HPLC System Preventative Maintenance Frequency & Procedure (PM); Part 2, Overlooked HPLC Chromatography Standard Operating Procedures (SOP's)

As a scientific consultant, I often review overall laboratory operations and make recommendations regarding documentation and procedures which may improve their accuracy and results. Some of these recommendations come in the form of SOP's.

Here is the second example of a 'must have' SOP which should be in place for any laboratory performing HPLC analysis. 

Part 2 of 3:
HPLC System Preventative Maintenance Frequency & Procedure (PM):
 
Regular cleaning, inspection and maintenance of the modules which make up the HPLC system should be carried out on a regular schedule. The frequency will depend on how the system(s) are used and what part lifetimes are typical. Preventative maintenance must not be confused with repairs or servicing to restore operation of the system after damage has occurred. Many companies perform PM services every 4, 6 or at most, 12-months. 

• PM procedures should include the inspection, cleaning and replacement of normal wear and tear parts such as: filters, frits, valve seals, injection seats/seals, pistons, piston seals and lamps. The PM provides an opportunity to inspect the condition of the modules and parts to insure they are operating properly. The goal should be to replace worn parts before they contribute to poor reproducibility or a failure. If the parts are found to be damaged, then that would be considered 'service' not maintenance and indicates that previous data collected on the system may be unreliable. Don't delay performing PM services on your instruments. Regular maintenance is a requirement. Your related SOP's should address which modules require regular maintenance ( A policy SOP), how often it is performed (A frequency SOP), who performs it and what training have they had (Training Requirements SOP. Also document in the specific Instrument's Logbook), which parts and tools are required (The actual PM SOP for the module) and what tests are performed to insure that it was done correctly (Separate SOP's for each Test).

Make sure you have several people review the draft SOP's before approving. Sometimes what appears clear to you may in fact have a different meaning to someone else. Clear procedures should contain enough detail that people with different backgrounds will each carry out the procedure in the same manner. Often, these types of documents will go through many drafts and even after approved, should also be open to future suggestions to make them even better.

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 PART #3 of this three-part series can be found at this link: "Routine Backup of HPLC Data, Methods And Related Data:; Part 3, Overlooked HPLC Chromatography Standard Operating Procedures (SOP's)";

If you missed Part #1, the article can be found at this link: "Mobile Phase Preparation; Part 1, Overlooked HPLC Chromatography Standard Operating Procedures (SOP's)".

Mobile Phase Preparation; Part 1, Overlooked HPLC Chromatography Standard Operating Procedures (SOP's)

As a scientific consultant, I often review company overall laboratory operations and make recommendations regarding documentation and procedures which may improve their accuracy and results. Some of these recommendations come in the form of SOP's.

Here is the first example (of three installment posts) of a 'must have' SOP' which should be in place for any laboratory performing HPLC analysis.

Part 1 :  
Procedure(s) For the Preparation of MOBILE PHASE :

Proper documentation of HPLC methods shall always include all of the information that someone would need to reliably reproduce the method in another laboratory. This includes the instrument brand, model, module numbers, configuration, details of the column type with the dimensions and particle size, flow rate, mobile phase composition, all detection parameters including flow cell dimensions, path length/volume, wavelength(s) and bandwidth (if applicable), sampling rate, injection solution, injection volume, sample concentration and other critical information. 

• An area which is often overlooked is HOW the mobile phase solutions are prepared. In addition to stating the chemical grades used, pH measurement checks/adjustments and if any filtering is required (esp. for Aqueous Solutions), mobile phase preparation often includes weighing, dispensing and mixing steps, each of which needs to be described in detail if they are to be reproduced. Without clear directions, the composition of the mobile phase may be different. For example, do you weigh or measure (volume) out all liquids? What type of glassware are used to measure volumes? Volumetric flasks, beakers, graduated cylinders (and if so, what tolerance grade or class are they?) When mixing two solutions, do you measure and prepare them separately in two containers (if so, which containers?), then mix them (how do you mix them)? Do you fill one container with one liquid, then fill to the desired level with the second one? Do you need to check the pH of the solution (as well as how to adjust the pH of the solution? With what?)? When and how? Do you have a SOP for the pH meter and how/when to calibrate it? Number of standards used (usually 3 are used)? Is the final solution filtered, and if so, by what method (be specific)? I have seen people use different methods each time they prepare a solution. As you can see, each procedure results in a final composition which will be different. Different mobile phase compositions usually lead to different results. The important message here is to use the SAME method to prepare solutions and to document it in a SOP for the method (and for all methods). Additionally, be sure there is formal training to insure that everyone prepares solutions in the same manner. Most labs will need multiple SOP's for mobile phase preparation, but as a general guideline, you should have one master SOP for the preparation of mobile phase solutions. The SOP should also include detailed information regarding STORAGE time limits for each type.This will set the standard from which the other SOP's can be based on.

• Be sure to prepare FRESH, filtered aqueous mobile phase solution for use each day. Prepare only enough solution for one or two days use maximum (refrigerate the solution to get 2 days), then dispose of any remaining solution and start with fresh solution. Do not store aqueous solutions at room temperature or for extended periods.
  

• Some types of ORGANIC solvents may require special SOP notes regarding preparation (e.g. pre-filtering), stabilization additives (e.g. mol sieves, dry Nitrogen gas, etc), types of storage (e.g. amber glass bottles away from light), expiration dates after opening (e.g. Acetic Acid, DEA, TEA, THF, etc). Be sure to include these in your SOPs.
  

Notes on SOP creation and editing: Make sure you have several people review the draft SOP's before approving. Sometimes what appears clear to you may in fact have a different meaning to someone else. Clear laboratory procedures should contain enough detail that people with different backgrounds will each carry out the procedure in the same manner. Often, these types of documents will go through many drafts and even after approved, should also be open to future suggestions to make them even better.

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PART #2 of this three-part series, "HPLC System Preventative Maintenance Frequency & Procedure (PM); Part 2, Overlooked HPLC Chromatography Standard Operating Procedures (SOP's)" can be found at this link:

Vespel, Tefzel or PEEK Valve Rotor Seals ?

One of the most common HPLC preventative maintenance parts is the injector valve rotor seal. Worldwide, the majority of these chromatography parts are produced by Rheodyne (IDEX) or Valco Instruments (VICI) and used by the major instrument manufacturers in their products. These valve seals are critical to maintaining a leak free, high pressure seal inside the injector. They are subjected to a lot of wear and chemical exposure with use. They have a finite lifetime which may be very short, in some applications (a few months) or last for many years in others. *The most common reason for rotor seal damage is a lack of flushing when buffers are used. Regular flushing down of the flow path (without the buffer) is required to maintain a clean flow path. Buffer deposits and crystals scratch and damage the rotor surfaces. Depending on the specific use and application, rotor seals are often replaced as a preventative measure once every 6 or 12 months. They should also be replaced whenever they are scratched, heavily worn, no longer sealing well, leak or become contaminated. Failure to maintain your injector's parts may lead to HPLC carry-over contamination problems.

The choice of rotor seal material should be based on: (1) the types of chemicals it will come into contact with; (2) the working pH range; (3) the temperature range.

• Note: When choosing a valve rotor seal material, please refer to the valve manufacturer's information, compatibility and advice. Blends and properties may vary between vendors so always verify compatibility with them before use.

 
Common HPLC Rotor Seal Material Types:

 Vespel ^®: Chemically, this is a polyimide blend (DuPont). One of the most widely used materials for HPLC valve rotor seals. It has excellent chemical compatibility with most HPLC mobile phases, excellent temperature stability and a pH limit of 10. An excellent choice for most applications.

Tefzel ^®: Chemical name, ethylene-tetrafluoroethylene (aka, "ETFE"). It has excellent chemical compatibility with most HPLC mobile phases and a higher pH limit of 14. Tefzel's preferred applications are where very high (>9) or very low (<3) pH solutions or mobile phases are used. Not compatible with some chlorinated solvents and in most forms it has a temperature limit of 50°C.

PEEK: Chemical name, polyether-ether ketone. Known for applications where biocompatibility and / or high temperatures are of concern. Like Tefzel, it has excellent chemical compatibility at room temperature with most HPLC mobile phases. Most vendors report a working pH range between 1 and 14. Unlike Tefzel, it has a much higher temperature limit (i.e. 100°C or higher), but its resistance to some chemicals appear to degrade with increasing temperature. Contraindicated where it will come into contact with solutions of THF, methylene chloride, DMSO or concentrated acids (i.e. nitric or sulfuric). Some sources have observed problems with chloroform as well, especially with PEEK tubing, so it may not be recommended for those applications.

 

For more information on troubleshooting HPLC injector valves, please refer to this linked article, "Troubleshooting HPLC Injectors (Manual and Automated)".