Agilent Quaternary Pump (e.g. G1311A ) "Secret" Operator Tip to FLUSH the HPLC Pump in 1/2 the time!

One of the most popular "tips" taught in our Agilent 1100 and 1200-series HPLC training classes shows users how to speed up the daily priming and flushing process of the Quaternary Pump. Many people use these pumps without taking advantage of the Quaternary pump's higher flow capability. If you are not currently using the higher 10mL/min flow rate capability offered by this pump (vs. the Binary pump's 5 mL/min), then you are missing out on a free time saving feature. Please read on to learn how to use this feature.

Based on the HP 1050 pump and introduced in 1995 as the "1100-series" version, the G1311  "Quat" pumps are one of the most popular research grade HPLC pumps found in laboratories today. They are extremely reliable, rugged, easy to operate and service. The Quat pump is driven by an easily accessible, single pump head with an in-series, servo controlled dual plunger and Multi-channel Gradient Valve ('MCGV') for 4-channel solvent proportioning with an active inlet valve (known as the 'AIV', first used in the HP 1050 pump and the reason for this pump's high reliability. No more "sticking" inlet valve issues!). Unlike the Agilent Binary pump (G1312), which uses two separate dual plunger pumps (2-channel) at up to 5.0 mL/min (maximum), the Quat pump offers an extended flow range, up to 10.0 mL/min (maximum). However, most users are not aware of this or do not know how to utilize this higher flow rate feature because the Quat pump defaults to a maximum flow rate of 5 mL/min at initialization. The ability to program the pump to operate at flow rates greater than 5 mL/min requires a "trick" to activate it (which apparently is a secret as we rarely encounter customers who are aware of how to use it). 

Let me share with you why you would want to use this feature, why the feature is hidden to most and of course HOW TO ACTIVATE IT on the Quat pump.

• Q: Why would you want to run the pump at 5 to 10 mL/min? Semi-prep columns can be run within this flow rate range, but a more common reason to operate at 10 mL/min is for daily system start-up. Anytime you replace or change the mobile phase bottle/solution OR when you startup the HPLC system (each day) one of the very first things you need to do is prime or flush each of the mobile phase channels, one-at-a-time through the system to waste. Air bleeds into the system when it is not used and this procedure primes the lines and pump head with fresh mobile phase preparing it for use. The system's flow path is directed to waste (via the open, prime-purge valve) during this step so back-pressure is not a concern. The higher the flow rate you can use for this flushing step, the sooner you can complete it. If you run the pump at 10 mL/min vs 5 mL/min, then flushing can be completed in half the time. This is especially useful if you have a model G1322A degasser module installed as the internal volume of each degassing channel in the G1322A is 10-12 mLs, requiring extended flushing times (4x channels = 30+ mLs flush per channel) before moving on to the next channel.

• Q: Why does the Quat pump initialize with a reduced, 5 mL/min maximum flow rate? The Quat pump was designed to meet two different operating pressure ranges. From 0 to 5 mL/min the permitted operating pressure range is 0 - 40 MPa (0 - 400 bar). Above 5 mL/min, the operating pressure range is reduced, 0 - 20 MPa (0 - 200 bar). As most analytical chromatography is performed at flow rates below 5 mL/min, the system initializes using the more practical, 0 - 400 bar range, limiting flow rates to 5 mL/min maximum. The default maximum pressure field is set to 400 bars. You should always change the maximum pressure value from 400 bars to a more realistic maximum pressure (lower value) for your method. Use a maximum value that is appropriate for your own method. *The only time you will want to set it to the maximum value is when conducting a Pump Pressure/Leak test (it must be set to max pressure for testing).
  

• Q: When I try and enter a pump flow rate larger than 5 mL/min, the system does not accept it. How do I program the pump to increase the flow rate past 5 mL to 10 mL/min? In order for the system to accept a flow rate of greater than 5 mL/min, you must FIRST set the maximum pressure limit to a value that is 200 bars or less (within the allowed "0 - 20 MPa (0 - 200 bar)" range). Once the maximum pressure limit has been reduced in the method, the system will then allow you to enter a higher flow rate such as 9.999 mL/min (10 mL/min). As long as the maximum pressure alarm is set within this window (200 or less), the pump will allow flow rates above 5 mL/min to be used. Now you can program the pump to flush lines or prime the system at twice the speed of the Binary pump equipped systems (10 mL/min).

Please share this "trick" with other users of the G1311A, G1311B, G1311C versions of this pump so they can maximize their time and productivity. Let us know if you find this tip useful.

Resetting or Clearing the TCP/IP stack to Resolve Connection Problems

• Warning: These commands should only be used by experienced users who both accept and understand the risks involved. Before using any system commands or utilities, please backup all systems, programs, applications, data and files.

BACKGROUND: A TCP/IP Stack Reset or 'clearing' will flush the computer’s stored Internet Protocol settings back to their default values. Flushing or resetting these values may solve some types of Internet connection issues where screen messages such as; "This Site Can’t be Reached” or “Unable to Connect to the Internet” are observed. 

EXAMPLE 1: To reset the TCP/IP stack in Windows O/S, you can use the Command Prompt. You MUST use and run the Command (CMD) prompt screen with Administrator's Privileges (or it may not work). 

   In Windows 10 O/S, from the CMD prompt screen, type and run the following commands:

•     netsh winsock reset and press Enter.
•     netsh int ip reset and press Enter. (*this command rewrites two registry keys)
•     ipconfig /release and press Enter.
•     ipconfig /renew and press Enter.
•     ipconfig /flushdns and press Enter.

•  Exit out of the CMD screen (exit) and recheck your connection. A re-boot, restart should not be needed.

  

   In Windows 7 O/S, from the CMD prompt screen, type and run:

• netsh int ip reset resetlog.txt 
• Exit the CMD.
• Restart the computer and check your connection.

EXAMPLE 2: For most MacOS, the TCP/IP stack reset can be run by renewing the DHCP Lease.

• Click on System Preferences then Network. Click on the Internet connection with status Connected. Next, click the Advanced button. Open up the TCP/IP menu and click on Renew DHCP Lease followed by the OK button.

Notes on Cleaning bound Protein from RP HPLC columns:

First, a few comments:

• ·         Before proceeding with any column regeneration or cleaning procedures, always refer to the specific advice provided by the column manufacturer. Approved maintenance and cleaning instructions can often be found in the product guide which comes with the new column. Their guidelines supersede these!

• ·         Columns are consumable items. After a suitable amount of use, the time and materials required to regenerate them may cost more than the purchase of a replacement column. Always have a new, spare column on hand.
  

•        Do not overload the column! This is the most common reason for column fouling, flow path contamination and sample carryover issues. In most cases, injection volume should be less than 1% of the column volume (maximum).
  

• ·         Protect your detector. Before washing or cleaning the column, disconnect the column outlet line and direct the column to waste only.

•        Column Storage solutions are not the same as column wash solutions. Never store a column in buffer or ion pairing containing solutions.

For RP supports, if buffers have been used, always start by washing the column down with ultra-high purity water and some organic solvent (e.g. Water/MeOH, 95%/5%) to remove all salts. Use about 10 column volumes to flush these off. Do not wash the column with organic solvents until you have first washed it thoroughly with high-purity filtered water.

Polymeric resins (e.g. PS-DVB) from many manufacturers can effectively be cleaned using 0.1 M Sodium Hydroxide solution or a mobile phase solution containing equal parts of isopropanol (IPA) and 1 to 3 M Guanidine hydrochloride at ~ 50 °C. Optionally, some success has been reported using other solutions such as: 5M Urea (pH 7) buffer solution; 1 M NaCl (pH 7) and even mixtures containing some methylene chloride solvent. Check with the manufacturer first as column damage/plugging may result if their directions are not followed.!

For RP silica based supports (non-SEC), we often start with a series of wash solutions. In most cases, pure water or pure organic solvents such as MeOH or ACN will not remove bound protein (common novice mistakes). An acid, base or even an ion pairing reagent is often needed to clean them. Start simple and monitor.

 

For RP silica based supports (SEC), a high salt buffer solution often releases bound proteins quickly. A mobile phase containing water plus an alcohol (methanol, IPA or ethanol) may also prove effective too.  Optionally, a solution of 0.5 M guanidine hydrochloride may effectively remove bound material.

General Advice: One of the first general wash solutions to start with (especially to remove basic compounds) is a 1% Acetic acid solution in Methanol (50/50). If desired a stronger acid such as 0.1 % Trifluoroacetic acid (TFA) or 0.1 % Formic Acid can be swapped for the acetic acid (where possible, start with a weaker acid). Use a low concentration of acid to achieve a pH of ~ 2.5. This acidic wash can be followed with a neutral solution, or if needed, a later solution where IPA or ACN replaces the MeOH used (50/50).

For extreme cases where the column has been overloaded with protein, a 5 M Urea solution has been proven effective in removing bound protein from silica and polymeric supports too. A word of caution, as the resulting pH of this strong solution may be greater than or equal to pH 9. Many types of silica based RP columns can not withstand strongly basic solutions and the silica inside may dissolve (plugging the column). Start with a lower concentration wash  first. You can always increase it later. Always read the instruction sheet carefully which came with the specific HPLC column to determine if it can be used at these high pH levels. Another salt solution that has shown some promise is 1 M sodium phosphate solution, pH 7.0. Run the salt solutions for about one hour at a moderate flow rate. Follow up all washes with rinses of mixtures of water and MeOH (80/20), then 90% MeOH/Water. 

Please remember that in ALL cases, HPLC columns are consumable items with a limited lifetime. Dispose of them properly when they are damaged or contaminated and replace with a new column. Once you have a fresh clean column to work with, prevent column fouling by developing better quality methods which utilize frequent, properly developed wash methods (using a wash solution which is stronger than your analysis mobile phase), filter all samples and be sure they fully dissolve in solution (100%). *Column fouling is not normal and can be prevented with proper training.