Second-dimension column

Efforts have been made, however, to extend the range or extent of samples that can be analysed by using a two-dimensional separation when used in heart-cut mode. This has been reported to include the use of numerous parallel micro-traps to essentially store the primary column eluent fractions ready for second-column separation, and the use of parallel second-dimension columns. 

Unlike the continuous zone development mechanism utilized in a planar separation experiment, comprehensive MDLC is a sequential operation in which finite volumes of eluant are injected into the next dimension column. Because of this finite volume aspect, the mechanism and consequences of sampling eluant from one column with subsequent injection into the next column must be understood. Undersampling would lead to a loss in two-dimensional resolution and oversampling would lead to excessively long run times as the second dimension column would be used in a very inefficient way. 

FIGURE 5.1 Heart-cut 2DLC where a zone from the first-dimension separation is reinjected onto a second-dimension column for improved resolution of three analytes coeluting on the... 

The instrumentation used to implement this comprehensive sampling is the main topic of this chapter. The most common comprehensive mode uses a sampling valve so that second dimension elution can begin as soon as a sampling loop has stored the necessary amount of first column solute. In this case, there is no need for storing the effluent from the first column it is continuously allocated to a sampling loop with subsequent injection into the second-dimension column. 

A 12-port valve was used for the periodic sampling of the first column onto multiple second-dimension columns for the 2DLC analysis of aromatic amines and other species (Venkatramani and Zelechonok, 2003). The utility of the 12-port valve is that two columns can be utilized in the second dimension and flow is kept constant through both columns. This configuration requires three sample loops for implementation. The output of the second-dimension columns are connected so that both columns continuously feed the detector. 

FIGURE 5.11 Timing diagram for comprehensive 2DLC with either a two-position valve (hottom) or four-position valve (top). Repetitive sampling of the first dimension at each time (7i, T2, T3, T4, ,) results in an injection onto the second-dimension column. 

Mass spectrometers that use electrospray ionization (ESI) do not function well if the eluent contains low volatility salts. This is a major concern when an ion-exchange column is used as a first-dimension column and the salt concentration is used to modulate the retention in this column. In this case, another valve can be connected between the second-dimension column and the detector so that any salt from the second-dimension elution process that is either unretained or weakly retained can be diverted prior to feeding zones to the mass spectrometer. 

Sampling The second column method must be as fast as possible to allow for optimal sampling of the first dimension. The method development of the second dimension should be done first. The sampling rate for the second-dimension column should maintain three to four samples across the narrowest peak in the first dimension for optimum 2DLC resolution. Less than three samples across the narrowest peak in the first dimension allow for faster analyses with lower 2DLC resolution. 

S ample loop volumes The volumes of the sample loops that store eluent from the first dimension and inject eluent into the second-dimension column system must be determined. The loop volume divided by the second-dimension elution time range determines the first-dimension flow rate in comprehensive 2DLC. If the dilution factor is small in the second column, a flow splitter can maintain a small loop volume even with a substantial flow rate from the first-dimension column. 

First-dimension optimization The flow rate, elution time range, and the efficiency of the first-dimension column must be carefully controlled and matched to the second-dimension column, the sample loop volume, and the sampling rate. 

For isocratic LC, the solute does not need to fully elute from the second-dimension column prior to the next sampling period. This is illustrated in Fig. 6.4, where it is shown that more than one sample can be resident in the column at one time. Using the chromatograms shown in Fig. 6.5, which show the effect of various injection volumes that will be discussed later, it is not necessary to wait for the full 2 min of sampling time. This significantly helps to speed up the sampling process and is most useful for SEC, where short elution time ranges are typically found and short times between the injection and nonretained peaks are typical of column operation. 

FIGURE 6.4 Zone evolution on the second column showing zones for two different times at each sampling number. Note that there is more than one injection of the sample loop on the second-dimension column after the first injection. 

In complex samples, when the range of elution times may not be known beforehand, there is the possibility of wraparound where components from the previous run are still eluting on the next second-dimension elution (Micyus et al., 2005). This situation is of concern and should be eliminated in the method development process for all but the most exploratory of work. This may require collecting fractions and injecting these fractions into the second-dimension column to determine the most retained compound retention time as part of the method development process. 

In the previously described approaches, different HPLC pumps are used in the two dimensions. A different approach, where the flow from one single pump was splitted and introduced to one first- and two second-dimension columns, was used by Venkatramani and coworkers [45,47]. In this approach, a 12-port valve equipped with three loops [45] or with three guard columns [47] was used as an interface (Figure 4.8). 

Another approach to improve the effective speed of the second-dimension separation is the use of an array of second-dimension columns, used in parallel [10,11,15,24,25,45,47,52-55,57], especially with 1.5 J,m i.d. pellicular columns [10,15,54,55,58]. This approach is more complicated due to the fact that different columns are rarely identical, and it is critical to achieve precision of retention time in consecutive second-dimension separation containing the same analyte peak. 

Most of the traditional HPLC detectors can be applied to LCxLC analyses the choice of the detectors used in comprehensive HPLC setup depends above all on the nature of the analyzed compounds and the LC mode used. Usually, only one detector is installed after the second-dimension column, while monitoring of the first-dimension separation can be performed during the optimization of the method. Detectors for microHPLC can be necessary if microbore columns are used. Operating the second dimension in fast mode results in narrow peaks, which require fast detectors that permit a high data acquisition rate to ensure a proper reconstruction of the second-dimension chromatograms. 

Comprehensive 2D HPLC can be also operated under stop-flow mode. In this case, after transferring a desired fraction volume onto the secondary column, the flow of the mobile phase in the first dimension is stopped and the fraction analyzed in the second dimension. When the separation is finished, the mobile-phase flow in the first dimension is switched on and the whole procedure is repeated again for the analysis of all the transferred fractions. The disadvantage of this procedure is the long analysis time, while the advantage can be that the second-dimension column can give higher plate numbers if compared to the continuous approach [23]. 

The combination of normal (silica) and reversed (C18) phase HPLC in a comprehensive 2D LC system was used for the first time for the analysis of alcohol ethoxylates [64] the NP separation was run using aqueous solvents, so the mobile phases used in the two dimensions were miscible, resulting in the easy injection of the entire first-dimension effluent onto the second-dimension column. 

As a rule, the sequence in which the columns are placed in a column-switching system has a marked effect on the results of a 2D separation. The final choice is dictated by the specific separation objectives. When subsequent fraction cuts have to be performed on the effluent from the first dimension, the column with a higher peak capacity should be placed into the first-dimension system and the flow rate should be matched to the fraction transfer switching period. The fractions transferred to the second-dimension column should be completely eluted before the subsequent fraction is transferred from the first to the second dimension. To increase the peak capacity in the first dimension, gradient elution is preferred to isocratic conditions. 

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