals can accept an input range of 10V to 10V witha 1 uV resolution Typically when the time constant is set to 1V a noise level of less than 10uV is observed To attach an Auxiliary connector do the following 1 Attach a cable to the appropriate port on the rear panel of the HELEOS Aux 1 and 2 are on one connector Aux 3 and 4 are on another 2 Connect the wires of the cable to your other device as shown in Table 2 1 Usually when connecting the AUX channels one need only connect AUX and AUX to the signal source If there is unacceptable noise pickup you can connect the GND connection to either the Chassis or the AUX connector of the source instrument Table 2 1 Back Panel Wiring dL Black SS 9 seen fave MWH Rev A 2 Chapter 2 Installing the DAWN HELEOS Table 2 1 Back Panel Wiring Continued Connector abel Pin Color Signal Comments TS emn mas alwe Ae pd oele fa o aes we SS a Frees fae SSCS sew menw S ooo y oe we O Autolnjectin 1 Whte ne O O To o oo o E 2 PT 8 Red Injecting P Green ee ee po Be NC 2 Inject_Out Po Red NG Po Green ENC Pf Yellow FNC Po Be NC Back nc Red m O afo m o f sew NC f ofw wo O f ofe amon o T shra nc o l emn pe Od sew wo f ofw wo i o y oe ooo y afon eenn soto pe sae nc ee oje ee 2 8 MWH Rev A C C C C C C C C C Connecting Auxiliary Devices Table 2 1 Back
36. Hardware Manual for the DAWN HELEOS Light Scattering Instrument eeineteey 6300 Hollister Ave Santa Barbara CA 93117 MWH Rev A Copyright 2005 Wyatt Technology Corporation WYATT TECHNOLOGY Corp makes no warranties either express or implied regarding this instrument computer software package its merchantability or its fitness for any particular purpose The software is provided as is without warranty of any kind Furthermore Wyatt Technology does not warrant guarantee or make any representations regarding the use or the results of the use of the software or written materials in terms of correctness accuracy reliability currentness or otherwise The exclusion of implied warranties is not permitted by some states so the above exclusion may not apply to you All rights reserved No part of this publication may be reproduced stored in a retrieval system or transmitted in any form by any means electronic mechanical photocopying recording or otherwise without the prior written permission of Wyatt Technology Corporation DAWN is a registered trademark and DAWN HELEOS is a trademark of the Wyatt Technology Corporation A variety of U S and foreign patents have been issued and or are pending on various aspects of the apparatus and methodology implemented by this instrumentation Table of Contents Chapter 1 INtrodUCTION ccsseecceeeeecenseeeeeeeeeeeneeseeneeneeaes 1 1
37. Index 3 Index disconnecting B 10 installation with B 7 operation with B 9 potential problems B 9 overview 1 2 P packing list 2 2 particles detecting 3 8 dislodging 5 5 from columns B 10 parts list 2 2 PCBs 3 17 Peltier cooler A 3 Peltier Heated Cooled HELEOS C 2 PL210 disconnecting B 10 installation with B 7 operation with B 9 potential problems B 9 polarization option installation D 2 power supply voltages viewing 2 4 power switch 3 2 printed circuit boards 3 17 pump operation B 10 Q QELS A 1 correlation function A 2 sample rate A 3 R ramp rate B 3 raw data 4 7 read head 3 9 detectors 3 10 thermocontroller B 3 read head temperature 4 13 sync 4 14 rear laser monitor values 3 8 viewing 2 4 recycle 4 6 delayed activation 4 14 refractive index 3 14 removing flow cell 5 7 temperature controlled B 5 restart instrument 4 15 restart ISI 4 15 RS 232 1 2 Index 4 S scattering angles G 7 scintillation vial B 12 serial number location 1 7 serial port input output 1 2 set temperature B 3 setting cell temperature 4 6 shipping list 2 2 Snell s Law 3 14 3 15 software 1 3 ASTRA V 1 3 1 4 1 7 2 4 3 15 4 14 5 2 5 21 A 3 B 7 B 9 B 18 H 1 H 20 ASTRA V for Windows 1 6 establishing communications 2 4 solvent filtering B 9 scattering angles G 7 temperature control C 3 solvent offsets 2 4 specifications flow cell G 2 laser F 2 Static IP H 19 static IP address 4 16 sync button 4 14 syste
38. MMEDIATELY 2 Unpack the instrument Place the HELEOS on a level surface and inspect the cabinet for damage If you see any damage CONTACT THE SHIPPING CARRIER IMMEDIATELY 4 Check that the boxes contain the all of the items listed as included with your instument shipment in addition to the instrument the packing slip sent with the instrument contains the most up to date list 2 2 MWH Rev A Installing the Instrument 2 2 Installing the Instrument MWH Rev A The installation procedure for the HELEOS involves some initial tests to see that everything is working properly To install the HELEOS do the following 1 Place the HELEOS on a flat clean surface standing on its feet and positioned to allow air flow through the back of the instrument to keep its electronics cool See Chapter 5 HELEOS Maintenance for more information about the HELEOS environment and how to keep it in peak condition The HELEOS is designed to stack with the ViscoStar or rEX It can be installed either at the top or bottom of the stack If the optional batch conversion is to be used we recommend that the HELEOS be installed on the top of the stack so that the batch holder is easily accessible through the top cover Make sure the supplied power plug is correct for the local power outlet The HELEOS is equipped with a universal power supply which operates anywhere in the world It accepts inlet voltages between 90 250V and line freque
39. ON PAN BN GW pasior a E N T A EE 3 2 BACK Mell VION sen E EEE E E 3 3 Changing a FUSE eee eee eee eee ee ee 3 4 WO COV EE E EAE 3 5 Removing the Cover oo ceecccccceseesccceeceaeeeceeeceeaeeeceeeeseeeeeeeeesseeeeeeeeeeseaeeseeeeesaaeseeeeeeas 3 6 E EE E pune P EE A E EE E E AE EE EY 3 7 Laser Beam Warning ccccccccssecccesececseseeeceseeeceueeceeseessuseecsaueceseuseessaseessaeeessaueeesees 3 7 AS SUNLESS reuters a E E buthadeoeaamediniatonaisanien 3 8 Read Head and Detectors sonahevecesesstiesnnexcntcieneced cnenecncsmeenssccenianeneasenceceresciewenciogo 3 9 Read Head STUCWUTG sssesiicintnrobi niin inina en ET E r EE 3 9 Detector Placement aca dtccanatemspcenannen ctteataasunceeosnanesnctateaMncttaansnesatataidadsradesinegetectsdacnnmeed 3 10 FOW GEM a E E E sedi 3 12 FOW GC Cll DESIGN cscs cesercceceesescasecensessedeceengssed oeesassactanesatesecaneteescewerstoccsexteeseteegesets 3 12 Laser Beam Orientation cccccccccsssecccceeeceeeeseeeeeeeaeeecessueaeeeesseueeessaeeeeeessaaeeeeesaaes 3 12 CEU VIIGOWS eenaa oldu uoceeactcavaeatenieaoaeanacesitedauneriiaieceuneawaeauiansanmatonnerdnes 3 13 Refractive Index Differences Liquid VS Glass ccccceecseeeeeeeeeeeeeeseeeeeeeeaeeeeeens 3 14 Accessible Available Detectors ccccccccccseeeececeeeeeeesaeeeeeeeeeeeeeeseeeeeeessaeeeesaaaeeeees 3 15 AAO c E E EE 3 16 Yao ORSO ei ee E E E E ee 3 16 Liquid Level Leak Sensor ccccccccccccceseseceeeeeeeeeeeeeeeeeeee
40. ONGINI seer vate aetancnortatenietise E E 1 2 Me WMS UHUMGTAN poaren E EE E ENE A E EA 1 2 TNE OMY Ale a E A EEA E 1 3 About This Manual s srrrcisirisinrcetesinn ena ian 1 4 Manual COnv ntiONS este weatice nse ncnys a tensandes cae sevatnnstasntvnceseteuneiednescadvaraneseaiepesastericstanes 1 4 How the Manual IS Organized cccceccccecceeeeeeeeesceeeeeceeeeeeeeeeeeeeeseaeseeeeeesaeeeeeeeeeas 1 4 How to Contact Wyatt Technology Corporation ccccccceeseeeeeseeeeeeeeeeeeeeeeeeas 1 6 Corporate Headquarters ccccccccccsseseccceeceeeeeeeeeceeeeeeeeeesseeeseeeeeeseeaeeeeeessaaaeeeeesseaaees 1 6 dle S Depe siiente Eeee Ea eE a O a Enisa Eai 1 6 Technical SUPPO sccasi senaince5 fs 5esacdec0sersaedscbdebitensdediabaieictteaetenstdadideds santas ondbadatelesotiaseeds 1 6 Where to Go from Here cccccecceeeeeeeeseeeeeeeeeeeeeeeseeeeaesseeeeeeeeessasaeeagaeeeeeeeesnnags 1 8 Chapter 2 Installing the DAWN HELEOG ceeee 2 1 Unpacking the IMISUFOMIGNIL scesscincstecarcece vast oussccvesenccedens EEE EEEE 2 2 STAN ANG MAS UMMIVGING cectawenedsun cs cusgstatin danse vnctasiueai nentons eaimepelouapunemnbehanteretaus 2 3 Connecting Auxiliary Devices ccccccccseeecceeceeeeeeeeceeeeeeeeaeeeceesaaseeeessuaeeeesanees 2 7 Attaching Auxiliary Device Connectors cccceeecceceeeeeeeeeeeeeeeeeeeeeeeeeeeeeseeeeessaeeeeeees 2 7 Chapter 3 HELEOS Components c csccceseeseeseneeeeenseneeaes 3 1 P
41. Rev A Connecting to a LAN H 2 Connecting to a LAN If an instrument is connected to a LAN it can be accessed by any computer plugged into the same LAN H 2 1 One instrument to LAN Plug the instrument into a LAN wall socket using a standard Ethernet cable The computer that is to communicate with the instrument must be on the same LAN Figure H 8 Connection for one instrument to LAN Note This view is the back panel of the ViscoStar but the same connection method is used by the HELEOS MWH Rev A H 9 Chapter H Connecting to Network or PC H 2 2 One instrument and computer to LAN If there is only one LAN wall socket available for both the instrument and computer it is necessary to use an Ethernet switch to connect both the computer and instrument to the LAN In this configuration the computer can access the LAN and the instrument and the instrument can be accessed from any other computer on the LAN Figure H 9 One instrument and a computer can both be connected to the LAN using an Ethernet switch H 10 MWH Rev A Connecting to a LAN H 2 3 Multiple instruments to LAN If there is only one LAN wall socket available two or more instruments can be connected to the LAN via an Ethernet switch The instruments can be accessed via any computer on the LAN MWH Rev A Figure H 10 Two instruments connected to the LAN via an Ethernet switch Chapter H Connecting to Network or PC H 2 4 Multiple in
42. TED LINE fitting contains live 48 VDC pins Keep the dust cap on this connector whenever the heated lines are not connected Disconnect the heated lines from the flow cell Remove the insulating read head cover from the HELEOS 10 Flush the cell with a suitable solvent such as toluene 11 Continue flushing with methanol ethanol and then cap the cell 12 Remove the cell assembly from the read head disassemble the cell clean it and replace all O ring seals 13 If the disconnection is only temporary do the following a Leave the heated lines connected to the oven b Attach the small piece of tubing with the unions at the HELEOS side of the lines B 11 Appendix B Ultra High Temperature Option B 5 Temperature Controlled Flow to Batch Conversion The Heated HELEOS Flow to Batch conversion differs slightly from the ambient model because of the extra cover plate for the read head an additional vial insulation ring and an insulated cap B 12 What you need for flow to batch conversion Two Crescent wrenches 2 5 mm Ball driver Phillips screw driver Heated Batch Conversion kit To convert from flow to batch operation do the following 1 2 Remove the bib from the cover of the instrument Remove the read head cover the flow cell cover plate and the cell assembly from the instrument See the section on Removing the Cell Assembly on page B 5 in this chapter Insert the batch manifold and secure it wi
43. Using the HELEOS with an OVEN cccccccecceseeeeeeeeeeeeeeeeeeeaeaees B 7 B 4 1 General Setup ProCedure cccccccccccccceeseeeeeeeeeeeeseeeeeeeeseseees B 7 B 4 2 Connecting the Heated Lines and Heating the HELEOG B 8 B 4 3 Operating the HELEOS with an OVEN ccccccseeeeeeeseeeeeeeeees B 9 B 4 4 Potential Problems ccccccseccccseececceeceeceeeeeseeseeseneeesenseeeeees B 9 B 4 5 Disconnecting the HELEOS from an Oven cccceeeeesseeeeeees B 10 B 5 Temperature Controlled Flow to Batch Conversion B 12 MWH Rev A B 1 Appendix B Ultra High Temperature Option B 1 Overview With the Ultra High Temperature option the read head may be heated from approximately 10 C above ambient temperature to 210 C The temperature can be controlled to within 0 01 C and is accurate to 1 C The temperature controlled read head is comprised of three distinct shells of material e The outer aluminum detector ring which contains the photodiode detectors e A layer composed of two insulating materials that keep the flow cell at a stable temperature while at the same time keeping the photodiodes as close to ambient temperature as possible e The innermost shell is the aluminum flow cell cavity The heater cartridges are located inside the read head Directly underneath the flow cell is a platinum temperature sensor Insulated read head cover Insulating layers inside
44. assed The optional recycle valve can be controlled by either the HELEOS ViscoStar or Optilab rEX but is always plumbed after the last instrument in the chain Figure 2 1 The HELEOS in line with a chromatography system 2 6 MWH Rev A Connecting Auxiliary Devices 2 3 Connecting Auxiliary Devices You can connect the HELEOS to various other devices using the connectors on the back panel Three cables are provided for such connections These cables have a HELEOS connector on one end and four wires on the other end Because devices have a variety of connector types you will need to attach these wires to the connector used by your devices The auxiliary device connectors on the back of the HELEOS are e AUX 1 AUX 2 AUX 3 and AUX 4 You can connect the HELEOS to up to four external detectors These are usually concentration sensitive detectors AUX 1 and AUX 2 are in one connector and AUX 3 and AUX 4 are wired in a second connector e AUTO INJECT You can use this connector to sense an injection from an auto injector This signal is then monitored by the ASTRA V software e 90 DET You can use this connector to send the 90 output signal to your existing data collection system or to a chart recorder e VAPOR INTERLOCK You can use this connector to shut down any external device such as a pump when the instrument detects either a vapor or liquid leak 2 3 1 Attaching Auxiliary Device Connectors Unlike the DAWN EOS the
45. ating cover onto the read head by pressing it 1n place and rotating it until it engages the shoulder bolts 9 Replace the instrument cover bib with the batch cover bib Batch cap M3x12 Batch a O spacer M3x20 Scintillation vial Ci Ball plungers i Batch manifold 4 M3x12 nm s bolts Batch spacer plate Ambient only Figure 5 11 Flow to batch conversion kit exploded To replace the flow cell reverse the previous process 5 20 MWH Rev A Flow to Batch Conversion Note When making measurements with scintillation vials take great care to keep the outside of the vials clean and free of fingerprints scratches etc as this can severely distort the measurement We also advise you rotate the vial in the read head to find the position where the laser beam enters the cell with the minimum amount of scattering at the air glass interface The batch cover includes a mechanism to rotate the scintillation vial in place until the signal is minimized Simply press down on the knob on the top of the batch cover and turn to the right while monitoring the results on the Batch page of the Display window The ASTRA V for Windows User s Guide has additional information MWH Rev A 5 21 Chapter 5 HELEOS Maintenance 5 22 MWH Rev A QELS Quasi elastic or dynamic light scattering is an internally installed option the measures time dependent fluctuations in the scattered light signal using a
46. be programmed for delayed activation see Recycle on page 4 14 for programming the delay MWH Rev A Batch Panel 4 4 Batch Panel In the Batch panel you can choose to display raw data or normalized data e Raw Data tThis is the data gathered by the data collection procedure For a light scattering experiment this 1s the detector voltages e Normalized Data For a light scattering experiment this is the detector voltages multiplied by the empirically determined normaliza tion coefficients for the particular solvent being used The Batch panel displays information and helps you set baseline and normalization coefficients while using prepared cuvettes of known sample concentrations Each LS detector has a slightly different sensitivity and views a slightly different illuminated volume LS detectors at low and high angles look along the beam and see a larger illuminated volume while the intermediate LS detectors look across the beam and see a smaller illuminated volume see Figure 4 5 LS Detector 90 es ae Laser bear ee Illuminated Volumes a i Detector 22 5 LS Detector 90 Figure 4 5 LS detector angles In order to compensate for these inherent differences you must set a baseline using a cuvette of pure solvent then you normalize using a normalization standard any sample in the same solvent which is small compared to 10nm Small samples scatter light isotropically the same in all dire
47. both ends of the cleaned window for any parti cles With the loupe look straight through the window from end to end See the Note at the end of this section Window face This is the surface with the anti reflective coating Window face This is the end that protrudes into the flow stream Figure 5 7 Cell window 5 12 MWH Rev A Note MWH Rev A Cleaning the Flow Cell and Windows By examining the flow cell through bore and the windows using a bright light you can with some practice easily find where any residue has accumulated Examine them with a jeweler s loupe while back lighting the glass at a slight angle The area next to the light should be dark to provide good contrast The bright light will illuminate any particles on the glass which when viewed against the dark background will show up clearly If you have a UV light you may shine it on surfaces at a slight angle to make certain types of dust particles especially clothing fibers more visible Since fingerprints on the glass cell circumference will alter the light scattering characteristics of a sample significantly we urge you to use great care when handling the cell Its role is vital in the measurement process and you must be certain to wipe it clean with high quality lens tissue before inserting it again in the cell assembly 5 13 Chapter 5 HELEOS Maintenance 5 3 4 Step 4 Reassembling the Flow Cell As you reassemble the flow cell you wil
48. ccseseeeeseeeeceeseeeaeeeesaeess A 6 To move the HELEOS QELS fiber positioner to a different detector location A 7 Appendix B Ultra High Temperature Option 00 5 B 1 OVEIVICW eciuspacecsonatindwanseasunonahenunesianinesdienaetinue Shales DEEE tn B 2 Heating te Gel niaaa Nericr cer etree nr eaten are torrie ger erere amet Aert Reee ec anenre ts nena en B 3 ADOUL TMS TMETMOCOMIFONGIS sosgsce ew Sect ace E cs darned e elect S B 3 Setting the Operating Temperature ccccscecccccsseeeeeeeeeeeeceeeseeeeeeeeeeeseegseeesseaseees B 3 Heated HINES sctacket ae altos canst bead aaa a a B 4 Operating Precautions sicsctisennticdectelannecteuthcoaenl idea te cseveeaieloaeceeeliet oeuakGenseate B 4 Removing the Cell ASSCMDINY scciciicssavtcdeessugdcetawenscinaheaicadeviees a ied asides ane B 5 Using the HELEOS with an OVEN ou ceecccccceeeeceeceeeeeeeseaeeeeeeeaeeeeeeseaeeeeesssaaeeees B 7 General Setup Procedure ceccccseescccceesececceeecececeuseceeseaceeeeeueeeessuausesssageeessaneeees B 7 Connecting the Heated Lines and Heating the HELEOS cccceecceseeeeeeeeeeees B 8 Operating the HELEOS with an OVEN ccccccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeaeaeeeeeessaeses B 9 Potential PrODISmS ccctis ses ecet Sec ce ee iets eed aceite Sines 8a Bien caabetateaca seared danceeaett B 9 Disconnecting the HELEOS from an Oven ccccccsseeeeeeeeeeeeeeeeeeeeeeseeeeeseasseeeees B 10 Temperature Control
49. cedures for keeping the flow cell clean described next For instructions on connecting the HELEOS to an HPLC system see the ASTRA V for Windows User s Guide MWH Rev A Flow Cell Maintenance 5 2 Flow Cell Maintenance The flow cell structure is critical to the operation of the HELEOS If you follow the guidelines here you may never need to delve deeper into the instrument If the flow cell is not cared for properly you will need to remove it from the read head for cleaning described in the next section This is a procedure that while not complicated can be circumvented if you can successfully clear contaminants such as particles from the bore of the flow cell while it is still installed 5 2 1 On line Cleaning To keep the flow cell free of contaminants we recommend regular maintenance as described here At All Times e Use solvents including water that are HPLC grade and filtered through a 0 02 um filter e Ifthe instrument is connected to a chromatography system keep pure filtered solvent pumping continuously through the cell e Ifthe instrument is in stand alone mode batch setup store the flow cell filled with filtered solvent e When you do not plan to use the HELEOS for some time check it about once a month for solvent in the cell Add more filtered solvent as needed Before and After Completing Experiments e With the flow cell still in place disconnect the HELEOS from your HPLC system Inject
50. ceeeeseeeeeeceeeseeeeessaseeeeeessaas 3 18 MWH Rev A 3 1 Chapter 3 HELEOS Components 3 1 Front Panel View 3 2 IN OUT Fluid LCD Display Keypad Connectors The front panel see Figure 3 1 contains the main power switch On Off provides fluid connections for the HELEOS along with the display window and display controls for operating the instrument and monitoring data On Off switch Figure 3 1 HELEOS Front Panel LCD Display The LCD display provides a full color high resolution user interface to the HELEOS It allows you to monitor control and configure the instrument Chapter 4 Using the Display Window describes all of the tabs available on the LCD display and their functions Keypad The keypad allows you to control the LCD display Navigating the Display Panels on page 4 2 describes how to use the keypad to navigate through the LCD display panels IN OUT Fluid Connectors Fluid comes into the HELEOS through the IN port and exits through the OUT port If the HELEOS is stacked on top of the Optilab rEX the drain system is designed to cascade so that only a single drain tube needs to be connected at the bottom of the instrument stack MWH Rev A Back Panel View 3 2 Back Panel View The back panel contains the AC power module auxiliary and serial connectors nitrogen purge connector and cooling fan The electronics fuse is also located on the back panel the main power fuses are located in t
51. condense on the read head The DAWN HELEOS eliminates all of the switches and jumpers of the older DAWN EOS All functions are now controlled by the embedded microprocessor for greatly improved ease of use Additionally the COMET and QELS options are now embedded to reduce the system footprint and reduce lab clutter Lastly the temperature control options are now entirely air cooled eliminating the need for an external water bath The Software Wyatt Technology offers the ASTRA for Windows software package for collecting and analyzing data from the DAWN HELEOS instrument ASTRA V for Windows collects and processes chromatography data from dilute polymer solutions From polymers fractionated by size or molecular weight ASTRA V calculates the molecular weight moments number weight and z average along with the rms radius moments of the molecules in solution From unfractionated polymers ASTRA V displays Zimm Debye or Berry plots 1 3 Chapter 1 Introduction 1 2 About This Manual 1 2 1 1 2 2 The DAWN HELEOS Hardware Manual describes how to set up and use the DAWN HELEOS laser photometer Please see the ASTRA V for Windows User s Guide for details on data analysis Manual Conventions We will refer to the DAWN HELEOS simply as the HELEOS Whenever we point out internal components your orientation is from the front of the instrument The IUPAC Definition Committee specifies the term molar mass for the sum of the at
52. cted See Attaching Auxiliary Device Connectors on page 2 7 Liquid Level Leak Sensor The HELEOS also has a liquid level leak sensor The vapor sensor is not sensitive to aqueous solvents but the liquid level leak sensor is sensitive to both aqueous and organic solvents However approximately 2ml of liquid must leak into the reservoir before the liquid level leak sensor will activate and therefore the liquid level leak sensor is much less sensitive to small leaks than is the vapor sensor Turning Off the Alarm When either the vapor or liquid sensor activates there 1s an audible alarm and the alarm button on the main page and the alarm page turns red When this occurs you can turn off the audible alarm but the red indicator will remain lit Even when the audible alarm is turned off the back panel alarm output will remain active MWH Rev A Printed Circuit Boards To turn off the audible alarm e Display the Alarm panel Tab to the Audio Alarm checkbox and press Enter to uncheck the Audio Alarm box To enable the audible alarm e Press Enter again to check the Audio Alarm box 3 8 Printed Circuit Boards Note 3 8 1 3 8 2 3 8 3 3 8 4 MWH Rev A There are no user adjustable settings inside the HELEOS this section is for reference only The HELEOS has six internal printed circuit boards PCBs e Front panel computer and display board e Flexible detector board cable e KOS instrument controll
53. cted size times the concentration divided by the flow rate If one has a concentrated sample a slow flow rate and a large size one should choose a sample rate of 1 second Otherwise longer sample times should be chosen The integration time selects the time for each measurement The correlation function measurement is averaged for a time equal to the integration time The longer the integration time the more accurate is the result However there are a couple of caveats If the sample is flowing through the cell as in chromatography the integration time cannot be made too long or one will get an average over the changing composition of the sample Also if one sets a long integration time the probability of the measurement being contaminated by dust increases As an aid to setting the integration time intermediate results are displayed in red every one second They get progressively more accurate less noisy as time progresses After the measurement is complete it is plotted in blue and the new intermediates are plotted The slider on the bottom shows the percent complete of the measurement Delay Time The delay time is the horizontal axis of the correlation function graph It is always less than the integration time APD Status The avalanche photodiode APD contains an internal Peltier cooler that cools the active element to provide improved performance When it is first powered on the detector is especially susceptible to da
54. ctions Normalizing in this way allows the instrument to adjust the response of each detector so that they are all on the same scale as the 90 degree detector MWH Rev A 4 7 Chapter 4 Using the Display Window 4 4 1 Setting the Baseline and Normalization Coefficients Note For each sample rotate the cuvette to find a clean spot on the glass When you have found a clean spot the signal will be minimized 1 Press Esc 2 select Raw Data and press Enter 2 Insert a cuvette containing pure solvent 3 Rotate the cuvette until the LS detector signal is minimized 4 Tab to the Set Baseline button and press Enter This allows the instrument to subsequently display only the additional scattering due to the sample 5 Insert a cuvette with a normalization standard for the solvent you are using Rotate the cuvette until the LS detector signal is minimized Tab to the Normalize button and press Enter Note Use the check boxes next to the Normalization Coefficients to turn off uncheck any associated detector in the plot When working with samples that have dust in them the forward angles are often contaminated with extra light Turning them off in the plot makes it easier to view the plot Main Batch QELS Alarm System Raw Data 7 z Set Baseline Normalize Normalization M Auto Scale Set Scale Coefficients 000 000 000 000 000 000 000 000 000 000 000 t a ji ic iz E 2 ce
55. e For these instructions the standard cable is always black H 6 MWH Rev A Components H 1 6 Ethernet to USB adapter This device can be used to connect an Ethernet cable to a USB port on the computer Using this adapter it 1s possible to have the computer connected to a LAN via the computer s Ethernet port and the instruments connected to the computer via USB The Ethernet to USB adapter supplied by Wyatt Technology will look similar to this The first time you connect an Ethernet to USB adapter to your computer you may be prompted to install USB drivers for the device To do so use the CD supplied with the Ethernet to USB adapter and follow the Microsoft Windows instructions p j 9 mi Ja Figure H 6 Standard Ethernet to USB adapter The Ethernet cable is plugged into the port with the yellow arrow and the USB plug green arrow is plugged into a USB port on the computer MWH Rev A H 7 Chapter H Connecting to Network or PC H 1 7 Ethernet switch Ethernet switches are used to connect several Ethernet cables to one resource such as the LAN socket in Figure H 2 The Ethernet switch supplied by Wyatt Technology will look similar to the five port switch shown below Please note that Ethernet cables can be connected to the switch in any order or position Also the switch has an external AC adapter not shown to provide power to the switch Figure H 7 Five port Ethernet switch H 8 MWH
56. e G 7 Appendix H Connecting to Network or PC 2 000008 H 1 COMPONGONO ev RRR RE tie eee neem Te nen H 2 NSTEUMENTCOMMECCHIONS enoe a a aea H 2 LAN CONNGCHON sindro ara a a ar H 3 COMPUTE CONMECHONS agarrar a E a tw seng caliamcceend H 4 CIOSSOVElCADIC ssrin a E H 5 EI NCIC CAI soist dan iaaea aaa O E H 6 Ethemetto USB adapter carea a ddan Ma italia H 7 Ethernet SWIC senen et cnlennbedtaneauato cad han us aiiuiak Souneub saxon ueeetulancs H 8 Connec TO aQ EAN iisen a ta teenie E H 9 One mstraument to EAN sisse iei a a N N a eee ANN H 9 One instrument and computer to LAN ccccccceeecceeeeeeeeeeeeseeeeeeeeeeeeseeeeeeesessaeeses H 10 M ltiplemstumemMms tO LAN s 6 veven weedeat raa aa ra a H 11 Multiple instruments and computer to LAN esassssnsennssssnnsnenrnnennnrrnnnrnreenrnnsrnrernenne H 12 CONMECING va USB assrrssrstsessn i Eea TERE H 13 One instrument to USB via a crossover cable ssssssnseensenreeeserrrrreesrrrrrresrrn renee H 13 One instrument to USB using an Ethernet switch 0 0 0 0 ccccceeccsseeeeeeeeeeeeeeeeeeeaeees H 14 Multiple instruments to USB ccccccsececcseeeceeseeeceueecseuseeesaseessoueeessseeesaseeesseees H 15 Connecting via Ethernet when not on a LAN aessssssssssssssssrssssrrrrnrrrrresrrrreesrnree H 16 One instrument to computer not on LAN using crossover Cable c cccceeeee H 16 One instrument to computer not on LAN using an Ethernet switch
57. e doing when the problem occurred e How you tried to solve the problem Wyatt Technology Corporation Technical Support Phone Number 805 681 9009 1 7 Chapter 1 Introduction 1 4 Where to Go from Here Continue now to Chapter 2 to check out your shipment and make some necessary initial checks and adjustments If you have purchased special options such as the Peltier Heated Cooled version of the HELEOS you will also want to read the appropriate appendix for a description and instructions for setting up and working with the option 1 8 MWH Rev A Installing the DAWN HELEOS This chapter helps you get the HELEOS unpacked tested and connected You will also make some first time adjustments CONTENTS PAGE 2 1 Unpacking the INStruMeNt cccccceccseceeseeeeeseeeeeseeeeesseeeeseeeeenees 2 2 2 2 Installing the Instrument c ccc cece sseeeeceeeeceeeeeceeseeseeeeeseneeeseees 2 3 2 3 Connecting Auxiliary Devices cccccccccccceesseeeeeeeeesaeeeeeeeessaaeeeeess 2 7 2 3 1 Attaching Auxiliary Device Connectors cceeeeeeeeeeeeeteeeeees 2 7 MWH Rev A 2 1 Chapter 2 Installing the DAWN HELEOS 2 1 Unpacking the Instrument Please read the shipping parts list packing slip included with your instrument shipment and check that everything arrived in good condition 1 Carefully examine the shipping container If it is damaged or shows signs of mishandling CONTACT THE SHIPPING CARRIER I
58. e flow cell tube on the inlet side has Cell 1 6mm OD tubing on one assembly side and 0 8mm OD tubing on the other Always disconnect the larger of the two tubes since it is more mechanically robust and less likely to be damaged from over tensioning when Figure B 3 Heated Lines Flow Cell Assembly reinstalled B 5 Appendix B Ultra High Temperature Option Disconnect here A Figure B 4 In Line Unions Note There should be two short lengths of 1 6mm OD tubing plumbed into the cell assembly ports when this procedure 1s completed 6 Use the 2 5 mm Ball driver to remove the two M3x10 screws then lift the cell assembly up and out of the read head Lift the assembly out using the connecting tubing The cell assembly is the same as described in Chapter 3 under Flow Cell Design Cell disassembly and cleaning is described in Chapter 5 under Cleaning the Flow Cell and Windows Note Whenever you clean the flow cell you should replace the O rings They become brittle when heated B 6 MWH Rev A Using the HELEOS with an Oven B 4 Using the HELEOS with an Oven B 4 1 MWH Rev A It typically takes two days to install a heated HELEOS in an oven such as the Waters 150C Waters GPC2000 or the Polymer Labs 210 The internal plumbing of the oven should be done beforehand with the HELEOS heated lines connected between the columns and the RI detector This list summarizes the installation process ass
59. e of the Wyatt COMET flow cell cleaning system and the QELS dynamic light scattering option The flexibility versatility and built in redundancy of the DAWN instruments make them exceptional measuring systems Read on to learn more about the DAWN HELEOS CONTENTS PAGE g Nes 2719 ae ee Se i ee eee me 1 2 1 1 1 VRS IS WEA CIA iriiria aaa 1 2 1 1 2 The Software co cic cnn cose nrerioe noes dias anticnaraaenrssestinntnnacsteenntantannnsisenieces 1 3 1 2 About This Manual ccc ccccccccescesceseeeeceseeeeeeeeueeeeeeeaeuaneeaneaeeags 1 4 1 2 1 Manual Conventions ccccccccccccecceeceeeeeseeseeeeeeeueeeeeeeeaeeaaeees 1 4 1 2 2 How the Manual Is Organized ccccccecseeeeeeeeeceeeeeeeeeeaeeees 1 4 1 3 How to Contact Wyatt Technology Corporation cccccceeeeeeeees 1 6 1 3 1 Corporate Headquarters ccccccccccccsssseeeeeeeseeeeeeeeesaseeeeeeeeseaas 1 6 1 3 2 Sales DepartMent ccccccccecceeccecceseeeeeeeeeseseeeeeeesseaeaeeeessaaeaees 1 6 Lo lechnical SUDDOM wusecge iececssiacanscciazsnatesesssnadoessGaestacaieedgsadaxccsnasies 1 6 1 4 Where to Go from Here cece ceccceceeeeseueseueseueeseeueeeueeueeeneeenauaes 1 8 MWH Rev A 1 7 Chapter 1 Introduction 1 1 Overview 1 2 1 1 1 The Instrument The DAWN HELEOS combines the proven features of photometers nephelometers turbidimeters and goniometers into a single light scattering instrument It can be used as a detector fo
60. eeeeeeseeeeeeeessaaseeeeessuaaseeeeess 3 16 Turning Off the Alarm ccccccccccseeeeeeeseeeeeeseeeeeeeeeeeeeeeeseeseeeseeeeeseeaeseesseaeeesesaeseeeeas 3 16 MWH Rev A Contents iii Contents Paned Circuit BO ANOS eres ee scet aloes sees dered N added ouetelenmusnase ss 3 17 Front Panel Computer and Display Board ccccsccecccseeeeceeeecseeeeesaeeeeseaeeesaeeees 3 17 FIGXIDIG WETECLOF BOAO aaxewtadtecriatearetyisat atest tara a al ual et etaa vas Manton ud aan 3 17 EOS Instrument Controller Board sosanna oaie a a a 3 17 UHV DO ANG aen a a shal yates 3 17 vVapor Sensor Doar essesi E a 3 18 Nitrogen Sensor Doar sein a nsivnnadene teats 3 18 Chapter 4 Using the Display WINdOW s cseseeeeeeeees 4 1 Navigating the Display Panels ccccccccccsecccescecseececeeeeseeeeeseueessaesesseeeeseeeess 4 2 Front panel button description cece ccccseeeeeceeeeeceecaeeceeeeeaeeeeeeaeeeeeesseaeeeesaeeeeeaseeees 4 2 Warming Lights and Alarms scrinio aein e a Da 4 3 Haze aaee a a S 4 3 A di Alam anena a a an mE Re ene 4 3 Man Panel srnocsiion a E 4 4 Selecting Display Settings for the X Y AXeS ccccsecececeseeeeeeeeeeeeeeeaeeeeeeseeeeesaaaeeees 4 4 Adjusting the Display Range nnnnoennnneenneonennnosrnnensnrnnnnnrnrsnnrrrnrnrnrsrnrrrssnnresrnrrennne 4 5 Setting Cell Temperature cccccccccescccccssececcceeeeeeeceeceeseeeueeeeeeseeeseeeueeeesegeeeessaeeess 4 6 LISE aurea a a a
61. er board EIC e Utility board on the subchassis e Vapor sensor board e Nitrogen sensor board Front Panel Computer and Display Board This contains a 8300MhZ Pentium class computer that is used to control the instrument It collects the data from the EIC and QELS and runs the front panel graphical user interface Flexible Detector Board The flexible detector board is the cable that wraps around the read head and connects to all of the light scattering detectors This PCB sends signals from the detectors to the EOS instrument controller for amplification and digitizing EOS Instrument Controller Board The EOS instrument controller EIC board contains 48Mhz microprocessor and 29 channels of amplifiers and 24 bit analog to digital converters It also contains provisions for temperature measurement and control as well as the laser driver feedback circuitry Utility board The utility board is responsible for DC power distribution and control of the Peltier temperature control system It also contains circuitry to process the keypad and send the resulting signals to the front panel computer 3 17 Chapter 3 HELEOS Components 3 18 3 8 5 3 8 6 Vapor sensor board The Vapor sensor board drives the vapor sensor transducer and the liquid leak sensor It has two LEDs which indicate the status of the two detectors Nitrogen sensor board The Nitrogen sensor board measures the pressure of the N2 port This is used t
62. ersion is triggered if the read head ever exceeds 220 C Vapor Vapor sensor detected a leak Leak Liquid sensor detected a leak Recycle Open Back panel recycle valve input requested the recycle valve be actuated External Back panel external alarm input is activated Comet failed to actuate This can occur when the cable is discon nected or if the transducer needs replacement N2 pressure not in Ambient version is triggered when the tempera ture is set to less than 20 C but the nitrogen pressure is less than 20psi In this case the alarm activates and resets the system tempera ture to 20 5 C This prevents condensation from damaging the optics if the nitrogen connection is not made or if the tank runs empty Flow Cell temperature lock shows if the flow cell temperature is locked MWH Rev A System Panel 4 7 System Panel The System panel contains additional options for some of the selections on the Main panel Main Batch GELS Alarm System Comm Flow Cell Temperature 25 00 C Heated Line Temperature MiA Setto 25 00 C Syne Iw Setto 25 00 C Laser QELS Power COMET Laser Dither GA AlarnRelay OFF Recycle Recycle COMET start atter minutes OFF start atter minutes OFF Run continuoushy ICE Run for minutes Cance Analog Output En lish Time Constant s 1 01 J LS Detector 11 w ai Restart ISI Serial Number WTC HELEOQS 10 EE inv Software Version 2 0 2 0 Load Factory De
63. esceasdteness 4 16 MWH Rev A 4 1 Chapter 4 Using the Display Window 4 1 Navigating the Display Panels You navigate through the Display Panels using the buttons to the right of the Display Window Panel tabs Temperature Instrument Lock Alarm Alarm Status Figure 4 1 Main display panel 4 1 1 Front panel button description Esc left and right arrows navigate through the Panel tabs Esc and the number of the Panel tab 1 through 6 selects the first field in that Panel Tab cycles through various fields in the current Panel Esc Tab selects the first field in the current Panel Enter displays the options of the selected field with the current option selected Use the arrow keys to change the option and then Enter to select If the field is a check box Enter toggles the option Tip If you miss a field press Esc and restart Tabbing through the fields 4 2 MWH Rev A Warning Lights and Alarms 4 2 Warning Lights and Alarms 4 2 1 4 2 2 Note MWH Rev A Color Meaning Yellow Not ready Green Ready Red Hazard Hazards The HELEOS will sound an audible alarm when a potential hazard is detected Hazards include e vapor or liquid leak is detected e an over temperature condition is detected e external alarm input is activated signal from associated equipment Audio Alarm Even when the audible alarm is turned off the back panel alarm output will remain active To turn off the audible alar
64. ext measurement MWH Rev A Aligning the Optical Fiber A 2 Aligning the Optical Fiber Before any measurements can be taken the optical fiber must be aligned to the laser beam The alignment is set at the factory but may change during shipping 1 Turn off the system power to the HELEOS 2 Remove the top cover of the HELEOS instrument Make sure that the flow cell is filled and has no bubbles in it Turn on the unit Navigate to the QELS panel of the GUI Set the collection interval to 1 second By default the QELS fiber is installed in detector 12 5 Using a 2 0 mm hex driver rotate the adjustment screw on the top of the QELS fiber mount see figure Figure A 3 This drives the fiber up and down so that its field of view sweeps past the laser beam The fiber is positioned correctly when the count rate is maximized This is accomplished by rotating the fiber counter clockwise until the fiber is at the top of the mount Stop when the adjuster screw is about 3mm above the top of the mount If turned too far the adjuster screw will come out If this happens simply screw it back into place taking care not to cross the threads Temperature Probe Fiber Optic Cable HELEOS QELS C aia m a Figure A 3 Adjusting the fiber positioner for the HELEOS QELS when connected to the DynaPro only 6 Once the screw has been raised to its maximum position slowly rotate the hex driver clockwise while monitoring t
65. fast photon counter QELS measurements can determine the hydrodynamic radius of macromolecules or particles CONTENTS Pat OEEO FP ee oa A ase A 2 PTW OU TA A atte eae oteceecaee tsnsencacatesecnngs E A 2 WMS res tee src ane sar AAEE E E santero ieee ene A 2 A 1 2 Correlation Function cccccccccecceesececceeeceeeseseceeseeeeeeeeeseeeeeas A 2 Paleo miegraton TIME esisin S A 3 OVA WMG E T E E E E A EE A 3 PA Ge ARO UU r A E A 3 Pel TOW CN rre E A RE A 4 A 2 Aligning the Optical Fiber ccccccccccssssseceeseeeeeeeeeeeseeeeeeeeeeeeaas A 5 A 3 Removing and Installing the Optical Fiber Receiver 008 A 6 A 3 1 To remove and reinstall the optical fiber receiver 000 A 6 A 3 2 To move the HELEOS QELS fiber positioner to a different detector location A 7 MWH Rev A A 1 Chapter A Using QELS A 1 QELS Panel A 1 1 This section describes the QELS Panel in the HELEOS display window Count Rate The Count Rate contains the raw signals for each of the light scattering detectors and the photon count rate for the QELS detector Time The Time field sets the time range of the X axis Main Batch GELS Alarm System CountRate l a l n M Auto Scale Change axis WYATT QELS Tie 1 0x10e5T o 8 0x10e4 6 0x10e4 E iz ce i i 4 0x10e4 2 0x10e4 10 06 AM 10 10 AM 10 12 AM 10 14 AM 10 16 AM Integration Time sec 7 E APD Status OK Power Figure A 1
66. faut a0 0y Set Time IE i aste ti Figure 4 9 System 4 7 1 Read Head Temperature and Heated Line Temperature The read head temperature is displayed for all instrument configurations However the ambient instrument configuration does not actively control the temperature and has no provisions for attaching a heated transfer line Read Head Temperature and Heated Line Temperature not in Ambient version displays the measured temperature of the read head for all instrument configurations and the heated lines However the ambient instrument does not actively control the temperature and has no provisions for attaching a heated transfer line If you have these options you can set these temperatures by selecting the Set to field typing the desired value then Enter The thermocontrollers are programmed to change the temperature at a rate of 1 C per minute to ensure that the flow cell glass does not crack due to thermal stresses For example if you wish to operate your system at 150 C and your system is initially at 25 C it will take about two hours for the temperature to reach 150 C MWH Rev A 4 13 Chapter 4 Using the Display Window 4 14 4 7 2 4 7 3 4 7 4 4 7 5 4 7 6 4 7 7 If you are using the Peltier Heated Cooled model the read head can be cooled or heated but the heated lines can only be heated Using a setpoint below ambient temperature will cool only the read head it won t cool the
67. gen sensor Flexible detector board Laser assembly et he Figure 3 5 The HELEOS uncovered 3 6 MWH Rev A Laser 3 4 Laser Ambient read head assembly Forward laser monitor The 50 mW linearly polarized GaAs gallium arsenide laser provides the light source for the system The laser system provides very high power density at the illuminated sample by means of a narrow beam diameter the 1 e diameter of the Gaussian beam profile is 0 2 mm This small beam diameter also helps reduce the noise contributions of larger particulate contaminants such as dust The laser is positioned so that the incident beam is vertically polarized A beam monitor rear laser monitor is incorporated into the laser assembly The output of this monitor can be displayed on the Main panel in the display window Photodetectors total of 18 Laser monitor detector Laser power Laser mbl connector aser assembly Flow cell cover Flexible detector cable Figure 3 6 Read head and laser assemblies 3 4 1 Laser Beam Warning MWH Rev A It is good laboratory practice with any laser source irrespective of its power to AVOID LOOKING INTO THE BEAM Figure 3 7 shows the warning label affixed to the read head Appendix F gives the laser specifications Chapter 3 HELEOS Components DANGER LASER RADIATION WHEN OPEN AVOID DIRECT EXPOSURE TO BEAM Figure 3 7 Laser beam warning label 3 4 2 Laser Monitors
68. gned both will be automatically updated on the front panel and should no longer read 0 0 0 0 At this point it should be possible to connect to the instrument from the computer Static If you wish to use a static IP address and subnet mask please contact your IT department to obtain a valid address and mask Enter the information into the IP address and subnet mask fields 4 16 MWH Rev A HELEOS Maintenance The HELEOS photometer requires little maintenance When you remove parts for cleaning or convert between flow and batch modes you will find they are easy to access and disassemble This chapter gives guidelines for keeping the instrument clean and in good working order It also has the procedure for converting from the flow cell to Batch mode measurements with scintillation vials CONTENTS PAGE 5 1 General Maintenance cccccccccccseecceeceseeeeceeeceeeaeeseeeseeeeeeeeseaeees 5 2 5 2 Flow Cell Maintenance ccccccescceceesseceeceeeeceseeneecesseeeeeeeseaees 5 3 5 2 1 On line CICANING cccceeeccccceesseeeeeeecceeeeeeeeeseauaeseeeeeseueeeeeeeeaaas 5 3 5 2 2 Particles in the Cell cccccccccccssseceecesseceeeeeeeeeesesseeeteeeeeeeeas 5 5 5 3 Cleaning the Flow Cell and WinNdOWS ccccceeeeeeeessseeeeeeeees 5 6 5 3 1 Step 1 Removing the Flow Cell Assembly ccccceeeeees 5 7 5 3 2 Step 2 Disassembling the Flow Cell cccccceeeeeeeeeeeeeeeees 5 9
69. hannel values with the solvent offsets on the Certificate of Performance If the readings for detectors 1 18 are different from those on the Cer tificate of Performance check your laboratory temperature The dark offsets for the detectors may differ from the Certificate of Performance by as much as 10 mV per C For example if your laboratory tempera ture is 20 C and the QC laboratory temperature was at 28 C your current dark offsets may be 30 mV different If you see a greater differ ence monitor the dark offsets for a few days to see if they remain stable at this voltage If they do not contact Wyatt Technology Techni cal Support The rear and forward laser monitors are set to 5 volts at the factory and should be within 0 1 Vdc Before measuring the dark offsets make sure that the laser is turned off by navigating to the laser button on the main page and pressing Enter to turn it off Before shipment the solvent offsets were measured with toluene and the flow cell was filled with toluene and capped so the solvent offsets you see should be very close to those on the Certificate of Performance More than 200 uV difference between your values and those on the Certificate of Performance may indicate air bubbles in the manifolds in which case you will need to flush the cell with filtered toluene and MWH Rev A MWH Rev A 11 12 13 Installing the Instrument recheck the solvent offsets If your dark off
70. he AC power module and are described below Aux In Auxlin Recycle Alarm Auto Inject 1 amp 2 Analog Out Ethernet MWH Rev A Ayu in Rasya Alarm Asio Inject prz In In 3 amp 4 In In In Recycle Out Alarm Auto Inject Out Out a ter In vile wiv v R Alarm Ato Inject oo Analog Ethemet Out Gut Out Heated Ling es s Pe DAWN HELEOS LASER PHOTOMETER ooo0000000 felete elelelelelelelelelelelelelelelele a a Tinea dont A 900000000 ocooeocoocoo0oo00o0o00000 6 426 704 6 452 672 DES 330 821 oo00000Cc0 eoojoocoocccoo0000000D EUROPEAN PATENT NOS 600000000 eodlooc0GCQ0000000000 M a A smia 000000000 oooeocooco0ooo0o0co00000 s THIS PRODUCT COMPLIES ooo00co0ceo oocpoocooccoooocoocooo hae OJ WITH DHHS RADIATION oO0000000D 0 o0 PERFORMANCE STANDARD OGOOGOOOOG olele slelelelelelelelelelojelelelelele 0 21 CFR SUBCHAPTER J oo000000D eoobococcccccoa00000 X Heated Fuse Power Nitrogen Line Holder Plug Purge Figure 3 2 Back panel 3 3 Chapter 3 HELEOS Components 3 4 3 2 1 Changing a Fuse What you need to change a fuse Tool for prying the AC Power module cover off such as a small bladed screwdriver Fuses from the spares supplied in the accessory kit To replace the fuses do the following 1 2 Disconnect the power cord Open the cover of the AC Power module using a small blade screwdriver or similar tool Replace the burned out fuse
71. he count rate on the computer display As the fiber s field of view passes the beam the MWH Rev A A 5 Chapter A Using QELS count rate should grow reach a maximum and then decay After passing through the peak once again raise the mount by rotating counter clockwise 7 Repeat the above procedure but stop when the count rate reaches a maximum The fiber is then aligned Do not adjust the lock screw on the side of the mount The tension is adjusted in the factory and should not be changed A 3 Removing and Installing the Optical Fiber Receiver The HELEOS optical fiber receiver can be removed and re installed in any detector location By default it is mounted in detector 12 which is a scattering angle of 100 3 degrees for water when using the K5 cell A 3 1 To remove and reinstall the optical fiber receiver 1 Turn off the HELEOS system power 2 Remove the top cover by removing the four M3x6 button head screws in the four corners 3 Unscrew the fiber collet see Figure A 4 The fiber collet grips the optical fiber and holds it in the receiver assembly The optical fiber will have a tendency to turn with the collet as 1t is removed or installed This is normal Take care to rotate the rest of the fiber to prevent it from becoming twisted Adjuster Screw Lock Tension Nut Fiber Collet Figure A 4 Optical fiber receiver and positioner for the HELEOS 4 Once the fiber collet has been unscrewed all the way
72. he instrument must be connected to a computer running the ASTRA V software for laser emission to occur While the DPSS laser is not intensity stabilized it does not suffer from sudden longitudinal mode hopes Therefore we accurately monitor the laser intensity and correct for slow intensity drifts in software The laser specifications are CONTENTS PAGE F 1 Electrical and Optical Specifications cccccceesseeeeeeeeeeeeeeeeeeaes F 2 F 2 Environmental Specifications and Safety Notes cccee F 2 MWH Rev A F 7 Appendix F Laser Specifications F 1 Electrical and Optical Specifications Table F 1 Electrical and optical specifications nts ss Polarization Ratio gt 100 1 gt 100 1 Max Power Stability Typical Optical Noise Typical Operating Voltage Typical Operating Current F 2 Environmental Specifications and Safety Notes Table F 2 Laser Environmental specifications 40 to 85 C 15 to 50 C 10 to 35 C 10 to 60 C Relative 0 95 10 85 10 85 10 85 Humidity Shock 1500 G 0 5 ms 1500 G 0 5ms 1G 11 0ms 1G 11 0 ms Vibration 5 to 500Hz 2 0G 2 0G 0 3 G 0 3 G sinusoidal The lasers used in the HELEOS are Class IIIb lasers However the HELEOS itself is classified as a Class 1 Laser Product according to IEC60825 1 1993 A1 A2 and CFR Title 21 Subchapter J Note these environmental specifications apply to the laser subsystem and not to the instrument as a whole This means tha
73. heir polarizing properties so it is necessary to take into account this attenuation There are two strategies to deal with calibration with the polarization strips Calibrate using toluene with the polarization strips installed Then correct the calibration constant by using the Cabannes factor for toluene at the wavelength of the laser light Calibrate without the polarization strips installed Then make a measurement of the scattered light on the 90 degree detector for a known sample that does not depolarize the scattered light e g polystyrene or any other random coil Install the polarization strips then measure the amount of scattered light on the 90 degree detector using the same sample at the same concentration The ratio of the measurements with the polarization strips installed and absent gives the attenuation factor of the polarization strips Use this factor to correct the calibration constant measured without the strips Either strategy should work Contact Wyatt Technology Corporation if you have further questions MWH Rev A D 3 Appendix D Polarization Option D 4 MWH Rev A Interference Filter Option Interference filters may be used to prevent light of wavelengths other than the laser s to reach the photodiodes This can be useful when the sample fluoresces Without these filters too high a molecular weight is obtained since both scattered light and fluorescence are detected CONTENTS PAGE E 1 Installing
74. his manual Internet You can use the Internet to ask questions and receive answers via e mail as well as visit Wyatt Technology s world wide web site World Wide Web URL http www wyatt com Electronic mail address support wyatt com FAX Please fill in a copy of the problem report form at the end of this chapter then fax it You can fax your questions or comments to us at any time Wyatt Technology Corporation Technical Support Fax 805 681 0123 Mail Please fill in a copy of the problem report form at the end of this chapter then mail it to our corporate headquarters MWH Rev A MWH Rev A How to Contact Wyatt Technology Corporation Telephone You can reach the voice mail for Wyatt Technology Corporation Technical Support at any time To speak to our support personnel directly please call between 8 30 A M and 5 00 P M Pacific Time Monday through Friday When you call you should be at your instrument and have the documentation at hand Please be prepared to provide the following information e HELEOS instrument serial number located on the back panel e Ifthe problem is software related Microsoft Windows version number ASTRA V version number exact wording of any messages that appear on your computer screen The software version number is located on the original distribution diskette s or you can view it by selecting About from the Help menu e The type of computer hardware you are using e What you wer
75. iagnostics H 7 Trouble shooting and diagnostics If you are experiencing instrument connectivity problems please go over these steps If you still cannot connect to your instrument after going over this section please contact Wyatt Technology for assistance or visit www wyatt com for the latest troubleshooting guides H 7 1 Verifying instrument connections Please verify that the instrument is communicating with the computer Open a Windows cmd prompt as shown in Figure H 19 At the command line type ping plus the IP address of the instrument as shown on the instrument front panel see Figure H 18 If the instrument is connected properly the result should be similar to that shown in Figure H 19 Gis ping 1 72 20 1 244 Pinging 172 20 1 244 with 32 bytes of data Reply from 172 20 1 244 hytes 32 time lt ims ITL 128 Reply from 172 246 1 244 bytes 32 time lt ims TTL 128 Reply from 172 26 1 244 bytes 32 time lt ims TTL 128 Reply from 172 26 1 244 bytes 32 time lt ims TTL 128 Ping statistics for 172 20 1 244 Packets Sent 4 Received 4 Lost Bz loss Approximate round trip times in milli seconds Minimum Gms Maximum ms Average ms Figure H 19 Using ping to verity the instrument connection If the instrument is not connected properly the result should be similar to that shown in Figure H 20 Co gt ping 172 26 1 243 1 72 2H 1 243 with 32 bytes of data timed out timed out timed out
76. ing the Cell earlier 9 Remove the dust cap from the HEATED LINE connector on the side panel of the HELEOS Be careful not to touch the electrical contact 10 Connect the electrical connector on the heated line to the HEATED LINE connector 11 Turn the HELEOS on and adjust the read head temperature setpoint This will increase the temperature of both the read head and the heated lines by 1 C per minute Check for leaks around the connections every 30 minutes or so Operating the HELEOS with an Oven Always increase the flow rate slowly 0 1 mL min increases every 1 10 minutes initially slower Watch the baseline of detector 11 in ASTRA V for changes whenever you increase the flow rate Check for leaks at all connections You will need to temporarily remove the insulated read head cover to do this If everything is working well the baseline noise on the HELEOS detectors with booster board jumpers removed should be random and less than 20 uV If the baselines have regular oscillations check the pump You may also want to try further insulating the lines next to the read head To calculate the inter detector delay volume and to normalize the HELEOS follow the instructions in the ASTRA V for Windows User s Guide The First Chromatography Run but use a 400 000 Dalton narrow polystyrene standard and a 32 000 Dalton polyethylene standard respectively Potential Problems Excessive baseline noise could have severa
77. ins an internal solenoid valve that switches between waste and recycle When this connector is actuated via the System tab or the Recycle In input the connector supplies current to drive a 12 V solenoid valve The valve is actuated MWH Rev A 2 9 Chapter 2 Installing the DAWN HELEOS with 12 V up to 1 Amp depending upon resistance of the solenoid held for 0 1 second and then dropped down with 12 V across an internal 51 Ohm resistor Ethernet Ethernet connection for connecting the instrument to an Ethernet network This connector is a standard RJ 45 wiring for a 10Base T 100Base TX connection 2 10 MWH Rev A HELEOS Components This chapter gives you a guided tour of the HELEOS components starting with the front panel and ending with the printed circuit boards If you have just installed the HELEOS read this chapter to become familiar with the various instrument parts and their functions CONTENTS PAGE Sl Front Panel VIEW sccstericecassicencdse dex nneneadionzcaueecmadcaenteeneserbentaettoxencaduadee 3 2 32 Back Fanell VIC W cnca A NATA 3 3 3 2 1 Changing a FUSE ix ccceusuvnenmrnsieancuiatseunoccencitnamecamaanmnccrticeeeacadores 3 4 TOI CO a E E E 3 5 3 3 1 Removing the COVEDS ccccccccccssseeeseeeeeeeecesaaaeeseeeeeeeeeesaeasaeses 3 6 DA ESO a AAE AA 3 7 3 4 1 Laser Beam WAarning cccssscecsseceeseeseeceeeesseeeeeseeeeeseeeeenseeeess 3 7 3 42 Laser VION OM 6 oeieo a enced aE aaa 3 8 3 5 Read Head a
78. installed If your instrument is configured for temperatures below 80 C and you decide to operate at temperatures above 80 C you must change the O ring configuration Failure to do so may cause the flow cell glass to crack 3 6 4 Refractive Index Differences Liquid vs Glass The difference in refractive index between the solvent and the surrounding glass cell results in some of the most important features of the flow cell design As long as the refractive index of the solvent is less than that of the cell glass it will be possible to obtain measurements of light scattered at relatively small angles with minimized background contributions Figure 3 12 shows a detail of the liquid glass interface and rays scattering from the laser illuminated sample s ATARnIAnR P OTO D Wy a f z g J y f yy y Jo JS Sg J J ff WA a a va S A gt 4 SJZ J l K XI Z YX Sy P S XX ww i gf NN 4 K YV NON 3 no F N i NY Pa N Jf LN ff Z Sy SS ye F ff N a ae S J a d Z vA yee oN SN GOG hws y if N N See toy ee deren B A N 5 Z p PN 9 y H SLA T E f be hee he S x 2 A NI G ra lt lt gt S m ae S A Z Y Pii LASI a ia X Beam Solvent m Fa Figure 3 12 Flow cell refractions Applying Snell s Law the refraction of a ray scattering at angle 0 may be determined from 1 Niguid sin 2 2 0 N lass sin 7 2 0 3 14 MWH Re
79. inum flow cell cavity Mounted directly underneath the cell cavity between the read head and the circuit board is a Peltier heat pump e The temperatures of the cell and optional heated lines are controlled by the front panel computer to a stability of 0 01 C and the accuracy is 1 C C 2 MWH Rev A Recommended Procedures C 2 Recommended Procedures MWH Rev A Be sure all chosen operating temperatures are between the freezing point and boiling point of the solvent you are using Make sure the solvent is close to the same temperature as the flow cell The heated cooled instrument has an integrated heat exchanger to bring the sample fluid to the same temperature as the flow cell However the closer the fluid is to the cell temperature the more stable the results will be Always have the insulating cover plate locked in place before heating or cooling the flow cell Check for leaks each time the HELEOS has been heated above 80 C Replace the cell O rings whenever the instrument is brought down from an elevated temperature They conform to the geometry of the cell and when brought down from an elevated temperature may not seal reliably The flow cell is initially configured for use at the temperature you indicate you will be using when you purchase the instrument If you decide to operate at a different temperature you may need to reconfig ure the flow cell O rings If your instrument is configured to operate at or
80. ions here Figure 5 4 Flow cell tubing and unions 6 Use the 2 5 mm Ball driver to remove the two M3 screws then lift the cell assembly up and out of the read head using the tubing 5 8 MWH Rev A Cleaning the Flow Cell and Windows IMPORTANT DO NOT PRY THE CELL OUT WITH A SCREW DRIVER OR ANY OTHER TOOL 7 Remove the short stainless steel tubing from the manifolds before proceeding with the disassembly and cleaning The inlet tube has white insulation and an interior diameter of 0 005 The outlet tube has blue insulation and an interior diameter of 0 010 5 3 2 Step 2 Disassembling the Flow Cell MWH Rev A 2 Use the Aperture Installation The different parts that make up the flow cell assembly are shown in Figure 5 5 1 Separate the stainless steel manifolds from the flow cell a Use the 1 5 mm Ball driver to unscrew the two M2 screws holding the bottom cell retainer in place Remove the bottom cell retainer taking care not to lose the two tiny screws and the bottom retainer O ring and cord b Use the 2 5 mm Ball driver to remove the M3 screws that connect the two manifolds c Gently pull apart the manifolds taking care not to drop the glass cell or touch its curved optical surfaces d Place everything on your paper covered work surface taking care not to lose the O rings sealing the manifolds to the cell If the HELEOS is configured for use below 80 C there is a backing ring outside each 6 mm flow ce
81. l clean the washers and O rings Note Assemble the flow cell in a laminar flow hood if there is one available 1 Carefully replace the windows with their O rings washers and retainers so that the windows are not chipped or over tightened See Figure 5 9 a Holding the window O ring with the tweezers put a drop of alcohol on it then dry with a burst of pressurized air Check for particles with the loupe b Insert the O ring into the manifold Lift the window with the tweezers Pick up the window near its back edge as shown in Figure 5 8 Figure 5 8 Holding the cell window for reinsertion into the manifold d Holding the manifold and window square with one another gently push the window into the O ring e Let go of the window with the tweezers pivoting them before you lift them out of the manifold The fit is tight enough that you could dislodge the window if you were to just lift the tweezers straight up f Inspect the tip of the Aperture Installation tool WTC 119033 with the loupe for any particles and if necessary clean with an alcohol moistened lens tissue before proceeding g Clean the window retainer with alcohol and pressurized air then place it in the manifold and tighten with the Aperture Installation tool h Inspect the window mount with the loupe If any particles appear on the window you need to remove it and its seals and clean again i Repeat steps la through 11 f
82. l sources Particulates and or air bubbles in the solvent Use only degassed high purity HPLC solvents Always filter your solvent using a 0 2 um filter or smaller With TCB 0 2 um is the smallest practical filter size With other solvents it may be possible to use smaller pore filters An in line filter after the pump but before the injector may help Appendix B Ultra High Temperature Option B 10 B 4 5 Particulates from the columns With time several days under operating conditions the noise should decrease 1f it does not choose another type of column Take care not to change temperature or pressure too rapidly Pump not operating properly Ensure that the pump is operating properly If spikes corresponding to the pump strokes are observed in the baseline the pump should be rebuilt with new check valves and new seals Operating the pump with sufficient back pressure 1s important to achieve this always work with at least two columns in line or place a restrictor immediately after the pump Also we strongly recommend a pulse dampener after the pump Problem with temperature regulation Check that the temperature output on the oven is calibrated within 1 C Heating and cooling effects between the two instruments could ruin the baseline stability You may check the reading from the oven by placing a temperature probe under the top lid Make sure the probe is not in contact with any metal objects within the oven If noise s
83. le Make sure to keep it vertical as the positioner clamp is tightened The clamp should hold the positioner firmly in place Install the fiber collet and fiber and tension until firm Do not over tighten When installed tug gently If the fiber is properly seated it will not slide out Align the fiber as described above Reinstall the MALS photodetector into the position vacated by the QELS detector First remove the small O ring from around the photodetector Moisten it slightly with some water and place it in the detector hole Press the detector into the O ring MWH Rev A Ultra High Temperature Option The Ultra High Temperature DAWN option has some differences from the ambient HELEOS This appendix describes those differences and supplies instructions for making adjustments and operating the Ultra High Temperature HELEOS CONTENTS PAGE BT VEVI W seesi E E E EE B 2 B2 Meaig ihe CON acs ccectiscninsecohcucenasactiecenaecnselaseanenseecunauctisteriandetadeccoaee B 3 B 2 1 About the Thermocontrollers ccccccceeeeeseeeeeeeeeeeeeeeeeeeenaees B 3 B 2 2 Setting the Operating Temperature ccccccsseeeeeeeeeeeeeeeeees B 3 B 2 3 Heated Lines cccccccccccssseceecceeeeeeseeeeceeeeeeceeseeaeeessaeeeeessaaseees B 4 B 2 4 Operating PreCautions cccccssssssceecceessseeeeceeeesseeeeeseanssseeess B 4 B 3 Removing the Cell ASSEMDI cccccccsseeseeeeeeeeeseeeeeeesaeeeeeeeeeaas B 5 B 4
84. led Flow to Batch Conversion cccccsccecsseeeseeeeeeeeees B 12 Appendix C Peltier Heated Cooled Option ccsssseseeees C 1 OV Cl VICW selaiiansrcasensasnatderancbaeniauesatnanersonsetun noauiasveugid dois oanse en eeesay eee C 2 Recommended Procedure Saaressa ar C 3 Appendix D Polarization Option ccccsssesseseeseneeeeneeeeneenes D 1 WISTAMATION eaaa E a e D 2 Normalization and Calibration with Polarization Strips Installed D 3 Normalization 234 teh cnseccct aaa ni so heanemiSscet state us easacn case a a a i D 3 ANNO AO NN cruinn e e2 tS sind tessa ants ieee alee Dap N D 3 Appendix E Interference Filter Option ccsssseeseseeeeeees E 1 installing IMTEHErONGe Fillers sai Scott oe hoa aig a bee E 2 MWH Rev A Contents v Contents Appendix F Laser Specifications ccssccsssssssseeseeeeeeeeeees F 1 Electrical and Optical Specifications cceccccccsseeeceeeeeeeeeseeeeeeeseeeeeeeseeeeeeeees F 2 Environmental Specifications and Safety Notes ccccceceeeeeeeeeeeeeeeeeeeeeeaes F 2 Appendix G Flow Cell Properties ccccsscsesseseseeeeesenees G 1 AST MAP TODS CS eee EE acansanmererncs Oconee acneraeedlntacase G 2 Bic ige el Youll e olctcgeeemme me tem reneme tines cme nr tcieie ter freer a TN G 3 GIeMiCal PrODCINCS oerni ar N IS G 4 DP STIMUOM Ol LOTS serso a aa G 5 Scattering ANgIES inacternce ose ivacaiwhe tahun tastwnadadelnebie a
85. lines Sync Click the Sync button to synchronize the Read Head Temperature and Heated Line Temperature This means that changing the read head temperature automatically changes the heated line temperature to match When Sync is unchecked you can set the cell and heated line temperatures independently Recycle A timer setting on the Systems Panel lets you program it for delayed activation Enter the time delay in minutes in the Start After field Comet You can set the start time and run time for the Comet option This is useful when setting the COMET to run at the end of the day Use the Start after field to provide delayed activation so the COMET activates after the last data run completes Alternatively you can schedule the COMET activation as part of an ASTRA V sample set see the ASTRA V for Windows User s Guide for more details The Run for field sets the time for which the COMET runs once activated Typically you set the COMET to run for an hour or two after the last data run completes Analog Output The analog output on the back panel can be used to transmit any data channel to a third party instrument Select the data channel from the drop down menu and type in the scale settings The output is always 10 to 10 V The two fields show which analog values will be mapped to these values If the upper field is set to 1 and the lower field is set to 1 then the output will be 10x the data channel signal Time Constant
86. ll O ring If the HELEOS is config ured for use at or above 80 C there is a 9 mm flow cell O ring but no backing ring on each side tool WTC 119033 shown at the right to remove one window retainer at a time Figure 5 5 9 illustrates the window mount and how it 1s housed in the manifold 3 Lightly tap the assembly ONCE against a flat clean surface The cell window and O ring should fall out if the cell is dry If the window does not fall out easily you could carefully apply a very mild burst of pressurized air to dislodge it or you could try gently pushing it out from the opposite side with a small piece of Teflon tubing If necessary put some filtered alcohol in all the manifold openings and soak overnight 4 Repeat Step 2 and Step 3 for the other window Chapter 5 HELEOS Maintenance A J Figure 5 5 Flow cell assembly exploded view Table 5 1 Flow cell assembly parts list Item P N Description 200694 Manifold out 3 P6504 2006 Flow cell O ring 2 P6504 2009 if HELEOS is configured for use above 80 C 4 200609 Backing ring 2 not used if HELEOS is configured for use above 80 C oe 212095 Flow cell 200690 Manifold in ST P6504 2004 Window O ring 2 116007 Flow cell window 2 9 212078 Window retainer 2 5 10 MWH Rev A Item P N Cleaning the Flow Cell and Windows Description 10 S5002 3030 M3 screw 211048 Bottom flow cell retainer S6501
87. long term time dependent studies bioassays depolarization and aggregation studies As well if you believe the sample may contaminate the flow cell it can be measured in a disposable vial Because sample measurements can be repeated easily and rapidly the batch method is often the fastest for determining molecular weight and radius of gyration from a static sample What you need for flow to batch conversion e 2 5 mm Ball driver e Two Crescent wrenches e Ambient Batch Conversion Kit To convert from flow to batch operation do the following 1 Remove the bib from the top cover of the instrument Batch Cover Bib MWH Rev A 5 19 Chapter 5 HELEOS Maintenance 2 Remove the read head cover and the flow cell assembly from the instrument If you need instructions follow Step 1 of flow cell cleaning described in Cleaning the Flow Cell and Windows on page 5 6 in this chapter 3 Insert the batch spacer plate into the bottom of the read head cavity and secure it with the two M38x12 screws Insert the batch manifold and secure it with the two M38x12 screws Insert the batch vial spacer and secure it with the two M3x12 screws Put a sample scintillation vial in the batch manifold cavity Note The vial should fit snugly in its mount but still be able to rotate If it does not slightly loosen or tighten the ball plungers as required Install 4 shoulder bolts into the batch spacer plate 8 Install the insul
88. m e Display the Alarm panel Tab to the Audio Alarm checkbox and press Enter to uncheck the Audio Alarm box To enable the audible alarm e Press Enter again to check the Audio Alarm box 4 3 Chapter 4 Using the Display Window 4 3 Main Panel 4 4 The Main panel contains the most commonly used HELEOS functions The display shows graphical representations of two of the data streams collected by the instrument One data stream is displayed in red on the right axis and other is displayed in blue on the left axis 4 3 1 Selecting Display Settings for the X Y Axes You can select the data channel you want displayed in each axis Left y axis selector x axis selector Right y axis selector Batch QELS Alarm System Comm Read Head Temperature Heated Line Temperature Ne Pressure Laser Current Rear Laser Monitor Forward Laser Monitor LS Detector 1 LS Detector Z LS Detector 3 LS Detector 4 LS Detector 4 LS Detector 6 LS Detector LS Detector amp LS Detector 4 LS Detector 10 LS Detector 11 LS Detector 12 LS Detector 13 ro a g pl 10 40 AM 10 42 AM 10 44 AM LS Detector 14 Ok oN GFF LS Detector 15 Laser Comet Recycle Figure 4 2 Main panel Left and Right Y axis Selectors e Tab to the field and press Enter to display the data channels e Use the up and down arrow keys to scroll through the parameters Press Enter to select The left Y axis data channel di
89. m panel 4 13 T technical support 1 6 temperature effect on dark offsets 2 4 temperature control 1 2 B 3 autotuning B 3 calibration B 10 flow to batch conversion B 12 heated lines B 4 heating flow cell B 3 operating precautions B 4 option B 2 C 2 ramp rate B 3 temperature set B 3 thermocontroller heated lines B 4 read head B 3 time constant 4 14 top cover 3 5 transformation temperature G 5 transmitted light 3 8 troubleshooting www wyatt com H 21 tweezers 5 14 MWH Rev A U Ultra High Temperature option B 2 unpacking the instrument 2 2 USB adapter H 7 USB connection multiple instruments H 15 one instrument H 14 via crossover cable H 13 Utility board 3 17 UV light 5 13 V Vapor Interlock connector 2 7 Vapor sensor board 3 18 ventilation 5 2 volume of flow cell 3 12 W warm up time 2 3 5 2 warning Heated Line connector B 8 high temperature B 4 laser 3 7 warning lights and alarms 4 3 Waters 150C disconnecting B 10 installation with B 7 operation with B 9 potential problems B 9 MWH Rev A Index window 3 138 5 12 mounting diagram 5 15 O ring 5 10 removing 5 9 retainer 5 9 5 10 wire colors 2 7 wrist strap 5 7 Wyatt Technology Corporation corporate headquarters 1 6 Internet 1 6 sales department 1 6 technical support 1 6 X X axis set time 4 14 selector 4 4 Y Y axis data channel 4 4 Z zoom pan buttons 4 5 Index 5 Chapter Index 6 MWH Rev A
90. mage from over illumination The APD is extremely sensitive to light and must be protected at all times Never expose it to room light with the power on It must either have the dust cover or light fiber connected to it at all times The Wyatt QELS is equipped with a protection circuit that will shut off the APD in the event of over illumination but it is intended as an emergency shutoff Chapter A Using QELS Main Batch GELS Alarm System Correlation Function ha ys TA mea A Auto Scale Change axis WYATT QELS Ma 5 S z iL S i T im S td 1e 006 1e 005 0 0004 0 001 0 01 Delay Time s Integration Time sec 7 APD Status OR ae Figure A 2 Correlation function A 1 5 Power CAUTION The power switch on the QELS panel turns off the power to the QELS option There is no external switch This is included in case you are not using the QELS for some time or if you want to open the flow cell to look inside to determine if there is dust or dirt Room light can damage the QELS detector so it is important to power it off if there is the potential to expose it to room light The QELS hardware includes an APD protection system The correlator hardware continuously monitors the count rate of the APD If at any point it exceeds 1OMHz for more than 1 msec it will shut down the detector to prevent damage This is referred to as an APD alert It will automatically restart during the n
91. ment The ASTRA V software will select the appropriate detectors based on these considerations See Table G 1 in Appendix G for more examples 3 15 Chapter 3 HELEOS Components 3 Alarms Note 3 16 3 7 1 3 7 2 3 7 3 The HELEOS will sound an audible alarm when a potential hazard is detected Hazards include e vapor or liquid leak sensor activates e cell protection thermostat activates indicating an over temperature condition e external alarm input activates When a potential hazard is detected the alarm output on the back panel also activates This is so that this signal can be used to control other instruments For example this signal can be used to turn off the chromatography pump Vapor Sensor The HELEOS has a vapor sensor to aid in safe operation of the instrument especially at high temperatures The vapor sensor is not intended as a protection device but as a convenience to alert the operator to the possibility of flammable liquid or vapor inside the HELEOS The alarm activates within 15 to 30 seconds after vapor is present The alarm should reset within 30 seconds after all solvent disappears from the flow cell cavity The sensitivity of the vapor sensing device is different for each solvent The sensor is set to a sensitivity level that works for both toluene and tetrahydrofuran You can use the VAPOR INTERLOCK connector to shut down the pump system or activate an external alarm if a leak is dete
92. ncies from 50 60Hz Connect one end of the supplied ethernet cable to the ethernet port on the back of the HELEOS and the other end to your local area network Alternatively you can use the supplied ethernet to USB converter and connect to the USB port on the host computer When the HELEOS is on the local area network it may be accessed and controlled from any machine on the network When using the USB converter it can be accessed only by the host computer See Appendix H for more details about implications for network security from the two different configurations Switch on the instrument and let it warm up for 30 minutes before beginning step 6 The power switch is on the front panel For Nitrogen Purge Option for Peltier Cooled Instruments Only this step is not necessary for ambient or heated instruments While the instrument is warming up attach a filtered dry air or chromatographic grade nitrogen line to the Nitrogen Purge connector on the back of the HELEOS Use the 90 degree fitting and the 10 inch Polyethylene tubing provided The dry gas will flow into the cell cavity and will minimize the amount of dust in the cell cavity The pressure in the dry air or nitrogen line should be 20 psi or less If you are using a Peltier Heated Cooled HELEOS and operating below ambient temperature it is particularly important to use the nitrogen purge line to prevent condensation At ambient or high temperatures the nitrogen purge line is n
93. nd Detectors cc cceeeceeceecneceeceeceeeeeeeeneceeceeeeeneeneeaes 3 9 3 5 1 Read Head Structure cece cccccccceccecceceeceeeeeeeeseeseeeeeeneeneeaeeens 3 9 3 5 2 Detector PIACEMENT ccccceceeceeeeceececeeceeeeceeeeceeeeeeeneeeeneeneness 3 10 236 POG CI seee vchasnena arcu aine nut EEE ented dautenianuners 3 12 3 6 1 Flow Cell DESIGN sccinszsacco nesseanenatcueadadnoacdessanodseutssadseesdtadaavenenteass 3 12 3 6 2 Laser Beam Orientation cccecceceeceeceeceeceeceeeeeeeeeeeeeeeeneeneens 3 12 3 6 3 Cell WINGOWS ccccceccecceeceeceecceeceeceeceeeeeeneeaeeeeeeaeeaeeaeeaeeseseees 3 13 3 6 4 Refractive Index Differences Liquid vs Glass ee 3 14 3 6 5 Accessible Available Detectors 00 0 0 cciccccecceccesseesseseeseeeeeeeaeees 3 15 Sigel VIPO Oe ISOM erie concenaiatanstinaies E 3 16 3 7 3 Turning Off the Alarm c2ccesirseaenciorsenancenssaatinesenaiandiecderdsaniennatons 3 16 3 8 Printed Circuit BOardS ccc cceccceceeeceseeceeeeeeeeeeeeeeeeeeeueeeeeaeeeeeeeeees 3 17 3 8 1 Front Panel Computer and Display Board ccccseeceeeeeees 3 17 3 8 2 Flexible DElECIOl DOAN id csccesccssmncccdencasswnucraconnsanecedaaendseeineanaeiet 3 17 3 8 3 EOS Instrument Controller Board cceeeeccseceeeseeeeneeeneeeees 3 17 S02 UNY DO AIC oserei E 3 17 3 8 5 Vapor sensor board sesissstcninvssincasewasasanvansniveidesseadedsanessssavsunennenes 3 18 3 8 6 Nitrogen sensor DOAIC cccccsesseccee
94. nect the heated lines e Two 14 Crescent wrenches e Stainless steel nuts and ferrules To connect the heated lines and heat the HELEOS do the following 1 Make sure the HELEOS is switched off using the switch on the front panel of the instrument Remove the cover bib from the top of the HELEOS On the HELEOS end of the heated lines connect the two pieces of tubing in series using a short piece of stainless steel tubing and two unions This effectively bypasses the cell and allows you to keep a small flow through the chromatography columns while the cell is being serviced Make sure you first seat the ferrules using the HELEOS flow cell manifolds Shorten the exposed stainless steel tubing if needed 4 Connect the heated lines to the column outlet using the tubing marked with metal tape Which one you connect depends on whether the HELEOS is placed to the right or the left of the oven MWH Rev A B 4 3 B 4 4 MWH Rev A Using the HELEOS with an Oven 5 Run the pump at 0 1 mL min until the lines are completely filled with solvent then attach the unmarked tubing of the heated lines to the RI inlet 6 Turn the pump off and disconnect the short piece of tubing at the HELEOS side of the heated lines 7 Connect the tubing from the columns to the rear inlet of the HELEOS cell and the other tubing to the front outlet of the cell 8 Run the pump at 0 1 mL min and replace the insulated read head cover see Heat
95. nected via a flex cable to the EOS Instrument Controller EIC which converts the analog signals to digital values with individual 24 bit analog to digital converters Note that the instrument s major components are mounted on the steel sub chassis which also contains all power supplies laser meters electronics and fan assembly Flow cell cover Forward laser monitor Flexible detector board as Photodetector 18 total Figure 3 8 Ambient read head With the read head covers removed to reveal the flow cell assembly you can view the cell bore through an opening in the cell manifolds Figure 3 10 3 9 Chapter 3 HELEOS Components 3 5 2 Detector Placement The 18 detectors are placed as shown in Figure 3 9 Channel 1 is available only during Batch measurements using scintillation vials 14 10 8 16 6 18 4 Laser Light i 5 15 E 13 11 Figure 3 9 Detector locations The angles are measured with respect to the direction of the laser beam Since the observed angle changes with solvent refractive index small scattering angle measurements are possible To include at least some small scattering angles for all solvents we have chosen the set of fixed detector angles See Flow Cell on page 3 12 in this chapter 3 10 MWH Rev A Read Head and Detectors Table 3 1 Positions of the 18 detectors relative to the incident laser beam E woo foe aoo fo 108 0 117 0 126
96. o determine if a source of dry gas is connected for operation below ambient or if the gas cylinder has emptied MWH Rev A Using the Display Window This chapter describes how the navigate and change settings in the HELEOS Display Window CONTENTS 4 1 Navigating the Display Panels cccccsecceesseeeeeeeeeseeeeeeeeeeseees 4 2 4 2 Warning Lights and Alarms cccccceceeeeeeeeeeeeeeeeaeeeeesaaeeeeeesaneees 4 3 MeN WAZA GC S A E E E E E IA E E A T 4 3 4 2 2 Audio AlA WW sicccSciesccexviexsnaceeeustensseduebianetsceesteecnoessSsexheecewhediengaseace 4 3 43 Man TING Mi esrss ea coun eh nenudninesdinaimundnausies 4 4 4 3 1 Selecting Display Settings for the X Y AX S ccccecseeeeeeees 4 4 4 3 2 Adjusting the Display Range ccceeeeeeeeeeeeeeeeeeeeeeeeeeeeaaeees 4 5 4 3 3 Setting Cell Temperature ccccccccccceceessseseeeeeeeeeeeeeaesseeeeeeeees 4 6 AoA LASO eee nn Pe oe ne ee ee ee ee A 4 6 AGS COME eapi E A E EER 4 6 A RC CY ClO en E ea eaceesneancsstacaaee 4 6 44 Balch Paneles Eai 4 7 4 4 1 Setting the Baseline and Normalization Coefficients 4 8 4o QELS AME aisnean E e i p nE s 4 10 4 6 Alarm FANE sic cncacttacsvcaccesnsnncntatemensasittancniadaxaedascliumunqasdbimsnintiiunes 4 11 4 7 System Panel cctnstivsnnlevncencsatdanaiissadaacunetnsedessdtctanaacanctdncnveataneneutavniaes 4 13 AB Comm AMC esiccdhectaesncacanicewawedeencstvadnadechneetaandicmnssinscctvadoencced
97. ocket on the flexible detector board b Gently remove the detector from the read head by grasping it on the sides and sliding it out of the read head You do not need to remove the detector from the flex board If it does come out of its socket simply reinsert 1t Typically the detector will come out of the read head with the O ring around the side of its can MWH Rev A A 7 Chapter A Using QELS 4 10 c The MALS detectors will not be damaged from room light and can be left on the flex board after the QELS detector has been installed Simply bend the flex board aside when installing the QELS fiber Remove the fiber mount from the read head by inserting a 4mm hex driver into the mount and loosening the interior screw as shown in Figure A 6 Adjuster screw Age Positioner driver clamp Figure A 6 Adjusting the EOS fiber positioner clamp Adjust the top adjuster screw to the center of its range to allow the 4mm driver to engage the nut on the positioner clamp This is when the top of the adjuster screw is nearly flush with the top of the fiber positioner Use the 4mm driver to loosen the positioner clamp It should slide free from the HELEOS detector hole Move the fiber positioner to the new detector location and press it firmly into the detector hole Use the 4mm driver to tighten the positioner clamp The clamp fingers will expand to grip the detector hole The fiber positioner can rotate in the detector ho
98. of the cell See Figure 5 6 a Pick up the cell with a folded lens tissue touch only the flat sur faces Wipe the curved optical surfaces with another folded lens tissue moistened with alcohol If needed wipe the alcohol off the optical surfaces with dry lens tis sue Do not repetitively rub the surfaces since this creates static electricity which attracts particles Using a magnifying loupe examine the optical surfaces for any dust See the Note at the end of this section Also check the bottom and top surfaces for dust and finger marks 5 11 Chapter 5 HELEOS Maintenance Top surface You can hold the cell by the top and bottom surfaces Step amp Optical surface Do not JN touch other than to clean with an ethanol dampened lens tissue Figure 5 6 Flow cell 4 Clean the window faces This is the most important step 1n cell cleaning Even the smallest particle left on the window faces will induce stray light and cause signal distortion especially at low angles a Take a folded lens tissue moistened with a couple of drops of alco hol and hold it against the side of your forefinger with your thumb and third finger b Pick up the window with the tweezers Hold the window by the sides not by the window faces c Smoothly wipe both window faces across the tissue d Carefully examine
99. omic weights of all atoms in a mole of molecules The term molecular weight means the same thing You will see both terms used in this manual How the Manual Is Organized The chapters and appendices in this manual are organized as follows Chapter 1 Introduction introduces the HELEOS and this manual and describes the support options available from Wyatt Technology Chapter 2 Installing the DAWN HELEOS takes you through the necessary first steps for unpacking connecting and testing the HELEOS Chapter 3 HELEOS Components takes you on a guided tour of the HELEOS Chapter 4 Using the Display Window shows you how to navigate and change settings in the HELEOS Display Window Chapter 5 HELEOS Maintenance has procedures for keeping the HELEOS in good working order and includes flow cell cleaning Appendix A Using QELS describes procedures for using the QELS option Appendix B Ultra High Temperature Option describes the Ultra High Temperature version of the HELEOS and its operation Appendix C Peltier Heated Cooled Option describes the Heated Cooled version of the HELEOS and its operation Appendix D Polarization Option tells about the installation and use of polarization filters Appendix E Interference Filter Option describes the use of interference filters for keeping non laser wavelengths from reaching the photodiodes Appendix F Laser Specifications
100. or the second window 5 14 MWH Rev A Manifold S Flow cell 2 a S gt i j f A Z P KAA J GF LG Cell O ring oe KNS Cleaning the Flow Cell and Windows s Cell window lt Window retainer Window O ring acking ring Bottom cell retainer Da below 80 C only MWH Rev A Figure 5 9 Window mount detail Install the cell in the manifolds a Insert the cell O rings followed by the backing rings if they were removed in Step 2 1 If you have an Ambient HELEOS or will be operating below 80 C install both the 6 mm O rings and the backing rings If you will be operating at or above 80 C use only the 9 mm O rings and do NOT use the backing rings The cell O rings need room to expand when heated above this temperature Using back ing rings at high temperatures could cause the glass to crack Holding the cell with lens tissue place it in the inlet manifold the larger manifold A step is machined into the top surface of the glass cell the mani fold has two pins to help align the cell properly Push the cell step against the manifold pins Make sure that the glass step and manifold pins are matched up well Place the outlet manifold next to the inlet manifold and push them firmly together Insert the short M3 screw into the outlet manifold and tighten with the 2
101. ork with all of the above connection schemes As shown in Figure H 18 there is a choice of DHCP or Static IP for the IP settings In general this setting can be left to DHCP With DHCP once the instrument is connected to a computer or LAN the IP address and subnet mask will be assigned automatically This will even work with the USB connections described in Connecting via USB on page H 13 When using DHCP it might take several minutes for the IP address to be assigned During this time the IP address and subnet mask on the front panel will read 0 0 0 0 Once the IP address and subnet mask have been assigned both will be automatically updated on the front panel and should no longer read 0 0 0 0 At this point it should be possible to connect to the instrument from the computer If you wish to use a static IP address and subnet mask please contact your IT department to obtain a valid address and mask Main Batch QELS Alarm System Comm Ethernet Communication DHCP gt Instrument Name IP Address age Be 1 BES Wyatt 100 HELEOS SubnetMask 255 255 0 0 Apply Figure H 18 Standard settings on instrument front panel for instrument connectivity H 19 Chapter H Connecting to Network or PC H 6 Accessing instruments with ASTRA V To access an instrument connected via Section H 2 H 3 or H 4 above using ASTRA V please refer to the ASTRA V for Windows User s Guide H 20 MWH Rev A Trouble shooting and d
102. ossover cable H 13 Chapter H Connecting to Network or PC H 3 2 One instrument to USB using an Ethernet switch Connect the instrument to the Ethernet switch using a standard Ethernet cable Then connect the Ethernet switch to the Ethernet to USB adapter using a standard Ethernet cable Plug the Ethernet to USB adapter into an available USB port on the computer You may be prompted to install drivers for the Ethernet to USB adapter the first time it is plugged into the computer To install the drivers insert the CD that came with the adapter and follow the Windows instructions sians Figure H 13 Connecting one instrument to USB using an Ethernet switch H 14 MWH Rev A Connecting via USB H 3 3 Multiple instruments to USB Two or more instruments can be connected to USB using an Ethernet switch Use a standard Ethernet cable to plug each instrument into the Ethernet switch Then connect the Ethernet switch to the Ethernet to USB adapter using a standard Ethernet cable Plug the Ethernet to USB adapter into an available USB port on the computer You may be prompted to install drivers for the Ethernet to USB adapter the first time it is plugged into the computer To install the drivers insert the CD that came with the adapter and follow the Windows instructions TTILE Figure H 14 Connecting two or more instruments to USB using an Ethernet switch and Ethernet to USB adapter MWH Rev A H 15 Chapter H Connecting
103. ot required but may be used to exclude dust from the instrument by creating positive pressure inside the cell 2 3 Chapter 2 Installing the DAWN HELEOS Note 2 4 6 The HELEOS has been shipped with chromatography grade toluene in the flow cell that can be used to verify that the instrument was not damaged during shipping Cycle through the light scattering graphs and check that the solvent offsets are consistent with the Certificate of Performance COP supplied with each instrument Sometimes when the instrument has been in storage or been subjected to extreme temperatures during transit the cell will have bubbles If this is case fill the cell with fresh toluene before checking the solvent offsets against the COP Use a glass syringe with a 0 02um filter and inject toluene directly into the flow cell through the in port You may wish to use a syringe pump to drive the syringe and to help prevent introducing bubbles into the flow cell Using the supplied ASTRA V software perform the appropriate steps to configure the instrument to communicate with the software The laser in the HELEOS is software controlled and can be turned on and off from the main page 8 10 After establishing communications wait at least 30 minutes for the laser to warm up and stabilize Start the Diagnostic Manager and select the numeric real time channel values see the ASTRA V for Windows User s Guide for details Compare the c
104. other interference filters Installing a filter on every other diode should be sufficient For example you might install filters on the odd numbered detectors only Replace the instrument cover and switch the instrument and laser back on Repeat the calibration if you installed a filter on detector 11 normalization and for Batch mode solvent offset measurements MWH Rev A Laser Specifications The HELEOS contains either a GaAs laser operating at a nominal wavelength of 658nm or Diode Pumped Solid State DPSS laser operating at 532nm The GaAs laser is a single transverse mode heterojunction that emits light at 658nm at a power of 50 mW delivered to the flow cell Typically diode lasers undergo periodic mode hops between different longitudinal modes which have slightly different efficiencies giving rise to sudden changes in intensity however Wyatt Technology utilizes a patented intensity stabilization method which achieves a typical long term intensity stability of 0 1 The DPSS laser consists of a diode pumped Nd YVO gain medium coupled with a nonlinear doubling crystal The time from unit power on to laser emission is approximately 10 seconds The beginning and end of laser emission 1s accompanied by an audible click It takes an additional 5 minutes after laser emission begins for the laser to reach thermal stability It is suggested that the laser be allowed at least 30 minutes to warm up before taking data Note that t
105. oving the Flow Cell Assembly ccceecccccceeeeeeeeeeeeeeeaeneeeeesaeeeesaaaneees 5 7 Step 2 Disassembling the Flow Cell ccccceccccccseececeeeseeeeceeeeeesseeeeeesaseeesseaaeees 5 9 Step 3 Cleaning the Flow Cell and Windows ccccseceeeeeeeeeeeeeeeeeeeeseeeeeeenaees 5 11 Step 4 Reassembling the Flow Cell ccccccccsseceecceseeeeeseeeeeeeeeeseeeeseeeeesseegeees 5 14 Step 5 Reinstalling the Flow Cell Ambient Version cccccccesseeeeeeeeeeeeeeseeees 5 17 step 5 Reinstalling the Flow Cell Heated Cooled Version cseeeeeeeeeeees 5 18 Flow to Batch Conversion icc ccseaimcanscetvanssntageawoudncatetneeelnsavaaidea sa vaoweneoeereantoass 5 19 Appendix A Using QELS 10 ceeececeseeeeenseecenseeseenseseeneeseenee A 1 OBIS PAREN siierspata poate sis E A etgneiaanneeetnnens A 2 COUNTE ALC zna has a acini ta a eterna aut aoe clad aw seed A 2 Correlation FUNCTION cccccccesseccccsecececeeseceeceeceeeseasceeeeeaueeesseaseeesegeeeessaeeeeesseeess A 2 mMtegraton Bina cumernemetesr meters mien tte a many rene anne nari i are OP re A 3 ARD Sta ogee ere en ce ee en eee ee ee A 3 OWE aineosat a T E E neue cae cee aetiee ee wate A 4 Aligning Ihe Optical IDEM 3265 4 cec5 vet cdenttcaeate ce anchuesy E EN A 5 Removing and Installing the Optical Fiber Receiver cccseeeceecesseeeeeeeeeeeees A 6 To remove and reinstall the optical fiber receiver cccsecc
106. pikes appear in the RI signal after each injection look for insulation problems at the heated line connections If further insulation does not remove the spikes try heating the HELEOS another five degrees The spikes are likely due to partial polymer precipitation and a higher temperature may be helpful Alternatively you can break the sync connection between the heated line and the read head so that you can run the heated line at 5 degrees higher than the read head Disconnecting the HELEOS from an Oven When you decide to disconnect the HELEOS from the oven be aware that you must replace all the O rings in the flow cell assembly after the instrument has cooled down If this is not done the flow cell may leak upon reheating To disconnect the HELEOS do the following 1 Decrease the pump speed slowly to 0 1 mL minute 2 Set the read head temperature setpoint on the HELEOS to room temperature The temperature will slowly decrease to room temperature 2 3 hours 3 When the cell is close to room temperature turn off both the HELEOS and the pump Unscrew the bolts holding on the heated line spacer Slide the heated line up on the capillaries by several centimeters 6 Remove the heated line spacer MWH Rev A DANGER MWH Rev A Using the HELEOS with an Oven 7 Disconnect the heated line electrical connector from the HEATED LINE connector and replace the dust cap on the HEATED LINE connector The HEA
107. ption The Peltier Heated Cooled HELEOS option has some differences from the ambient HELEOS This appendix describes those differences and supplies instructions for making adjustments and operating this version of the instrument CONTENTS PAGE CM OVS ecne A N REELE C 2 C 2 Recommended Procedures cccccscececcececccecececccecucccceceueuaececass C 3 MWH Rev A C 1 Appendix C Peltier Heated Cooled Option C 1 Overview The read head on the Peltier Heated Cooled HELEOS can be heated up to 150 C or cooled down to 30 C The Heated Cooled HELEOS uses a solid state Peltier device and a cartridge heater to operate over the whole temperature range Cell cavity Outer cover Inner cover Outer cover insulation Detector ring SIN Z Upper and lower a yaa eT ae insulation DA ct eeeee p NL ha ZUA Cell orc NS gH HTR RTD Cc LEE Base insulation d AE AAEE AIL i Heat pump heat exchanger assembly Figure C 1 Heated cooled HELEOS read head cross section The temperature controlled read head is comprised of three distinct shells of material e The outer aluminum detector ring which contains the photodiode detectors e A layer composed of two insulating materials that keep the flow cell at a stable temperature while at the same time keeping the photodiodes as close to ambient temperature as possible e The innermost shell is the alum
108. r continuous flow GPC SEC HPSEC detection or as a stand alone unit in a batch or micro batch mode The read head and the laser system of the DAWN HELEOS anchor to the base plate and the flow cell and manifolds are mounted directly into the read head to provide a single stable optical bench The laser beam is aimed into the flow cell and passes in the same direction as the flowing stream The windows that let light pass through the flow cell are recessed in the manifolds minimizing sample volumes and stray light The 18 discrete photodetectors spaced around the flow cell enable simultaneous measurements over a range of angles typically 15 160 depending on solvent glass refractive indices Each photodetector has its own DSP Digital Signal Processor chip for processing the analog signal In addition four auxiliary analog inputs with their own DSP chips enable interfacing to external detectors such as differential refractive index and ultra violet absorption detectors or differential viscometers Electronic filters within the DSP chips and within the embedded computer processes each light scattering and auxiliary signal for the greatest possible noise rejection without peak distortion Since the analog to digital conversion is performed onboard the DAWN HELEOS low light scattering signals are not prone to environmental noise or pickup The digital output transmits to your computer through its Ethernet port or USB with the s
109. rization Strips TST CT D 2 Normalization and Calibration with Polarization Strips Installed D 2 1 Normalization To normalize the HELEOS it is necessary for each detector to receive light from an isotropic scatterer With the polarization strips in however half of the detectors are receiving light that has passed through a horizontal polarizer while the other half are receiving light that has passed through a vertical polarizer In the case where the normalization standard does not depolarize the scattered light the detectors with the horizontal polarizer will receive no scattered light at all Therefore it is not possible to normalize the HELEOS with the Polarization strips installed Normalization should be performed without the Polarization strips Then install the Polarization strips after normalization D 2 2 Calibration For calibration the 90 degree detector must receive a known amount of light Typically toluene is used as the calibration standard However toluene depolarizes the scattered light so that there are horizontally and vertically polarized components that reach the detector Therefore installing a vertically polarized filter in front of the 90 degree detector blocks some of the horizontally polarized scattered light that is necessary for an accurate calibration It might seem necessary therefore to remove the polarization strips before calibrating The strips however attenuate some of the light regardless of t
110. rt 3 2 installation heated lines B 8 HELEOS 2 3 with PL210 B 7 with Waters 150C B 7 installation tool aperture 5 9 instrument connections H 2 verifying H 21 interference filters E 2 Internet 1 6 K K5 cell specifications G 1 keypad 3 2 L LAN connection multiple instruments H 11 multiple instruments and computer H 12 one instrument H 9 one instrument and computer H 10 language 4 14 laser beam orientation 3 12 channel monitors 2 4 current monitor 3 8 description 3 7 MWH Rev A Index forward monitor 3 8 monitor values 3 8 monitors 3 8 On Off 4 6 rear monitor 3 8 specifications F 2 warning 3 7 laser drive current 3 8 viewing 2 4 LCD display 3 2 load factory defaults 4 15 location 2 3 M maintenance flow cell 5 3 general 5 2 manifold 5 10 manifolds 5 9 5 10 manual conventions 1 4 N network settings H 19 Ninety Degree Detector 2 7 Nitrogen Purge fitting connecting 2 3 Nitrogen sensor board 3 18 noise sources B 9 normalization after setup with oven B 7 polarization option D 3 normalization coefficients 4 7 setting 4 8 normalization standard 4 7 normalized data 4 7 O On Off switch 3 2 optical fiber aligning A 5 removing reinstall A 6 options Comet 4 6 Interference filters E 2 Peltier Heated Cooled C 2 Polarization D 2 QELS A 1 Ultra High Temperature B 2 O rings ambient and low temperature configuration 5 15 C 3 high temperature configuration 5 15 OUT port 3 2 oven
111. rument H 17 ethernet crossover cable H 5 ethernet port H 2 ethernet switch H 8 F F2 cell specifications G 1 filtering solvent B 9 filters interference E 1 polarized D 2 flow cell 3 12 5 10 chemical properties G 4 cleaning 4 6 5 6 exploded diagram 3 13 heating B 3 maintenance 5 3 O ring 5 10 particles 5 5 refractive index 3 14 G 3 removing temperature controlled B 5 removing the assembly 5 7 retainer 5 11 scattering angles G 7 thermal properties G 2 volume 3 12 window 5 10 5 12 window mounting 5 15 windows 3 13 flow cell temperature lock 4 12 flow to batch conversion 5 19 temperature controlled B 12 fluid connections 3 2 fluorescing samples E 1 forward laser monitor 3 8 viewing 2 4 front panel 3 2 Front Panel Display board 3 17 fuses replacing 3 4 G grounding 2 7 H heated HELEOS C 2 heated line temperature 4 13 sync 4 14 heated lines B 4 installation B 8 thermocontroller B 4 heating B 3 MWH Rev A HELEOS adjusting the display range 4 5 alarm signal state 4 11 connecting to H 1 connecting via USB H 13 display window 4 1 flow cell temperature lock 4 12 functions 4 4 LAN connection H 3 load factory defaults 4 15 network settings H 19 recycle 4 6 restart instrument 4 15 restart ISI 4 15 setting cell temperature 4 6 time constant 4 14 warning lights and alarms 4 3 zoom pan buttons 4 5 help technical support 1 6 where to get 1 6 high temperature option B 2 hydrodynamic radius A 1 I IN po
112. sets differed from the Certificate of Performance in step 6 the solvent offsets should differ by the same amount Calibrate the HELEOS using the ASTRA V software See the software user s guide for instructions to configure communication with the instrument and perform the calibration measurement Compare your calibration result with the value from the Certificate of Performance Your calibration result should be within 5 of the value on the Certificate of Performance When you have confirmed that the instrument is in good working order connect the HELEOS to any other devices for your application Auxiliary cable connection is described in the next section The ASTRA V for Windows User s Guide describes how to connect the HELEOS to your chromatography system 2 5 Chapter 2 Installing the DAWN HELEOS THIRD PARTY UV DETECTOR OPTIONAL ETHERNET HUB LS DAWN HELEOS ETI G 0000000 H VISCOSTAR OPTIONAL mn N _ SSS HH SSEE el COLUMNS gt SOLENOID VALVE B os ats Solvent DE GASSER PUMP INJECTOR Je B Reservoir B WASTE RECYCLE RESERVOIR This configuration requires the ASTRA V software The dark black lines denote the electrical data connections The thin black lines show the fluid connections Several optional instruments are shown If not con necting one of the optional instruments the fluid connections are byp
113. splays in blue the right Y axis data channel displays in red X axis Selector The X axis selector sets the time range from 10 minutes to 2 hours To change the time see the Set Time field under System Panel on page 4 13 MWH Rev A 4 3 2 Adjusting the Display Range Main Panel You can adjust the range displayed in the graph in a variety of ways This also applies to the Change Y axis button in the Batch Panel To use the zoom and pan buttons 1 Tab to the Change Y axis button The zoom pan buttons are displayed Click the left arrow to zoom in Click the right arrow to zoom out Click the up arrow to pan up a Click the down arrow to pan down Pan up Zoom in 3 amp H Zoom out Tab to here T to display the zoom Pan down pan buttons Tab to here to display the zoom pan buttons MEE Nive Main Batch GELS Alarm System Comm Service HELEOS amp pa Read Head Tempfaoture hi Me Pressure Auto Scale axi pp M Auto Scale Change axis 22 0 26 2 aa a T E E T E amp Eo m E amp En 10 16 AM 10 16 AM 10 20 AM 10 22 AM 10 24 AM Cell Temp ZE Lon OFF 25 83 C ean ok Ok Laser Comet Recycle Figure 4 3 Zoom and pan buttons To change the scale numerically 1 Tab to the Change Y axis button 2 Press Enter The Set Scale window is displayed MWH Rev A OFF F Sd 1nsselq ZN
114. struments and computer to LAN If there is only one LAN wall socket available for multiple instruments and a computer it is necessary to use an Ethernet switch to connect both the computer and instruments to the LAN In this configuration the computer can access the LAN and the instruments and the instruments can be accessed from any other computer on the LAN Figure H 11 Two instruments and a computer connected to the LAN via an Ethernet switch H 12 MWH Rev A Connecting via USB H 3 Connecting via USB H 3 1 MWH Rev A If it is not possible or desired to have the instruments connected to a LAN it is possible to connect to the instruments via USB In this way the instruments can be isolated from the LAN even while the computer maintains its own Ethernet connection with the LAN One instrument to USB via a crossover cable Connect the yellow crossover cable from the instrument to the Ethernet to USB adapter Plug the Ethernet to USB adapter into an available USB port on the computer You may be prompted to install drivers for the Ethernet to USB adapter the first time it is plugged into the computer To install the drivers insert the CD that came with the adapter and follow the Windows instructions Please note that the network communication setting in the Communications tab of the instrument display is Ethernet for this configuration and not USB Virtual Ethernet Figure H 12 One instrument to USB via yellow cr
115. supplies the electrical optical and environmental specifications for the GaAs and Diode Pumped Solid State DPSS laser head MWH Rev A MWH Rev A About This Manual Appendix G Flow Cell Properties lists thermal and chemical properties refractive indices and scattering angles of solvents for the K5 and F2 flow cells Appendix H Connecting to Network or PC covers connecting the HELEOS to either a network through the ethernet or to a host PC through the ethernet to USB converter is Chapter 1 Introduction 1 6 1 3 1 1 3 2 1 3 3 1 3 How to Contact Wyatt Technology Corporation If you have a question about your HELEOS first look in this manual or consult the online help that comes with ASTRA V for Windows If you cannot find an answer please contact Wyatt Technology Technical Support Corporate Headquarters Wyatt Technology Corporation 6300 Hollister Ave Santa Barbara CA 93117 USA Sales Department Wyatt Technology Corporation Sales Hours are 8 30 A M to 5 00 P M Pacific Time Sales Phone 805 681 9009 Sales Fax 805 681 0123 Technical Support Wyatt Technology Corporation offers a variety of support options to help you get the most from your HELEOS You can also contact the Wyatt Technology Distributor in the country where you bought your product Before contacting technical support try to resolve any problems through the ASTRA V for Windows on line help system and t
116. t under normal operation no laser radiation should escape from the instrument and no protective equipment must be worn However the follow warning applies Caution Use of controls or adjustment or performance of procedures other than specified herein may result in hazardous radiation exposure The instrument also bears the following warning label Danger Laser Radiation when open Avoid direct exposure to beam Note Laser safety labels are in English If you need safely labels in a language other than English please contact Wyatt Technology F 2 MWH Rev A Flow Cell Properties This appendix contains tables listing thermal and chemical properties of the two types of flow cells and their refractive indices Except for Table G 2 all data and descriptions are from the Schott Glass Optical Glass Catalog CONTENTS PAGE G 1 Thermal Properties ccccccceccccceceeesseeeeeeesesseeeeeseaeaeeeeeeseaseeeeees G 2 G2 R fractive INGICES s assisia aaa aAA Eara G 3 G 3 Chemical Properties ccccssesssseeceeeeeceeeesseeeeeeeeeseeaeassseeeeeseesenegs G 4 GA Delinin OL leris seinri a aE G 5 G 5 Scattering Angles 00 cccsseeeeecceceeseeeeeeeeeeeeeeeeessseeeeeeeeeaeseeeeees G 7 MWH Rev A Appendix G Flow Cell Properties G1 Thermal Properties Glass Classification Thermal Expansion 30 to 70 C 20 to 300 C Ke 82x106 96x10 K 543 C 0 783 F2 82x10 K 93x10 K
117. te around 50 kHz and clean by creating cavitation bubbles in the solvent which scrub surfaces when they collapse This can damage the fine polish on the optical surfaces of the flow cell It is not recommended that you clean either the flow cell or the windows in traditionally ultrasonic baths The COMET by contrast operates between 600 900kHz and avoids cavitation completely It works by creating resonate sound waves in the flow cell bore These sound waves help suspend dirt in the solution which is then flushed out by the flowing mobile phase Since it is permanently installed you can activate as needed Many customers operate it every night as part of a standard cleaning regimen Others schedule COMET activation between runs in a autosampler collection It is intended to be operated while the mobile phase is flowing through the flow cell You can also use it in conjunction with detergents for more effective cleaning Protease Cocktail Some users have found that a simple protease cocktail rinse is effective in removing protein deposits from glass flow cell surfaces You might be able to use this rinsing treatment rather than disassembling the flow cell Ingredients For 3 ml of protease cleaning solution All enzymes are sequencing grade preparations from either Boerhringer Manheim or Roche e Trypsin modified 25 ug lyophilized e Chymotrypsin 25 ug lyophilized e Pepsin 25 ug lyophilized You might be able to get away
118. ted line controller is slaved to the cell controller That is when the temperature of the cell is changed the temperature of the heated line is changed in sync On the System panel it is possible to break this slave relationship by unchecking the Sync button Then you can set the temperature of the heated line independently of the cell If you are using the Peltier Heated Cooled model the read head can be cooled or heated but the heated lines can only be heated Using a setpoint below ambient temperature will only cool the read head it won t cool the lines Operating Precautions Keep in mind these important points e Always have the insulating cover plate locked in place when bringing the cell up to temperature or cooling it down e If possible keep the instrument at operating temperature at all times e Replace the cell O rings whenever the instrument is brought down from an elevated temperature They conform to the geometry of the cell and when brought down from an elevated temperature may not seal reliably e The system will issue a warning if a heated cooled instrument is set to a temperature above 80 C This is to remind you that the O rings must be changed to the high temperature configuration before setting any temperature above 80 C No such warning is issued on the ultra high temperature instrument since it 1s typically configured with the high temperature O rings However if the O ring set is ever changed to the
119. th the two M38x20 screws Install the batch spacer plate with four M3x20 shoulder bolts ambient only Install the Batch vial spacer with two M38x10 Install the insulated batch cap Heat the read head to the required temperature then put the pre heated sample scintillation vial in the read head cavity and cover it with the insulated cap MWH Rev A Temperature Controlled Flow to Batch Conversion Batch cap M3x12 Batch ia spacer M3x20 Scintillation vial Cc Batch manifold lt _ Ball plungers it _M3x20 Ambient shoulder bolts spacer plate Figure B 5 Temperature Controlled flow to batch conversion kit exploded To replace the flow cell cool down the read head then reverse the previous process Notes The sample should be initially heated in an oven then filtered and transferred to the HELEOS Wait 15 20 minutes before taking a measurement When making measurements with scintillation vials take great care to keep the outside of the vials clean and free of fingerprints scratches etc as this can severely distort the measurement We also advise you to rotate the vial in the read head to find the position where the laser beam enters the cell with the least amount of scattering at the air glass interface See the ASTRA V for Windows User s Guide for further instructions MWH Rev A B 13 Appendix B Ultra High Temperature Option B 14 MWH Rev A Peltier Heated Cooled O
120. the flow cell is dirty light scatters excessively which shows up as high voltage unstable baselines and distorted chromatography peaks The flow cell cleaning procedure can be broken down into five major steps Step 1 Removing the flow cell Step 2 Disassembling the flow cell Step 3 Cleaning the flow cell and windows Step 4 Reassembling the flow cell Step 5 Reinstalling the flow cell What you will need for flow cell cleaning e A sheet of clean white paper taped down to your work surface e Anti static wrist strap e Ball drivers 1 5 mm and 2 5 mm e Aperture Installation tool WTC 119083 e Two Crescent wrenches e Lens tissue Fold several pieces in finger width strips for handling the cell and cleaning e lint free gloves e Oral B SuperF loss e Inert dusting gas Photographic supply stores carry this At Wyatt Technology we use Tech Spray from Com Kyl distributors in Santa Barbara 805 520 1731 e Filtered methanol ethanol or isopropanol e Tweezers e Optional Sonicating bath to clean stainless steel flow cell manifolds e Optional UV light The flow cell you are about to remove constitutes a substantial amount of the purchase price of the HELEOS Its parts are carefully machined and are expensive If you have any doubts whatsoever about the safest procedure for handling the cell structure do not hesitate to call Wyatt Technology We also offer a flow cell cleaning service for those who do not
121. uming the oven remains at operating temperature The following pages provide more details about these steps DAY 1 1 Calibrate the HELEOS using toluene Connect the heated lines to the HELEOS flow cell Set the pump rate to 0 1 mL min 2 a Install the flow cell cover plate and the insulated read head cover plate Bring the HELEOS to 185 C about 2 hours Slowly bring the flow rate to 1 mL min or other desired rate Check for leaks Check the calibration using TCB oe ee E Measure the flow rate 10 Prepare standards for the next day DAY 2 1 Inject a narrow polystyrene standard we suggest 400 000 Dalton calculate the inter detector delay volume 2 Inject alow molecular weight polyethylene we suggest 32 000 Dalton normalize the HELEOS and calibrate the refractometer 3 Inject two or three standard polymers 4 Process the data 5 Check normalization and calibration The ASTRA V for Windows User s Guide provides more details on calibration normalization and determination of the delay volume General Setup Procedure Calibrate the instrument Calibrate the instrument with toluene before connecting it to the oven Once at temperature and if the baseline signal is stable and free of particulate noise typically less than 20 uV at detector 11 you can check the calibration with the mobile phase if using 1 2 4 trichlorobenzene TCB near 135 C Appendix B Ultra High Temperature Option
122. upplied ethernet to USB converter Instrument Options The DAWN HELEOS is available with the following heating and cooling options e Ambient Operates at room temperature only This is the base model described in Chapters 1 through 4 e Ultra High Temperature The read head may be heated from approximately 10 above ambient temperature to 210 C Temperature can be controlled to within 0 01 C and is accurate to 1 C This option is described in Appendix B e Peltier Heated Cooled The read head may be cooled down to 30 C or heated up to 150 C Temperature can be controlled to within 0 01 C and is accurate to 1 C This option is described in Appendix C MWH Rev A 1 1 2 MWH Rev A Overview DAWN HELEOS vs DAWN EOS The DAWN HELEOS uses a 60mW laser operating at 658 nm which after intensity stabilization delivers 50mW to the sample It also includes a new electronics package and features an embedded microprocessor It includes a graphical user interface and provides two times the signal to noise ratio and one hundred times the dynamic range of the DAWN EOS A nitrogen purge connector is included on all models of the DAWN HELEOS In addition to preventing condensation in a cooled instrument s read head the nitrogen purge keeps the flow cell and read head cleaner at all temperatures The nitrogen pressure 1s monitored by the embedded microprocessor to insure that even when the nitrogen tank runs out no water will
123. v A 3 6 5 MWH Rev A Flow Cell where the angle of incidence is 7 2 and the angle of refraction is mz 2 6 Expanding the sine functions in Equation 1 results in 2 Niguid cos 0 N elass cos a The detectors are set to detect light at an angle 0 collimated to be centered in the cell As a result of refraction the light detected is the light scattered at an angle 6 In this way a greater angular range of scattered light can be detected Table G 1 in Appendix G lists the flow cell scattering angles Accessible Available Detectors Because of the refraction of scattered light passing from the solvent into the glass cell some fixed detector angles are inaccessible Consider water for example With a refractive index of 1 830 and the K5 cell refractive index of 1 51876 at 690 nm the smallest scattering angle must be 0 0 Substituting into Snell s Law Equation 2 we find the smallest fixed detector angle viz 1 332 cos 0 152064 cos 0 0 cos 1 332 152064 28 8 We see that for water the first accessible detector is the third corresponding to 32 0 Since this is the lowest detector for a water solvent system theoretically no scattered light should enter Channels 1 and 2 note that Channel 1 is actually blocked by the flow cell Although there may be signals on these channels their source is not light scattered from the sample but rather stray light outside the range of the experi
124. value the DAWN HELEOS will switch from a constant intensity mode to a constant current mode In the constant current mode the laser intensity will begin to decrease and the signal to noise ratio will begin to degrade The instrument will still provide accurate data but it indicates that the laser is nearing its maximum usable lifetime and the instrument should be serviced When the instrument switches to a constant current mode the rear laser monitor value which is proportional to the laser intensity will drop from the typical value of 5 volts MWH Rev A Read Head and Detectors 3 5 Read Head and Detectors 3 5 1 MWH Rev A The next major assembly is the read head Figure 3 8 Here the sample cell is held precisely scattered light 1s collimated and the detectors are aligned in their proper angular positions Read Head Structure The read head structure holds the 18 hybrid trans impedance photo detectors limits the sample field of view at each detector and minimizes stray light effects by means of its special structure Since each detector s field of view is limited by its own collimator only the center of the illuminated sample scatters light into a given detector A heavy aluminum mounting plate supports both the laser and the read head and is attached to the instrument sub chassis providing a single stable optical bench The optics have been aligned at the factory and should need no adjustment The detectors are con
125. with a 4 amp 250V slow blow fuse The fuse block contains two fuses Both of them must be installed for the instrument to operate correctly Replace the cover of the AC Power module and reconnect the power cord Figure 3 3 Fuseholder and Fuses MWH Rev A Top Cover 3 3 Top Cover There are two covers The standard cover for chromatography applications has no openings If the customer has purchased the optional flow to batch conversion kit or a heated transfer line a new cover is included that has removable pieces These pieces can be removed to allow access to the read head assembly and for introducing scintillation vials or the microcuvette Batch Cover Figure 3 4 Top Cover MWH Rev A 3 9 Chapter 3 HELEOS Components 3 3 1 Removing the Cover For normal operation and maintenance you should not need to open the top cover If you do need to open the top cover follow these instructions What you need to remove the cover e 2 0 mm Ball driver To remove the cover do the following 1 Make sure the HELEOS has enough space above it to lift up the cover 2 Disconnect the power cord 3 Remove the four screws that fasten the top cover to the instrument using the 2 0 mm Ball driver The screws are shown in Figure 38 4 4 Slide the cover up to remove it You can now see the HELEOS components as shown in Figure 38 5 Vapor detector Flow cell and read head Main circuit board Nitro
126. with just pepsin alone as it s so non specific Procedure 1 Reconstitute each with 1 mL of PBS 25 mM Na phosphate 150 mM NaCl pH 7 25 2 Mix the three solutions and vortex load syringe fitted with 0 02 um filter for LS detector 3 Flush detector with 20mL pure water then infuse 1 mL of cocktail via syringe pump MWH Rev A Flow Cell Maintenance Stop flow turn on Comet if you have one and leave it for a few hours or overnight The following morning remove syringe flush with fresh 20 mL of water then mobile phase 5 2 2 Particles in the Cell Here is a list of symptoms of particles in the cell and what you can do to dislodge them MWH Rev A Some Symptoms of Particles in the Cell Bright stationary spots when viewing the cell bore from above An increase in baseline voltage at all angles Unstable fluctuating baselines Distorted chromatography peaks dips below baseline shoulders on low angle peaks Some Suggestions for how to Dislodge Particles Change to a solvent with a different polarity Try injecting a small air bubble If the particle s move repeat until they are flushed out Flush the cell with 0 02 um filtered HPLC grade water Fill a syringe with a few mL of 6 M nitric acid inject and leave the acid in the cell for 10 minutes then flush with 0 02 um filtered HPLC grade water again 5 5 Chapter 5 HELEOS Maintenance 5 3 Cleaning the Flow Cell and Windows Caution When

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