Column back pressure

Several compromises are involved in the selection of the correct particle size. On one hand, one desires the highest possible resolution in the shortest amount of time. Therefore, the smallest particle size should be chosen that still gives resolution of the polymer without causing excessive column back pressure. On the other hand, there are constraints on both the strength of the particle and the strength of the polymer. This section discusses the selection of the best particle size. 

In the pneumatic pumping system, the pressure (and not the flow rate) is maintained constant as variations in chromatographic conditions occur. Thus, a change in mobile phase viscosity (e.g. gradient elution) or column back pressure will result in a change in flow rate for these types of pumps. The gas displacement pump in which a solvent is delivered to the column by gas pressure is an example of such a pneumatic pump. The gas displacement system is among the least expensive pumps available and is found in several low cost instruments. While the pump is nonpulsating and hence, produces low noise levels with the detectors in current use, its flow stability and reproducibility are only adequate. In addition, its upper pressure limit is only 2000 psi which may be too low in certain applications. 

FIGURE 7.4 (a) Plots of column back pressure against linear velocity of mobile phase (29,... 

A typical HPLC system set to a flow rate of 1 ml/min, indicating a column back pressure of 1265 psi and connected to a UV/visible detector which is set to monitor the column effluent at 260 nm. 

While pressure has no effect on the eluent strength when liquid solvents are used, under supercrihcal conditions the solvent density increases greatly with increased pressure. This offers an additional degree of freedom when selecting the chromatographic conditions of the process. The impact of the column back pressure is illustrated in Figure 12.21. The impact of the pressure on retention... 

Recovery of only 20 % was observed in the separation of influenca virus components on a RP column (250x4.6 mm dG = 30 nm) with a water/acetonitrile gradient. The virus consists of three protein components with molar mass alues ranging from 28 to 55 kg/mol. After several runs, the column back pressure increased from 60 to 90 bars. Overnight treatment with 0.1 % sodium dodecylsulfate and 0.05 % TFA helped to regain approximately the initial permeability 67 ... 

These techniques are useful for the separation of complex mixtures. Quantitative analyses have been reported on malathion pesticide in vegetable matter, limonin in grapefruit peel, and additives in compound rubber by means of SEC and RPC126>. SEC was performed with unstabilized THF and monitored at 215 nm. Working so near to the UV cut-off (210 nm) of THF was possible through the use of a detector with a flowing reference installed between the pump and the sample valve. Then the eluent passed the column and finally the analytical cell. Since the column back pressure never exceeded 10 MPa, this eluent line caused no problems. 

Beside column dimension the size of stationary phase particles is a matter of recent progress. More traditional columns are packed with 3.0-5 pm particles enabling satisfying resolution and reasonable column back pressure of solvent suitable to be processed by conventional HPLC pumps. In contrast, sub 2-pm particles (e.g. 1.7 and 1.8 pm) as applied in rapid or fast LC or ultra high-performance LC (UHPLC) allow better resolved separations in shorter run times. Column back pressure (>12,000 psi) is remarkably high demanding more robust solvent pumps. 

The main disadvantages of micellar chromatography are the observed diminished chromatographic efficiency, higher column back pressure, and in preparative work, the need to separate the final resolved analyte from the surfactant (95) (a later section of this review will discuss this latter problem and its resolution in further detail). The higher column back pressure and part of the decreased efficiency stem from the fact that surfactant-containing mobile phases are more viscous compared to the usual hydro-organic mobile phases employed in conventional RP-HPLC (refer to viscosity data in Table X)... 

The pressure experienced by the pump as it forces mobile phase through the column is known as the column back pressure. This will vary of course, from column to column (according to dimensions and particle size) and with the flow rate and viscosity of the mobile phase used. The relationship between these parameters is expressed in the following equation ... 

Typical flow rates for analytical columns (4.6mm i.d.) are 0.5-2mL/min. Operating at higher flow rates (F) increases column back pressure (AP)6 but reduces retention time and analysis time ... 

Higher column temperatures (T) lower the viscosity of the mobile phase (thus, column back pressure, see Eq. 2.19) and usually have significant effects on retention (k), efficiency (N), and selectivity (a). Some of these effects are discussed further in Sections 2.4 and 2.5 of this chapter and in Chapter 8. 

Particle size and size distribution define the quality of the support material and are the key determinants of efficiency and back-pressure of the column.1-3 The effect of dp on H is discussed in Chapter 2. For a well-packed column, Hmin is approximated to 2-2.5 dp. Also, since the van Deemter equation C term is proportional to dp2, columns packed with small particles have much less efficiency loss at high flow rates.14 However, since column back-pressure is inversely proportional to dp2, columns packed with sub-3-pm particles can easily exceed the pressure limit of most HPLC instruments at 6,000 psi. Note that decreasing particle size while keep the L constant can increase column efficiency and peak resolution (Figure 3.5A) and also increase peak height and sensitivity (Figure 3.5B). 

Monitor the column back-pressure and efficiency performance and take corrective action immediately don t wait until the column is plugged. If pressure is abnormally high, try back flushing the column as soon as... 

The principal merits of such systems were the ability to vary sample size the efficiency of sample usage and that they were relatively inexpensive. However, on-column injection has a number of disadvantages principal amongst which is that they cannot be used at the high column back pressures associated with modern packing materials and thus this technique is simply of historical interest. 

General Considerations, When particles are packed into a column, they offer a restriction to solvent flow. The longer the column and the smaller the particles, the greater the restriction. If flow is forced through the column, it generates a back pressure. The relation of this column back pressure AP to the other chromatographic variables is given by... 

Since efficiencies for microparticulate columns are very high (optimum H values of 0.01-0.03 mm), only short columns (15-25 cm) are required for analytical HPLC, as can be seen in Figure 21.14C. The use of 5- m particles implies greatly increased column back-pressures compared to those produced by the larger porous or pellicular particles, as suggested by Equation 21.13. However, these short columns exhibit moderate back-pressures (less than 200 atm) when used at flow rates of... 

Initially, the solvent reservoirs should be checked to ensure that they contain sufficient mobile phase for the proposed chromatographic separation and so prevent air being drawn into the pumps. The mobile phase can then be pumped through the column until a steady baseline signal is obtained from the detector. This would usually entail a flow rate of between 0.5 and 4 ml/min for a 4.6 mm I.D. column for anything between 5 and 60 min. Care should be taken to ensure that the column back pressure does not exceed the recommended limitations for the column as this could cause irreversible damage. 

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