Capillary Electrophoresis (CE) Troubleshooting Tips:

What follows is a short list of problems, "observations" followed by a list of areas that should be investigated, as appropriate in parenthesis (), to troubleshoot common problems seen when using the analytical technique of capillary electrophoresis, CE, CZE.

 Observation (Investigate for cause):

            Excessive Baseline Drifting up or down

·         Temperature is not stable (stabilize room and/or capillary temperature).

·         Fouling of capillary (replace or clean and wash capillary with fresh, filtered solution).

·         Current levels unstable (loose connections, partial obstruction in capillary or running out of buffer solutions).

·         Capillary may have poorly cut ends resulting in poor connections or flow (replace capillary).

Excessive Signal Noise

·         Detector has air in flow cell (purge capillary and wash flow path).

·         Current level may be too high (reduce current).

·         Detection parameters, wavelength and bandwidth, may be inappropriate for buffer solution (select appropriate detection settings which are appropriate for the buffer used and selective for the analyte).

Loss of Signal

·         Voltage/Current has turned off (turn ON or investigate if system is in “alarm” state due to an error).

·         Detector parameters not selected.

·         Capillary has not been fully equilibrated (equilibrate capillary and auto-zero the scale).

·         Baseline offset may be off-scale (after equilibration, adjust scale or auto-zero).

·         Detector lamp(s) off, not ignited or due for replacement (verify lamp operation).

Signal Peak Shape Issues

·         Truncated, clipped or ‘square’ peaks (sample overload, reduce concentration 10x, shorten load time and re-evaluate).

·         Tailing peaks often result from very high current or when the concentration of buffer is too high (lower the current and/or reduce the buffer concentration, then re-evaluate).

·         Sampling rate may be too low (measure the peak width in units of time (i.e. seconds), then configure the detector to insure that the sampling rate allows for at least 20 points to be collected per average peak width (30 points is a better target # to use).

·         No peaks observed (Many possible causes, including: Partially or fully obstructed capillary, broken capillary, out of buffer, no injection, detector settings inappropriate for analysis, current too low, pressure too low. Look for a small peak from the injection along the start of the baseline to confirm that an analysis was started, then troubleshoot the method and settings).

            General Stability and Noise Issues 

·   When the CE system has not been used in a few days, salts from the buffer solution(s) may deposit on and clog the capillary line, flow cell and/or sensors. To avoid these problems, be sure to thoroughly clean, flush and wash down the flow path before use. Take the time to prepare fresh filtered solutions (each day) and allow time for the system to equilibrate. Taking these basic steps will avoid many hours/days of frustration.

HPLC Mobile Phase Composition and LC-MS Electrospray Voltage

I am often asked about the importance of selecting and optimizing the LC-MS Electrospray Ionization Interface (ESI) voltage. To better understand why it is necessary to do so and how it effects the results obtained, let us review some key facts about ESI first.

• While a gas sheathed flow of volatile mobile phase is directed into the MS source, a strong positive or negative electric field (KV) is applied across the MS inlet. The effluent is atomized and evaporated to form ions (voltage polarity determines positive/negative mode).
• Too high of a capillary voltage may produce electrical arcing resulting in damage to the system (e.g. PEEK needle may melt, burn and/or clog).
• Too low of a capillary voltage and ion evaporation will not occur.
• The voltage needed to produce efficient desolvation and ion evaporation are directly related to the sheath gas flow rate, the mobile phase composition and the flow rate.

What Can You Do To Insure Finding A Suitable ESI Capillary Voltage?

1. High quality HPLC methods which utilize fully volatile mobile phases and first retain, hold, then elute all samples are needed to generate LC-MS or LC/MS-MS methods. Optimize the HPLC column type, dimensions, MS compatible mobile phase composition and flow rate before optimizing the MS settings. If you have enough sample available, use an infusion method (continuous flow injection) to establish the initial MS settings needed to detect the sample before continuing with the LC/MS method development optimization. Infusion (with a syringe pump) provides the needed time to makes changes, observe how they change the signal for fastest optimization.
2. The HPLC mobile phase and any dissolved additives or buffers used for LC/MS analysis must be of high purity and fully volatile.
3. Make sure your sample is fully dissolved in the mobile phase and filtered (0.22 u filter) before injecting into the system.
4. Basic samples can be protonated to form [M+H]+ clusters in acidic mobile phases.
5. Acidic samples can be deprotonated to form [M-H]- clusters in basic mobile phases.
6. The electrospray ionization (ESI) process used in LC/MS or LC/MS-MS analysis is affected by the surface tension of the HPLC mobile phase used. Water has a higher surface tension than most organic solvents (i.e. Methanol, Acetonitrile, Ethanol, IPA). Using conventional flow rates with highly aqueous mobile phases requires a higher initial voltage for ion evaporation to occur. IOW: Mobile phase mixtures high in water content will require a higher capillary voltage.
7. Higher organic solvent content usually leads to better atomization / droplet formation and require less capillary voltage to maintain.
8. Lower HPLC flow rates usually lead to better atomization / droplet formation and require less capillary voltage to maintain.
9. To optimize the ESI capillary voltage it is necessary to carry out experiments trying different voltages and monitoring the signal (S/N of a standard or sample) to find the best voltage which results in good signal quality and low noise. This process requires experience to know which settings are likely to enhance the signal and a great deal of skill operating the Mass Spectrometer.

Optionally, ESI signal output may be enhanced using: Adducts or changing the solution chemistry with other mobile phase additives.

Power and Surge Protection for Computers & Analytical Instruments (e.g. Uninterruptible Power Supply AKA UPS)

(1) When selecting an Uninterruptible Power Supply (UPS) for your computers and analytical instruments, only purchase one which outputs a true sine wave voltage (just like real AC power). Most of the UPS systems sold at the local office and electronic supply stores output either square wave A/C or pseudo-sine wave (which is not the real thing). Some brands will tell you they offer true sine wave outputs right in the model number or package, but always double-check the specifications (e.g. APC's "Smart-UPS" offer sine wave outputs, but NOT the Smart-UPS SC models). Using a UPS that outputs something other than true sine wave power can damage the power supply of what it is attached to and this can lead to premature failure of system you are trying to protect.

(2) Calculate the maximum load of the system being protected in Volt Amps (Line Voltage x Amps = VA) and purchase a system that can provide power well above that load for the time period you want to protect (*I think 20 or 30 minutes of run time, at load, is a realistic guideline to go by). A 700 VA UPS will usually power most desktop computers and a monitor for that amount of time. Most brownouts last for just seconds, but that is enough time to shut everything down or loose communication. If a power outage is more than five minutes in length, then it may be a while before it is restored. In this case, be sure to safely shut everything down manually before you run out of battery reserve power. Once everything has been turned off, remember to turn OFF the UPS system to stop it from slowly draining the battery (most computers are still turned ON and using power when they are OFF). Doing so will preserve the remaining battery capacity. Just turn the UPS back 'ON'  when power has been restored. 

(3) Unless you want to spend really BIG dollars, just put a high quality UPS system on the computer. A high quality UPS system designed for a single high current draw instrument like a chromatograph may cost thousands of dollars. If the system must be maintained at all times, then a serious back up source of energy should be invested in. High current UPS systems are available from a number of manufacturers so it would be wise to investigate these for your application. For everything else, use a dedicated, high quality power conditioner and surge suppressor on the analytical instrument circuits ($85 and up each, not the $20 models sold at most box stores). The computer system is really what you want to protect first. If the computer gets knocked out you loose communication and control of the analytical system. This usually means it will continue to run on and on without stopping. If the computer is protected by a UPS and the power gets glitched (such as a voltage drop/brown-out) or goes out entirely, then the computer may sense this and abort the run or shut the system down (if it has not been shut down). This is the type of approach we have used in our labs for twenty years and it works great during brown outs and black outs. The computers stay on long enough to properly and safely shut down everything without loosing data (which is the key). The instruments are all connected to individual power conditioners/surge suppressors (Tripp lite brand units in our case) so are protected from brown outs and surges. To protect our equipment from large surges, our electrician installed a high amperage DELTA lightning arrestor (100,000 AMP, 3000 joules per pole, unlimited number of surge capacity) on the breaker panel and individual Delta surge capacitors on each critical 20A circuit that supplies power to laboratory equipment. These extras steps provide protection from direct lightning strikes and surges to the panel and equipment. 

(4) The UPS Batteries: The lead acid batteries in your UPS system have a finite life. Mark them with a date and keep records on battery changes. Depending on the quality, temperature and load over time, they may need to be replaced in as little as one year or perhaps last as long as four years. When the batteries loose their ability to hold a charge your on-battery run time will drop dramatically so replace them early rather than late. The battery pack can be (and should be) tested on a regular basis. To test the UPS refer to the Operator's Manual for the correct procedure [*the test often involves selecting a time when the computer is not running any critical applications or providing any networked services (such as right before you would normally shut it off for the day). Once it is safe to do so, go ahead and remove the UPS’s A/C plug from the wall. This will remove the UPS system’s source of input power just as if a real power failure had occurred. Your computer system should behave as if nothing has happened. Monitor how long the system stays on until the available UPS run time shows 15 to 20% remaining time left and you will know how long the system can handle a complete power outage. The system will need overnight to fully recharge after this test and you should repeat this test a few times each year to monitor the status of the system]. *Many of the modern UPS systems provide a software application that continuously monitors the UPS modules which are connected to your computer or entire network. This can provide a record of the systems used, battery change dates and current charge levels right from your desktop.