HPLC-Hardware Requirements

Much of the recent impetus for temperature control has focused on exploiting the effects of elevated temperature on viscosity and diffusion coefficients [2], These lead to faster separations and also allow smaller particle diameters to be employed with conventional HPLC hardware. As the viscosity of solvents decreases, the column pressure drops. This can be exploited by using faster flow rates and smaller particle diameters. All of this leads to faster separations. In one experiment in this laboratory, a separation which required 8 min at room temperature was reduced to 2 min at 50°C without changing the column. Speed enhancements of as much as 50-I00-fold have been reported [13] as shown in Figure 9.1. 

The first commercial SEC instruments were composite instruments with all the components contained within a box . With the advent of HPLC in the early 1970s, hardware requirements changed somewhat. Columns became... 

This level of control requires a controlling script from the instrument vendor and is for more serious programmers. Direct HPLC control by the MS and multiple staggered LC capabilities provided by several autosampler and instrument vendors fulfill the function but may limit the hardware used or require purchase of software/hardware. 

Note As is often the case, the HPLC system will be under computer control, which is likely to be part of a data-handling system. Since the data generated from the OQ hardware tests typically require chromatographic data handling, the data-handling software should be validated beforehand. The data-handlingfLC control software installation and IQ/OQ implementation, which are not addressed in this chapter, may take a considerable amount of time. This is often the case since this process typically involves an initial client/server implementation. 

Typical hardware functional requirements for a gradient HPLC system with UV-Vis detection are listed in Table 3 as an example. 

Functional Requirements. Based on the user requirements, more detailed functional requirements can then be defined. Take as an example a gradient HPLC system with UV-Vis detection required to run a stability-indicating method. The functions of each of the hardware and software components required to perform the tasks in the user requirements should be specified. The functional specifications typically include ... 

The required instrument hardware was only recently commercialized this may explain why the IPC literature concerning U-HPLC is very limited and contemporary. For example, ion-pair U-HPLC coupled with electrospray time-of-flight mass spectrometry played a crucial role in development of rapid and sensitive analytical... 

Validation of HPLC instrumentation, also called qualification, is the procedure that ensures that the instrument is qualified that is, that its performance complies with the method s predetermined requirements, providing reliable and valid results. Modern HPLC systems are computerized, generally consisting of analytical hardware, computer hardware, peripherals, and software. 

Because SFC hardware is somewhat more complex, it is also more costly. In addition, at the semiprep scale, hardware cost should also include a gas delivery system to minimize barrel rolling. On the other hand, a typical SFC uses much less expensive solvent and has roughly 3.5 times higher throughput. To do the same quality work, in the same time, by HPLC, 3.5 times more chromatographic equipment would be required. 

The HPLC of large biomolecules such as proteins and DNA often requires specialized columns packed with wide-pore polymer or silica-based bonded phase with extra-low silanol activity.1215 Alternate approaches are pellicular materials or very small nonporous particles. Some of these columns are packed in PEEK or titanium hardware to allow the use of high-salt mobile phase and to prevent possible protein denaturing by metallic leachates. Further details on bio-separations and application examples are discussed in Chapter 7. 

The price that must be paid for faster, more efficient separations using finer col-unm packings is pressure and the special hardware to handle this. Pressures of 1000 to 3000 psi are required to provide flow rates of 1 to 2 mL/min in columns of 3- to 5-mm diameter and 10 to 30 cm long, although in certain instances pressures up to 6000 psi may be required. Probably 80 to 90% of HPLC separations are performed with pressures of less than 1200 psi, and even some polyurethane column materials require very low pressures near atmospheric pressures. 

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