Columns back pressure limitation

Fig. 3. Here, we have created the same diagram as discussed above for a series of 5 cm columns packed with 5 tm, 3.5 tm, and 2.5 im particles. As we can see, for slow analyses the separation power increases with decreasing particle size. With the 5 im column, we reach a peak capacity of nearly 150 for a 30 min analysis, while with the 3.5 im column a value of around 180 is reached. This performance is surpassed by the 2.5 pm column, with a peak capacity of roughly 220 for this half-hour analysis. However, for very rapid analyses, the column back-pressure limits the performance of the column. For a 1 min separation, the 3.5 pm column shows a somewhat better performance than the 5 pm column, but the peak capacity for the 2.5 pm column is lower than what was achievable with the 3.5 pm column. The reason for this is that the 2.5 pm column reaches the pressure limit imposed by the instrument. The main explanation for this is that, at a fixed column length, smaller particles reduce the flow rate that can be used. This can also be seen in Fig. 3. On the other hand, the 5 cm 3.5 pm column is ideal for analysis times in the 2-4 min range. Under these conditions, the 3.5 pm column exhibits a better separation power than the 5 pm column and still exceeds the performance of the 2.5 pm column.

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. 

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). 

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. 

Note 7. A well-packed column is essential to achieve high-efficiency elutions. There are two different methods to pack a column the dry and the wet method. In the first case the column is filled with the dry stationary phase, closed and the mobile phase is then pumped in. This method is suitable for silica-based beads, but not for polymeric beads, because this kind of material is prone to swelling when wet. Thus, high column back pressures, preferential channels and flow limitations due to a nonhomogeneous wetting of the stationary mobile phase could deteriorate the chromatographic performances of the column. When the wet packing method is used, the stationary phase is swollen and suspended in a solvent of similar density, and introduced into the column as a slurry. In this case the main drawback is the necessity to provide a reservoir for the column to keep the beads in suspension. 

Usually, the UPLC conditions with scaled gradient (accounting for particle size) are selected for method verification however, the UPLC conditions with scaled gradient (disregarding particle size) are not correct. The UPLC conditions for shortest analysis time at original peak capacity usually have column back pressure close to or over the instmment limit. The conditions with maximum peak capacity at original... 

The solvents most commonly used for the mobile phase are mixtures of an alkane (hexane or the less toxic heptane) with a solvent of medium or high polarity. Their ratio will determine retention and, therefore, the possibihty for the analyte to interact with the CS in the stationary phase. Given their compatibility with a range of CSPs either bonded or coated, alcohols are frequently included as polar components of the mobile phase (Figure 54.12a). Among them, 2-propanol is more extensively used than ethanol or methanol because of its full miscibility with alkanes. Viscosity, which impacts column back-pressure, is another of the reasons for limiting the use of high ratios of certain alcohols. 

As the porosities of PDVB gels increase above 10 A, the pressure limits drop, with 2500 psi being the maximum usable pressure for 10 A, 10 A, and mixed-bed columns. Because the normal operating pressures in most solvents for these columns tend to be in the range of 1000 psi or less for a 10 X 500-mm column, there is seldom an operational problem. Figure 13.8 shows the resolution of a typical mixed-bed column run in chloroform at 1.5 ml min yielding a back pressure of 700 psi and running polystyrene standards. 

A longer column is preferred because of a greater processing capacity nd an increased number of plates, as long as the back pressure does not exceed the upper limit and the nonuniform displacement of the solution and the solvent is not serious. The theoretical plate in HOPC is defined as a section in the column in which equivalently full exchange of all of the polymer components... 

One potential problem associated with column coupling in reversed phase is relatively high back-pressure ( 2600 psi at 1 mL miir ). This will place a limit on the flow rate, which in turn limits the further reduction of analysis time. Also, compared to the new polar organic mode, the retention in reversed phase on coupled columns is deviated more from the average retention on the individual stationary phases. 

Implementation of SFC has initially been hampered by instrumental problems, such as back-pressure regulation, need for syringe pumps, consistent flow-rates, pressure and density gradient control, modifier gradient elution, small volume injection (nL), poor reproducibility of injection, and miniaturised detection. These difficulties, which limited sensitivity, precision or reproducibility in industrial applications, were eventually overcome. Because instrumentation for SFC is quite complex and expensive, the technique is still not widely accepted. At the present time few SFC instrument manufacturers are active. Berger and Wilson [239] have described packed SFC instrumentation equipped with FID, UV/VIS and NPD, which can also be employed for open-tubular SFC in a pressure-control mode. Column technology has been largely borrowed from GC (for the open-tubular format) or from HPLC (for the packed format). Open-tubular coated capillaries (50-100 irn i.d.), packed capillaries (100-500 p,m i.d.), and packed columns (1 -4.6 mm i.d.) have been used for SFC (Table 4.27). 

Bayliss and co-workers [10] combined ultra-high flow rates, parallel LC columns, a multiplex electrospray source, and mass spectrometric detection for the rapid determination of pharmaceuticals in plasma using four narrow bore (50 mm x 1 mm, 30 pm Oasis HLB) or capillary (50 mm x 0.18 mm, 25 pm Oasis HLB) HPLC columns with large particle sizes (to avoid high system back-pressure) in parallel with a multiple probe injector and a MUX MS interface. Small sample aliquots were injected directly into the system without sample pre-treatment procedure, obtaining very low limits of quantification (from 1 to 5 ng/mL). 

The smaller the particle size the more efficient a separation will be. However, the smaller the particle the higher the back pressure in the packed column, which limits the practical size that can be used due to pressure limitations of the hardware. Typically, most equipment will be capable of withstanding the back pressures generated by 5 xm particles at optimized flow rates. 

Eluent pH is limited to a maximum of 7 to 8 due to the reduced chemical stability of a chromatographic bed in an alkaline medium. The nucleophilic attack of Si-0 bonds by hydroxide ions leads to the erosion of the silica surface as shown by back pressure increases caused by the formation of Si(OH)4. With polystyrene-divinyl-benzene-based stationary phases, pH stability is not an issue and a very wide mobile phase pH range can be used, thereby providing additional selectivity [1]. Several silica-based and polymeric columns claimed to be stable in pH ranges from 1 to 13 are commercially available, however, they are not commonly used. 

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