Instrumental bandwidth

An overview of HPLC instrumentation, operating principles, and recent advances or trends that are pertinent to pharmaceutical analysis is provided in Chapter 3 for the novice and the more experienced analyst. Modern liquid chromatographs have excellent performance and reliability because of the decades of refinements driven by technical advances and competition between manufacturers in a two billion-dollar-plus equipment market. References to HPLC textbooks, reference books, review articles, and training software have been provided in this chapter. Rather than summarizing the current literature, the goal is to provide the reader with a concise overview of HPLC instrumentation, operating principles, and recent advances or trends that lead to better analytical performance. Two often-neglected system parameters—dwell volume and instrumental bandwidth—are discussed in more detail because of their impact on fast LC and small-bore LC applications. 

FIGURE 16 The chromatogram of an injection of a caffeine solution without the column showing the instrumental bandwidth of a Waters Alliance HPLC system with a 966 PDA detector with a standard flow cell. 

The bandwidth values in the table are those calculated for the total system the instrument plus the column. The values for number of plates are for the number of plates realized in the total system. It can be seen that the optimized system does not greatly impact column efficiency, the total loss in plates being only about ten percent at total exclusion for a 24,000 plate column. This is consistent with an instrumental bandwidth equal to a third of the bandwidth of the column. The conventional system, with a bandwidth equal to or greater than that of the column, exhibited a severe loss in realized efficiency, particularly at or near exclusion. 

Table III. Effect of instrumental bandwidth on column efficiency...

Until quite recently, direct measurements of o(>d2)(X) were limited by the very real experimental difficulties associated with the highly efficient deactivation of O ( D2) by O3, as well as the need to provide a sensitive probe for atomic oxygen atoms in the ground Pj state as well as in the electronically excited D2 state. The development of resonance spectroscopic techniques for time-resolved detection of O ( Pi) has permitted monitoring of this state at densities of ca. 10 cm with an instrumental bandwidth in excess of 10 MHz. When combined with the use of high intensity photolysis sources such as the excimer lasers and frequency quadrupled Nd/YAG, it has proved possible to measure directly the yield of 0( D2) and O( Pj) at several discrete wavelengths in the middle ultraviolet. 

This instrumental bandwidth can be physically measured as follows ... 

This chapter provides an overview of modern HPLC equipment, including the operating principles and trends of pumps, injectors, detectors, data systems, and specialized applications systems. System dwell volume and instrumental bandwidth are discussed, with their impacts on shorter and smaller diameter column applications. The most important performance characteristics are flow precision and compositional accuracy for the pump, sampling precision and carryover for the autosampler, and sensitivity for the detector. Manufacturers and selection criteria for HPLC equipment are reviewed. 

Operational specifications Pump precision of retention time <0.5% RSD Composition accuracy <1% absolute Detector noise, <+2.5 x 10"5 AU Auto sampler precision <0.5% RSD, <0.1 carryover System dwell volume <1 mL Instrumental bandwidth <40 pL (4o)... 

Low-dispersion HPLC systems are necessitated by the increasing trend of using shorter and narrower HPLC columns, which are more susceptible to the deleterious effects of extra-column band-broadening. HPLC manufacturers are designing newer analytical HPLC systems with improved instrumental bandwidths compatible with 2-mm i.d. columns by using micro injectors, smaller i.d. connection tubing, and detector flow cells. A new generation of ultra-low dispersion systems dedicated for micro and nano LC is also available. 

If the central plane of the near-confocal FPI is imaged by a lens onto a circular aperture with sufficiently small radius b < (Ar ) / only the central interference order is transmitted to the detector while all other orders are stopped. Because of the large radial dispersion for small p one obtains a high spectral resolving power. With this arrangement not only spectral line profiles but also the instrumental bandwidth can be measured, when an incident monochromatic wave (from a stabilized single-mode laser) is used. The mirror separation d = r - is varied by the small amount e and the power... 

In contrast, the natural bandwidth is an intrinsic property of the sample, independent of the instrument bandwidth, and is defined as the width (in nm) at half the height of the sample absorption peak, as shown in Figure 11. For example, the value for the natural bandwidth of the 340 nm peak of NADH is 58 nm, whereas for most cytochromes at room temperature the natural bandwidths in the a-region are of the order of 10 nm. It is easy to conceive that having too broad a spectral bandwidth would result in an apparent decrease of sample absorption. This is because the incident light would contain a large fraction of radiation with wavelengths poorly absorbed by the sample. 

For grating instruments, this characteristic is formally defined as the convolution of the dispersion of the grating and the slit functions for both the entrance and exit slits. This term is not equivalent to resolution but determines the resolution of an instrument. Bandwidth is sometimes equated to slit width or bandpass. The bandwidth or bandpass can be defined as the full width at half maximum (FWffM) of the bandshape of monochromatic radiation passing through a monochromator. With high-quality optics, the smaller the bandwidth, the higher the resolution... 

Altogether, the results deliver a clear answer to the question, what is the optimal instrumental bandwidth for CS AAS. The bandwidth should never be chosen smaller than two times the FWHM of absorbing lines. Higher values for AAinstr will not significantly reduce the shot-noise limited Amin- Nevertheless they should be avoided to allow the best possible background correction (BC). With respect to the results presented in Section 2.1.5, an optimized value for the instrumental resolving power / = A / AA is between 50 000 and 150000. 

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