Column backpressure

FIGURE 13.24 A mixture of hydrocarbons and some polyethylene standards at I45°C. Column backpressure was approximately 5500 psi. Plate counts calculated on the hexane and heptane peaks yield 204,000 plates. 

Reciprocating-piston pumps deliver a constant flow at si fixed backpressure. At high pressures some minor flow variability ziay arise due to the compressibility of the mobile phase. Soms instruments incorporate a flow controller which provides a fixadi backpressure for the pump to work against, independent of the column backpressure. The influence of pressure fluctuations, solvent compressibility, and solvent viscosity on the volumetrie output of the pump are thereby eliminated. Reciprocating-piston pumps can provide continuous solvent delivery, fast solvent change--... 

FIGURE 3.10 Development of column backpressure when a series of incompletely equilibrating methods are applied (illustration). After an initial phase of blank runs, the pressure curve shows a continuous pattern (illustration). 

Another interesting consequence arises from the fact that diffusion and viscosity both depend on temperature. As the viscosity decreases, the diffusivity increases. As a consequence of this, the column performance as measured by the plate count is exclusively determined by the column backpressure. While this is not 100% accurate, it is at least a good rule of thumb. To get about the same column performance, you should increase the flow rate when you increase the temperature such that the column... 

An example of a fast, well-executed separation is shown in Figure 8. A 2-mm i.d. 3 cm 3.5- J,m column was used in a rapid, 1-min gradient. In order to maximize the peak capacity, a high flow rate was used 2mL/min. To reduce the column backpressure, the separation was carried out at 60°C. A fast detector sampling rate was employed to keep up with the high speed of the separation. The injection was coordinated with the arrival of the gradient at the top of the column in order to eliminate the... 

Although a flow rate of 50 ml/mln was used in these preparative runs (typical column backpressure was 20 psi), it Is acceptable to use flow rates as high as 100 ml/mln with the 57 mm I.D. Styragel columns. Such Increased flow rates would afford reduced analysis times, especially when using several columns in series for improved fractionation. Although the pumping system used for the preparative work had a maximum flow rate of 80 ml/mln, other systems are commercially available if higher flow rates are desired. 

Very detailed separations have been obtained by numerous authors (61-66) based upon the method originally developed by Christie (67). This method is based mainly on iso-octane (similar to hexane), 2-P, water containing 500 /jlM serine adjusted to pH 7.5 with ethylamine, and trace amounts of tetrahydrofuran (THF) as a mobile-phase modifier. Lutzke and Braughler modified slightly the mobile-phase system proposed by Christie by including a flow rate gradient to maintain low column backpressure (62). According to the authors, this positively affected detector response to PLs. Markello et al. used the procedure described by Christie, albeit without the addition of serine or ethylamine (65). Melton proposed the use of two solvent mixtures only, but they included exactly the same solvents as proposed by Christie (66). However, PI and PA were not resolved. 

Watch the recorder or computer baseline. When it is stable, slow the pump flow to 0.1 mL/min, remove the column blank, and connect the Cl8 column to the injector. Do not connect the column to the detector yet. Wash the column solvent into a beaker (start a slow flow ramp up from 0.1 to l.OmL/min) for six column volumes (12-18mL). Pressure should slowly increase to around 2000 psi at 1 mL/min due to column backpressure. (Lab note Always hook up a column with solvent running to prevent introducing air from the column head into the column.)... 

Due to the high sensitivity it is favorably to couple a nanoHPLC to an ESI-source. As mass spectrometers are concentration dependent detectors, the sensitivity of an instrumental setup is mostly determined by the peptide concentration of the eluate but not by the peptide amount. Thus a nanocolumn with a flow rate of 300 nL/min provides an about thousand times higher sensitivity than a microbore column with a flow rate of 300 (xL/min. As an alternative to buying a nanoHPLC system it is also possible to use a relatively inexpensive flow splitter after the pump and before the injection valve and the column. Thereby the flow rate can be reduced to use a capillary column (flow rate 4 (xL/min) on an analytical HPLC system or a nanocolumn on a capillary HPLC system. Instead of a flow-splitter it is preferred to couple a nanoHPLC to an ESI-source. Thereby, the flow rate is split according to the column backpressure, i.e., mostly the column volumes if the same packing materials are used. However, these low-cost setups are less reliable than a nanoHPLC and the reproducibility is worse. 

At low selectivity to achieve the same resolution, one has to use a longer column to increase efficiency and consequently operate under higher-pressure conditions. The relationship between the column length, mobile-phase viscosity, and the backpressure is given by equation (2-17), which is the variation of the Kozeny-Carman equation. Expression (2-17) predicts a linear increase of the backpressure with the increase of the flow rate, column length, and mobile phase viscosity. The decrease of the particle diameter, on the other hand, leads to the quadratic increase of the column backpressure. 

Figure 3-24. Column backpressure as a function of the flow rate. Backpressure comparison on conventional and monolithic column (sihca) Circles denote Purospher-C18 packed column closed squares denote equipment pressure drop without the column other symbols denote different monolithic columns, all in one line. (Reprinted from reference 96, with permission.)...

Column backpressure gives a good indication of how the column and/or system are operating. The initial backpressure of the column should be... 

Figure 11-10. Flow spUtting is controlled exquisitely independent of the individual column backpressures, as shown in the scheme.

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