10


August/September 2010


Excel files and further processed to obtain a 3D-plot using Matlab 6.5.


Results and Discussion Orthogonal systems


RP-systems differing in stationary phase and/or mobile phase and/or temperature were compared using a mixture of 17 ionisable compounds, which were separated by gradient runs using a normalized gradient slope of 5%. The degree of orthogonality between two given RP-systems was assessed by the Pearson regression coefficient r² calculated from the 2D-retention plot of compositions at peak elution. Furthermore, as the interest of 2D-LC mainly relies on its high resolution power, the practical peak capacity was also considered. Only few studies have dealt with this critical parameter [13, 14]


. In this study a novel method


was proposed to determine the practical peak capacity from the 2D retention plots using the confidence envelopes of the regression straight line. The peak capacity in each dimension is derived from the Snyder’s sample peak capacity (Equation 2) [15]


which is related,


for a given sample, to both the retention range Δtr,i


and the mean peak width according to (Equation 2)


2D systems 1 2 3 4


Dimension 1 Dimension 2


Gemini pH 2.7 30°C Acquity Shield pH 2.7 30°C Gemini pH 2.7 30°C Acquity Shield pH 2.7 90°C Gemini pH 2.7 30°C


Hypercarb pH 2.7 90°C Gemini pH 2.7 30°C Acquity Shield pH 6.8 90°C r² 0.97


nc, total 850


0.82 1700 0.02 1846 0.01 5700


Table 2. Combinations of different RP-systems characterized by their degree of orthogonality (regression coefficient r2 practical peak capacity (nc, total)


When comparing two different silica-based stationary phases in the same conditions of both temperature and mobile phase (2D- system #1), the degree of orthogonality is very poor (r² = 0.97) and the resulting peak capacity is therefore disappointing (nc = 850). When the temperature is raised (2D-system #2), the orthogonality is not much better (r²=0.82 vs. 0.97) but the peak capacity is significantly increased (nc


= 1700 vs. 850). This is mainly due where w10%,i , Ce,i , Pi and t0,i are respectively


the mean peak width at 10% of the peak height, the range of composition at elution, the normalised gradient slope and the column dead time in the ith dimension. The practical peak capacity is given by the product of the sample peak capacity in each dimension as illustrated in Figure 3 which displays an example of a 2D retention plot, showing the confidence envelopes and the composition range in each dimension


to the improvement in peak shape at elevated temperature and hence the decrease in the ratio of the peak width to the column dead time (Equation 2). For combinations involving a stationary phase based on another type of material such as the Hypercarb column (2D- system #3), it can be observed that although the orthogonality is excellent (r² = 0.02), the practical peak capacity is not significantly higher than the one obtained with the preceding 2D system. In case of such materials, the peak efficiency is very poor for ionisable compounds and as a result the ratio of the peak width to the column dead time is dramatically high. The variation of the mobile phase pH is also a very efficient way to get orthogonal combinations (2D-system #4). As soon as the pH is different from one dimension to another, the regression coefficient becomes close to 0. In addition, due to the very good peak efficiency with silica- based columns, especially when the temperature is increased, the practical peak capacity is impressive (nc


= 5700).


Influence of the injection volume In 2D-LC, the mobile phase of the first dimension becomes the injection solvent of the second separation. Eluent compatibility is critical for the peak shapes and resulting efficiencies. When using RPLC in both dimensions, the eluents are of the same type and hence highly compatible. However, the maximum possible injection volume has to be determined for a proper design of the instrumentation.


Figure 3. Representation of a 2D retention plot comparing compositions at elution in two selected systems.


Regression coefficients and practical peak capacities of different combinations of RP- systems for a sample of 17 ionisable compounds are given in Table 2.


We investigated which volume can actually be injected relative to the column volume when the compositions at elution for a given solute are the same in both dimensions. The study was conducted on a typical second dimension column (Acquity BEH C18 50 x 2.1mm; 1.7 µm). As can be seen in Figure 4, the peaks superimpose up to 15% of the


critical in HILIC x RPLC, is under investigation in our laboratory.


Instrumental design


The optimisation of the instrumental design was performed assuming orthogonal combinations of two RP-systems. The Van Deemter coefficients were determined with neutral compounds. The objective was to maximize the practical peak capacity, taking into account numerous constraints. Some of them are inherent to successful 2D-LC separations and include (1) a suitable sampling rate (around 3) as well as (2) an injection volume compatible with the second column dimensions (Vinj


<0.15 x V0 ). In


addition, many constraints result from the instrument: (1) the minimal flow-rate for reproducible gradients in the first dimension (20 µL/min); (2) the maximal allowable


) and their


column dead volume (V0) injected. Beyond


this limit, the peak shape is significantly affected and the loss of resolution becomes unacceptable. Consequently, in these conditions, an injection volume up to 0.15 x V0


is appropriate. In isocratic elution, it


was observed that due to the lack of focusing effect, much smaller volumes have to be injected otherwise a strong deformation of peaks occurred (results not shown). It should be underlined that when the eluent strength of the injection solvent is higher than the composition at elution in the second dimension, smaller injection volumes are required. This issue which is


Figure 4. Peak shape of butylparaben depending on the injection volume (expressed as a percentage of the column dead volume). Acquity BEH C18 50 x 2.1mm; 1.7 µm, 30 °C, 0.5 mL/min, 5-85% methanol in 1.65 min, The injection solvent is the same as the composition at elution in the second dimension (78% methanol). The injected quantity is constant irrespective of the injection volume


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