The Connection Matters
Simple and Intuitive Connection for Micro-LC Columns
The connection technique plays an important role when micro-LC columns are used instead of analytical HPLC columns. Just a few years ago, it was very difficult to get user-friendly “tools and accessories” that enable a simple and intuitive connection. At present, some suppliers offer user-friendly, biocompatible and high-pressure stable components to realize a low dead volume connection between the individual modules and the HPLC column.
Micro-LC columns with an inner diameter (ID) of 300 µm are available with a connection technology of 1/16 or 1/32 inch. For the connection of micro-LC columns, the thread diameter and the type of thread (turns per inch or mm) of the input port must be considered. Commercially available columns based on stainless steel hardware are generally designed for thread diameters of 1/16 or 1/32 inches. For columns with a 1/32 inch connection, these are usually 6-40 or 6-32 threads, while 1/16 inch columns with 10-32 threads can be connected. In order to minimize dead volume and thus also the band broadening, it is recommended to use columns with 1/32 inch hardware. The reason for this is the adjustment or expansion of the outer diameter of the capillary with the aid of so-called sleeves, which can also contribute to band broadening if the connection is incorrect. However, these only should be used if fused-silica capillaries with an outer diameter of e.g. 360 µm are utilized. Since the 1/32 inch ports have a significantly smaller diameter compared to 1/16 inch, fewer sleeves have to be used, which in turn has a positive effect on dead volume and peak broadening.
However, many manufacturers now offer capillaries that allow direct connection without the use of sleeves. These are stainless steel capillaries with an outer diameter of 1/32 inches or capillaries with a 1/16 inches fitting. These capillaries eliminate the use of sleeves. Another alternative is PEEKSil capillaries, which are polymer-coated fused silica capillaries. They are available with an outside diameter (OD) of 1/16 and 1/32 inches in various lengths. The polymer for the coating is polyether-ether-ketone (PEEK), which gives the capillaries mechanical stability.
PEEKSil capillaries are generally considered robust and very pressure-resistant. According to the manufacturer, they also have a lower roughness of the inner surface compared to stainless steel capillaries and the inner diameter can be adjusted more precisely (± 1-6 µm). Furthermore, they are compatible with most organic solvents in the pH range of 0-10 and are considered to be largely inert due to their adsorption characteristics. Figure 1 shows a schematic drawing of such a capillary.
Cutting of Capillaries
It is not recommended to adjust the length of the PEEKSil capillaries with conventional cutting devices, but to select the appropriate commercially available capillary length to avoid cracks and fractures in the fused-silica capillaries. Incorrect cutting can cause a large number of problems. On the one hand, a narrowing can occur if the capillaries are cut with high mechanical pressure. If such a capillary is inserted into the flow path of the HPLC system, the result is usually a significant increase in pressure. Furthermore, the cut itself is problematic, because the user is not able to achieve planar cut at the capillary ends with classical tools such as a capillary cutter. This in turn leads to additional dead volumes, which reduce the separation performance of the system. These effects are reflected in distorted and disproportionately broad peaks with pronounced tailing. This problem also occurs when cutting stainless steel capillaries. Here too, capillaries pre-cut by the manufacturer should be used.
In addition to the already mentioned steel hardware columns, filled fused-silica capillary columns (monolithic or particulate packing) can be purchased. These usually consist only of a fused-silica capillary in which the stationary phase is introduced. Such capillary columns can be coupled directly to the injection valve and the detector without connectors, which counteracts peak broadening. Furthermore, special valves and detector connections with 360 µm ports can be used, eliminating the need for sleeves.
Steel ferrules with suitable screws can be used to connect the capillaries to the columns. Care must be taken to ensure that the angle of the input port is wider than the angle of the ferrule. This is necessary to fix the capillary at the ferrule tip by compression with the screw. If the angle of the input port is identical or narrower, a leaky connection may occur. The use of one-piece or two-piece PEEK fittings is also common but has the disadvantage of lower pressure stability compared to steel.
Steel ferrules are usually irreversibly pressed with the connecting capillaries. If the connection between capillary and column is disconnected, a new ferrule must be used for a new connection. PEEK-Titan Hybrid Ferrules are a good alternative because they can be used several times. These have a PEEK side that adapts universally to almost any port angle and a titanium side that ensures a pressure-resistant connection. Furthermore, these ferrules can be used several times if handled correctly, e.g. when changing columns.
It should therefore be noted that the user now has almost all possibilities for simple and intuitive connection of micro-LC columns at his disposal. All these options offer advantages and disadvantages. In principle, the recommendation would be to use separation columns with a 1/32 inch connection and pre-cut capillaries with universally applicable fittings.
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Thorsten Teutenberg1, Terence Hetzel2, Juri Leonhardt3
1Institut für Energie- und Umwelttechnik e. V., IUTA, Duisburg, Germany
2Bayer AG, Wuppertal, Germany
3Currenta GmbH & Co. OHG, Dormagen, Germany