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. 

Unattended assays have unique requirements that manual techniques do not. On-instrument stability is an issue, particularly if the environment of the instrument is hotter than the laboratory bench, the typical case. For example, CK is unstable at 37°C, is light sensitive, and is easily oxidized during storage such concerns impact the validity of CK assays (35). Specimen evaporation can be a serious problem, particularly for small specimens held in containers with large, exposed surfaces for prolonged periods of time (36, 37). Evaporation lids are desirable, but they cannot always be used owing to instrument constraints on the specimen probe. 

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