Column selectivity chart

Figure 3.25. RPLC column selectivity chart. X-axis is an indication of the hybrophobicity and Y-axis is an indication of the silanophilic activity of the bonded phase. This comparative chart is useful for selecting equivalent or dissimilar columns from various vendors. Diagram courtesy of Waters Corporation.

Consult column selectivity chart to select similar (equivalent) or dissimilar (with different selectivity) columns based on its hydrophobicity or silanol activity (Figure 3.25). 

Waters Reversed Phase Column Selectivity Chart (also for columns of other manufacturers)... 

TABLE 11.6 Eluent Selection Chart for Ultrahydrogel Columns... 

It follows these steps (1) select the design criteria from a worksheet [Table 7-18(a)] and check off only the major criteria across the worksheet, keeping it simple but realistic (2) refer to the selection chart [Table 7-18(b)] and transfer the bold-faced numerical rating in each selected criteria column to the worksheet, for example, if toughness is one criterion, list 6,4,1, 2, 4, and 2 on the worksheet from top to bottom in the toughness column (3) add up the numbers across the worksheet... 

The procedural guidance to the operators also involves deciding on the format of the new EOPs. The format has been shown to affect the reliability of the control room team in upset situations. This has been the subject of many studies in the past. Different EOP formats are commonly used in the world one column (mostly used for abnormal procedures), two column (flow chart, etc.), and the selection between them should be based on factors like ... 

If time is at a premium, a selectivity chart need not be established - just injecting ethylbenzene/fluorenone at 80/20 methanol/water and phenol/benzylamine at 40/60 methanol/water may sometimes yield quite enough information. Figures 2 and 15 show the hydrophobic characteristics and indirectly the suitability of the phases for certain compound categories, while the peak shape of benzylamine in connection with its elution compared to phenol reveals whether the column in question carries strongly polar/ionic groups on its surface or not. 

If, for example, in the case of a column of group A an unsatisfactory selectivity is obtained, for the next trial one should not use a column from the same part of the selectivity map as this column. It can be expected to have similar properties and thus be equally unsuitable for the test substances. A better choice would probably be a column of group B or C with completely different properties. Various cross experiments have shown that selectivity charts such as Figs. 19 and 20 very well represent the similarity of stationary phases. 

In this section, we explain the principles that influence the selectivity of a reversed-phase column. The first parameter is the hydrophobicity of the stationary phase, which can be measured with purely hydrophobic probes. The second value describes the silanol activity, which is of special importance for basic analytes. Naturally, the silanol activity is best measured using a basic compound, using a correction for the hydrophobic contribution of the structure of the analyte to its retention. The third value is the polar selectivity, which measures the formation of hydrogen bridges between analytes and the stationary phase. With these values, one can create selectivity charts that can help in the selection of the best stationary phase for a particular separation problem. These charts help in method development, whether one would like to find a stationary phase that is drastically different or one that is rather similar to one that is available or in use. At the end of the chapter, we briefly touch on the subject of stationary phase reproducibility. 

When one develops new reversed-phase (RP)-HPLC methods, one usually uses the selectivity of the mobile phase as the primary method development tool. The chromatographic separation can be influenced by the choice of the organic solvent (mainly methanol and acetonitrile), or by variation of pH or buffer type. Schemes for method development using these parameters have been described in the literature [1,2]. Most important are the selectivity changes caused by pH changes, which are well-understood and easily predictable (3). It is well known that the stationary phase influences the selectivity as well, but this effect is often not very well understood. The primary reason for this is the fact that reliable methods for the description of the stationary phase selectivity have only become available fairly recently. In the last few years, several papers have been published that deal with the subject of selectivity in a fimdamental way [4—9] or represent a data collection based on older methods [10-15]. In this chapter, we describe in detail the method used in our laboratory. We then look at our selectivity charts and discuss our results. It needs to be pointed out in advance that selectivity charts only accurately represent the properties of a stationary phase under the conditions of the measurement. If we depart from the mobile phase composition of the test, the relationships between different columns will change, since selectivity arises from a combined effect of the mobile phase and the stationary phase. 

How well these selectivity groupings work, and how these similarities translate into the everyday separations of the chromatographer s work is left to the judgement of the reader. There are many separations in many laboratories that can be carried out on more than one stationary phase. The column selection used for the creation of the charts described here is rather large, and the reader will surely find at least some of the columns that he or she is commonly using. With the information provided here, the interested user can then test the described classification scheme and see how well it applies to his or her separation problem. 

In general, one can be sure that the hydrophobidty of the stationary phase will play the major role, no matter what the analyte is. If the analyte bears basic functional groups, then the silanol activity will surely affect the retention. However, if the analyte does not contain basic functions, then the silanol activity is not very important see also Chapter 2.1.1. The same is true for the polar selectivity. The similarities and differences of stationary phases depend upon the nature of the analytes. For example, the stationary phases Hypersil Elite Cjg, 89, and Synergi MAX RP, 78, are practically identical with respect to hydrophobicity and polar selectivity, but the silanol activity at neutral pH is much lower for the Hypersil Elite Cjg column. Therefore, we would expect very similar separations on both columns if the silanol activity does not play a significant role, but more different separations if the opposite is true. On the other hand, if one is working on a new separation and would like to efficiently exploit the selectivity differences of different columns in an automated method development scheme, then the selectivity charts presented here will certainly be useful in the selection of columns of different selectivity properties. 

The selectivity of a chromatographic method is determined by the column stationary phase chemistry and the mobile phase composition consequently, it is advisable to keep the mobile phase and column chemistry the same when the method is transferred between HPLC and UHPLC. This means that in labs where exchange of methods between instrument types is anticipated, chromatographers are advised to choose column brands where both UHPLC and HPLC columns with the same stationary phase chemistry are available. Table 3.7 lists the column brands currently provided by the major manufactures. It can be seen that not all brands carry both columns packed with the conventional 3-5 xm and the sub-2 xm particle sizes. Within each brand, not all stationary phases are available in both platforms. In such situations, column equivalence assessment needs to be performed to find the best alternative column by using tools such as the reversed-phase colunm selectivity charts available from column vendors. 

Solvent selection and conversion chart for Waters Styragel columns. (Courtesy of... 

Separation of components occurs as the analytes and mobile phase are pumped through the column. Eventually, each component elutes from the column as a narrow band or peak on the recorder. Detection of the eluting components is important, and this can be either selective or universal, depending upon the detector used. Tlte response of the detector to each component is displayed on a chart recorder or computer screen and is known as a chromatogram. To collect, store, and analyze chromatograms, computers, integrators, and other data processing equipment are frequently used. 

Other properties that are heavily influenced by the choice of monomer include cure speed (in general higher functional monomers cure more rapidly), viscosity, and durability of the film. Table 1 lists some monomers, their viscosities, and the properties that they enhance (reprinted with permission from Sartomer). it is important to note several trends on the chart. Cure speed increases with an increase in functionality (all of the recommended monomers in that column are at least trifunctional and several are tetra- or penta-functional). Viscosity also increases as the functionality of the monomer is raised (all of the low viscosity diluents are diacrylates). The adhesion promoting monomers are all di- or mono-functional. Most formulas contain several different monomers and sometimes also oligomers as there is often a balancing act that must be performed when selecting materials that will provide the required performance properties while still maintaining the correct viscosity and surface tension. 

You can use the Chart Wizard toolbutton fl to create a chart. To use the Chart Wizard, first select the data to be plotted, e.g., a column of x values and a column of y values. The data can be in rows or columns. If the rows or columns... 

If more than two columns (or rows) of data are selected for plotting. Excel uses the leftmost column or uppermost row as the independent variable (plotted on the X Axis) and the remaining rows or columns as the dependent variables (plotted on the Y Axis). Figure 5-3 illustrates one column of x data and two columns of y data to be selected for a chart. If the data series are non-adjacent. 

First, click on the outer border of the chart to select the Chart Area. All data series in the chart will be indicated on the worksheet by means of color-coded ranges purple for x values, blue for y values, as shown in Figure 5-9. (If you selected cells containing labels for the columns, they will be color-coded green.)... 

The Chart Wizard always uses the leftmost column (of those that you selected) for the x values when it creates an XY chart. To change the plotting order, so that values from a column other than the leftmost one are used as the x values, you must first create a chart in the normal way, then either ... 

Step 1 Select the data in columns A2 B8. Then choose Insert/Chart and choose the scatter plot with no lines. Follow the instmctions, adding titles, and so on, and place the chart on the spreadsheet. 

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