Instrumentation, for HPLC

Shodex Packed Columns for HPLC and Shodex Instruments for HPLC, 95/96, p. 10, Showa Denko. 

This equation can be used to calculate the expected pressure drop across the column. The present instrumentation for HPLC usually allows for column backpressures up to 30-40 MPa, which means that short columns should be used with small-diameter particles not to exceed the pressure limits. 

Although it is beyond the scope of this review to discuss the advances in instrumentation for HPLC, the authors feel it might be helpful to the reader if some recent references to this area were described. It should be realised that in some cases the author of the paper quoted may have a commercial affiliation where this is known it is indicated. 

For many analytical methods using HPLC as the end step, the samples presented to the analyst cannot be injected directly into the instrument. For example, the sample may be a large volume of water where the analytes are present at low concentrations, a solid such as soil or foodstuffs, or a biological specimen where numerous other compounds are present. Sample preparation is therefore needed to isolate the analytes of interest, to pre-concentrate them in order to lower detection levels and also to protect the analytical column from substances which may potentially damage the bed of packing material. Despite many advances in instrumentation for HPLC, only rarely are samples (particularly complex samples) injected without some form of sample pre-treatment. 

HPLC columns contain, usually, spherical particle packings, which are carefully sorted to fractions with narrow size distribution to provide high separation efficiency. Totally porous packing materials most frequently used for separations of small molecules in contemporary HPLC have pore sizes of 7-12 nm and specific surface area of 150-400 m /g, but wide-pore particles with pore sizes of 15-100 nm and relatively low specific surface area of 10-150 m /g, or nonporous materials are used for separations of macromolecules. Perfusion materials, designed especially for the separation and isolation of biopolymers, contain very broad pores (400-800 nm) throughout the whole particle, which are interconnected by smaller pores. Column efficiency and flow resistance increase with small particles, and a high pressure has to be used to maintain required flow rate and to keep an acceptable time of analysis. However, the maximum operating pressure is 30-40 MPa, with common instrumentation for HPLC. Hence, short columns should... 

Profile Gilson is a manufacturer of specialized analytical instrumentation for scientific research and industrial markets since the early 1950s. The company has developed software and instrumentation for HPLC, LC, and sample preparation technologies. It also produces high-precision pipettes and tips and automated instruments and systems for increasing throughput and productivity in the laboratory. 

It is quite useful to view the instrumentation for HPLC in terms of zones according to the following schematic (3) ... 

As mentioned earlier, the pressures and temperatures required for creating supercritical fluids derived from several common gases and liquids lie well within the operating limits of ordinary HPLC equipment. Thus, as shown in Figure 29-1, instruments for SFC are similar in most aspects to the instruments for HPLC described in Section 28C. There are two important differences between the two techniques, however. First, a thermoslatted column oven, similar to that used in GC (Section 27B-3), is required to provide precise temperature control of the mobile phase second, a restrictor. 

The starting point for this chapter is that of the definition of HPLC (high-performance liquid chromatography) adopted by the author namely, that this is a method of separation of the components of a solution effected by the chromatographic process involving a column of solid stationary phase material and a liquid mobile phase in which all of the various components of the system have been designed so as to optimize the separation. In this context, optimization means first and foremost the complete (quantitative) separation of the components but it can also include such separations that are rapid and which have been automated. The key word in this treatment of instrumentation for HPLC is that of design. 

FIGURE 7 Diagram of a photodiode array instrument. For HPLC, the cuvette is replaced by a flow cell similar to that used for the standard UV-VIS detector. [From Siouffi, A-M.. Chapter 1, in Food Analysis by HPLC. (L. M. L. Nollet, ed.), Marcel Dekker, New York. 

A brief account of the individual components will be given here, but specialised texts should be consulted for a detailed description of the instrumentation available for HPLC.51 The manufacturer s instructions must be consulted for details of the mode of operation of particular instruments. 

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. 

Stanley, P. E. (1992). A survey of more than 90 commercially available luminometers and imaging devices for low-light measurements of chemiluminescence and bioluminescence, including instruments for manual, automatic and specialized operation, for HPLC, LC, GLC and microtiter plates. Part I descriptions. T. Biolumin. Chemilumin. 7 77-108. 

Major advances have occurred over the past few years in equipment for HPLC. Figure 2 presents a block diagram of a basic LC instrument. We shall now discuss individual components. ... 

In modern times, most analyses are performed on an analytical instrument for, e.g., gas chromatography (GC), high-performance liquid chromatography (HPLC), ultra-violet/visible (UV) or infrared (IR) spectrophotometry, atomic absorption spectrometry, inductively coupled plasma mass spectrometry (ICP-MS), mass spectrometry. Each of these instruments has a limitation on the amount of an analyte that they can detect. This limitation can be expressed as the IDL, which may be defined as the smallest amount of an analyte that can be reliably detected or differentiated from the background on an instrument. 

At this point it is worth considering the demands made on the instrumentation for operation with wide bore columns and, in particular, the adaptation of analytical Instruments for this purpose [596,597]. The pump requirements for preparative separations differ from those in analytical HPLC as the ability to generate high flow rates at moderate backpressures is crucial to the efficient operation of wide bore columns. A flow rate maximum of 100 ml/min with a pressure limit of 3000 p.s.i. is considered... 

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). 

Instrumentation for fluorescence spectroscopy has been reviewed [8]. For standards in fluorescence spectroscopy, see Miller [138]. Fluorescence detection in HPLC has recently been reviewed [137], Phosphorescence detection of polymer/additive extracts is not being practised. 

In on-line multidimensional HPLC (MDHPLC) two relatively high-efficiency columns are coupled in an instrument, via the use of valves, traps and other means. In LC-LC the precolumn is used for sample cleanup and prefractionation, before introduction of the fraction of interest to the analytical column. Much of the instrumentation for MDHPLC is the same as that in conventional one-dimensional experiments. However, the additional complexity of MDHPLC experiments leads to greater difficulties than those found in conventional HPLC ... 

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. 

Bushey, M.M., Jorgenson, J.W. (1990). Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography of proteins. Anal. Chem. 62,161-167. Cabrera, K. (2004). Applications of silica-based monolithic HPLC columns. J. Sep. Sci. 27, 843-852. 

He et al. (2002) used an off-line HPLC/CE method to map cancer cell extracts. Frozen ovarian cancer cells (containing 107 cells) were reconstituted in 300 pL of deionized water and placed in an ultrasonic bath to lyse the cells. Then the suspension was centrifuged and the solubilized proteins were collected for HPLC fractionation. The HPLC separation was carried out on an instrument equipped with a RP C-4 column, 250 mm x 4.6 mm, packed with 5-pm spherical silica particles. Extracted proteins were dissolved in 300 pL of DI water, and lOOpL was injected onto the column at a flow rate of 1 mL/min. Buffer A was 0.1% TEA in water and buffer B was 0.1% TFA in acetonitrile. A two-step gradient, 15-30% B in 15 min followed by 30-70% B in 105 min, was used. The column effluent was sampled every minute into a 96-well microtiter plate with the aid of an automatic fraction collector. After collection, the fractions were dried at room temperature under vacuum. The sample in each well was reconstituted before the CE analysis with 10 pL deionized water. The... 

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