Known Concentration 0.86 mM 13.09 70.26 4.14 28.47 22.98 in .NET framework

Creating PDF417 in .NET framework Known Concentration 0.86 mM 13.09 70.26 4.14 28.47 22.98
Known Concentration 0.86 mM 13.09 70.26 4.14 28.47 22.98
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Calculated Concentration 0.85 mM 12.38 72.63 4.23 28.42 22.06
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% Error 0.7 5.4 3.4 2.3 0.2 4.0
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Data from reference 31.
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strategies for quantitative analysis
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provides no interference for samples that must also be analyzed by HPLCUV-MS to con rm structural and purity data obtained from NMR. Once stability of BTMSB was demonstrated (about 1 month in solution) as well as the precision, accuracy, and linearity of the quantitation method validated, 314 combinatorial products were quantitated individually with this standard.33 1.4.2. Residual Protonated Solvent
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Another convenient alternative is to simply quantitate using the residual protonated solvent signal as an internal standard. This can be challenging in aqueous (D2O) or hygroscopic (DMSO) solvents since environmental instabilities (e.g., humidity) make it dif cult to know the exact concentration of the solvent. Furthermore solvents like DMSO give rise to multiplets in a spectral region (~2.5 ppm) where sample signals may exist. Chloroform, which exhibits a singlet at about 7.2 ppm, down eld of some aromatic protons, is a better solvent standard and solubilizes many organic compounds. However, since expensive deuterated solvents are required, this method may be less useful for routine analyses. 1.4.3. ERETIC Method
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For complex analytes or mixtures, it may be dif cult to select a standard that contains resonances resolved from those of the sample. The ERETIC (Electronic Reference To access In vivo Concentrations) method overcomes this challenge by using an electronic signal as a reference.34 Nothing is physically added to the sample or NMR tube, in contrast to traditional internal or external standards. The 13C coil generates a pseudo-FID producing a signal that can be placed in a transparent region of the spectrum.34 The ERETIC signal must be calibrated monthly against a sample of known concentration to provide the most accurate results. Table 1.5 shows the precision and accuracy of this method, as re ected by similar lactate concentrations determined using the ERETIC method and quantitation using trimethylamine hydrochloride (TMA) as an internal reference. 1.4.4. Special Issues Related to the Flow-Probe Format
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The ERETIC method may be most useful for split-and-pool syntheses where it is undesirable to further complicate the NMR spectrum of complex mixtures by adding a standard. In both split-and-pool and parallel synthetic strategies, micro- to nanoscale reactions generate small amounts of product. For example, solution-phase libraries can be generated in 96-well microtitre
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quantitative analysis in organic synthesis with nmr spectroscopy
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Table 1.5. Accuracy (D) and Precision (d) of Lactate Concentrations Determined by the ERECTIC Method versus the Use of TMA as an Internal Standarda Known Lactate Concentration 5.25 mM 10.92 16.13 25.55 54.11
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[Lactate] d ERETIC 5.19 0.05 mM 11.01 0.07 16.27 0.07 25.74 0.14 54.55 0.20
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[Lactate] d TMA 5.46 0.05 10.88 0.05 16.56 0.15 25.51 0.06 53.84 0.22
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D (mM) ERETIC -0.06 0.04 0.14 0.09 0.23
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D (mM) TMA 0.21 -0.04 0.43 -0.14 -0.27
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Data from reference 34.
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plates and the volumes contained in the individual wells are too small for analysis in standard NMR tubes. Hyphenation of HPLC with NMR (HPLCNMR) has revealed the potential to analyze compounds such as peptides in a owing system.7 Commercially designed ow probes typically contain a total volume of about 120 mL while the active region is roughly half that value, making it possible to analyze library compounds dissolved in the volume of a single well. The development and commercialization of microcoil NMR probes capable of measuring spectra for nL to mL volumes greatly reduces the sample volumes needed for NMR analysis and especially facilitates measurements for mass limited samples.5,35,36 Using NMR ow probes, spectra can be acquired in on- ow (for concentrated samples) or stopped- ow (for minimal sample that requires signal averaging) formats.The low drift rate of most modern high- eld magnets permits NMR ow analyses to be performed on-the- y and unlocked, eliminating the need for expensive deuterated solvents to provide the deuterium lock. This is an advantage for quantitation of samples containing exchangeable protons (e.g., amide protons in peptides). In addition multiple components can be separated rst by HPLC, stored individually in loops, and then ushed to the NMR probe for analysis. Unique challenges exist for quantitation in owing systems. Band broadening dilutes the sample to varying degrees depending on the ow rate and length of tubing connecting the HPLC to the ow probe. Multiple solvents create additional spectral interferences, and although solvent signals may be suppressed from the spectrum, signals underneath or nearby will likely be affected. Solvent gradients also change the composition of the solution (and in some cases the solubility of analytes), which will affect integration if the changes are signi cant on the time scale of the measurement. Direct-injection NMR (DI-NMR) capitalizes on the small-volume capacity of the ow probe and averts the disadvantages encountered in the
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