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Relaxation Time (T1 and T2) Measurements Bruker minispec

1. pulses sp 90 0 1 y sd duration while CNT loop count count 0 while count lt echo sp 180 90 1 sd 2 duration count count 1 endwhile sp 180 90 1 sd duration wait 2 asd wait sd duration wait 2 CNT CNT 1 endwhile endpulses 9 2 T1 Relaxation Time Test Program program setup par scans 1 rd 3 000000 gain 88 dbw 20000 00 abw broad off_comp off det_mode real dig res high endpar program measure int no int Loop Count int cnt int CNT int ExpOrder Order int dec int store loop int id int out int sample int modi Bruker Relaxation Time Manual TI Experiment 180 90 Pulse sequence digital filter bandwidth kHz analogue bandwidth number of data loop counter help variable number of loops exponential order results mode data pairs storing help sample identification output actual data point sample identification data modifications dk dk dk db Hb db Hb db db db db ehm 35 Z ninispec real sx 10 sy 10 real x 500 y 500 real a b c f g h real DUR D real DF real WIN real DSW real Result 5 real Error 5 real helptau helpamp char ResS 16 ErrS 16 char text1 100 char str buf 200 char name 20 pointer textp pointer txt strcpy str buf 1 data po
2. Relaxation Time T and T2 Measurements minispec Operator s Manual 1 INTRODUCTION The Bruker minispec is a low field pulsed NMR spectrometer used for a wide range of routine NMR applications Data is usually but not necessarily analyzed in the time domain form This guide describes the use of the minispec with Bruker pre programmed applications to determine relaxation parameters T and T In particular this manual discusses the use of Bruker pre programmed applications t ir mb t sr mb t2 cp mb and 12 se mb The fitting routines included in these applications are suitable for processing data that are described by or that can be simplified to mono and bi exponential decay Thus appropriate samples are simple single component systems neat liquids solutions and two component systems dispersions suspensions Three and more component systems can sometimes be treated if some of the components are relatively fast decaying and can be ignored by using suitable experimental parameters More complex systems that cannot be so simplified may require tri and quad exponential decay fits available in minispec TestApps Additionals and named fit rel or other third party modeling software in order to analyze the experimental data Instructions in this manual were developed for the minispec mq series software Depending upon the versions of the minispec software different variables or other setup options may be used 2 COMPONENT LIST
3. Z ninispec ehm 6 T1 MEASUREMENT PROCEDURES 6 1 Application t1 ir mb Options Application t7 ir mb Inversion Recovery Pulse Sequence RD 180 IR Delay 90o RDT asd ws N with increment in IR Delay IR delay IR Delay Factor where RD is the recycle delay user defined in the PARAMETER menu IR Delay is an incremented inversion recovery delay interval user defined starting value in the Application Configuration Table RDT is a delay for receiver dead time user defined value in the Application Configuration Table NS is the number of scans for signal averaging user defined in PARAMETER F4 menu N is the number of points to be collected user defined value in the Application Configuration Table FACTOR After each measurement IR Delay is multiplying by the duration factor to determine the IR Delay for the next experiment 6 2 Configuring t1 ir mb Options Configure pulse sequence variables and user options through the Application Configuration Table This table looks as follows might look different in later application versions the minispec APPLICATION CONFIGURATION TABLE 171 Curve Fit Results qu Ej LI LI M LI iv LI m GHG A w AD SE IU IUE Bruker Relaxation Time Manual 13 Z ninispec Mise Sample Identification Check mark this option if the user should be prompted for a corresponding sample ID at the time of measurement Th
4. 2 1 System This manual refers to the use of the minispec mq series 2 2 Probe head In principle any probe head can be used to make relaxation measurements However due to the dependence of relaxation on sample temperature a probe head that permits variable temperature control type V is recommended This is especially true for T measurements which require a relatively long time to complete compared to T experiments A room temperature probe head may also be used if the temperature of the sample is preconditioned to a defined value before the measurement However the duration of the measurement should be short to avoid temperature change during the measurement Bruker Relaxation Time Manual 3 tipinispec Mise 2 3 Circulating Heater Cryostat Bath If a type V probe head is used then an external circulating heater cryostat bath is needed to control the temperature of the sample in the magnet sample chamber Many manufacturers exist Some of the prominent brand names include Haake Julabo and Neslab 2 4 Software The following Bruker pre programmed applications are discussed in this manual e Application t7 ir mb for determination of T e Application t _sr_mb for determination of T e Application t2_cp_mb for determination of T e Application 2 se mb for determination of T 3 INSTALLATION OF the minispec 3 1 Electrical Requirements The electrical requirements of the minispec are as follows
5. Automatic Daily Results Saving To control results storing If this option is selected the program automatically creates a saved copy of the results on the hard disk This option is useful for archiving after acquisition and results output Beep after Analysis To generate a PC noise after termination of the measurement This option is useful if not too much time after measurements should be wasted Results according to Fitting Formula Check mark this option to cause the fit amplitude results the amplitudes at the origin for each component to be listed according to the signal reading at the origin as projected by the fit regardless of the fraction of signal this component constitutes in the total signal Do not check mark to cause the fit amplitude results to be given as a fraction of the total signal i e if two components A and B are detected at a ratio of 2 1 respectively Component A will be listed as 66 6 component B will be listed as 33 3 Bruker Relaxation Time Manual 23 tipinispec Mise Data Points File Storing To control data points storing If this option is selected the program automatically creates a saved copy of the X Y data pairs on the hard disk This option is useful for archiving after acquisition and or for fitting with other functions not available in t _ir_mb refer to the test application fit rel Monoexponential Curve Fitting To select either mono or bi exponential fitt
6. Decide how many points will be collected Decide what working application copy will best fit the time X diff Calculate the tau value from the appropriate equation Tau sampling period number of points x dummy echos 1 x 2 e g if dummy echoes 3 every 4 echo is sampled If set up T2 determination shows a sampling period of 800 ms is necessary and 100 points will be collected tau 1 ms If tau is longer than 3 ms you may have to shorten tau and collect more points instead avoid diffusion cross relaxation T2 When analyzing a component that decays within 100 us solids the effect of the magnet inhomogeneity is minimal In this case T2 T2 For this component the FID measured after a 90 degree pulse provides the T2 decay constant by direct fitting To find appropriate parameters Tau must be short enough to avoid errors due to diffusion between pulses Set tau appropriately The time taken by the total number of echoes must describe most of the decay curve The maximum number of points is 256 Fitting is faster with fewer points Fitting 100 points is commonly enough for a mono exponential decay For bi exponential decays 200 to 250 points may be beneficial Bruker Relaxation Time Manual 26 Z ninispec Mise Set the DE so that the number of points collected adequately describes the relaxation curve The user may select either mono or bi exponential fitting If bi exponential is selected and the fitting rout
7. Factor Calculate the final delay by considering the Initial SR Delay the Factor and the Number of Points collected This could be accomplished with a simple spreadsheet that determines the next entry in a column of values by multiplying the present value by a constant value see also section 6 2 Bruker Relaxation Time Manual 19 Z ninispec Mise Delay Sampling Window To set the Delay of Sampling Window in ms From within this window 16 data points are collected and averaged to produce the data point for that particular SR Delay Sampling Window To set the Sampling Window width in ms From this window 16 data points are collected and averaged to produce the data point for that particular SR Delay Factor for Duration s Expansion To set the saturation recovery delay multiplication Factor This factor controls how closely spaced in time the data points will be Note that while the Factor does not have limits it impacts on the calculation of the SR Delay and the SR Delay does have a limit Please refer to the discussion on SR Delay above for guidelines Number of Data Points for Fitting To set the Number of Points which is equal to the number of experiments to be repeated and corresponding points that will be collected Fitting as few as 10 points is common for a mono exponential decay For bi exponential decays 20 to 100 points may be beneficial The maximum number of points is 256 Note that the N
8. y no Result Error Order display_fit SHOW_FIT Result Error x y no ms endif endif if modi 1 if ExpOrder 2 Order display_fit BIDECAY Result Error x y no ms 96 else Order display_fit MONODECAY Result Error x y no ms endif endif if modi 0 if ExpOrder 2 Order fit_bidecay x y no Result Error else Order fit_monodecay x y no Result Error endif if Order 0 print line FLASHBOX No Exponential Functions Switch to Modifications 1 endif if Order 1 Order display fitt MONODECAY Result Error x y no ms endif if Order 2 Order display_fit BIDECAY Result Error x y no ms 96 endif endif Bruker Relaxation Time Manual 44 Z ninispec ehm 10 REFERENCE VALUES AND T1 PULSE PARAMETER COMBINATIONS Refer also to the Contrast imaging agent literature for a known relaxation standard Check with Nycomed Mass Gen Hospital Epix for standard recipe NOTE A calibrated thermometer is recommended for measuring the actual temperature in the probe chamber 90 formamide in DMSO Dioxane in 60 C6D6 Dodecane 40 deg C T2 approx 70 msec T2 approx 5 4 sec T2 approx 1 2 msec Examples of allowed combinations for DUR DURFACTOR and NP see T1 applications Starting DUR ms 5 0 5 0 3 5 3 5 0 5 0 5 0 5 0 05 0 05 0 05 0 05 Bruker Relaxation
9. 17 result 4 A O Offs result 5 Ti result 6 Offs Error array Uncertainty errors corresponding to each position in the result array The command fit quaddecay fits a 4 exponential curve to the input data The fit equation has the form y A exp x tj Az exp x t As exp x t Ag exp x 7 O where Ag amplitude of component 1 to 4 ty decay constant component 1 to 4 O offset Prior to the command the program must declare the necessary input and output arrays and assign values to the input arrays To assist in the fitting process the user can be prompted for estimates of the component amplitudes and corresponding relaxation times A A A3 A and corresponding T starting values and the estimated signal offset Previous variable declarations int order Order of the fit see explanation below real result 10 error 10 Arrays for results output and their uncertainties real x_array positions y array positions Arrays for input data int number_of_data Number of data pairs Assign values to the input parameter number_of_data and input arrays by direct copy of the NMR signal into x and y array Bruker Relaxation Time Manual 41 tipinispec Mise Pre assign starting values for component amplitudes and relaxation time constants i e Quad exponential Fit pre definitions Result 0 input real Input Amplitude Start Value of
10. 7 4 Application t2 se mb Op tlOns eoo coercere eoru to o eoe e Seo edo eo cu ego ue Fo cu ea one Eo ke as ne arae aene done 27 7 5 Configuring t2 se mb Options eeeeeee eerie esee e eene eene sensns sense tasto senses setas ense tns en se tnseon 27 7 6 Routine Measurements with Soft EDM t2 se mb ssccscssssssssssssssscsscssessesesessscecsssscessneees 30 8 ADVANCED TOPICS icssose sesstuss s dee PDA NER IARE FPEXYREEF EARS SE ERENE IN ER DR RAN IER ARA AS EUER Od Kb s 32 8 1 T2 Relaxation Time Physical Principles eee eee essere eene eee eene enean tns tn senatus sense tnsna 32 9 PULSE PROGRAMMING scsssssusssscaasiasacsnnsvsnpcasnancsdssveinciaeniaeveasnadssveaneaasassianissaanaseaqaseansessaneas 34 9 1 T2 Relaxation Time Pulse Program sessessessesosssssosoesossoosossossosoossessesossossessossossoesessssossossessosos 34 9 2 T1 Relaxation Time Test Program eee eese eee eren eene eene tasto natns sense tasto setas tassa sense ta sa 35 9 3 Program the Data Fitting mm M S 37 9 4 T1 Relaxation Time Program Fitting ssessessesosssssesoesossoosossossesossoesossossessosossoesoosossesoososssesossos 44 10 REFERENCE VALUES AND TI PULSE PARAMETER COMBINATIONS 45 II TROUBLE SHOOTING Gioia HARE RR DI BER ENS TO DUI AMEN LR I TINTE KH A EIN DR I EIRIE 46 Bruker Relaxation Time Manual 2 Z ninispec Mise
11. Input 110 V 090V 132 V AC 50 Hz 60 Hz 4 0 A 230 V 220 V 250 V AC 50 Hz 60 Hz 2 5 A Power 400 Watts maximum Grounding is vital for safe operation of the instrument Never operate the minispec with the grounding conductor disconnected If local wiring does not provide a ground an earth to chassis connection must be added at the user s location For best performance e Do not locate the minispec near high voltage power cables e Avoid plugging the minispec into the same circuit as appliances that have a heavy and variable draw on the line voltage e g heating ovens microwaves air conditioners etc e Ensure a stable clean power supply If power conditions are suspect due to the potential for brownouts sudden power outages voltage spikes etc consider the installation of a line filter and or UPS The UPS unit should be at least a 500W type preferably based on the saturated transformer principle 3 2 Operating Environment The minispec should be located so as to avoid exposure to direct sunlight and direct heating cooling sources e g heating cooling vents Recommended Operating Temperature Range 17 C to 28 C Recommended Operating Relative Humidity Range 20 to 80 non condensing Bruker Relaxation Time Manual 4 Z ninispec Mise For proper ventilation a minimum distance of 10 cm should be allowed between the back of the electronics box and adjacent objects DO NOT OPERATE the minispec WITH THE CLEAR
12. area of best B and B field homogeneity This is most simply accomplished by calibration using a substance of known 7 gt e g water at 25 C Application 12 cp mb is the application most commonly used to measure NMR relaxation due to spin spin interactions as characterized by the time constant T Equations Mono exponential fit y A exp x t O where A amplitude at time zero T T decay constant O offset Bi exponential fit y A exp x T Az exp x 5 O where A Az amplitudes of components 1 and 2 at time zero T T decay constants of components 1 and 2 O offset Bruker Relaxation Time Manual 33 Z ninispec 9 PULSE PROGRAMMING 9 1 T2 Relaxation Time Pulse Program program setup int CNT counter int count counter int loop count loop counter number of data points int ExpOrder exponential order int store data pairs storing int dummy_echos dummy echoes real duration duration real wait waiting for data acquisition loop_count 50 duration 1 5 dummy_echos 1 par scans 1 rd 2 00 gain 56 dbw 100 00 abw narrow off comp off det mode magnitude dig res high endpar dk dk dt db ehm echo input int Input Number of Echoes without Data Acquisition 0 20 echo if ESC return FALSE endif wait duration 10 CNT 0 Bruker Relaxation Time Manual 34 Z ninispec
13. but not least if multiple CPMG experiments are repeated in a short period of time significant RF energy can be absorbed by the sample cause heating Therefore increased relaxation times are to be expected However a few minutes later the sample should have recovered to its usual temperature and then the correct relaxation time should be found again Problem C Expected relaxation time values cannot be reached In case relaxation time values are expected from previous measurements or other information sources and those values cannot be reached care has to be taken on different aspects Was the sample previously analyzed at exactly the same temperature and also at the same field frequency If not the same relaxation times should not be expected Also it is important to be sure that the sample investigated is the same as before For instance mineral oils will have significant relaxation time differences An unsealed doped water sample will change its relaxation time when some of the water evaporates Care has to be taken also in the choice of the applications and their parameters Results from a sample previously analyzed with inversion recovery t1 ir mb sequence should not be compared directly with results from a saturation recovery t1 sr mb sequence The CPMG application t2 cp mb should always be executed with the same pulse sequence parameters like tau value number of data points etc if results must be closely compared If absolu
14. can be measures all together without tuning Very different samples should be run after tuning the instrument Press Measure to start the sample series If the sample ID option is turned on the user will be prompted to enter a two part identifier The first part accepts alphanumeric entries of up to ten characters The second part must be a numeric entry of up to 3 characters If T is poorly known it is normal to perform a survey measurement to decide what conditions will be needed to determine the true relaxation time constant PARAMETERS can be changed to suit the particular sample For example Recycle Delay may need to be adjusted normally to at least 5 times the anticipated value of the longest T If Gain needs to be changed because of a SIGNAL CLIP error and real detection mode is being used it may be necessary to tune the detection angles at the new gain settings select Update Settings and uncheck Update all Settings afterwards check mark Detection Angles If data storing is selected see section 7 5 but sample ID labels are not data is stored with time coordinates in the filename YYMMDDHRMNSD The actual title will be listed in the Resultbox after measurement is complete If data storing and sample ID labels are turned on then the file name is set to the two part sample ID The actual title will be listed in the Resultbox after measurement is complete Bruker Relaxation Time Manual 30 Z ninispec Mise Genera
15. eae i fes onse eo nel sv gt t2_cp_mb sig 3 t2_cp_mb mdt Expon Order Modifications Storing Data 64 3 0 5 1890 ms DIAGNOSTICS _iojxj 1000 2000 3000 4000 5000 ms Continue c disk_old nms new ap t2_cp_mb a EY View Settings Instrument Status Ready NB1269 149 236 42 201 0 ANS Astan CAWINDOWS Intemet Mail BY Microsoft Word relax ma iS BRUKER WIN NMS jue yas 12 34 The NMR signal fit above indicates that the mono exponential fitting function is not suitable for this decay curve In this case a oil water mixture has been measured therefore bi exponential fitting should be used The results of the fit above will vary from one measurement to the other The examples above always demonstrated the influence of one certain error However it might be possible that the signals of primary analyses will be influenced by multiple errors Therefore inexperienced users should follow this trouble shooting guide point by point in order to get rid of all unexpected signal behavior Bruker Relaxation Time Manual 56
16. exp x O where Ay amplitude of component 1 to 3 t4 decay constant component 1 to 3 O offset Prior to the command the program must declare the necessary input and output arrays and assign values to the input arrays To assist in the fitting process the user should be prompted for estimates of the component amplitudes and corresponding relaxation times A Az A and corresponding 7 starting values and the estimated signal offset Bruker Relaxation Time Manual 39 Z ninispec Mise Previous variable declarations int order Order of the fit see explanation below real result 10 error 10 Arrays for results output and their uncertainties real x array positions y array positions Arrays for input data int number_of_data Number of data pairs Assign values to the input parameter number_of_data and input arrays by direct copy of the NMR signal into x and y array Pre assign starting values for component amplitudes and relaxation time constants i e Tri exponential Fit pre definitions Result 0 input_real Input Amplitude Start Value of FIRST Component 20 1 if ESC return TRUE endif Result 1 input_real Input Relaxation Time Start Value of FIRST Component 100 1 if ESC return TRUE endif Result 2 input_real Input Amplitude Start Value of SECOND Component 20 1 if ESC return TRUE endif Result 3 input_real Input Relaxation Tim
17. inside the magnet box and the temperature to which this probe head sample compartment will passively be heated if the cap is left on the sample chamber Note however that after successive measurements the chamber of a probe head will cool off as room temperature air is introduced during sample exchange 2 For variable temperature probe heads a circulating heater cryostat bath is used to control the temperature in the probe head sample compartment Samples can be preconditioned in an Aluminum block placed in the bath reservoir or in the probe head chamber leave 5 to 20 minutes for the sample to equilibrate to temperature depending on the volume and temperature differential Bruker Relaxation Time Manual 5 tipinispec Mise 4 2 Circulator Fluid Variable temperature probe heads are equipped with a dewar that is shielded from the NMR coil These probe head accept all types of circulating fluids such as protic circulating fluids e g distilled water ethylene glycol mixtures However the viscosity of the liquids should be always comparable to distilled water 4 3 VT Connections The fluid necessary to heat or cool the variable temperature probe head is carried from an external connector situated on the back of the magnet box to the probe head dewar by flexible tygon tubing The external brass fittings are designed for slip on connection to hoses that carry fluid to and from the circulating water bath Rubber tubing can be slipp
18. seen setas sissoo usive 6 tasto sense esee ea sense ta s stas etas tne so 7 5 2 Preparation of the Temperature Conditioner creer eee eee esee esee eese nennen tnstnaae 7 5 3 Instrument Tuning and Pulse Length Calibration e eeeeee eee ee e eese tenete senes aetas tn une 7 5 3 1 Tuning Calibration Sample 1 erede iieeteete tede teet rue cted ett ege tete bd bo dose ste Ub det eee setaa 8 5 3 2 Tuning Calibration Procedure sessssssssseeeeseeee ertet ertet eter enet nenne ten treten nee nenne 8 5 4 Application Selection and Curve Fitting Rules e eeeeee esee eese eese eee enses sensns toan ne 9 DAL Application Selection ooi er eee DR ra e eee YR EQ EEEE EE eR EUR ERR TEE ETE E EXE ENSE FORES EE VE 9 25 4 2 Curve Fitting Rules cde roe rr e EHE CHR EE EEEXRI EEO EEEN EENEN ERE OEE E EES ESOS TEA cuss FO ER ERE VR 9 5 5 Preparing the Applications 4 eee eee eee eene eee eene eene tns tn setas tn natns seats sets setas tns s tassa seta sna 10 5 5 1 Preparing the Application 1 ir mb sessssssesseseee enne ener nennen nennen nennen ens 10 20 2 Preparmg the Application sr HDi ederet RR eee E E HERR EFE YR FE Ea EE 11 5 5 3 Preparing the Application 2 cp mb enne nennen nennen nennen nenne eren 12 5 5 4 Preparing the Application 2 se mb enne ennt nennen nennen treten ennt 12 Bruker Relaxation Time Manu
19. the initial IR Delay After the first measurement and each time the pulse sequence is repeated the IR Delay is lengthened and the new value is used in the next determination e g First IR Delay initial IR Delay Second IR Delay z initial IR Delay Factor all points thereafter Next IR Delay present IR Delay Factor Calculate the final delay by considering the Initial IR Delay the Factor and the Number of Points collected This could be accomplished with a simple spreadsheet that determines the next entry in a column of values by multiplying the present value by a constant value Microsoft Excel IRdelaycalculate xls n x File Edit View Insert Format Tools Data 2 Ai Edi view resect Window Help z e x Window Help alal siasi Haas sls Zel ala Delaj alal Helea ale xL ea a jo s u lela pz 1 3 12 Sample Point IR Delay B2 C5 B 1 C6 B 1 C7 B 1 C8 B 1 C9 B 1 C10 B 1 C11 B 1 C12 B 1 C13 B 1 C14 B 1 16 C15 B 1 i i e nf Sheet Sheet2 Sheer Si ia gt Sum 0 J NUM 2 Sample Font IR Diele 5 KI 6 50 8 45 10 38 15 25 18 56 24 13 1 2 3 4 5 6 7 8 31 37 40 78 23 02 BB ga 83881 2 3 a E 3 j B HL Bruker Relaxation Time Manual 15 Z ninispec Mise Delay Sampling Window To set the Delay of Sampling Window in ms From within this window 16 data points are collected and averaged to produce t
20. to ten characters The second part must be a numeric entry of up to 3 characters If T is poorly known it is normal to perform a survey measurements to decide what conditions will be needed to best determining the relaxation time constant Initial values for IR Delay and Factor should be set so that approximately half of the measured amplitudes are negative and the rest are positive Recall that the maximum IR Delay 100 sec Refer to section 6 2 for tips on how to ensure that the IR Delay will not exceed the 100 sec limit PARAMETERS can be changed to suit the particular sample For example Recycle Delay may need to be adjusted normally at least 5 times the anticipated value of the longest T If Gain needs to be changed because of a SIGNAL CLIP error it is recommended to tune the detection angles at the new gain settings select Update Settings and uncheck Update all Settings afterwards check mark Detection Angles Progress during measurements is given by message in the MESSAGEBOX Measuring Point No lt gt and by the Number of Scans countdown in the right lower corner of the display If data storing is selected see section 6 2 but sample ID labels are not data is stored with time coordinates in the filename YYMMDDHRMNSD The actual title will be listed in the Resultbox after measurement is complete If data storing and sample ID labels are turned on then the file name is set to the two part sample ID The actual tit
21. FIRST Component 20 1 if ESC return TRUE endif Result 1 input_real Input Relaxation Time Start Value of FIRST Component 100 1 if ESC return TRUE endif Result 2 input_real Input Amplitude Start Value of SECOND Component 20 1 if ESC return TRUE endif Result 3 input_real Input Relaxation Time Start Value of SECOND Component 100 1 if ESC return TRUE endif Result 4 input_real Input Amplitude Start Value of THIRD Component 20 1 if ESC return TRUE endif Result 5 input_real Input Relaxation Time Start Value of THIRD Component 100 1 if ESC return TRUE endif Result 6 input_real Input Amplitude Start Value of FOURTH Component 20 1 if ESC return TRUE endif Result 7 input_real Input Relaxation Time Start Value of FOURTH Component 100 1 if ESC return TRUE endif Result 8 input_real Input Offset Start Value 0 1 if ESC return TRUE endif The default is to allow the function to include the offset as a fit variable One can assign the fit Offset to a pre defined value using off_presetting 0 fixed_offset_value To release the fixed Offset fit use off presetting 1 0 Syntax of the command order fit quaddecay x array y array number of data result error ERROR is set if determination fails not enough data pairs matrix singularity After the fit the routine sets a return value to
22. PLASTIC DUST COVER OVER THE ELECTRONICS BOX Place the two units on a flat level surface capable of supporting their weight 110 kg Placing the minispec magnet on insulating rubber pads can dampen excessive floor vibration Since iron containing objects can influence the magnetic field inside the magnet box do not locate the magnet box within 2 m of moving or moveable metallic objects Vibration caused by a circulating heater cryostat bath located on the same bench top as the magnet can degrade performance Suggested location for the bath is on the floor below and to the side of the magnet The bath should be on a separate electrical circuit from the minispec 4 SAMPLE TEMPERATURE CONTROL 4 1 General It is recommended that T and T be measured at a defined temperature A calibrated thermometer is recommended for measuring the actual temperature in the probe head sample compartment Sample temperature can be controlled in two ways 1 For magnet temperature probe heads precondition the temperature in an external heating device such as a heating block or circulating heater cryostat bath WARNING the duration of the measurement should be short to avoid temperature change during the measurement A large sample will resist temperature change better than a small sample due to heat capacity If a magnet temperature probe head will be used the sample temperature is most conveniently conditioned to 40 C since this is mostly the temperature
23. Time Manual DURFACTOR 1 5 5 3 1 1 1 5 5 3 1 5 1 3 NP 25 5 108 25 30 46 35 152 45 Z ninispec Mise 11 TROUBLE SHOOTING This chapter deals with typical errors that may be encountered specifically with relaxation time analysis A user might refer to the troubleshooting part of this manual only after encountering some problems with relaxation time results If this is the case it is recommended that the user read this entire manual and the minispec User s Manual for full information regarding operation of the minispec Problem A Results are unstable In this section the expected stability of relaxation results is discussed The accuracy of a fit depends upon the number of data points to be fitted the shape of the decay curve and the available dynamic range of the data points For all Bruker relaxation time applications relaxation time standard deviations below 5 are possible The default values of the Bruker applications are optimized for doped water samples distilled water with 0 5 CuSO4 5 H2O filling height 1 cm and this type of sample can be measured with relaxation time standard deviations below 3 T2 measurements on the doped water sample mentioned above by the CPMG sequence using Bruker Soft EDM t2 cp mb with default parameters or more data points should produce even better results 2 If the relaxation time results of numerous analyses have greater scatter about the mean
24. Z ninispec Mise Relaxation Time T and T2 Measurements Bruker minispec Relaxation Time Manual CONTENTS 1 INTRODUCTION 3 2 COMPONENT LIST aided ter oO OVE Eb ECHO E AL MH pn aaeeei easain CER CD 3 2 1 System SD 3 2 2 Probe Tcr 3 2 3 Circulating Heater Cryostat Bath ecce ee eee Lees ee eee eee ee eese seen ase en asta asta ases e setas seas esas 4 2 4 SOFtWALE mM 4 3 INSTALLATION OF the minispec cossiesv cont ivsss sie cusccutervavechbvnssiecucscsiseavssadessstaesudeduarssvibuntssbiesiene 4 3 1 Electrical Requirements ccsccccsccssccsscscssscsscessccsssccsccsccceccsscessessscssccssccsscscsccsesscsssssessseescess 4 3 2 Operating Environm nl sicccessccerseseoseseoseseessseetssecssoncsionsssoncsionsssonseiacceiaccedaccedeecsiasceseucessons cosdesorseeas 4 4 SAMPLE TEMPERATURE CONTROL e eee ee eene ete ee eene thee tta o eto eee pa eee ea e eene tpa eta o 5 LNMECDT2U 5 4 2 Circ lator diio c PE 6 DNMBAMWMOD ITO DI P 6 4 4 Temperature LOTTP EERERROETTETTER 6 5 PREPARATION FOR MEASUREMENTS eee e esee eene eee eene ee ee aeta nae a sete sa tease tasse tese nae 7 5 1 Preparation of the minispec 4 eere eee eese eese eee en
25. a 1 1 sx ordinate 1 1 sy x Loop_Count sx 0 y Loop_Count sy 0 Loop_Count Loop_Count 1 endwhile sample sample 1 endwhile 9 3 Program the Data Fitting The command fit_monodecay fits a mono exponential decay curve to the input data The fit equation has the form y A exp x t O where A amplitude T decay constant O offset Prior to the command the program must declare the necessary input and output arrays and assign values to the input arrays Pre assign the Order to 1 Previous variable declarations int order Order of the fit see explanation below real result 10 error 10 Arrays for results output and their uncertainties size of the arrays in brackets real x_array positions y array positions Arrays for input data int number_of_data Number of data pairs Assign values to the input parameter number_of_data and input arrays by direct copy of the NMR signal into x and y array OR from some combination of user input and signal evaluation i e known concentration for x array vs weight normalized NMR signal amplitude for y array Bruker Relaxation Time Manual 37 Z ninispec Mise The default is to allow the function to include the offset as a fit variable One can assign the fit Offset to a pre defined value using off presetting 0 fixed offset value To release the fixed Offset fit use off presetting 1 0 Syntax of the command orde
26. airs on the hard disk This option is useful for archiving after acquisition and or for fitting with other functions not available in t _ir_mb refer to the test application fit rel Monoexponential Curve Fitting To select either mono or bi exponential fitting If no exponential decay is detected the average value of the amplitudes is calculated If bi exponential is selected monoexponential curve fitting is not check marked and the fitting routine detects only one exponential the routine defaults to mono exponential fit Raw Data Modifications To select whether or not the fit routine should allow the user to preview data points decide if any data points should be deleted and or re fit the data Title of Results not more than 24 digits If desired a custom title can be entered Up to 24 characters will fit in the space allotted Normally accept the default T1 Curve Fit Results First 90 90 Pulse Separation ms To set the initial Saturation Recovery SR Delay delay between the 90 and 90 pulse default value is 5 ms The first saturation recovery pulse sequence is executed using the initial SR Delay After the first measurement and each time the pulse sequence is repeated the SR Delay is lengthened and the new value is used in the next determination e g First SR Delay initial SR Delay Second SR Delay initial SR Delay Factor all points thereafter Next SR Delay present SR Delay
27. al 1 Z ninispec 6 TI MEASUREMENT PROCEDURES vissssissssvesssesssssssscosssvnssssetsecssosassvcaseassssosessoustssasscseneasoees 13 6 1 Application t1 ir mb Options crece ee eee ente eren eene nns tn sena tn setas tn stas teas tensa aseo s sens tasto 13 6 2 Configuring t1 ir mb Options eese escis cete e eene eene ennt natns sense tasto sensns teas ta asta s stas sensn 13 6 3 Routine Measurements with Soft EDM t1 ir mb eeeeeeeee eese esses eene tne tn sens enata sas tnsno 16 6 4 Application t1 sr mb Options ecce eene eee ee eene ense tan etos stans stans etae tane tane tane eaae eoa eee 17 6 5 Configuring t1 sr mb Options eese esee eese eene eene tentur natns sense tasto estas etas esatta asta asta seen sena 18 6 6 Routine Measurements with Soft EDM t1 sr mb e eeeeee sees eene enses en enne tn tosta sense tn snas enano 20 7 T2 MEASUREMENT PROCEDURES scssssossssssecssonnssexasvannasenssssvasnscesdsounasvsnassactaacesssaansonnayes 22 7 1 Application t2_cp_mb Options sessessssessossessosoososssesossosseosossosoosossoesossossessososssesessossossossossessesos 22 7 2 Configuring t2 cp mb Options eere eese e eene eene esee tasses setae etae setas e ease ease tasse ease seas eaa 22 7 3 Routine Measurements with Soft EDM t2 cp mb e eeee cesse eese tete eene toast tne tas toaeno 24
28. al components are parallel If the field distribution is rectangular i e there is a linear field gradient across the sample then the signal is described to a first approximation by M sin y AB t 3 y AB t where A B is the largest deviation of the field from the average value Provided that the individual nuclei do not change their positions there is a fixed relationship between their phases In other words the dephasing of the magnetization is under the above condition a reversible process With a 180 pulse applied after a time 7 one can reverse the motion of the spins and cause the nuclear moments to refocus after a time 2 T They then dephase again This process is referred to as a spin echo For non viscous liquids the condition that the nuclei do not change their positions in order to ensure that they are in phase again at time 27 is often not met As a result of diffusion the nuclei move in the time between the 90 and 180 pulses and may in so doing reach a position at which the field strength is different The precession frequency is thus changed and the phase relationship irreversibly altered After 2T these nuclei are no longer in phase with the others and the signal amplitude is therefore reduced Bruker Relaxation Time Manual 32 Z ninispec Mise Under this condition the measured T is smaller than the true T2 To minimize this error the sample size should be restricted to 1 cm so the sample is restricted to the
29. ameters used to vary the pulse sequence of the relaxation time applications Calibrate Sample routine The number of data points and the distances between them have to be adjusted to ensure that the analyzed time period is matched to the decay of the signal For T2 measurements it is important to reach almost zero signal on the right side of the time window If this is not the case the fit returns an offset that might vary from one fit to the next altering the real value of T2 Therefore the pulse sequence parameters have to be carefully adjusted It should also be kept in mind that the larger the number of data points the more accurate the relaxation values will be delivered by the fitting routine Note also that in the case of T2 measurements delay values longer than 2 msec should be avoided because external sources might disturb the NMR signals In case mono exponential fit is chosen and the sample contains more than one component unstable and inaccurate results will be received If bi exponential fit is active and both components have quite close relaxation times the fitting results can be unstable Results will also vary if bi exponential fit is selected and the sample contains more than two components It was discovered that sample tubes filled more than 1 cm and therefore not located in the center of the probe coil might show unusual behavior on the left side of the time period investigated Fill height should be reduced in those ca
30. are the units of the data pair arrays in quotations The maximum number of characters for each unit is nine Additionally order 1 indicates that no fit has been executed finally when this utility has been left The Error array value for LIN REG is of no meaning but the above syntax still has to be fulfilled These commands operate the same as the previously described forms used for calculation only Therefore previous variable declarations are required values must be assigned to the input arrays and where applicable values must be pre assigned to the fixed input parameters or starting value assigned to the variable input Results and Error arrays are also follow the same pattern Please see Section for details of each command Menu Items DEL POINT Deletes one point on cursor position and executes the fitting procedure The results of the fit are displayed in the result box not available on decay fits MARK REGION gt DEL REGION and FIT Handles data points deletion and refitting of decay curves using a graphic cursor CONTINUE Leaves the display of the data pairs and executes the next ExpSpel command Bruker Relaxation Time Manual 43 Z ninispec Mise 9 4 T1 Relaxation Time Program Fitting label calc no Loop Count if modi 2 if ExpOrder 2 Order fit_bidecay x y no Result Error Order display_fit SHOW_FIT Result Error x y no ms else Order fit_monodecay x
31. can therefore avoid running into too long experiments 6 3 Routine Measurements with Soft EDM t1 ir mb Load the application that contains the parameters that are most nearly suitable for the T in your sample If you would like a hard copy of results set the blue Windows banner on the results window and select Print from the File menu Remember to condition the samples to a defined temperature Care must be taken to use the correct 90 and 180 pulse Samples of similar type e g all aqueous vs organic volume and molarity can be measured all together without tuning in between Samples that differ considerably will require prior tuning of the instrument using Update Settings Incorrectly set pulse lengths will result in X Y magnetism and an error in the estimated T due to super imposed T relaxation Tune calibrate the instrument by running Update Settings using a suitable test sample or an experimental sample if T is reasonable short e g 400 ms or less If no sample is available with suitably short T then the Recycle Delay for Update Settings must be set longer 5 to 10 times than the estimated T of the available sample and Update Settings should be run with this longer delay Bruker Relaxation Time Manual 16 tipinispec Mise Press MEASURE to start the sample series If the sample ID option is turned on the user will be prompted to enter a two part identifier The first part accepts alphanumeric entries of up
32. cans countdown in the right lower corner of the display If data storing is selected see section 6 5 but sample ID labels are not data is stored with time coordinates in the filename YYMMDDHRMNSD The actual title will be listed in the Resultbox after measurement is complete If data storing and sample ID labels are turned on then the file name is set to the two part sample ID The actual title will be listed in the Resultbox after measurement is complete Bruker Relaxation Time Manual 21 tipinispec Mise 7 T2 MEASUREMENT PROCEDURES 7 1 Application t2 cp mb Options The pulse sequence employed is the Carr Purcell Meiboom Gill CPMG spin echo method CPMG Pulse Sequence RD 90o tau 1 8000 tau DE 18090 tau asd tau u us where RD is the recycle delay user defined in the PARAMETER menu tau is a delay interval user defined value in the Application Configuration Table DE is the number of dummy echoes before an echo is sampled user defined value in the Application Configuration Table e g if DE is 1 every second echo is sampled if DE is 3 every fourth echo is sampled etc N is the number of points to be collected user defined value in the Application Configuration Table NS is the number of scans for signal averaging user defined in the PARAMETER menu Further explanation The pulse sequence begins with a 90 excitation pulse followed by a waiting interval referred to as tau in which spins e
33. comparisons are possible 5 5 Preparing the Applications Application files are available to the user via the button minispec Applications Browsing to the directory APPV400x later versions Application Pool Version x x will display the complete pool of minispec applications In this pool also the relaxation time applications are included The sub directory Additionals in minispec TestApps contains applications which are mainly intended for programming language ExpSpel teaching purposes Application files app are viewed and edited by pressing the lt EXPSPEL gt editor button empty sheet of paper 5 5 1 Preparing the Application t7 ijr mb It is highly recommended that you work with a copy of an application Do not work with the original file 7 ir mb 1 Open a Copy of the Original Application 7 ir mb Bruker Relaxation Time Manual 10 Z ninispec Mise To copy an existing application to a new name make sure that the application that should be copied is active Use the File Copy Application File as function and select the new name Return to the minispec software and load the new copy of your application as described in the minispec applications manual The minispec is now ready for adjustment and calibration with this application 2 Set Parameters for the Working Application After the new application is loaded prepare it for use through the following steps see below for details e Adjust the applicati
34. cquired using several tau values in order to detect chemical exchange or diffusion See also M C Vackier D N Rutledge Journal of Magnetic Resonance Analysis 2 1996 311 32 311 320 If data storing is selected see section 7 2 but sample ID labels are not data is stored with time coordinates in the filename YYMMDDHRMNSD The actual title will be listed in the Resultbox after measurement is complete Bruker Relaxation Time Manual 25 Z ninispec Mise If data storing and sample ID labels are turned on then the file name is set to the two part sample ID The actual title will be listed in the Resultbox after measurement is complete General Rules of Thumb for T2 set up For most reproducible results Tau and number of points should be chosen so that each sample is measured over the same intensity range i e if the first data point collected is measured at 80 full scale the last point measured should be at the same intensity offset for each sample Results may be best for offsets from 1 to 5 96 not higher than 5 Start by doing a set up run where the last signal collected will be much longer than T2 at least 5 x T2 Note the Y axis intensity on the left side of the display Y left Move to the data point where the Y axis intensity is only 1 to 5 96 of the Y left intensity Y right Take note of the time distance between Y left and Y right This is the required sampling period X diff Calculate the tau value
35. e Start Value of SECOND Component 100 1 if ESC return TRUE endif Result 4 input_real Input Amplitude Start Value of THIRD Component 20 1 if ESC return TRUE endif Result 5 input_real Input Relaxation Time Start Value of THIRD Component 100 1 if ESC return TRUE endif Result 6 input_real Input Offset Start Value 0 1 if ESC return TRUE endif The default is to allow the function to include the offset as a fit variable One can assign the fit Offset to a pre defined value using off_presetting 0 fixed_offset_value To release the fixed Offset fit use off presetting 1 0 Syntax of the command order fit tridecay x array y array number of data result error ERROR is set if determination fails not enough data pairs matrix singularity After the fit the routine sets a return value to order which is the actual order used e g if tri exponential fit is selected but only one component is detected order will be set to 1 after the fit order 3 means the data fit a tri exponential function order 2 means the data fit a bi exponential function order 2 1 means the data fit a mono exponential function order 0 means the average value of the amplitude was calculate Bruker Relaxation Time Manual 40 Z ninispec Mise Results array order 3 2 1 0 result 0 A A A average value result 1 Ti T T result 2 A2 A2 O Offs result 3 h
36. e deleted and or re fit the data Phase Cycling not in Application Versions lower than V5 0 Phase cycling is a feature to reduce instrumental errors to a minimum Therefore it is recommended to enable this option Note that the number of scans will always be a multiple of 4 when phase cycling is active Title of Results not more than 24 digits If desired a custom title can be entered Up to 24 characters will fit in the space allotted Normally accept the default T2 Curve Fit Results First 90 180 Pulse Separation To set the delay tau between the two pulses See explanations above Sampling Window ms To set the window which is used for data acquisition As both values sampling window and tau are not independent from each other the software always verifies whether the entered combinations are possible Factor for Duration s Expansion To define the duration factor and therefore the durations between the two RF pulses of the different experiments The default value of the duration factor is 1 30 Number of Data Points for Fitting To set the number of data points or experiments This value fixes the number of Hahn Echo sequences to be executed Therefore a rough estimation of the total time of analysis can be done as follows Total Time of Analysis Number of Scans x Recycle Delay Time x Number of Points Bruker Relaxation Time Manual 29 tipinispec Mise In Application Ver
37. e sample Detection angles are gain dependent and therefore need to be tuned at the gain to be used for measuring the sample of interest Refer also to any directions listed in the corresponding application notes for more information in this matter Bruker Relaxation Time Manual 46 tipinispec Mise The parameters of an application that are accessible through the Parameter menu particularly receiver gain recycle delay or number of scans have to be adjusted as well The recycle delay time allows for the nuclei e g Hydrogen nucleus in the sample to develop initial magnetization and return to this initial state between measurements i e repeat scans for signal averaging In effect the recycle delay makes sure that the second scan will give the same signal as the first one and so on Usually at least 5 times the T1 relaxation time must be entered in order to fulfill this condition The number of scans must be set so as to ensure adequate signal to noise is attained before the data points are fitted Number of scans will have to be adjusted according to signal strength and according to the user s expectation for result repeatability In general more scans will need to be collected a higher gains The receiver gain fixes the dynamic range of the data points The operator has to maximize the gain without allowing the signal to clip in order to maximize dynamic range otherwise results will be unstable Very important also are the par
38. e sample ID will be printed with the results and if data storing is chosen will be used as the data filename Do not check mark this option to use the default sample numbering The first sample measured will be labeled sample 1 and the sample number will be incremented after each sample is measured Automatic Daily Results Saving To control results storing If this option is selected the program automatically creates a saved copy of the results on the hard disk This option is useful for archiving after acquisition and results output Beep after Analysis To generate a PC noise after termination of the measurement This option is useful if not too much time after measurements should be wasted Results according to Fitting Formula Check mark this option to cause the fit amplitude results the amplitudes at the origin for each component to be listed according to the signal reading at the origin as projected by the fit regardless of the fraction of signal this component constitutes in the total signal Do not check mark to cause the fit amplitude results to be given as a fraction of the total signal i e if two components A and B are detected at a ratio of 2 1 respectively Component A will be listed as 66 6 component B will be listed as 33 3 Data Points File Storing To control data points storing If this option is selected the program automatically creates a saved copy of the X Y data pairs on the hard d
39. ed over the fitting and clamped into place Tubing from the circulator should be insulated to prevent thermal loss and should be long enough to allow the circulating heater cryostat to be located at least 0 5 m from the magnet If the circulator has flow rate control adjust the flow rate to obtain the necessary temperature stability 4 4 Temperature Range The normal operation range of the variable temperature probe head is from 10 to 70 C Operation outside this temperatures range is possible but the minispec may not be able to properly regulate magnet temperature Bruker Relaxation Time Manual 6 Z ninispec Mise 5 PREPARATION FOR MEASUREMENTS 5 1 Preparation of the minispec Check cable connections before powering ON and operating the minispec Turn ON the minispec at least three hours prior to use The temperature in the magnet chamber must be at stable operating temperature 40 0 C for best results If temperature is not yet stabilized the minispec Status Box Instrument Status will indicate the temperature error magnet temperature not check marked The actual magnet temperature is displayed there as well NOTE When restarting the minispec do not toggle the POWER switch OFF and ON too quickly or the power supply will not have a chance to reset and may not come on Allow about 5 seconds for a total reset The minispec is normally left ON if it will be used from time to time during the course of a work week It is rec
40. ent by running the Update Settings routine using a suitable test sample or an experimental sample if the T is reasonable short e g 400 ms or less If no sample is available with a suitably short T then the Recycle Delay for Update Settings must be set longer 5 to 10 times than the estimated T of the available sample and Update Settings should be run with this longer delay Like samples can be measures all together without tuning Very different samples should be run after tuning the instrument Press Measure to start the sample series If the sample ID option is turned on the user will be prompted to enter a two part identifier The first part accepts alphanumeric entries of up to ten characters The second part must be a numeric entry of up to 3 characters If T is poorly known it is normal to perform a survey measurement to decide what conditions will be needed to determine the true relaxation time constant PARAMETERS can be changed to suit the particular sample For example Recycle Delay may need to be adjusted normally to at least 5 times the anticipated value of the longest T If Gain needs to be changed because of a SIGNAL CLIP error and real detection mode is being used it may be necessary to tune the detection angles at the new gain settings select Update Settings and uncheck Update all Settings afterwards check mark Detection Angles It is recommended that for unknown systems the CPMG data should be a
41. gn values to the input parameter number of data and input arrays by direct copy of the NMR signal into x and y array OR from some combination of user input and signal evaluation i e known concentration for x array vs weight normalized NMR signal amplitude for y array The default is to allow the function to include the offset as a fit variable One can assign the fit Offset to a pre defined value using off presetting 0 fixed offset value To release the fixed Offset fit use off presetting 1 0 Syntax of the command order fit bidecay x array y array number of data result error ERROR is set if determination fails not enough data pairs matrix singularity After the fit the routine sets a return value to order which is the actual order used If bi exponential fit is selected but only one component is detected order will be set to 1 after the fit order 2 means the data fit a bi exponential function order 1 means the data fit a mono exponential function order 0 means the average value of the amplitude was calculated Results array order 2 1 0 result 0 Ay A average value result 1 T T result 2 A O Offs result 3 T result 4 O Offs Error array Uncertainty errors corresponding to each position in the result array The command fit_tridecay fits a tri exponential curve to the input data The fit equation has the form y A exp x t Az exp x t As
42. he data point for that particular IR Delay Sampling Window To set the Sampling Window width in ms From this window 16 data points are collected and averaged to produce the data point for that particular IR Delay Factor for Duration s Expansion To set the inversion recovery delay multiplication Factor This factor controls how closely spaced in time the data points will be Note that while the Factor does not have limits it impacts on the calculation of the IR Delay and the IR Delay does have a limit Please refer to the discussion on IR Delay above for guidelines Number of Data Points for Fitting To set the Number of Points which is equal to the number of experiments to be repeated and corresponding points that will be collected Fitting as few as 10 points is common for a mono exponential decay For bi exponential decays 20 to 100 points may be beneficial The maximum number of points is 256 Note that the Number of Points impacts on the number of IR Delays that must be calculated and the IR Delay does have a maximum value Please refer to the discussion on IR Delay above for guidelines In Application Versions V5 0 or higher the Factor for Duration s Expansion is no longer requested Instead the value for the last desired duration has to be entered Afterwards the program calculates the duration factor and also the total measurement time is displayed The operator has to confirm the total measurement time and
43. imental sample if T is reasonable short e g 400 ms or less If no sample is available with suitably short T then the Recycle Delay for Bruker Relaxation Time Manual 20 tipinispec Mise Update Settings must be set longer 5 to 10 times than the estimated T of the available sample and Update Settings should be run with this longer delay Press MEASURE to start the sample series If the sample ID option is turned on the user will be prompted to enter a two part identifier The first part accepts alphanumeric entries of up to ten characters The second part must be a numeric entry of up to 3 characters If T is poorly known it is normal to perform a survey measurements to decide what conditions will be needed to best determining the relaxation time constant Initial values for SR Delay and Factor should be set so that approximately half of the measured amplitudes are negative and the rest are positive PARAMETERS can be changed to suit the particular sample For example Recycle Delay may need to be adjusted normally at least 3 times the anticipated value of the longest T If Gain needs to be changed because of a SIGNAL CLIP error it is recommended to tune the detection angles at the new gain settings select Update Settings and uncheck Update all Settings afterwards check mark Detection Angles Progress during measurements is given by message in the RESULTBOX Measuring Point No lt gt and by the Number of S
44. iments have proven that the results for samples with two components and small differences in relaxation times may scatter Two components with significant differences in relaxation times maybe factor 10 can be analyzed very reproducible 3 Fitting Offset and Dynamic Range of Data Points Adjust parameters in a way that the dynamic range of the data points becomes as big as possible Adjust the time base so that the remaining offset on the right side of the time axis becomes a minimum Note that small dynamic ranges and big offsets lead to unstable relaxation time results 4 Comparison of Different Samples If a number of samples should be analyzed with the minispec and distinguished e g by their relaxation times fix the parameters of the application in a way that makes them suitable for all samples of interest Analyze all samples with one and the same set of parameters This allows to check for differences in relaxation time parameters best 5 Amplitudes of Components All relaxation time applications deliver also amplitudes of components They can be used for quantitative analysis Note that those amplitudes depend also upon sample weights and tube positions in the probe Therefore calculations with amplitudes require the knowledge of the sample weights and the tubes have to be positioned in the linear responding area of the probe head It is essential that for comparisons of amplitudes the application parameters remain unchanged otherwise no
45. ine detects only one exponential the routine defaults to mono exponential fit In the case of mono and bi exponential fits if no exponential decay is detected the average value of the amplitudes is calculated 7 4 Application t2 se mb Options The pulse sequence employed is the well known Hahn Spin Echo method Hahn Spin Echo Sequence RD P 900 tau 1800 tau asd tau ns n with increment in tau tau tau Factor where RD is the recycle delay user defined in the PARAMETER menu tau is an incremented delay interval user defined starting value in the Application Configuration Table NS is the number of scans for signal averaging user defined in the PARAMETER menu N is the number of points to be collected user defined value in the Application Configuration Table Factor After each measurement tau is multiplying by the duration factor to determine the tau for the next experiment Further explanation The pulse sequence begins with a 90 excitation pulse followed by a waiting interval referred to as tau in which spins evolve A 180 pulses that is phase shifted in the X Y plane by 90 relative to the 90 excitation pulse reverses the sense of the evolving vectors and after another period tau causes an echo to form Acquisition of a single sample point occurs at sampled echoes Afterwards the recycle delay time passes and the same experiment is repeated NS times Now the first data point is acquired and the measure
46. ing If no exponential decay is detected the average value of the amplitudes is calculated If bi exponential is selected monoexponential curve fitting is not check marked and the fitting routine detects only one exponential the routine defaults to mono exponential fit Raw Data Modifications To select whether or not the fit routine should allow the user to preview data points decide if any data points should be deleted and or re fit the data Phase Cycling not in Application Versions lower than V5 0 Phase cycling is a feature to reduce instrumental errors to a minimum Therefore it is recommended to enable this option Note that the number of scans will always be a multiple of 4 when phase cycling is active Title of Results not more than 24 digits If desired a custom title can be entered Up to 24 characters will fit in the space allotted Normally accept the default T2 Curve Fit Results 90 180 Pulse Separation tau To set the delay tau in ms This factor controls how closely spaced in time the data points will be Note that tau does have limits 0 04 tau 100 Number of Data Points for Fitting To set the Number of Points which is equal to the number of echoes to be sampled Fitting as few as 100 points is common for a mono exponential decay For bi exponential decays 200 to 250 points may be beneficial The maximum number of points is 256 Number of not Fitted Echoes To
47. ints measured data variables for determinations pulse sequence parameters pulse sequence parameter pulse sequence parameter pulse sequence parameter result from fit result errors help variables rounded results resultbox title sample identification datapairs filename resultbox title sample identification id 2 sample zd CNT 20 Loop Count 10 ExpOrder 1 DUR 5 DF 1 3 WIN 0 01 DSW 0 05 dec afl store 0 ExpOrder zl modi 2 loop 0 out 1 print line CALIBRATION FILE if ERROR DUR get real CALIBRATION FILE DUR CNT get int CALIBRATION FILE LC ExpOrder get int CALIBRATION FILE EO WIN get real CALIBRATION FILE WIN DSW get real CALIBRATION FILE DSW while TRUE while Loop Count lt CNT and Loop Count lt 500 pulses pulse sequence generation sp 180 0 1 a 180 degree pulse sd D dur milliseconds sp 90 0 1 a 90 degree pulse sd DSW wait for receiver dead time asd WIN get a single data point D D DF next duration endpulses pulse sequence end is_sample_in Bruker Relaxation Time Manual dk Hb dt db Hb db db db db Hb db db db db dt db ehm 36 Z ninispec Mise if ERROR print line FLASHBOX No Sample Inserted return TRUE endif print line RESULTBOX Measuring Point No lt gt Loop Count 1 endif measure absciss
48. isk This option is useful for archiving after acquisition and or for fitting with other functions not available in t _ir_mb refer to the test application fit_rel Monoexponential Curve Fitting To select either mono or bi exponential fitting If no exponential decay is detected the average value of the amplitudes is calculated If bi exponential is selected monoexponential curve fitting is not check marked and the fitting routine detects only one exponential the routine defaults to mono exponential fit Raw Data Modifications To select whether or not the fit routine should allow the user to preview data points decide if any data points should be deleted and or re fit the data Bruker Relaxation Time Manual 14 Z ninispec Phase Cycling not in Application Versions lower than V5 0 Phase cycling is a feature to reduce instrumental errors to a minimum Therefore it is recommended to enable this option Note that the number of scans will always be a multiple of 4 when phase cycling is active Title of Results not more than 24 digits If desired a custom title can be entered Up to 24 characters will fit in the space allotted Normally accept the default T1 Curve Fit Results First 180 90 Pulse Separation values in ms To set the initial Inversion Recovery IR Delay delay between the 180 and 90 pulse default value is 5 ms The first inversion recovery pulse sequence is executed using
49. ith short inter pulse durations is used The decay behaves fully mono exponential These conditions should result in stable and reproducible T2 relaxation time analysis Bruker Relaxation Time Manual 50 Z ninispec FS BRUKER WIN NMS t1 ir mb mdt File Edit Measure Parameter View Window Help a R p amp Be ss E a mb sig T1 CURVE FIT RESULTS 12 02 1998 11 13 Pulse Separ Dur Factor Sam Window Del Sam Win Data Points Expon Order Modifications Data Storing The screen dump above shows a tl_ir_mb analysis As described for the T2 analysis before parameters are selected optimally Again the full dynamic range of the system is used and the T1 signal can reach the M magnetization near the end of the sampling period Note that for both above shown measurements the same sample has been used As expected from theories the T1 value of this liquid distilled water with CuSO4 is longer than the T2 value Bruker Relaxation Time Manual 5 Z ninispec Below you will find a few examples of non optimized analysis parameters FJ BRUKER WIN NMS tl_ir_mb_sig L a Lx Eie Edit Measure Parameter View Process Window Help ICA AEA tl mb sig LLO xd Ges Pulse Separ Dur Factor Sam Windouw Del Sam Win Data Points Expon Order Modifications Data Storing For Help press F1 NB1302 149 236 42 200 0 iB sten Sc swindowssintemet Mail GS BRUKER WIN NMS T Micros
50. l Rules of Thumb for T2 set up For most reproducible results Tau and number of points should be chosen so that each sample is measured over the same intensity range i e if the first data point collected is measured at 80 full scale the last point measured should be at the same intensity offset for each sample Results may be best for offsets from 1 to 5 96 not higher than 5 Start by doing a set up run where the last signal collected will be much longer than T2 at least 5 x T2 Note the Y axis intensity on the left side of the display Y left Move to the data point where the Y axis intensity is only 1 to 5 96 of the Y left intensity Y right Take note of the time distance between Y left and Y right This is the required sampling period X diff Calculate the tau value Decide how many points will be collected Decide what working application copy will best fit the time X diff To find appropriate parameters The time taken by the total number of echoes must describe most of the decay curve The maximum number of points is 256 Fitting 100 points is commonly enough for a mono exponential decay amp for bi exponential decays 200 to 250 points may be beneficial However due to long experimental times 20 respectively 40 data points should be measured at least Set the pulse sequence parameters so that the number of points collected adequately describes the relaxation curve The user may select either mono or bi exp
51. le will be listed in the Resultbox after measurement is complete 6 4 Application t1 sr mb Options Application t sr mb Saturation Recovery Pulse Sequence RD 90 SR Delay 909 RDT asd ys N with increment in SR Delay SR delay SR Delay Factor where RD is the recycle delay user defined in the PARAMETER menu SR Delay is an incremented saturation recovery delay interval user defined starting value in the Application Configuration Table RDT is a delay for receiver dead time user defined value in the Application Configuration Table NS is the number of scans for signal averaging user defined in the PARAMETER menu N is the number of points to be collected time user defined value in the Application Configuration Table FACTOR After each measurement SR Delay is multiplying by the duration factor to determine the SR Delay for the next experiment Bruker Relaxation Time Manual 17 Z ninispec eh sn 6 5 Configuring t1 sr mb Options Configure pulse sequence variables and user options through the Application Configuration Table This table looks as follows the minispec APPLICATION CONFIGURATION TABLE T1 Curve Fit Results e TOT a we iv is a n z cd E n r inputi IF put Sample Identification Check mark this option if the user should be prompted for a corresponding sample ID at the time of measurement The sample ID will be printed with the re
52. les e g 1 cm for under filled coil 4 cm for over filled coil 5 3 2 Tuning Calibration Procedure For under filled coils samples should be centered in the coil so that the maximum amplitude is obtained using the test application FID Position the under filled sample for maximum signal amplitude by observing the signal with the test application FID If a consistent sample height is used all the time the sample lower limit can be set by adjusting the pedestal inside the probe head chamber so that the e g 1 cm sample is in the optimum position This adjustment has been preset in the factory for a 4 cm sample over filled coil on ratio type probe heads Sample positioning is not an issue for over filled coils Bruker Relaxation Time Manual 8 Z ninispec Mise Start the tuning calibration procedure by selecting Update Settings from the the minispec menu Follow the instructions that come up The update routine automatically sets the correct Gain Magnetic Field Detection Angles and Pulse Lengths while the calibration sample is in the sample chamber Make a note of the correct Gain setting found by the update routine for later use 5 4 Application Selection and Curve Fitting Rules 5 4 1 Application Selection 1 T1 Relaxation Time Analysis As indicated later the data points of the tl_ir_mb application are spread upon a range that is a factor 2 bigger than the dynamic range of the t1 sr mb data points Therefore fiting the da
53. mb_mdt oj xi ES Order 1 Storing Data D Amplitudel 62 0 0 8 53 ms 7 l ms 3 2 0 2 m HH Ic Ed E EDIECDDEHTE L cd x Above T2 Determination by t2_cp_mb Detection Mode magnitude minispec out of Resonance Besides the totally unusual behavior of the signal signal strength is partially left 54 Bruker Relaxation Time Manual Z ninispec Fo BRUKER WIN NMS t2 cp mb sig f x File Edit Measure Parameter View Process Window Help EJS EJA slal pj amp alas t2_cp_mb sig 3 t2_cp_mb mdt Nms Applications Pulse Separ Data Points Dummy Echoes Expon Order Modifications Storing Data Instrument Status Amplitudel Taul 500 1000 1500 2000 2500 3000 3500 Continue c disk_old nms new cp_mb For Help press F1 NB1269 149 236 42 201 0 EES ifi Start x C WINDOWS Internet Maii TRY Microsoft Word rela ma a BRUKER WIN NMS YS ad 12 30 In the example above quite long values for tau have been chosen External sources influence the NMR signal significantly Reduction of the tau value is absolutely necessary In order to reach the base line on the right side of the signal more data points or dummy echoes have to be acquired Bruker Relaxation Time Manual 55 Z ninispec f BRUKER WIN NMS t2_cp_mb sig File Edit Measure Parameter View Process Window Help Es Saloy eJ eoo ss
54. ment continues with the same sequence but increased tau value The number of data points defines the number of experiments This means that the execution of this measurement with 20 data points will take approximately 20 times longer than a corresponding CPMG pulse sequence However for polymers this sequence is a must 7 5 Configuring t2 se mb Options Configure pulse sequence variables and user options through the Application Configuration Table This table looks as follows Bruker Relaxation Time Manual 27 T2 Curve Fit Results put Be putra IF Sample Identification Check mark this option if the user should be prompted for a corresponding sample ID at the time of measurement The sample ID will be printed with the results and if data storing is chosen will be used as the data filename Do not check mark this option to use the default sample numbering The first sample measured will be labeled sample 1 and the sample number will be incremented after each sample is measured Automatic Daily Results Saving To control results storing If this option is selected the program automatically creates a saved copy of the results on the hard disk This option is useful for archiving after acquisition and results output Beep after Analysis To generate a PC noise after termination of the measurement This option is useful if not too much time after measurements should be wasted Resul
55. nent should always be checked by the operator whether they make really sense As different combinations of relaxation times and their amplitudes might lead to comparable fit qualities it is possible to measure a multi component sample several times and receive completely different results Bruker Relaxation Time Manual 49 Z ninispec Problem G NMR Signals do not show expected curve behavior In the following example a couple of NMR relaxation time curves are presented Whereas the first curves show the desired and expected behaviors the rest exhibit unexpected relaxation behaviors Explanation is given regarding the sources of the errors fF BRUKER WIN NMS t2_cp_mb sig File Edit Measure Parameter View Process Window Help m Ep rz amp e E3 3 se lan Hui E rex EX IA pe ies Sx Ne uox Pulse Separ Data Points Dummy Echoes Expon Order Modifications Storing Data 50 100 150 200 250 300 350 400 450 500 ms Ready INBi3O2 i48236 42200 0 IgA Start xzcs wiNDOwSsIntemet Maill iS BRUKER WIN NMS __ Ce Above is shown a t2_cp_mb application with well chosen parameters The gain is chosen so that the full dynamic range of the system is used signal on the left side starts at about 80 90 of the full display The total acquisition time is adjusted so that data is sampled from the signal over the entire decay period as the signal approaches base line A high number of data points w
56. nual 7 Z ninispec Mise 5 3 1 Tuning Calibration Sample For rigorous measurement of absolute values of T or T samples should be restricted to the homogeneous field region of the magnet B field and of the coil B field This is accomplished by ensuring the proper positioning of the probe head in the magnet pole gap see Section 3 2 and by under filling the probe head coil Recommended under filling heights for the following probe heads are SARI ratio type lt 1cm cl AD a absolute type lt 3cm If relative values of T or Tz are sufficient and absolute relaxation values are not really important it is advantageous to use an over filled coil because less care needs to be take in preparing samples and optimizing conditions Suggested over filling heights are IRI ratio type 4cm ed Alon absolute type lt 6cm For measurements of aqueous samples the calibration sample should have a composition similar to the samples of interest especially with regards to the molarily of salt or buffer The following sample is suggested Phosphate buffer or physiologic saline with approximately 1 25 g L CuSO4 5H2O to shorten T relaxation time Use the same sample tube filling height as will be used with real samples e g 1 cm for underfilled coil 4 cm for overfilled coil For oil samples the following sample is recommended White light mineral oil Use the same sample tube filling height as will be used with real samp
57. of t1 ir mb application chapter 5 5 1 Adjust the application parameters through the Acquisition Parameter Table Number of Scans normally 1 to 4 but depends on signal to noise Recycle delay set to 5 to 10 times longer than the anticipated longest T Analogue filter narrow to reduce noise broad if early sampling is required see Delay Sample Window below Digital filter 20000 Detection mode real Offset compensation off Configure pulse sequence variables and user options through the Application Configuration Table 5 5 4 Preparing the Application t2 se mb See preparation procedure of t1 ir mb application chapter 5 5 1 Adjust the application parameters through the Acquisition Parameter Table Number of Scans normally 1 to 4 but depends on signal to noise Recycle delay set to 5 to 10 times longer than the anticipated longest T Analogue filter narrow to reduce noise broad if early sampling is required see Delay Sample Window below Digital filter 20000 Detection mode magnitude Offset compensation off Configure pulse sequence variables and user options through the Application Configuration Table Note that for all applications minimum application version 5 0 an enabled Phase Cycling Mode see Application Configuration Table may have influence on the number of scans performed If Phase Cycling is active the number of scans is always a multiple of 4 Bruker Relaxation Time Manual 12
58. oft Word pic rela The same T1 application and the same sample analyzed with non optimized parameters The dynamic range of the system is poorly used the time scale does not reach the M magnetization the number of data points acquired is rather low As a result of those non optimized settings a big T1 error range is received 80 msec with an error of 3 msec On the optimized analysis before an error of only 0 1 msec could be reached Bruker Relaxation Time Manual 52 A non optimized T2 screen is presented below Explanations as above in the case of non perfect T1 parameters RUKER WIN NMS t2_ Edit Measure Parameter View Window Help Modifications Storing Data JAmplitudel e Taul Re For Help press F1 ST T md KA yd 12 502 EH I 1 1 Edi Measure Parameter jew Process Window Help l x 25 125 150 175 200 225 250 275 300 325 350 375 400 ms For Help press F1 NB1269 149 236 42 201 0 581371 3454634 3 eQS i star xL csvwiNDOwSsntemetr Maill is BRUKER WIN NMS BY Microsoft Word relax ma BISA 1231 Above T2 Determination by t2_cp_mb application Detection Mode real minispec out of Resonance Besides the unusual behavior at the signal beginning signal strength is significantly lost Bruker Relaxation Time Manual 53 Z ninispec ES BRUKER WIN NMS DIAGNOSTICS File Edit Nms Setup View Window Help Bl f t2_cp_
59. ommended to turn OFF the power to the minispec when it will not be in use for a number of weeks The minispec power should be left OFF when power interruptions are likely e g during building maintenance heavy thunder storms or if the minispec is being cleaned 5 2 Preparation of the Temperature Conditioner Turn on the temperature conditioning apparatus at least 30 minutes before use and let the temperature stabilize If a circulating heater cryostat bath is used with a variable temperature probe head to regulate temperatures below 10 C consider ventilating the probe head sample compartment with dry temperature conditioned air or Nitrogen gas to prevent condensation in the sample compartment 5 3 Instrument Tuning and Pulse Length Calibration Before using relaxation time applications for measurements the instrument must be tuned and the 90 and 180 pulse lengths must be calibrated using a representative sample ideally at a Gain that will be appropriate for experimental samples T experiments in particular require very accurate setting of 90 and 180 pulse lengths because miss set values result in incomplete inversion Residual signal that results in the X Y plane will undergo T or T2 relaxation which decreases the signal intensity by a mechanism other than T relaxation Since the length of time for this to occur is variable because of the variable tau a variable error in signal amplitude will result Bruker Relaxation Time Ma
60. on parameters Acquisition Parameter Table from the Parameter menu e Configure the pulse sequence variables and other settings through the Application Configuration Table from the Parameter menu Adjust the application parameters through the Acquisition Parameter Table Number of Scans normally 1 to 4 but depends on signal to noise Recycle delay set to 5 to 10 times longer than the anticipated longest T Analogue filter narrow to reduce noise broad if early sampling is required see Delay Sample Window below Digital filter 20000 Detection mode real Offset compensation off Configure pulse sequence variables and user options through the Application Configuration Table 5 5 2 Preparing the Application t1 sr mb See preparation procedure of t1 ir mb application chapter 5 5 1 Adjust the application parameters through the Acquisition Parameter Table Number of Scans normally 1 to 4 but depends on signal to noise Recycle delay set to 5 to 10 times longer than the anticipated longest T Analogue filter narrow to reduce noise broad if early sampling is required see Delay Sample Window below Digital filter 20000 Detection mode magnitude Offset compensation off Configure pulse sequence variables and user options through the Application Configuration Table Bruker Relaxation Time Manual 11 Z ninispec Mise 5 5 3 Preparing the Application t2 cp mb See preparation procedure
61. onential fitting If bi exponential is selected and the fitting routine detects only one exponential the routine defaults to mono exponential fit In the case of mono and bi exponential fits if no exponential decay is detected the average value of the amplitudes is calculated Bruker Relaxation Time Manual 3 Z ninispec Mise 8 ADVANCED TOPICS 8 1 T2 Relaxation Time Physical Principles For a sample containing hydrogen atoms in a magnetic field Bo the NMR resonance frequency 0 is given by y Bo 1 where y is the gyromagnetic ratio of the hydrogen nucleus If all the nuclei are in the same field i e in a completely homogeneous field then the NMR signal after a 90 pulse decays exponentially with time constant T according to the equation M Mo exp t T 2 2 In practice the perfect field condition is almost never achieved The field is much more often inhomogeneous i e some nuclei see a higher field By A B and some a lower field Bo A B than the average Bo Thus within the sample there are nuclei with various resonance frequencies 0 0 A 0 0 Aw After the 90 pulse is switched off all nuclei have the same phase However after a time f phase differences are present as a result of the different precession speeds These differences reduce the signal since this depends on the vector sum of the magnetic moments of all the rotating nuclei and the maximum signal is only obtained when all the individu
62. order which is the actual order used e g if quad exponential fit is selected but only three component are detected order will be set to 3 after the fit order 4 means the data fit a quad exponential function order 3 means the data fit a tri exponential function order 2 means the data fit a bi exponential function order 1 means the data fit a mono exponential function order 0 means the average value of the amplitude was calculate Bruker Relaxation Time Manual 42 Z ninispec Mise Results array order 4 3 2 1 result 0 Aj Aj Aj A result 1 Ti T T T result 2 A z A gt O Offset result 3 h 7 7t E result 4 A3 A3 O Offs result 5 Ts 5 result 6 Ay O Offs result 7 T4 result 8 O Offs Error array Uncertainty errors corresponding to each position in the result array The command display_fit FIT calculates the specified fit displays the input data pairs with the fit and executes a utility that provides cursor control and menu options so that the user may highlight and delete data points from the input data and fit the remaining pairs again The utility may be used in connection with the fit functions discussed in Section Syntax of the command order display fit FIT result error x y n x unit y unit where FIT can be one of the following fit types MONODECAY BIDECAY TRIDECAY or QUADDECAY X unit y unit These
63. r fit monodecay x array y array number of data result error ERROR is set if determination fails not enough data pairs matrix singularity After the fit the routine sets a return value to order which is the actual order used If mono exponential fit is selected but no exponential decay is detected the average of ordinate values will be calculated and order will be set to 1 after the fit order 1 means fit function y A exp x 7T O was used order 0 means the average value of the amplitude was calculated Results array for order 1 0 result A average value result 1 T result 2 O Offset Error array Uncertainty errors corresponding to each position in the results array The command fit bidecay fits a bi exponential curve to the input data y A exp x T Az exp x 5 O where Aj Az amplitudes of components 1 and 2 T 5 decay constants of components and 2 O offset Prior to the command the program must declare the necessary fit parameters input and output arrays and assign values to the fit parameter and input arrays Previous variable declarations int order Order of the fit see explanation below real result 10 error 10 Arrays for results output and their uncertainties real x array positions y array positions Arrays for input data int number_of_data Number of data pairs Bruker Relaxation Time Manual 38 tipinispec Mise Assi
64. ses Instruments not operated on resonance have shown decay curves T2 analysis that contain negative data points real detection mode or that reflect from the zero line magnitude detection Of course also a defective instrument could be responsible for scattered results In one case a total collapse to zero of the data points somewhere in the time period was reported The problem was investigated by running repeat measurements in LIVE mode set SETUP PROGRAM Acquisition mode to LIVE PARAMETER Number of Scans to 1 and initiate repeat scans with lt SHIFT gt R This will show if the above described collapse of the signal appears at random or systematically A defective probe head could lead to such a behavior Bruker Relaxation Time Manual 47 Z ninispec Mise Problem B Results are drifting If results are stable in the short term but drift to higher or lower values over time this usually indicates that one part of the experiment set up has not stabilized Often this will be the temperature of the sample itself Relaxation times are strongly temperature dependent and therefore sample temperature is quite important If an external water bath is connected the temperature stability of this bath should be verified Also a non stabilized magnet temperature magnetic field change could cause a results drift Non stabilized magnetic fields are indicated on the lower left side of the LC display by a T symbol Last
65. set the number of Dummy Echoes A combination of the size of tau the number of dummy echoes and the number of points controls the length of the acquisition period 7 3 Routine Measurements with Soft EDM t2 cp mb The CPMG pulse sequence is used in order to eliminate the effect of slightly miss set 180 pulse widths The sequence also minimizes errors due to diffusion effects and J modulation However care must be taken not to set tau too long relative to the likely rate of diffusion Bruker Relaxation Time Manual 24 Z ninispec Mise A tau limit of 1 msec is recommended to eliminate J mod and diffusion effects However tau is not actually limited in the application and can be set much longer if desired This is particularly true when measuring long T2s in aqueous solutions but can also be a problem in organic solvents that have high self diffusion coefficients see also table of self diffusion coefficients in the Bruker Almanac Diffusion within the sample between pulses causes the signal to decay more rapidly than decay due to T alone Hence diffusion causes an error lower value for the estimated T Load the application that contains the parameters which are most nearly suitable for the T in your sample If you would like a hard copy of results set the blue Windows banner on the results window and select Print from the File menu Remember to condition the samples to a defined temperature Tune calibrate the instrum
66. sions V5 0 or higher the Factor for Duration s Expansion is no longer requested Instead the value for the last desired duration has to be entered Afterwards the program calculates the duration factor and also the total measurement time is displayed The operator has to confirm the total measurement time and can therefore avoid running into too long experiments 7 6 Routine Measurements with Soft EDM t2 se mb The spin echo pulse sequence is used in cases where the CPMG sequence cannot be applied Please note that the sequence is sensitive to sample diffusion Therefore liquids will be examined with CPMG In the case of fast relaxation diffusion and other effects can be neglected and the t2 se mb software can be executed Load the application that contains the parameters which are most nearly suitable for the T in your sample If you would like a hard copy of results set the blue Windows banner on the results window and select Print from the File menu Remember to condition the samples to a defined temperature Tune calibrate the instrument by running the Update Settings routine using a suitable test sample or an experimental sample if the T is reasonable short e g 400 ms or less If no sample is available with a suitably short T then the Recycle Delay for Update Settings must be set longer 5 to 10 times than the estimated T of the available sample and Update Settings should be run with this longer delay Like samples
67. sults and if data storing is chosen will be used as the data filename Do not check mark this option to use the default sample numbering The first sample measured will be labeled sample 1 and the sample number will be incremented after each sample is measured Automatic Daily Results Saving To control results storing If this option is selected the program automatically creates a saved copy of the results on the hard disk This option is useful for archiving after acquisition and results output Beep after Analysis To generate a PC noise after termination of the measurement This option is useful if not too much time after measurements should be wasted Bruker Relaxation Time Manual 18 Z ninispec Mise Results according to Fitting Formula Check mark this option to cause the fit amplitude results the amplitudes at the origin for each component to be listed according to the signal reading at the origin as projected by the fit regardless of the fraction of signal this component constitutes in the total signal Do not check mark to cause the fit amplitude results to be given as a fraction of the total signal i e if two components A and B are detected at a ratio of 2 1 respectively Component A will be listed as 66 6 component B will be listed as 33 3 46 Data Points File Storing To control data points storing If this option is selected the program automatically creates a saved copy of the X Y data p
68. ta points will cause more accurate results in the case of the t1 ir mb application On the other side the repetition delay of the t1 sr mb application needs to be only half compared to the delay for the t1 ir mb application Thus the measurement time of the t1 ir mb application is twice the time for the t1 sr mb analysis Therefore the user has to select whether accuracy tl ir mb or short measurement time tl sr mb is required 2 T2 Relaxation Time Analysis Liquids with T2 relaxation times in the msec range are usually investigated with the t2 cp mb application Whereas the t2 se mb application requires analysis times which are comparable to T1 applications the t2 cp mb investigations require only a few seconds This makes the t2 cp mb application very attractive However it is known that samples with shorter T2 relaxation times like polymers or solids cannot be measured with the fast t2 cp mb software but have to be analyzed with the t2 se mb application 5 4 2 Curve Fitting Rules The following recommendations are valid for all relaxation time applications 1 Data Points Spacing It has been shown in an unchanged time period that always those data point fits have delivered best accuracy and stability which could operate with the highest number of data points Therefore the operator should always try to chose a high number of data points 2 Bi Exponential Fitting Bruker Relaxation Time Manual 9 tipinispec Mise Multiple exper
69. te relaxation time numbers are expected the sample has to be placed in the middle of the probe head coil and the fill height should be around 1 cm Higher fill heights will lead to relaxation time shifts Another trouble might occur if someone measures T2 times with application t2 se mb If the sample has is characterized by significant diffusion this application will lead to shortened relaxation times Samples that may exhibit diffusion behavior should be examined with the CPMG application t2 cp mb On the other hand scientists have shown that polymer samples cannot be analyzed with CPMG but require the Hahn Echo pulse sequence as used in the application t2 se mb Besides this it is also possible that the measurement parameters are not fixed perfectly More details about choosing the parameters properly are listed above Bruker Relaxation Time Manual 48 Z ninispec Mise Problem D T2 relaxation times bigger than T1 relaxation times According to the definition of the relaxation times the T1 times are always longer or at least equal to the T2 relaxation times If this is not the case parameters of the two applications are not correctly adjusted The user might refer to the above described parameter adjustments Problem E Only a mean value is presented as the result When the measured data points decay curve cannot be fitted with the expected number of components the fitting software automatically reduces the fit order For e
70. ts according to Fitting Formula Check mark this option to cause the fit amplitude results the amplitudes at the origin for each component to be listed according to the signal reading at the origin as projected by the fit regardless of the fraction of signal this component constitutes in the total signal Do not check mark to cause the fit amplitude results to be given as a fraction of the total signal i e if two components A and B are detected at a ratio of 2 1 respectively Component A will be listed as 66 6 component B will be listed as 33 3 Bruker Relaxation Time Manual 28 tipinispec Mise Data Points File Storing To control data points storing If this option is selected the program automatically creates a saved copy of the X Y data pairs on the hard disk This option is useful for archiving after acquisition and or for fitting with other functions not available in t _ir_mb refer to the test application fit rel Monoexponential Curve Fitting To select either mono or bi exponential fitting If no exponential decay is detected the average value of the amplitudes is calculated If bi exponential is selected monoexponential curve fitting is not check marked and the fitting routine detects only one exponential the routine defaults to mono exponential fit Raw Data Modifications To select whether or not the fit routine should allow the user to preview data points decide if any data points should b
71. umber of Points impacts on the number of SR Delays that must be calculated and the SR Delay does have a maximum value Please refer to the discussion on SR Delay above for guidelines In Application Versions V5 0 or higher the Factor for Duration s Expansion is no longer requested Instead the value for the last desired duration has to be entered Afterwards the program calculates the duration factor and also the total measurement time is displayed The operator has to confirm the total measurement time and can therefore avoid running into too long experiments 6 6 Routine Measurements with Soft EDM t1 sr mb Load the application that contains the parameters that are most nearly suitable for the T in your sample If you would like a hard copy of results set the blue Windows banner on the results window and select Print from the File menu Remember to condition the samples to a defined temperature Care must be taken to use the correct 90 pulse Samples of similar type e g all aqueous vs organic volume and molarity can be measured all together without tuning in between Samples that differ considerably will require prior tuning of the instrument using the automatic Update Settings routine An incorrectly set pulse length will result in unexpected X Y magnetism and an error in the estimated T due to super imposed magnetization Tune calibrate the instrument by running Update Settings using a suitable test sample or an exper
72. value than mentioned above the measurement parameters or the sample condition may have to be optimized 1 First the user must ensure that the sample inside the probe compartment is kept at a constant temperature The temperature should be checked for stability at the actual sample position i e inside the probe head A thermometer can be placed inside the sample chamber and should be checked over a period of time a few hours If no external water bath is connected the temperature is stable as long as there is no T symbol on the lower left corner of the minispec LC display an indication of wrong temperature If an external bath is connected the temperature of the bath and the probe head chamber should be checked If prior to measurement the sample is not pre conditioned to the temperature of the sample compartment a delay of at least 15 minutes should be allowed after the sample is inserted into the minispec before the first acquisition is started 2 Non optimized parameters are the main cause of unstable relaxation results Parameters that must be considered include those set in the instrument calibration table pulse lengths magnetic field and detection angle those set using the Parameter menu and those set through the menu Calibrate Sample in order to vary the pulse sequence Instrument calibration parameters depend on the type of sample and may need to be optimized using the sample of interest or a representativ
73. volve A 180 pulses that is phase shifted in the X Y plane by 90 relative to the 90 excitation pulse reverses the sense of the evolving vectors and after another period tau causes an echo to form Acquisition of a single sample point occurs at sampled echoes This sequence of phase shifted 180 pulses with evolution interval tau and sampling is repeated to collect all the points necessary for describing the signal decay In most cases the data is not sampled at each echo Dummy echoes are echoes in which no sampling point is collected Use of dummy odd number echoes causes the program to sample at every e g 2 4 g or 20 echo The user may set the number of dummy echoes in the Application Configuration Table 7 2 Configuring t2 cp mb Options Configure pulse sequence variables and user options through the Application Configuration Table This table looks as follows Bruker Relaxation Time Manual 22 T2 Curve Fit Results PUL ae IH pU Be putra Inputs Sample Identification Check mark this option if the user should be prompted for a corresponding sample ID at the time of measurement The sample ID will be printed with the results and if data storing is chosen will be used as the data filename Do not check mark this option to use the default sample numbering The first sample measured will be labeled sample 1 and the sample number will be incremented after each sample is measured
74. xample if a bi exponential fit order 2 does not detect two components the order will be reduced to 1 and a mono exponential fit will be calculated instead A mono exponential fit if no exponential decay is found will be reduced to a calculation of mean value Such a result indicates that either no sample is inserted the signal is too low receiver gain requires increased value or the time period is not chosen correctly In most cases the correct adjustment of the parameters will help It is also obvious that samples must contain the investigated nuclei in order to yield an NMR signal Problem F Results make no sense Multiple component analysis do not work When samples with more than one component are examined the user must judge whether the results really make sense The more components the software considers the more fitting parameters are available A fit might have a number of different solutions although only one makes sense It was already indicated above that problems might also appear when the relaxation times of the different components are too close Unstable results might be caused Fitting routines for more than two components are included in the application fit rela found in the directory Test Applications For the previously mentioned reason the user is expected to enter the fitting start parameters This increases the chance of finding meaningful results However the results of fits on signals with more than one compo

Relaxation Time (T1 and T2) Measurements Bruker minispec

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