HPLC terms/concepts

Chapter 3 provides an overview of physicochemical factors that impact analysis and purification of polypeptides and proteins by HPLC techniques. The current status and some of the future challenges facing this major field of separation sciences are considered from both didactic and practical perspectives (Chapter 3). This chapter attempts to provide an overview of terms, concepts, principles, practical aspects, and primary references that underpin the recent developments in this field. Where appropriate, key relationships and dependencies that describe the interactive behavior of polypeptides and proteins with chemically immobilized ligands are discussed. This understanding is central to any subsequent exploration of alternative avenues now available for further research and development into the field of polypeptide or protein purification and analysis. 

This chapter provides an overview of basic terminology and essential concepts in HPLC including retention, selectivity, efficiency, resolution, and peak symmetry as well as their relationships with key column and mobile phase parameters. The resolution and van Deemter equations are discussed. The concepts of peak capacity and method orthogonality as well as key gradient parameters such as gradient time and flow rate are described. An abbreviated glossary of HPLC terms is listed. 

Compare HPLC with GC in terms of (a) the force that moves the mobile phase through the stationary phase, (b) the nature of the mobile phase, (c) how the stationary phase is held in place, (d) what types of chromatography are applicable, (e) application of vapor pressure concepts, (f) sample injection, (g) mechanisms of separation, (h) detection systems, (i) recording systems, and (j) data obtained. 

Meanwhile, the use of bonded reversed-phase columns in HPLC was common, and there were applications for the concentration of organic solutes directly onto the head of the HPLC columns with the term trace enrichment being used for this process (Little and Fallick, 1975). However, interferences also concentrated at the head of the column, such as proteins in biological samples and humic substances in water samples thus, column life was greatly shortened. The answer was a small precolumn positioned ahead of the analytical column to retain contaminants or to retain analytes in trace enrichment. The concept of the precolumn quickly led to the use of separate or off-line low-pressure SPE cartridges. 

The focus is on concepts in reversed-phase liquid chromatography (RPLC), though the same concepts are usually applicable to other modes of HPLC. International Union of Pure and Applied Chemistry (IUPAC)10 nomenclature is used. The term sample component is often used interchangeably with analyte and solute in the context of this book. As mentioned in Chapter 1, the most common stationary phase is a hydrophobic C18-bonded phase on a silica support used with a mixed organic and aqueous mobile phase. The terms packing and sorbent often refer to the bonded phase whereas solid support refers to the unbonded silica material. 

Thin-layer chromatography has often been considered to be a manual method. With this in mind, users tend to consider this technique to be of low quality and unreliable, thus disregarding its high potential. The instrumental development of new techniques such as HPLC reinforced this idea. Therefore, the TLC concept was reviewed and fully automated to alter this perception of the method. First, the basic performance of TLC had to be improved, in terms of ... 

---