Modem HPLC Column

The separation of analyte mixtures in modern HPLC is performed in the device called the column. Current HPLC columns in most cases are a stainless steel tube packed with very small (1-5 pm) particles of rigid porous material. Packing material is retained inside the column with special end-httings equipped with porous frits allowing for liquid line connection (to deliver mobile phase to the column). Stainless steel or titanium frits have a pore size on the level of 0.2-0.5 pm, which allows for the mobile phase to pass through while small particles of packing material are retained inside the column. 

The column is the heart of the chromatographic system and it is the only device where actual separation of the analyte mixture takes place. Detailed discussion of HPLC columns and stationary phases is given in chapter 3. 

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When installing the column, make sure that the mobile-phase flow is in the direction indicated on the column. Most modem HPLC columns, especially those based on hard spherical particles, can be used in both directions without detrimental effect but it is nevertheless good practice to have a standardized direction of flow. 

The relationship between particle size and column efficiency is now well understood, although the exact form of the equations, including the Knox equation [see Equation (3)], is still debated (47). The 3-5 (xm particle size of modem HPLC columns allows fast analysis of small molecular weight compounds at near optimal column efficiency. As discussed, larger molecular weight compounds, because of their smaller diffusion coefficients, require much lower flow rates to elute with maximum column efficiency. Because of the usual variation in polymer molecular weight, it is not possible to operate the column at the optimal speed for all components in the sample. 

I 4 Comparison and Selection of Modem HPLC Columns Test 2... 

In-line Detector It broadly helps to sense the separated solutes, after they exit through the column. Invariably the detector is an electrical signal whose variation is displayed on a potentiometer recorder or a computing integrator or a video-screen. Modem HPLC units are provided with detectors having selective-devices thereby categorically restricting the response to all the solutes present in a mixture. 

Whether eluted from columns or from thin-layer plates, the quantitative determination of sugars was traditionally based on colorimetric reactions involving the use of chemical reagents, e.g., anthrone. These detection methods have been largely replaced in modem HPLC by the refractive index detector, although ultraviolet detectors are also employed. Recently we have also seen the introduction of other types of detector (e.g., the mass detector), as will be discussed later. 

A modem HPLC system is shown schematically in Figure 2. The equipment consists of a high-pressure solvent delivery system, a sample auto injector, a separation column, a detector (often an UV or a DAD) a computer to control the system and display results. Many systems include an oven for temperature control of the column and a pre-column that protects the analytical column from impurities. The actual separation takes place in the column, which is packed with chemically modified 3.5-10 pm (often silica) particles. A mobile phase is pumped through the column with the high-pressure pump and the analytes in the injected sample are separated depending on their degree of interaction with the particles. A proper choice of stationary and mobile phase is essential to reach a desired separation. 

Also known as gradient delay volume, system dwell volume is the liquid holdup volume of the HPLC system from the point of solvent mixing to the head of the column. This includes the additive volumes of the injector, the sample loop, all fluidic connection tubing, and any internal pump volumes of a low-pressure mixing system. The typical dwell volume of a modem HPLC is 0.5-2 mL, but can be as high as 5-7 mL in older systems. 

This technique is now used extensively to assess enantiomeric excesses in organic reactions and separate small quantities of enantiomers. Closely related chiral corands are particularly useful in assessing the optical purity of amino acids, although modem chiral columns for HPLC may cost in excess of US 2000. 

For complete characterization of the column performance the efficiency has to be tested over the vhole operation range of linear velocities. The injection of a test mixture at different fio v rates can be easUy automated with modem HPLC equipment and yields the pressure drop versus flow curve of the adsorbent as well as its HETP curve. By using marker substances with different molecular weights the influence of the mass transfer resistance (C term) can be investigated. Figure 4.34 shows the HETP curve for two similar adsorbents, which exhibit large differences at high flow rates. 

Most bonded phase ion-exchangers are stable up to 60 ° C and the speed of modem HPLC minimises the possibility of solute degradation. The k values for solutes frequently respond differently to changes in temperature and consequently this parameter can be used very effectively to modify the selectivity of the column. 

In general, in the development of a separation of a sample mixture, the sample is initially injected onto the HPLC column in a solvent of low elution strength which is then steadily increased until the compounds of interest elute from the column. The main limitation of this method is that it may take a long time to effect elution and consequently it is often quicker to use this procedure in reverse, with discrete jumps in eluent strength (Parris, 1978). Modem equipment allows gradient development to be carried out unattended. 

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