Column dimensions selection

Based on the requirements of the separation, media of suitable pore size, particle size, and surface properties are selected as well as column dimensions and column material. In some cases a suitable combination of media type and column dimensions may be available as a prepacked column. In most cases, this is a more expensive alternative to preparing the column yourself but will provide a consistent quality as assured by the manufacturing and testing procedures of the vendor. The consistent quality may be critical in obtaining reproducible results and may thus be a cost-effective solution. Also, the fact that smaller particle-sized media are more difficult to pack and require special, and expensive, equipment has resulted in that gel filtration media of small particle size, e.g. smaller than 15 /zm, are predominantly supplied as prepacked columns. 

The flow rate in SEC significantly affects the resolution. Depending on the selectivity wanted, linear flow rates have to be adapted to the column dimensions. In general, running the column at a low flow rate results in higher resolution, but diffusion may produce diminishing resolution when the flow rate is too low. The flow rates recommended for a particular column diameter should not be increased. In the case of Superformance columns, the best results can be obtained by applying linear flow rates of about 30-80 cm/hr. Of course, linear flow rates below 30 cm/hr can contribute to further increased resolution. 

In Excel, the cells comprising the prospective result Y have to be pre-selected as we have already seen for matrix transposition. For this, we need to predict the dimensions of Y from the row dimension of C and column dimension of A. Also, there is no direct operator for matrix multiplication in Excel. The function MMUUI in conjunction with the SHIFT+CTRL+ENTER key... 

In the development and optimization of a comprehensive LCxLC method, many parameters have to be taken in acconnt in order to accomplish snccessfnl separations. First of all, selectivity of the columns used in the two dimensions must be different to get maximum gain in peak capacity of the 2D system. For the experimental setup, column dimensions and stationary phases, particle sizes, mobile-phase compositions, flow rates, and second-dimension injection volumes should be carefully selected. The main challenges are related to the efficient coupling of columns and the preservation of mobile phase/column compatibility. 

FIGURE 11.7 Selective displacement of AVP on DEAE Sepharose Fast Elow. Column dimensions were 10 X 290mm load was 6.5 mL AVP feedstock/mL resin. The flow rate was 1 mL/min. (Reprinted with permission from Elsevier from Barnthouse, K.A. et ah, J. BiotechnoL, 66, 125, 1998. Copyright.)... 

The factors that control separation and dispersion are quite different. The relative separation of two solutes is solely dependent on the nature and magnitude of the Interactions between each solute and the two phases. Thus, the relative movement of each solute band would appear to be Independent of column dimensions or particle geometry and be determined only by the choice of the stationary phase and the mobile phase. However, there is a caveat to this statement. It assumes that any exclusion properties of the stationary phase are not included in the term particle geometry. The pore size of the packing material can control retention directly and exclusively, as in exclusion chromatography or, indirectly, by controlling the access of the solute to the stationary phase in normal and reverse phase chromatography. As all stationary phases based on silica gel exhibit some exclusion properties, the ideal situation where the selective retention of two solutes is solely controlled by phase interactions is rarely met in practice. If the molecular size of the solutes differ, then the exclusion properties of the silica gel will always play some part in solute retention. 

Method development for mass spectrometry portion of LC-MS/MS assay is relatively simple and straightforward. For an experienced method development (MD) scientist, the optimization typically takes only hours to complete. In contrast, method development for optimal liquid chromatography conditions can be one of the most challenging tasks. Chromatography development can be very time-consuming. The task is further complicated by the nearly infinite choices in chromatography options such as vendor, sorbent, solvent selection, particle size, and column dimensions. 

In the case of cephalosporin C extraction, chemical engineers played the major role in designing the large plant needed to meet the projected tonnage off-takes. Plant design was based on the results of resin evaluation and selection, hydraulic studies on the resin bead size and column dimensions, Cephalosporin C loading capacities,... 

FO equals unity this becomes quite impossible. In other words, given the final column for routine analysis, very large values of At are unattractive, since they do not increase the value of FO, but do lead to an increase in analysis time. If, however, we can tailor our column to the result of the optimization procedure (i.e. to the number of plates required), then large values of At leading to very large values of Rs are indeed significant. Hence, in the case where the column dimensions can be chosen after completion of the optimization of selectivity, the use of Rs or S is preferred, because of the clear and simple relationship between these criteria and the required number of theoretical plates. 

Chromatogram c appears to show a much longer analysis time than does chromatogram a. However, if we are free to define the column dimensions after the selectivity optimization process, chromatogram c can be the basis for a very quick separation on a very short column. 

Only the first factor is influenced by the physico-chemical separation process (the selectivity), while the other two factors are determined by the column and the operating conditions, respectively. If C is a continuous criterion (see table 4.7), then both C and C, can be transferred from one column to another. Both column dimensions and flow rate have trivial effects on the analysis time tm. However, if the final analysis is to be run on a different (optimized) column, then it is more logical to use the dimensionless, column-independent factor (1 + km) in eqn.(4.31) instead of tm ... 

The situation in programmed analysis is similar to the one described above for chromatographic elution under constant conditions, in that retention and selectivity may be optimized more or less independently. However, under constant elution conditions the optimization of the retention only involves adapting the primary parameters such that all capacity factors fall into the optimum range (1 programmed analysis the optimization of the retention involves optimizing the characteristics of the program (initial and final composition, slope and shape) in conjunction with the physical parameters (e.g. flow rate and column dimensions, see section 3.6). 

The required number of plates (Nne) is the most relevant factor for the selection of the type of column and the column dimensions. However, there are various other factors which we need to consider in the selection of the most suitable column for a given analysis ... 

A column consisting of a deactivated silica-based stationary phase is used for the packed-column mode. A packed column allows larger volumes of sample solvent to be injected, thus improving sensitivity. Generally, the column dimensions are 1 x 100-250 mm and the particle size is 5 / m. Commercial SFC instruments are also available that will handle the classical 4.6 x 150-mm or 250-mm columns. With the introduction of electronically controlled variable restrictors to control the back pressure, the packed columns are becoming increasingly more popular. This feature allows the independent flow and pressure control of mobile phases, thus helping in rapid optimization of selectivities. Some of the commonly used packed columns are as follows ... 

Figure 4-1. Effect of column type on selectivity. Mobile phase Low pH. (A) 0.1 v/v % TFA. (B) 0.1 v/v% TFA in MeCN. Linear gradient from 5% B to 80% B in 40min, 220nm. Temperature, 40°C flow rate, l.OmL/min column dimensions, 150 x 3.0mm particle sizes, 3.5 pm for Symmetry Shield and Atlantis and 3.0pm for YMC ODS AQ. (Courtesy of Markus Krummen, Novartis Pharmaceuticals.)...

Advances in column technology have improved the selectivity, stability, and reproducibility of LC analytical columns. For example, analytical columns are packed with a variety of stationary phases, providing enormous versatility in the separation process. This section describes (1) column dimensions, (2) particulate column packings, (3) monolithic column packings, and (4) the use of guard columns. 

The sample solution may contain over 100 chemically similar compounds, so an accurate control of the experimental parameter is crucial for a good separation. In addition to the selection of the most suitable stationary phase and column dimension and optimization of the oven temperature program, other experimental parameters are decisive for the band broadening such as a proper injection procedure and a proper carrier gas flow rate. Instead of helium, hydrogen can be used as a carrier gas, thus allowing an increase in the gas flow rate without a loss in column efficiency. ° ° 2° i49 i50... 

Table I summarizes the adsorption selection incorporated into the experimental studies. Four pre-purified synthetic resin sorbents obtained from Alltech Associates (Deerfield, IL) were used along with an activated carbon sample (Union Carbide Grade 6GC) procured from our pilot plant. The coarse particle size of the selected adsorbents in conjunction with the column dimensions and experimental flow rates yielded very small pressure drops (< 2 atm) across the sorbent bed, as ascertained by inlet and outlet pressure gauges before and after the column, respectively. Because of this small pressure gradient within the sorbent column, the recorded experimental breakthrough data were taken essentially at isobaric conditions.

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