Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, January 9, 2021

Speed Up HPLC Analysis Time Using Higher than "Normal" Flow Rates with SMALLER Particles

Column efficiency (as described by Van Deemter) in HPLC is largely a function of dispersion, column particle size and the flow rate of the mobile phase. Flow rate should be optimized for all methods (linear velocity). Once the optimum flow rate range is achieved, little to no advantage in analysis time or solvent savings is found by increasing it (as column efficiency normally decreases at higher flow rates).

From a practical point of view, columns packed with porous 3 to 5 micron diameter supports show only small differences in efficiency as flow rate is varied above the initial, optimum level. Running at too low a flow rate serves no purpose, increases dispersion/diffusion and delays the peaks from eluting off the column in a timely manner. Once the flow rate has been set within the 'optimized zone', it no longer becomes a variable in HPLC method development. 

Many 3 micron supports do demonstrate some ability to maintain optimum efficiency at slightly higher flow rates (e.g. with linear velocities > 1 mm/second), but significant advantages in using higher flow rates to save time and solvent are not obvious. 

However, with the much smaller diameter ~ 2 micron particles, column efficiency can be further optimized using higher than "typical" flow rates on standard columns. Columns packed with these smaller porous particles show optimized flow rates at higher linear velocities (2x normal or ~ 2 mm/second for standard analytical sized columns. If your method currently runs at 1.000 mL/min, you may be able to run the same method at 2.000 mL/min using one of the very small particles). This increased available efficiency coupled with proper optimization of the flow path to reduce dispersion allows for a doubling of the flow rate without a loss of efficiency (or loss of resolution). Depending on the scaling used, a two-fold savings in analysis time over conventional methods using larger particles may be observed. There may be a corresponding increase in system back-pressure too (if only the particle size is changed, but the column dimensions are unchanged. *Some of this can be countered using proper scaling of the column dimensions too. Optimization of method resolution, overall time and solvent usage should be considered). The increased efficiency gained from the smaller particle size also allows for scaling down the column dimensions (i.e. length, ID or both) too, though a trade-off between overall column efficiency vs analysis time and/or back-pressure must be addressed to optimize the method to meet the application goals.

Summary: HPLC analytical column flow rate is often ignored in method development (esp after it has been adjusted to the initial optimum, often 1.0 mL/min for a 4.6 mm ID column), but IF you are using porous HPLC particles that are smaller than 3.5 micron diameter, please be sure to re-optimize the flow rate for your application so you can take advantage of any increases in column efficiency and/or scaling. As with ALL applications using these small particles, pre-optimization of the HPLC flow path is often needed to achieve any of the possible benefits.

Saturday, October 31, 2020

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.

 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).

 

Saturday, September 5, 2020

Tips and Advice for Priming your HPLC PUMP (or similar pumps, FPLC or UHPLC Pump)


The single most important component of any HPLC system is the Pump module. We often refer to it as "the heart of the HPLC system". 

  • You may have the most sensitive HPLC detector, the best column, a perfect method of analysis, but none of this will matter unless the HPLC pump(s) that provide mobile phase to the system operates perfectly, all of the time. If you have a poor quality (or poorly maintained) system, then you will spend much of your time trying to establish reliable flow through the HPLC system, not running samples. 
  • Before using an HPLC system, you should prime all of the lines in your HPLC pump. This is needed to purge any air from the tubing, introduce fresh mobile phase to each line and then to VERIFY that each channel delivers the reported amount of fluid (measure it).
  • This is a LONG, detailed article with lots of information, Hints and Tips. It is available in PDF format for download, here.

The HPLC pump's ability (stability) to provide reliable operation depends on: 

(1) The Chemical, Physical and Miscibility properties of the Liquid(s) being pumped;

(2) The Amount of dissolved gas inside the liquid must be minimized;

(3) The Temperature of the room (or HPLC) must be stable;

(4) The Position of the mobile phase bottles (relative to the pump, above or below);

(5) The Solvent Pickup Filters used (are appropriate in material & porosity);

(6) The Fittings used are correctly installed & tightened;

(7) The types of Tubing used are chemically, temperature and pressure compatible (esp. the Inside Diameter of the tubing);

(8) The Selected Flow Rate(s) are within the optimal range of the pump;

(9) All mobile phase solutions are Degassed, Filtered and Fresh;

(10) How often the Pump is properly Inspected, Cleaned & Serviced.

 The HPLC pump is the most important part of your HPLC system. Take care of it. Neglect it, and you will lose time and money. Almost every problem you experience using an HPLC will be related in some way to the pump. Make sure you understand the flow path of the system in detail, and have the training to setup and use it properly. Take a hands-on training class to learn how to use the pump on your specific system. Learn how to run simple verification tests to check the flow rate (with a graduated cylinder). Never rely on the software values, check and verify. Priming and flushing are needed any time air bubbles are present, mobile phase solutions are changed or the system has sat unused overnight. Always flush multi-channel pumps (i.e. Binary, Ternary, Quaternary...) using a setting of 100% channel composition. Run one channel at a time at 100%, not 25% or 50% to flush channels (a common novice mistake).


OK, so what can you do to make sure your HPLC pump is properly primed with fluid and operating to the best of its ability?


Start, by reading the operator's manual for your pump. Review the procedures for connecting it to the system, become familiar with the flow path and understand the procedures to prime the pump heads. Practice these procedures.


If an inline vacuum degasser is used, become familiar with the specifications, chemical compatibility (some are not compatible with solvents such as strong acids, strong bases, THF, chloroform, fluorinated additives and so on) and internal channel volume of each chamber used. You will need to know what the degasser chamber volume is to figure out what the total channel priming volume is. This may be different for similar systems. Check, measure, verify, do not assume.


Priming Volume: The total volume contained in each channel's low-pressure line from the mobile phase bottle to the degasser + the degasser chamber channel volume + the total volume in the line from the degasser to the pump head (or multichannel valve) = the total volume you must flush out before using the system. Because flushing just the minimum of volume (1x) of fluid through the channel is unreliable, flush 2x or 3x this total volume, per channel (or as much fluid as it takes), to prime each channel. *If no degasser is present, then just calculate the volume contained in the low pressure tubing from the bottle to the pump head/valve. Set the pump to direct the flow to waste and use a high flow rate to speed up the process.


Use fresh mobile phase (prepared daily and filtered). Make sure the solvent pickup filters are clean. If possible, have the bottles placed higher than the pump's inlet (once flow has been established, this will allow natural siphoning to push liquid towards the pump head). Prime all of the lines used. The pumps run on liquid, not air so try and fill any of the lines with pure mobile phase before you connect them to the pump and/or degasser (If all of the lines are prefilled with fresh liquid, you can skip this part).  


There are two ways to PRIME EACH line (Flush the Channel).

  • *First, open any Prime/Purge or Waste Valve so the mobile phase is directed to waste, not the injector, column or detector. Our goal is to initially fill the lines with liquid, quickly, and we do not want these fluids to go through the system (i.e. column), just the pump.

(1) Wet Priming use a syringe fitted with a Luer-to-threaded fitting adapter (usually 1/4-28) to draw liquid through the tubing in the mobile phase bottle and into the pump's degasser and/or pump head's inlet. Be sure to have this type of syringe available (very useful). Never push fluid, only draw fluid through the tubing, just like the pump does. Connect the syringe to the mobile phase bottle lines, degasser ports and/or pump head multichannel valve or pump head inlet, as needed, to draw liquid through until all lines are filled.


(2) Dry Priming using the HPLC pump to draw the mobile phase out of the bottles, through the lines, degasser channels and to the pump head or multichannel valve. Always do this one channel at a time (e.g. A=100%). This insures no miscibility or mixing problems and is standard procedure. Start with a modest flow rate to get the fluid moving through the lines, then increase the flow rate to speed up the process. The low pressure Teflon tubing is transparent so you can watch this process. Repeat with each channel. Note: Some HPLC pumps will struggle to perform this type of dry priming, as they will be unable to draw the liquid up from the bottle and/or pump the air out of the system. Pre-priming the lines using a syringe (as in #1 above) will help solve this. Running the pump with just air inside the lines may result in increased wear on the system (piston seals) so if the system struggles to fill with liquid after one minute, discontinue and manually prime.


NOTES: 

  • The back-pressure shown on the system readout should be very low during this priming process (e.g. < 15 bars) as the HPLC system should not be plumbed with the column or detector inline, during the priming process. Only the viscosity of the solution, the selected flow rate and the internal diameter of the tubing going into and out of the pump will contribute to the observed back-pressure, and this should be very low.

  • Once you have verified that liquid is exiting through the pump head waste port, you can increase the flow rate to speed up the priming process, but pay attention to the back-pressure. It should increase as the flow rate increases and drop as the flow rate drops. Continue to prime each channel in this way, one-at-a-time, until all channels are primed and flushed with liquid.

  • If liquid has been drawn to the pump head, but the pump head still is not pumping liquid through it, it may be experiencing cavitation (air locked). If there is an outlet port on top of the pump head, try drawing liquid through this port, while it is running, to gently fill the pump head chamber and remove the air. Alternatively, the outlet fitting above the pump head can sometimes be briefly loosened allowing the system to push the air out more easily (open it slightly with a wrench, then quickly close it). Have a towel ready to soak up any fluid that comes out.
  • The outlet check valve can also become "stuck" open in some cases and drawing liquid out of the pump head's outlet port with a syringe (or pushing it through the pump head) may remove the air bubble and prime the valve, restoring function. 
  • In more extreme cases, you can change the mobile phase going into the pump head to a more viscous intermediate solvent to get things moving (an alcohol such as IPA might work). 
  • Degas all eluents / mobile phase solutions used. All of them. Degassing will help reduce the formation of bubbles inside the pump head. Failure to properly degas the solutions may result in loss of prime, baseline and pressure instability. Make sure your degasser is operating properly (electronic vacuum degassers only last ~ 5 years at most. Be sure to have them professionally serviced). 
  • Verify the flow rate. It may be unwise to rely on the indicated flow rate shown on the instrument screen or display. It is wise to measure the flow rate of each channel, separately, using a graduated cylinder and a timer. This is the most reliable way to determine what the actual flow rate is through the system (and is also the method we use during performance verification or qualification testing too). To check the flow, make sure the system has been primed and flushed. Install a flow restriction capillary in place of the column (to provide the required back pressure). Set the flow rate to a value which is appropriate for the pump and measure/record the volume delivered vs. time. Example: Using a flow rate of 1.000 mL/min obtain a 10 mL volume, glass laboratory grade graduated cylinder. At time zero, direct the flow from the restrictor's outlet into the graduated cylinder. Measure the volume of fluid collected in 8 minutes. *It should be 8.00 mLs.

If you continue to have priming problems and/or air bubbles disrupting the flow there are three more things you can check. 


  1. Make sure the solvent pickup filters/frits are clean and unobstructed (these are maintenance items). If the filters are obstructed, then a vacuum may form on the line resulting in pump cavitation and loss of prime. One quick way to check if this might be a problem is to remove the suspect solvent pickup filter from the line, then try again. If flow is restored w/o the filter in place, then the filter may have been clogged. Install a new solvent filter as soon as possible. *Never run the HPLC without solvent filters installed. Those filters perform a very important job and protect the flow path of the system.  
  2. Service the Pump Heads. Regular cleaning, inspection and replacement of worn parts must be done to maintain the function of the pump. Worn parts will result in failures, instability, lost time, plus invalid data. The pump has many mechanical parts which wear out and require replacement. Most pumps should be serviced every 6 months. Keep the pumps clean and fully serviced (replace: piston seals, pistons, frits, check valves as needed). Depending on the brand, model and applications, the types of parts needed and the frequency of repairs varies widely. *This is discussed in another article. 
  3. If your HPLC system has an inline vacuum degasser (either a standalone or integrated module), it may be damaged, contaminated or broken. The typical service life of an electronic inline vacuum degasser is only five years. Internal damage may result in air or other contaminates leaking into the mobile phase. A failing or damaged HPLC vacuum degasser may directly contribute to analysis problems (ghost peaks, pressure instability, poor baseline stability...). Have your degasser professionally diagnostically tested and serviced often.  
  • Additional Troubleshooting Info can be found here:

Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)