Elution peaks

Figure 3. A Composite Peak Formed by Two Closely Eluting Peaks of Different Size...

Figure 5. Curves Relating Apparent Separation Ratio Relative to Actual Separation Ratio for Two Closely Eluting Peaks...

Considerable care must be taken when accessing closely eluting peaks. If the resolution is inadequate, measurements must be taken on the individual solutes, chromatographed separately on the column. 

Equation (16) was first developed by Purnell [3] in 1959 and is extremely important. It can be used to calculate the efficiency required to separate a given pair of solutes from the capacity factor of the first eluted peak and their separation ratio. It is particularly important in the theory and practice of column design. In the particular derivation given here, the resolution is referenced to (Ra) the capacity ratio of the first... 

Having established that a finite volume of sample causes peak dispersion and that it is highly desirable to limit that dispersion to a level that does not impair the performance of the column, the maximum sample volume that can be tolerated can be evaluated by employing the principle of the summation of variances. Let a volume (Vi) be injected onto a column. This sample volume (Vi) will be dispersed on the front of the column in the form of a rectangular distribution. The eluted peak will have an overall variance that consists of that produced by the column and other parts of the mobile phase conduit system plus that due to the dispersion from the finite sample volume. For convenience, the dispersion contributed by parts of the mobile phase system, other than the column (except for that from the finite sample volume), will be considered negligible. In most well-designed chromatographic systems, this will be true, particularly for well-packed GC and LC columns. However, for open tubular columns in GC, and possibly microbore columns in LC, where peak volumes can be extremely small, this may not necessarily be true, and other extra-column dispersion sources may need to be taken into account. It is now possible to apply the principle of the summation of variances to the effect of sample volume. 

Now, from the plate theory, this transient concentration change will be eluted through the column as a concentration difference and will be sensed as a negative or positive peak by the detector. The equation describing the resulting concentration profile of the eluted peak, from the plate theory, will be given by... 

The theoretical treatment given above assumes that the presence of a relatively low concentration of solute in the mobile phase does not influence the retentive characteristics of the stationary phase. That is, the presence of a small concentration of solute does not influence either the nature or the magnitude of the solute/phase interactions that determine the extent of retention. The concentration of solute in the eluted peak does not fall to zero until the sample volume is in excess of 100 plate volumes and, at this sample volume, the peak width has become about five times the standard deviation of the normally loaded peak. 

The curves show that the peak capacity increases with the column efficiency, which is much as one would expect, however the major factor that influences peak capacity is clearly the capacity ratio of the last eluted peak. It follows that any aspect of the chromatographic system that might limit the value of (k ) for the last peak will also limit the peak capacity. Davis and Giddings [15] have pointed out that the theoretical peak capacity is an exaggerated value of the true peak capacity. They claim that the individual (k ) values for each solute in a realistic multi-component mixture will have a statistically irregular distribution. As they very adroitly point out, the solutes in a real sample do not array themselves conveniently along the chromatogram four standard deviations apart to provide the maximum peak capacity. 

It is clear that for this type of detector to be effective and produce the true Gaussian form of the eluted peak, then (Ca) must, at all times, be unity and, consequently, the... 

The column is a small bore column and, thus, the eluted peaks have a relatively small peak volume, which is commensurate with that of the sensing cell. It is seen that even a sensor volume of 1 pi has a significant effect on the peak width and it is clear that if... 

Column Length 15 cm, Column Diameter 1 mm Particle Diameter 5 micron, k of First Eluted Peak, 1... 

Column design involves the application of a number of specific equations (most of which have been previously derived and/or discussed) to determine the column parameters and operating conditions that will provide the analytical specifications necessary to achieve a specific separation. The characteristics of the separation will be defined by the reduced chromatogram of the particular sample of interest. First, it is necessary to calculate the efficiency required to separate the critical pair of the reduced chromatogram of the sample. This requires a knowledge of the capacity ratio of the first eluted peak of the critical pair and their separation ratio. Employing the Purnell equation (chapter 6, equation (16)). 

Capacity Ratio (first eluted peak of the Critical Pair) (k ) Capacity Ratio (first eluted peak of the Critical Pair) (k") Viscosity of the Mobile Phase (r])... 

Capacity Ratio (first eluted peak of the Critical Pair) (k ) 2.5... 

Employing equation (1), curves relating maximum sample volume to the capacity ratio of the first eluted peak for different separation ratios were calculated and constructed and the results are shown in Figure 2. 

If unidentified peaks are detected the stability of the protein under the chromatographic conditions should be checked. In all analytical investigations of proteins on SEC columns it is desirable to be able to monitor the eluted peaks at a very high sensitivity of the ultraviolet detector. Therefore, very pure (analytical grade) salts and buffers should be used. 

Figure 2.7 Gas clnomatogram obtained for 500 jl1 of diluted gasoline in -pentane inti O-duced by concunent eluent evaporation, using w-heptane as the co-solvent. Reprinted from Journal of High Resolution Chromatography, 11, K. Grob and E. Muller, Co-solvent effects for preventing broadening or loss of early eluted peaks when using concunent eluent evaporation in capillary GC. Part 2 w-heptane in w-pentane as an example , pp. 560-565, 1988, with permission from Wiley-VCH.

The non-intrusive manipulation of carrier gas effluent between two columns clearly has significant advantages in two-dimensional GC. In addition, a pressure-driven switch between the columns introduces no extra band broadening to an eluting peak. 

The ability of a GC column to theoretically separate a multitude of components is normally defined by the capacity of the column. Component boiling point will be an initial property that determines relative component retention. Superimposed on this primary consideration is then the phase selectivity, which allows solutes of similar boiling point or volatility to be differentiated. In GC X GC, capacity is now defined in terms of the separation space available (11). As shown below, this space is an area determined by (a) the time of the modulation period (defined further below), which corresponds to an elution property on the second column, and (b) the elution time on the first column. In the normal experiment, the fast elution on the second column is conducted almost instantaneously, so will be essentially carried out under isothermal conditions, although the oven is temperature programmed. Thus, compounds will have an approximately constant peak width in the first dimension, but their widths in the second dimension will depend on how long they take to elute on the second column (isothermal conditions mean that later-eluting peaks on 2D are broader). In addition, peaks will have a variance (distribution) in each dimension depending on... 

As described above, resolution can be improved by variations in plate number, selectivity or capacity factor. However, when considering the separation of a mixture which contains several components of different retention rates, the adjustment of the capacity factors has a limited influence on resolution. The retention times for the last eluted peaks can be excessive, and in some cases strongly retained sample components would not be eluted at all. 

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