34 May / June 2014


Considerations for the Use of LC x LC


A. Soliven1 1


2 , T. Edge2


Australian Centre for Research on Separation Science (ACROSS), School of Science and Health, University of Western Sydney, Parramatta, NSW, Australia. Thermo Scientific, Runcorn, UK


The resolution improvement of a 2DLC system makes it an extremely powerful separation tool. Exploiting 2DLC is vital for separations demanding higher peak capacities than afforded by 1D approaches. Valve operation is a crucial aspect of 2DLC and achieving the best out of various configurations available to operate different modes of 2DLC (heart cutting and comprehensive) will be discussed. We will also highlight specific applications to demonstrate the power of 2DLC to separate complex samples.


1. Introduction


Two-dimensional liquid chromatography (2DLC) is a high-resolution separation tool best exploited for complex samples where peak capacity powers beyond the grasp of conventional 1D methods are required. The power of 2DLC to practically resolve chemically similar species is achieved by transferring aliquots from the first dimension separation system via a sampling/fractioning device into the second dimension separation system [1].


The peak capacity (maximum number of resolvable peaks) of a 2D separation (nc,2D


nc )


defined by Guiochon et al. [2] and Giddings [3] is equal to the multiplication of the first dimension peak capacity (1 second dimension peak capacity (2


) and the nc


in Eq. (1) using the nomenclature from Stoll et al. [1]:


(1) (4)


Comprehensive online 2DLC (LC × LC) is the most powerful 2D separation modes with respect to peak capacity per unit time 2D [1]. During LC × LC the sampling time (ts


) of


the first dimension is equal to the second dimension total cycle time (2


tc tg ), which


equates to the second dimension separation time (2


equilibration time (2


) and the second dimension re- treeq


) shown in Eq (2) [4]: (2)


Eq. (1) over-estimates the LC × LC practical peak capacity. Stoll et al. defined the effective peak capacity of a LC × LC separation as follows [5]:


2. The resolving power of LC × LC


The “crossover” time (τ) is the best way to illustrate the power of 2D over a 1D method by calculating the analysis time


(3) where fcoverage takes into account the use of


the entire 2D separation space and β the under-sampling of the first dimension [5]. Under-sampling often occurs, as most LC × LC studies do not abide the Murphy-Schure- Foley rule, that the loss of resolution can be avoided if the first dimension peak width is sampled at least 4 times across a 81 width, where 1 [6].


σ peak σ = peak standard deviation ); shown


In cases where under-sampling occurs, the Davis-Stoll-Carr under-sampling correction factor [7] must be applied, shown in the re-written form in Eq. (4) [4,7], and is used in various forms by other prominent 2D research groups [9-11]:


when peak capacity of a LC × LC and 1D optimised approach are equal for gradient separations (LC elution conditions for maximum peak capacity). The analysis time includes the practical aspects of a gradient separation: the system dwell time, column re-equilibration and the separation window (actual gradient time).


A study by Huang et al. experimentally investigated the overall resolving power of LC × LC (using Eq. (5)) and the effect of the sampling time of the first dimension [12]. Their findings experimentally validated that the maximum resolving power of LC × LC is achieved at an intermediary sampling rate in line with previous theoretical studies [4,13,14], of 12-21 s for all 1


tg runs [12]. For their intermediary sampling rates (when ts =


12 and 21 s) crossover times were calculated to be in the range of 5-7 min in line with previous experimental and theoretical findings [5, 15].


where 1 dimension and 1


w is the 4σ peak width of the first tg


is the first dimension gradient time. Assuming that fcoverage was 1.0


and that severe under-sampling occurred Li et al. approximates that the corrected peak capacity shown in Eq. (5) [4]:


(5)


Most recently a study by Potts and Carr theoretically compared the performance of an optimised 1D vs. LC × LC method [16], the crossover time and the effect of 1 2tc, 2


nc nc fcoverage and fcoverage , were studied. When the = 0.4-0.7 (based on previous results


that studied the effect of the first dimension phases and mobile phase eluent strength for LC × LC [17,18]) the crossover time was calculated between 3-8 min. With the increase of fcoverage


(a more “orthogonal”


separation) a direct and fast reduction of the crossover time occurred.


The effect the fraction of time dedicated to the separation (gradient time/analysis time) denoted by λ for the first dimension λ was


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