Column parameters

The Smith-Brinkley Method uses two sets of separation factors for the top and bottom parts of the column, in contrast to a single relative volatility for the Underwood Method. The Underwood Method requires knowing the distillate and bottoms compositions to determine the required reflux. The Smith-Brinkley Method starts with the column parameters and calculates the product compositions. This is a great advantage in building a model for hand or small computer calculations. Starting with a base case, the Smith-Brinkley Method can be used to calculate the effect of parameter changes on the product compositions. 

Recalling that a separation is achieved by moving the solute bands apart in the column and, at the same time, constraining their dispersion so that they are eluted discretely, it follows that the resolution of a pair of solutes is not successfully accomplished by merely selective retention. In addition, the column must be carefully designed to minimize solute band dispersion. Selective retention will be determined by the interactive nature of the two phases, but band dispersion is determined by the physical properties of the column and the manner in which it is constructed. It is, therefore, necessary to identify those properties that influence peak width and how they are related to other properties of the chromatographic system. This aspect of chromatography theory will be discussed in detail in Part 2 of this book. At this time, the theoretical development will be limited to obtaining a measure of the peak width, so that eventually the width can then be related both theoretically and experimentally to the pertinent column parameters. 

Column design involves the application of a number of specific equations (most of which have been previously derived and/or discussed) to determine the column parameters and operating conditions that will provide the analytical specifications necessary to achieve a specific separation. The characteristics of the separation will be defined by the reduced chromatogram of the particular sample of interest. First, it is necessary to calculate the efficiency required to separate the critical pair of the reduced chromatogram of the sample. This requires a knowledge of the capacity ratio of the first eluted peak of the critical pair and their separation ratio. Employing the Purnell equation (chapter 6, equation (16)). 

The person who has tested the column collects all necessary columns parameters and prepares the PSS column quality certificate. 

Most size exclusion chromatography (SEC) practitioners select their columns primarily to cover the molar mass area of interest and to ensure compatibility with the mobile phase(s) applied. A further parameter to judge is the column efficiency expressed, e.g., by the theoretical plate count or related values, which are measured by appropriate low molar mass probes. It follows the apparent linearity of the calibration dependence and the attainable selectivity of separation the latter parameter is in turn connected with the width of the molar mass range covered by the column and depends on both the pore size distribution and the pore volume of the packing material. Other important column parameters are the column production repeatability, availability, and price. Unfortunately, the interactive properties of SEC columns are often overlooked. 

Kessler D.P and Wankat P.C (1988) Correlations for Column Parameters, Chem Eng, Sept 72. 

This expresses tR as a function of the fundamental column parameters t0 and k tR can vary between t0 (for k = 0) and any larger value (for k > 0). Since to varies inversely with solvent velocity u, so does tR. For a given column, mobile phase, temperature, and sample component X, k is normally constant for sufficiently small samples. Thus, tR is defined for a given compound X by the chromatographic system, and tR can be used to identify a compound tentatively by comparison with a tR value of a known compound. 

The important column parameter t0 can be measured in various ways. In most cases, the center of the first band or baseline disturbance, following sample injection, denotes t0. If there is any doubt on the position of t0, a weaker solvent (or other unretained compound) can be injected as sample, and its tR value will equal t0. A weaker solvent provides larger k values and stronger sample retention than the solvent used as mobile phase (see Table 15.1 for a list of solvents according to strength). 

S.No. Name of Substance Column Parameters Solutions Calculations... 

It is of course possible to generalise the above equations for any number of parameters. Having a column parameter vector p of length np, we can write ... 

It is seen that the separation ratio is independent of all column parameters and depends only on the nature of the two phases and the temperature. Thus providing th sam phase system is used on two columns, and the solutes are chromatographed at the same temperature, then the two solutes will have the same separation ratio on both columns, The separation ratio will be independents the phase ratios of the two columns and the flow-rates. It follows, that the separation ratio of a solute can be used reliably as a means of solute identification. ... 

The Rb-82 generator permits serial studies in the same patient as often as every 10 minutes with 20-60 mCi of Rb-82 for rapid bolus intravenous infusion. Inherent in the administration of high levels of Rb-82 activity is the need for precise flow control from an automated system to deliver the desired amount of radioactivity. The development of the alumina column parameters and the elution protocol as well as the automated microprocessor system controller are presented here. Some of the details of this system have been discussed in earlier publications (15,21). Generator produced Rb-82 is used as a diffusible flow tracer in myocardial perfusion studies and as a nondiffusible tracer in brain studies to assess blood brain barrier permeability changes in patients with brain tumors or Alzheimer s type dementia. 

The three experimental parameters A, B and C are related to column parameters and also to experimental conditions. If H is expressed in cm, A will be expressed in cm, B in cm2/s and C in s (where velocity is measured in cm/s). The function is a hyperbolic function that goes through a minimum (//mjn) when ... 

One of these factors can be improved usually at the expense of another. Thus, a number of column parameters need to be discussed so that we can arrive at an efficient operation of a column. We now look at several of these and illustrate with appropriate relationships. 

Equations 2.109 and 2.111 illustrate that the required number of plates will depend on the partition characteristics of the column and the relative volatility of the two components, that is on K and 3. Table 2.9 gives the values of the last term of Equation 2.109 for various values of k. These data point up a few interesting conclusions If k <5 the plate numbers are controlled mainly by column parameters if k >5 the plate numbers are controlled by relative volatility of components. The data also illustrate that k values greater than 20 cause theoretical number of plates, n, and effective number of plates, N, to be of the same order of magnitude, that is,... 

It is important to specify detectors independent of column parameters and of sample size. One parameter that does this is minimum detectable level, MDL. It is the "level" of sample in the detector at the maximum of the peak, when the signal-to-noise ratio is two. The term detectability is sometimes used for MDL. Variations of this definition are sometimes given which require the signal-to-noise ratio to be either one, three, or five. The parameter is also defined sometimes in terms of root-mean square (rms) noise. Peak-to-peak noise can be taken as six times rms noise. 

The maximum value of CRF-4 will be obtained for a separation that provides the required resolution in the shortest amount of time, assuming that column parameters remain the same. Note therefore, that the retention factors (k s) of equation 9 must be converted to retention times (t s) via the simple relationship tR = (1 + k ). Direct use of retention factors instead of times in equation 10a is not generally recommended unless it is known that t0 is constant over the range of densities and temperatures employed. 

The efficiency can be varied by changing physical column parameters such as the length, diameter, and construction material of the container of the column. It can also be varied by changing chemical parameters such... 

Column Parameters in Thermal Field-Flow Fractionation, M. E. Hovingh, G. E. 

The quality of LC columns and LC separations can be measured in several ways. A recent paper96 summarizes some testing procedures, and Table 21 is taken from that publication. Four commercial columns are compared using the column parameters we have defined and described n, H, h, and values presented provide convenient comparisons against which one can judge his/her own column s performance. It is highly recommended that any new or repacked column be tested to evaluate its performance, and that it be periodically reevaluated to determine when it needs to be repacked or dis-... 

Several other parameters, apart from the nature of the reversed phase, are important in the determination of the exact column best suited for a given separation. Such parameters as particle size, shape of particles, range of particle size, pore size, surface area of the silica particles per gram, amount of reversed phase per gram, the presence of polymerized surface coating, the use of a secondary silanization reagent, the stability of the reversed phase to aqueous conditions, and the column efficiency are all important details. In the following discussion, we will attempt to clarify the relevance of these column parameters to a protein separation. 

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