Resolution baseline

Reiterating the conditions for a chromatographic separation once again, for two solutes to be resolved their peaks must be moved apart in the column and maintained sufficiently narrow for them to be eluted as discrete peaks. However, the criterion for two peaks to be resolved (usually defined as the resolution) is somewhat arbitrary and is usually defined as the ratio of the distance between the peak maxima to half the peak width (a) at the points of inflection. To illustrate the various degrees of resolution that can be obtained, the separation of a pair of solutes 2o, 3o, 4o, 5o and 6o apart are shown in Figure 12. Although, for baseline resolution, it is clear that the peak maxima should be separated by at least 6o for most quantitative analyses. 

The right chromatography column should separate the sample sufficiently to enable identification or quantitative measurement of the components within a reasonable period of time. The resolution factor (Rs) for two sample components is determined by the width of the two peaks and the distance between the peak maxima. In general, Rs values of 1.0 are required for good qualitative or quantitative work, whereas Rs values >1.5 indicate baseline resolution for two components (3). 

For the separation of large DNA fragments greater than 1000 bp, a four-column system is typically required. The baseline resolution of DNA fragments... 

The resolution obtainable with a UTI-100C quadrupole mass analyser is m/Am 2m (Jjt)). These three peaks are also separated to baseline resolution In Figure 7a however, they appear as one peak due to the wide mass range which is displayed. 

Potassium dichromate was used as a molecular marker to measure total system volume and plate count (1). The three column sets exhibited the equivalent of approximately 250-300 plates per foot. This was adequate, even with Set I, to give near baseline resolution of an equal weight blend of the 103 nm and 312 nm samples as shown in Figure 2. This Is approximately an 84 1 number ratio. 

A value of S, 1.0 corresponds to a peak separation on the order of 94% and is generally considered an adequate goal for an optimized separation. Baseline resolution corresponds to an R, value of 1.5. 

A typical hplc column (25 cm x 4.6 mm, packed with a 5 jum bonded silica stationary phase) will have an efficiency corresponding to a plate number of 10 000-15 000. For many separations, this efficiency is far more than is needed, as often a plate number of 3000-5000 will give baseline resolution of all solutes. If this is the case, using a conventional column will waste analysis time and sol-... 

A 10-pL sample of the a-methoxy ester is dissolved in 0.5 mL of CDCI3 and transferred to an NMR tube. A 5-pL portion of (+)-Eu(dppm)3 (30 w/v% CCI2FCCIF2 solution) is added to the a-methoxy ester sample. The mixture is shaken well, and the 1H NMR spectrum is recorded. Additional portions of the shift reagent solution are added in 5-pL portions until the methyl ether resonance shifts downfield beyond that of the methyl ester and shows baseline resolution of the methyl ester and methyl ether resonances from the two enantiomers. (Four singlets should be observed). In total 15-20 pL of the shift reagent solution should be required to achieve the desired shift. At... 

Compared with GC and HPLC, the most important advantage of CE is its high peak efficiency. It can give a baseline resolution of peaks even when the separation factor is low. Volatile chiral samples are best analyzed by GC, whereas HPLC and CE are more suitable for nonvolatile samples. CE is the best choice for a charged compound or for a high-molecular-weight sample. 

A calculated value for resolution greater than 1.5 indicates that the adjacent peaks exhibit baseline resolution the signal has fully returned to the baseline from the first peak before the second peak begins. Often, a minimum acceptable resolution of 2 is used in method development to ensure that acceptable resolution is maintained, even as the method is transferred among instruments, analysts and laboratories. 

Bossio, R.E. Marshall, A.G. Baseline Resolution of Isobaric Phosphorylated and Sulfated Peptides and Nucleotides by ESl-FT-lCR-MS Another Step Toward MS-Based Proteomics. Anal. Chem. 2002, 74, 1674-1679. 

In a recent study, chiral separations for pyrethroic acids, which are the chiral building blocks of synthetic pyrethroids and the primary metabolites of the acid part of these potent ester insecticides, have been developed [62], For example, a polar-organic mobile phase allowed the complete baseline resolution of all four stereoisomers of chrysanthemic acid (2,2-dimethyl-3-(2-methylprop-l-enyl)-cyclopropanecarboxylic acid) on a 0-9-(tcrt-butylcarbamoyl)quinine-based CSP(acjj = 1.20, oLtrans = 1-35, critical Rs = 3.03) (Figure 1,32a). This chiral acid is the precursor of pyrethroids like allethrin, phenothrin, resmethrin, and tetramethrin but not excreted as metabolite. The primary acid metabolite of these pyrethroids is chrysanthemum dicarboxylic acid (3-[(l )-2-carboxyprop-l-enyl]-2,2-dimethylcyclopropanecarboxylic acid) the stereoisomers of which could also be resolved with a reversed-phase eluent (acetonitrile— 30-mM ammonium acetate buffer 90 10, v/v pHa = 6.0) and employing an O-9-(2,6-diisopropylphenylcarbamoyl)quinine-based CSP ads = 1-09, atrans = 1-50,... 

Note that not all enantioseparations in SFC are better than in HPLC [34], Bernal et al. [62] described the enantiomeric separation of several pharmaceutical-related compounds on a polysaccharide-based column using HPLC and SFC. They showed that most of the separations obtained by SFC are better, in terms of resolution and analysis time, than the separations obtained by HPLC. However, one compound could not be resolved using SFC, but LC provided baseline resolution. 

System suitability tests for chromatographic impurities procedures, such as precision, resolution factor, calibration standard, and tailing factor, should be considered as appropriate. In the presence of multiple peaks, a resolution factor between the two closest peaks should be proposed. For reliable quantitation, baseline resolution of the impurities will provide accurate measurement of the... 

Figure 3.10 exemplifies the application of packed-column SFC for the rapid determination of the enantiomeric purity of pharmaceutical materials. The method employed a CHIRALCEL OD CSP with a carbon dioxide/methanol/ isopropylamine mobile phase under isocratic conditions. A flow rate of 4 mFmin was used to generate a high linear velocity whilst maintaining an outlet pressure of 200 bar by use of a back-pressure regulator to ensure reproducible chromatography. Baseline resolution of the enantiomers of propanolol was achieved in approximately 3 min with an overall cycle time of 4 min. Detection was by UV... 

The effect of the electroosmotic flow on the resolution is also evident from Eq. (16). A high electroosmotic flow in the direction of the moving ions can significantly diminish resolution. Theoretically, infinite resolution of two peaks could be reached when jlEOF is equal but opposite to the average mobility m.Av. In this case one of the solutes would migrate in the direction of the detector and the other one in the opposite direction. In other words, the separation run would be infinitely long. Thus, for a practical separation the electroosmotic flow should be controlled in a way to achieve baseline resolution (R = 1) at minimal separation time. 

PQQ was successfully separated from three closely related isomers by capillary electrophoresis with UV detection <2000JCH(876)193>. Rapid and efficient separation of all four compounds 17-20 as their negatively charged carboxylate ions with baseline resolution was achieved by the addition of 1-5 mM R4N salts to the capillary buffer. Detection limits of PQQ and its three isomers were in the range of 7-15 xM with mass detection limits of 98-210 fmol. 

In contrast to the analytical approach where the emphasis is placed on reagents with only sufficient chromatographic diastereoselectivity for baseline resolutions combined with highly sensitive detection properties (e.g., chromophore, fluorophore, electrochemical sensitive groups), the preparative technique requires different and additional criteria to be fulfilled by the reagents and the reaction products. In this case it is mandatory that the separation products can be cleaved under reasonable conditions to obtain the resolved optically pure parent compounds (selectands). Briefly summarized, the requirements are the following ... 

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