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When evaluating column mass overloading, consideration should also be taken of the retention mechanism. For purely hydrophobic interactions, the above statements are valid, however where a molecule has an associated charge, the charge interactions also have to be considered. Charged molecules typically load at levels10-50 times less than comparable neutral molecules. The understanding here is that if the analyte is adsorbed to the surface through a hydrophobic interaction then the charge will be exposed, effectively repelling other analyte molecules from the surface. For porous stationary phases the repulsion effect could result in analyte molecules being effectively excluded from the pore structure. In a purely hydrophobic retention mechanism, this clearly does not happen. To alleviate this issue, altering the pH to ensure that the analyte is in a neutral form will result in a larger loading capacity.
Another consideration is the elution mechanism and whether an isocratic or gradient system is being employed. A gradient system allows for refocussing of a peak as it progresses down the column, whereas an isocratic elution does not. In terms of overloading, both elution techniques will ultimately suffer from too much mass loading, however the refocussing that is observed in a gradient elution will help to alleviate some of the observed peak effects.
Volume overloading
The second type of overloading that can occur is due to injecting too much solvent. This results in tailing peaks, Figure 2. The tailing is caused by the analyte molecules having different elution start times due to the injection solvent taking time to load onto the column. This phenmomena is more noticeable under isocratic conditions, where there are no focussing effects, but can to a certain extent be disguised when using gradient elution.
which can happen through diffusional processes or through any form of retention on the stationary phase. Reducing the injection volume will favour the retention, also improving the peak shape. An added benefit of improving the peak shape is the increase in peak height and hence better sensitivity. An example of the effect of injection volume and solvent composition on the peak height is given in Figure 3. It should be noted that the injection solvent may be a very relevant consideration when there are solubility issues or if some form of sample preparation is being performed on the original sample. An example of this could be protein precipitation or SPE, which could result in the injection solvent having a highly elutropic solvent.
Figure 3. The effect of varying the injection volume on peak height for two types of injection solvent. Methanol / phosphate buffer (50/50 v/v), Indomethacin (100
ng/mL) injected onto C18 column (120 x 1.0 mm), data taken from [2] Scaling injection volumes
Equation 1 allows the phase ratio of two different columns to be determined which will give guidance on the relative amount that can be loaded onto each column. This equation is quite complicated and a different equation can be used if the same type of column is being scaled down. Thus if the same packing material substrate and the same column length are being used then the following equation can be employed;
Figure 2. Schematic of column overloading caused by an increase in injection volume
The type of injection solvent employed can also limit the amount of analye that can be loaded onto a column. For an efficient separation to occur, the analyte molecules must immediately partition into the stationary phase upon injection, and then be controllably eluted by the B solvent. If the injection solvent has too high of an elutropic strengththe analyte molecules will start to migrate down the column before the loading of the sample is complete. The injection solvent at that point is controlling the elution and not the the bulk mobile phase. The most common reason for use of too strong of an injection solvent is samples with poor solubility in weaker solvetns. To reduce this effect the analyte molecules have to migrate away from the injection solvent,
Equation 2
Since the concentrations of the samples are nominally the same, the Amount term can be replaced with an injection volume term. It can be seen therefore that there is an inverse square relationship between the column radius and the amount that can be loaded onto the column. This would suggest that there are substantial sensitivity disadvantages to using capillary columns, however since most of the detectors that are employed are concentration sensitive detectors, there is also an increase in the sensitivity of the instrumentation which will offset the loss in sensitivity. The HelpDesk [3] has discussed this in a previous article. In summary the following equation determines the relative response that is observed for different column i.d.’s
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