Calibration of columns

Use of Kq Instead of Vg in the calibration of columns produces a calibration curve that is independent of column dimensions and pore volume. To obtain Kq for any species requires the determination of Vg and Vj in addition to Vg. Vg is usually taken as the elution volume of an excluded polymer while Vj is equal to V-j - Vg. The volume V-j is the total permeation volume of the column and is measured with a low molecular weight compound that totally permeates particle matrices. 

The variance for the weight fraction w(i) can be obtained from equation 1H and the variance for M(i) can be calculated directly from equation 18. Note that the propagation of error analysis can be readily extended to other averages and and it can also be used to account for the errors associated with the calibration of columns and detectors. 

Figure 2. Calibration of column set with THF as eluant using polystyrenes ( ), polymethylmethacrylates ( ) and Igepals (Q)-...

Elution of Lignin Sulfonates with Electrolyte Solution and Calibration of Columns... 

Calibration of Columns. Figure 2 demonstrates that branched dex-... 

Some disadvantages include (1) possibility of colunm-blocking (2) mass balance (particle deposition) (3) calibration of columns required (4) peak-spreading (5) requires removal of large outside or agglomoated particles (6) corrections to detector response are needed. 

Calibration of Gauges Simple liquid-column manometers... 

Liquid level above seal pan in bottom of column. Poor stripping of bottoms product. High pressure drop across section. Board mounted instrument improperly calibrated. Level gauge in field not properly blown down or even checked. Operation problem. 

TABLE 7.3 Selection of Calibration Proteins Commonly Used for the Calibration of Fractogel EMD BioSEC Columns ... 

FIGURE 11.12 Calibration curves of the Ultrahydrogel family of columns. (Courtesy of Waters Corp.)... 

Gels made in this way have virtually no usable porosity and are called Jordi solid bead packings. They can be used in the production of low surface area reverse phase packings for fast protein analysis and in the manufacture of hydrodynamic volume columns as well as solid supports for solid-phase syntheses reactions. An example of a hydrodynamic volume column separation is shown in Fig. 13.2 and its calibration plot is shown in Fig. 13.3. The major advantage of this type of column is its ability to resolve very high molecular weight polymer samples successfully. 

Normally a calibration curve—molar mass against the total retention volume—exists for every GPC column or column combination. As a measure of the separation efficiency of a given column (set) the difference in the retention of two molar masses can be determined from this calibration curve. The same eluent and the same type of calibration standards have to be used for the comparison of different columns or sets. However, this volume difference is not in itself sufficient. In a first approximation the cross section area does not contribute to the separation. Dividing the retention difference by the cross section area normalizes the retention volume for different diameters of columns. The ISO standard method (3) contains such an equation... 

It is evident from these results that the interactive properties of the investigated SEC PS/DVB or DVB gels are very different. Because polar electroneutral macromolecules of PMMA were more retained from a nonpolar solvent (toluene) than from polar ones (THF, chloroform), we conclude that the dipol-dipol interactions were operative. Columns No. 1 and No. 2 were very interactive and can be applied successfully to LC techniques that combine exclusion and interaction (adsorption) mechanisms. These emerging techniques are LC at the critical adsorption point (18), the already mentioned LC under limiting conditions of adsorption (15,18), and LC under limiting conditions of desorption (16). In these cases, the adsorptivity of the SEC columns may even be advantageous. In most conventional SEC applications, however, the interactive properties of columns may cause important problems. In any case, interactive properties of SEC columns should be considered when applying the universal calibration, especially for medium polar and polar polymers. It is therefore advisable to check the elution properties of SEC columns before use with the... 

The precision of SEC must be established before a comparison of columns and calibration standards can be made. Consistency in flow rate or elution time is the first requirement to obtain precision in SEC. Consistency in flow rate or elution time can be monitored by the elution time of the PEO standards, which are run before and after the samples. Elution time or flow rate can be considered consistent if the elution times of the PEO standards before and after the samples agree within 0.1 min. 

A commercially available cationic standard that can be used for the calibration of CATSEC columns is poly(2-vinyl pyridine), or PVP. Cationic PVP can be characterized easily on CATSEC columns over a broad range of molecular weight. DRI chromatograms of two cationic PVP standards using a bank of CATSEC columns (100-, 300-, 1000-, and 4000-A pore size) and a mobile phase of 0.05 N NaNOi/0.1% TFA are shown in Fig. 20.10. 

For ILS, the number of degrees of fieedom is equal to the number of samples minus the number of wavelengths, w, used in the calibration, (i.e. the number of columns in the P matrix) minus 1. 

CYANlDE.dat Section 4.13 Two calibration series over the same range, and one over a short range (three groups of columns Concentration/Signal), and a fourth group that combines all of the above data the data can he fitted to a parabola Y = -0.002125 + 0.005211 X - 0.0000009126 jc 2 with a residual standard deviation of 4.5 mAU. Use with LINREG, TESTFIT. 

Unrecognized shifts in the calibration of the columns will affect the raw data, which are in terms of elution time of the components of the specimen from the chromatographic columns. Use of log hydrodynamic volume allows the operator to correct for such... 

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