Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, June 26, 2021

Repair Corrupted Windows 7, 8 & 10 System Files Using the System DISM Tool

Time to share another useful Microsoft Windows command-line utility tool. If you have experienced the "Blue Screen of Death", a crash, service pack installation failure, windows update failures or observed general Windows system file corruption error messages, then this utility tool might be useful to you.

Many common Windows Update errors which result from new system file corruption can be corrected using the Windows DISM.exe tool. Failed application or update installs often result in corruption of some of the system files. These utilities are designed to detect corrupted file and repair them. It targets the currently running operating system for repair. The DISM tool must be run from the Windows Command prompt (cmd), with an account that has administrator privileges using " Run as administrator ".

DISM = Windows "Deployment Image Servicing and Management" tool.

  • Before using any software utility program, make sure you first have permission and authorization to do so. Most Importantly of all: Back up your system programs and any data files before using any utilities such as this one. Create a Restore Point to protect the basic settings too. Do this now. Take precautions before using any utility programs and do so at your own risk. You are responsible for your data, programs and computer.
  • Please make sure you have reviewed my earlier article on how to run the System File Checker (SFC) tool first. The SFC tool scans Windows operating system files for corruption AND restores any found corrupted files, all automatically! The SFC often quickly corrects many system errors and I always run that utility first. DISM is more thorough, but takes more time to run.
  • Before using the DISM (or SFC) utilities, set a new restore point using the very useful "Restore Point" feature found in Windows (discussed in an earlier post). Make sure you have enough time available for the computer to run this utility (overnight is best). Once started, it will show a progress bar. Do not interrupt the process.

To run the DISM.exe utility, close down all applications for now. Make sure your account has Administrator privileges, then open up the Command Prompt using the " Run as administrator " option (you must do this so the system32 path is selected). 

At the command line prompt, Type the command line below (make sure to include the spaces, exactly as shown): 

 DISM /Online /Cleanup-image /Restorehealth  

 Optionally, for some O/S enter:  DISM.exe /Online /Cleanup-image /Restorehealth 

The screen should show " Deployment Image Servicing and Management Tool " with the version #. Image Version plus some [ ] showing the progress. When it is finished, it should report that "The operation completed successfully". Type 'EXIT' to close the Command Prompt screen, then Reboot your computer.

 

 

References:

1.  Microsoft Windows Support Page:
 
2.  If you encounter "error 87", then please confirm you have: typed the command line as shown; are running the cmd "as administrator"; have applied all new or pending Windows Updates and have run the SFC tool first. If you continue to see the error, then refer to Microsoft's knowledge base for additional tips.

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.After a column has been selected, the Flow rate should be optimized for all methods (start with the nominal 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 the flow rate is varied above the initial, optimum level (linear velocity). 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. Higher rates often decrease column efficiency. 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 unless the particle size is reduced further. 

With the much smaller diameter ~ 2 micron particles, column efficiency can be further optimized using higher than "normal" flow rates on standard columns. Columns packed with these smaller porous particles show optimized flow rates at much higher linear velocities (e.g. 2x normal or ~ 2 mm/second for standard analytical sized columns, but experiment using 2 to 5x the normal linear velocity to compare results). 

  • For example: If your method currently runs at 1.000 mL/min, you may be able to run the same method at 2.000 mL/min OR if your method currently runs at 0.200 mL/min, you may be able to run the same method at 0.400 mL/min or higher using one of the 2.5 or smaller particles. 
This increased efficiency coupled with proper optimization of the HPLC's 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 and the column dimensions are unchanged). *Some of this can be countered using proper scaling of the column dimensions too). 

NOTE: Do Not Optimize HPLC Methods for "Pressure". This goes against basic chromatography fundamentals. Back Pressure is a result of pushing mobile phase through the tubing and column and is not a method development tool or variable. As mobile phase composition changes, so does the pressure. Flow rates should be stable. Work within a pressure range that is high enough to permit the pump(s) to function properly, but below the point in which frictional heating interferes with the method.

Optimization of method resolution, overall analysis time and solvent usage should be considered. The increased efficiency gained from the smaller particle size supports 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 too high a back-pressure must be addressed to optimize the method and 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 investigate if you should re-optimize the flow rate used in your method / application so you can take advantage of any increases in column efficiency and/or scaling. As with ALL applications using these very small particles, pre-optimization of the HPLC flow path is often needed to achieve many of the available 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, 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.

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 operate 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 to the column (measure it).
  • NOTE: 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 clean and 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) and Back-pressure are within the optimal range of the pump;

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

(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 or abuse it, and you may 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 (not a video or web based tutorial) to learn how to use the pump on your specific HPLC system. Learn how to run simple verification tests to check the flow rate (best done with a graduated cylinder). Never rely on the software values, check and verify everything yourself. Priming and flushing are needed any time air bubbles are present, mobile phase solutions are changed or the system has sat unused (this includes overnight). Always flush multi-channel pumps and valves (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). Flush ALL channels on a regular basis.


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. It is useful 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 minimum 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, 3x or more times 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 initial flow rate to speed up the priming 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 (Flushing the Channels).

  • *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 entire HPLC system (i.e. column), just the HPLC 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. Note: "Dry" because the lines (low pressure tubing) are initially dry when we start. 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 (esp the piston seals) so if the system struggles to fill with liquid after one minute, discontinue and manually wet prime each line.


NOTES: 

  • The back-pressure shown on the system readout should be very low during this initial  priming process (e.g. < 15 bars) as the HPLC system should not be plumbed with the column or detector inline, during the priming process (it should be by-passing those parts). 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 value.

  • 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. You can gradually slow the flow rate down as you stop, to transition from one channel to another.

  • 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, the outlet fitting above the pump head can sometimes be briefly loosened with a wrench, allowing the system to push the air out (open it slightly with a wrench, then quickly close it after liquid comes out). Have a towel ready to soak up any fluid that comes out. Keep the area clean and dry. Alternatively, try drawing liquid through this port, while it is running, to gently fill the pump head chamber and remove the air.
  • In some case, the inlet or especially the outlet check valve(s) can also become "stuck" open. When buffers are left in the system (they should be flushed out with water), crystals and particulate matter may deposit on the valve resulting in poor sealing, leaks or air being drawn through. Drawing liquid out of the pump head's outlet port with a syringe (or gently pushing it through the pump head) may remove the air bubble, debris and prime the valve, restoring function. Note: If needed, shut down the pump and clean/replace any contaminated or worn check valves before proceeding.
  • 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 well. If buffers have been used, then always first flush with pure water). 
  • 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 used may result in loss of prime, baseline and/or 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). Sonicating fluids at the bench or using vacuum filtration to initially remove gas from the solution will only degas the solution for a short time (minutes). Gas will slowly diffuse back into the solution resulting in baseline noise, drift and pump problems (for HPLC, only use inline degassing or Helium sparging).
  • 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 four 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 tubing, 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 inspected/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 (some models have even shorter lifespans). Degasser's with internal damage may result in contamination of 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.  
  4. Clean and/or replace any worn or damaged inlet or outlet pump head valves. Not flushing buffers out of the HPLC system on a regular basis or remain in contact with the solution for long periods of time can damage the valves. In some cases, cleaning is all that is needed, but in others, replacement is required to restore function. Be sure to have the system professionally serviced on a regular basis.
  • Additional Troubleshooting Info can be found here:

Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)