4.2. Limitations of manifold design due to column properties
The main limitation is due to flow resistance of columns that require high pressure and long-term stability of flow rate.
The peristaltic pump, still widely used in flow analysis, is suitable for low pressure applications. Known weaknesses are inability to generate sufficient pressure to overcome column resistance, and low inter-day repeatability of flow rates, thus pumped volumes. Therefore, peristaltic pumps are suitable only for very short columns, monolithic columns, and discs.
Syringe pumps deliver a stable flow rates, and higher pressures corresponding to the power of the stepper motor and the seal between the syringe and piston. Common is a combination of stainless steel piston and glass syringe. Advanced is the syringe made of durable plastic or stainless steel syringe that can operate at a pressure of 1000 psi / 7.0 MPa. The use of a continuously refilling milliGAT type pump (Section 3.2.6.) is advantageous. Recently its working force increased up to four times in compared to previous generation now operating at 500 psi / 3.5 MPa.
The pressure rating of the valves and connectors must match the expected pressures, so it is advisable to use new pressure-tight or even HPLC parts, including fasteners and tubing. These parts have a smaller internal volume due to typically lower inner diameters.
The column, in particular the solid phase/sorbent, determines the backpressure of the flow system. Lab-made columns are typically packed with particles of 25-150 µm size and are used for rapid sample preparations - extraction or reaction - before the next analytical step. Extraction columns can also be filled with (nano) fibers, membranes, or monolithic structures. On the other hand, commercial columns are mainly used for chromatography - monolithic or 2-5 µm particle-packed columns with dimensions up to 100 mm in length and 1-4.6 mm in diameter. The possibilities of using chromatographic columns are mainly determined by the pressure limit of the flow system.
Efficient High-Pressure Liquid Chromatography operates at pressures well over 1000 psi. It offers multicomponent separation and, therefore it is widely used. Still, it is worthwhile to consider designing hybrid systems comprising low pressure pFI system, interfaced with HPLC instrument by a two-position injection valve.
/4-2-Limitations-of-manifold-design-due-to-column/4_2-1-(1).png.aspx?width=800&height=478 "4_2-1-(1).png")
pF-HPLC is a hybrid system comprised of low pressure pFI and high pressure HPLC. It is based on injection of sample that travels upstream from injection point into a reactor where target analyte are is processed. Following flow reversal moves reaction mixture into loop of two position valve that serves as interface between low and high pressure systems. A high pressure pump (P3) moves sample trough a column, where sample components are separated due to difference in migration velocities, resulting in a multicomponent response curve that yields quantitative information on content of target analytes. SPE column is optional, while flow cell 1 is used to monitor positioning of sample within loop of the valve.
Since flow cells are positioned in low-pressure part of a flow path (upstream and downstream from HPLC column), they do not need to be pressure, but should be chemically resistant. The flow cell of low internal volume and its placing as close as possible behind the column reduces zone dispersion and yields faster response.