Preloaded Gradient System Components

The gradient UHPLC is composed of both commercially available and custom

components. An overall schematic is shown in Figure 2-2. The system can be broken down into three individual pieces: hydraulic amplifier, gradient capillary LC (CapLC), and high pressure union & valves. Each piece plays a specific role in the overall gUHPLC.

2.2.1.1 Hydraulic Amplifier

Commercially available pumps with pressure capability over 15 kpsi are currently not available, and any gUHPLC will require some form of customization. It is desirable to use as many commercial components as possible to minimize development of the pump itself. Therefore, a commercial Waters 1525 Binary Gradient pump (Waters Corp., Milford, MA) was modified to pump motor vehicle brake fluid (Castrol N.A., Wayne, NJ), which serves as

an hydraulic fluid in this application. The 1525 pump is connected to a custom-built syringe- driven hydraulic amplifier with a 30:1 amplification, allowing for the generation of ultrahigh pressures from the nominal pressures of the Waters 1525 pump. A diagram of the hydraulic amplifier is shown in Figure 2-3. It should be noted from this diagram that while the

hydraulic fluid is doing the work, it is physically isolated from the mobile phase solvents. Early versions of the amplifier used differing solvents in pistons A & B for real-time gradients, however, this preloaded configuration uses only water in the piston heads.

Commercially available seals capable of holding ultrahigh pressures were obtained from Bal Seal Engineering (Foothills Ranch, CA) and were made out of ultra-high molecular weight polyethylene (UHMWPE). The seals were modified slightly with the addition of a #13 neoprene o-ring (McMaster-Carr, Atlanta GA) on the outside of the seal. This o-ring served as a static seal and increased the pressure capabilities to 40 kpsi. The final seal design was found to have a long lifetime, with the o-ring static seal being the main component to fail. Over three years of consistent use, each piston head was only rebuilt once and the problem was traced to catastrophic o-ring failure. This long lifespan is likely due to the fact that the seal only experiences DI water and is never exposed to a harsher organic solvent. O- ring failure is likely due to absorption of water which ultimately softens the seal. Lifetime of the o-ring could possibly be improved with the use of a different material, but this was not explored.

Several different check valve designs were initially explored by Link. A specially fabricated ball & seat cartridge design by Waters Corp. was eventually settled upon. Earlier designs suffered from poor reproducibility in low pressure sealing and were prone to clogs. This design was improved, but problems still arose with low-pressure sealing. Careful pump

operation normally alleviates the problem, but evaluation of newer designs is ongoing. Finally, all stainless tubing used in this system was brazed with silver solder before use since Waters fittings are only capable of 6 kpsi. As noted by Link, we have yet to see a brazed fitting fail at our operating pressures.

2.2.1.2 Capillary LC

The second major component to this system is a commercial Capillary LC (Waters Corp.). This component is unmodified and serves two main purposes. First, by using a commercial gradient system the gradient generation was found to be significantly more reproducible. Second, the CapLC incorporates an auto sampler, which was a significant limitation of earlier designs. Work with proteins typically involves numerous samples, and some form of run-to-run automation is required. Overall, few problems were encountered with the CapLC and autosampler.

2.2.1.3 High-Pressure Union & Valving

The hydraulic amplifier and CapLC are coupled via a high pressure 4-port union (custom made, Waters Corp.). This union contains a 400 μm through hole to which various capillary and stainless steel tubing connections can be made. The outlet of the hydraulic amplifier is connected to the inlet of the union via 6 m of 0.020” i.d. (~1.5ml) stainless steel gradient storage tubing (GST). This large dead volume of tubing serves to hold the preloaded gradient from the CapLC before ultrahigh pressures are applied.

An open-tubular 120-cm x 10 μm i.d. splitter capillary, packed chromatographic capillary and gradient inlet capillary from the CapLC are attached to the remaining ports on the union to create a closed system capable of ultrahigh pressures. A picture and internal layout of the 4-port union are shown by Figure 2-4a & b. The column was positioned ~17 mm in front of the splitter outlet to create a narrow injection plug. Pressure at the head of the column is

controlled by the length and inner diameter of the splitter capillary and the volumetric flow rate of the amplifier syringe pump, typically 4 μl/min.

A novel freeze-thaw valving design has been implemented to reduce the dead volumes that are present in the system. A freeze-thaw valve (FTV) utilizes liquid CO2 to freeze a

small volume of liquid inside a capillary column.6 Once frozen, this plug is capable of withstanding UHPLC pressures in narrow i.d. (< 50 μm) capillary columns, essentially creating a “closed” valve. By heating the capillary, the plug can be thawed and the valve “opened”. The valve, shown in Figure 2-5, consists of a capillary sandwiched between two copper plates to which CO2is applied. A resistive thermofoil heater is also integrated into

the design in order to open the valve by thawing the frozen plug. Since the capillary is acting as the valve, only the width of the copper plates, typically 2 cm, introduce dead volume. For a 30 μm i.d. capillary, ~14 nl of dead volume would be introduced, which is considered minimal for μl flow rates.

This configuration has also proven to be quite robust. Few leaks are introduced by the various ports, with the typical source being the GST connections. The FTV are even more reliable as a single capillary has undergone hundreds of F-T cycles with no breakage. Additionally, since no mechanical wear takes place as in a typical valve, the FTV never develops leaks.