Isocratic methods

FIGURE 11.32 Densitogram illustrating various techniques of development of Heracleum sphondylium fruit extract on silica with AcOEt + H (1 9) (a) by isocratic method, (b) incremental multiple development with 10-mm steps. For abbreviations, see Figure 11.1. (For details, see Wawrzynowicz, T., Czapinska, K., and Markowski, W., J. Planar Chromatogr., 11, 388-393, 1998.)... 

Du, C. M., Valko, K., Bevan, C., Reynolds, D., Abraham, M. H. Rapid gradient RP-HPLC method for lipophilicity determination a solvation equation based comparison with isocratic methods. Anal. Chem. 1998, 70, 4228-4234. 

For each compound the log k values were obtained by isocratic methods at three different methanol water ratios and the logkw value was derived by extrapolation to 100% water. 

Some LC/MS users adhere to isocratic separation because of the myths around gradient elution (it is complex to develop and transfer between instruments and laboratories, it is inherently slower than isocratic methods because of re-equilibration, and other reasons summarized by Carr and Schelling6). A researcher may have a very good reason to use an isocratic method, for example, for a well defined mixture containing only a few compounds. The isocratic method would certainly not be useful in an open access LC/MS system processing varying samples from injection to injection. 

Using initial scouting gradient experiments for efficient development of final gradient or isocratic methods... 

Isocratic method. Whenever possible, use isocratic HPLC condition, as this is affected less by the variation in flow rate, temperature, and dwell volume. If gradient HPLC conditions have to be used, a simple linear gradient is preferred over multistep gradients. Complicated gradient conditions are more susceptible to differences between HPLC instruments (e.g., flow rate, dwell volume). 

Figure 25-28 Isocratic method development for HPLC, using solvent composition, % B. and temperature, T, as independent variables. % B and T are each varied between selected low and high values. From the appearance of chromatograms resulting from conditions A-D, we can select intermediate conditions to improve the separation.

SU Rehman. Rapid isocratic method for the separation and quantification of major phospholipid classes by high performance liquid chromatography. J Chromatogr 567 29-37, 1991. 

A review of HPLC methods for antiepileptic drug analysis was published in 1987 by Juergens.18 Standard analytical separations were compared with narrow-bore separations, and a 70% reduction in the cost of solvents was possible, owing to a reduction in flow rate from 1 ml/min for the analytical column to 0.3 ml/min for the narrow-bore column. Gayden et al.19 developed an isocratic method, using narrow-bore columns, to quantify adenosine (Ado) release by dispersed rat renal outer medullary cells under conditions of normoxia and hypoxia. Standard HPLC with UV detection has been the predominant method for studying the metabolic pathways of adenosine and measuring the Ado breakdown products inosine (Ino) and hypoxanthine (Hyp). However, the conventional methods lack reliability... 

Lombardo et al. use also different octanol concentration in their isocratic method. They use an LC-ABZ column (Pagliara). This method is capable to cover a wide lipophilicity range with good correlation with log Pow-... 

The product quality was analysed using silver-ion-HPLC and gradient elution [14]. This method was developed from an isocratic method [15]. The kind (cis/ trans) and the amount of the FAME was determined. From these data the iodine value (IV) was calculated. 

For development of a weight percent assay, a short isocratic method can be implemented based on observations from the gradient method used for the impurity profile. One can use a shorter column such as a 5-cm column and keep retention of the solute of interest to around a capacity factor of 3 as long as it is still resolved from impurities observed in the impurity prohle. Additionally, the elution of more hydrophobic species should not co-elute with the drug substance in later injections. During the method development of an isocratic method, the compound should be injected and then a suitable number... 

A similar approach using an isocratic method can be applied to in-process monitoring, where the goal is to monitor the disappearance of the starting material and appearance of the product. In-process methods will be discussed in greater detail in Section 14.6. 

Figure 7.5 Optimisation of a separation of degradation products produced in a stability trial. Originally the separation of the active agent and a number of its impurities and degradation products was achieved using a complex ternary gradient over 30 min. Using Diamond, an isocratic method was developed taking only 20 min to run. This reduced the analysis time and removed the need for re-equilibration between runs. The method also had better resolution, as one of the major degradation products was completely resolved from the major component.

Another group of methods in analytical affinity chromatography are those that examine the kinetics of biological interactions. Band-broadening measurements (also known as the isocratic method) represent one such approach. This is really a modification of the zonal elution method in which the widths of the eluting peaks are measured along with their retention times. 

Gradient elution is ideal for separating certain kinds of sample which cannot be easily handled by isocratic methods, because of their wide k range. Nevertheless, there is a strong bias against the use of gradient elution in many laboratories. 

FIGURE 14 RP-HPLC-UV chromatogram (using an isocratic method) of an aqueous solution of drug substance A after stressing at 70°C for 14 days at pH 7. 

---