locate and correct. A drop in system pressure usually indicates a leak. Leaks at fitting unions can often be fixed by tightening the fitting. However, a fitting may need to be disassembled and cleaned prior to tightening to remove particulates or buffer residues. If this does not correct the leak, it is best to replace the fitting. Leaks at other locations such as detector cells, pump heads and injection valves may require rebuilding or replacing seals. Check the instrument manual or contact the manufacturer for hplc service information.

, mobile phase composition and flow rate before trying other corrective measures. Particulates typically originate from samples, mobile phase, or wear of system components such as pump seals. The fastest way to locate an HPLC problem causing high pressure is to start with the components that contribute most to system pressure. These include first any inline filter or guard column, and then the analytical column. Replace each component in sequence with one known to be in good working order. If an inline filter or guard column is causing the problem, replace it. If the analytical column is blocked, try back flushing the column or replacing the inlet frit. Replace the analytical column if these procedures do not correct the problem. When pressure problems persist, begin at the detector and loosen connecting fittings one at a time, working upstream until the pressure drops dramatically. This can help to isolate the fitting or component causing the problem. Check tubing ends to make sure they are clean and free of filings. Metal fragments from an improperly finished tubing cut can create blockage and cause increased pressure. Precipitation of buffers within the system can cause pressure increases. This HPLC problem is common in buffered aqueous mobile phases when the organic concentration exceeds 50%. If buffer precipitation is suspected, remove the HPLC column and begin flushing the system with a buffer-free 5% organic aqueous mobile phase at a low flow rate. After system pressure stabilizes, reinstall the column and rinse it thoroughly at a low flow rate with the same flushing solvent. Re-equilibrate the system with the standard mobile phase and begin analysis. Premixing and filtering the mobile phase may help prevent buffer precipitation. In general, however, conditions that may lead to buffer precipitation should be avoided because in many cases, if it occurs, buffer precipitation can irreversibly damage an expensive HPLC column. Low pressure often indicates the presence of a leak, a reduction in the flow rate, or a problem with a system component. First, check for leaks throughout the system and ensure the solvent delivery flow rate setting is correct. Verify proper flow rate and pump operation with an in-line electronic flowmeter, or by filling a graduated cylinder from the detector or column outlet with a measured volume of mobile phase over a measured time interval. (Note: Measuring flow in the system with the column disconnected may cause erroneously low readings. Most high-pressure hplc pumps require flow restriction at the outlet for the check valves to operate properly.) If the measured flow rate is correct, check the mobile phase composition. The wrong organic content or the wrong organic solvent, for example acetonitrile instead of methanol, can cause reduced pressure. Column problems are more likely to increase pressure than to cause a decrease, but as a last resort, check the column by removing the inlet frit and observing any loss of packing. If this has occurred, replace the hplc column. If the measured flow is too low, be sure the pump is not being starved for mobile phase. Check reservoir inlet frits and any pump inlet filters for clogging. Also ensure that mobile phase reservoirs are not sealed tightly or below the level of the pump inlet. Check for air bubbles in the pump inlet lines, and if found reprime the pump. Outgassing of mobile phase can produce air bubbles in the pump which result in variable flow and pressure, especially in the case of low-pressure-mixing gradient systems. Be sure all solvents have been degassed properly and recently by sonication, vacuum filtration, helium sparging (preferably continuous at very low flow rate), or by using an inline vacuum degasser. Flush and prime the pump with degassed solvent according to recommended procedure. If this fails to correct the problem, replace the pump check valves. Wear or particulate contamination can cause improper check-valve operation. Either the inlet or outlet check valves can cause the problem. Change the pump seals if seal leakage is detected or if the pressure continues to cycle or remains erratic. Note: Seal leakage may be hidden in pumps with seal-washing head designs.

may result from changes in mobile phase flow rate, mobile phase composition, column stationary phase, and column temperature. If the retention time for the column void volume, t0, changes and there have been no changes in the column configuration, the problem is related to flow rate. System leaks and air bubbles in the pump cause t0 to increase. To troubleshoot, follow the recommendations in the previous paragraph for low-flow problems. Increased flow rates cause t0 to decrease. Verify proper flow rate selection on the pump controller and check the pump flow rate accuracy as described in the “Low Pressure” section.

in one direction without a change in t0 represents a change in the mobile phase. Such changes are often caused by the reduction of at least one liquid mobile phase component due to evaporation or improper mixing procedures. When mobile phase is suspect, mix a new batch and determine if this corrects the problem. Degassing solvents using helium sparging may lead to the evaporation of volatile solvents, thus affecting the mobile phase concentration. Solvent degassing using an Inline HPLC Solvent Degasser (see listing in Instrumentation Section of this catalog) is the recommended alternative to helium sparging.

,the most common source of problems includes mobile phase pH and buffer concentration. Depending on the pKa, the retention time of ionizable compounds can shift up to 10% when the mobile phase pH changes by as little as 0.1 pH unit. Remember to measure the pH of the aqueous component only, as the addition of organic solvents will alter pH readings. Buffer concentrations should be typically 20-50 mM. Lower levels may provide irreproducible results and higher levels may reduce column life.

with the starting mobile phase before sample injection is essential for reproducible retention times. Determine adequate HPLC column equilibration by allowing longer equilibration times to see if the problem improves. Allow at least 10 to15 column volumes of solvent to pass through the column before injecting a sample. When using on-line solvent mixing instrumentation, compare isocratic results with a mobile phase that has been mixed manually. In gradient separations, exchange solvent inlet lines and make appropriate program adjustments. If the system is operating correctly, the results should remain the same in both configurations. Retention time drift may be observed during the first few samples of a given analysis. If retention times move and then stabilize after several sample injections, deactivation of the column may be taking place through sample loading onto the column. This may not be a problem if sample supply is not limited and the resultant chromatography is acceptable. As an alternative, a different type of column chemistry changes in sample preparation procedures, or mobile phase modifiers may help eliminate this phenomenon. If mobile phase modifiers such as ion-pairing reagents are being used, drifting retention times may also be the result of incomplete column equilibration.

such as contaminant buildup or general column deterioration can manifest as retention time drift. A reduction in HPLC column efficiency, or plate number (N), may result in broad peaks with poor retention. HPLC Column contamination that contributes to retention time drift is sometimes accompanied by changes in relative peak spacing and usually takes place over a large number of samples or over a long period of time. If a defective analytical column is suspected, replace or remove the guard column and run several more samples. If this does not correct the problem, or if a guard column is not being used, flush the analytical column with strong solvent to remove any contaminants. If the problem persists, replace the analytical column. Generally, a guard column should always be used to protect an analytical column. Periodic cleaning of the analytical column using strong solvents helps extend useful column lifetime and reduces long-term contamination.

may show up as shifts in retention times that cycle with changes in ambient laboratory temperature. Retention times can change as much as 2% with each 1°C change in ambient temperature, depending on the analysis. It is difficult and expensive to control the ambient air temperature through control of the laboratory air conditioning or heating system. As a cost effective alternative, column thermo stating can be used to either maintain the column at ambient temperature, or slightly elevate the column temperature above ambient. This helps to maintain reproducible retention times without affecting the HPLC chromatography. For a listing of thermo stated HPLC column heaters and HPLC chillers, see the HPLC Instrumentation Section of this catalog.

can also negatively impact retention times. If the sample is injected in a large volume of strong solvent, poor peak shape and irreproducible retention times may result for early eluting peaks. To avoid this problem, samples should be injected using the starting mobile phase solvent whenever possible.

can cause rapid contamination buildup on the guard and analytical columns, and this can reduce column life as well as affect retention times. Using an appropriate sample preparation technique such as solid phase extraction (SPE) may alleviate this problem.