System suitability relative retention

System suitability specifications and tests, Capacity factor (k ), Preci-sion/injector repeatability (RSD), Relative retention (a), Resolution (Rs),Tailing factor (T),Theoretical plate number (N)... 

Chromatographic System The HPLC system is equipped with a 240-nm detector and a 4.6-mm 25-cm column that contains packing LI (octadecyl silane bound to porous silica particles). The flow rate is about 0.8 mL/min. System Suitability Chromatograph the Standard Preparation, and record the peak responses as directed in the Procedure section. The relative retention times are about 0.8 for benzoylformic acid, 1.0 for mandelic acid, 2.5 for benzoic acid, 2.8 for benzaldehyde, and 3.7 for acetophenone. The tailing factor for each peak is not more than 2.0. The resolution between the benzoylformic acid peak and the mandelic acid peak, and between the benzoic acid peak and the benzaldehyde peak, is not less than 3.0. The relative standard deviation for replicate injections is not more than 1% for the mandelic acid peak. 

Chromatographic System (See Chromatography, Appendix IIA.) Use a liquid chromatograph equipped with a refractive index detector that can be maintained at a constant temperature of 25°, a 25-cm x 4.6-mm (id) column packed with 10- im porous silica gel bonded with aminopropylsilane (Alltech 35643, or equivalent), and a guard column that contains the same packing. Maintain the column at a constant temperature of 25° 2°, and the flow rate at about 2.0 mL/min. Inject 20 pL of System Suitability Preparation into the chromatograph, and record the peak responses as directed under Procedure. The relative standard deviation for replicate injections is not more than 2.0%, and the alpha-Cyclodextrin and beta-Cyclodextrin peaks exhibit baseline separation, the relative retention times being about 0.8 and 1.0, respectively. 

System Suitability Test Obtain chromatograms of duplicate 20-p.L injections of the Standard Preparation. Ensure that the retention time of Sucralose is approximately 9 min. It may be necessary to adjust the Mobile Phase composition to obtain the desired retention time. Ensure that the relative... 

System Suitability Chromatograph a suitable number of injections of the Assay Preparation, as directed under Calibration, to ensure that the resolution factor, R, between the major peaks occurring at retention times of approximately 0.50 (8-tocopheryl propionate) and 0.63 ((3- plus y-tocopheryl propionates), relative to hexadecyl hexadecanoate at 1.00, is not less than 2.5 (see System Suitability in High-Performance Liquid Chromatography under Chromatography, Appendix IIA). 

System Suitability Test Chromatograph five injections of the System Suitability Preparation, and measure the peak responses as directed under Procedure (below). The relative standard deviation for the peak response does not exceed 2.0%, and the resolution between mms-cholecalciferol and pre-cholecalciferol is not less than 1.0 (see System Suitability under Chromatography, Appendix IIA). The chromatograms obtained in this test exhibit relative retention times of approximately 0.4, 0.5, and 1.0, for pre-cholecalciferol, trans-cholecalciferol, and cholecalciferol, respectively. 

It has been shown that gas-Hquid chromatographic methods are particularly suitable for a quantitative characterization of the polarity of solvents. In gas-liquid chromatography it is possible to determine the solvent power of the stationary liquid phase very accurately for a large number of substances [98, 99, 259, 260]. Many groups of substances exhibit a certain dependence of their relative retention parameters on the solvation characteristics of the stationary phase or of the separable components. In determining universal gas-chromatographic characteristics, the so-called retention index, I, introduced by Kovats [100], is frequently used. The elution maxima of individual members of the homologous series of n-alkanes (C H2 +2) form the fixed points of the system of retention indices. The retention index is defined by means of Eq. (7-41),... 

Prior to performing a formal validation, the analytical chemist should have performed some prevalidation during method development. The expectation is that a well-developed HPLC method should subsequently be validated with no major surprises or failures. Prior to validation, specificity and some degree of robustness should be demonstrated. In addition, some form of system suitability criteria will have been established. System suitability evaluates the capability of an HPLC system to perform a specific procedure on a given day. It is a quality check to ensure that the system functions as expected and that the generated data will be reliable. Only if the system passes this test should the analyst proceed to perform the specific analysis. System suitability can be based on resolution of two specified components, relative standard deviation, tailing factor, limit of quantitation or detection, expected retention times, number of theoretical plates, or a reference check. 

The responses of main interest are different during both applications. In optimization, responses related to the separation of peaks (Section 6.2) are modelled. In robustness testing the quantitative aspect (the content determination) of the method is of most interest, since it is the one that should remain unaffected by small variations in the variables. Responses related to the separation (resolution, relative retention) or describing the general quality of the chromatogram (capacity factors, analysis times, asymmetry factors, and column efficacy) are often also studied. As recommended by the ICH guidelines the results of a robustness test can be used to define system suitability test limits for some of the responses [82]. 

A comprehensive semi-empirical description of reversed-phase HPLC systems, for predicting the relative retention and selectivity within a series of analytes, has been developed by Jandera and co-workers [72,73]. The approach consists of determining the interaction indices and the structural lipophilic and polar indices. A suitable set of standard reference analytes is necessaiy to calibrate the retention (or selectivity) scale. 

Relative retention time fRJ. The main practical disadvantage of using k to describe retention arises from the difficulties associated with the accurate measurement of The accurate measurement of t, requires correct choice of a reference compound that is neither retained by the column nor excluded from its pores. Problems with the accurate and reproducible measurement of k have led to the use of a retained compound for system suitability tests for liquid chromatography and related techniques when it has been checked that the column, mobile phase and equipment are performing identically to when the method being used was first developed. The relative retention time of a solute j may be defined by... 

System suitability is guaranteed if both apparatus test and validation match their requirements. It is best performed on a routine basis and can be done very easily if the HPLC apparatus is equipped with a computer data system. Then during each run or in well-defined intervals a number of parameters are acquired plate number, resolution, precision, retention time, relative retention time (i.e. k value) and peak asymmetry-also if necessary linearity and limits of quantification. The results are followed by statistical tools, including easy-to-monitor graphical documentation with control charts. ... 

Chromatograph this system suitabUity solution, the standard solutions, and dissolution test samples by separately injecting equal volumes (about 50 p.1) of the solutions into the chromatograph, record the chromatograms, and measure the areas for the major peaks. For the system suitability solution, the relative retention times are about 0.7 for isonicotinic acid, 1.0 for pyrazinamide, and 1.8 for isoniazid and the resolution, R, between isonicotinic acid... 

One of the first non-formaldehyde fluorescent dye carrier systems for polyolefins was based on the reaction of polyfunctional amines with polyfunctional carboxylic acids to form relatively short chain polyamides [5].These linear thermoplastic resins showed good solubility and friability making them suitable for the incorporation of dyes, which offered increased protection from thermal and UV degradation. This fluorescent resin showed a dramatic increase in color retention upwards of 288°C, even after a 10 minute hold period at this temperature. Due to its non-ide-alized polymeric nature, the polyamide chemistry suffered from preferential plating out or migration of polar oligomeric species not incorporated into the polymer chains. 

The utility of HDC is somewhat limited by the relatively poor resolution and particle-size discrimination of the method, which restrict the precision of HDC in silica sol characterization. In principle, accurate particle-size distributions of silica sols also are possible with the HDC method. However, for such characterizations special software with corrections for the extensive band dispersion in HDC is required, along with a suitable band deconvolution method (28). Commercial HDC apparatus with this sophisticated software package apparently is no longer available. Standards are generally required, although quantitative retention relationships have been reported for capillary HDC systems in characterizing polymers (37). As with all of the other separation methods, careful selection of the mobile phase is required in HDC. Mobile phases generally are the same as those used for the FFF methods and SEC. 

A second aspect of analytical methodology that concerns me is the lack of suitable standards. In my laboratory and certainly in many other laboratories the application of HPLC, glass capillary GC, and glass capillary GC-mass spectrometry-computer systems allows us to separate relatively easily hundreds of individual aromatic compounds, e.g., 9 to 12 isomers of C-3 phenanthrenes. However, there are no commercial sources for standards to verify our identifications or calibrate the quantification of these compounds. Synthesis of all isomers is clearly a monumental task. In the interim, perhaps the analytical chemists interested in this problem should be encouraged to develop systematic rules for interpreting glass capillary GC and HPLC retention indices, subtle mass spectral differences, and UV-fluorescence spectra. 

The suitability of a stationary phase for a particular application depends on the selectivity and the degree to which polar compounds are retarded relative to what their retardation would be on a completely non-polar stationary phase. Since retention time is a function of temperature, flow-rate, stationary phase type and loading or film thickness it cannot be used to relate the retention characteristics of one column to another. Various retention index methods have been described such as evaluating the partition and separation properties of solute-stationary phase systems. Kovats (1958) devised a system of indexing chromatographic retention properties of a stationary phase with respect to the retention characteristics of n-alkanes, alkanes being used as reference materials since they are non-polar, chemically inert and soluble in most common stationary phases [8-10]. The retention index (RI) for the n-alkanes is defined as... 

The solvent delivery system is responsible for delivering the pressurized mobile phase with the desired composition and chosen flow rate to the head of the column. To achieve this goal a number of components work together under the supervision of the system computer or microprocessors to achieve the tight specifications typical of a modem liquid chromatograph. Table 5.2 [15-19]. Suitable tests for performance evaluation of solvent delivery systems are briefly described at the end of section 5.2.2. In retention terms a relative standard deviation of better than 0.15% for retention factors under normal operating conditions is expected. 

A study on alkaloid production by nongrowing (owing to the absence of 2,4-D) cell suspension cultures of C. roseus in a continuous flow reactor with cell retention was performed by Pareilleux and Vinas (626). It was shown by the authors that part of the produced alkaloids were excreted into the culture medium. Although the continuous flow system seemed to be suitable for secondary metabolite production, the reported specific production rate was relatively low (0.012 mg ajmalicine/g DW/day). To obtain ajmalicine production 1.2 mM tryptamine had to be added to the infiuent medium. The duration of the continuous flow experiment was only... 

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