Normal bonded-phase chromatography

In NPLC, which refers to the use of adsorption, i.e. liquid-solid chromatography (LSC), the surface of microparticulate silica (or other adsorbent) constitutes the most commonly used polar stationary phase normal bonded-phase chromatography (N-BPC) is typified by nitrile- or amino-bonded stationary phases. Silica columns with a broad range of properties are commercially available (with standard particle sizes of 3, 5 and 10 im, and pore sizes of about 6-15nm). A typical HPLC column is packed with a stationary phase of a pore size of 10 nm and contains a surface area of between 100 and 150m2 mL-1 of mobile phase volume. 

The stationary phases available for HPLC are as numerous as those available for GC. As mentioned previously, however, adsorption, partition, ion exchange, and size exclusion are all liquid chromatography methods. We can therefore classify the stationary phases according to which of these four types of chromatography they represent. Additionally, partition HPLC, which is the most common, is further classified as normal phase HPLC or reverse phase HPLC. Both of these are bonded phase chromatography, which was described in Chapter 11. Let us begin with these. 

What is meant by bonded phase chromatography Would such a name describe normal phase, reverse phase, neither, or both Explain. 

Bonded phase chromatography is a type of liquid-liquid chromatography in which the liquid stationary phase is chemically bonded to the support material (as opposed to being simply adsorbed). The stationary phase can be either polar or nonpolar, and thus both normal phase and reverse phase are possible. 

If these small silica particles are used, then the chromatography is called normal phase, and the polarity of the stationary phase is higher than that of the mobile phase this is what happens, for example, when silica is used in adsorption chromatography. However, almost all the work in analytical HPLC is now carried out with chemically modified silica, which is the bonded phase. In a bonded phase, the highly polar surface of silica is modified by the chemical attachment of various functional groups. Bonded-phase chromatography is experimentally much easier, more versatile, and quicker it also has better reproducibility than the older modes. When a nonpolar-bonded phase is used, the operation is performed in an RP mode, which means that the polarity of the stationary phase is less than that of the mobile phase. These columns, contrary to normal silica columns, elute polar compounds more rapidly than nonpolar compounds. 

Bonded phase chromatography (BPC) takes place either under normal phase or reverse phase conditions. In reverse phase mode the stationary phase is non-polar while the eluant is polar, e.g. methanol or acetonitrile with aqueous buffers. Bonded phase packings have superceded the classical packings where the stationary phase was distributed over the surface of the support particles and bound simply by physical forces of attraction. However, due to the problems of solvent stripping and limited hydrolytic stability, these classical systems, though developed for a few specialised applications, have been replaced by organo-bonded stationary phase materials. 

Normal bonded phase packing materials have been used as an alternative to adsorption chromatography because column deactivation due to contamination of polar contaminants injected is less frequently seen in the normal bonded phase than in adsorption chromatographic systems. 

Similar to adsorption chromatography, also on normal bonded phase packing materials, cis- and traws-vitamin Ki(20) can be separated. 

BPC (bonded-phase chromatography) A hquid chromatographic technique based on the use of support materials that are chemically modified to produce stationary-phases of varying polarity and functionahty. Bonded phases run the range from reversed phase to normal phase to chiral to affinity, etc. 

The most common technique used for agrochemicals is reversed-phase SPE. Here, the bonded stationary phase is silica gel derivatized with a long-chain hydrocarbon (e.g. C4-C18) or styrene-divinylbenzene copolymer. This technique operates in the reverse of normal-phase chromatography since the mobile phase is polar in nature (e.g., water or aqueous buffers serve as one of the solvents), while the stationary phase has nonpolar properties. 

Liquid-solid chromatography (LSC), sometimes referred to as normal phase or straight phase chromatography, is characterized by the use of an inorganic adsorbent or chemically bonded stationary phase with polar functional groups and a nonaqueous mobile phase... 

Although SPE can be done in a batch equilibration similar to that used in LLE, it is much more common to use a small tube (minicolumn) or cartridge packed with the solid particles. SPE is often referred to as LSE, bonded phase or sorbent extraction SPE is a refinement of open-column chromatography. The mechanisms of retention include reversed phase, normal phase, and ion exchange. 

The TLC process is an off-line process. A number of samples are chromatographed simultaneously, side-by-side. HPTLC is fast (5 min), allows simultaneous separation and can be carried out with the same carrier materials as HPLC. Silica gel and chemically bonded silica gel sorbents are used predominantly in HPTLC other stationary phases are cellulose-based [393]. Separation mechanisms are either NPC (normal-phase chromatography), RPC (reversed-phase chromatography) or IEC (ion-exchange chromatography). RPC on hydrophobic layers is not as widely used in TLC as it is in column chromatography. The resolution capabilities of TLC using silica gel absorbent as compared to C S reversed-phase absorbent have been compared for 18 commercially available plasticisers, and 52 amine and 36 phenolic AOs [394]. 

Microparticulate silica can be used in a number of modes for hplc of these, reverse phase chromatography using bonded phases is the most widely used. In normal and reverse phase chromatography the retention times and selectivities of solutes can be altered by adjustment of the nature and composition of the mobile phase. 

---