Every single separation method suffers from limited chromatographic resolution with respect to peak purity, limited peak capacity, determination of multiple property distributions and more. This separation dilemma cannot be resolvend, even if powerful and expensive detection methods like mass spectrometry or light scattering are added to a chromatographic system.
However the combination of different separation techniques into a single experiment (multi-dimensional chromatography; also called 2D Chromatography, orthogonal chromatography and cross-fractionation) allows for tackling the core of limited chromatographic resolution by vastly improving the peak capacity. This is specifically important for the analysis of complex polymeric materials. One feature of all polymers is heterogeneity, which arises from the statistics involved in every polymerization process. Even homopolymers prepared by (ideal) living polymerization techniques contain chains of different molar masses, giving rise to a molar mass distribution (MMD). Additionally, in copolymerizations the different comonomers are distributed over the different polymer chains, resulting in individual chains, which will vary in composition (chemical composition distribution). Other heterogeneities arise from differences in end-group functionalization, branching, microstructures etc. Thus in polymer samples the number of different structures easily reaches a few hundreds or even thousands so a high peak capacity would be beneﬁcial for a separation.
In order to separate such complex polymers 2-dimensional chromatography is the method of choice. In 2-dimensional chromatography the sample is fractionated in a 1st dimension by one structural parameter and the fractions are collected. These fractions are then subsequently separated in a second chromatographic experiment separating according to a second structural feature.
2D Chromatography solutions are available from manual and offline to fully automated online solutions and for the transfer of just a few selected fractions to comprehensive transfer of all fractions.
Typical Techniques applied in 2D-LC
The choice of separation techniques for 2D setups is done specifically for the samples to be characterized. Very typical techniqes are GPC/SEC for size separation in one dimension togther with an IPC technique for composition or end group separation in the other dimension. However, also FFF techniques, HDC, SFC, CE or others are used. Orthogonality, i.e. completely independent retention mechanisms for both separations, is not required for useful two-dimensional separations. A substantial increase in information is gained even if retention in both dimensions depends on the same structural features, but to different degrees.
The proper sequence of separations methods is important for highest resolution and accurate determination of property distributions. It has been shown that it is best to apply the method with the highest selectivity for one property as the first dimension.
A comprehensive characterization can be achieved by direct coupling of two liquid chromatographs using a software controlled 8- or 10-port 2-way valve. The valve, which is equipped with two identical sample loops, thereby assumes the function of the autosampler for the second chromatographic dimension. While one loop is filled with a fraction from the first dimension, the contents of the other sample loop is simultaneously analyzed in the second dimension. After each filling of the loop in the first dimension the valve is switched, whereby the loop that has just been filled is now analyzed and the loop, that was just analyzed before, is now filled with the next fraction.
Heart-Cutting is useful when co-elution might be present in just a few fractions of the first dimension. Only those fractions will be analyzed in the second dimension. A typical application is the analysis of polymer blends: after the first dimension separation, chemically uniform peaks are obtained which differ only with respect to molecular weight. Again a valve (here a collecting valve) allows for an easy-to-use solution without to much manual operation. Only those fractions of the first chromatographic separation, which should be characterized further, are stored in the loops of the collection valve.
The simplest approach for the realization of two-dimensional chromatographic separations is the use of a fraction collector. The fractions are separated by the first chromatographic separation column, collected and then analyzed by changing the separation column and/or the eluent using the same chromatograph. Only the separation conditions are different.