Different modes of HPLC

In RP-HPLC, the stationary phase is less polar than the mobile phase and is usually comprised of spherical silica particles (typically, 3-5 pm in diameter). The acidic functionalities on the silica material have been modified by deriv-atisation with alkyl (C2 to C18), phenyl, cyano and amino groups. Typical mobile phases used in RP-HPLC consist of mixtures of aqueous buffers mixed with water-miscible organic solvents, such as methanol and acetonitrile. In addition to modified silica stationary phases, other new developments in RP-HPLC are now available, e.g. porous polymeric, carbon and mixed modal phases. 

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Fig. 3.1e. Use of different modes of hplc. Source Analytical Chemistry Reviews, April 1984...

Figure 31.1. Elution behavior of macromolecules in the different modes of HPLC of polymers.

Over the past few years, there has been considerable interest expressed in materials that can serve as alternatives to silica as the basis of supports in different mode of HPLC. Zirconia is chemically and mechanically stable relative to silica and polymeric phases. It is resistant to chemical degradation from pH 1 to pH 14. The material s thermal stability is excellent over any liquid chromatographically accessible temperature, leading to increased flexibility when designing separation. ... 

Individual stationary supports for different modes of HPLC have been described in Table 11.16. 

Except for the high molecular weight range, nearly all substances can be separated by reversed-phase (RP) HPLC. The many different separation mechanisms in RP HPLC, based on hydi ophobic, hydi ophilic and ion-pairing interactions, and size exclusion effects together with the availability of a lai ge number of high quality stationary phases, explain its great populai ity. At present approximately 90% of all HPLC separations are carried out by reversed-phase mode of HPLC, and an estimated 800 different stationai y phases for RP HPLC are manufactured worldwide. 

Sample Injection System There are in all three different modes of sample injection system that are used in HPLC, namely ... 

The process for introducing the HPLC-separated analyte peaks into the NMR probe can be configured using two different modes of operation on-flow (continuous flow) or stopped-flow. 

Simplex Optimization. The sequential simplex method is an example of a sequential multivariate optimization procedure that uses a geometrical figure called a simplex to move through a user-specified of experimental conditions in search of the optimum. Various forms of the simplex have been successfully used in different modes of chromatography, particularly HPLC (40-42) and GC (43-46). 

HPLC techniques have occupied a dominant position for over two decades in peptide and protein chemistry, in molecular chemistry, and in biotechnology. These techniques with their various selectivity modes (listed later) can be considered the bridges that link cellular and molecular biology (viz., structural proteomics and atomic biology) and industrial process development associated with the recovery and purification technologies that turn these opportunities into realities. Different dominant interactive modes of HPLC are as follows ... 

From the view point of the assessment, the quality of an HPLC separation in response to changes in different system variables, such as the stationary phase particle diameter, the column configuration, the flow rate, or mobile phase composition, or alternatively, changes in a solute variable such as the molecular size, net charge, charge anisotropy, or hydrophobic cluster distribution of a protein, can be based on evaluation of the system peak capacity (PC) in the analytical modes of HPLC separations and the system productivity (Peff) parameters in terms of bioactive mass recovered throughput per unit time at a specified purity level and operational cost structure. The system peak capacity PC depends on the relative selectivity and the bandwidth, and can be defined as... 

Like gas chromatography (GC), HPLC employs a chromatographic column for the separation. It differs from GC in that the sample components need not be volatile and stable at elevated temperatures, they must only be soluble in a suitable single-component or mixed solvent. Various modes of HPLC can be applied to the analysis of a large variety of sample types containing non-polar, moderately or strongly polar and ionic compounds, either simple species or high-molecular mass synthetic polymers or biopolymers. These features of HPLC are especially useful in pharmaceutical and clinical analysis. 

Figure 7. Examples of different modes of chromatography (a) calibration curves (schematic), (b) SEC separation of oligomeric polyesters with different molar mass, (c) critical chromatogram (LACCC) of polyester diole terminated sample is main compound, and (d) gradient HPLC run of the same polyester sample showing multiple distributions.

One of the main reasons for the widespread use of silica as an HPLC packing material is that it may be used either unmodified or after chemical derivatisation of the silanol groups, allowing the surface chemistry to be altered to suit different modes of chromatography. 

There are six modes of HPLC currently in use for secondary metabolite analysis, namely, HP-RPC, HP-NPC, HP-IEX, HP-HILIC, HP-SEC, and HP-AC. The principles of these different modes are explained below. All of these various chromatographic modes can be operated under isocratic (i.e., fixed eluent composition), step gradient, or gradient elution conditions (variable step or continuous changes in eluent composition), except for SEC, which is usually performed under isocratic conditions. All modes can be used in analytical, semipreparative,8 or preparative9-14 situations. 

HPLC provides reliable quantitative precision and accuracy, along with a linear dynamic range (LDR) sufficient to allow for the determination of the API and related substances in the same run using a variety of detectors, and can be performed on fully automated instrumentation. HPLC provides excellent reproducibility and is applicable to a wide array of compound types by judicious choice of HPLC column chemistry. Major modes of HPLC include reversed phase and normal phase for the analysis of small (<2000 Da) organic molecules, ion chromatography for the analysis of ions, size exclusion chromatography for the separation of polymers, and chiral HPLC for the determination of enantiomeric purity. Numerous chemically different columns are available within each broad classification, to further aid method development. 

In normal-phase HPLC, solute retention is based on the distribution of solute between a polar stationary phase and a nonpolar mobile phase (typically a mixture of hexane and a more polar solvent such as isopropanol). Elution may be promoted by increasing the amount of polar solvent in the mobile phase. In reversed-phase HPLC, retention is based on distribution between a nonpolar stationary phase and a polar mobile phase (typically a mixture of water and acetonitrile or methanol), and elution is promoted by addition of the less polar solvent to the mobile phase. With the exception of extremely polar or ionized compounds, which are not amenable to normal-phase HPLC, and extremely nonpolar compounds such as certain steroids and natural products, which are not amenable to reversed-phase HPLC, both modes of HPLC are potentially applicable to APIs and related substances. However, about 75% of current HPLC analyses are performed using the reversed-phase.This is due not only to safety considerations using nonpolar solvents but also to the differences in sample preparation procedures required for normal-phase versus reversed-phase HPLC. 

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