Second-dimension elution time

Second-dimension elution time range The second-dimension elution time range must be determined. The flow rate needs to be optimized for maximum resolution and speed. This will establish the performance of the second dimension. The elution time range can be tuned with either gradient elution and/or by flow rate to determine the sampling rate. 

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. 

Elevated temperature can also be a very effective way to increase column performance and reduce the second-dimension elution time range as retention is generally reduced at higher temperatures. This has been utilized by Carr and coworkers (Stoll et al., 2006, 2007) to perform very fast second-dimension elution time ranges, and it should be considered for faster chromatographic analysis in general. 

All times are referenced to the start of the second-dimension elution period. Sampling time is I min. 

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. 

Elution with salt pulses A multiple step elution is performed by the introduction of, for example, 5%, 10%, 25%, 50%, and 100% of 1.5 M sodium chloride in 19 mM phosphate buffer (pH 2.5) containing 5% methanol. Each step is for 10 min and run at 0.5 mL/min. This elution method compromises analytical system dimensionality, as the peak capacity of the ion-exchange chromatography (IEX) step is equal at most to the number of salt steps. However, in the second dimension only one or two columns are needed and there is no particular limitation in the second dimension separation time as peptides are eluted in portions in a controlled manner. However, the number of salt steps is limited by the total analysis time. In this case the multidimensional system is relatively simple. 

The HPLC elution time was typically under 260 min, and the CZE analysis took place in 60 s, which led to an overall run time of about 4 h. The 1 min CZE sampling interval was problematic, as the LC column was probably slightly undersampled. A shorter CZE analysis time, which would provide a more frequent sampling rate, would improve this system a great deal. The second-dimension analysis time must be short relative to the first dimension, lest resolution in the first dimension be sacrificed. 

Wraparound The occurrence of second-dimension peaks in subsequent elution sequences, caused by second-dimension retention times that exceed the modulation time of a comprehensive two-dimensional system. 

Figure 5 Colour plot of the GCxGC high-resolution separation of a diesel [32]. Because of the clustering, all the hydrocarbon classes can be identified, from n-C7 through n-Cjg and the branched alkanes in between, up to toluene through C2o-monoaromatics. From naphthalenes (second-dimension retention times 10 s) through the triaromatics in the top of the plot. The insert depicts one single second-dimension chromatogram, showing that in a single one-dimensional peak at least thirty compounds may co-elute.

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... 

Figure 4.8 The GC X GC experiment can be considered to be a series of fast second clno-matograms conducted about five times faster than the widths of the peaks on the first dimension. The ID elution time is the total chromatograpliic run time, wliile the 2D time is the modulation period (e.g. 4-5 s). This figure shows two overlapping peaks A and B, with the zones of each peak collected together. When these slices are pulsed to the second column, they are resolved. Here, we show peak B eluting later on column 1, but earlier on column 2, with the 2D peak maxima nacing out a shape essentially the same as the original peak on 1D.

An example of the results obtained in the form of a chromatoelectropherogram can be seen in Figure 9.6. The contour type data display showed the three variables that were studied, namely chromatographic elution time, electrophoretic migration time, and relative absorbance intensity. Peptides were cleanly resolved by using this two-dimensional method. Neither method alone could have separated the analytes under the same conditions. The most notable feature of this early system was that (presumably) all of the sample components from the first dimension were analyzed by the second dimension, which made this a truly comprehensive multidimensional technique. 

Elution in the second dimension need not be executed until solute is present in the first sample loop. This predelay period allows the first-dimension column void solvent to be dumped to waste collection prior to the arrival of the first retained component. After some initial time, the regular sampling interval 7j = 7) = 7 3 is started. 

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 synthetic and naturally occurring polymers, a few well-established techniques have proven useful. The first column pair to try is RPLC, followed by SEC. As SEC has a limited elution range, it can be used as a very fast second-dimension technique with run times on the order of 1-2 min. There are many examples of fast second-dimension SEC columns in the literature (Murphy et al., 1998a van der Horst and Schoenmakers, 2003). If molecules are small and polar and if the number of different solutes is large, RPLC and NPLC can be combined into a very powerful 2DLC separation system (Murphy et al., 1998b) see Chapter 18. 

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. 

We use the second-dimension separation from Fig. 6.6 with a 25 pL injection volume and 2.5 min sampling time the separation is an RPLC method that uses a monolithic column. Thus, 10 pL/min is the maximum flow rate in the first-dimension. Fig. 6.7 shows the development of the first-dimension column that utilizes a hydrophilic interaction (or HILIC) column for the separation of proteins at decreasing flow rates. The same proteins were separated in Fig. 6.6 (RPLC) and 6.7 (HILIC) and have a reversed elution order, which is known from the basics of HILIC (Alpert, 1990). It is believed that HILIC and RPLC separations are a good pair for 2DLC analysis of proteins as they appear to have dissimilar retention mechanisms, much like those of NPLC and RPLC it has been suggested that HILIC is similar in retention to NPLC (Alpert, 1990). Because the HILIC column used in Fig. 6.7 gave good resolution at 0.1 mL/min and no smaller diameter column was available, the flow was split 10-fold to match the second-dimension requirement... 

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