Types of HPLC

The four main types of HPLC techniques are NP, RP, lEX, and SEC (Section 1.2). The principal characteristic defining the identity of each technique is the dominant type of molecular interactions employed. There are three basic types of molecular forces ionic forces, polar forces, and dispersive forces. Each specific technique capitalizes on each of these specific forces  

Polar forces are the dominant type of molecular interactions employed in normal-phase HPLC (see Chapter 5). 

Dispersive forces are employed in reversed-phase HPLC (see Chapter 4). 

The fourth type of HPLC technique, size-exclusion HPLC (see Chapter 6), is based on the absence of any specific analyte interactions with the stationary phase (no force employed in this technique). 

They represent four separate mechanisms for the interaction of sample molecules with the stationary phase. Size-exclusion chromatography is used for sample molecular weight over 2000, whereas the other types do not have this limit. Usually these other types are used for sample molecular weights below 2000. 

The differences between the types of chromatography lie basically, among others, in the different columns that are used. 

Liquid-Solid (Adsorption) Chromatography This type of chromatography is based on the competition between the molecules of the sample and the molecules of the mobile phase for adsorbent sites on the active adsorbent surface of the stationary phase. To alter adsorptive activity, two solvents are used in the mobile phase the principal solvent and the modifying solvent. In most cases, the principal solvent is hexane or dichloromethane and the modifying solvent is water, alcohol, or dimethyl sulfoxide. A modifying solvent is added to the principal solvent to control the absorptive activity of the samples. 

Two kinds of adsorbents are used for column packings polar adsorbents and nonpolar adsorbents. The acidic polar adsorbents, such as silica, are used for aliphatic nitrocompounds or aromatic amines. The basic adsorbents, such as alumina, are used for pyrrole derivatives, phenols, and carboxylic acids. The nonpolar adsorbents, such as charcoal, are used for high-molecular-weight homologs and aromatic compounds. 

Liquid-Liquid (Partition) Chromatography If a third component is dissolved in the immiscible layers of the solvents, there is a distribution of the third component between the two layers. Liquid-liquid (partition) chromatography is based on a multistage distribution of a sample between two solvents within a column. The mobile phase is a liquid. The stationary phase is also a liquid (another liquid), which may be dispersed onto a finely divided inert support. The separation is attributed to the different distribution of the sample compounds between the two liquid phases. There are two kinds of partition chromatography normal systems in which the mobile phase is less polar than the stationary phase and reversed-phase systems in which the mobile phase is the more polar hquid. Table 13.1 compares the two different types of partition chromatography. 

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Hplc techniques are used to routinely separate and quantify less volatile compounds. The hplc columns used to affect this separation are selected based on the constituents of interest. They are typically reverse phase or anion exchange in nature. The constituents routinely assayed in this type of analysis are those high in molecular weight or low in volatility. Specific compounds of interest include wood sugars, vanillin, and tannin complexes. The most common types of hplc detectors employed in the analysis of distilled spirits are the refractive index detector and the ultraviolet detector. Additionally, the recent introduction of the photodiode array detector is making a significant impact in the analysis of distilled spirits. 

A number of different types of HPLC detector have been discussed in the previous chapter. In comparison to these, a mass spectrometer is a relatively expensive detector and there need to be considerable advantages associated with its use to make the significant financial investment worthwhile. What are these advantages In order to answer this question, we must first consider what it is we are trying to achieve when using chromatography ... 

It is not unexpected that the choice of the type of HPLC column, the temperature at which it is maintained and the choice of buffer added to the mobile phase are likely to have an effect on the separation obtained, even for a simple mixture as in this case. 

Figure 4.11 Schematic classification of different types of HPLC detectors...

The operating principles of three types of hplc pump are described, together with their advantages and limitations. Techniques for the production of solvent gradients and for the introduction of samples are considered. 

TABLE 13.1 Summary of Applications of the Different Types of HPLC... 

What type of HPLC should be chosen for each of the following separation applications ... 

Name two types of HPLC columns that might be used to separate nucleotides. 

The HPLC method as originally described (5 t ) lacked adequate sensitivity for several of the PSP toxins and to obtain a complete toxin profile, duplicate injections on two different types of HPLC columns were necessary. To solve these problems, modifications were made that included developing the PSP toxin separations on a different column and the utilization of a more efficient reaction/detection system. Incorporation of these changes enables complete separation of all the toxins with the exception of Cl and C2 which still co-elute (Figure ). In addition, the improved HPLC... 

Each type of HPLC instrument has its own characteristics and operating directions. It is not feasible to describe those here. However, it is appropriate to outline the general approach taken when an HPLC analysis is desired. The following items must be considered ... 

MIPs have been frequently used as stationary phases in HPLC. It is useful to distinguish two types of HPLC experiments with MIPs ... 

There are many types of HPLC detectors available today with the most popular ones including UV and UV-photodiode array (PDA), fluorescence, refractive index, evaporative light scattering (ELSD), charged aerosol (CAD), and the mass spectrometer. Of these, the most commonly used detector for pharmaceutical analytical methods is the UV detector since a majority of pharmaceutical compounds have some type of chromophore. Multiple detectors in series can also be utilized in order to obtain more information per chromatographic run. For example, a PDA detector can... 

The sample preparation of endohedral metallofullerenes was done by Shino-hara and details are described in the review article [16]. The soot containing M C2 (M=Sc and La) was produced in direct-current (300-400 A) spark mode under He flow at 50 torr and collected under totally anaerobic conditions. The target fullerenes were separated and isolated from the various hollow fullerenes (C60-C110) and other metallofullerenes by the two-stage high-performance liquid chromatography (HPLC) method by using two complementary types of HPLC columns. The purity of the metallofullerenes used for structure analysis relative to other fullerenes was always more than 99.9%. 

Artemisinin was produced by some A. annua cultures, as were three other electrochemically reducible compounds, based on the type of HPLC detector used for analysis of the peroxide-bridged, functionally active materials in the... 

The first type of HPLC column investigated was a polymeric octadecylsilane column. A number of different mobile phase systems were examined as shown in Table VI For these isocratic studies a flow rate of 40 ml/hr and 100 ng of VI in mobile phase as the analyte was used. 

Derivatization of solute molecules can be utilized to modify properties of analytes of interest such that they may be more readily identified by a specific type of HPLC detector. 

The amino acid composition of peptides is generally assayed by carrying out acid hydrolysis with 6 M hydrochloric acid, followed by determination of the individual amino acids by HPLC. Various types of HPLC have been employed to separate amino acids, but reversed-phase chromatography on Cigcoiumns is the most commonly system used. Detection of amino acids normally involves derivatization, since their maximum absorbance is at 214 nm in which many other compounds also can absorb. The most frequently used derivatizing agents are dansyl chloride, phenyl isothiocyanate and o-phthaldialdehyde. 

Based on the major goal of preformulation—identification of possible failure in future development—numerous studies are performed to fully characterize prospective drug candidates. The major analytical technique in each preformulation group is liquid chromatography. Ninety percent of all analytical equipment in preformulation groups are HPLC systems equipped with UV and MS detection systems. HPLC is a fast and reliable method for concentration and identity determination by UV and/or MS detection, respectively. The type of HPLC methods differ based on the specific preformulation tests that will be described below. 

Fig. 12.18. The nonlinear map of the normalised regression eoellicienls of Ihc general solvalion equation. Eq. (12.14), obtained for several reversed-phase types of HPLC systems, (a) Nonlinear map of all columns, (b) Nonlinear map of only the C-18 phases. (From Ref. 64 )...

There are several different types of HPLC pump available. However, these all fall into two main classes those which deliver mobile phase at a constant pressure, and those that pump at constant flow rates. In the vast majority (almost 90%) of current analytical HPLC work, it is the latter type of pump that is used. However, both types of pump have their place in modern chromatography, as discussed in the following sections. 

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