Modes of HPLC

HPLC suffers from several well-known disadvantages or perceived limitations. First, there is no universal detector, such as the equivalence of flame ionization detector in GC, so detection is more problematic if the analyte does not absorb UV hays or cannot be easily ionized for mass spectrometric detection. Second, separation efficiency is substantially less than that of capillary GC, thus, the analysis of complex mixtures is more difficult. Finally, FIPLC has many operating parameters and is more difficult for a novice. As shown in later chapters, these limitations have been largely minimized through instrumental and column developments. 

In this section, the four major separation modes of HPLC are introduced and illustrated with application examples, each labeled with the pertinent parameters column (stationary phase), mobile phase, flow rate, detector, and sample information. These terminologies will be elaborated later. 

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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. 

Also for analysis of some pharmaceutical substances a normal-phase mode of HPLC and a lot of organic solvents are needed, especially if it is used in routine analysis. 

With notable exceptions, the application of HPLC to clinical chemistry has not as yet been extensive. This is somewhat surprising in view of the potential the method has for this area. This potential arises, in part, from the fact that HPLC is well suited to the types of substances that must be analyzed in the biomedical field. Ionic, relatively polar species can be directly chromatographed, without the need to make volatile derivatives as in gas chromatography. Typically, columns are operated at room temperature so that thermally labile substances can be separated. Finally, certain modes of HPLC allow fractionation of high molecular weight species, such as biopolymers. 

Instrumentation requirements for SEC are somewhat simpler than those of other modes of HPLC, since mobile phase gradients are not used however, adequate computer support for data acquisition and processing is essential. Method development involves finding a suitable solvent for the sample and choosing a mixed bed column or, more often, a set of columns in series to match the pore size of the column(s) with the size distribution of the sample. 

The experiments below use reverse phase chromatography with bonded silica columns and uv absorbance detection. If more extensive experimental facilities are available, some additional experiments are suggested. These are concerned with the preparation and evaluation of columns, and with the use of other detectors and modes of hplc. It should be possible to complete each experiment within a three hour practical period. 

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

Which mode of hplc would you choose lor each ol the following ... 

The use of HPLC in all its forms is growing steadily and may eventually exceed that of GC. This is because all four sorption mechanisms can be exploited and the technique is well suited to a very wide range of compound types including ionic, polymeric and labile materials. The most appropriate choice of mode of HPLC for a given separation problem is based on the relative molecular mass, solubility characteristics and polarity of the compounds to be separated and a guide to this is given in Figure 4.43. 

Size exclusion (gel filtration or permeation) chromatography (SEC) is suitable for solutes with molecular weights of 2000 or more and is also useful for the preliminary investigation of unknown samples. Separated fractions can then be subjected to one of the other modes of HPLC. Exclusion chromatography is discussed in section 4.3.6. 

Note that our primary focus is on reversed-phase HPLC (RPLC) since it is the predominant mode for pharmaceutical analysis. Many of these concepts, however, are applicable to other modes of HPLC such as ion-exchange, adsorption, and gel-permeation chromatography. 

The selected methods are presented in tables and have been grouped by analyte of interest, type of column, and detection method, which are often the principal criteria governing method selection. Because of the water-soluble nature of the vitamins, reversed-phase chromatography is the most common mode of HPLC. Ion exchange is used occasionally. When attempting to reproduce a published HPLC method, attention should be paid to both the type of column and the manu-... 

The purpose of this section is to provide a review of HPLC methods available for the determination of sweeteners in foods. First, general information of the various modes of HPLC and on sample preparation procedures available for the determination of intense sweeteners is described. Then information is given on each individual sweetener. 

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 ... 

In the case of gel permeation or size-exclusion HPLC (HP-SEC), selectivity arises from differential migration of the biomolecules as they permeate by diffusion from the bulk mobile phase to within the pore chambers of the stationary phase. Ideally, the stationary phase in HP-SEC has been so prepared that the surface itself has no chemical interaction with the biosolutes, with the extent of retardation simply mediated by the physical nature of the pores, their connectivity, and their tortuosity. In this regard, HP-SEC contrasts with the other modes of HPLC, where the surfaces of the stationary phase have been deliberately modified by chemical procedures by (usually) low molecular weight compounds to enable selective retardation of the biosolutes by adsorptive processes. Ideally, the surface of an interactive HPLC sorbent enables separation to occur by only one retention process, i.e., the stationary phase functions as a monomodal sorbent. In practice with porous materials, this is rarely achieved with the consequence that most adsorption HPLC sorbents exhibit multimodal characteristics. The retention behavior and selectivity of the chromatographic system will thus depend on the nature and magnitude of the complex interplay of intermolecular forces... 

FIGURE 11 Schematic illustration of several common modes of protein - ligate interaction used in these adsorption modes of HPLC, namely the electrostatic interaction (I), immobilized metal ion affinity interaction (2), hydrophobic interaction and reversed phase (3), and bis-macrocydic metal ion affinity interaction with the psuedo-cation exchange features typical of the MN6 system. 

These various contributions to the overall free-energy change for the ligand-ligate interaction, given by AG°SS0C, in all modes of HPLC of polypeptides and proteins can thus be expressed in terms of the relevant solvophobic considerations29,30,42,47,53,62,215,216 such that... 

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... 

As noted, and as detailed in Table 2, a large variety of stationary-phase and mobile-phase factors influence the selectivity, recovery, and stability of proteins and other biomacromolecules in the adsorptive modes of HPLC. Batch adsorption pilot experiments provide an expedient approach to ascertain the effect of many parameters, such as the pH, nature, and concentration of organic solvent or ionic additives in the mobile phase, the temperature- or the static-binding capacity with a defined sorbent. Similarly, the influence of... 

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