HPLC method development solvent selection

Snyder L.R., Dolan J.W., Molnar I., and Djordjevic, N.M., Selectivity control in reversed-phase HPLC methods development varying temperature and solvent strength to optimize separations, LC-GC, 15 (2), 136, 1997. 

The selection of starting conditions is the first step in HPLC method development. Instead of starting with arbitrary conditions or conditions derived from the chromatographer s experience or intuition, the software can predict suitable initial conditions for reversed-phase methods from analyte structures and properties of the sorbent/solvent system [1-3]. If structures are known, the theoretical approach has the potential to save time and effort, since in this way the experimental method development process will start under the theoretically predicted optimum conditions. 

The software has been equipped with a fimction such that it is not only able to conduct experiments with one column/organic modifier/buffer combination, but to automatically optimize a method by trying different column/organic modifier/ buffer combinations. The system provides imattended HPLC method development and performs autonomous development and optimization of isocratic and gradient methods for selection of the best variant column, pH value, solvent Two typical hardware configurations and other mixed combinations are supported by ChromSword standard and powerful. 

In the development of a SE-HPLC method the variables that may be manipulated and optimized are the column (matrix type, particle and pore size, and physical dimension), buffer system (type and ionic strength), pH, and solubility additives (e.g., organic solvents, detergents). Once a column and mobile phase system have been selected the system parameters of protein load (amount of material and volume) and flow rate should also be optimized. A beneficial approach to the development of a SE-HPLC method is to optimize the multiple variables by the use of statistical experimental design. Also, information about the physical and chemical properties such as pH or ionic strength, solubility, and especially conditions that promote aggregation can be applied to the development of a SE-HPLC assay. Typical problems encountered during the development of a SE-HPLC assay are protein insolubility and column stationary phase... 

Immunoaffinity procedures have also been developed to selectively extract corticosteroids from different sample matrices. Thus, Seymour et al. demonstrated the higher efficiency of the immunoaffinity methods compared with the conventional extraction procedures using organic solvents [177]. Immunosorbents have also been used for online procedures followed by HLPC-UV [178, 179], HPLC-APCI-MS [179,180], GC-MS [176,181], or capillary electrophoresis [182]. Poly(hydroxyethyl methacrylate) (HEMA) was evaluated as a support material for the anti-dexamethasone antibodies used in IAC. The online IAC-HPLC-MS allowed determination of dexamethasone and flumethasone in equine urine with LODs in the range 3-4 ng mL-1 [180]. The cross-reactivity values obtained in the ELISA and the recoveries of an IAC-HPLC procedure are presented in Table 7. Bagnati et al. developed an immunoaffinity extraction... 

A simple and rapid RP-HPLC method was developed for the determination of retinoid in galenicals. Commercial preparations were diluted, filered and used for separation. Measurements were carried out in an ODS column (150 X 4.6 mm i.d. particle size 3 /xm). Solvents A and B were methanol-10 mM ammonium acetate (75 25, v/v) and methanol-THF (84 16, v/v), respectively. The flow rate was 0.8ml/min. Gradient conditions were 0-25 min, 0 per cent B 35 min, 100 per cent B, isocratic for 10 min. Typical chromatograms are shown in Fig. 2.37. The repeatability of peak area ranged between 0.48 -3.2 per cent for UV-DAD and 0.57 - 3.1 per cent for fluorescence detection. The reproducibility varied between 0.26 - 4.6 per cent. It was found that the method is precise, selective, sensitive and linear, therefore, it can be employed for the routine quality control of this class of drags [85],... 

To apply a screening approach to proactive method development, analyses of selectivity samples under a variety of mobile phase conditions are conducted on different HPLC columns. HPLC columns should be as orthogonaT as possible and variations in solvent composition should be designed to maximize the probability of selectivity differences. Alternate separation techniques, such as ion exchange chromatography (IC), supercritical fluid chromatography (SFC), or capillary electrophoresis (CE) may also be used to obtain orthogonality. 

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.

More recently, a highly selective, sensitive, and rapid HPLC method has been developed and validated to quantify tadalafil in human plasma [44]. The tadalafil and internal standard (loratadine, I.S.) were extracted by liquid-liquid extraction technique followed by an aqueous back-extraction allowing injection of an aqueous solvent in the HPLC system. The chromatographic separation was performed on a reverse phase BDS Hypersil C18 column (250 mm x 4.6 mm, 5 pm. Thermo Separation Co., USA) with a mobile phase of acetonitrile and aqueous solution containing 0.012 M... 

HPLC separation of polymers requires the removal of polymer prior to the analysis, low molecular weight SEC can operate without such need, as the polymer will Just be exeluded from the system. SEC presents a quick and easy method development strategy the process involves the selection of a suitable solvent and the use of any small pore size eolumns 50 A and 100 A. All eomponents will elute within a predetermined interval, amounting to one eolumn volume, and the elution volume of any material ean be predicted for a given eolumn provided a ealibration curve is available. 

Since the functionality of most silica stationary phases is the same, the selection of the mobile phase is very important during the screening and method development of a normal-phase silica separation. As mentioned above, typical mobile phases involve a nonpolar solvent like heptane and a polar solvent like ethanol, but the combination of solvents is almost endless. A good screening (using TLC or analytical HPLC) should examine numerous solvent combinations utilizing different types of solvents, such as THF instead of alcohols, ethyl acetate or methylene chloride instead of heptane and should also examine the use of additives, e.g., acetic acid or triethylamine, to adjust the pH to either suppress or enhance interactions with the stationary phase. More information on the method development is provided in Section V. 

Whereas the other separation methods have been demonstrated to also provide the requisite performance for release and stability testing for select drug substances and drug products, more typically the techniques are applied as supportive methods for HPLC during early-phase development and in niche areas during late-phase development. Because each separation method provides a different mechanism of separation to HPLC, utilization in early-phase development can be used to confirm specificity of HPLC methods. In later phases, both SFC and CE have shown applicability to chiral separations, and GC remains as the unique technique for the determination of residual solvents. 

The first concern in the selection of the sample preparation solvent is to optimize recovery. However, a secondary consideration is the sample solvent s effect on the analysis. This is true whether the analytical technique is ultraviolet spectroscopy (UV), high-performance liquid chromatography (HPLC), or gas chromatography (GC). The method development sequence can be described as (a) development of the chromatographic separation, (b) development of the sample preparation method, and then (c) evaluation and optimization of the interaction of the sample preparation with the instrumental method. 

Although not a requisite for the selection of SFC as the purification technique of choice, highly lipophilic samples are preferable so as to avoid solubility problems often encountered in reverse-phase HPLC processing. It should be noted, however, that highly polar materials can be purified by SFC so long as materials can be dissolved at a level of 50m mL of methanol, along with co-solvents such as acetonitrile or dichloromethane. Further, when a synthetic chemist specifically requests that samples be returned in their non-TFA salt form, SFC is selected as it can be frequently developed in methanol/carbon dioxide or, alternatively, volatile small amine modifiers such as triethylamine. Finally, it is generally possible to achieve better resolution with an SFC rather than the HPLC method of our present setup, so in instances where a difficult separation of desired product is anticipated based on LC/MS analysis, SFC may be preferentially chosen over HPLC. 

When one develops new reversed-phase (RP)-HPLC methods, one usually uses the selectivity of the mobile phase as the primary method development tool. The chromatographic separation can be influenced by the choice of the organic solvent (mainly methanol and acetonitrile), or by variation of pH or buffer type. Schemes for method development using these parameters have been described in the literature [1,2]. Most important are the selectivity changes caused by pH changes, which are well-understood and easily predictable (3). It is well known that the stationary phase influences the selectivity as well, but this effect is often not very well understood. The primary reason for this is the fact that reliable methods for the description of the stationary phase selectivity have only become available fairly recently. In the last few years, several papers have been published that deal with the subject of selectivity in a fimdamental way [4—9] or represent a data collection based on older methods [10-15]. In this chapter, we describe in detail the method used in our laboratory. We then look at our selectivity charts and discuss our results. It needs to be pointed out in advance that selectivity charts only accurately represent the properties of a stationary phase under the conditions of the measurement. If we depart from the mobile phase composition of the test, the relationships between different columns will change, since selectivity arises from a combined effect of the mobile phase and the stationary phase. 

Method development in SFC is significantly easier, since only one mobile phase (CO2) with a few modifiers (methanol, ethanol, isopropanol, acetonitrile) instead of a multitude of solvent mixtures has to be tested. If the first experiments do not show at least a partial resolution, it is recommended that one should switch to another CSP. To optimize SFC separations, the amount of the modifier can be varied between 5 and 25%, as well as varying its nature. As in HPLC, acidic and basic modifiers can be used. Pressure changes have a greater influence on retention than on selectivity. 

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