Mobile phase special additives

The mobile phase reservoir is made of an inert material, usually glass. There is usually a cap on the reservoir that is vented to allow air to enter as the fluid level drops. The purpose of the cap is to prevent particulate matter from falling into the reservoir. It is very important to prevent particulates from entering the flow stream. The tip of the tube immersed in the reservoir is fitted with a coarse metal filter. It functions as a filter in the event that particulates do find their way into the reservoir. It also serves as a sinker to keep the tip well under the surface of the liquid. In addition, in specially designed mobile phase reservoirs, this sinker/filter is placed into a well on the bottom of the reservoir so that it is completely immersed in solvent, even when the reservoir is running low. This avoids drawing air into the line under those conditions. These details are shown in Figure 13.3. 

The first step in method development is selecting an adequate HPLC mode for the particular sample. This choice depends on the character of the sample compounds, which can be either neutral (hydrophilic or lipophilic) or ionic, low-molecular (up to 2000 Da) or macromolecular (biopolymers or synthetic polymers). Many neutral compounds can be separated either by reversed-phase or by normal-phase chromatography, but a reversed-phase system without ionic additives to the aqueous-organic mobile phase is usually the best first choice. Strongly lipophilic samples often can be separated either by non-aqueous reversed-pha.se chromatography or by normal-phase chromatography. Positional isomers are usually better separated by normal-phase than by reversed-phase chromatography and the separation of optical isomers (enantiomers) requires either special chiral columns or addition of a chiral selector to the mobile phase. 

Soluble enzymes are employed in a wide variety of substrate and enzyme activity assays, and specialized instrumentation has been developed to automate reagent addition and quantitation. However, several disadvantages exist in the analytical use of soluble enzymes for substrate assays. Soluble enzymes are not reused or recycled, unless the cost of the enzyme justifies the lengthy repurification procedure. Furthermore, the activities of soluble enzymes decreases significantly with time, so that fresh assay solutions are frequently required. For these reasons, many assays that employ soluble enzymes have been adapted for use with immobilized enzymes. These immobilized enzymes are usually incorporated onto or into a stationary phase in a flow system substrate is introduced via a mobile, or buffer phase, and conversion into products occurs as the mobile phase flows through a column containing immobilized enzyme. A postcolumn detector allows product quantitation. 

For the separation of basic analytes it is recommendable to use a stationary phase which is specially designed for such samples. " Otherwise severe tailing may occur (which perhaps can be suppressed by additives to the mobile phase, but this approach is less elegant). An example can be found in Figure 10.10. 

To obtain micro flow rates (for example, 1 pL/min), necessary for a packed capillary column, the same pumps are used, though with the addition at the outlet of a bypass which divides the flow into two fractions of which only the smaller is directed towards the column. To resist the low pHs of many elution mixtures, which can be more corrosive when pressure is high, the components and surfaces that come into contact with the mobile phase need to be inert. The pistons and valves of the pumps are made of sapphire, agate. Teflon or special alloys. 

The evaluation of some samples is sometimes not possible after the first development. This can be caused by a wrong choice of solvent or by special properties of the sample. However, it is possible to develop the thin film one or more additional times. There are two methods for developing the thin-film card more than one time one-dimensional and two-dimensional development. The thin-film card must be dried so that there are no residues of the mobile phase on the thin film before the development in the second step. 

Special additives are used to improve properties of mobile phases for chromatography in biochemical separations. Antimicrobial reagents are the most often applied a list of their characteristics is given in Ref. 98 (Table 10.8) and in Ref. 6 (Table... 

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