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 Baseline Stabilization Tips for Refractive Index Detectors (RI or RID)

If you use refractive index detection (RID) for your HPLC samples, then you are already familiar with the very long equilibration periods needed to stabilize the system and associated baseline drift. Initial equilibration can take several hours. In fact, re-equilibration takes far longer to achieve with this detection mode than most others (i.e. UV/VIS, FLUOR, EC). While there is no quick cure for these delays, there are a number of things that you may be able to do to minimize or reduce these wait times. Here are a few to consider.

• ROOM TEMPERATURE: Locate the RID in a quiet, stable location. If the room temperature in which your HPLC system with RID system is located fluctuates by even one degree C, that can effect the stabilization of the system. The ideal room to use RID will be away from any windows, drafts, doorways, direct sunlight and HVAC vents or ducts. It should be located in a quiet area away from people walking by it. All of these things can contribute to temperature instability, which is what you want to avoid.

• INSULATION of HPLC CAPILLARY LINES: All of those stainless steel capillary lines leading from your column outlet to the RID's flow cell are loosing heat to the surrounding air (cooling). To reduce this thermal effect, insulate any metal lines with plastic tubing to reduce the heat loss. Most any type of laboratory grade, thick walled plastic tubing can be used. Pass the SS tubing through the plastic insulated tubing or use a section of split-tubing to cover it. Cover as much of the exposed tubing as possible, right up to the fittings. - Note: Sometime the HPLC system's solvent bottles may be subjected to varying temperature changes too. In these cases you can wrap the bottles with an appropriate insulating material to reduce the effects.

• FLOW CELL TEMPERATURE: Modern RID units have a heated flow cell with thermostat to control the temperature of the flow cell. This helps stabilize the temperature inside the flow cell as well as minimize the unintended effect that the heat given off by the RID's electronics has on the temperature inside the flow cell. If the flow cell temperature does not stabilize, then the baseline will drift in response to it. For most methods, select a flow cell temperature which is at least 10 degrees C above ambient (since most of these units can heat only, not cool). Factor in any column temperature used too. If you are maintaining your column at 40C, then try to maintain your flow cell at the same temperature to minimize any differences. Feel free to experiment to find the best temperature for your flow cell. Try different temperatures (in 5 degree C intervals), wait for the system to fully equilibrate, then measure the baseline S/N ratio. You may find best results using different column and flow cell temperatures. Sometimes the room temperature effect can be countered by using an optimized flow cell temperature (higher or lower). Always factor in your mobile phase boiling point (b.p.) into your method and keep the column and flow cell temperatures well below the b.p.

• DEGASSING / DECREASING DISSOLVED OXYGEN: Reduce and stabilize the amount of dissolved gas inside the mobile phase and you may achieve faster equilibration times with a RID. You do not need to remove all the dissolved gas (in fact, a reduction of 50% is often enough). The amount of dissolved gas inside the mobile phase effects the measured refractive index. As it changes, so does your baseline. High percentages of mobile phase dissolved gas = lower RI; Less dissolved gas = higher RI. Now water holds less dissolved gas than non-polar organic solvents (e.g. THF) so this effect is more pronounced when you are running non-aqueous GPC separations, but maintaining a stable dissolved gas level for all mobile phase types is important to reduce baseline drift. Stability is our goal. Continuous degassing of the mobile phase either through sparging with high purity helium gas (best for non-aqueous separations) OR using an inline vacuum degasser should provide you with a way to control the amount of dissolved gas in the solution and reduce drift.

These are a few of the factors which can effect the equilibration and drift times of an HPLC system equipped with a refractive index detector (RID). Careful selection of the instrument module's location, insulating the exposed capillary lines and bottles, optimizing the column and flow cell temperatures, maintaining a steady and controlled temperature in the environment, plus removing dissolved gas from the mobile phase may all contribute to more stable baselines and better quality peak integration. It is also a good idea to review training in the correct operation of the RI detector too. Learning to correctly operate the flush and optional recycle valves on these detectors is critical to their operation. Failure to properly flush the reference cell before each analysis with fresh mobile phase may lead to baseline changes or artifacts.

Another article which may help you improve your analysis method can be found on this site. "Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)".

UHPLC TIP: Reducing the Column Temperature to Offset Frictional Heating Effects (Causing Poor Resolution)

HPLC column temperature is a critical variable that we adjust and optimize during method development. We use it as a variable during the method development process to improve solubility, optimize peak shape and increase resolution. Once established, it must be carefully controlled during the method analysis to provide reliable and reproducible analysis results. Change the column temperature and you may also change the results obtained. This is a fundamental method development tool and must not be forgotten.

If you are developing a new UHPLC method OR perhaps scaling an HPLC method to utilize 2.5 micron or smaller support particles, then you may observe a loss of resolution or poor peak shape in the new method. There are many reasons why this may occur, and the most common ones relate to not optimizing all of the method parameters correctly when scaling the method (e.g. dwell volume too large, flow cell volume too large, injection volume too large, sample rate too slow, flow rate not optimized, mobile phase composition changes not in scale with the gradient...). But there is another reason...

Resolution may be reduced or lost when all of the initial scaling and instrument set-up parameters are optimized. What is the most likely reason for this? In many cases the use of substantially higher flow rates (relative to linear flow rates) and the use of smaller diameter particles results in much higher backpressures (you may recall that if you halve the particle size, the backpressure increases 4x). The resulting backpressure might be 2, 3 or even 4 times higher than observed in the original method. While these higher backpressures were well within the operating parameters of the HPLC system used, the results obtained were poor. The possible cause? The much higher backpressure increased the amount of frictional heating inside the column, raising the actual analysis method temperature and changing the separation conditions. 

Pushing mobile phase (liquid) through a chromatography column generates heat and pressure. The heat generated increases the actual temperature of the column and reduces the viscosity of the fluid. In conventional columns (i.e. 4.6 x 150 mm, 5u) at 1.00 ml/min, this heating effect is minimal, but at much greater column pressures, > 400 bars, the frictional effects may be substantial. These types of very high pressures may be seen with methods which utilize columns containing the smallest particles (1.9 to 2.5 micron). Enough to change the temperature in the column by several degrees (e.g. >5 degrees C) and result in different method conditions. So, what can you do about this? The most direct way to address the problem is to run the same method at a lower temperature (perhaps decrease by 5 C to start with). This will slightly raise the backpressure (lower temperature equals higher viscosity), but it should cool the column and restore the original temperature conditions used. Additionally, we suggest that you always start column equilibration using a flow ramp to gradually increase the flow over time and reduce the overall heating effect and resulting "shock" placed on the column. An initial delay at equilibration may help reduce these effects (gradually ramp up to the regular flow rate and hold). You may need to try several temperatures and this may be easiest to do if your HPLC has a column compartment with heating and COOLING capabilities. Optimizing the temperature and internal pressures may increase the column lifetime and result in better overall data reproducibility.

Column Temperature in HPLC / UHPLC / LC-MS

Let us not forget the role of temperature in liquid chromatography. Just as mobile phase composition changes are used to develop better methods, column temperature is an important chromatography variable which must be addressed. I would like to call to your attention to a few different ways temperature can change your chromatography in this "hint and tip".

(1) Stability & Reproducibility of the Method: 
Maintaining a stable column temperature during a separation is important. Excellent temperature stability can lead to a high degree of reproducibility (*Their are of course many other factors to consider as well). For a typical analysis, temperature stability of 1.0 °C / hour (over the course of the analysis) is usually enough. If you are not using a thermostatted column compartment to perform your chromatography you may have already noticed the hour-to-hour or day-to-day fluctuations which can result from running samples under ambient temperature conditions. The normal changes in room temperature can be several degrees C over an eight hour period. These types of temperatures changes can make it impossible to achieve reproducible results for some samples. It is for this reason that it is critical that you include some type of temperature control as part of your method. Always record the temperature at the start and end of each run and include this data with your report. Most of the automated chromatography data systems provide this data as standard today and it is very valuable in reproducing the data as well as for troubleshooting, if needed.

(2) Back Pressure:
Column back pressure is directly changed by temperature. As the temperature rises, the column back pressure decreases. As the temperature decreases, the back pressure increases. This can be a useful variable when working with some of the newest sub-two micron particles on the market. The very high back pressures produced by these particles can be significantly reduced by increasing the column temperature [See "Pressure Drop Across an HPLC Column" http://www.hplctools.com/Tip%20114%20Pressure%20Drop%20Across%20an%20HPLC%20Column.htm]. 

When practical, try experimenting with your method by increasing the temperature, in increments of 5°C, to measure the change. You may discover an improved method with lower back pressures, a shorter run time and sharper peaks.

(3) Viscosity: 
Viscous mobile phase systems can take advantage of using higher temperatures to reduce the overall system back pressure. Since efficiency often improves with higher temperatures a double bonus of higher efficiency (sharper peaks) and lower back pressure can be achieved just by increasing the column temperature (peaks sometimes change elution order too so use standards to check this). 

(4) Practical Considerations:
Their are limits to using higher temperatures in chromatography which must be respected. The stability and solubility of your sample, the boiling point of your solvent, the maximum temperature setting of your column heater (mobile phase, flow cell and the rest of the HPLC system) and the stability of your column over time will determine how far you can safely push this.

(5) Specifications: 
One other issue worth mentioning here is that many traditional silica columns can loose their bonded phase at temperatures above 60°C. Some specialty silica phases (i.e. Waters XBridge & Zorbax StableBond) have temperature ratings to ~ 90°C. The more exotic non-silica based supports (e.g. Zirconium, graphitized carbon and/or PSDVB) often provide poor efficiency compared to the silica based products, but can handle temperatures in excess of 100°C. 

*Always consult with the column and/or instrument manufacturer to determine what the correct and safe operating conditions are before using any instrument, column or chemical.

Pressure Drop Across an HPLC / UHPLC Column

Many of you prefer tables of data over equations that you must work out. So, instead of providing you with another equation, I have done some basic measurements for you to provide a general overview of how particle size (porous) effects System backpressure.

For simplicity, let us start with a few parameters. Pore Volume = 0.70; Linear Velocity = 1.44 mm/sec; Solvent Viscosity = 0.89 cP at 25C (Water). 

Pore Volume and Flow Resistivity will vary by column type. Obviously the back pressure will be higher with more viscous solvents (e.g. EtOH is 1.20 cP) and lower with less viscous solvents (e.g. ACN is 0.34cP). A Table of HPLC Solvent Viscosity values can be found here [ http://www.hplctools.com/lcsolvent.htm ]. Linear flow rates have been used for all column I.D.'s to better illustrate the relationship between column dimensions and flow rate. If you double the flow rate, then the pressure will approximately double as well. 

Note that when run at traditional linear velocities, most 2.5u particles are within the maximum pressure limits of most HPLC systems (under 400 bars). Only the newer sub 2.0 micron particles used in long columns exceed the 400 bar limit. The higher maximum pressure limits of many UHPLC systems allow the use of higher flow rates with these particles. Naturally, you should optimize both column efficiency and system dwell volume when developing any UHPLC method. Failure to optimize the dwell volume (and minimize all volumes) may result in very poor chromatography separations. Meeting any/all backpressure requirements to run a method does not translate to success in sample analysis. Successful ultra-fast separations require ultra-low system dwell volumes, higher sampling rates and usually smaller flow cell volumes.

HPLC Column I.D. (mm)	Particle Size (u)	Column Length (mm)	Flow Rate (mL/min)	Observed System Back Pressure (Bars)
4.6	5	250	1.000	89
4.6	5	150	1.000	54
4.6	5	100	1.000	36
4.6	5	50	1.000	18
4.6	3.5	250	1.000	182
4.6	3.5	150	1.000	109
4.6	3.5	100	1.000	73
4.6	3.5	50	1.000	36
4.6	2.5	250	1.000	357
4.6	2.5	150	1.000	214
4.6	2.5	100	1.000	143
4.6	2.5	50	1.000	71
4.6	1.9	250	1.000	618
4.6	1.9	150	1.000	371
4.6	1.9	100	1.000	247
4.6	1.9	50	1.000	124
3.0	5	250	0.430	90
3.0	5	150	0.430	54
3.0	5	100	0.430	36
3.0	5	50	0.430	18
3.0	3.5	250	0.430	184
3.0	3.5	150	0.430	110
3.0	3.5	100	0.430	74
3.0	3.5	50	0.430	37
3.0	2.5	250	0.430	361
3.0	2.5	150	0.430	217
3.0	2.5	100	0.430	144
3.0	2.5	50	0.430	72
3.0	1.9	250	0.430	625
3.0	1.9	150	0.430	375
3.0	1.9	100	0.430	250
3.0	1.9	50	0.430	125
2.1	5	250	0.210	90
2.1	5	150	0.210	54
2.1	5	100	0.210	36
2.1	5	50	0.210	18
2.1	3.5	250	0.210	184
2.1	3.5	150	0.210	110
2.1	3.5	100	0.210	73
2.1	3.5	50	0.210	37
2.1	2.5	250	0.210	360
2.1	2.5	150	0.210	216
2.1	2.5	100	0.210	144
2.1	2.5	50	0.210	72
2.1	1.9	250	0.210	623
2.1	1.9	150	0.210	374
2.1	1.9	100	0.210	249
2.1	1.9	50	0.210	125

* The results obtained in this table from are from one of our HPLC systems and reflects the total system backpressure (what the pressure gauge reads), with the column inline. Your results may vary due to differences in HPLC system used, flow path, tubing ID, column choice and mobile phase selected.