/* gNhsqc.c HSQC gradient sensitivity enhanced version for N15, with options for TROSY on N15/H1 and for T1, T1rho, and T2 relaxation measurements of the N15 nuclei. Coherence transfer gradients may be z or magic-angle (using triax probe). Standard features include optional 13C sech/tanh pulse on CO and Ca to refocus 13C coupling during t1; 1D checks of N15 and H1 pulse times using calN and calH; one lobe sinc pulse to put H2O back along z (the sinc one-lobe is significantly more selective than gaussian, square, or seduce 90 pulses); preservation of H20 along z during t1 and the relaxation delays; option of obtaining spectrum of only NH2 groups; 2H decoupling option for partially-deuterated (ie NHD) groups. pulse sequence: Kay, Keifer and Saarinen, JACS, 114, 10663 (1992) sech/tanh pulse: Silver, Joseph and Hoult, JMR, 59, 347 (1984) Bendall, JMR, A116, 46 (1995) relaxation times: Kay et al, JMR, 97, 359 (1992) Farrow et al, Biochemistry, 33, 5984 (1994) TROSY: Weigelt, JACS, 120, 10778 (1998) Written by MRB, December 1997, starting with ghn_co from BioPack. Revised and improved to a standard format by MRB, BKJ and GG for the BioPack, January 1998, so as to include calculation of the above standard features within the pulse sequence code and associated macro. Minor additions during 98, TROSY added Dec 98. (Version Dec 1998). ************************************************************************************ Dealing with Radiation Damping Use at very high magnetic fields (>600 MHz) and/or with cryogenically-cooled probes can present unique problems arising from fast radiation damping. Theoretically- calculated power levels may be incorrect. For this reason, fine power control is present prior to H2O-selective shaped pulses. The amplitude of the flipback pulse(s) (pwHs) is adjustable around theoretical value (tpwrs). If tpwrsf_t < 4095.0 the value of tpwrs is increased 6db and values of tpwrsf_t(_d,_n) of 2048 or so should give equivalent amplitude. Severe radiation damping( which happens during pwHs) can cause the optimal flip angle to differ from 90 degrees, so fine power should be adjusted to a value to minimize the observed single-scan experiments (with ssfilter='n'). Added control of phase of flipback pulses via H2Osinc_t.RF (TROSY='n') and H2Osinc_n.RF/H2Osinc_d.RF (TROSY='y'). The flipdown pulse H2Osinc_n.RF has fine power adjustment tpwrsf_n. The flipdown pulse H2Osinc_t.RF has fine power adjustment tpwrsf_t. The flipdown pulse H2Osinc_d.RF has fine power adjustment tpwrsf_d. The shaped pulses are automatically generated via the AutoCalibration process in BioPack. They have internal phase corrections for optimal water suppression. The tpwrsf_* values are determined in the AutoCalibration process and are stored in the active probefile. The experiment setup macro "gNhsqc" retrieves these values into the gNhsqc parameter set as part of the experiment setup. (GG sept03) ************************************************************************************ CHOICE OF DECOUPLING AND 2D MODES Set dm = 'nnn', dmm = 'ccc' Set dm2 = 'nny', dmm2 = 'ccg' (or 'ccw', or 'ccp') for N15 decoupling. Set dm3='nyn', dmm2='cwc' for 2H decoupling using 4th channel Must set phase = 1,2 for States-TPPI acquisition in t1 [N15]. The flag f1180 should be set to 'y' if t1 is to be started at halfdwell time. This will give 90, -180 phasing in f1. If it is set to 'n' the phasing should be 0,0 and will still give a perfect baseline. Thus, set f1180='n' for (0,0) in N15. f1180='y' is ignored if ni=0. DETAILED INSTRUCTIONS FOR USE OF gNhsqc 1. Obtain a printout of the Philosopy behind the BioPack development, and General Instructions using the macro: "printon man('BioPack') printoff". These Detailed Instructions for gNhsqc may be printed using: "printon man('gNhsqc') printoff". 2. Apply the setup macro "gNhsqc". This loads the relevant parameter set and also sets ni=0 and phase=1 ready for a 1D spectral check. 3. Centre H1 frequency on H2O (4.7ppm), C13 frequency on 100ppm, and N15 frequency on the amide region (120 ppm). 4. CALIBRATION OF pw AND pwN: calN and calH are multipliers of the first N15 and H1 90 degree pulses in the sequence. They can be used to calibrate the pulses using a 1D spectrum. Setting either calN or calH = 2.0 should give a S/N null (or small dispersion signals) corresponding to the pulses being set at 180 degrees. Adjust pwN or pw, respectively, until this occurs. An array of calH or calN = 1.8, 2.0, 2.2 is also convenient to judge the null at 2.0. calN and calH are automatically reset to 1 for 2D spectra if you forget to do so. (Note that compH and compN can be calibrated using calH and calN in an analogous way to compC as in point 6, gChsqc manual). 5. CALIBRATION OF dof2: Set ni=ni2=0 and phase=phase2=1 and obtain a well=phased absorption mod 1D spectrum. Set d2=0.0001 and phase=1,2, sum the two resulting spectra and display in exp5 with a 90 degree phase shift: clradd select(1) spadd select(2) spadd jexp5 full rp=rp+90 ds dof2 will be in the middle of the NH region when the signals between 7.4 and 9ppm in the 1H spectrum are roughly balanced between +ve and -ve signals. Note that the same method with d2=0 should give a zero spectrum. Don't forget to reset d2=0 when you have finished. 6. Splitting of resonances in the N15 dimension by C13 coupling in C13 enriched samples can be removed by setting C13refoc='y'. 7. H2O preservation is achieved according to Kay et al, except that a sinc one-lobe selective pulse is used to put H2O back along z. This is much more selective than a hard, Seduce1, or gaussian pulse. H2O is cycled back to z as much as possible during t1, and during the relaxation delays for the following relaxation time measurements. 8. T1 MEASUREMENTS OF NH GROUPS: Implemented automatically by setting the flag T1='y'. An array of 1D spectra is obtained by arraying the relaxation time parameter, relaxT, to a multiple of 10 milliseconds. relaxT corresponds exactly to the relaxation time of the N15 spins. The method uses 180 degree H1 pulses every 5ms during relaxT as according to Kay et al. An alternative method using waltz decoupling was tested and discarded. It gave exactly the same results using alphalytic protease, but has the comparative disadvantage of delivering more H1 RF, so more heating. 2D spectra should be acquired in a separate run for each of the desired values of relaxT. (maxrelaxT as in point 10 is not used when T1='y'.) 10. T1rho MEASUREMENTS OF NH GROUPS: Implemented by setting the flag T1rho='y' and arraying relaxT as above. relaxT corresponds exactly to the relaxation time of the N15 spins. Spin lock power is limited to 1.5 kHz for a 600 Mhz spectrometer as this delivers about the same sample heating as the T2 method below. Increasing this RF (by changing the number 1500 in the DECLARE AND LOAD VARIABLES section of gNhsqc.c) causes substantial sample or coil heating as indicated by deterioration of the lock signal). Care should be taken for relaxT times greater than 0.25 seconds, and times greater than 0.5 seconds are automatically aborted. Gives very similar results to the T2 method as determined using alphalytic protease. A dummy period of spinlock RF is delivered at the beginning of each pulse sequence to ensure constant average sample heating - the code determines this from the maximum relaxT you have set in your array of relaxT. The spectrum at relaxT=0 may be erroneous because of insufficient dephasing of unlocked spins. 2D spectra should be acquired in a separate run for each of the desired values of relaxT. If T1rho measurements are to be made in different experiments with different relaxTs as for 2D, including arrays of relaxT's, set the parameter maxrelaxT to the maximum relaxT you have set in all experiments - this will ensure constant average sample heating in all experiments. maxrelaxT does not need to be set for a single array in a single exp. 11. T2 MEASUREMENTS OF NH GROUPS: Implemented by setting the flag T2='y' and arraying relaxT as above. relaxT corresponds exactly to the relaxation time of the N15 spins. As for T1rho, care should be taken for relaxT times greater than 0.25 seconds, and times greater than 0.5 seconds are automatically aborted. The method is according to Kay et al, with N15 180's every 625 us, and H1 180's every 10 ms. 625us was used instead of approx 500us used by Kay et al to reduce sample heating. An alternative method using the same no. of N15 and H1 pulses for all relaxT times (by decreasing the 625us delays to obtain short relaxT) to test whether large nos. of pulses decreases S/N because of pulse imperfection, gave exactly equivalent results for alphalytic protease, and was discarded. Another alternative, with waltz H1 decoupling and a single N15 180 at the middle of relaxT (credited to Wagner et al by Kay et al), gave substantially shorter apparent T2 times, and was discarded. A dummy period of N15 180's is delivered at the beginning of each pulse sequence to ensure constant average sample heating. 2D spectra should be acquired in separate runs with maxrelaxT set as for T1rho='y' in point 10. 12. NH2 GROUPS: A spectrum of NH2 groups, with NH groups cancelled, can be obtained with flag NH2only='y'. This utilises a 1/2J (J=94Hz) period of NH J-coupling evolution added to t1 which cancels NH resonances and inverts NH2 resonances (normal INEPT behaviour). A 180 degree phase shift is added to a N15 90 pulse to provide positive NH2 signals. The NH2 resonances will be smaller (say 80%) than when NH2only='n'. 13. RELAXATION MEASUREMENTS OF NH2 GROUPS: NH2 resonances are supressed for the methods described in points 9-11. To obtain relaxation times of NH2 groups, set the flag NH2only='y' and proceed as for points 9-11. In these cases the 1/2J delays described in points 8 and 12 are changed to 1/4J delays by the pulse sequence code. Although NH groups are completely cancelled in point 12, when measuring relaxation times NH groups appear at 50% intensity. 14. The coherence-transfer gradients using power levels gzlvl1 and gzlvl2 may be either z or magic-angle gradients. For the latter, a proper /vnmr/imaging/gradtable entry must be present and syscoil must contain the value of this entry (name of gradtable). The amplitude of the gzlvl1 and gzlvl2 should be lower than for a z axis probe to have the x and y gradient levels within the 32k range. For any value, a dps display (using power display) shows the x,y and z dac values. These must be <=32k. 15. 1/4J DELAY TIMES: These are are determined from the NH coupling constant, JNH, listed in dg for possible change by the user. lambda is the 1/4J time for H1 NH coupling evolution, tNH is the 1/4J time for N15 NH coupling evolution. lambda is usually set a little lower than the theoretical time to minimise relaxation, but tNH should be as close to the theoretical time as possible. 16. TROSY and COUPLED 2D SPECTRA: Set TROSY='y' and dm2='nnn' for a TROSY spectrum of the bottom right peak of the 2D coupled quartet (high-field H1, low-field N15). For comparison, TROSY='n' and dm2='nnn' gives a 2D spectrum coupled in both dimensions. The TROSY spectrum retains the N15 magnetization as well as the H1 magnetization and so gives a little more than 50% S/N than decoupled HSQC for a small peptide. For TROSY, H2O flipback is maintained throughout t1 and t2 using two sinc one-lobe pulses (H2O is inverted for half of t1 for normal HSQC, ie TROSY='n' as described in point 7). To select any of the other three peaks of the 2D coupled quartet, in a clockwise direction from bottom right, change t4/t10 from x/y to x/-y to -x/-y to -x/y. NOTE, the Varian phases are of opposite sense to those in the Weigelt paper, ie swap y and -y. The same phases as written by Weigelt also work provided the +/- gzlvl1 signs for phase=1,2 are swapped to -/+, and the phase of one of the first two H1 90 degree pulses is swapped to ensure that the N15 magnetization adds rather than subtracts. The phase list used for this sequence code is yet another equivalent which permits the least change from the normal SE train in the BioPack sequences. Relaxation time measurements have not yet been implemented for TROSY spectra (requires customer demand !). 17. Radiation Damping: At fields 600MHz and higher with high-Q probes, radiation damping is a factor to be considered. Its primary effect is in the flipback pulse calibration. Radiation damping causes a rotation back to the +Z axis even without a flipback pulse. Hence, the pwHs pulse often needs to be reduced in its flip-angle. This can be accomplished by using the parameter tpwrsf_t. If this value is less than 4095.0 the value of tpwrs (calculated in the psg code) is increased by 6dB, thereby permitting the value of tpwrsf_t to be optimized to obtain minimum H2O in the spectrum. The value of tpwrsf_t is typically lower than 2048 (half-maximum to compensate for the extra 6dB in tpwrs). Added control of phase of flipback pulses via H2Osinc_t.RF after INEPT and H2Osinc_d.RF in TROSY. These pulses are generated by Pbox in the autocalibrate process and have internal small-angle phase corrections for best solvent suppression. */ #include static int /* T is for TROSY='y', phx etc also enable TROSY phase changes */ phx[1]={0}, phy[1]={1}, ph_x[1]={2}, phi3[2] = {0,2}, phi9[8] = {0,0,1,1,2,2,3,3}, rec[4] = {0,2,2,0}, recT[2] = {1,3}; static double d2_init=0.0, relaxTmax; pulsesequence() { /* DECLARE AND LOAD VARIABLES */ char f1180[MAXSTR], /* Flag to start t1 @ halfdwell */ mag_flg[MAXSTR], /* magic-angle coherence transfer gradients */ C13refoc[MAXSTR], /* C13 sech/tanh pulse in middle of t1*/ NH2only[MAXSTR], /* spectrum of only NH2 groups */ T1[MAXSTR], /* insert T1 relaxation delay */ T1rho[MAXSTR], /* insert T1rho relaxation delay */ T2[MAXSTR], /* insert T2 relaxation delay */ TROSY[MAXSTR]; /* do TROSY on N15 and H1 */ int icosel, /* used to get n and p type */ t1_counter, /* used for states tppi in t1 */ rTnum, /* number of relaxation times, relaxT */ rTcounter; /* to obtain maximum relaxT, ie relaxTmax */ double tau1, /* t1 delay */ lambda = 0.91/(4.0*getval("JNH")), /* 1/4J H1 evolution delay */ tNH = 1.0/(4.0*getval("JNH")), /* 1/4J N15 evolution delay */ relaxT = getval("relaxT"), /* total relaxation time */ rTarray[1000], /* to obtain maximum relaxT, ie relaxTmax */ maxrelaxT = getval("maxrelaxT"), /* maximum relaxT in all exps */ ncyc, /* number of pulsed cycles in relaxT */ /* the sech/tanh pulse is automatically calculated by the macro "proteincal", */ /* and is called directly from your shapelib. */ pwClvl = getval("pwClvl"), /* coarse power for C13 pulse */ pwC = getval("pwC"), /* C13 90 degree pulse length at pwClvl */ rf0, /* maximum fine power when using pwC pulses */ rfst, /* fine power for the stCall pulse */ compH = getval("compH"), /* adjustment for H1 amplifier compression */ compN = getval("compN"), /* adjustment for N15 amplifier compression */ compC = getval("compC"), /* adjustment for C13 amplifier compression */ calH = getval("calH"), /* multiplier on a pw pulse for H1 calibration */ tpwrsf_t = getval("tpwrsf_t"), /* fine power adustment for first soft pulse(TROSY=n)*/ tpwrsf_n = getval("tpwrsf_n"), /* fine power adustment for first soft pulse(TROSY=y)*/ tpwrsf_d = getval("tpwrsf_d"), /* fine power adustment for second soft pulse(TROSY=y)*/ pwHs = getval("pwHs"), /* H1 90 degree pulse length at tpwrs */ tpwrs, /* power for the pwHs ("H2Osinc") pulse */ pwNlvl = getval("pwNlvl"), /* power for N15 pulses */ pwN = getval("pwN"), /* N15 90 degree pulse length at pwNlvl */ calN = getval("calN"), /* multiplier on a pwN pulse for calibration */ slNlvl, /* power for N15 spin lock */ slNrf = 1500.0, /* RF field in Hz for N15 spin lock at 600 MHz */ sw1 = getval("sw1"), gt1 = getval("gt1"), /* coherence pathway gradients */ gzcal = getval("gzcal"), /* dac to G/cm conversion */ gzlvl1 = getval("gzlvl1"), gzlvl2 = getval("gzlvl2"), gt0 = getval("gt0"), /* other gradients */ gt3 = getval("gt3"), gt4 = getval("gt4"), gt5 = getval("gt5"), gstab = getval("gstab"), gzlvl0 = getval("gzlvl0"), gzlvl3 = getval("gzlvl3"), gzlvl4 = getval("gzlvl4"), gzlvl5 = getval("gzlvl5"); getstr("f1180",f1180); getstr("mag_flg",mag_flg); getstr("C13refoc",C13refoc); getstr("NH2only",NH2only); getstr("T1",T1); getstr("T1rho",T1rho); getstr("T2",T2); getstr("TROSY",TROSY); /* LOAD PHASE TABLE */ settable(t3,2,phi3); settable(t4,1,phx); if (TROSY[A]=='y') {settable(t1,1,ph_x); settable(t9,1,phx); settable(t10,1,phy); settable(t11,1,phx); settable(t12,2,recT);} else {settable(t1,1,phx); settable(t9,8,phi9); settable(t10,1,phx); settable(t11,1,phy); settable(t12,4,rec);} /* INITIALIZE VARIABLES */ /* maximum fine power for pwC pulses (and initialize rfst) */ rf0 = 4095.0; rfst=0.0; /* 180 degree adiabatic C13 pulse from 0 to 200 ppm */ if (C13refoc[A]=='y') {rfst = (compC*4095.0*pwC*4000.0*sqrt((30.0*sfrq/600.0+7.0)/0.35)); rfst = (int) (rfst + 0.5); if ( 1.0/(4000.0*sqrt((30.0*sfrq/600.0+7.0)/0.35)) < pwC ) { text_error( " Not enough C13 RF. pwC must be %f usec or less.\n", (1.0e6/(4000.0*sqrt((30.0*sfrq/600.0+7.0)/0.35))) ); abort(1); }} /* selective H20 one-lobe sinc pulse */ tpwrs = tpwr - 20.0*log10(pwHs/(compH*pw*1.69)); /*needs 1.69 times more*/ tpwrs = (int) (tpwrs); /*power than a square pulse */ /* power level for N15 spinlock (90 degree pulse length calculated first) */ slNlvl = 1/(4.0*slNrf*sfrq/600.0) ; slNlvl = pwNlvl - 20.0*log10(slNlvl/(pwN*compN)); slNlvl = (int) (slNlvl + 0.5); /* use 1/8J times for relaxation measurements of NH2 groups */ if ( (NH2only[A]=='y') && ((T1[A]=='y') || (T1rho[A]=='y') || (T2[A]=='y')) ) { tNH = tNH/2.0; } /* reset calH and calN for 2D if inadvertently left at 2.0 */ if (ni>1.0) {calH=1.0; calN=1.0;} /* CHECK VALIDITY OF PARAMETER RANGES */ if ((TROSY[A]=='y') && (gt1 < -2.0e-4 + pwHs + 1.0e-4 + 2.0*POWER_DELAY)) { text_error( " gt1 is too small. Make gt1 equal to %f or more.\n", (-2.0e-4 + pwHs + 1.0e-4 + 2.0*POWER_DELAY) ); abort(1); } if((dm[A] == 'y' || dm[B] == 'y' || dm[C] == 'y' )) { text_error("incorrect dec1 decoupler flags! Should be 'nnn' "); abort(1); } if((dm2[A] == 'y' || dm2[B] == 'y')) { text_error("incorrect dec2 decoupler flags! Should be 'nny' "); abort(1); } if( dpwr2 > 50 ) { text_error("don't fry the probe, DPWR2 too large! "); abort(1); } if( pw > 50.0e-6 ) { text_error("dont fry the probe, pw too high ! "); abort(1); } if( pwN > 100.0e-6 ) { text_error("dont fry the probe, pwN too high ! "); abort(1); } /* RELAXATION TIMES AND FLAGS */ /* evaluate maximum relaxT, relaxTmax chosen by the user */ rTnum = getarray("relaxT", rTarray); relaxTmax = rTarray[0]; for (rTcounter=1; rTcounter relaxTmax) relaxTmax = maxrelaxT; if ( ((T1rho[A]=='y') || (T2[A]=='y')) && (relaxTmax > d1) ) { text_error("Maximum relaxation time, relaxT, is greater than d1 ! "); abort(1);} if ( ((T1[A]=='y') && (T1rho[A]=='y')) || ((T1[A]=='y') && (T2[A]=='y')) || ((T1rho[A]=='y') && (T2[A]=='y')) ) { text_error("Choose only one relaxation measurement ! "); abort(1); } if ( ((T1[A]=='y') || (T1rho[A]=='y')) && ((relaxT*100.0 - (int)(relaxT*100.0+1.0e-4)) > 1.0e-6) ) { text_error("Relaxation time, relaxT, must be zero or multiple of 10msec"); abort(1);} if ( (T2[A]=='y') && (((relaxT+0.01)*50.0 - (int)((relaxT+0.01)*50.0+1.0e-4)) > 1.0e-6) ) { text_error("Relaxation time, relaxT, must be odd multiple of 10msec"); abort(1);} if ( ((T1rho[A]=='y') || (T2[A]=='y')) && (relaxTmax > 0.25) && (ix==1) ) { printf("WARNING, sample heating will result for relaxT>0.25sec"); } if ( ((T1rho[A]=='y') || (T2[A]=='y')) && (relaxTmax > 0.5) ) { text_error("relaxT greater than 0.5 seconds will heat sample"); abort(1);} if ( ((NH2only[A]=='y') || (T1[A]=='y') || (T1rho[A]=='y') || (T2[A]=='y')) && (TROSY[A]=='y') ) { text_error("TROSY not implemented with NH2 spectrum, or relaxation exps."); abort(1);} if ((TROSY[A]=='y') && (dm2[C] == 'y')) { text_error("Choose either TROSY='n' or dm2='n' ! "); abort(1); } /* PHASES AND INCREMENTED TIMES */ /* Phase incrementation for hypercomplex 2D data, States-Haberkorn element */ if (TROSY[A]=='y') { if (phase1 == 1) icosel = -1; else { tsadd(t4,2,4); tsadd(t10,2,4); icosel = +1; } } else { if (phase1 == 1) {tsadd(t10,2,4); icosel = +1;} else icosel = -1; } /* Set up f1180 */ tau1 = d2; if((f1180[A] == 'y') && (ni > 1.0)) { tau1 += ( 1.0 / (2.0*sw1) ); if(tau1 < 0.2e-6) tau1 = 0.0; } tau1 = tau1/2.0; /* Calculate modifications to phases for States-TPPI acquisition */ if( ix == 1) d2_init = d2; t1_counter = (int) ( (d2-d2_init)*sw1 + 0.5 ); if(t1_counter % 2) { tsadd(t3,2,4); tsadd(t12,2,4); } /* Correct inverted signals for NH2 only spectra */ if ((NH2only[A]=='y') && (T1[A]=='n') && (T1rho[A]=='n') && (T2[A]=='n')) { tsadd(t3,2,4); } /* BEGIN PULSE SEQUENCE */ status(A); obspower(tpwr); decpower(pwClvl); decpwrf(rf0); dec2power(pwNlvl); txphase(zero); decphase(zero); dec2phase(zero); delay(d1); /* xxxxxxxxxxxxxxxxx CONSTANT SAMPLE HEATING FROM N15 RF xxxxxxxxxxxxxxxxx */ if (T1rho[A]=='y') {dec2power(slNlvl); dec2rgpulse(relaxTmax-relaxT, zero, 0.0, 0.0); dec2power(pwNlvl);} if (T2[A]=='y') {ncyc = 8.0*100.0*(relaxTmax - relaxT); if (ncyc > 0) {initval(ncyc,v1); loop(v1,v2); delay(0.625e-3 - pwN); dec2rgpulse(2*pwN, zero, 0.0, 0.0); delay(0.625e-3 - pwN); endloop(v2);} } /* xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx */ rcvroff(); if (TROSY[A]=='n') dec2rgpulse(pwN, zero, 0.0, 0.0); /*destroy N15 magnetization*/ zgradpulse(gzlvl0, 0.5e-3); delay(1.0e-4); if (TROSY[A]=='n') dec2rgpulse(pwN, one, 0.0, 0.0); zgradpulse(0.7*gzlvl0, 0.5e-3); decpwrf(rfst); txphase(t1); delay(5.0e-4); if ( dm3[B] == 'y' ) /* begins optional 2H decoupling */ { lk_hold(); dec3rgpulse(1/dmf3,one,10.0e-6,2.0e-6); dec3unblank(); dec3phase(zero); delay(2.0e-6); setstatus(DEC3ch, TRUE, 'w', FALSE, dmf3); } rgpulse(calH*pw,t1,0.0,0.0); /* 1H pulse excitation */ txphase(zero); dec2phase(zero); zgradpulse(gzlvl0, gt0); delay(lambda - gt0); sim3pulse(2.0*pw, 0.0, 2.0*pwN, zero, zero, zero, 0.0, 0.0); txphase(one); zgradpulse(gzlvl0, gt0); delay(lambda - gt0); rgpulse(pw, one, 0.0, 0.0); txphase(two); if (tpwrsf_t<4095.0) { obspower(tpwrs+6.0); if (TROSY[A] == 'n') { obspwrf(tpwrsf_t); shaped_pulse("H2Osinc_t", pwHs, two, 5.0e-5, 0.0); } else { obspwrf(tpwrsf_n); shaped_pulse("H2Osinc_n", pwHs, two, 5.0e-5, 0.0); } obspower(tpwr); obspwrf(4095.0); } else { obspower(tpwrs); if (TROSY[A] == 'n') shaped_pulse("H2Osinc_t", pwHs, two, 5.0e-5, 0.0); else shaped_pulse("H2Osinc_n", pwHs, two, 5.0e-5, 0.0); obspower(tpwr); } zgradpulse(gzlvl3, gt3); dec2phase(t3); delay(2.0e-4); dec2rgpulse(calN*pwN, t3, 0.0, 0.0); txphase(zero); decphase(zero); /* xxxxxxxxxxxxxxxxxx OPTIONS FOR N15 RELAXATION xxxxxxxxxxxxxxxxxxxx */ if ( (T1[A]=='y') || (T1rho[A]=='y') || (T2[A]=='y') ) { dec2phase(one); zgradpulse(gzlvl4, gt4); /* 2.0*GRADIENT_DELAY */ delay(tNH - gt4 - 2.0*GRADIENT_DELAY); sim3pulse(2.0*pw, 0.0, 2.0*pwN, zero, zero, one, 0.0, 0.0); zgradpulse(gzlvl4, gt4); /* 2.0*GRADIENT_DELAY */ delay(tNH - gt4 - 2.0*GRADIENT_DELAY); } /* xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx */ if (T1[A]=='y') { dec2rgpulse(pwN, one, 0.0, 0.0); dec2phase(three); zgradpulse(gzlvl0, gt0); /* 2.0*GRADIENT_DELAY */ delay(2.5e-3 - gt0 - 2.0*GRADIENT_DELAY - pw); rgpulse(2.0*pw, zero, 0.0, 0.0); delay(2.5e-3 - pw); ncyc = (100.0*relaxT); initval(ncyc,v4); if (ncyc > 0) {loop(v4,v5); delay(2.5e-3 - pw); rgpulse(2.0*pw, two, 0.0, 0.0); delay(2.5e-3 - pw); delay(2.5e-3 - pw); rgpulse(2.0*pw, zero, 0.0, 0.0); delay(2.5e-3 - pw); endloop(v5);} dec2rgpulse(pwN, three, 0.0, 0.0); } /* xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx */ /* Theory suggests 8.0 is better than 2PI as RF */ /* field multiplier and experiment confirms this.*/ if (T1rho[A]=='y') /* Shift evolution of 2.0*pwN/PI for one pulse */ { /* at end left unrefocused as for normal sequence*/ delay(1.0/(8.0*slNrf) - pwN); dec2rgpulse(pwN, zero, 0.0, 0.0); dec2power(slNlvl); /* minimum 5ms spinlock to dephase */ dec2rgpulse((2.5e-3-pw), zero, 0.0, 0.0); /* spins not locked */ sim3pulse(2.0*pw, 0.0, 2.0*pw, zero, zero, zero, 0.0, 0.0); dec2rgpulse((2.5e-3-pw), zero, 0.0, 0.0); ncyc = 100.0*relaxT; initval(ncyc,v4); if (ncyc > 0) {loop(v4,v5); dec2rgpulse((2.5e-3-pw), zero, 0.0, 0.0); sim3pulse(2.0*pw, 0.0, 2.0*pw, two, zero, zero, 0.0, 0.0); dec2rgpulse((2.5e-3-pw), zero, 0.0, 0.0); dec2rgpulse((2.5e-3-pw), zero, 0.0, 0.0); sim3pulse(2.0*pw, 0.0, 2.0*pw, zero, zero, zero, 0.0, 0.0); dec2rgpulse((2.5e-3-pw), zero, 0.0, 0.0); endloop(v5);} dec2power(pwNlvl); dec2rgpulse(pwN, zero, 0.0, 0.0); delay(1.0/(8.0*slNrf) + 2.0*pwN/PI - pwN); } /* xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx */ if (T2[A]=='y') { dec2phase(zero); initval(0.0,v3); initval(180.0,v4); ncyc = 100.0*relaxT; initval(ncyc,v5); loop(v5,v6); initval(3.0,v7); loop(v7,v8); delay(0.625e-3 - pwN); dec2rgpulse(2.0*pwN, zero, 0.0, 0.0); delay(0.625e-3 - pwN); endloop(v8); delay(0.625e-3 - pwN - SAPS_DELAY); add(v4,v3,v3); obsstepsize(1.0); xmtrphase(v3); /* SAPS_DELAY */ dec2rgpulse(2.0*pwN, zero, 0.0, 0.0); delay(0.625e-3 - pwN - pw); rgpulse(2*pw, zero, 0.0, 0.0); delay(0.625e-3 - pwN - pw ); dec2rgpulse(2.0*pwN, zero, 0.0, 0.0); xmtrphase(zero); /* SAPS_DELAY */ delay(0.625e-3 - pwN - SAPS_DELAY); initval(3.0,v9); loop(v9,v10); delay(0.625e-3 - pwN); dec2rgpulse(2.0*pwN, zero, 0.0, 0.0); delay(0.625e-3 - pwN); endloop(v10); endloop(v6); } /* xxxxxxxxxxxxxxxxxx OPTIONS FOR N15 EVOLUTION xxxxxxxxxxxxxxxxxxxxx */ txphase(zero); dec2phase(t9); if ( (NH2only[A]=='y') || (T1[A]=='y') || (T1rho[A]=='y') || (T2[A]=='y') ) { delay(tau1); /* optional sech/tanh pulse in middle of t1 */ if (C13refoc[A]=='y') /* WFG_START_DELAY */ {decshaped_pulse("stC200", 1.0e-3, zero, 0.0, 0.0); delay(tNH - 1.0e-3 - WFG_START_DELAY - 2.0*pw);} else {delay(tNH - 2.0*pw);} rgpulse(2.0*pw, zero, 0.0, 0.0); if (tNH < gt1 + 1.99e-4) delay(gt1 + 1.99e-4 - tNH); delay(tau1); dec2rgpulse(2.0*pwN, t9, 0.0, 0.0); if (mag_flg[A] == 'y') magradpulse(icosel*gzcal*gzlvl1, gt1); else zgradpulse(icosel*gzlvl1, gt1); /* 2.0*GRADIENT_DELAY */ txphase(t4); dec2phase(t10); if (tNH > gt1 + 1.99e-4) delay(tNH - gt1 - 2.0*GRADIENT_DELAY); else delay(1.99e-4 - 2.0*GRADIENT_DELAY); } else if (TROSY[A]=='y') { if ( (C13refoc[A]=='y') && (tau1 > 0.5e-3 + WFG2_START_DELAY) ) {delay(tau1 - 0.5e-3 - WFG2_START_DELAY); /* WFG2_START_DELAY */ decshaped_pulse("stC200", 1.0e-3, zero, 0.0, 0.0); delay(tau1 - 0.5e-3);} else delay(2.0*tau1); if (mag_flg[A] == 'y') magradpulse(icosel*gzcal*gzlvl1, gt1); else zgradpulse(icosel*gzlvl1, gt1); /* 2.0*GRADIENT_DELAY */ delay(2.0e-4 - 2.0*GRADIENT_DELAY); dec2rgpulse(2.0*pwN, t9, 0.0, 0.0); txphase(three); /* now do a "flipdown" pulse (has different phase correction internally)*/ if (tpwrsf_d<4095.0) { delay(gt1 + 2.0e-4 - pwHs - 1.0e-4 - 2.0*POWER_DELAY -2.0*PWRF_DELAY); obspwrf(tpwrsf_d); obspower(tpwrs+6.0); shaped_pulse("H2Osinc_d", pwHs, three, 5.0e-5, 0.0); obspower(tpwr); obspwrf(4095.0); } else { delay(gt1 + 2.0e-4 - pwHs - 1.0e-4 - 2.0*POWER_DELAY); obspower(tpwrs); shaped_pulse("H2Osinc_d", pwHs, three, 5.0e-5, 0.0); obspower(tpwr); } txphase(t4); delay(5.0e-5); } else { /* fully-coupled spectrum */ if (dm2[C]=='n') {rgpulse(2.0*pw, zero, 0.0, 0.0); pw=0.0;} if ( (C13refoc[A]=='y') && (tau1 > 0.5e-3 + WFG2_START_DELAY) ) {delay(tau1 - 0.5e-3 - WFG2_START_DELAY); /* WFG2_START_DELAY */ simshaped_pulse("", "stC200", 2.0*pw, 1.0e-3, zero, zero, 0.0, 0.0); delay(tau1 - 0.5e-3); delay(gt1 + 2.0e-4);} else {delay(tau1); rgpulse(2.0*pw, zero, 0.0, 0.0); delay(gt1 + 2.0e-4 - 2.0*pw); delay(tau1);} pw=getval("pw"); dec2rgpulse(2.0*pwN, t9, 0.0, 0.0); if (mag_flg[A] == 'y') magradpulse(icosel*gzcal*gzlvl1, gt1); else zgradpulse(icosel*gzlvl1, gt1); /* 2.0*GRADIENT_DELAY */ txphase(t4); dec2phase(t10); delay(2.0e-4 - 2.0*GRADIENT_DELAY); } if (T1rho[A]=='y') delay(POWER_DELAY); /* xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx */ if (TROSY[A]=='y') rgpulse(pw, t4, 0.0, 0.0); else sim3pulse(pw, 0.0, pwN, t4, zero, t10, 0.0, 0.0); txphase(zero); dec2phase(zero); zgradpulse(gzlvl5, gt5); if (TROSY[A]=='y') delay(lambda - 0.65*(pw + pwN) - gt5); else delay(lambda - 1.3*pwN - gt5); sim3pulse(2.0*pw, 0.0, 2.0*pwN, zero, zero, zero, 0.0, 0.0); zgradpulse(gzlvl5, gt5); txphase(one); dec2phase(t11); delay(lambda - 1.3*pwN - gt5); sim3pulse(pw, 0.0, pwN, one, zero, t11, 0.0, 0.0); txphase(zero); dec2phase(zero); zgradpulse(1.5*gzlvl5, gt5); delay(lambda - 1.3*pwN - gt5); sim3pulse(2.0*pw, 0.0, 2.0*pwN, zero, zero, zero, 0.0, 0.0); dec2phase(t10); zgradpulse(1.5*gzlvl5, gt5); if (TROSY[A]=='y') delay(lambda - 1.6*pwN - gt5); else delay(lambda - 0.65*pwN - gt5); if (TROSY[A]=='y') dec2rgpulse(pwN, t10, 0.0, 0.0); else rgpulse(pw, zero, 0.0, 0.0); delay((gt1/10.0) + 1.0e-4 +gstab - 0.65*pw + 2.0*GRADIENT_DELAY + POWER_DELAY); if ( dm3[B] == 'y' ) /* turns off 2H decoupling */ { setstatus(DEC3ch, FALSE, 'c', FALSE, dmf3); dec3rgpulse(1/dmf3,three,2.0e-6,2.0e-6); dec3blank(); lk_autotrig(); /* resumes lock pulsing */ } rgpulse(2.0*pw, zero, 0.0, 0.0); dec2power(dpwr2); /* POWER_DELAY */ if (mag_flg[A] == 'y') magradpulse(gzcal*gzlvl2, 0.1*gt1); else zgradpulse(gzlvl2, 0.1*gt1); /* 2.0*GRADIENT_DELAY */ delay(gstab); rcvron(); statusdelay(C,1.0e-4); if (dm3[B] == 'y') {delay(1/dmf3); lk_sample();} setreceiver(t12); }