Retention time reproducibility

The solvent delivery system is one of the most important components of the liquid chromatograph since its performance directly affects retention time reproducibility d detector baseline stability [1,2,39,40]. As well as the pump, other components such as check valves, flow controllers, mixing... 

Retention time reproducibility less crucial than for SFC... 

Figure 6.18 presents example chromatograms obtained during the evaluation of retention time and peak area reproducibility across the 24 columns (within a single composite run). Table 6.1 lists the average retention time reproducibility and peak area reproducibility values obtained across 24 columns for a total of 24 composite runs (column to column within run). Reproducibility values are expressed as percentage relative standard deviation (%RSD). 

As demonstrated by the results summarized in the table, retention time reproducibility values below 1.0% were consistently obtained during all evaluations. Peak area reproducibility values... 

Other matters to consider in column choice are column length, column diameter, and particle size. Column efficiency (theoretical plate count) is determined by a ratio of column length to particle size. A shorter column with the same particle size may give a shorter run time but at a loss of resolution. A shorter column with a smaller particle size with a lower flow rate may give a similar resolution in a shorter time. Retention time reproducibility improves in systems where column temperature can be controlled, especially in cases where ambient room temperature varies. 

Figure 4 Dual detector SECs Solution of the two equations for the two unknown concentrations leads to plots of the concentration ratio versus retention time. (Reproduced frcni Ref. 6. Copyright 1983, American Chemical Society.)...

The discussion of IL-based stationary phases up to this point has centered around ILs that are either coated as a thin film on a capillary wall or on a solid support. Although ILs exhibit a variety of properties that allow them to be unique stationary phases, their most significant drawback lies with their drop in viscosity with increasing temperature. This results in an increased propensity for flowing of the IL within the capillary, which often produces pooling of the stationary phase and nonuniform film thickness throughout the column. These factors often contribute to diminished analyte retention time reproducibility as well as detrimental effects on separation efficiency. 

Janssen et al carried out a comparison of TLC and HPLC methods to identify various cinnamons [24]. TLC methods were found to have a better retention time reproducibility relative to HPLC methods. In addition, solvent costs and time efforts were also less when using TLC. 

Spherisorb ODS-1 (3 pm) Retention time reproducibility/ parameters effecting... 

It is difficult to reproduce water content on a silica column when using dry solvent directly from a supplier s bottle. Therefore, water-saturated solvents are useful in attaining an equilibrium with the silica surface while also eliminating very active sites. This will reduce tailing of solute peaks. A 50% water-saturated solvent will make retention times reproducible. 

Cooper et al. [86] analyzed environmental matrices derived from soil, plant, and animal extracts to study an insecticide, a fungicide, and their metabolites. The clean-up steps consisted simply of removal of the solid debris by centrifugation and filtration. The authors recognized that while retention time reproducibility was satisfactory for a study of pesticides metabolism, in order to achieve the sensitivity required for this type of analysis a sample preconcentration step was required before the CEC separation. 

Retention time reproducibility is also enhanced with a new HPLC system, especially in the shallow region of the gradient profile. Analysis of collagen hydrolysates and cell culture supernatants are shown as representative applications. 

Retention Time Reproducibility Standard Mixture II Amount Reproducibility Ham s F-10 ... 

The method of stationary-phase preparation has a major effect on the resolution, column stability, retention time, reproducibility, and peak shape. When preparing Cjg or Cg, for example, it is important that the residual silanol groups are capped to prevent peak tailmg. The extent of capping must be consistently maintained between different batches of the stationary phase for reproducible results. 

The selection of an appropriate mobile phase is another aspect of concern. Most often the commonly used LC mobile phase is not compatible with thermospray LC/MS, for instance owing to the use of non-volatile buffers can be left out or replaced by volatile ones. In other cases the buffers are present for retention time reproducibility, which mostly is not very important for identification. In other applications however a correspondence between UV or fluorescence peaks and MS identification is obligatory, which makes mobile phase changes unattractive. In this respect it is often overlooked that LC-UV and LC/MS give different responses as a result of different detection principles. For not too complex samples a UV photo-diode array detector can be used to link up the chromatographic peaks obtained under different mobile phase conditions. To cut short, despite many successes also many potential problems are met in LC/MS to which tailor-made creative solutions are needed. 

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. 

Generally, the smallest amount of sample injected will give the best separation performance on thin-film columns. Thin films of stationary phase are easily destroyed or their polarity altered by overheating or by injecting "dirty samples that contain nonvolatile and/or particulate residues. However, many workers point out that it is easier and faster to break off the first few coils of the column and reinstall it rather than to do extensive sample cleanup. For best separation efficiency, good retention time reproducibility, quantitative accuracy and precision, and to... 

At high pH, the stability of the packing is severely affected by the dissolution of the bonded phase [10]. Typically, reversed-phase packing materials that are currently available are stable up to about pH 8.5. Silica is soluble at high pH, and this may cause a steady dissolution of the column packing. Therefore, retention time reproducibility will be affected by the steady degradation of the stationary phase. For acidic and basic components there may be a decrease in retention on a column that has been exposed to high pH. 

It should be noted that retention-time reproducibility is not necessarily the most important parameter of an assay, but that the selectivity of a separation is more important. The influence of retention time shifts on quantitative results can be prevented by using peak area integration instead of peak-height measurements. 

Table 5.1 Molecular interaction energy values for saccharides on graphitized carbon calculated using the MM2 program. MIPS, MIVW, MIHB and MIES represent the molecular interaction energy value of the final (optimized) structure, the van der Waals energy, the hydrogen-bonding energy, and the electrostatic energy (kcal mol ), respectively. tR represents the retention time. Reproduced by permission of Elsevier, ref. 24.

Limited commercial libraries Poor retention time reproducibility... 

IGC data recorded for hydrated alumina treated with an organosilane adhesion promoter when five probes are injected simultaneously to illustrate the effect of probe characteristics on retention time (Reproduced from Chehimi et al. 2001 with permission of The Royal Society of Chemistry)... 

---