Elution solution

Time or volume of eluting solution passed through Figure 15.1. Ion-exchange graph lor kimhanidi . 

Besides resolution, another important factor in chromatography is the amount of time required to elute a pair of solutes. The time needed to elute solute B is... 

The process of changing the column s temperature to enhance the separation of both early and late eluting solutes. 

An eluted solute was originally identified from its corrected retention volume which was calculated from its corrected retention time. It follows that the accuracy of the measurement depended on the measurement and constancy of the mobile phase flow rate. To eliminate the errors involved in flow rate measurement, particularly for mobile phases that were compressible, the capacity ratio of a solute (k ) was introduced. The capacity ratio of a solute is defined as the ratio of its distribution coefficient to the phase ratio (a) of the column, where... 

If the corrected retention volume in the pure strongly eluting solute is very small compared with the retention volume of the solute in the other pure solvent, i.e., V"a V"b, which is very often the case in practical LC, then equation (12)... 

Under these conditions the reciprocal relationship fits the data extremely well, particularly at volume fractions below 0.5 of the strongly eluting solute. In addition, under these conditions the inverse will also apply, i.e.,... 

Figure 4. The Effect of Peak Asymmetry on the Apparent Composition of Closely Eluting Solutes...

As the peak represents the concentration profile of the eluting solute, the fraction of the peak height at which the points of inflexion are located will be the same as the ratio of the solute concentration after (n - Vn ) plate volumes of mobile phase has passed through the column to the solute concentration after (n) plate volumes of mobile phase have passed through the column. 

It is seen that the chromatographer can arrive at the minimum (aA/e) value for a pair of solutes that the column can resolve directly, from either the resolution, as defined by Giddings, or from a simple function of the number of effective plates. However, again it must be emphasized that this will not be a unique value for any column, as it will also depend on the (k ) of the eluted solute. 

Equation (33) shows that the maximum capacity ratio of the last eluted solute is inversely proportional to the detector sensitivity or minimum detectable concentration. Consequently, it is the detector sensitivity that determines the maximum peak capacity attainable from the column. Using equation (33), the peak capacity was calculated for three different detector sensitivities for a column having an efficiency of 10,000 theoretical plates, a dead volume of 6.7 ml and a sample concentration of l%v/v. The results are shown in Table 1, and it is seen that the limiting peak capacity is fairly large. 

The choice of variables remaining with the operator, as stated before, is restricted and is usually confined to the selection of the phase system. Preliminary experiments must be carried out to identify the best phase system to be used for the particular analysis under consideration. The best phase system will be that which provides the greatest separation ratio for the critical pair of solutes and, at the same time, ensures a minimum value for the capacity factor of the last eluted solute. Unfortunately, at this time, theories that predict the optimum solvent system that will effect a particular separation are largely empirical and those that are available can be very approximate, to say the least. Nevertheless, there are commercially available experimental routines that help in the selection of the best phase system for LC analyses, the results from which can be evaluated by supporting computer software. The program may then suggest further routines based on the initial results and, by an iterative procedure, eventually provides an optimum phase system as defined by the computer software. 

It is also clear from equation (2) that the sample mass can also be extended by increasing the capacity ratio (k ) of the eluted solutes (i.e, by careful phase selection). In this case the maximum load will increase linearly with the value of (k ) but so will... 

An alternative elution technique is to transfer the powder (e.g. for bromophenol blue) to a glass column fitted with a glass-wool plug or glass sinter, and elute the dye with ethanol containing a little ammonia. The eluted solution, made up to a fixed volume in a small graduated flask, may be used for colorimetric/ spectrophotometric analysis of the recovered dye (see Chapter 17). A calibration curve must, of course, be constructed for each of the individual compounds. 

Some detectors can give additional information about the elutes (the eluted solutes). One example is the gas chromatograph—mass spectrometer (GC-MS), which produces a mass spectrum of each compound as well as its mass and location in the chromatogram. This powerful means of detection can be used when standard samples are not available to help determine the identities of the solutes. A beam of ions bombards each compound as it emerges from the chromatograph. The compound breaks up into ions of different masses, providing a spread of narrow peaks instead of one peak for each compound. The relative amount of each fragment is determined and used to help identify the compound. 

However, consider the separation of solutes that are more polar than the aromatic hydrocarbons, for example mixtures of ethers or aliphatic esters. If it were attempted to separate a series of aliphatic esters on silica gel employing n-heptane as the mobile phase it would be found that the retention of the later eluting solutes was inordinately long. If a slightly stronger solvent, such as chloroform was used as an alternative to n-heptane, it would be found that the less polar esters... 

Detector Sensitivity or the Minimum Detectable Concentration has been defined as the minimum concentration of an eluted solute that can be differentiated unambiguously from the noise. The ratio of the signal to the noise for a peak that is considered decisively identifiable has been arbitrarily chosen to be two. This ratio originated from electronic theory and has been transposed to LC. Nevertheless, the ratio is realistic and any peak having a signal to noise ratio of less than two is seriously obscured by the noise. Thus, the minimum detectable concentration is that concentration that provides a signal equivalent to twice the noise level. Unfortunately, the concentration that will provide a signal equivalent to twice the noise level will usually depend on the physical properties of the solute used for measurement. Consequently, the detector sensitivity, or minimum detectable concentration, must be quoted in conjunction with the solute that is used for measurement. 

Thus, the minimum value of (a) for any pair of solutes can be calculated for any given column. The minimum values of (a) required for a pair of solutes that will be separated on a column having 25,000 theoretical plates (the efficiency of the standard ASTEC column 25 cm long, 4.6 mm in diameter and packed with spherical particles 5 p in diameter) is shown plotted against the (k ) of the first eluted solute is shown in figure 5. 

Graph of Minimum Separation Ratio for a Solute Pair that Can Be Separated on a Column of 25,000 Theoretical Plates against Capacity Ratio of the First Eluted Solute... 

The chemical constituents of the backwash water (which may be acidified) and of the various eluants (recycling, new and special) are specific to the problem of displacing the captured ions from the resin and reactivating its sites. The final stage of IX, namely, the recovery in a solid form of the values from the eluting solution, is effected by chemical methods which yield the desired precipitate. This is settled out, filtered and dried for despatch. 

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