Selecting an HPLC System

The selectivity (separation capability) of an HPLC system is dependent upon the combination of mobile and stationary phases. Since ions are being generated directly from the mobile phase by electrospray, its composition, including the identity and concentration of any buffer used, and its flow rate are important considerations. 

The absolute difference in polarity between the mobile phase and the stationary phase may be defined as the general selectivity of an HPLC system. 

Prepared samples can be passed through an ion-exchange column attached to an HPLC system to selectively retain metals present in the samples while the remaining sample is eluted from the column. The separation of organometallic or inorganic ions is favoured by ion exchange columns and the operating conditions are different from the analysis of non-metallic compounds particularly for retention and separation of trace elements. 

Retention factor A measure of the amount of time an analyte spends in the stationary phase relative to the mobile phase Retention time The time taken for an analyte to travel from the point of injection to the point of detection within an HPLC system Reverse phase chromatography Describes the chromatographic separation in which the stationary phase is nonpolar and the mobile phase is composed of an aqueous, moderately polar liquid Robustness A measure of a method s ability to withstand small but deliberate changes in the method parameters it provides an indication of its reliability during normal usage Selectivity factor See separation factor... 

The last but not least part of an HPLC system is the detector. There are several ways to detect when a certain compound elutes from the colunm. Detection systems based on molecular absorption (UV, Visible, or DAD), fluorescence (FLD) or chemiluminescence, EC or MS are the most popular. It is worthy to highlight the high sensitivity and selectivity provided by detection systems based on fluorescence or chemiluminescence. 

With the acquisition of an HPLC system for micro- and nano-LC, significant influence on the robustness and stability of the subsequent analyses can be exercised through the selection of the system. 

The volume between the solvent mixing point and column inlet is defined as dwell volume, and the corresponding time it takes for the selected composition of mobile phase to reach the column is called dwell time. Although the dwell volume is not the part of the extra-column volume, it is discussed in this section, as it impacts chromatographic performance. Under gradient conditions, the dwell volume for an HPLC system can have a significant effect on separation parameters including retention times (tr) and apparent retention factors fc. ... 

Where the use of multiple spectroscopic analysis on a single HPLC separation is an advantage, the benefit of using the simplest possible mobile phase for separations is manifest. While selecting compatible solvent systems for NMR and MS is sometimes complex, addition of IR (even off-line) places even more constraints on solvent composition. For SEC-NMR-IR CDCR is a suitable eluent [666] for RPLC-FTIR-UV-NMR-MS D2O-CD3CN is recommended. Superheated D20 has been proposed as the mobile phase [670],... 

Bergstrom et al. [63] used HPLC for determination of penicillamine in body fluids. Proteins were precipitated from plasma and hemolyzed blood with trichloroacetic acid and metaphosphoric acid, respectively, and, after centrifugation, the supernatant solution was injected into the HPLC system via a 20-pL loop valve. Urine samples were directly injected after dilution with 0.4 M citric acid. Two columns (5 cm x 0.41 cm and 30 cm x 0.41 cm) packed with Zipax SCX (30 pm) were used as the guard and analytical columns, respectively. The mobile phase (2.5 mL/min) was deoxygenated 0.03 M citric acid-0.01 M Na2HP04 buffer, and use was made of an electrochemical detector equipped with a three-electrode thin-layer cell. The method was selective and sensitive for mercapto-compounds. Recoveries of penicillamine averaged 101% from plasma and 107% from urine, with coefficients of variation equal to 3.68 and 4.25%, respectively. The limits of detection for penicillamine were 0.5 pm and 3 pm in plasma and in urine, respectively. This method is selective and sensitive for sulfhydryl compounds. 

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