Practical Protein Chemistry - A. Darbre 1989

Current state of automated liquid-phase amino acid sequence analysis
Key challenges encountered when using an automated sequencer
Vacuum system

Most difficulties encountered in the automated determination of Peptide and Protein structures are related to vacuum system malfunctions. Troubleshooting leaks in this system and maintaining the required vacuum accounts for the majority of the working time of both the operator and the service engineer. When clearing a leak, the primary task is to locate and isolate the depressurized section. This involves a set of Procedures that enable the operator to pinpoint the exact part of the instrument where the leak has occurred: the Reactor, the line connecting the reactor to the low-vacuum distribution manifold, or the section between the manifold and the vacuum pump.

In most cases, a leak is detected after connecting the entire low-vacuum line to the reactor. This usually helps to determine whether the reactor is the cause of the malfunction. If the result is negative, troubleshooting should proceed from the reactor toward the pump, rather than in the reverse direction.

15.6.1.1. Vacuum leaks in the reactor. There are two potential sources of leaks when a reactor loses its seal. First are the reagent supply lines, cracks in diaphragms, and improper placement of connecting lines; these are the most frequent causes of significant vacuum degradation. Second, leaks (although much less common) can occur in the lines used to withdraw Solvents from the reactor and in the reactor nitrogen purge lines. Finally, O-rings must be thoroughly inspected for distortion, tears, and excessive Swelling.

15.6.1.2. Leaks in the low-vacuum manifold. Three lines branch off from the manifold: one leads to the ballast nitrogen source, the second to the Valves, and the third to the low-vacuum solenoid valve. Each of these lines should be shut off sequentially and the system vacuum checked. We typically use a grease-coated rubber stopper. If the leak is found in the line leading to the ballast nitrogen valve, it is usually caused by a failure of the three-way nitrogen valve connected to the nitrogen distribution manifold. This defect is particularly common in instruments heavily utilized for protocols involving DMAA. A second frequent cause of vacuum deterioration is an excessively high nitrogen actuation pressure in the valve control line, which keeps the valve constantly open. If the leak traces all the way back to the reagent supply valve, it indicates that one of the valve diaphragms has ruptured. This issue is usually identified only by experienced engineers. However, most commonly, the leak traces to the low-pressure solenoid valve; this occurs in instruments that have undergone prolonged operation with DMAA. The low-pressure solenoid valve should be replaced regularly.

15.6.1.3. Leaks between the pump and the low-vacuum manifold. Diagnostics for such failures are performed by disconnecting the line leading to the low-vacuum manifold and sealing it with a grease-coated rubber stopper. The vacuum in this compartment should be ≤50 mm Hg; if this condition is not met, either There is a leak between the manifold and the pump, or the pump itself requires repair or replacement. Replacement can often be avoided by checking the vacuum directly at the pump. Naturally, one should also replace the pump oil, both vacuum gauges, the lines connecting the gauges to the pump, and the connections between the manifold and the pump.