Retention time precision

SEC offers several advantages that make it a desirable technique for both preparative and analytical applications. First, separations are rapid with an 8 x 300 mm analytical column operated at 1 ml/min, all analytes elute in about 10 min. Second, because the stationary phase is designed to eliminate interactions with the sample, SEC columns exhibit excellent recovery of mass and biological activity. Third, because all separations are performed under isocratic conditions, peak area and retention time precision are high. 

In reversed-phase HPLC, column temperature is a strong determinant of retention time and also affects column selectivity. A column oven is therefore required for most automated pharmaceutical assays to improve retention time precision, typically at temperatures of 30-50°C. Temperatures >60°C are atypical due to concerns about thermal degradation of the analytes and column lifetimes. Exceptions are found in high-throughput screening where higher temperatures are used to increase flow and efficiency. Ambient or snb-ambient operation is sometimes found in chiral separations to enhance selectivity. Column ovens... 

B. Practical Guidelines for Improving Retention Time Precision... 

A theoretical equation on retention time precision has been proposed by Eli Grushka and Zamir ... 

According to the equation, column temperature is expected to be the dominant factor in controlling retention time precision because changes of retention are proportional to the inverse of the square of the absolute temperature. 

The interferences created other chromatographic problems besides poor retention-time precision. These problems included the presence of a large rising and falling base line (a hump between C-10 and C-17), an unexpected rise in the level of noise from the flame ionization detector, and contamination of the stationary phase of the chromatographic column. These problems were especially severe for extracts of 150-L samples of chlorinated drinking water. The contamination of the... 

The removal of chromatographic analysis interference increases the reliability of the broad spectrum approach to organic analysis. The improved retention-time precision and lower background noise level make it possible to use statistical significance-level testing of the broad spectrum data (26). 

Since the response of the detector (and the separation) is a function of a flow rate, it is essential that the standard response curve be determined at the same flow rate as the tablet assay. If retention times differ significantly from the runs of the standards, there is a need to troubleshoot the HPLC to determine where the problem resides. Refer to Chapter 3 for a discussion of retention time precision. 

Precision in retention times and peak area or height is a major criterion of a separation system. Retention-time precision is important because Ri is the primary means for peak identification. It is also an important performance criterion and diagnostic for an LC pump and a column. 

Solvent Line Filter (Sinker) The solvent line sinker, typically a 10-pm sintered stainless-steel filter, can be replaced by direct connection to the Teflon solvent line (Figure 10.2a). The sinkers should be replaced annually. A partially plugged solvent sinker can restrict solvent flow, which can result in poor retention time precision. 

Table 10.2. Peak Area and Retention Time Precision Data Before and After Replacing the HPLC Column...

Case study 4 shows several examples of problems caused by equipment malfunctions and their subsequent diagnosis and solution. The first one involved a situation of poor retention time reproducibility of a gradient assay. It involved the analysis of a complex natural product, using a narrowbore column (2-mm i.d.) at 0.5 mL/min. System suitability test showed retention times to be erratic and could vary by 1-2 minutes without any obvious trends. Flow rate accuracy was found to be acceptable, however, the compositional accuracy test failed (see Chapter 9 on HPLC calibration). The tentative diagnosis was that of a malfunctioning of the proportioning valve. After its replacement, the retention time precision performance was re-established. 

The attractive features of splitless injection techniques are that they allow the analysis of dilute samples without preconcentration (trace analysis) and the analysis of dirty samples, since the injector is easily dismantled for cleaning. Success with individual samples, however, depends on the selection of experimental variables of which the most important sample size, sample solvent, syringe position, sampling time, initial column temperature, injection temperature and carrier gas flow rate, often must be optimized by trial and error. These conditions, once established, are not necessarily transferable to another splitless injector of a different design. Also, the absolute accuracy of retention times in splitless injection is generally less than that found for split injection. For splitless injection the reproducibility of retention times depends not only on chromatographic interactions but also on the reproducibility of the sampling period and the evaporation time of the solvent in the column inlet, if solvent effects (section 3.5.6.2) are employed. The choice of solvent, volume injected and the constancy of thermal zones will all influence retention time precision beyond those for split injection. For quantitative analysis the precision of repeated sample injections is normally acceptable but the method is subject to numerous systematic errors that may... 

Unambiguous confirmation of identity is a critical part of forensic toxicology and provides the foundation for all subsequent quantitative results, interpretations, and ultimately court reports and testimonies. Despite the advances in mass spectrometric detection discussed here, chromatography is still regarded as critical. Confirmation that the retention time of a compound in a sample matches that of an authentic reference standard is considered a critical identification parameter and necessitates the contemporaneous analysis of the authentic standard. On modem instmmentation, intraday retention time precision should be well below 2%, and potential positives outside the expected tolerance should be excluded or explained, regardless of other confirmatory mass spectral data. 

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