Column instrument constraints

The information contained in the three data bases provides the necessary information required to design the optimum column. In addition, once the column has been designed, and its properties defined, a complementary set of Analytical Specifications can also be calculated. Thus, the design protocol contains three data bases. Performance Criteria, Elective Variables and Instrument Constraints. 

Finally, the speed of response of the detector sensor and the associated electronics once played an important part in optimum column design. The speed of response, or the overall time constant of the detector and associated electronics, would be particularly important in the analysis of simple mixtures where the analysis time can be extremely short and the elution of each peak extremely rapid. Fortunately, modern LC detector sensors have a very fast response and the associated electronic circuits very small time constants and, thus, the overall time constant of the detector system does not significantly influence column design in contemporary instruments. The instrument constraints are summarized in Table 2... 

There are a number of limitations on the use of extremes of temperature in HPLC. Clicq et al. [91] note that instrumental issues become increasingly limiting as one goes to very high temperatures and flow rates. They suggest that most separations will occur below 90°C where there are less instrumental constraints. As detailed below, column bleed can limit the selection of columns. Highspeed separations require a faster detector response than many systems allow and constrain extra column volume. This is especially true for narrow bore columns and sub-2 jam particles. In many cases, the additional speed gained above the temperature limits of commercial HPLC ovens will not be worth the additional expense and complexity required. For macromolecules, the effect of extreme pressure can also impact retention time as noted by Szabelski et al. [92]. 

Finally, the analyst is left with some choice in the strategy that can be used In the analysis by way of the chromatographic media selected, and in the level of some operating variables that may be considered appropriate or necessary. The range of variables left to the choice of the analyst constitutes the the third data base necessary for optimum column design and this will be termed the elective variables. However, as most of the conditions that need to be specified will be defined under performance criteria and determined under instrument constraints, the analyst is not left with a very wide choice of variables from which to choose. This might be considered advantageous, however, as the fewer the decisions that are left in the hands of the operator, the less skill and experience will be required and fewer mistakes will be made. 

The column design protocol, therefore, consists of three data bases, performance criteria, elective variables and instruments constraints. These data bases will provide, firstly, the column specifications and finally, the analytical specifications. A diagram representing the overall design protocol is shown in figure (1). The four different components of the column design protocol will now be discussed in detail. 

COI.ITMNST7F. Analytical reversed phase columns for proteins and peptides range from 50 to 250 mm in length having 4 to 5mm i.d. Columns with 2mm i.d. have also been employed for peptide separations in microsequencing applications. The use of microbore columns with less than 2 mm i.d. is severely hampered by instrumental constraints due to small extra-column dead volumes and difficulties in obtaining accurate... 

The instrumental constraints, such as the maximum acceptable pressure drop over the column. 

There are probably several different methods that could be developed to solve the same problem. The decision to use one method over the other is frequently based on the availability of a certain column or detector. The method chosen may not even be the best available in a perfect world, but it may be the best given financial, time or instrumental constraints. Certain sacrifices may have to be made on sensitivity, accuracy, and the ease of analysis, e.g., the number of different runs needed. It is even possible a decision may have to be made on whether the total goal can be accomplished. 

Si(Li) detectors without Be windows ("windowless") or with thin metal-coated polymer films (Ultra-Thin Window UTW) have become an important peripheral to modern-day AEMs for the qualitative detection of elements with 5vacuum requirements because the removal of the Be window increases the probability of detector contamination (from the specimen or column environment) and consequent degradation of performance [12]. Windowless and UTW Si(Li) detectors are commonly installed with additional airlock mechanisms and only on instruments with acceptable levels of vacuum cleanliness. Thus, design constraints on modern AEMs preclude placement of the UTW detector close to the sample. In addition, loss of detection efficiency at low energies (light-element K-lines with the L-lines of transition metals all conspire to limit windowless or UTW EDS analysis of minerals to a qualitative basis only. 

Preferably, however, we may still optimize the dimensions of the column after we have established an optimum phase system. The available instrumentation puts constraints on the column that may be used and hence, ideally, we should also have the possibility to select the most appropriate instrumentation for a given application. 

In this section we will identify other constraints imposed by the instrumentation on the dimensions of the column. Two factors need to be considered in this respect ... 

The ultimate in selectivity in HPLC detection is seen with the use of mass-spectrometric detection, and for many applications this could be seen as the ideal detection method. However, more mundane considerations such as size of the instrumentation and limited budgets combine to reduce HPLC-MS to a relatively small number of applications which most effectively exploit its unique properties. When such practical constraints are taken into account, the real detector coimected to the HPLC system usually turns out to be a device that is a compromise, and its performance characteristics need to be taken into account during the development of many analyses just as much as the performance of the column or any other component of the HPLC system. For example, lack of detection selectivity may require extra method development to completely resolve an interfering peak, or lack of sensitivity could force the inclusion of an extraction-concentration step in an analytical method to achieve detectable levels of analyte. 

Many reports have been given dealing with the constraints of open-tubular column LC Instrumentation. There Is no doubt that open-tubular column LC poses the most stringent requirements on the pump (e.g., 0.01 til/min controlled flow... 

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