Mobile phase system choice

Measurement of labelling yield and subsequent radiochemical purity requires a suitable analytical technique, and the method of choice for radio-labelled peptides is reversed phase HPLC with on-line UV and radiometric detection. It is important to use as stringent a separation method as possible with isocratic or slow mobile phase composition gradients over the peptide peak. Ideally, more than one mobile phase system should be used (e.g. a phosphate buffer-methanol system in addition to the standard water-acetonitrile system), since these may show the presence of new impurities. It is important to recognize that HPLC analyses only measure those components that elute from the column. Insoluble, highly lipophilic or positively charged species may bind to the solid phase. It is very important to verify the absence of these species by a complimentary technique such as thin layer chromatography (TLC) and to ensure that the two techniques produce similar results. 

The flexibility of TLC relative to HPLC is enhanced by the greater choice of solvents available for preparing TLC mobile phases. The choice of solvents for HPLC is limited by the requirements for their chemical and physical properties imposed by the nature of the method. HPLC is a closed system operated under high pressure with on-line detection, most often using a UV monitor, and the column is continually reused. Solvent components with high vapor pressure (e.g., ethyl ether) or UV absorbance (benzene) or those that might degrade the column (NaOH) are difficult to use in HPLC but are readily applicable to TLC. 

Major organic components of the mobile phase Minor components of the mobile phase Choice of mobile phase system Achieving and Modifying a Separation Sample preparation Initial conditions Improving separation Other problems Apres Chromatography Scale-up... 

Two variations of the technique exists isocratic elution, when the mobile phase composition is kept constant, and gradient elution, when the mobile phase composition is varied during the separation. Isocratic elution is often the method of choice for analysis and in process apphcations when the retention characteristics of the solutes to be separated are similar and not dramaticallv sensitive to vei y small changes in operating conditions. Isocratic elution is also generally practical for systems where the equilibrium isotherm is linear or nearly hnear. In all cases, isocratic elution results in a dilution of the separated produces. 

It is clear that the first challenge facing the analyst is the choice of the phase system that is appropriate for the particular sample to be analyzed. Only after the phase system has been chosen, can the correct column be selected. It is therefore necessary to know the types and properties of the different stationary phases that are available and how to formulate the pertinent mobile phases that must be used with them. 

Apart from the choice of an appropriate stationary and mobile phase, the essential problem for PLC is to attain equilibrium in a three-phase system — between the stationary, mobile, and gas phases. In a nonequilibrated system, the velocity of the mobile phase in a thicker layer (i.e., the effect of solvent evaporation) is less in a lower part of an adsorbent. Such a situation leads to the diffusion of bands and deterioration of the adjacent bands separation. This can be minimized or avoided by prerunning the plate with the mobile phase before spotting of the sample and the saturated chromatographic chambers. 

The choice of the chromatographic system depends on the chemical character of the extracts being separated. The mobile phase should accomplish all requirements for PLC determined by volatility and low viscosity, because nonvolatile components (e.g., ion association reagents and most buffers) should be avoided. It means that, for PLC of plant extracts, normal phase chromatography is much more preferable than reversed-phase systems. In the latter situation, mixtures such as methanol-ace-tonitrile-water are mostly used. If buffers and acids have to be added to either the... 

Liquid chromatography has a number of different configurations with regard to technical (instrumental) as well as separation modes. The HPLC system can be operated in either isocratic mode, i.e. the same mobile phase composition throughout the chromatographic ran, or by gradient elution (GE), i.e. the mobile phase composition varies with run time. The choice of operation... 

In both cases, either conventional FTIR transmission or diffuse reflection detection may be used. Because TLC and the postspectroscopic evaluation are not linked directly, few compromises have to be made with regard to the choice of the solvent system employed for separation. Chromatographic selectivity and efficiency are not influenced by the needs of the detector. The TLC plate allows the separation to be made in a different site from the laboratory where the separated analytes are evaluated. The fact that the sample is static on the plate, rather than moving with the flow of a mobile phase, also puts less demand on the spectrometer. The popularity of TLC-IR derives in part from its low cost. 

There is a very wide choice of pairs of liquids to act as stationary and mobile phases. It is not necessary for them to be totally immiscible, but a low mutual solubility is desirable. A hydrophilic liquid may be used as the stationary phase with a hydrophobic mobile phase or vice versa. The latter situation is sometimes referred to as a reversed phase system as it was developed later. Water, aqueous buffers and alcohols are suitable mobile phases for the separation of very polar mixtures, whilst hydrocarbons in combination with ethers, esters and chlorinated solvents would be chosen for less polar materials. 

Adequate resolution of the components of a mixture in the shortest possible time is nearly always a principal goal. Establishing the optimum conditions by trial and error is inefficient and relies heavily on the expertise of the analyst. The development of computer-controlled HPLC systems has enabled systematic automated optimization techniques, based on statistical experimental design and mathematical resolution functions, to be exploited. The basic choices of column (stationary phase) and detector are made first followed by an investigation of the mobile phase composition and possibly other parameters. This can be done manually but computer-controlled optimization has the advantage of releasing the analyst for other... 

Some, uPLC systems are equipped with UV absorbance detection, and other systems allow for both UV absorbance and fluorescence detection. Fluorescence detection increases the sensitivity and selectivity of certain applications and is the method of choice in many separation-based assays. The liquid (mobile phase + sample) leaving the individual flow cells designated for UV detection is transferred through capillaries to a bank of 24 flow cells designated for fluorescence detection. 

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