Reverse-phase liquid chromatography RPLC

The column methods are much faster and are automated so that a much larger number of samples can be processed per unit time. An example of this technology, described in more detail in Chapter 10 by Lubman and coworkers, is shown in Figure 1.2, where the first dimension is from a chromatofocusing column, which gives separations in pI much like isoelectric focusing, only here the p/ axis is in bands instead of continuous pI increments. The second dimension is by reversed-phase liquid chromatography (RPLC). 

Although relatively unknown, the instrumentation for 2DLC was conceived and implemented by Emi and Frei (1978). They reported the valve configuration presently used in most comprehensive 2DLC systems. However, they automated neither the valve nor the data conversion process to obtain a contour map or 2D peak display. They used a gel permeation chromatography (GPC) column in the first dimension and a reversed-phase liquid chromatography (RPLC) column in the second dimension and studied complex plant extracts. 

Others have examined the necessary parameters that should be optimized to make the two-dimensional separation operate within the context of the columns that are chosen for the unique separation applications that are being developed. This is true for most of the applications shown in this book. However, one of the common themes here is that it is often necessary to slow down the first-dimension separation system in a 2DLC system. If one does not slow down the first dimension, another approach is to speed up the second dimension so that the whole analysis is not gated by the time of the second dimension. Recently, this has been the motivation behind the very fast second-dimension systems, such as Carr and coworker s fast gradient reversed-phase liquid chromatography (RPLC) second dimension systems, which operate at elevated temperatures (Stoll et al., 2006, 2007). Having a fast second dimension makes CE an attractive technique, especially with fast gating methods, which are discussed in Chapter 5. However, these are specialized for specific applications and may require method development techniques specific to CE. 

Pursch, M., Sander, L.C., and Albert, K., Understanding reversed-phase liquid chromatography (RPLC) through sohd state NMR spectroscopy. Anal. Chem., 71,733A,... 

Reversed-phase liquid chromatography (RPLC) with ICP-MS detection has been used for speeiation studies of environmental and more general analytical samples. Butyltin compounds, used in polyvinyl chloride production and in fungicides and insecticides, have been separated by Dauchy et al. [25] using a methanol/water/ acetic acid mobile phase (80 14 6) that had previously been optimized for this separation. Monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) compounds... 

Method Reversed Phase Liquid Chromatography (RPLC) ... 

Reversed phase liquid chromatography (RPLC) allows the separation of analytes with different hydrophobicity and polarity characteristics. It has good selectivity mobile phases used in the technique contain organic solvents and small amounts of inorganic salts [149]. The effectiveness of the process depends on the hydrophobicity of the separated analytes. Charged substances must first be transformed into neutral derivatives (e.g., by adding appropriate anti-ions into the mobile phase). 

The chromatographic procedure intially investigated was reverse-phase liquid chromatography (RPLC) using Cig-bonded silica gel. This technique has been used previously for the preconcentration of trace metals from seawater prior to analysis by atomic absorption spectrophotometry (23, 24) and for the isolation of organically bound copper from seawater (4). These methods were modified and adapted for automation. 

The first report on the analytical use of an aqueous solution of a surfactant, above its critical micellar concentration (CMC), as mobile phase in reversed-phase liquid chromatography (RPLC) was published in 1980. The technique, named micellar liquid chromatography (MLC), is an interesting example of the modification of the chromatographic behavior taking advantage of secondary equilibria to vary both retention and selectivity. 

In reversed phase liquid chromatography (RPLC) silylated silicas are preferred. The surface of these silicas is covered with chemically bonded non-polar groups such as alkyl chains or polymeric layers (Chapter 3.2.3). Silica modified with medium polar groups such as cyano, diol or amino might be used in NP as well as RP mode. Alternatively, cross-linked polymers such as hydrophobic styrene divinyl benzene-copolymers can be used (Chapter 3.2.4). Polymer packings show stability in a pH range 2-14 while silica based packings show limited stability for pH > 7. 

In addition to the three classical separation methods mentioned above, reversed-phase liquid chromatography (RPLC) is becoming increasingly popular for the separation of highly polar and ionic species, respectively. Long-chain fatty acids, for example, are separated on a chemically bonded octadecyl phase after protonation in the mobile phase with a suitable aqueous buffer solution. This separation mode is known as ion suppression [18]. 

---