HPLC separation modes

When using NP the chromatographer must remember to convert then-system to NP mode if RP mode was used previously. Any aqueous/buffer left in the system could precipitate out when the normal-phase solvents are pumped than the system. Water contamination in the mobile phase lines can also lead to water absorption on the column and change the chromatography significantly. It is generally recommended, that if the system was previously in RP mode, to flush the system with pure water for about 15 minutes at 2 mL/min. Then use IPA to flush the system for an additional 10 minutes at 2 mL/min. The system should then be flushed with the desired NP mobile phase for 5 minute at 2mL/min. Then the NP column can be installed and equilibrated with the NP mobile phase. 

Despite the popularity of RP chromatography, NP has its usefulness in the analysis of compounds during drug development. It can be used for polar 

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In the case of peptide separation by HPLC, separation modes are combined in series. This approach is called tandem LC. For instance, ion exchange associated with RP is used for peptide separation. Multidimensional protein identification technique (MudPIT) involving use of microcapillary columns (SCX cationic column and RP column) linked in series and eluted into MS is preferred for separation of complex peptide mixtures (Figure 5.4). 

In the past 5 years the frequency of reports on the use of HPLC technology for the determination of trace organic compounds in aqueous environmental samples has been steadily increasing. Many innovative approaches to sample cleanup and analyte isolation have been reported. Reversed-phase separation, with its many mobile-phase adaptations, has been and continues to be the most popular HPLC separation mode. The development of fast columns and microbore columns should provide optimal configurations for particular applications. The operating characteristics of microbore columns also make... 

The most common HPLC separation mode is based on separating by differences in compound polarity. A good model for this partition, familiar to most first-year chemistry students, is the separation that takes place in a separatory funnel using immiscible liquids such as water and hexane. The water (very polar) has an affinity for polar compounds. The lighter hexane (very nonpolar) separates from the water and rises to the top in the separating funnel as a distinct upper layer. If you now add a purple dye made up of two components, a polar red compound and a nonpolar blue compound, and stopper and shake up the contents of the funnel, a separation will be achieved (Fig. 1.2). 

Once a suitable HPLC separation mode has been selected, the experimental conditions should be adjusted to suit the objective of the separation. To proceed in this way, either empirical or systematic (statistical or predictive) HPLC method development strategies can be used. Any method development necessitates a convenient measure of the quality of separation. The separation of two sample compounds is most often measured either by resolution or by peak separation function (see Section 1.1.2). The resolution is an especially useful criterion of separation as its definition Eq. (1.3) can be transformed to another expression relating directly to the experimental conditions of separation v/A k... 

Once a suitable HPLC separation mode has been selected, experimental conditions can be adjusted using either an empirical method or a systematic method development approach. The separation of two sample compounds is conveniently characterized by resolution Rs. ... 

Besides the four major HPLC separation modes, several others often encountered in HPLC or related techniques are noted below. 

HPLC Separation Modes, Column-Packing Materials, and Solvents... 

Figure 5.4-5. Online MDLC using salt-plugs. This HPLC separation mode uses salt-plugs with increasing salt concentrations that are injected by the autosampler onto the lEX-column to elute stepwise stronger bound peptides.

Derivatized polysaccharide CSPs are operational, quite well, in all three HPLC separation modes as well as under super- or sub-critical fluid conditions [153, 160, 166, 170, 176-178]. A previous study based on a collection of more than 100 pharmaceutically important compounds with diverse structures clearly showed that polysaccharide CSPs generally had much higher success rate in resolving enantiomers under normal-phase and SFC conditions, followed by RP and polar organic modes (Fig. 17) [167]. This study also revealed that amylose tris(3,5-dimethylphenylcarbamate) AD phase was more effective than the other studied polysaccharide CSPs in polar organic mode and SFC, whereas cellulose tris(3,5-dimethylphenylcarbamate) phase is more applicable in reversed-phase mode. This observation is consistent with two other studies [159, 160]. 

A variety of pharmaceutical drugs or intermediates, such as -blockers, P-agonists, benzodiazepines, nonsteroidal anti-inflammatory drugs, and sulfoxides, can be enantioresolved on the same polysaccharide CSP in all HPLC separation modes and in SFC. A common structural feature of these compounds is that they all contain at least one aromatic group and multiple hydrogen-binding sites in... 

A general trend of enantioseparation among the three HPLC separation modes is that the highest enantioselectivity was commonly observed under normal-phase conditions (an example is shown in Fig. 18), while the shortest retention was obtained in polar organic mode. This is not an absolute rule since a great number of exceptions have been noticed. In the case of Nutlin-3, the enantioselectivity and resolution were much higher in reversed-phase mode than in polar organic and normal-phase mode (Fig. 16). Another example is Naproxen, whose enantiomers were baseline resolved on a Chiralpak AD-RH column in RP mode, while no enantioselectivity... 

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