A Simple and Effective Binomial Block Based Pulse Sequence

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A Simple and Effective Binomial Block Based Pulse Sequence Capable of Suppressing Multiple NMR Signals Scott A. Willis, Gang Zheng, Allan M. Torres, Timothy Stait-Gardner, and William S. Price J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.8b08160 • Publication Date (Web): 26 Nov 2018 Downloaded from http://pubs.acs.org on December 4, 2018

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The Journal of Physical Chemistry

A Simple and Effective Binomial Block Based Pulse Sequence Capable of Suppressing Multiple NMR Signals

Scott A. Willis,* Gang Zheng, Allan M. Torres, Timothy Stait-Gardner and William S. Price

Nanoscale Organisation and Dynamics Group, Western Sydney University, Locked Bag 1797, Penrith NSW, 2571, Australia

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

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ABSTRACT: A binomial-like block based multiple suppression NMR pulse sequence, termed MULTI-GATE-FSB, that is simple to implement with outstanding suppression performance for multiple solvent signals (or multiple resonances) is investigated. The sequence was tested on two water-alcohol solvent systems, and a standard lysozyme sample, and with suppression of three or four regions (though it is extendable to any number of regions). The suppression of all solvent signals was possible in the alcohol-water systems tested with both long and short recycle delays, and without the requirement for lengthy pre-saturation pulses. Such a sequence holds promise not only for LC-NMR applications and solvent suppression but for multiple suppression applications in general (e.g., analysis of impurities/components).

INTRODUCTION

Several approaches to solvent/signal suppression in NMR exist with most concentrating on suppressing a single peak (e.g., water).1,

2, 3, 4

However, techniques such as LC-NMR5 often

require multiple solvents – giving rise to multiple intense solvent signals. Solute signals might still be significantly smaller than the solvent signals for LC-NMR even with deuterated solvents.5 Additionally, deuterated solvents with exchangeable groups can hinder analysis.5 Solvent and signal suppression is also important for many other NMR experiments from chemical component analysis/profiling (e.g., Refs.6, 7, 8, 9) to diffusion (e.g., Refs.3, 10, 11) and biomolecular studies (e.g., Refs.3, 4, 12, 13, 14). Possibly the first use of multiple solvent suppression for LC-NMR was by Smallcombe et al.15, 16

who expanded the Water suppression Enhanced through T1 effects (WET) sequence by Ogg et

al.17 They used four shaped selective radiofrequency (rf) pulses (shifted laminar pulses (SLPs)18 and SEDUCE pulses19, 20) with gradients and a spatially selective composite 90° rf pulse.21 WET


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is suited to multiple solvent suppression for LC-NMR,5,

15, 16, 22

e.g., two resonances in

acetonitrile/water (D2O)15, 16 and five resonances in acetonitrile/water (D2O)/triethylammonium acetate solutions.23 Multiple suppression WET has also been used to suppress water and ethanol signals for component analysis/profiling of Greek spirits.6 However, WET requires modification for high quality factor probes or for samples with significant radiation damping,24, 25, 26 and other improvements are possible.27 Multiple suppression for analysis of alcoholic beverages is also possible using a 1D-NOESY type experiment with multiply selective pre-saturation and/or multiple selective pre-saturation pulses.7, 8, 9, 12 Dalvit et al.14,

28, 29

introduced a multiple suppression sequence based on the WATER-

suppression by GrAdient-Tailored Excitation (WATERGATE) sequence30,

31

using multiply

selective SLPs – termed MULTIGATE.28 While suppression of two solvent peaks worked well for water/acetonitrile29 or DMSO/water14, this was not possible for peaks in alcohol/water systems (e.g., methanol/water, ethanol/water) without pre-saturation and/or trim pulses.29 Parella et al.32 modified a double echo WATERGATE for two-site suppression in acetonitrile/H2O/D2O solutions (a modification of their multisite selective excitation sequence33). Instead of multiply selective SLPs, Parella et al.32 used several singly selective 180° shaped pulses and hard 180° rf pulses. Prost et al.34 briefly introduced a multiple suppression sequence using WATERGATE with multiple double echoes each using singly selective shaped rf pulses (i.e., globally antisymmetric selective pulses (GASP)) applied at different frequencies for each double echo. Two peak suppression was demonstrated using acetonitrile/H2O/D2O. Prost et al.34 suggested that a ‘W5’35 rf pulse was not as good as their shaped rf pulses but noted that GASP rf pulses were not suitable for single echo sequences.



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For LC-NMR it is desirable to have an easily implemented multiple suppression sequence that is as short as possible so that the solutes can be analyzed effectively.15, 16 Typically shaped rf pulses are long for multiple suppression, e.g., ~20 ms were used for the shaped pulses in Ref.15, 16 to maintain selectivity. Those used in the sequences of Dalvit et al.14, 28, 29 and Parella et al.32 were shorter, ~2 - 5 ms, but an additional pre-saturation pulse on the order of 1 - 2 s and/or a trim pulse may be needed. Smallcombe et al.15,

16

demonstrated that pre-saturation may not be

suitable when the solvent is flowing as in LC-NMR.15, 16 An improvement to the above sequences is to use binomial or binomial-like rf pulses. In this work we have implemented a block based WATERGATE30 sequence using ‘W5’35 rf pulses suitable for suppressing several frequencies by changing the offset frequency for each block in the sequence (Figure 1). This can be seen as an expansion on the work of Prost et al.34, but is somewhat more flexible as it uses binomial-like rf pulses, e.g., even single echoes can be used or combinations of echoes. Also, if all or some of the resonances to be suppressed are evenly spaced, i.e., in chemical shift, then the number of selective pulses can be limited accordingly (if suitable, i.e., no other losses due to the periodicity in the excitation profiles). This sequence will be referred to as the MULTI-GATE-FSB (MULTIple signal suppression via GrAdient Tailored Excitation and Frequency Shifted Binomial pulses) sequence (Figure 1). Here we present results obtained with the MULTI-GATE-FSB using two alcohol-water solutions and one biomolecular sample.

EXPERIMENTAL METHODS

Ethanol (Sigma-Aldrich, 200 proof, anhydrous, ≥ 99.5%), methanol (Sigma-Aldrich, anhydrous, 99.8%), D-glucose (Chem-Supply, anhydrous, laboratory reagent grade), L-alanine


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(Sigma-Aldrich, Fluka BioChemika, ≥ 99%), glycine (Sigma-Aldrich, Reagent Plus®, ≥ 99%) and D-(−)-fructose (Sigma-Aldrich, ≥ 99%) were used as received. The water used to make the samples was of MilliQ grade. The following samples were used in this work: A ‘3-peak suppression’ sample was 0.5 mL of a solution made by dissolving 30.67 mg D-glucose, 26.05 mg L-alanine, 20.2 mg glycine and 26.95 mg D-(−)-fructose in 2 mL MilliQ H2O + 2 mL ethanol (i.e., 42.6 mM D-glucose, 73.1 mM L-alanine, 67.3 mM glycine and 37.4 mM D-(−)-fructose in a water/ethanol solution). A ‘4peak suppression’ sample was 0.5 mL of a solution made by mixing 3 mL from the ‘3-peak suppression’ stock solution with 1.5 mL methanol (i.e., final composition: 28.4 mM D-glucose, 48.7 mM L-alanine, 44.9 mM glycine and 24.9 mM D-(−)-fructose in a water/ethanol/methanol solution). A lysozyme sample was 2 mM lysozyme in ‘90% H2O with 10% D2O’ (Bruker) – although this sample has only one solvent peak multiple suppression of different regions shows that the sequence still works with biomolecular samples or samples with short spin-spin relaxation decay constants. Additional experiments were also performed with a second set of ‘3peak suppression’ and ‘4-peak suppression’ samples prepared similarly to those above (details are provided in the Supporting Information). Experiments were performed on a Bruker Avance 400 MHz NMR equipped with a BBFO probe or a Bruker Avance II 500 MHz NMR equipped with a BBI probe or a Bruker Avance IIIHD 600 MHz NMR equipped with BBI probe. The 400 MHz and 500 MHz were operating with Bruker Topspin 2.1 (patch level 5) and the 600 MHz was operating with Bruker Topspin 3.5 (patch level 6). Experiments were performed at 298 K. The maximum gradient strengths, g, available on the BBFO probe (400 MHz) were gx = 0.428 T/m, gy = 0.439 T/m and gz = 0.558 T/m and for the BBI probe (500 MHz) was gz = 0.553 T/m and for the BBI probe (600 MHz)



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was gx = 0.474 T/m, gy = 0.480 T/m and gz = 0.628 T/m, for gradients along the x, y or z gradient directions (where z corresponds to the spectrometer/probe/NMR tube axis). Experiment specific parameters for each dataset acquired are provided in the associated figure captions and a general description of the experiments performed is given here. Where receiver gain is specified, the minimum receiver gain (i.e., lowest amplification of the signal) for the Bruker systems used in this work were: 1 (400 MHz), 0.25 (500 MHz and 600 MHz). Experiments were performed with recycle delays ≥5 × T1 (i.e., longest T1) and short recycle delays (i.e., 1 or 2 s) for the 3-peak and 4-peak suppression samples and for a recycle delay of 10 s for the lysozyme sample. Note, although not necessary when quoting the 90° rf pulse durations, the pulse powers used were included in the figure captions as this is useful for when the pulse durations and power did not correspond to a 90° rf pulse or an equivalent 90° rf pulse as in some of the experiments performed. Of course pulse powers are hardware/system dependent and users should adjust these based on their 90° rf pulse duration/power and hardware limitations.

Figure 1. MULTI-GATE-FSB pulse sequence. Black rectangles represent rf pulses, rectangles with diagonal lines are gradient pulses and the signal acquired is shown by the grey ‘triangle’. The first rf pulse is a π/2 (i.e., 90°) pulse and this is followed by multiple loops of gradients and binomial pulses. The f-loop is the number of frequencies to suppress and the n-loop is the


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number of times each frequency is repeated (e.g., ‘single echo’, ‘double echo’…; NB: this could be different for each f). For each suppression frequency the binomial rf pulse (e.g., here it is a ‘W5’ pulse35) is centered on that frequency and has the effective final pulse angle of θBin, f and τBin, f are the binomial inter-pulse delays (the same pulse angle and inter-pulse delays can be used for all f-loops or otherwise optimized). The gradients used for each suppression frequency have strength gf and duration δf (for the experiments of this work each f-loop used a different gf but the same gf was used for each n-loop – but other experiments were attempted with differences). There was a 100 µs delay before the first gf of each n or f-loop (for the first n or f-loop this is the delay between the π/2 rf pulse and gf pulse), a 100 µs delay after the first gf pulse of each n or floop prior to the first rf pulse of θBin, f, a delay of 50 µs between the last rf pulse of θBin, f and the second gf pulse of each n or f-loop and a 104 µs delay prior to the next loop. NB gradient blanking and frequency change commands occurred during these delays and were each contributed a 50 µs delay (e.g., the first 100 µs was made up of a frequency change command + 50 µs delay and a gradient blanking command + 50 µs delay). The phase cycle used was π/2: [0º, 180º], pulses in the first half of θBin, f: [0º, 0º, 180º, 180º] for all loops, pulses in the second half of θBin, f: [180º, 180º, 0º, 0º] for all loops, and receiver: [0º, 180º].

Spectra were obtained for the 3-peak and 4-peak suppression samples using the MULTIGATE-FSB sequence. Typically, the order of the suppression frequencies (i.e., f loops) were done with the first on the water resonance, the second on the ethanol quartet and the third on the ethanol triplet for the 3-peak suppression sample, and the first on the water resonance, the second on the ethanol quartet, the third on the methanol singlet and the fourth on the ethanol triplet. But an additional experiment was also performed to exploit a secondary null point of one binomial so



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that four peaks could be suppressed with only three suppression frequencies. Several experiments were performed to investigate the effect of n, τBin, f, the shape and duration of the gradients, recycle delay, and the length and power of the reference pulse used to calculate the rf pulses of the binomial rf pulse and are provided in the Supporting Information. Experimental excitation profiles were also obtained using the MULTI-GATE-FSB sequence (Figure 1) and the second 3-peak and 4-peak suppression samples (see Supporting Information). Spectra for the lysozyme sample were also acquired with the MULTI-GATE-FSB sequence with all three suppression frequencies on the water signal or using multiple suppression regions at various locations (i.e., with at least one on the water signal). The effects of repeating the suppression multiple times on one region, τBin,

f

and the shape and duration of the gradients were also

considered for the lysozyme sample (see Supporting Information). Results obtained from the MULTI-GATE-FSB sequence were compared to results obtained using three ‘Bruker standard’ suppression sequences: i) Pre-saturation,36 and the ii) WET sequence15, 16, 17 using shaped (selective) rf pulses for multiple suppression for the 3-peak and 4peak suppression samples, and the iii) WATER-suppression by GrAdient-Tailored Excitation (WATERGATE) sequence30, 31 with ‘W5’ binomial rf pulses35 with double echo (i.e., excitation sculpting2, 37) to suppress the water peak in the lysozyme spectrum. A schematic for these pulse sequences are provided in Figure S1. The shaped pulses for multiple suppression via pre-saturation and WET were automatically generated (i.e., shapes and phases) using the multiple suppression tool/macro in Bruker Topspin 2.1 (patch level 5) software for each sample. The shaped pulses in the WET sequence used were 20 ms duration and were each the same shape. The shaped pulses in the pre-saturation sequence used were 100 ms duration and were repeated to occupy the entire recycle delay. The pulse


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powers for the shaped pulses in the WET and pre-saturation sequences were optimized by observing the spectra as a function of shaped pulse power(s). For the WET sequence this was the pulse power of a reference shaped pulse such that the power level used for the four shaped pulses in the WET sequence were calculated from this according to the pulse program. NB the maximum pulse powers used for the shaped pulses during optimization were limited for hardware protection. Signal to noise ratios (SNRs) for selected peaks in the spectra for the 3-peak and 4-peak suppression samples were obtained using the Bruker Topspin command sino and Topspin 3.0 (patch level 4) software. Details for sino can be found in the Bruker Topspin – Processing Commands and References Manual (Topspin 3.0, Manual Version 3.0.0). The spectral width acquired in the experiments was large enough so a noise only region could be selected and used in the SNR calculations. The noise only region used for the calculations of signal to noise for the 3-peak and 4-peak suppression samples was from 11 - 13 ppm.

RESULTS AND DISCUSSION

The pulse-acquire spectra, i.e., without suppression, were acquired for all samples and are provided for reference in the Supporting Information (Figure S2, Figure S27, Figure S28 and Figure S45). The solvent signals (and residual solvent signals in the spectra with suppression) are: ~4.79 ppm (water/hydroxyl); ~3.6 ppm (ethanol quartet) ~3.3 ppm (methanol singlet); ~1.13 ppm (ethanol triplet). The results for multiple suppression for the 3-peak and 4-peak samples is shown in Figure 2 and Figure 3, respectively, for long recycle delay with the corresponding results for short recycle delay in Figure S14 and Figure S15. These figures provide an overarching summary of the experiments performed and a direct comparison of the results for



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three multiple suppression sequences (WET, pre-saturation and MULTI-GATE-FSB), i.e., with equivalent scaling and the same number of scans and receiver gains. Although some scaling in the figures (i.e., Figure 2 and Figure 3, and others in the Supporting Information) results in some spectra appearing featureless, this is only because they are shown with equivalent scaling and allows the full/unclipped residual solvent signals to be compared.

Figure 2. Comparison of the results obtained for multiple suppression of the three solvent peaks in the 3-peak suppression sample. Spectra in A) are to the same scale, i.e., relative to one another, for the experimental parameters used and are the ‘×1’ scaled reference spectra. The spectra in A) are scaled to best show the residual solvent signals for all suppression sequences. Spectra in B) are scaled relative to the ‘×1’ spectra as indicated. Multiple suppression was achieved via i) WET, ii) Pre-saturation and iii) MULTI-GATE-FSB sequences. Symbols indicate


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frequencies suppressed:  (water),  (ethanol quartet) and  (ethanol triplet). The order of the symbols in the caption indicate the order of the three f loops in the MULTI-GATE-FSB sequence. The spectra were acquired at 400 MHz with 8 scans (with 4 dummy scans), hard π/2 rf pulse length/power = 15.6 μs/32.05 W for the hard rf pulses, acquisition time = 1.5 s, receiver gain = 32, and recycle delay = 15 s. Additional parameters: i) gradients were along the z-axis and were half-sine shaped with strengths g1 = 0.446 T/m, g2 = 0.223 T/m, g3 = 0.112 T/m and g4 = 0.056 T/m, and durations δ1 = δ2 = δ3 = δ4 = 1 ms, the four 20 ms shaped rf pulses were the same shape but were at different power levels for θS1 – θS4: 0.00104 W, 0.00162 W, 0.00076 W, and 0.00408 W; ii) 150 repetitions of the 100 ms shaped rf pulse at 0.00227 W; iii) τBin, f was the same for all binomial pulses and was 0.4 ms, three f loops were used, n = 2 for each f loop, the reference pulse length/power used to calculate the binomial pulses was 20 μs/8.052 W. Gradient pulses were along the z-axis and were rectangular shaped with δf1 = δf2 = δf3 = 2 ms and gf1 = 0.502 T/m, gf2 = 0.435 T/m, and gf3 = 0.062 T/m where f1, f2, and f3 are for the first, second, third and fourth suppression frequencies. f1 was set to the water resonance and f2 was set to f1 – 484.59 Hz for the ethanol quartet and f3 was set to f1 – 1466.24 Hz for the ethanol triplet.



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Figure 3. Comparison of the results obtained for multiple suppression of the four solvent peaks in the 4-peak suppression sample. Spectra in A) are to the same scale, i.e., relative to one another, for the experimental parameters used and are the ‘×1’ scaled reference spectra. The spectra in A) are scaled to best show the residual solvent signals for all suppression sequences. Spectra in B) are scaled ×8 (still relative to one another) to better show the residual solvent signals in the MULTI-GATE-FSB results. Spectra in C) are scaled relative to the ‘×1’ spectra as


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indicated. Multiple suppression was achieved via i) WET, ii) Pre-saturation and iii) MULTIGATE-FSB sequences. Symbols indicate frequencies suppressed:  (water),  (ethanol quartet),  (methanol singlet), and  (ethanol triplet). The order of the symbols in the caption indicate the order of the four f loops in the MULTI-GATE-FSB sequence. The spectra were acquired at 400 MHz with 8 scans (with 4 dummy scans), hard π/2 rf pulse length/power = 15.15 μs/32.05 W for the hard rf pulses, acquisition time = 1.5 s, receiver gain = 32, and recycle delay = 16 s. Additional parameters: i) gradients were along the z-axis and were half-sine shaped with strengths g1 = 0.446 T/m, g2 = 0.223 T/m, g3 = 0.112 T/m and g4 = 0.056 T/m, and durations δ1 = δ2 = δ3 = δ4 = 1 ms, the four 20 ms shaped rf pulses were the same shape but were at different power levels for θS1 – θS4: 0.00117 W, 0.00182 W, 0.00085 W, and 0.00458 W; ii) 160 repetitions of the 100 ms shaped rf pulse at 0.00227 W; iii) τBin, f was the same for all binomial pulses and was 0.4 ms, four f loops were used, n = 2 for each f loop, the reference pulse length/power used to calculate the binomial pulses was 20 μs/8.052 W. Gradient pulses were along the z-axis and were rectangular shaped with δf1 = δf2 = δf3 = δf4 = 2 ms and gf1 = 0.502 T/m, gf2 = 0.435 T/m, gf3 = 0.062 T/m, and gf4 = 0.116 T/m where f1, f2, f3 and f4 are for the first, second, third and fourth suppression frequencies. f1 was set to the water resonance, f2 was set to f1 – 480.23 Hz for the ethanol quartet, f3 was set to f1 – 591 Hz for the methanol singlet, and f4 was set to f1 – 1459.7 Hz for the ethanol triplet.

MULTI-GATE-FSB showed good suppression of all solvent peaks in the 3-peak and 4-peak suppression samples (Figure 2 and Figure 3). Unlike the sequence of Dalvit et al.14, 28, 29 and the results from WET in Figure 2 and Figure 3 (and Figure S12 and Figure S13), the broad water/hydroxyl signal could be significantly, if not completely, suppressed with MULTI-GATE-



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FSB without pre-saturation or trim pulses. Increasing n for MULTI-GATE-FSB improved suppression (see Supporting Information) but n = 2 gave the best solute signal intensity compared to the suppression level. However, quite reasonable suppression could still be achieved with n = 1 (e.g., Figure S44). Pre-saturation provided reasonable suppression for all peaks in the 3-peak suppression sample when the recycle delay was 15 s (i.e., ≥ 5 × T1, longest) (Figure 2) but expectedly did not perform as well when this was reduced to 1 s (Figure S14). Pre-saturation failed to suppress all four peaks in the 4-peak suppression sample even with a recycle delay of 16 s (i.e., ≥ 5 × T1, longest) (Figure 3; Figure S15 shows similar results with short recycle delay). It could be expected from the results of optimizing the pulse power for the pre-saturation shaped pulses (Figure S7 and Figure S8), that applying pre-saturation at higher pulse powers or for longer should further reduce the residual solvent signals observed for the pre-saturation sequence in Figure 2 and Figure 3. However, the qualitative appearance of the suppression for the water signal was similar for both short and long recycle delays (compare Figure 2 and Figure 3 to Figure S14 and Figure S15) and as mentioned the methanol signal was not adequately suppressed in the 4-peak sample with presaturation. Note the shaped pulse powers for pre-saturation (and WET) were optimized (within the limited power range) using the long recycle delay and as such pre-saturation with short recycle delay may simply need more power but the lack of suppression of the methanol signal is not expected to be improved based on the optimization results (i.e., Figure S8). The optimization of the shaped pulse powers used for the WET sequence (Figure S3 and Figure S5) showed that there was a value for the power levels optimal for the suppression of the either the water signal or the alcohol signals. The results with WET pulse powers optimized so that the residual water signal was qualitatively the smallest (Figure 2 and Figure 3; see also


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Figure S3 and Figure S5) showed some baseline distortion around the water signal but there are also other notable effects. For the 3-peak sample using WET pulse powers optimized for the best suppression of the water signal resulted in significant residual signals from the ethanol triplet/quartet (Figure 2 and Figure S3). For the 4-peak sample and WET pulse powers optimized of for the best suppression of the water signal, the peaks at ~3.44 ppm and ~3.48 ppm in the 4peak suppression sample were inverted (Figure 3 and Figure S5). However, they can be acquired without inversion using the WET power levels optimized for the suppression of the ethanol triplet/quartet and methanol singlet only (Figure S5 and Figure S13) but the residual water signal was larger. Similarly, the suppression of the ethanol triplet/quartet could be improved for the 3peak sample using alternate WET pulse powers but again the residual water signal is larger (Figure S3 and Figure S12). The distortion of the baseline observed in the WET results around the residual water signal in Figure 2 and Figure 3 (more significant in the 4-peak sample results) could potentially be alleviated by using the pulse powers optimized for suppression of only the ethanol triplet/quartet and methanol singlet (e.g. Figure S12 and Figure S13) but other optimizations, e.g., Ref. 27, may be needed to further reduce the residual water signal. The results from WET with short recycle delays (Figure S14 and Figure S15) were similar to the results with long recycle delays (Figure 2 and Figure 3). Multiple suppression of ethanol and water signals in Greek spirits has been reported using WET in a metabolomics study,6 however the spectra acquired with suppression were not shown with complete residual ethanol/water signals so it is difficult to compare the performance with respect to this. Each block in the MULTI-GATE-FSB was ~8.1 ms long for Figure 2 and Figure 3, and ~6.3 ms for Figure 4 (i.e., mostly dependent on τBin, f and δf). The block time corresponds to the delay preceding the binomial pulse, the two gradients associated with the W5 and the delays after each



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gradient, the W5 pulse durations and inter-pulse delays. So for the experiments of this work, the block time = 2 × 50 µs + δf + 100 µs + 9 × τBin, f + 2 × (0.087 × tRef + 0.206 × tRef + 0.413 × tRef + 0.778 × tRef + 1.491 × tRef) + 50 µs + δf + 100 µs + 4 µs where tRef is the duration of the reference pulse used to calculate the durations of the pulses in the binomial rf pulse. For a block of ~8.1 ms the total sequence length with n = 2 for 3 peaks was ~48.6 ms (neglecting recycle delay/acquisition time). WET for Figure 2 and Figure 3 was ~100 ms long (neglecting recycle delay/acquisition time) but it is acknowledged that there are several different pulse shapes and optimizations that could be used and so this time could be reduced. WET experiments were attempted with 8.5 ms shaped pulses (automatically generated using a modified multiple suppression tool/macro) and results are shown in Figure S4 and S6. The time for this sequence with these pulses was ~54 ms (neglecting recycle delay/acquisition time). However, while the solvent signals of the 3-peak sample could be suppressed (Figure S4), this was not the case for the 4-peak sample within the range of pulse powers tested (Figure S5). Further, the suppression is less selective with the 8.5 ms shaped pulses. The pre-saturation sequence used 100 ms shaped pulses repeated to occupy the entire recycle delay period. Prost et al.34 used GASP pulses to suppress two peaks with a total time for four echoes of 16 ms. Such a time and shorter are likely achievable with MULTI-GATE-FSB (and may be further reduced since even n = 1 can be used). Both a short recycle delay (e.g., 1 s) and a long recycle delay (i.e., ≥5 × T1, longest) provided good suppression with MULTI-GATE-FSB (compare Figure 2 and Figure 3 to Figure S14 and Figure S15). Short block times are suited to fast single scan acquisitions and automation of this pulse sequence should also be possible as done for WET.15, 16 Typically, τBin, f must be chosen so that the periodic nulls from each binomial pulse are outside the desired spectral range so that no unwanted suppression occurs (e.g., consider Figure S9 and


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Figure S10 showing unwanted suppression with τBin, f = 0.4 ms) but this also has an impact on the selectivity of the suppression35 (see also the experimental excitation profiles in the Supporting Information, Figure S16 – S26). This is a limitation with binomial type pulses, though a suitable τBin, f should exist for most situations. However, another possible means of further reducing the acquisition time is to exploit the secondary null points of the W5 binomial rf pulses to suppress peaks. For the special case of evenly spaced solvent peaks with solute signals not extending too far beyond the ‘terminal’ solvent signals, then one binomial pulse may suffice for the suppression of multiple peaks (although optimization of the inter-pulse delays may be needed to maintain good excitation profiles38). Otherwise if there are ‘terminal’ solvent signals with solute signals that don’t extend too far beyond these, then this may still reduce the number of binomials needed. For example, the 4-peak suppression sample used in this work still allows exploitation of the secondary null points to suppress all four solvent peaks with only three f loops (e.g., Figure S11). In this experiment the block size for the first binomial was ~8.4 ms, and the second and third were ~5 ms, and so for three blocks each repeated twice this was ~36.7 ms neglecting recycle delay/acquisition time. Compare this to the total time for the blocks used in Figure 3 which was ~64.8 ms (i.e., ~8.1 ms blocks each repeated twice for four frequencies). This optimization was even shorter than the total time for the blocks used for the 3-peak sample in Figure 2 and such exploitation is potentially possible for the 3-peak sample (i.e., using only two f loops). However, it is accepted that exploitation of the periodicity of the suppression nulls may not be possible for all cases. SNR values for selected peaks (Table 1) in the spectra of Figure 2 and Figure 3 show that the MULTI-GATE-FSB sequence suffers from a loss of signal compared to WET and pre-saturation. This is also easily seen in Figure 2 and Figure 3 (and Figure S14 and Figure S15) in the



Page 18 of 37

additional scaling of the MULTI-GATE-FSB results. Although, all sequences can be expected to have different signal intensities because of differences in the applied pulse sequences, however it can be seen in Figure 2 and Figure 3 that the residual solvent signals are also far less than those obtained from WET and pre-saturation. Some modulation of the signals can be seen when comparing the SNR values in Table 1 but there is also a difference between the WET and presaturation results for some of the peaks. WET and pre-saturation results may also be artificially enhanced by the residual solvent signals (e.g., the tails of the residual water signal add to the magnitude of the effected solute signals). Experimental excitation profiles for the MULTIGATE-FSB sequence and the second set of 3-peak and 4-peak suppression samples are provided in the Supporting Information (Figure S16 – S26). The experimental excitation profiles show modulation of the unsuppressed regions and this is likely related to the durations and inter-pulse delays of the binomial rf pulses which can be corrected or reduced.38

Table 1 Signal to Noise Ratios for Selected Peaks in the Suppression Spectra for WET, PreSaturation and MULTI-GATE-FSBa 3-Peak Suppression Sample ~1.42 ppm

~3.15 ppmb

~3.45 ppm

~4.03 ppm

~4.54 ppm

~5.14 ppm

MULTIGATEFSB

916.3

96.7

283.1

145.0

88.7

89.0

WET

4808.7

480.1

1005.7

559.0

501.5

407.2

PreSaturation

4696.7

276.2

2868.2

405.0

581.0

400.2

~4.51 ppm

~5.14 ppm

4-Peak Suppression Sample ~1.45 ppm

~3.16 ppmb

~3.44 ppm

~4.03 ppm


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MULTIGATEFSB

765.3

27.6

57.5

119.3

69.8

61.7

WET

4394.5

496.6

177.6

417.3

470.9

266.5

PreSaturation

3750.2

424.8

2454.1

343.0

426.9

389.6

a

Results for the data presented in Figure 2 and Figure 3.

b

This multiplet was partially overlapped with one of the satellites from the methanol in the 4peak suppression sample but the central peak of the multiplet was used in the SNR calculation.

To further the discussion of signal modulation with respect to quantification, e.g., which may be important for metabolomics (e.g., Refs.13,

39, 40

), some selected integral ratios were also

compared to those expected from sample composition. Based on the composition of the samples used in Figure 2 and Figure 3, the expected ratios of the integral sum for the anomeric protons41, 42

of α-D-glucose and β-D-glucose (i.e., ~5.14 ppm (α) and ~4.53 ppm (β)) to the integral of the

CH3 protons of L-alanine (i.e., ~1.43 ppm), and of the integral of the CH2 protons of glycine (i.e., ~3.44 ppm) to the integral of the CH3 protons of L-alanine (i.e., ~1.43 ppm) are 0.194 and 0.614, respectively (for both the 3-peak and 4-peak suppression samples). Integrals ratios were calculated for the data in Figure 2 and Figure 3, and pulse-acquire experiments with long recycle delays. Baseline corrections around each peak integrated were necessary for pulse-acquire, WET and pre-saturation spectra, to remove the effects of baseline distortion/added signal due to the large solvent signals or residual solvent signal phase distortion effects. No baseline correction was done for each solute signal for the MULTI-GATE-FSB results, i.e., only an overall spectrum baseline correction was necessary. NB baseline corrections for each peak were not done for the SNR ratios in Table 1 and the sino command uses magnitude data in the calculations. For the 3peak suppression sample, the ratio of the anomeric protons of glucose to the CH3 protons of L-



Page 20 of 37

alanine was 0.171 (pulse-acquire), 0.247 (MULTI-GATE-FSB), 0.202 (WET) or 0.242 (presaturation). The ratio of the CH2 protons of glycine to the CH3 protons of L-alanine in this sample was 0.661 (pulse-acquire), 0.111 (MULTI-GATE-FSB), 0.086 (WET) or 0.292 (presaturation). For the 4-peak suppression sample, the ratio of the anomeric protons of glucose to the CH3 protons of L-alanine was 0.181 (pulse-acquire), 0.269 (MULTI-GATE-FSB), 0.213 (WET) or 0.260 (pre-saturation). The ratio of the CH2 protons of glycine to the CH3 protons of L-alanine in this sample was 0.568 (pulse-acquire), 0.034 (MULTI-GATE-FSB), -0.017 (WET) or 0.236 (pre-saturation). The negative ratio here for the WET result is due to the inverted signals in the WET spectrum for the parameters used in Figure 3 as discussed. Compared to the expected ratios, i.e., 0.194 and 0.614, the integral ratios from the pulse-acquire spectra – free from the modulation of a multi-suppression sequence – are in reasonable agreement given the baseline corrections performed. While some values from the multi-suppression experiments are similar to the expected values, overall the multi-suppression sequences show modulated values (noting the negative value for one result). Relaxation effects, i.e., longitudinal (T1) and transverse (T2), were ignored for the comparative ratios calculated above but for metabolomics studies and fast acquisitions, the relaxation rates of a metabolite and a reference compound may become important for the calculation of metabolite concentration.39 While the results above were from data acquired with long recycle delays (≥5 × T1,

longest),

T2 weighting and diffusion may also

become important, e.g., as in WATERGATE type sequences.4,

13, 38, 43

T1, T2 and diffusion,

however, offer a means to edit spectra in metabolomics studies.44 To complement the above ratios, the value of α-D-glucose/(α-D-glucose + β-D-glucose) was estimated from the anomeric proton signals of D-glucose. For the 3-peak sample this was 0.374 (pulse-acquire), 0.496 (MULTI-GATE-FSB), 0.403 (WET) or 0.375 (pre-saturation). For the 4-peak sample this was


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0.450 (pulse-acquire), 0.497 (MULTI-GATE-FSB), 0.395 (WET) or 0.435 (pre-saturation). For aqueous solutions the equilibrium value is expected to be 0.364.42 The values obtained from WET and pre-saturation, at least for the 3-peak sample, are better comparable to the results from pulse-acquire experiments and the aqueous solution value. However, from both composition ratios and anomeric composition values, the modulation of the integrals is easily noted for all multi-suppression sequences. Parameters would need to be optimized/improved to enable full quantification with any of these multi-suppression sequences (e.g., as is the case for single suppression sequences13). For qualitative experiments or experiments where relative changes in the signals are all that is needed the multi-suppression sequences are still expected to be useful provided the same suppression parameters are used for each experiment. A demonstration of the ability to suppress multiple regions in a biomolecular sample, i.e., lysozyme sample, is shown in Figure 4 with suppression of the water peak and different regions in the lysozyme spectrum. Hence, MULTI-GATE-FSB still proves useful for biomolecular samples. It can be seen from Figure 4 that repetition of the same frequency resulted in a broadening of the suppressed region (i.e., using more than one f loop for the same frequency). Spectra of the lysozyme sample with only single suppression WATERGATE and additional MULTI-GATE-FSB results are provided in the Supporting Information (Figure S46 and Figure S47). Additional results for the lysozyme sample and MULTI-GATE-FSB are also provided in the Supporting Information (Figure S48 – S53). The maximum SNR was found for the spectrum in Figure S46B, i.e., using a double-echo WATERGATE sequence, and Figure S48C, i.e., using MULTI-GATE-FSB with three f loops (and n = 2) were 140.8 and 50.1, respectively. These two experiments were selected for the SNR calculation since they had similar parameters. The maximum SNR was found using sino and the noise region used was from 14 – 18 ppm (note the



Page 22 of 37

full spectral width acquired was large enough but was not shown in the figures for best display of the data). The signal range used for sino was -2 – 12 ppm and the maximum SNR was for the signal at 0.89 ppm for the WATERGATE results and at 0.845 ppm for the MULTI-GATE-FSB results. Although the SNR is lower for the MULTI-GATE-FSB results this is somewhat expected since the MUTLI-GATE-FSB results were from a pulse sequence with six binomial pulses.


Page 23 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Suppression results using MULTI-GATE-FSB with three suppression frequencies and a standard lysozyme sample. Spectra in A) are scaled to show the full residual water signal and spectra in B) are scaled to show the lysozyme signals (NB: Spectra in i) – iv) have the same scaling factor for A) or B)). Symbols ,  and  indicate frequencies suppressed and f-loop order ( first and  last). Spectra were acquired at 400 MHz with receiver gain = 456.1, 16 scans (with 4 dummy scans), hard π/2 rf pulse length/power = 16.6 μs/14.3 W, acquisition time = 3 s, recycle delay = 10 s, reference pulse length/power used to calculate the binomial pulses was 20 μs/3.6 W, n = 2 and τBin,

f

= 0.2 ms. Gradient pulses were along the z-axis and were

rectangular shaped and δf1 = δf2 = δf3 = 2 ms and gf1 = 0.502 T/m, gf2 = 0.435 T/m and gf3 = 0.062 T/m where f1, f2 and f3 are for the first, second and third suppression frequencies. f1 was set to the water resonance and i) f2 and f3 were set to f1 + 983 Hz or ii) f2 and f3 were set to f1 – 1508 Hz or iii) f2 was set to f1 – 1508 Hz and f3 was set to f1 + 983 Hz or iv) f2 and f3 were set to f1.

Although the simplest implementation of this sequence provides outstanding suppression results, optimizations are possible. While the duration/power of the pulses in a ‘W5’ are based on a π/2 reference pulse to give the desired flip angles in Ref.35, this can be used to optimize suppression/signal (i.e., using a different reference length and power that may not be a π/2 rf pulse as in experiments of this work; see Supporting Information). Further modifications may be beneficial, e.g., different binomial pulses for each f loop (e.g., Figure S11) or n-loop or all loops. The ratios of the gradients used for each f-loop could be optimized or a different g in each n-loop (i.e., not only in each f-loop) and/or different δ for each n-loop (i.e., not using one δf for the entire sequence) could be used (e.g., Figure S11). The order of the f-loops, e.g., should the broad peaks be first or the strongest signals etc., and the phase cycle also deserves more consideration.



Page 24 of 37

Optimizations and modifications found elsewhere might also be useful, e.g., adapted binomial inter-pulse delays to help with excitation profile distortions38 or alternate binomial-like pulses.45 J-modulation is well known for spin-echo sequences, e.g., Refs.

43, 46, 47

, and some slight

modulations for some of the multiplets in the spectra for the 3- and 4-peak suppression samples could be seen (e.g., Figure S42 at ~4.5 ppm and ~1.4 ppm, and to a lesser extend in Figure S41B at ~3.15 ppm and ~4.5 ppm, Figure S9, and Figure S10). These modulations could be suppressed with appropriate selection of τBin, f, n and reference pulse power used for the binomial rf pulses (i.e., the rf pulses that make up the W5 binomials were not based on a 90° rf pulse or an equivalent 90° rf pulse). The modification presented in Ref.

43

to suppress J-modulation in the

WATERGATE sequence may be required if J-modulation effects prove significant and other parameters cannot be optimized to reduce/remove modulations in the spectra. Residual

13

C

satellites might be removed via decoupling during the selective pulses15, 32 or other 13C filter.14

CONCLUSION

MULTI-GATE-FSB showed excellent multiple solvent suppression characteristics. The sequence was shown to work well with alcohol-water systems, with both long and short recycle delays without the need for a long pre-saturation pulse, and a biomolecular sample. The block nature of this sequence means that it is easily extended to any number of suppression regions and is easily implemented.

ASSOCIATED CONTENT

Supporting Information Available: The following files are available free of charge.


Page 25 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Schematics of the Bruker standard suppression sequences used and additional data (pulse acquire spectra, shape pulse power optimizations for the WET and pre-saturation experiments, suppression spectra with short recycle delays, spectra showing the effect of recycle delay, n, τBin, f,

the shape and duration of the gradients, the length and power of the reference pulse used to

calculate the rf pulses of the binomial, and results of using WATERGATE sequence with ‘W5’ rf pulses with double echo for the lysozyme sample) (PDF)

AUTHOR INFORMATION

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT

This research was supported by Western Sydney University (WSU) and the National Imaging Facility WSU Node, and was performed in the Biomedical Magnetic Resonance Facility at WSU.

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MULTI-GATE-FSB pulse sequence. Black rectangles represent rf pulses, rectangles with diagonal lines are gradient pulses and the signal acquired is shown by the grey ‘triangle’. The first rf pulse is a π/2 (i.e., 90°) pulse and this is followed by multiple loops of gradients and binomial pulses. The f-loop is the number of frequencies to suppress and the n-loop is the number of times each frequency is repeated (e.g., ‘single echo’, ‘double echo’…; NB: this could be different for each f). For each suppression frequency the binomial rf pulse (e.g., here it is a ‘W5’ pulse35) is centered on that frequency and has the effective final pulse angle of θBin, f and τBin, f are the binomial inter-pulse delays (the same pulse angle and inter-pulse delays can be used for all f-loops or otherwise optimized). The gradients used for each suppression frequency have strength gf and duration δf (for the experiments of this work each f-loop used a different gf but the same gf was used for each n-loop – but other experiments were attempted with differences). There was a 100 µs delay before the first gf of each n or f-loop (for the first n or f-loop this is the delay between the π/2 rf pulse and gf pulse), a 100 µs delay after the first gf pulse of each n or f-loop prior to the first rf pulse of θBin, f, a delay of 50 µs between the last rf pulse of θBin, f and the second gf pulse of each n or f-loop and a 104 µs delay prior to the next loop. NB gradient blanking and frequency change commands occurred during these delays and were each contributed a 50 µs delay (e.g., the first 100 µs was made up of a frequency change command + 50 µs delay and a gradient blanking command + 50 µs delay). The phase cycle used was π/2: [0º, 180º], pulses in the first half of θBin, f: [0º, 0º, 180º, 180º] for all loops, pulses in the second half of θBin, f: [180º, 180º, 0º, 0º] for all loops, and receiver: [0º, 180º]. 80x41mm (300 x 300 DPI)

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Comparison of the results obtained for multiple suppression of the three solvent peaks in the 3-peak suppression sample. Spectra in A) are to the same scale, i.e., relative to one another, for the experimental parameters used and are the ‘×1’ scaled reference spectra. The spectra in A) are scaled to best show the residual solvent signals for all suppression sequences. Spectra in B) are scaled relative to the ‘×1’ spectra as indicated. Multiple suppression was achieved via i) WET, ii) Pre-saturation and iii) MULTI-GATE-FSB sequences. Symbols indicate frequencies suppressed: ▽ (water),  (ethanol quartet) and △ (ethanol triplet). The order of the symbols in the caption indicate the order of the three f loops in the MULTI-GATEFSB sequence. The spectra were acquired at 400 MHz with 8 scans (with 4 dummy scans), hard π/2 rf pulse length/power = 15.6 μs/32.05 W for the hard rf pulses, acquisition time = 1.5 s, receiver gain = 32, and recycle delay = 15 s. Additional parameters: i) gradients were along the z-axis and were half-sine shaped with strengths g1 = 0.446 T/m, g2 = 0.223 T/m, g3 = 0.112 T/m and g4 = 0.056 T/m, and durations δ1 = δ2 = δ3 = δ4 = 1 ms, the four 20 ms shaped rf pulses were the same shape but were at different power levels for θS1 – θS4: 0.00104 W, 0.00162 W, 0.00076 W, and 0.00408 W; ii) 150 repetitions of the 100 ms


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shaped rf pulse at 0.00227 W; iii) τBin, f was the same for all binomial pulses and was 0.4 ms, three f loops were used, n = 2 for each f loop, the reference pulse length/power used to calculate the binomial pulses was 20 μs/8.052 W. Gradient pulses were along the z-axis and were rectangular shaped with δf1 = δf2 = δf3 = 2 ms and gf1 = 0.502 T/m, gf2 = 0.435 T/m, and gf3 = 0.062 T/m where f1, f2, and f3 are for the first, second, third and fourth suppression frequencies. f1 was set to the water resonance and f2 was set to f1 – 484.59 Hz for the ethanol quartet and f3 was set to f1 – 1466.24 Hz for the ethanol triplet. 80x106mm (300 x 300 DPI)



Comparison of the results obtained for multiple suppression of the four solvent peaks in the 4-peak suppression sample. Spectra in A) are to the same scale, i.e., relative to one another, for the experimental parameters used and are the ‘×1’ scaled reference spectra. The spectra in A) are scaled to best show the residual solvent signals for all suppression sequences. Spectra in B) are scaled ×8 (still relative to one another) to better show the residual solvent signals in the MULTI-GATE-FSB results. Spectra in C) are scaled relative to the ‘×1’ spectra as indicated. Multiple suppression was achieved via i) WET, ii) Pre-saturation and iii) MULTI-GATE-FSB sequences. Symbols indicate frequencies suppressed: ▽ (water),  (ethanol quartet), ○ (methanol singlet), and △ (ethanol triplet). The order of the symbols in the caption indicate the order of the four f loops in the MULTI-GATE-FSB sequence. The spectra were acquired at 400 MHz with 8 scans (with 4 dummy scans), hard π/2 rf pulse length/power = 15.15 μs/32.05 W for the hard rf pulses, acquisition time = 1.5 s, receiver gain = 32, and recycle delay = 16 s. Additional parameters: i) gradients were along the z-axis and were half-sine shaped with strengths g1 = 0.446 T/m, g2 = 0.223 T/m, g3 = 0.112 T/m and g4 = 0.056 T/m, and durations δ1 = δ2 = δ3 = δ4 = 1 ms, the four 20 ms shaped rf pulses


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were the same shape but were at different power levels for θS1 – θS4: 0.00117 W, 0.00182 W, 0.00085 W, and 0.00458 W; ii) 160 repetitions of the 100 ms shaped rf pulse at 0.00227 W; iii) τBin, f was the same for all binomial pulses and was 0.4 ms, four f loops were used, n = 2 for each f loop, the reference pulse length/power used to calculate the binomial pulses was 20 μs/8.052 W. Gradient pulses were along the zaxis and were rectangular shaped with δf1 = δf2 = δf3 = δf4 = 2 ms and gf1 = 0.502 T/m, gf2 = 0.435 T/m, gf3 = 0.062 T/m, and gf4 = 0.116 T/m where f1, f2, f3 and f4 are for the first, second, third and fourth suppression frequencies. f1 was set to the water resonance, f2 was set to f1 – 480.23 Hz for the ethanol quartet, f3 was set to f1 – 591 Hz for the methanol singlet, and f4 was set to f1 – 1459.7 Hz for the ethanol triplet. 80x159mm (300 x 300 DPI)



Suppression results using MULTI-GATE-FSB with three suppression frequencies and a standard lysozyme sample. Spectra in A) are scaled to show the full residual water signal and spectra in B) are scaled to show the lysozyme signals (NB: Spectra in i) – iv) have the same scaling factor for A) or B)). Symbols , △ and ▽ indicate frequencies suppressed and f-loop order ( first and ▽ last). Spectra were acquired at 400 MHz with receiver gain = 456.1, 16 scans (with 4 dummy scans), hard π/2 rf pulse length/power = 16.6 μs/14.3 W, acquisition time = 3 s, recycle delay = 10 s, reference pulse length/power used to calculate the binomial pulses was 20 μs/3.6 W, n = 2 and τBin, f = 0.2 ms. Gradient pulses were along the z-axis and were rectangular shaped and δf1= δf2 = δf3 = 2 ms and gf1 = 0.502 T/m, gf2 = 0.435 T/m and gf3 = 0.062 T/m where f1, f2 and f3 are for the first, second and third suppression frequencies. f1 was set to the water resonance and i) f2 and f3 were set to f1 + 983 Hz or ii) f2 and f3 were set to f1 – 1508 Hz or iii) f2 was set to f1 – 1508 Hz and f3 was set to f1 + 983 Hz or iv) f2 and f3 were set to f1. 80x152mm (300 x 300 DPI)


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49x43mm (300 x 300 DPI)


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A Simple and Effective Binomial Block Based Pulse Sequence

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