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PDExpert Manual - The Molecular Materials Research Center

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1. where nis the refractive index of the solvent c is the concentration of polymer Na Avagadro s number A 1s the wavelength of light in vacuo is the wavelength of light in the medium O is the scattering angle f is a correction factor A is a constant PDExpert Appendix D D 1 Re is the reduced scattered intensity which is defined in equation D 2 I Ro r D 2 Io where ris the distance between the sample and the point where the intensity Ip is recorded and I is the intensity of the incoming light This equation allows for separate determination of Mw and lt r gt via equations D 3 and D 4 D 3 Ke 1 1 16 r 460 im PE 1 aT D 4 0 Re Mw Mw3 62 2 From an operational perspective data is collected at a number of angles for several concentrations and then plotted as shown in Figure D 1 the Zimm plot The molecular weight and the radius is obtained by extrapolating the data to concentration 0 and O 0 and applying equations D 3 and D 4 Ro Ro sin 8 2 be Figure D 1 A Schematic Zimm Plot D 2 PDExpert Appendix D index A Additional Information 1 4 Alignment Laser 1 3 3 4 Autozero 6 10 Attenuator 1 3 B Beam Dump 1 3 Cc Cell Maintenance 6 3 Collecting Data 3 4 Cooling Requirements 2 3 Detector 1 2 Detector Settings 2 7 Diode Laser 1 3 Discovery32 1 3 DLS 2000 Photon Detector Module 2 4 Dynamic Detector Module 1 2 Dynamic Light Scattering 4 1 Layout 4
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3. If it is not adjust the Laser Scale to obtain the desired reading in most cases it will be necessary to decrease the Laser Scale Note When you change the Laser Scale it is necessary to change the Laser Threshold by the same amount As an example if the Laser Power is found to be 12 mW instead of 15 mW when the Laser Scale is 0 677 and the Laser Threshold is 0 378 it will be necessary to decrease the Laser Scale In this case you might lower the Laser Scale to 0 650 and add the value that you subtracted 0 027 from the Laser Scale to the Laser Threshold which would now become 0 405 w Repeat step v until the observed Laser Power is 15 mW x In the software set the Laser Power to 25 and measure the laser power with the laser power meter The observed reading should be 7 5 mW if it is not adjust the Laser Scale to obtain the desired reading in most cases it will be necessary to decrease the Laser Scale When you change the Laser Scale recall that it is necessary to change the Laser Threshold by the same amount as described above y Check the laser power at 10 25 50 75 and 100 If the values are not correct adjust the Laser Scale and Laser Threshold until the readings are correct 3 4 PD Expert System Chapter 3 3 3 MAIN LASER ALIGNMENT To align the main laser a Remove the Cell Assembly the Beam Dump Monitors and the Steering Lens from the optical platform b Loosen the screws that attach the laser to
4. and Diffusion Constant measurements The workstation is capable of measuring the scattered light at a number of angles in a single experiment This data can then be used for advance molecular weight measurements such as Zinn plots A short discussion of these measurements 1s presented in Appendix D The user can configure the system to meet the exact requirements of the laboratory with powerful data acquisition processing reporting storage and retrieval programs such as Precision Detectors PrecisionMALS and Precision Detectors PrecisionIlluminte Detailed information about these programs is included in the manuals provided with those software packages This manual is designed to provide information about the use of the detector sample handling troubleshooting and related topics It contains information about all modules that are available for the PDExpert system For additional information about detector modules that are not installed in your detector please contact Precision Detector or your local representative PDExpert Preface ix This page intentionally left blank PDExpert Preface Table of Contents WV AE ANI Y E E A tesdass cus vees ads E I E S E E iii Warnings and Satety Precautions ssrissssoi a e E E EEA REE v Warmin LaADEl Sessea EE E E EE TE vii Pre aCei eroaa r E E rE E E O E aE E ix Chapter l Mtroduc honasi e cas suas pcan soda eckson ace im eas 1 1 RERE E ian ean neem nee ere er eae en A T ae emt site E Meat eee n
5. Performance Liquid Chromatography HPLC Size Exclusion Chromatography SEC and Gel Permeation Chromatography GPC This chapter presents oa information about using the system to collect light scattering data and focuses on sample handling and general operation of the system For additional information the user is referred to the manuals describing the application software 4 2 POWERING UP THE SYSTEM To power up the system turn on the switches on the PD4001 Electronics Module the PD2000 DLS Module and the personal computer Open the application software and select the appropriate settings for the desired analysis Note For optimum results a warm up period of 20 30 minutes is recommended 4 3 SAMPLE HANDLING 4 3 1 Using the Flow Cell When the flow cell is installed the sample is delivered by an HPLC SEC GPC system via the inlet fitting on the rear panel of the optical module The following precautions should be followed e The mobile phase should be filtered through a 0 22 um Nylon or PVDF membrane Make certain that the filter is compatible with all constituents of the sample e The sample to be analyzed should be dissolved in a suitable solvent and must be soluble in the mobile phase used for the separation If a gradient is employed the sample should be soluble in the entire range of the gradient that is employed It is recommended that you check the solubility of the sample in each mobile phase using a beaker rather than
6. list is dependent on the configuration of the system A typical system will include the items indicated in Table 2 1 the user should refer to the shipping list for the precise list of components Table 2 1 Shipping List for PDExpert Diskette or CD with applications program 1 Flow Cell or Cuvette Holder Static Detector S a Dynamic Detector rs ee ee Cables from Electronics Module to Light Scattering Platform UserManual OOOO S o a Dependent on user configuration PD Expert System Chapter 2 2 1 WARNING WARNING WARNING Carefully inspect the shipping carton and all components If there is any damage to the carton or to any components or if any components are missing contact both the shipping agent and Precision Detectors or its representative immediately to make a claim If any parts are missing please contact Precision Detectors customer service department or your local representative and indicate the missing items via the part numbers Warning If there is any evidence that the Precision Detectors PDExpert system has been damaged in shipping do not plug the unit into the line Contact Precision Detectors or its representative for advice The shipping cartons should be retained as they can be used if it becomes necessary to transport the system 2 3 POWER AND WATER REQUIREMENTS 2 3 1 Power Requirements Warning The Precision Detectors PDExpert System uses a three prong power cord that includ
7. lt 1 Low efficiency makes the quality of measurements vulnerable to fluctuations in the average intensity caused by the presence of large dust particles in the sample or instability of the laser intensity C 4 PDExpert Appendix C C 4 4 Determination of the Correlation Function In PDI instruments the correlation function is determined digitally The number of photons registered by the photodetector within each of a number of short consecutive intervals is stored in the correlator memory Each count in a given interval termed the sample time and denoted represents the instantaneous value of the photocurrent The series of counts held in the correlator memory is termed the digitized copy of the signal According to Equation 1 to obtain the correlation function at the average product of counts separated by sample times should be determined The number is referred to as a channel number Upto channels in principle can be measured simultaneously but usually a smaller subset of equidistant or logarithmically spaced channels is used Clearly the shortest ce delay time at which the correlation function is measured by the procedure described above is channel 1 The longest delay time cannot exceed the duration of the digitized copy Thus it is important that the correlation time fit into the interval This condition determines the choice of the sample time for the particular measurement To increase the statistical accuracy with
8. or Realign laser contact service erratic 5 10 PDExpert Chapter 5 Appendix A Specifications Sample Cell 35 uL flow through or 6 mm test tube others available Light Scattering Focused Volume 20 x 60 microns Sequential Static Angle Measurements up to 24 angles available Available Measurement Angles Both sides of the plain 5 degree increments excluding 0 and 180 degrees DLS Hydrodynamic Radius Option 0 5 to 1000 nm 0 10 degrees C 9 000 hours in normal operation Stand alone Weight a 45 kg 100 Ib 5 VA 120 240 V 50 60 Hz a Approximate weight depending on configuration PDExpert Appendix A A 1 This page intentionally left blank PDExpert Appendix A Appendix B Spare Parts and Replacement Parts Consumables Part Number KIT PDE 5001 6 mm Test Tube pack of 10 KIT PDE 5002 6 mm Test Tube pack of 50 pt Index Matching Fluid Replacement Parts Part Number ASY PDE 2021 Cell Ring Assembly Cuvette AST PDE 2022 Cell Ring Assembly Flow Cell Accessories Part Number AS Y PDE 3030 Static Detector Assembly AST PDE 3032 DLS Detector Assembly PDExpert Appendix B B 1 B 2 This page intentionally left blank PDExpert Appendix B Appendix C General Principles of Dynamic Light Scattering C 1 WHAT IS LIGHT SCATTERING ce The propagation of light may be considered as a continuous rescattering of the incident electromagnetic wave from every point of the illum
9. over the eye piece and make sure you do not see a red laser spot If you see ared spot determine the cause and rectify the condition before continuing e Look through the eye piece You should see the dark edges of the cuvette and the beam across the middle Use the screw on the top of the detector body to adjust the focus The beam should be a then bright line and you will see some dust spots when properly focused Secure the focus screw f Look through the eye piece Use the screw on the side of the detector lens to center the vertical eye piece cross hair between the 2 sides of the cuvette g Use the screw on the top of the detector lens to line up the horizontal eye piece cross hair with the laser beam h Remove the eyepiece and replace the aperture holder 3 12 PD Expert System Chapter 3 3 9 STATIC DETECTOR ALIGNMENT If a flow cell is employed the cell should be full of toluene If a cuvette is employed it should be filled with a strong scattering sample ex 5mg mL BSA and should be centered To align a static detector a Place the detector at the desired angle and secure to the optical platform b Remove the rear aperture holder and insert the eye piece 38 c Power up the main laser and set the power to 10 d Hold a piece of white paper over the eye piece and make sure you do not see a red laser spot If you see a red spot determine the cause and rectify the condition before continuing e Look through
10. the mobile phase over time This process will slowly reduce the efficiency of the cells and can be minimized by ensuring that e The sample is filtered before injection e The mobile phase is filtered before use with a 0 2 micron filter membrane e The stationary phase is stable with regard to the mobile phase e The overall system is kept clean Removal of extraneous materials can frequently be performed by flushing the system with a solvent such as methanol or acetonitrile the selection of the solvent will depend on the nature of the sample 5 3 REPLACING SYSTEM COMPONENTS 5 3 1 Filter Elements When a flow cell is employed filter elements may become clogged and must be replaced on a periodic basis The frequency of replacement is dependent on the level of particulate matter present in the mobile phase and the sample as well as the production of fine particles from the column If the filter must be replaced very frequently e g more than once a week it may be worthwhile to modify the composition of the mobile phase and or switch to a more stable column e g a column from a different manufacturer which might create fewer fines Microbial growth may occur in mobile phases with low levels lt 3 of organic solvents unless suitable precautions are taken PDExpert Chapter 5 5 3 CAUTION WARNING A daily log of system pressure should be kept so that any pressure fluctuations can be monitored as this is a good indication of cl
11. the optical platform and move the laser so that is at the maximum position from the center of the plate Retighten the screws c Place the Alignment Target Assembly at the 0 position directly across from the laser d Turn the laser on and set it to 10 using the application software 38 e Override the laser interlocks A Caution When the laser is on wear protective eyeglasses and do not look directly at the laser beam f Verify that the beam is e Collimated the diameter of the beam should be the same at the laser and the target e Level the height of the beam should be the same height from optical platform at the laser and the target A white business card is useful for this test The card should be placed about 10 cm from the laser and about 10 cm from the target e Straight the beam should be centered on the target If the laser is perfectly collimated ignore step g and continue with step h PD Expert System Chapter 3 3 5 g If the laser is not perfectly collimated 1 Loosen the two set screws on the side of the laser and the four screws on the front face of the laser Figures 3 3 Knurled Wheel Figure 3 3 Front of Laser Housing 2 Turn the knurled wheel on the laser to adjust the focus 3 Tighten the side set screws and the screws on the front and check the collimation I Note This adjustment may require a number of iterations may be a trial and error exercise as tightening the screws wi
12. via the chromatographic system PDExpert Chapter 4 4 1 ee CAUTION Ee Note If a gradient mobile phase is employed make certain that any buffers salts etc are soluble in all combinations of the mobile phase We recommend that you check the solubility using a beaker or test tube rather than via the detector as precipitation of the buffer or salt inside the detector cell may necessitate a tedious cleaning process or a service call e High purity solvents HPLC grade should be used when possible to prepare samples and for the mobile phase e The mobile phase should be is degassed so that air bubbles cannot form in the flow cell 4 3 2 Using the Test Tube Chamber When the test tube chamber is installed the sample 150 uL minimum is placed in a standard disposable 6 mm test tube and the tube is placed in the sample chamber The test tube holder is accessed by opening the hinged lid of the optical bench aution The laser can cause serious eye damage Always wear safety glasses when opening the cover Note Ensure that the lid is closed before collecting measurements ote It is critical that the sample and the test tube are clean and there is no particulate matter in the sample Dust undissolved sample and any extraneous matter will significantly affect the accuracy of your results The following actions will assist you in obtaining the best results e Make sure the sample is well dispersed e Filter out any dust part
13. 2 Principles C 1 Specifications 4 4 Enable T 3 4 Erratic Baseline 6 7 Expert Control Parameters Command 2 7 F Features 1 3 Filter Elements 6 3 Flow Cell 3 1 G Get Temp 3 4 H Hardware Configuration Dialog Box 3 3 High Background 6 8 High Back Pressure 6 9 HPLC Cell Installation with 2 4 PDExpert Index Installation 2 1 2 3 Software 2 5 Interfacing Modules 2 3 Introduction 1 1 L Laser Alignment 3 4 Power Setting 2 7 3 3 Line Fuse 6 4 Location in Laboratory 2 3 Loss of Response 6 10 M Maintenance 6 1 N Noisy Baseline 6 7 O Optical Bench 1 1 P PD Expert Control Dialog Box 2 7 PD Expert Tab 3 3 Peltier Controller 1 3 Power Up 3 1 Power Requirements 2 2 Photon Detector Module 2 4 PrecisionAcquire 2 5 Installation 2 5 Precision Deconvolve32 1 3 Installation 2 5 Preface 1x Record Temperature 3 4 Replacment Parts B 1 Replacing Detector Modules 6 5 Replacing System Components 6 3 S Safety Precautions v Sample Chamber 1 2 Sample Handling 3 1 Select Detectors for Display Dialog Box 2 6 Setting Conditions 3 3 Shutter Gain 3 4 Software Installing 2 5 Spare Parts B 1 Specifications A 1 Static Detector Module 1 2 Static Light Scattering 5 1 Layout 5 4 Specification 5 6 Static Sample Chamber 1 2 System Log System Verification 2 7 T Temperature Setting 2 7 30 3 Test Tube Sample Chamber 1 2 3 2 T Record 3 4 Troubleshooting 6 6 U Unpacking 2 1 Using
14. Figure C 1 The vector is called the scattering vector which is a fundamental characteristic of any scattering process The length of the vector is indicated in equation C 1 C 1 where 1s the refractive index of the medium is the wavelength of light is the scattering angle Partial cancellation of waves scattered by different parts of the large aggregate reduces the intensity of light scattering by a factor of where is an averaged value of the phase factors for all monomers The factor should be averaged over all possible orientations of the particle The result of this averaging yields the structure factor Expressions for the structure factors for particles of various shapes can be found elsewhere PMT LASER SAMPLE Ko C 2 PDExpert Appendix C Figure C 1 The Scattering Vector The path traveled by a wave scattered at the point with radius vector differs from the path passing through the reference point O by two segments 1 and 2 with lengths and respectively The phase difference is where is the absolute value of the wave vector or The segment is a projection of onthe wave vector of the incident beam i e Similarly and thus Vector is called the scattering vector C 4 METHOD OF QUASIELASTIC LIGHT SCATTERING SPECTROSCOPY QLS C 4 1 The Motion of Particles in Solution ce When light is scattered from a collection of N solute molecules at the observation point we also have a sum of waves scattered by
15. PDExpert Mult Angle Light Scattering Workstation User Manual Created by Precision Detectors Inc Notices This product is covered by a limited warranty A copy of the warranty is included in this manual No part of this document may be reproduced in any form or by any means electronic or mechanical including photocopying without written permission from Precision Detectors Inc Information in this document is subject to change without notice and does not represent a commitment on the part of Precision Detectors Inc No responsibility is assumed by Precision Detectors for the use of this detector or other rights of third parties resulting from its use Precision Detectors products are covered by US Patents 5 305 073 and 5 701 176 Additional patents applied for Precision Detectors PrecisionDeconvolve PrecisionDeconview Discovery32 PrecisionAcquire32 PDExpert PDDLS Batch and PDDLS CoolBatch are trademarks of Precision Detectors Inc All other brands and products mentioned are trademarks or registered trademarks of their respective holders Precision Detectors Inc 34 Williams Way Bellingham Massachusetts 02019 USA Tel 508 966 3847 Fax 508 966 3758 e mail PDI lightscatter com Web site www precisiondetectors com Copyright 2004 2006 by Precision Detectors Inc Printed in the United States of America Warranty Precision Detectors PDI will repair or replace any item that does not work as sp
16. PDExpert Appendix C C 5 C 4 6 Determination of the Diffusion Coefficient D As explained earlier temporal fluctuations in scattered light intensity are caused by the relative motions of particles in solution Two spherical waves scattered by a pair of individual particles have at the observation point a phase difference of where is the vector distance between particles As the scattering particles move over distance along the vector 7 the phases for all pairs of particles change significantly and the intensity of the scattered light becomes completely independent of its initial value The correlation time _ is thus the time required for a Brownian particle to move a distance along the vector 7 As stated above thus for Rigorous mathematical analysis of the process of light scattering by Brownian particles leads to the following expression for the correlation function of the ce scattered light C 5 C 4 7 Determination of the Sizes of Particles in Solution According to Equations C 4 and C 5 measurement of the intensity correlation function allows evaluation of the diffusion coefficients of the scattering particles The diffusion coefficient in an infinitely dilute solution is determined by particle geometry For spherical particles the relation between the radius and its diffusion coefficient is given by the Stokes Einstein equation C 6 where 1s the Boltzmann constant is the absolute temperature is the viscosity o
17. PDExpert 4 1 W Warning Labels vii Warnings v Warranty 111 Water Requirements 2 2 PDExpert Index
18. a specific analytical procedure it may be useful to use the standard sample to ensure that the system is functioning properly The user is encouraged to maintain a log of all operations of the detector maintenance activities and all observed problems should be entered into the log A discussion of the log is provided in Section 5 2 8 Note The information in this chapter is designed to provide general information about the detector Specific information about troubleshooting maintenance for a specific mode of operation is provided in the chapter that describes the detection mode 5 2 MAINTENANCE 5 2 1 Overview While the Precision Detectors PDExpert system requires little day to day maintenance we recommend that e Samples should be free of particulate matter filtering through a 0 22 um Nylon or PVDF membrane filter is a useful method Filters should be checked to ensure that extractable materials are not present and they are compatible with all constituents of the sample e The mobile phase should be filtered through a 0 22 um Nylon or PVDF membrane Make certain that the filter is compatible with all constituents of the sample e Ifthe Precision Detectors PDExpert system is used as a part of a chromatographic system and the output from the detector reflects the performance of the overall system it is important to perform all maintenance procedures for each of the various components e g the solvent delivery module injector etc
19. also be above the average within an area around this point where phases of the scattered waves do not change significantly this area is called the coherence area Within different coherence areas the fluctuations in intensity of light collected are statistically independent Increasing the size of the light collecting aperture beyond the size of a coherence area does not lead to improvement of the signal to noise ratio because the temporal fluctuations in the intensity are averaged out For a monochromatic source the scattered light is coherent within a solid angle of the order of where is the cross sectional area of the scattered volume perpendicular to the direction of the scattering Because the coherence angle is fairly small powerful 100 mW and well focused laser illumination and photon counting techniques are used in the PDI BATCH instrument C 4 3 The Correlation Function While the photodetector signal in QLS is random noise information is contained in the correlation function of this random signal The correlation function of the signal which in the particular case of QLS is the photocurrent is defined in equation C 2 C 2 The notation is introduced to distinguish the correlation function of the photocurrent from the correlation function of the electromagnetic field which is the Fourier transform of the light spectrum C 3 In the above formulae the angular brackets denote an average over time This time averaging an inh
20. am describe the application programs that are used to collect process store and report light scattering data collected with the system The documentation provided with Precision Detectors PrecisionMALS describes the application software for multi angle light scattering analysis 1 4 PD Expert System Chapter 1 CAUTION Chapter 2 Unpacking and Initial Installation 2 1 INTRODUCTION This chapter describes how the workstation should be set up in the laboratory and includes the following e Unpacking the workstation Section 2 2 e Power and water requirements Section 2 3 e Locating the workstation in the laboratory Section 2 4 Once the instrument is installed alignment of various components should be performed as described in Chapter 3 2 2 UNPACKING THE WORKSTATION Carefully unpack your shipment and inspect the contents to verify receipt of all components The Precision Detectors PDExpert system is normally shipped in two cartons Carton A contains the Optical Bench part number PD 2051 and the optical components that are installed on the optical bench The lasers the dynamic detectors static detectors beam dump and steering lens are mounted directly on the optical bench The cell must be installed as described in Chapter 3 Caution The optical bench is heavy and special care should be taken when unpacking it Carton B contains the Electronics Module part number PD2052 and a number of accessory items The precise
21. asers on and set the main laser power to 10 Caution When the laser is on wear protective eyeglasses and do not look directly at the laser beam CAUTION 1 Check that the main laser beam is centered on the target J Check that the alignment laser is aligned on the target If either laser beam is not centered slide the cell slightly on the copper plate until they are both centered k Tighten the 4 screws ae Note The beam may be a line with a bright center This is OK PD Expert System Chapter 3 3 9 CAUTION 3 6 MAIN BEAM DUMP MONITOR ALIGNMENT Note The cell must be properly aligned Section 3 5 before this operation is performed and the cell must be full of toluene To align the main beam dump monitor a Make sure the laser is off b Set the switch on the front of the PD4001 to Coarse c Place the Beam Dump Monitor over the set of holes closest to the cell do not secure with screws d Turn on the Main Laser and set the power to 10 Caution When the laser is on wear protective eyeglasses and do not look directly at the laser beam e Move the beam dump monitor away from the cell until you find the set of holes that gives you a solid red light on the LEDs that are perpendicular to the main laser beam D36 on the circuit board next to the optical platform Figure 3 5 Figure 3 5 Control Circuit Board f Secure the beam dump monitor g Loosen the 2 screws on the top of the beam dump monitor h S
22. at 85 across from the Alignment Laser e Turn the Alignment Laser on Caution When the laser is on wear protective eyeglasses and do not look directly at the laser beam f Verify that the beam is elliptical with the long side of the ellipse parallel to the plate If the beam needs to be rotated loosen the set screw on the top of the alignment laser and twist the laser cable slightly to rotate the beam g Verify that the beam is level it should be the same height at the laser and at the target slot and straight centered on the slot If it 1s not continue to move the laser with the cable until it is level then tighten the set screw h The beam should be focused in the center of the plate center of the copper piece when properly focused with the lens installed To focus the beam 1 Turn the laser off and place the lens in front of the laser as if it were being reattached but do not screw it in 2 Turn the laser on and check the focus If it focuses too soon before the plate adjust the focusing screw counterclockwise if it focuses too late past the plate adjust the focusing screw clockwise 3 To adjust the focusing screw turn the laser off and move the lens away from the laser assembly Inside the laser assembly there is a ring with 2 small holes in it this is the focusing screw 4 Use a pick or very small screw driver to turn the screw in the proper direction Put the lens back in front of the laser and turn t
23. ay down Turn the Output Current Adjust potentiometer until a power reading of 0 1 mW is obtained Measure and record the voltage between TP1 and GND J13 pin 1 Record this as the 0 Voltage Adjust the Output Current Adjust potentiometer until a power reading of 30 mW is obtained The power reading is obtained with the laser power meter Measure and record the voltage between TP1 and GND J13 pin 1 Record this as the 100 voltage Turn the Output Current Adjust potentiometer to the maximum counterclockwise position all the way down In the software set the Laser Threshold to the voltage that you obtained in step k In the software set the Laser Scale to the difference between the 100 voltage step m and the 0 voltage step k In the software set the Laser Power to 0 Measure the voltage between TP1 and GND This voltage should equal the 0 voltage that was found in step k If this voltage does not correspond to the voltage found in step k adjust the Laser Threshold value in the software until the voltage between TP1 and GND equals the 0 voltage In the software set the Laser Power to 100 Measure the laser power with the Laser Power Meter The reading should be 30 mW Tf it is not adjust the Laser Scale in the software until it does Use the application software to set the Laser Power to 50 PD Expert System Chapter 3 3 3 v Measure the laser power with the laser power meter The reading should be 15 mW
24. e connected to a common ground Temperature variation potentially due to a temperature change in laboratory If all channels are noisy or erratic Stir mobile phase Remove column and see if problem exists Clean the column and or replace Check AC line receptacle verify that you have a true ground Maintain cells at constant temperature Realign detector module contact service e e Realign laser contact service 5 5 4 High Background Signals This section describes situations where the background signal has noticeably increased in a short period of time Impurities in the mobile phase Select a new supply of mobile phase Check mobile phase reservoir filters column end frits replace if necessary Detector channel is misaligned If one channel is noisy or Realign detector module erratic contact service Laser is misaligned If all channels are noisy or Realign laser contact service erratic Contaminants leaching from system components Leaking Cell Check for wetness around cell and tighten fittings or replace cell 5 8 PDExpert Chapter 5 5 5 5 Increase in Back Pressure Accumulation of particulates Replace in line filter elements from the mobile phase or injected samples Ensure that the mobile phase and or samples are filtered through a 0 22 um Nylon or PVDF membrane filter Use a mobile phase with a substantial fraction of an organic solvent to prevent bacterial growth Use
25. ecified in our User s Manual for a period of 1 year from the date of delivery of your System This warranty does not cover damage from causes outside PDI s control if you have made any alteration to any item if you use any item except within a liquid chromatography system and within our specified temperature If we have delivered a computer and or other manufacturer s liquid chromatography equipment as part of your System you agree to accept the computer or chromatography equipment manufacturer s warranty in place of PDI s warranty THE WARRANTIES CONTAINED HEREIN ARE IN LIEU OF ALL OTHER WARRANTIES EXPRESS OR IMPLIED INCLUDING ANY WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WHICH ARE EXPRESSLY DISCLAIMED BY PDI Repair of malfunctioning items In the event that your system needs repair after the 1 year warranty period we agree to repair it for you at our then current labor and material rates if it is sent freight and insurance prepaid to our headquarters Maintenance For a period of 5 years following delivery of your system we agree to clean and maintain items for you at your request charging you our then current labor and material rates if the system is sent to our headquarters freight and insurance prepaid Limitations of liability PDI is not liable for any damages of any kind incidental or consequential and under no circumstance will damages be in excess of the amount paid by you to PDI for the System re
26. en EAT ee rena On ORR ONT TEE 1 1 E2 Feat res orhe CLC CIOL ich oh he dved ties fans Sedcacta aa a 1 3 Lo sebUCiiire On the NianW asst cis ccd 2s ence sinsancretreas ses taisaen ace vateaneshiee hans tace smiaaneddsoateesuatmecsedacaseuss 1 4 1 4 For Additional Information cccccccccccccccceececceceeeeaeaaeeeeesssesceeecsseeeeeeeeeeeeeeeeeaaaegeessseesseseeees 1 4 Chapter 2 Unpacking and Installation eeeeeeeeoccccscssssssescossssececccecccceccososssssececcosssssssssscecccccccessssssssse 2 1 Ze o O E A nemrenee nents me rorteeel 2 1 22 Unpackine the Workstation E A 2 1 2 3 Fowerand Water Requirements cisnienia a NA 2 2 23A POWER RCQUINE MC IIS cies ci ds cree cum caps sod a a a a a aa weleisialos 2 2 23 2 COONS REGU MENS assists cus ctnes Sod Gale dunotua tes duvoiees a Sines ted Gan sed Sadan E teeta tedoss 2 3 2 4 Locate the Detector in the Laborator y sccsssri e a ero 2 3 29 merice Ne Module Seaan a ehwsnittunca E 2 3 Chapters Aliens the Systemi ssriirse tses s a a E 3 1 E OR E e MO PCR Ne E E E EE Tern Ce nen nur 3 1 Siz Laser Power Camp Eamon nnee a ee E a 3 2 3r Manm Ea ser AN SiMe essre N 3 5 3 4 Aligning the Alignment Laser ccccccccccccccceeeeecceeeeeeeccaeeesssssseeeeeessseeeeeecceeeeeseesaeaggeggssnas 3 8 Z Wnitial Cell Ale TEN ea T T ee atid onal etGeted ee aacdaamaacc it 3 9 3 0 Mair Beam Dump Monitor Alionment srunsa a eek 3 10 3 7 Alignment Beam Dump Monitor Alignment
27. er First the lower are the statistical errors of the measurements the smaller the smoothing parameter can be without loss of stability This will yield finer resolution in the reconstructed distribution Second narrow distributions generally require much less smoothing and can be reconstructed much better than can wide distributions This is because oscillations in narrow distributions are effectively suppressed by non negativity conditions PDExpert Appendix C Cc 9 The moments of the distribution reconstructed by the regularization procedure coincide closely with those obtained by other methods However the regularization procedure in addition gives unbiased apart from smoothing information on the shape of the distribution This shape cannot be extracted through use of the direct fit method nor from cumulant analysis In a typical QLS experiment regularization analysis can resolve a bimodal distribution with two narrow peaks of equal intensity if the diffusion coefficients corresponding to these peaks differ by more than a factor of 2 5 FOOTNOTES 1 R Pecora Dynamic Light Scattering Applications of Photon Correlation Spectroscopy Plenum Press New York 1985 2 K S Schmitz An Introduction to Dynamic Light Scattering by Macromolecules Academic Press Boston 1990 3 H C van de Hulst Light Scattering by Small Particles Dover New York 1981 4 A N Tikhonov and V Y Arsenin Solution of Ill Posed Pr
28. er 2 Describes how the detector should be installed in the laboratory and provides information about initial checkout of the system General Operation of the Detector Chapter 3 Presents a discussion about the use of the detector in an HPLC system This chapter includes information about sample handling and related topics that are general in nature rather than specific to a given module Using PDExpert Chapter 4 Includes a discussion about sample handling and setting system parameters Maintenance and Troubleshooting Chapter 4 Describes a series of operations that the operator should perform to optimize detector performance and determine the cause of problems Specifications Appendix A Describes the physical characteristics of the major components and provides certification information Spare Parts and Replacement Parts Appendix B Presents a list of components to maintain the system General Principles of Dynamic Light Scattering Appendix C Describes the fundamental principles of Light Scattering Measurements from a mathematical perspective Advanced Calculations Appendix D Presents a discussion of the use of multi angle light scattering calculations to obtain additional information about the polymer FOR ADDITIONAL INFORMATION The documentation provided with Precision Detectors Discovery32 static measurements and Precision Detectors PrecisionDeconvolve32 dynamic measurements application software progr
29. erent feature of the QLS method is necessary to extract information from the random fluctuations in the intensity of the scattered light For very large delay times the photocurrents at moment and are completely uncorrelated and is simply the square of the mean current At is obviously the mean of the current squared Since for any _ the initial value of the correlation function is always larger than the value at a sufficiently long delay time The characteristic time within which the correlation function approaches its final value is called correlation time For example in the most practically important case of a correlation function that decays according to an exponential law P FTT the correlation time is the parameter In the majority of practical applications of QLS the scattered light is a sum of waves scattered by many independent particles and therefore displays Gaussian statistics This being the case there is a relation between the intensity correlation function and the field correlation function C 4 Here is the normalized field correlation function 1s the average intensity of the detected light and is the efficiency factor For perfectly coherent incident light and for scattered light collected within one coherence area the efficiency factor is 1 If light is collected from an area times larger than the coherence area fluctuations in light intensity are averaged out and the efficiency factor is of the order of I J lt
30. es a ground wire The unit must be connected to a properly grounded three prong power outlet to ensure safety and proper operation If there is any doubt about the power supply a qualified electrician should be contacted to ensure a properly operating and properly grounded power outlet Note The PDExpert system accepts all voltages from 90 to 250 V 50 60 Hz The 120 VAC power cable consists of a 3 prong receptacle for attachment to the power inlet on the back of the optical bench and a three prong plug for connection to a standard U S grounded output The 100 230 240 VAC power cable consists of a 3 prong receptacle for attachment to the power inlet on the back of the optical bench The other end of the cable has three color coded wires that are used to attach to the appropriate plug The color coding of the wires meets ISO and VDE conventions as follows Earth Ground Green with Yellow Stripe Neutral Blue Line Brown Warning The power plug should be installed by a qualified electrician and should be an approved plug e g CE TUV The power consumption of the system is approximately 5 VA If the workstation is used with an HPLC system it should be connected to an electrical line that shares a common ground with other components of the chromatographic system e g computers recorders the HPLC system controller pump autosampler etc This will avoid ground loops which can create erratic results e g varying background high n
31. et the switch on the front of the PD4001 Electronics Module to Fine 1 Slide and twist the top of the beam dump monitor until the center LED is green J While holding the top of the beam dump monitor in place set the switch to Coarse and make sure that the LED is still red If it is no longer red try to get it back to red by moving the top of the monitor k Set the switch back to fine Once you get the LEDs to be Green in the Fine and Red in Coarse settings secure the top of the beam dump monitor 3 10 PD Expert System Chapter 3 Caution The beam dump monitor should not move when you secure the screws Make sure that it is still Ld Green in Fine and Red in Course once it is secured 3 7 ALIGNMENT BEAM DUMP MONITOR ALIGNMENT IS Note The Cell Alignment must be completed first and the cell must be full of toluene To Align the Alignment Beam Dump Monitor a Make sure the alignment laser is off b Set the switch on the front of the PD4001 to Coarse 38 c Place the Beam Dump Monitor over the set of holes across from the alignment laser Secure the Beam Dump Monitor to the platform d Turn on the Alignment laser 6 Caution When the laser is on wear protective eyeglasses and do not look directly at the laser beam e Monitor the set of LEDs that are perpendicular to the alignment laser beam D35 on the circuit board next to the optical platform while adjusting the height of the beam using the screw on the top of the a
32. f the DLS 2000 Photon Detector Module To connect the modules a Connect the fiber optic cable that is attached to the Optical Module Figure 2 2 to the screw connector on the front of the DLS 2000 Photon Detector Module Figure 2 3 Screw the cable onto the connector by applying it to the connector and turn it back one turn until it mates with the thread and screw it in finger tight aution Do not over tighten this connector a finger tight connection is sufficient Do not use a tool to camion ighten this connector b Check that the Batch Mode Flow Switch on the rear panel of the DLS 2000 Photon Detector Module Figure 2 3 is set to the 256x position This configures the correlator to have 256 real channels in the 1024 channel space 2 4 PD Expert System Chapter 2 c d e Connect the USB cable from the Photon Detector Module Figure 2 3 to a USB socket on the personal computer Connect the communication cables between the Electronic Module and the Light Scattering Module There are two cables which are keyed to fit in the appropriate manner If you anticipate the use of the Peltier system to cool the sample cell connect a source of cold water to the inlet on the optical bench and connect the outlet to the waste line or the re circulating bath and connect the outlet to a suitable waste bottle It is recommended that the distance between the end If the HPLC flow cell is employed connect the end of the column to the inle
33. f the solution For non spherical particles it is customary to introduce the apparent hydrodynamic radius defined as C 7 where is the diffusion coefficient measured in the QLS experiment For non spherical particles it is important to note that the diffusion coefficient is actually a tensor the rate of particle diffusion in a certain direction depends on the particle orientation relative to this direction As small particles diffuse over a distance their orientation may be changed many times QLS measures the average diffusion coefficient for these particles Particles of a size comparable to or larger than essentially preserve their orientation as they travel a distance smaller than their size For these particles the single exponential expression of equation A 5 for the field correlation function is not strictly applicable For particles that are small compared to the hydrodynamic radius is calculated numerically and in some cases analytically for a variety of particles shapes The important analytical formula for the prolate ellipsoid with the long axis a and the ratio of lengths of the short axis to the long axis p is C 8 The above formulae connecting the diffusion coefficient or hydrodynamic radius to particle geometry are strictly applicable only for infinitely dilute solutions At finite concentrations two additional factors significantly affect the diffusion of particles viscosity and interparticle interactions Visc
34. freshly prepared mobile phase Bacterial growth in the mobile phase may lead to clogging of the filter Clogged injector or column Isolate suspect component Refer to manufacturer s cleaning directions or replace rotor seal and or stator face on injector Ensure that the mobile phase and sample are filtered before use Plugged Tubing ee Isolate plugged tubing and replace Clogged cell Remove cell from system and check back pressure Clean cell PDExpert Chapter 5 5 9 5 5 6 Loss of Response This section describes the abrupt loss of a peak or peaks from the chromatogram when using a set of analytical conditions which is known to provide a useful chromatogram Accidental change of a Check settings parameter in the software Compounds of interest not Some compounds will Check stability as a function of sufficiently stable decompose as a function of time and prepare fresh time standards If necessary change conditions Use a cooled autosampler Change in pH or mobile phase Mobile phases should be mobile phase are clean Detector channel is misaligned If one channel is noisy or Realign detector module erratic 5 5 7 Inability to Autozero the Signal Autozero has been performed Reduce the noise or increase on a very noisy signal the current gain range Detector channel is misaligned If one channel is noisy or Realign detector module erratic contact service Laser is misaligned If all channels are noisy
35. full power of a fraction thereof This value will be adjusted downwards if toomany photons are being received overload Overload may occur when the sample particles are large the sample concentration is high and or when low angle scattering is used This is to be performed by an authorized service representative aution Use caution when aligning the laser to prevent exposure of your eyes to the laser beam ote When a non ambient temperature is employed make certain that sufficient time is provided to vasure that the sample has attained the desired temperature This issue is especially important when the sample chamber is used with test tubes PDExpert Chapter 4 4 3 4 5 COLLECTING LIGHT SCATTERING DATA At a given time one dynamic light scattering measurement and up to eight static measurements can be performed Dynamic light scattering data is initially processed via the PD2000 DLS Module and static light scattering data is initially processed by the work station If desired you can independently monitor one channel of static light scattering data using the contacts on the right side of the electronics module The switch immediately to the left of the contacts is used to select the static detector to be monitored From time to time standards should be run to determine if the system is operating in an acceptable manner For dynamic measurements see Section 5 6 for static measurements see Section 4 6 2 If the signal for a give
36. gardless of the form of the claim PDExpert Warranty iil This page intentionally left blank PDExpert Warranty Warnings and Safety Precautions The Precision Detectors PDExpert Light Scattering Workstation can be used as a detector system for High Performance Liquid Chromatography Size Exclusion Chromatography and Gel Permeation Chromatography or it can be used as a static system with atest tube as a sample holder The following precautions should be followed to minimize the possibility of personal injury and or damage to property while using the instrument 1 Maintain a well ventilated laboratory The mobile phase or solvent typically contains a volatile organic solvent Ensure that the laboratory is well ventilated so that a buildup of vaporized solvent cannot occur 2 Avoid open flames and sparks Do not use an open flame in the laboratory and do not install any equipment that can cause sparks in the same room as the instrument 3 The instrument must be plugged into a grounded power line Ensure that all parts of the system are properly grounded It is strongly recommended that all parts of the system are connected to a common ground 4 Treat all samples and mobile phases as if they are capable of containing hazardous substances or transmitting disease The sample and or mobile phase may contain compounds which may present a health hazard If you are analyzing biological clinical samples treat them in accordance wit
37. h the infectious disease control program of your institution 5 Do not attempt to over ride High Voltage or Laser Safety Interlocks Do not run the instrumentation unless all protective devices are functioning properly 6 Do not remove the cover of the system while the power is provided to the laser The laser can cause serious eye damage PDExpert Warnings and Safety Precautions v vi This page intentionally left blank PDExpert Warnings and Safety Precautions Warning Labels A o l PDExpert Warning Labels Electrical Requirements 100 120 230 240 V 50 60 Hz To be serviced by factory authorized personnel only For assistance call 508 966 3847 or your local representative Protective conductor terminal Earth ground terminal Danger Laser radiation when open Avoid direct exposure to beam This page intentionally left blank viii PDExpert Warning Labels Preface The Precision Detectors PDExpert Multi Detector Workstation is a multi detector system for light scattering measurements Flowing samples High Performance Liquid Chromatography Size Exclusion Chromatography and Gel Permeation Chromatography as well as stationary samples in a test tube can be studied The system can provide the following types of measurements e Static Light Scattering to provide R and molecular weight measurements e Dynamic Light Scattering with on board correlators and photon counting to provide flow through R
38. he distribution that gives the average diffusion coefficient can be determined from the initial slope of the field correlation function Indeed using equation C 12 it is straightforward to show that C 12 The second cumulant moment of the distribution can be obtained from the curvature second derivative of the initial part of the correlation function As in the direct fit method the accuracy of the real QLS experiment allows determination of at most three moments of the distribution The first moment can be determined with better than 1 accuracy The second moment the width of the distribution can be determined with an accuracy of 5 10 The third moment which characterizes the asymmetry of the ce distribution usually can be estimated with an accuracy of only about 100 C 5 5 Regularization The regularization approach combines the best features of both of the previous methods The advantage of the cumulant method is that it is completely free from bias introduced by a priori assumptions about the shape of _ assumptions that are at the heart of the direct fit method On the other hand reliable a priori information on the shape of the distribution function in addition to the experimental data improves significantly the quality of results obtained by the QLS method The regularization method assumes that the distribution is a smooth function and seeks a non negative distribution producing the best fit to the experimenta
39. he laser on again 5 Recheck the focus Continue doing this until the focus is correct It may be necessary to perform this operation several times to optimize the beam focus 1 Reattach the focus steering lens The beam should still be centered on the target slot If it is not use the screw on the side of the lens to center the beam 3 8 PD Expert System Chapter 3 3 5 INITIAL CELL ALIGNMENT To perform the initial cell alignment a Turn off the main laser and the alignment laser b Place the alignment target at 0 and the Alignment Slot at 85 c Puta thin layer of thermal grease on the copper plate in the center of the optical bench this is the heat sink The layer should be just enough to coat the plate you should still see the copper color of the plate d Puta thin layer of thermal grease on the bottom of the cell 3 e Place the cell on the copper plate so that the thermistor wires are toward the main laser f Attach the cell to the copper plate with 4 screws but do not tighten them g Fill the cell with Toluene If the cell holder is for a flow cell use a syringe or pump to fill the cell and make sure there are no air bubbles If the cell holder is for a cuvette fill the cell with toluene using a pipette until the meniscus is just above the top of the quartz ring Caution Toluene is flammable and is toxic Wear gloves when handling the liquid and avoid getting it on your skin CAUTION h Turn both l
40. ibutions from closely spaced positive and negative spikes in the particle distribution cancel each other We discuss below three approaches for dealing with this i1l posed problem C 5 3 The Direct Fit Method The simplest approach is the direct fit method In this method the functional form of is assumed a priori single modal bimodal Gaussian etc and the parameters of the assumed function that lead the best fitof to then are determined This method is only as good as the original guess of the functional form of Moreover using the method can be misleading because it may confirm nearly any a priori assumption made It is also important to note that the more parameters there are in the assumed functional form of _ the better the experimental data can be fit but the less meaningful the values of the fitting parameters become In practice typical QLS data allow reliable determination of about three independent parameters of the size distribution of the scattering particles C 5 4 The Method of Cumulants The second approach is not to attempt to reconstruct the shape of the scattering particle distribution but instead to focus on so called stable characteristics of the distribution 1 e characteristics which are insensitive to possible fast oscillations In particular these stable characteristics are moments of the C 8 PDExpert Appendix C distribution or closely related quantities called cumulants 2 The first cumulant moment of t
41. icles and un dissolved materials using a filter size that will not remove your sample or dissolve in the solvent If you cannot use a filter because your sample adheres to it filter the solvent before preparing the solution As an alternative centrifuge the sample to remove particulate matter 15 min at 5000g should be sufficient e High purity solvents HPLC grade should be used when possible to prepare samples wash test tubes etc e Remove any air bubbles in the sample before measurement Air bubbles may form if the sample has been sitting for an extended period of time e Ifsample gets on the outside of the test tube rinse with clean solvent and dry with a lint free tissue Do not air dry the tube unless you are certain that the solvent is clean e Do not get fingerprints in the measuring area of the test tube lower 6 cm 1 5 If you have touched the test tube in the measuring area wipe the tube with a methanol soaked lint free tissue Fingerprints will scatter light and create scatter signals that are not related to your sample 4 2 PDExpert Chapter 4 CAUTION eS e Do not use scratched test tubes as the scratches can scatter light e Keep the sample covered at all times e Pour the sample into the test tube and place the test tube into the sample chamber do not pour the sample into the test tube when it is in the sample chamber as a spill could contaminate the sample chamber Use a narrow tipped syringe
42. inated medium The amplitude of each secondary wave is proportional to the polarizability at the point from which this wave originates if the medium is uniform rescattered waves will have the same amplitude and interfere destructively in all directions except in the direction of the incident beam If however at some location the index of refraction differs from the average value the wave that is rescattered at this location is not compensated for and some light will be observed in directions other than the direction of incidence and light scattering occurs Scattering of light can be viewed as a result of microscopic heterogeneities within the illuminated volume and macromolecules and supramolecular assemblies are examples of such heterogeneities C 2 LIGHT SCATTERING TECHNIQUES Static light scattering probes concentration molecular weight size shape orientation and interactions among scattering particles by measuring the average intensity and polarization of the scattered light Static light scattering measurements which are performed at different scattering angles provide information on the molecular weight size and shape of the scattering particles Measurements of the intensity of light scattering as a function of concentration yield the second virial coefficient which is the key characteristic of the strength of attractive or repulsive interactions between solute particles Quasielastic dynamic light scattering 2 probes the relati
43. individual particles Figure A 1 Each particle could be at any random location within the scattering volume the intersection of the illuminated volume and the volume from which the scattered light is collected Since the size of the scattering volume is much bigger thang with the exception of nearly forward scattering where _ the phases of the waves scattered by different particles will vary dramatically As a result the average amplitude of the scattered wave is proportional to and the average intensity of the scattered light is simply times the intensity scattered by an individual particle as expected The local intensity however fluctuates from one point to another around its average value The spatial pattern of these fluctuations in light intensity called an interference pattern or speckles is determined by the positions of the scattering particles As the scattering particles move the interference pattern changes in time resulting in temporal fluctuations in the intensity of light detected at the observation point The essence of the QLS technique is to measure the temporal correlations in the fluctuations in the scattered light intensity and to reconstruct from these data the physical characteristics of the scatterers PDExpert Appendix C C 3 C 4 2 Coherence Area There is a characteristic size for speckles in the interference pattern If the intensity of the scattered light is above average at a certain point it will
44. ioration of the sample In the first case the polydispersity itself is often an object of interest while in the second case it is an obstacle In both instances polydispersity significantly complicates data analysis For polydisperse solutions equation A 5 for the normalized field correlation function must be replaced with C 9 In this expression is the diffusion coefficient of particles of the i th kind and 1s the intensity of light scattered by all of these particles where is the number of particles of i th kind in the scattering volume and 1s the intensity of the light scattered by each such particle For a continuous distribution of scattering particle size equation C 10 is generalized as follows PDExpert Appendix C C 7 C 10 where is the intensity of light scattered by particles having their diffusion coefficient in the interval D D dD N D dD is the number of these particles in the scattering volume is the intensity of light scattered by each of them The goal of the mathematical analysis of QLS data is to reconstruct as precisely as possible the distribution function or from the experimentally measured function It should be noted that polydispersity is not the only source of non single exponential correlation functions of scattered light Even in perfectly monodisperse solutions interparticle interactions orientation dynamics of asymmetric particles and conformational dynamics or deformations of flexible particle
45. is possible that a fuse has blown Warning Disconnect the system from the line power before removing the cover from the power input module For continued protection against the risk of fire replace only with the same type and rating of fuse To replace a fuse a Remove the cover from the power input module using a small screwdriver or similar tool b Replace the fuse s c Return the cover to the power module 5 4 PDExpert Chapter 5 5 3 3 Replacing or Moving a Static Detector Module To replace or move a static detector module a Turn off power to the system b Disconnect the cable connecting the detector module from the system board c Remove the two screws that attach the module from the base plate 5 3 4 Replacing or Moving the Dynamic Detector To replace or move a dynamic detector module a Turn off the power to the system b Disconnect the fiber optic cable connecting the detector module from the PD2000 DLS Module 5 c Remove the two screws that attach the module from the base plate 5 4 ESTABLISHING A SYSTEM LOG A log that includes the usage and maintenance as well as any comments about operation of the system should be maintained This log should include the date time technician s name number of samples any maintenance activities and any relevant user comments about the performance of the system A typical sample log is presented as Figure 5 1 If a prescribed sample log format is provided by your o
46. l data As discussed above the ill posed nature of the deconvolution problem means that distributions differing by the presence or absence of a fast oscillating function produce very similar correlation functions The regularization requirement that the distribution should be sufficiently smooth eliminates this ambiguity allowing unique solutions to the minimization problem There are several methods that utilize this approach for reconstructing the scattering particle distribution function from QLS data All of these methods impose the condition of smoothness on the distribution but differ in the specific mathematical approaches used for this purpose One popular program originally developed by Provencher is called CONTIN Precision Detectors use a proprietary algorithm of superior quality All regularization algorithms produce similar results and incorporate the use of a parameter that determines how smooth the distribution has to be The choice of this parameter is one of the most difficult and important parts of the regularization method If the smoothing 1s too strong the distribution will be very stable but will lack details If the smoothing is too weak false spikes can appear in the distribution The rule of thumb is that the smoothing parameter should be just sufficient to provide stable reproducible results in repetitive measurements of the same correlation function Two facts are helpful for choosing the appropriate smoothing paramet
47. lignment laser lens until you get a Red LED f Loosen the 2 screws on the top of the beam dump monitor g Set the switch on the front of the PD4001 box to Fine h Slide and twist the top of the beam dump monitor until you get the center LED to be Green 1 While holding the top of the beam dump monitor in place set the switch to coarse and see that it is still red If the led is no longer red move the top of the monitor and set it back to fine J Once you get the LEDs to be Green in Fine and Red in Course secure the top of the beam dump monitor amp Caution The beam dump monitor should not move when you secure the screws Be aware that it cannot move when you secure the screws Make sure that it is still Green in with the Fine setting and Red in Coarse setting once it is secured PD Expert System Chapter 3 3 11 CAUTION 3 8 DLS DETECTOR FOCUS AND ALIGNMENT If a flow cell is employed the cell should be full of toluene If a cuvette is employed it should be filled with a strong scattering sample ex 5mg mL BSA and should be centered in the cell holder To focus and align a DLS Detector a Place the detector at the angle you want and secure the detector to the optical platform b Remove the rear aperture holder and insert the eye piece c Turn the main laser and set the power to 10 Caution When the laser is on wear protective eyeglasses and do not look directly at the laser beam d Hold a piece of white paper
48. ll make small changes in the focus It may be necessary to position the beam slightly off focus when you start to tighten the screws so that the focus will be correct when the screws are tightened 3 6 PD Expert System Chapter 3 h When the beam is collimated level and straight turn the laser off 1 Place the steering lens on the optical platform on the set of holes nearest the center of the platform on the same side as the laser is positioned The curved surface of the lens should be facing the laser j Turn the laser on and view the focus The laser should be focused at the center of the optical platform Move the steering lens toward the laser until you find the set of holes that provides the best focus k Turn the laser off 1 Secure the steering lens to the optical platform m Turn the laser back on Although the beam is no longer level it still needs to be straight centered on a the center of the target Use the screw on the side of the steering lens Figure 3 4 to adjust the beam so it is centered on the target Top Screw Side Screw Figure 3 4 Steering Lens Adjustment Screws PD Expert System Chapter 3 3 7 CAUTION 3 4 ALIGNING THE ALIGNMENT LASER To align the Alignment Laser a Remove the Cell Assembly and the Alignment Beam Dump Monitor from the Optical Platform b Remove the front lens from the alignment laser c Place the alignment laser at the 95 position d Place the Alignment Slot
49. n sample has decreased it may be necessary to realign the corresponding detector module if the signal for all samples has decreased it may be necessary to realign the laser Contact Precision Detectors or your local representative for assistance The position of the detector modules is specified by the user and set at the time of manufacture If desired you can move detector modules as described in Section 5 3 3 and 5 3 4 The Expert workstation is capable of collecting light scattering data at a number of angles simultaneously A discussion of the use of the system is presented in the software manual and a description of how the multi angle data can be employed is presented in Appendix D 4 4 PDExpert Chapter 4 Chapter 5 Maintenance and Troubleshooting 5 1 INTRODUCTION Optimum performance of the Precision Detectors PDExpert detector system will be obtained when the user performs a series of routine maintenance activities on a periodic basis This chapter provides e A listing of various activities that should be performed on a routine scheduled basis Section 5 2 e A discussion about troubleshooting Section 5 3 When the system is initially installed the analyst should obtain data chromatogram from a well defined 5B sample before developing a new analytical procedure This data chromatogram can serve as a benchmark to be used to check the performance of the system Similarly if problems are observed in the use of
50. nce some problems from the pump or column are observed via the detector we include a detailed discussion of potential problems for a typical system which includes HPLC with light scattering detection 5 5 3 Erratic Noisy Baseline Dissolved gases in the pumphead Dissolved gases in the detector cell Pump Head Problem If dissolved gases come out of solution in the pumphead the flow rate will be variable This will cause cyclic noise The frequency of the pattern will increase as the flow rate is increased If dissolved gases come out of solution in the detector cell sharp noise spikes may be observed Check pump seals check valves for wear or leaks Sparge the mobile phase with a brisk flow of He for a few minutes or degas it via an ultrasonic bath Increase the flow rate for 30 min to remove gases Use a vacuum degasser Remove the detector from the system flush it with water and then with degassed MeOH and again with water Replace seals if worn Replace check valves if necessary Leaks in the system Check for leaks in system Tighten all fittings Mobile phase not properly mixed Contaminants eluting from column System not grounded Cell Temperature not held constant Detector channel is misaligned If one channel is noisy or erratic Laser is misaligned PDExpert Chapter 5 Trace levels of organic compounds may be tightly retained by the column All components of the system must b
51. ny particulate matter Replace solvent filters if they are discolored the mobile phase should be filtered with a 0 2 micron filter membrane g Runa test run using a standard or a well defined sample and ensure that the signal has not changed appreciably from day to day h Monitor the pressure and ensure that it has not changed significantly from the level that was observed on the previous day 5 2 2 2 Weekly Maintenance For systems with a flow cell the following should be performed on a weekly basis a Check and replace if necessary any filter elements in the HPLC system b Replace the pump washing solution if applicable c Perform a flow rate check on the pump d Perform all of the daily activities 5 2 PDExpert Chapter 5 5 2 2 3 Monthly Maintenance For systems with a flow cell the following should be performed on a monthly basis a Inspect the condition of the tubing to detect potential problems and replace if necessary b Perform all of the daily and weekly activities 5 2 2 4 Quarterly Maintenance For systems with a flow cell the following should be performed on a quarterly basis a Inspect and change the seals check valves and pistons in the solvent delivery system if necessary b Replace the 10 um mobile phase filters c Replace all filter elements in the HPLC system 5 d Perform all of the daily weekly and monthly activities 5 2 2 5 Cell Maintenance The cells may adsorb analyte or impurities in
52. oblems Halsted Press Washington 1977 5 D E Koppel J Chem Phys 57 4814 1972 6 S W Provencher Comput Phys Commun 27 213 1982 cC 10 PDExpert Appendix C Appendix D Multi Angle Light Scattering to Determine M and lt r gt The Zimm Plot The discussions about static light scattering and dynamic light scattering that are presented in Chapters 5 and 6 are based on a number of assumptions including DO e the particles are monodisperse e the particles are present at infinite dilution e the solvent does not affect the configuration of the polymer The analyst will note that in many cases there is at least some degree of polydispersity of the polymer and it is known that the configuration of many polymers is affected by the nature of the solvent In addition the particles are not present at infinite dilution and the light scattering phenomena may be affected re may be affected by interparticle interaction and or the interference between the light scattered by different particles While the assumptions are useful as they simplify data analysis more advanced approaches may be more valid Collecting light scattering data at several angles and using the Zimm plot can lead to the determination of more accurate physical properties The scattering of light by a solution of polymers as a function of the size of the random coil is presented as equation D 1 Ke 1 116 0 mi A ea in 2A c D 1 Ro My Mv 3 6 2 2
53. ogged filter elements To change a filter a Turn off the mobile phase flow and allow system pressure to drop to zero before disconnecting any components Caution Do not remove the system pressure by opening a fitting on the high pressure side of the column The rapid pressure drop can damage various components in the overall system b Remove the filter assembly from the chromatographic system by removing the nuts on either end of the assembly c Remove both end nuts from the filter assembly d Remove the used filter If necessary CAREFULLY insert a small wooden dowel or plastic rod to dislodge the filter e Rinse the filter housing with deionized water f Replace one end nut Insert a new filter element into the filter housing Ensure that the element is properly centered and seated against the surface of the end nut g Re install the filter housing in the chromatographic system Ensure that the direction of flow is as indicated on the filter housing Note Initially only the upstream end of the filter should be attached to the HPLC system Pump about 5 mL of the mobile phase through the filter to waste before attaching the downstream end of the filter to the cell this step will serve to wash the filter and ensure that particulate matter does not enter the cell 5 3 2 Changing the Line Fuse If the unit does not power up when the main power switch is turned on or if the display is suddenly no longer illuminated it
54. oise etc Use a power strip to plug all HPLC components into a common ground if necessary 2 2 PD Expert System Chapter 2 Although the workstation contains a built in line filter to reduce interference at any input voltage connection to an electrical line which also serves units with a large power drain or which may be subject to power surges typical systems of this type include centrifuges ovens refrigerators and fume hoods is not recommended In addition a surge suppressor or an uninterrupted power supply UPS should be used Surge suppressors or uninterrupted power supplies designed for personal computers are suitable 2 3 2 Cooling Requirements If you intend to operate the system below ambient temperature connect a source of cold running water to the cooling ports either from a faucet or a closed circuit source This running water will be used to cool the heat sink of the Peltier cooling electronics The temperature of the water is related to the achievable temperature when you are operating below ambient temperature In most cases cold water from the tap is satisfactory when operating in the normal range of the instrument If the desired temperature is not attainable using tap water it may be necessary to chill the cooling water 2 4 LOCATING THE DETECTOR IN THE LABORATORY I Note If the PDExpert is used with an HPLC SEC or GPC system place the PDExpert system in a location such that the distance between the end of the c
55. olumn and the flow cell of the PDExpert system is minimized This will reduce post column band broadening effects and optimize chromatographic resolution The PDExpert should be placed in an area that is free from drafts or significant temperature changes Avoid placing the system near air conditioning vents windows ovens etc The system and associated HPLC system if employed should be placed on a sturdy laboratory bench or table that provides access to all components and provides sufficient working space The weight of the system is approximately 45 kg 100 Ib depending on the system configuration 2 5 INTERFACING THE MODULES Caution Do not apply electric power to the system until instructed to do so in these instructions If power caution 4g connected while either end of the fiber optic cable is exposed to room light high light levels may cause excessive heating and damage the detectors and or the power supply This damage is not covered by the warranty I Note It is not necessary to remove the cover of the optical bench during the installation The rear panel of the Electronics Module is shown in Figure 2 1 the back panel of the Optical Module is shown in Figure 2 2 and the back panel of the PD2000 DLS Module is shown in Figure 2 3 PD Expert System Chapter 2 2 3 Water To Flow Cell From Flow Cell Communication Analog Analog Ports Sector Ports Figure 2 2 Rear Panel of the Optical Module Figure 2 3 Rear Panel o
56. on a routine basis The user should refer to the operating manuals for each part of the system and perform the necessary activities on a periodic basis PDExpert Chapter 5 5 1 5 2 2 Maintenance For Systems with a Flow Cell Note The frequency for doing the various activities is dependent on the sample type mobile phase composition sample cleanliness and a number of other factors The frequency indicated below should be considered as a guideline As the user gains experience with the system and the analytical procedure it is likely that a user generated protocol will be developed 5 2 2 1 Daily Activities For systems with a flow cell or every time that the unit is started up a Check that the pump is working properly and the solvent bottle s contain sufficient mobile phase for the expected analysis b There is sufficient pump seal wash solution if applicable c The pump seal wash system is primed and flowing properly if applicable d All connections are leak free Check for the presence of salt on joints and the base of all components If a salt deposit or leak is observed tighten the offending joint but do not overtighten if necessary make a new fitting e If an autosampler is in the system check that the tray temperature is correctly set the syringe is bubble free and the wash syringe has sufficient wash solution for the day s analyses f The filters in the solvent bottle and the mobile phase should not include a
57. on of the solute particles is much more straightforward when these particles are treated as individual scatterers Intensity of the light scattered by a single particle 1s dependent on the mass and the shape of the particle In this discussion we will consider an aggregate composed of monomers and the amplitude of the electromagnetic wave scattered by an individual monomer is at the point of observation If the size of the aggregate is small compared to the wavelength of light all waves scattered by individual monomers interfere constructively and the resulting wave has an amplitude Since the intensity of a light wave is proportional to its amplitude squared the intensity of the light scattered by the aggregate is proportional to the aggregation number squared where I is the intensity of scattering by a monomer The quadratic dependency of scattering intensity on the mass of the scatterer is the basis for optical determination of the molecular weight of macromolecules It is this dependency which is accounted for by the Mass Normalization function of PrecisionDeconvolve If the size of an aggregate particle is not small compared to the interference of the electromagnetic waves scattered by the constituent monomers is not all constructive and the phases of these waves must be taken into account If the phase of a wave scattered at the origin is used as a reference the phase of a wave scattered at a point with radius vector is as shown in
58. osity generally increases with the concentration of macromolecular solute According to equation A 6 this leads to a lower diffusion coefficient and therefore to an increase in the apparent hydrodynamic radius Interactions C 6 PDExpert Appendix C between particles can act in either direction If the effective interaction is repulsive which is usually the case for soluble molecules otherwise they would not be soluble local fluctuations in concentration tend to dissipate faster meaning higher apparent diffusion coefficients and lower apparent hydrodynamic radii If the interaction is attractive fluctuations in concentration dissipate slower and the apparent diffusion coefficients are lower Thus depending on whether the effect of repulsion between particles is strong enough to overcome the effect of increased viscosity both increasing and decreasing types of concentration dependence of the hydrodynamic radius are observed In this context it should be noted that the interaction between large particles as compared to generally leads to a non exponential correlation function that does not take the form of equation C 4 and therefore cannot be completely described by a single parameter C 5 DATA ANALYSIS C 5 1 Polydispersity and the Mathematical Analysis of QLS Data Polydispersity can be an inherent property of the sample for instance when polymer solutions or protein aggregation are studied or it can be a consequence of impurities or deter
59. pare Parts and Replacement Parts cccccccccssssssssssscscsssssssssssscsccccccccccsccssccccccssssees B 1 Appendix C General Principles of Dynamic Light Scattering ssescccssescsscccsssccessssscssssssssssssssssssso C 1 Appendix D Multi Angle Light Scattering to Determine M and lt r gt The Zimm Plot D 1 DINO ON E E E A E EEEE A A ATE A AE E E AE EE EAE AE E S I 1 xii PDExpert Table of Contents Chapter 1 Introduction 1 1 OVERVIEW The Precision Detectors PDExpert Light Scattering Workstation is a multi detector light scattering platform that can be employed for light scattering measurements on a static sample e g a test tube or for flow through measurements e g High Performance Liquid Chromatography Gel Permeation Chromatography and Size Exclusion Chromatography The workstation is designed to provide the ultimate in detection for characterization of polymers nanoparticles lipids colloids and proteins The optical bench of the workstation is the heart of the system Figure 1 1 Sample cell Detector Laser Figure 1 1 The Optical Bench of the Precision Detectors PDExpert Light Scattering Workstation PD Expert System Chapter 1 1 1 The sample holder is located in the center of the optical bench Two sample holders are available e A static sample chamber Figure 1 2 in which the sample is placed in a 6 mm disposable test tube which requires 150 uL of sample Sample chambers fo
60. r 3 mm and 5 mm tubes as well as NMR tubes 10 uL are available Figure 1 2 The Static Sample Chamber e A flow cell detector which is similar to the fixed sample holder The mobile phase is delivered through a port on the rear of the optical bench The base system includes a dynamic light scattering detector that is set at a user specified angle Additional static and dynamic detectors are available from Precision Detectors up to twenty four static and eight dynamic detectors can be accommodated Detectors can be set at any angle between 5 and 175 at 5 intervals as desired A typical dynamic detector module is shown in Figure 1 3 the static detector module is similar in design and is 1 longer than the dynamic detector module Figure 1 3 A Dynamic Detector Module Assembly 1 2 PD Expert System Chapter 1 1 2 FEATURES OF THE DETECTOR The PDExpert Light Scattering Workstation includes the following features e lt A diode laser 685 nm with 30 mW power is employed The laser is temperature controlled for stability e Up to 24 Static and 8 Dynamic Light Scattering angles can be selected for optimum data acquisition The 360 base plate has three rows of threaded holes on the laser path that are 0 5 apart on 0 5 centers and 2 concentric circles of holes that are 2 5 apart drilled every 5 These holes are precision drilled to ensure positional accuracy of the detector heads e A beam dump is mounted after
61. ration of the following guidelines a In almost all cases there is one proximate cause for the problem As an example if an increase in the baseline noise is observed the problem can be caused by one of the following e The pump e g the pump is not primed e The mobile phase the mobile phase is not suitably degassed e The column the column is contaminated and strongly eluted compounds are being eluted e The detector there is an electronic problem e The cell may be contaminated e A fitting a fitting may be leaking b A fundamental knowledge of the role of each component of the system is extremely useful in diagnosing the problem c The availability of spare parts to substitute is very useful in diagnosing the problem d Ifa problem is observed run a standard sample to determine if the problem is instrument related or analysis related e If any aspects of the analytical conditions is to be changed run a before and after to ensure that the effect of the change is well understood Do not consider any change as trivial As an example if you change the supplier of a buffer salt verify that the change has no effect on the analysis f To isolate the source of the problem it may be valuable to perform independent checks of each of the components in the HPLC system 5 6 PDExpert Chapter 5 A series of diagnostic procedures is presented below that will assist in pinpointing the cause of the problem Si
62. realign a single component remove only those components indicated in the appropriate section 3 2 LASER POWER CALIBRATION The following equipment is required to calibrate the laser power e Laser Power Meter e Miulti meter e Allen Wrench Set To calibrate the laser power a Remove the cover of the PD4001 Electronics Module b Check that the jumper on J14 connects pins 2 and 3 Figure 3 1 J14 Laser Diode Driver Module Figure 3 1 Electronics Module Printed Circuit Board 3 2 PD Expert System Chapter 3 CAUTION c d e f g h 1 J k m n 0 p q r s t u Use the application software to set the Laser Scale to 0 and the Laser Threshold to 0 Turn the Output Current Adjust potentiometer on the laser driver board to the maximum clockwise position all the way up Turn the Limit Current Adjust potentiometer on the laser driver board to the maximum counterclockwise position all the way down Override the laser interlocks Power up the laser the laser will not appear to be on Caution When the laser is on wear protective eyeglasses and do not look directly at the laser beam 38 Turn the Limit Current Adjust potentiometer until a power reading of 0 69 mW is obtained The power reading is obtained by monitoring the voltage between TP1 and GND J13 pin1 Turn the Output Current Adjust potentiometer to the maximum counterclockwise position all the w
63. rganization that format could also be used to capture the relevant information Name Samples Activities Comments Activities 2319 activities repaired 7 7 m 11 33PM Davis Compd 2301 Daily OK activities 7 ial 02 2 12 PM Jones Compd 2318 Daily Significant activities peak tailing replaced column DO Mala La ic gt activities Figure 5 1 A Typical Log PDExpert Chapter 5 5 5 5 5 TROUBLESHOOTING 5 5 1 Introduction to Troubleshooting Troubleshooting refers to the determination of the cause of an abnormal condition or abnormal results from the system The analyst should recognize that if a problem is observed it might be due to the control module the cells the column the solvent delivery system or to some other component of the system if an HPLC system is used to deliver the sample When the system is initially installed the analyst should obtain a chromatogram of a well defined sample before developing a new analytical procedure This chromatogram can serve as a benchmark to be used to check the performance of the system Similarly if problems are observed in the use of a specific analytical procedure it may be useful to use the standard sample to ensure that the chromatographic system is functioning properly 5 5 2 Troubleshooting Guidelines If the PDExpert system is connected to an HPLC system it 1s important to understand that the system consists of several components and troubleshooting can be simplified by conside
64. s will lead to a much more complicated correlation function than described by equation A 6 These effects are usually insignificant for scattering by particles small compared to the length of the inverse scattering vector but become important and often overwhelming for larger particles In those cases QLS probes not the pure diffusive Brownian motion of the scatterers but also other types of dynamic fluctuation in the solution C 5 2 Deconvolution of the Correlation Function an Ill Posed Problem The values of contain statistical errors We have described previously the features of the QLS instrument that are essential for minimizing these errors It 1s equally important to minimize the distorting effect that experimental errors in have on the reconstructed distribution function The distribution is a non negative function A priori then a non negative function should be sought that produces via Equations A 3 and A 10 the function which is the best fit to the experimental data Unfortunately this simplistic approach does not work The underlying reason is that the corresponding mathematical minimization problem is ill posed meaning that dramatically different distributions lead to nearly identical correlation functions of the scattered light and therefore are equally acceptable fits to the experimental data For example addition of a fast oscillating component to the distribution function does not change considerably since the contr
65. t fates carta Saat ac tasie use aclacaatia et asa a aaterean ema E ese ceed eaenteehetoeeee 5 1 5 2 2 Maintenance Systems with the Flow Cell oo ccccccccccccccaseeceeeeeeecaaeaesesseseseeeceeeseeeeeeeeeeeeeaaaas 5 2 F2 ADAMI ACO VAM S eaa a an beasts spade a a r 5 2 PL Weekly Ma ne Dan eanan aa a eoeMacoadssausiaathannsieadanaeene sees tenths 5 2 S22 Mony M antena NEC assa a a a E SROS 5 3 SaF Quarten y Maintenances eaea a EEE aS AEREE 5 3 Del 2D Cel IVT AIC AICS a ce a a a a Gap teudanseee Tesaxean cee 5 3 55 Replace System OMPONCMES usenge T 5 3 Jo PREE E EMOS aerae a r E T N 5 3 I Chanoine CENE PUS u a A 5 4 5 3 3 Replacing or Moving a Static Detector Module n nnensesssssseseeossssssseeererersssssssssssssceresssseerreeeo 5 5 5 3 4 Replacing or Moving the Dynamic Detector cccccccccccccccceceeeceeeaeaaeeseesseeeeeeeeeeeeeeeeeeeseseeaaaas 5 5 SA Establi hins as Sy Stemi 0 9 3 ction pit ohana titan E E E E NAR E 5 5 R AOU DICSNOO I ieran saetacasnasnasencimagacnaants a 5 6 55s Introduction to Frou ble smOO Ua sarsa a E A E a 5 6 33 2 Troubieshgoonn S Gude MEE eaa E E a E awhom eee 5 6 RIOM ENaC NOISY Basc iNeo r a a a a 5 7 5 5 4 ESM Backorownd Sinal Sou a a a a a oud 5 8 D955 sINCLEASe BACK PICS SUT rsen E E E 5 9 525 0 LOSS OLERE DONS xiri ronen aa r a a a a 5 10 IT AMADIS TO AWZ ro eS nala a a a E E 5 10 Appendix A Specifications sissien ianiai enasini Ee aaea iiei eines sai e Se R s A 1 Appendix B S
66. t on the optical bench 2 of the column and the inlet of the cell 1s minimized to reduce post column band broadening ote When you are fitting the inlet from the HPLC and the outlet line tighten the fittings finger tight and eck that they do not leak If leaks are observed tighten the offending fitting approximately 1 8 of a turn Note Do not over tighten the fittings If the fitting is over tightened it is possible that you will permanently distort the fitting rendering it useless g Once the fittings are properly made allow mobile phase to flow through the system for 15 30 minutes at a flow rate that is 25 greater than the flow rate that you expect to use for normal operation to ensure that there are no leaks Connect the power cords and power up each module The green light on each module will be illuminated PD Expert System Chapter 2 2 5 2 6 This page intentionally left blank PD Expert System Chapter 2 CAUTION CAUTION Chapter 3 Aligning the System 3 1 OVERVIEW This section document describes the alignment of various components of the Precision Detectors PDExpert system that are attached to the optical bench including the laser the cell the steering lens and the detectors The alignment should be performed by an individual who has been trained in the procedure Once a system is installed these various adjustments need not be performed unless a component is replaced or if significant de
67. terioration of the performance has been observed aution The procedures in this section are performed with the power on Take care that you do not ontact live components in the controller or in the optical module Caution The main laser and or the alignment laser must be powered up for certain of the procedures described in this section When a laser is on wear protective eyeglasses and do not look directly at the laser beam The alignment of the system includes the following steps e Laser Power Calibration Section 3 2 e Main Laser Alignment Section 3 3 e Alignment Laser Alignment Section 3 4 e Initial Cell Alignment Section 3 5 e Temperature Calibration Section 3 6 e Main Beam Dump Alignment Section 3 7 e Alignment Beam Dump Alignment Section 3 8 e DLS Detector Focus and Alignment Section 3 9 e Static Detector Focus and Alignment Section 3 9 e Cuvette Alignment Section 3 10 e Installing Replacing Apertures Section 3 11 PD Expert System Chapter 3 3 1 When the system is initially installed these procedures should be performed in the indicated order These procedures can also be used in conjunction with the maintenance and troubleshooting procedures described in Chapter 4 If a complete system alignment is performed remove all detectors and beam dump monitors from the optical table The various sections will indicate the components are to be installed If these procedures are employed to check and
68. the eye piece You should see the dark edges of the cuvette and the beam across the middle Use the screw on the top of the detector body to adjust the focus The beam shouldbe a then bright line and you will see some dust spots when properly focused Secure the focus screw f Look through the eye piece Use the screw on the side of the detector lens to center the vertical eye piece cross hair between the 2 sides of the cuvette g Use the screw on the top of the detector lens to line up the horizontal eye piece cross hair with the laser beam h Remove the eye piece and replace the aperture holder PD Expert System Chapter 3 3 13 CAUTION 3 10 CUVETTE ALIGNMENT Note The initial Cell Alignment Section 3 5 must be completed first To align the cuvette a Fill the cell with Toluene Caution Toluene is flammable and is toxic Wear gloves when handling the liquid and avoid getting it on your skin b Insert the cuvette so that it is just off the bottom of the cell and secure with the set screw c Turn on the Main Laser and set the power to 10 d Turn on the Alignment Laser e Set the Switch to Fine f Use the micrometers to move the cuvette until both LED banks are in the Green Figure 3 9 3 14 PD Expert System Chapter 3 Chapter 4 Using PDExpert 4 1 OVERVIEW PDExpert is a multi detector system which can be used for measuring light scattering from a static sample using a test tube or a flowing sample High
69. the sample cell so that the laser beam is attenuated to essentially zero power e An attenuator to define the physical field of the detector and abeam monitor are available options e The sample temperature can be controlled from 0 80 C via a Peltier controller e The flow cell module can be readily removed and a temperature controlled sample holder can be place in the system to provide a batch analysis system full goniometer results The batch analysis sample holder permits the use of standard test tubes for samples e An alignment laser 689 nm 3 mW is included for use in aligning the optical components to optimize performance e The distance of the detectors from the scattering point in the cell is 6 which provides excellent discrimination and detection characteristics at any angle on the optical table e Control of the system as well as data processing storage and reporting is provided by PrecisionDeconvolve32 for dynamic light scattering measurements and Discovery32 for static light scattering measurements e A broad range of accessories and custom configurations are available meet the specific needs of the laboratory The system can be upgraded at any time PD Expert System Chapter 1 1 3 1 3 STRUCTURE OF THE MANUAL This manual contains general information about the system and detailed information about each specific detector module The structure is as follows 1 4 Unpacking and Installation Chapt
70. to deposit the sample at the bottom of the test tube e Take every other precaution to ensure that your sample is clean and free of particulate matter A variety of sample chambers are available including ones for 3 and 5 mm NMR tubes which require a 10 uL sample Please contact Precision Detectors for additional information 4 4 SETTING OPERATING CONDITIONS oa A detailed discussion about operating conditions is presented in the manuals supplied with the application software packages The PDExpert dialog box dialog box Figure 4 1 is used to select the temperature of the cell PD Expert Control Laser Controls Cell Temperature Controls Alignment Laser W Enable Temperature Control Main Laser Power J 50 0 Desired Temperature C 00 Range 0 100 Range 4C 50C Input Laser Power 0 026 Actual Temperature C 20 02 Vin 1 752 Vout 0 920 OK Cancel Aen Figure 4 1 PD Expert Control Dialog Box The Cell Temperature Controls field is used to control the temperature of the cell Enter the desired temperature and press Apply The temperature will be sent to the indicated temperature and the present temperature will be indicated in the Actual Temperature field The green indicator field will be illuminated from time to time to denote that the temperature is being updated The Laser Controls field is used to set the relative intensity of full power that the laser is generating it does not imply
71. vely slow fluctuations in concentration shape orientation and other particle characteristics by measuring the correlation function of the scattered light intensity Fast vibrations of small chemical groups which lead to significant changes in the frequency of the scattered light is the domain of Raman spectroscopy These latter two methods which probe the dynamics of the particles which cause light scattering are intrinsically more complicated than static light scattering since they involve measurements of spectral characteristics or related correlation properties of the scattered light PDExpert Appendix C C 1 C 3 LIGHT SCATTERING FROM MACROMOLECULES IN SOLUTION One may consider the solution as a homogeneous medium and ascribe light scattering to the spatial fluctuations in the concentration of a solute An alternative way is to consider each individual solute particle as a heterogeneity and therefore as a source of light scattering The first approach is more appropriate for solutions of small molecules in which the average distance between the center of the scatterers is small compared to the wavelength of light The second approach is more appropriate for solutions of large macromolecules and colloids when the average distance between particle centers is comparable to the wavelength of light When the size of the solute particles becomes comparable to the wavelength of light the description of the effects of orientational motion and deformati
72. which the correlation function is determined it is essential to maximize the number of count pairs whose products are averaged within the measurement time If the correlation function is being measured in channels simultaneously ideally products should be processed for each new count 1 e during sample time The instrument capable of doing this is said to be working in the real time regime The real time regime means that the information contained in the signal is processed without loss The PDI correlator works in real time with a minimal sample time of 1 microsecond and the length of the digital copy 1024 The number of channels processed in real time is determined by formula and cannot exceed 256 C 4 5 Brownian Motion Temporal fluctuations in the intensity of the scattered light are caused by the Brownian motion of the scattering particles The speed of the particles is related to the size small particles move faster than large particles Though each particle moves randomly in a unit time more particles leave regions of high concentration than leave regions of low concentration This results in a net flux of particles along the concentration gradient Brownian motion is thus responsible for the diffusion of the solute and is quantitatively characterized by the diffusion coefficient The laws of diffusive motion stipulate that over time the displacement of a Brownian particle in a given direction is characterized by the relationship

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