Multicapillary Columns for Chromatography

The principles and theory of high-speed capillary chromatography have been known since the 1960s. To realize the maximum speed capabilities, it is necessary to use capillaries with a diameter of approx. 5–50 μm. However, capillary columns with a diameter smaller than 100 μm are rarely used because they require very small sample quantities that are difficult to inject and detect. To overcome this problem, multicapillary columns (MCCs) composed of a large number of capillaries were developed.1 Due to much larger surface and cross-sectional areas, MCCs overcome the flow rate, volume, and sample capacity limitations associated with single-capillary columns. MCCs are compatible with standard chromatographic equipment and work with all common sample sizes and injection techniques. No extensive modifications of the injector and detector are needed.

Figure 1 - Cross-section of a multicapillary column with capillary diameters approx. 20 μm.

A cross-section of an MCC is shown in Figure 1. The capillaries in an MCC are highly uniform but are not identical, with a radius dispersion of about 2%. According to Poiseuille’s law, the mobile phase velocity in wider capillaries is higher than in smaller capillaries. Obviously, the efficiency of an MCC cannot be better than that of a single capillary. Assuming that the stationary phase film thickness in the capillaries is proportional to the capillary radius in power a, the height equivalent to a theoretical plate (H) of an MCC can be expressed by the following equation1

where HC is the H of an average radius capillary, His the H for a nonsorbing analyte, kis the retention factor of the analyte in the capillary of an average radius, and a is a constant that depends on the method used for the stationary phase deposition. The MCC efficiency is maximal at a = 3.

Multicapillary gas chromatography

Typically, an MCC for GC contains 1000–4000 capillaries of 10–40 μm i.d. in a monolithic glass rod of 1–3 mm o.d. in a 20–100 cm helix. The characteristics of MCCs in comparison to other types of GC columns are listed in Table 1.

The high sample capacity of an MCC decreases the chance of overloading the column. Samples with a wide range of concentration of analytes require large injection volumes for the accurate detection of the less-concentrated components. The sample capacity of an MCC with a 0.2-μm stationary phase film thickness exceeds that of a conventional 0.53- mm-i.d. capillary column with a 0.25-μm film thickness.When there is no need for extreme sample capacity, an MCC is also a superior alternative to a packed column.

A van Deemter graph demonstrates the efficiency of a chromatographic column expressed as height equivalent to a theoretical plate versus linear velocity of a mobile phase. The minimal region on the curve represents the optimal linear velocity that provides the highest efficiency. MCCs with 40-μm-diam capillaries produce more theoretical plates per meter than a conventional single-capillary or packed column, though their short length hinders their total efficiency. Compared to conventional single-capillary columns, MCCs have a much wider optimal mobile phase velocity window on a van Deemter curve. This allows one to use high flow rates in order to elute strongly retained analytes, and use low flow rates for fast-eluting components.

Figure 2 - a) Analysis of industrial solvent by multicapillary gas–liquid chromatography. b) Analysis of C1–C4 hydrocarbons by multicapillary gas–solid chromatography.

The greatest advantage of MCCs is the speed of analysis. Separations are achieved approximately ten times faster than on conventional single-capillary columns. The ultrarapid analysis of the industrial solvent by gas–liquid chromatography is shown in Figure 2a. Seven hydrocarbons were separated in less than 10 sec. Figure 2b illustrates the analysis of C1–C4 hydrocarbons by gas–solid chromatography. Ten hydrocarbons were separated in less than 15 sec.

Multicapillary liquid chromatography

The diffusion of molecules in liquids is much slower than in gases. For sufficient mass transfer, the diameter of capillary LC columns should be substantially smaller than that of the capillaries used in GC. Due to miniscule sizes and sample capacity, LC capillary columns are not compatible with standard chromatographic equipment and have very limited applications. Virtually all routine LC separations are conducted on packed columns. The MCCs overcame this limitation and have the potential to substantially shorten the analysis time of countless samples in a wide variety of LC applications.

The separation of the adjacent chromatographic peaks is quantitatively expressed by the resolution factor (RS) defined as the distance between the two peak centers divided by the average peak width. The resolution factor can be expressed by Eq. (2) via the major chromatographic parameters of selectivity (α), efficiency (N), and retention (k2) for the second peak2:

In HPLC, α ≥1.1 is common and k= 4–5 is optimal. It means that the baseline resolution of two analytes (RS ≥1.25) can be achieved on a column having N ≤5000. In other words, most mixtures can be separated with sufficiently good resolution on the column, providing a total efficiency of approx. 5000 theoretical plates.

The manufacture of MCCs for LC is complicated. A stationary phase, insoluble in common organic and water-organic solvents, must be deposited on the capillary walls in a manner that compensates for the polydispersity of the capillaries. At present, MCCs for LC provide up to 2000 theoretical plates per column of 15–25 cm length. However, progress in this area is rapid. MCCs for LC with an efficiency of 5000 theoretical plates per column will soon be commercially available.

Figure 3 - Separation of 1) uracil, 2) fluorene, and 3) phenanthrene by multicapillary LC.

MCCs are used for the following applications: fast HPLC separations, the fractionation of complex mixtures, desalting of biological samples, solid-phase extraction, and concentration of samples prior to instrumental analyses. Figure 3 illustrates a fast separation of a mixture of three organic compounds.

One of the most integral aspects in the preparation of a good biological sample for further instrumental analysis is the elimination of impurities. For example, the analysis of bovine serum albumin digest peptides by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) suffers greatly due to the presence of salts, buffers, and low-molecular-weight organic compounds commonly used in the preparation of biological samples. MCCs are highly desirable for quick and effective desalting, purification, and fractionation of digest peptides, and other biological samples prior to MALDI-MS and other instrumental analyses.