## 7 NMR Pulse Sequences: Recent Advances and Future Prospects
[124], resonances arising from these small molecules have shorter \(T_{2}\) values than those present in non-protein-containing solutions and hence are more influenced by the CPMG relaxation filter system. Further pioneering studies concerning development of the CPMG pulse sequence, and that of the alternative, albeit earlier, Hahn spin-echo Fourier-transform (SEFT) option, are reported in [125; 126], respectively. As long ago as 1988, [PERSON] et al. [124] reported the detection of a highly significant fraction of blood plasma lactate that was bound to plasma proteins (predominantly but not exclusively albumin present at a level of ca. 0.50 mmol./L) in both single-pulse and Hahn spin-echo \({}^{1}\)H NMR spectra acquired on this biofluid. This so-called 'NMR-invisible' lactate may to mobilized from the protein bound to the Hahn spin-echo spectrum-detectable 'free' form via the addition of charged reagents such as ammonium chloride (NH\({}_{4}\)Cl), with a maximal release extent observed at 0.50 mol./L of this reagent added, so presumably negatively charged lactate was tightly electrostatically bound to positively charged protein amino acid side-chains, such as lysine residues, in these plasma proteins, and displaceable by added Cl\({}^{-}\) anion. In corresponding spin-echo spectra of ultrafiltrates (<10 kDa cut-off) obtained before and after NH\({}_{4}\)Cl addition to whole plasma, large increases in the lactate-CH\({}_{3}\) and -CH resonance intensities of up to three-fold were observed when they were expressed relative to those of the endogenous amino acids alanine or valine. Following NH\({}_{4}\)Cl addition at a level of 0.50 mol./L, the \(\Delta v_{1/2}\) values of the lactate-CH\({}_{3}\) signal decreased from 3.5 to 2.2 Hz; those of alanine remained unmodified (2.3 Hz). The authors concluded that total plasma lactate concentrations were only reliably estimated following excess NH\({}_{4}\)Cl addition. Without this prior sample preparation step, \({}^{1}\)H NMR-determined lactate concentrations were approximately only one-third of those measured by an established enzyme assay system. However, we find that, for both metabolites, these added NH\({}_{4}\)Cl-liberated \(\Delta v_{1/2}\) values found for plasma in Hahn spin-echo spectra were actually rather higher than those measured in single-pulse spectra acquired on WMS supernatant samples, so this indicated that there is little or no'masking' of fractions of these metabolites (and hence also their \({}^{1}\)H NMR resonances), via their equilibria with protein binding-sites available in this relatively protein-deplete biofluid. The dependencies of these \(\Delta v_{1/2}\) values on biomolecule structure, participant donors, and doublet resonance line position (frequency) are investigated in Case Study 1 described in Section 7.3 below. In a related study, [PERSON] et al. [122] explored the NMR detection and quantification of low-molecular-mass species in deuterochloroform (C\({}^{2}\)HCl\({}_{3}\)) solutions containing synthetic polymers (polyethylene glycol and polystyrene), and experiments were conducted both with and without use of the \(T_{2}\) CPMG spin-echo pulse sequence filter. The effects exerted by differing acquisitional parameters (echo time, relaxation delay, and the number of cycles), along with sample compositional indices, i.e., molecular mass of polymer and concentrations, on analytical responses, were investigated with a highly recommendable analysis-of-variance (ANOVA)-based design of experiments model, which considered a first-order interaction and squared variable terms, in addition to a range of potential response parameter variance-contributing input factors. This design was employed in order to optimize the reliability of quantitative analysis performed. Mixtures containing variable concentrations of both low-molecular-mass and macromolecular molecules were analyzed both with and without application of the CPMG filter.