Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, August 3, 2019

Air Bubbles Exiting the HPLC Degasser, Reasons Why

A common question we are asked to solve relates to why air bubbles might be observed exiting out of an HPLC vacuum degasser module  (where the mobile phase leaves the degasser ports to go to the pump heads and/or gradient valve)? Troubleshooting and answering this question is most easily accomplished if you first have a solid understanding of the HPLC flow path, how to make proper connections and are familiar with performing routine maintenance on the HPLC system. 
  • Key Point: HPLC systems utilize Teflon low-pressure tubing to transfer the mobile phase from the mobile phase bottles to the HPLC pump. The Teflon lines are permeable to gas in the atmosphere. Gas is diffusing through the plastic tubing used to transport your solvents. This is one of the reasons why we purge the entire flow path of the HPLC system before use, each day. Overnight, gas has diffused into the system so we start by flushing (purge) the mobile phase from each bottle, through the degasser, through each channel all the way to the pump head, to waste.

To start the troubleshooting process to find the reason why air bubbles may be observed existing the HPLC vacuum degasser module, we examine the flow path.

Reasons For Air Bubbles Exiting The Vacuum Degasser:

  • Loose Connections: If the low pressure fittings (nuts and ferrules)  which secure the Teflon tubing to the degasser are damaged or loose, air may enter the system resulting in bubbles. Most vacuum degassers use plastic finger-tight style fittings 1/4-28 (or 5/16-24). The threads are soft and can be deformed. When access to these fittings is difficult, sometimes the fittings are left loose and will allow small amounts of air to be drawn in (such as found on many of the generic small benchtop degasser which use the micro-chambers or the HP/Agilent model G1379-series). Inspect the tubing and fittings used for proper seating depth, wear and/or damage. Replace parts as needed and re-install using the correct amount of torque.
  • Flow Rate Too High or Not Enough Degasser Equilibration Time: Degassing efficiency is directly related to the flow rate. Lower flow rates increase the residence time of the mobile phase in the degassing membrane or tubing, improving the gas removal. Higher flow rates provide less time for gas extraction and result in lower degassing efficiency. Check with the manufacturer regarding the optimal flow rate range for your degasser to insure you are working  within an acceptable range. Allow enough time for the degasser to reach its set-point and stabilize before use.
  • Choice of Mobile Phase Liquid: The solubility of air (gas) in the specific solution used also affects the efficiency of the vacuum degasser. Aqueous solutions usually hold less gas than popular organic solvents (though air bubbles can be harder to "push" through in water). The amount of dissolved gas inside the liquid relates directly to the time needed to reduce it to acceptable levels for use in HPLC.
  • Dirty or Obstructed Solvent Pickup Filters (Bottle filters): Bottle filters should be cleaned or replaced at regular intervals, following routine maintenance SOPs. When they become fouled or obstructed, a vacuum may form as the liquid is drawn into the system. This may result in air being sucked into the tubing or through a fitting (remember that the low pressure Teflon tubing used to connect the bottles to the degasser and pump is porous and allows gas to diffuse through it). The pickup filters should not obstruct the normal flow of solvent (typically they are 10-20 u in porosity).
  • Lack of Vacuum Degasser Preventative Maintenance: HPLC Vacuum degasser modules, like most other component parts of your HPLC system break down over time and require professional diagnostic testing, cleaning and repair. Under ideal conditions, most inline electronic vacuum degassers require diagnostic testing and cleaning or repair every 4 to 5 years. *Many show signs of contamination or failure before that time. The internal vacuum tubing becomes contaminated and worn over time. The vacuum pump is an electromechanical part which is exposed to all of the mobile phase additives and solvent vapors during use. Other internal component parts such as vacuum valves or restrictors may also become contaminated or worn over time. The vacuum degassing membranes (or tubing) themselves can stretch from use and wear out over time. The vacuum chambers may be exposed to incompatible chemicals or overpressured resulting in internal leakage. Certain chemicals may also attack and even dissolve the degassing membranes causing more internal damage and contamination of the mobile phase. These devices do not have any "contamination" detection alarms and the vacuum sensors sometimes become damaged over time leading to false vacuum levels being reported. Never rely on the module's built-in error alarm system as proof of compliance (no more than you would the reported flow rate shown on the computer screen. It must be measured to be known). Regular professional HPLC degasser testing and service are required to maintain the modules and meet compliance requirements.
 Any of the above causes may contribute to air being drawn into the degasser system. Troubleshooting should begin with the easiest and obvious areas first. Check the condition of the low pressure tubing used to make the connections to and from the mobile phase bottles and degasser. If it is kinked, twisted or damaged, replace it with new tubing. Check the fittings used (nuts and ferrules) for tightness and to insure they have been installed properly. Replace any damaged fittings with new ones. Check the solvent pickups to insure they are clean and not obstructed. Make sure the flow rate you are using is within the acceptable range for your degasser. Has your degasser module been professionally cleaned and serviced within the last 5 years? Are any degasser errors being generated? Is the degasser making any unusual sounds? If any of the answers to these questions are 'yes', then have the HPLC vacuum degasser professionally diagnosed for problems so that repairs can be made to restore function.

Saturday, June 29, 2019

Backpressure Changes, Pressure Drop from HPLC Tubing Selection (0.007, 0.005, 0.010")


In previous articles we have discussed how the choice of column particle size directly changes the system backpressure. Smaller particles generate higher back-pressures. We have also discussed the importance of HPLC tubing selection to minimize delay volume and diffusion within the HPLC system flow path. Let us now focus on how the tubing's internal diameter and length impacts the total backpressure (or pressure drop) observed. 

Key Point: Try to optimize the plumbing of your HPLC system. HPLC Tubing lengths between connections (or HPLC modules) should always be as short as possible.


Once the HPLC tubing connection lengths have been minimized, the next critical dimension which affects band broadening, delay volume and peak width is the internal diameter (ID) of the tubing. The tubing selected should be narrow enough to reduce the undesirable spread of the peak(s) inside the tubing, but not be so narrow or restricted to result in clogs or obstructions (which is why good chromatography guidelines should be followed insuring that each sample is fully dissolved and filtered before injection). For many analytical HPLC systems, commonly used tubing ID’s are 0.010” (0.25 mm), 0.007” (0.17 mm) or 0.005” (0.12 mm). *0.007” (0.17 mm) is the most commonly used size. However, in addition to the much lower internal volumes which accompany the narrower ID’s, the pressure drop across equivalent lengths of tubing also change dramatically and should be noted.

Let us compare the pressure drops measured across three popular HPLC tubing ID’s of the same length (40 cm) using common HPLC mobile phase liquids. This will help illustrate the observed backpressure changes that the tubing ID and liquid have on the pressure drop.

PRESSURE DROP (in bars):

SS Capillary Tubing, 40 cm length, flow rate 1.000 mL/min.

Mobile Phase / Tubing ID
Water
ACN
MeOH
MeOH/Water
IPA
0.010” (0.25 mm)
0.7
0.2
0.4
1.2
1.5
0.007” (0.17 mm)
2.7
1.0
1.6
5.1
6.2
0.005” (0.12 mm)
10.4
4.0
6.3
19.1
24

Note: Pressure drop is also a function of tubing length so if we halve the length of tubing used, we also will reduce the pressure drop by one half. 

Note the four-fold change that narrowing the tubing ID has at each ID reduction. The change is more dramatic when viscous solutions are used (i.e. MeOH/Water or IPA). If you re-plumb any part of your HPLC system with new tubing, then awareness of this physical change will assist you in troubleshooting many types of HPLC problems (to know which types of pressure changes indicate a real problem and which types of pressure changes are normal). Changes to the overall length or ID may result in noticeable changes to the total system backpressure.

Saturday, May 11, 2019

Useful Windows Command Line Programs and Shortcuts

  • Warning: These commands and shortcuts should only be used by experienced users who both accept and understand the risks involved. Please backup all systems, programs, applications, data and files before using any utility program or command line.
Command Names:


Command Line Shortcut (to exit from the command line, type exit):

            cmd



View Network Address (shows your local IP address)

ipconfig



View IP address Routes (shows Interface list with IPv4 and IPv6 Route Tables)

            netstat –r

Ping an Address or Host (From the command prompt, type "ping" followed by the IP or name)
           
           ping hostname     ( e.g. ping 192.168.254.01 )
           ping IP address    ( e.g. ping chiralizer.com )

Find Devices on Network (shows device IP and MAC address. *Useful when you know the MAC address but not the IP it was assigned to)

            arp -a



System Config:

            msconfig



Windows Version:

            winver



Add Hardware Wizard:

            hdwwiz



Control Panel Shortcut:

            control



Device Manager Shortcut:

            devmgmt



Disk Cleanup:

            cleanmgr



Display:

            dpiscaling



Print Manager Shortcut:

            printmanagement



Windows Explorer Shortcut:

            explorer