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Plumbing and Dead Volume Reduction Tips

It is always wise to follow good HPLC plumbing practices. At higher flow rates 5-8 µL/min the capillary conversion is robust and can take a lot of abuse, but if you want your new capillary HPLC to run at top performance take extra care to reduce unnecessary dead volume and leaks. While the capillary system will run with finger tight connections, we prefer to use the appropriate metal nuts and ferrules for all connections.  Metal connections last longer and it is easier to be sure that the manufacturer specifications are met for ferrule and receptacle. For example the tee and capillary column normally come with the appropriate nut and ferrule making the likelihood of connection dead volume due to incompatibility less likely.  Valco, Waters and Vydac replacement nuts and ferrules are readily available.  Also, take care that the tubing is bottomed out or flush when swaging and making connections.

HPLC Tubing

We construct the restriction capillary from 50 micron ID fused silica tubing. (see Figure 1). The tubing coming from the HPLC pumps can be made from any appropriate HPLC tubing (stainless steel, PEEK™, fsc etc...). For all connections to the capillary column we use 50 micron ID fused silica tubing (fsc).  The volume of 50 centimeters of a 50 µM ID fsc tube is approximately 1 µL, so apparently long lengths of tubing contribute relatively low dead volumes.  The fused silica with it's polyimide coating is very pliable and easily cut to size with a ceramic knife.  Fused silica, due to it's small size and pliability is also ideal for plumbing within an existing HPLC.

Swaging Ferrules to Fused Silica.

Care must be taken when swaging ferrules to fused silica to avoid creating dead volume at the nut.  Commonly a short length of sleeve tubing is fitted over the fsc before tightening the ferrule.  Bottom out the sleeve tubing within the ferrule and nut assembly then insert the fsc until it touches bottom. Swage the assembly tight, then loosen the assembly and inspect the end and make sure the fsc is flush with the end of the sleeve tubing. When the assembly is loosened the fsc can be adjusted in or out to adjust for gaps, also this allows for future "freshening" of the fsc end.  The polyimide coated fused silica capillary is robust and is not often crushed during the swaging process.  Care must be taken to choose sleeve material that has an internal diameter that is slightly larger than the outer diameter of the fused silica, too large and gaps may form between the fsc and the sleeve, too small and it becomes difficult to fit the fsc into the sleeve.  Special care must be taken when swaging fsc into injection valves.  If the fused silica protrudes past the PEEK sleeve the injection valve has the potential of being scratched when the valve is rotated.  A good practice is to tighten the entire fitting assembly (sleeve, fsc, nut, ferrule) into the switching valve until it is relatively tight then loosen and remove it for inspection, pull the fsc back flush with the PEEK sleeve.  After tightening the fitting the sleeve should provide some resistance on the fsc making precise positioning of the fsc possible when it is removed for inspection.   Fused silica, sleeves and other supplies can be found on the "order components page."

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Last updated:  Friday, April 29, 2005 08:17:36 AM