HPLC instrumentation

Ion-exchange columns can be substituted into the general HPLC instrument shown in Eigure 12.26. The most common detector measures the conductivity of the mobile phase as it elutes from the column. The high concentration of electrolyte in the mobile phase is a problem, however, because the mobile-phase ions dominate the conductivity, for example, if a dilute solution of HCl is used as the mobile phase, the presence of large concentrations of H3O+ and Ck produces a background conductivity that may prevent the detection of analytes eluting from the column. 

Size-exclusion chromatography can be carried out using conventional HPLC instrumentation, replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is the most common means for obtaining the chromatogram. 

The basic instrument required for packed-column unified chromatography is shown schematically in Figure 7.9. This is essentially a two-pump HPLC instrument utilizing high-pressure mixing with just a few new components. At least one pump must... 

Many HPLC instruments are already furnished with temperature controls for the column. Unified chromatography requires a much wider temperature range than is currently practiced in HPLC. Until better defined by experience, a temperature range from about —60 to about 350°C seems reasonable as a specification. Since this is well in the range of a GC oven with subambient temperature capability, no new technology is required. 

Other properties of solvents which need to be considered are boiling point, viscosity (lower viscosity generally gives greater chromatographic efficiency), detector compatibility, flammability, and toxicity. Many of the common solvents used in HPLC are flammable and some are toxic and it is therefore advisable for HPLC instrumentation to be used in a well-ventilated laboratory, if possible under an extraction duct or hood. 

The HPLC instrument is more expensive and thus costs more to run because it is used 12 to 16 hours per day, versus only 2 to 5 hours/day for the photometer, the hourly rates are not proportional to the initial investment and servicing needs. The cost structure given in Table 4.6 can now be derived. 

Small bore or microbore is a term used for hplc columns that have diameters less than about 2 mm. Columns of this type were first used as long ago as 1967, but at that time the influence of extra-column dispersion was not appreciated, so that the columns were not used in chromatographs of appropriate design. In 1977 there was a renewal of interest in the properties of small bore columns, but it is only in the last few years that systems have become commercially available that allow the potential of small bore columns to be realised. Several manufacturers are now marketing a range of small bore columns, and a number of recent hplc instruments are claimed to be compatible with them. 

Most of the hplc instrumentation now in use is unsuitable for small bore columns. At the moment, the technique is used mainly in the applications laboratories of some instrument manufacturers (they are interested in selling it ). The method is potentially attractive in areas where sample sizes are very limited, for example in biochemical or life sciences applications, but whether or not it becomes widely accepted remains to be seen. 

Many commercial hplc instruments optionally provide a forced air oven which will control temperature with a stability of typically 0.1 °C from ambient temperature to 100 °C. Because of the use of flammable solvents, safety considerations are important, so the ovens are usually provided with a facility for nitrogen purging and are designed to prevent the build up of solvent vapour in the event of a leak. If temperature control is used, it is important that the sam-... 

The earliest form of constant pressure pump in hplc (the coil pump) used pressurised gas from a cylinder to drive mobile phase from a holding coil through the column. This type of pump was used in some of the older hplc instruments, but is now only of historical interest. If you want to know any more about it, there are details in most textbooks. 

Most widely used is the two-dimensional combination of SEC and HPLC for copolymer characterization. The typical HPLC instrument is very similar to an SEC apparatus. While the ideal SEC separation is exclusively determined by entropy changes, in HPLC or adsorption chromatography it is assumed that no... 

FIGURE 3.6 Decreasing peak dispersion of a conventional Agilent 1100 Series HPLC instrument by stepwise optimization of components. System dispersion determined by injecting low volumes of acetone without a column. (Courtesy of Michael Woodman, Agilent Technologies, Inc.)... 

A standard curve was then generated by plotting the log k w data against log P values obtained for all compounds cited in the literature. The standard curve was then used to determine log P for the unknown compound. For comparison, the procedure was repeated using HPLC instrumentation with... 

Most modern HPLC instruments include a column oven that can thermostat the column to at least 100°C. A typical HPLC analysis can be done in half the time by elevating the column temperature from ambient to 50 or 60°C. At temperatures above 100°C, it is not uncommon to decrease analysis time by a factor of 5.26 Also, re-equilibration time for the column is much shorter, so it is possible to achieve ultra-fast gradient analysis with HTLC. 

Current commercial silica-based columns have two important characteristics (1) they can produce efficiency similar to that of columns packed with 3.5 /tm particles and (2) they typically produce a pressure drop of half that caused by a column packed with 5 /tm particles.35 Monolithic columns have been shown to exhibit flat van Deemter curves, resulting in little loss of efficiency at high flow rates.36 As a result, high-throughput separations on conventional HPLC instruments can be achieved by increasing flow rate up to nine times (up to 9 ml/min) the usual rate in a conventional packed column. Cycle times for HPLC analysis as short as 1 min (injection-to-injection) have been reported by users of monolithic columns. Additional benefits of monolithic columns cited include... 

FIA has also found wide application in pharmaceutical analysis.214,215 Direct UV detection of active ingredients is the most popular pharmaceutical analysis application of FIA. For single component analysis of samples with little matrix interference such as dissolution and content uniformity of conventional dosage forms, many pharmaceutical chemists simply replace a column with suitable tubing between the injector and the detector to run FIA on standard HPLC instrumentation. When direct UV detection offers inadequate selectivity, simple online reaction schemes with more specific reagents including chemical, photochemical, and enzymatic reactions of derivatization are applied for flow injection determination of pharmaceuticals.216... 

Column diameter is an important parameter to consider in life science applications in which sample amounts are very limited and the components of interest may not be abundant. Researchers have reviewed micro HPLC instrumentation and its advantages.910 Nano LC-MS offers 1000- to 34,000-time reductions in the dilution of a sample molecular zone eluted from nano LC columns of 25 to 150 [Mi IDs in comparison to a 4.6 mm ID column. This represents a large enhancement of ion counts in comparison to counts obtained for the same amount of sample injected into a conventional 4.6 mm column. Solvent consumption for an analysis run or sample amount required for injection in a nano LC application may be reduced 1000 to 34,000 times compared to amounts required by an analytical column operated at a 1 mL/min flow rate. 

The mobile phase in HPLC is called the eluent and is a liquid or a mixture of liquids. Common eluents are water, aqueous solutions, acetonitrile, and methanol. Almost any other common solvent compatible with the column packing and the detector may be used. In some cases, the HPLC instrument will be capable of making a mixture of eluents or changing the mixture of eluents during chromatography. If this is done, care must be taken to make sure that the eluent mixture is compatible with the detector. 

A simple system is comprised of an isocratic pump, a manual injector, a UV detector, and a strip-chart recorder. A schematic diagram of an HPLC instrument is shown in Fig. 15.4. This simple configuration is rarely used in most modern laboratories. A typical HPLC system is likely to consist of a multi-solvent pump, an autosampler, an on-line degasser, a column oven, and a UV/Vis or photodiode array detector all connected to and controlled by a data-handling workstation. Examples of modular and integrated systems are shown in Fig. 15.5. Some of the important instrumental requirements are summarized in Table 15.2. 

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