Column dimension

The optimum length of the column required for a particular separation is dictated by the number of theoretical plates needed to give the desired resolution. If the column is too short, then clearly the column will not have enough resolving power to achieve the separation and if it is too long, then analysis time is needlessly extended. The most common column 

Recently, there has been a revival in interest in the use of HPLC of columns with smaller internal diameters (i.e. 2 mm or less). Two major features of chromatography with small-bore columns are behind this trend a significant reduction in the consumption of mobile phase, and an increase in the detector signal for a particular mass of solute (i.e. increased sensitivity). 

There is another important benefit in using smaller bore columns. Because of the smaller geometries involved, the same amount of solute injected onto a small-bore column will elute in a smaller volume than that of a larger column. This leads to a higher response at the detector for a particular mass, and therefore to a significant improvement in sensitivity. The major disadvantage associated with the use of miniaturised HPLC systems is the need for certain specialised components. Because the 

The end-fittings of the column come in a number of designs, but all play the same role they provide a means via which the column is connected to the other components of the HPLC system. This simple role has to he achieved without introduction of unwanted dead volume into the system, to avoid uimecessary band broadening. 

In order to attain the best possible efficiency from the chromatographic system, it is necessary to exercise some care in the choice and use of the tubing used to coimect all of the individual components. In particular, the tubing and any coimections should not chemically interact with the solutes or mobile phases used, and should not contribute excessively to the dead volume in the system. In regular-bore HPLC, most analysts use precision engineered stainless steel tubing (which is often passivated to reduce interaction with solutes) with an outer diameter of 1/16 inch, and various internal diameters. 

it is no problem to manufacture a column with 2 Rm packing material, an i.d. of 50 Rm, and 125 mm in length. Regarding efficiency, resolution, and sensitivity, this column represents the optimum. However, provided that the packing material has suitable selectivity for the selected application, for most applications it makes no sense to go into these ranges. The choice of the individual parameters depends on the requirements of the equipment, demands in terms of sensitivity and resolution, and on the stability and robustness of the separation. 

The choice of the i.d. is mainly determined by the possibilities of the HPLC system to generate low flow rates and gradients with high accuracy and precision and the sensitivity needed. The use of 50 Rm i.d. columns with flow rates in the range of 100-200 nL min represents a considerable challenge for both the equipment and user. A further criterion is the choice of the detector, since the implementation clearly depends on the flow rate of the system (see Section 2.7.1.4.3). 

The length of the colutrm depends primarily on the type of chromatography. 

As a rule of thumb, for RPC the following is considered to hold true 1 cm of column length is required to determine one compound. When using 3 Rm particles and gradients, the column length can usually be halved. 

With suitable selectivity, ten compounds can usually be separated on a 50 mm long column see also Chapter 2.7.3 on UPLC. 

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Finally, we note that the size and shape of the particles of the packing, the packing technique, and column dimensions and configuration are additional factors which influence a GPC experiment. In addition, the flow rate, the sample size, the sample concentration, the solvent, and the temperature must all be optimized. Details concerning these considerations are found in analytical chemistry references, as well as in the technical literature of instrument manufacturers. 

Assuming the column dimensions are 320 pm I.D. (radius r=0.0160 cm), 30 m long, and it is operated at 120°C using nitrogen as the carrier gas which, at that temperature, has a viscosity of 129 x 10 Poises, then by using equation (3), the change in (y) can be calculated for different flow rates. The relationship between flow... 

Extra-column dispersion can arise in the sample valve, unions, frits, connecting tubing, and the sensor cell of the detector. The maximum sample volume, i.e., that volume that contributes less than 10% to the column variance, is determined by the type of column, dimensions of the column and the chromatographic characteristics of the solute. In practice, the majority of the permitted extra-column dispersion should... 

In the previous two chapters, equations were developed to provide the optimum column dimensions and operating conditions to achieve a particular separation in the minimum time for both packed columns and open tubular columns. In practice, the vast majority of LC separations are carried out on packed columns, whereas in GC, the greater part of all analyses are performed with open tubular columns. As a consequence, in this chapter the equations for packed LC columns will first be examined and the factors that have the major impact of each optimized parameter discussed. Subsequently open tubular GC columns will be considered in a similar manner. 

Column Bed volume (ml) Column dimensions [diameter (cm) x length (cm)] Column fittings Column materials" (Tube/Frit) Maximum operating pressure (kPa) Maximum linear velocity (cm/hr)... 

Column Column dimension (i.d. X L) (cm X cm) Bed volume (ml) Column materials° (Tube/Frit) Column fittings (inlet/outlet) Theoretical plates (N/m) Maximum operating pressure/flow rate (kPa)/(ml/hr)... 

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. 

TABLE 7.1 Recommended Maximum Sample Volumes for Different SEC Column Dimensions... 

Column dimension Bed height (cm) Maximum sample volume (mi)... 

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. 

FIGURE 7.17 Separation of a complex mixture on Fractogel EMD BioSEC (S) with a column dimension of 1000 X 50 mm (Superformance glass column). The sample contained ferritin (I), immunoglobulin G (2), transferrin (3), ovalbumin (4), myoglobin (5), aprotinin (6), and vitamin B, (7). Five milliliters of the mixture was injected onto the column at a flow rate of 3 ml/min (eluent 20 mAI sodium phosphate buffer, 0.1 M NaCI, pH 7.2). 

Column dimensions mainly determine the quantity of sample to be separated. However, because the SEC process is driven by size separation and is diffusion controlled, special care has to be taken to keep optimized separation conditions, especially when going to smaller internal diameter columns. Overloading and excessive linear flow rates can be observed quite often in these typese of columns. For this reason, standard 8-mm i.d. columns are commonly used, as they are rugged and have a good tolerance toward separation conditions. 

PSS SEC column dimensions were chosen to allow easy scaling of chromatography conditions without the need to optimize separations for each column dimension separately. The volume flow rate and the sample load can be calcu-... 

The volume flow rate between different column dimensions can be calculated easily (assuming constant linear flow velocity) according to... 

Table 9.8 shows examples of preparative separation conditions that allow a simple transfer of one method to a different column dimension (6). 

TABLE 9.8 Scaling of Experimental Conditions for Different Column Dimensions... 

Column dimension i.d. X L (mm) Typical flow rate (ml/min) Sample load (mg) Instrument requirements... 

Individual pore size columns have variable pore volume, and because the column dimensions are fixed, the combination of different columns must result in variable slope of the overall calibration curve and hence variable degrees of resolution as a function of molecular weight. 

The optimum flow rate for most SEC separations using conventional PLgel column dimensions (internal diameter 7.5 mm) is 1.0 ml/min. It may be of some benefit to work with lower flow rates, particularly for the analysis of higher molecular weight polymers where the reduced flow rate improves resolution through enhanced mass transfer and further reduces the risk of shear... 

Typical column dimensions i.d. 7—10 mm Length 25-30 cm Several columns may be coupled in series i.d. 0.1—3 fjLtn Length 100-500 cm Similar to SEC Thin channel Thickness 50-150 /mi Width 1-2 cm Length 30-50 cm... 

Each of the four columns was packed with CPG00120C d = 13.0 nm). The column dimensions and experimental conditions are listed in Table 23.1. The flow rates (solution and solvent) were set to be proportional to the cross section of the column, whenever possible. The number of drops collected in each test tube was almost proportional to the cross section, especially for the initial fractions that might show a shift in M. Figure 23.9 shows chromatograms for some the fractions separated using 2.1-, 3.9-, and 7.8-mm i.d. columns. The result with the 7.8-mm i.d. column is a reproduction of Fig. 23.2 (3). Chromatograms of the fractions obtained from the 1.0-mm i.d. column overlapped with the chromatogram of the injected polymer sample (not shown). 

C-1, first column C-2, second column dimensions given in mm. M-1, first mobile phase M-2, second mobile phase. 

Considering that the separation system is fully characterized, i.e., adsorbent and mobile phases, column dimensions, SMB configuration and feed concentration, the optimization of the TMB operating conditions consists in setting the liquid flow rates in each section and also the solid flow rate. The resulting optimization problem with five variables will be certainly tedious and difficult to implement. Fortunately, the... 

A matrix is a rectangular array of numbers, its size being determined by the number of rows and columns in the array. In this context, the primary concern is with square matrices, and matrices of column dimension 1 (column vectors) and row dimension 1 (row vectors). 

Units in SI system Si Stanton number h/Cf,pu Dimensions depend ort order of reaction. Suffixes 0 Value in bulk of phase 1 Phase 1 2 Phase 2 A Component A B Component B AB Of A in B b Bottom of column equilibrium with bulk of other phase G Gas phase / Interface value. L Liquid phase u Overall value (for height and number of transfer units) value in bulk of phase i Top of column Dimensions in in M. N, 1. T. 

Figure 5. Effect of column dimensions on the chromatographic separation (ordinate absorbance, 340 nm abscissa elution time, min). Column A 1.5 yi 25 cm 442 mL 397 theoretical plates/ft 325 theoretical plates 0.4 mL/min flow rate Column B three 0.9 X 25 cm 47.7 mL 112 theoretical plates/ft 275 theoretical plates 1.05 mL/min flow rate.

Dolan,. W., Snyder, L. R. Maintaining fixed band spacing when changing column dimensions in gradient elution. 

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