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)".

Repair Missing Or Corrupted Windows System Files Using the System File Checker Tool

It has been awhile since I posted any Microsoft Windows software tools or tips so here is a MS utility program. Most laboratory instruments operate under MS Windows software control and you will be able to keep things running smoothly if you take the time to learn and use many of the available utility programs. 

• Note: Before using any software utility program, make sure you first have permission and authorization to do so. Back up your system programs and any data files. Create a Restore Point to protect the basic settings too. Take precautions before using any utility programs and do so at your own risk.

In addition to the very useful "Restore Point" feature found in Windows (discussed in an earlier post, which is great for solving a number of issues), Microsoft has another time-saving utility tool built into most versions of Windows (i.e. XP, Vista, 7, 8 and 10). The Windows System File Checker (simply known as, SFC) offers users the option to scan their Windows operating system files for corruptions AND restore any found corrupted files, all automatically! Running the utility is very easy. First, make sure your account has Administrator privileges, then use the Run Command prompt to run the program SFC 'As Administrator' *Type:  sfc /scannow 

More information about this useful utility can be found on Microsoft's website support page.

Determine the HPLC System Dwell Volume (Gradient Delay Volume)

Note: The total HPLC gradient system dwell volume is different than the HPLC column’s void volume. Two different terms for two very different measurements.

When we perform gradient HPLC analysis, the mobile phase composition is changed over a period of time. The mobile phase is mixed in real time by the pump(s), mixer and/or valves, then transported to the injector and finally, on to the head of the HPLC column. The total volume of liquid contained between where the mobile phase is mixed and the head of the column helps us determine when the newly mixed solution arrives at the column head (it is not instantaneous). This delay is often referred to as the gradient delay time (or delay volume) and its value will vary for different HPLC systems due mainly to differences in tubing dimensions used, pumping system type and the design of the flow path. 

For example: If the system dwell volume is found to be 1 ml and the flow rate used is 1.000 ml/min, then the gradient delay time is one minute. 

So how do we know what the system dwell volume or gradient delay volume is? Well, we measure it of course!

Measure the ‘System Dwell Volume’ (aka: Gradient Delay Volume)*:

(1) REMOVE any HPLC column(s) and install a Zero Dead Volume Union (*ZDV) or a restriction capillary of know volume in its place.

(2) Prepare Two Different Mobile phase solutions:

Bottle ‘A’: HPLC grade Methanol (MeOH).

Bottle ‘B’: HPLC grade Methanol with 0.1% acetone added (v/v).

(3) Set your UV/VIS detector to 265 nm (8 nm Bandwidth, Reference OFF).

(4) Program a suitable system flow rate and create a simple Gradient Method (linear change) which starts at 0.0 minutes with 100% ‘A’ (HPLC grade Methanol) and 0% B (HPLC grade Methanol with 0.1% acetone added) and runs to 0% ‘A’ and 100% ‘B’ for about 10.0 minutes (actual times used will depend on your selected flow rate).

(5) Flush and degas both solutions, ‘B’ first, then ‘A’ through the system until you get a nice clean, flat baseline. Make sure their is enough backpressure on the pump (>40 bars) to obtain a stable signal (use a restrictor or back-pressure regulator if needed).

(6) No injection should occur during this method.

(7) Start the method (RUN) and observe the 265 nm signal over time. At some point you should observe the signal begin to rise. When you see this signal change occur, the acetone has finally made it from the pump head to the detector’s flow cell. Make note of the time this occurs. 

Using the known flow rate and observed signal change time, you can now estimate the total system dwell volume. 

Example: If you observe the signal start to rise steeply at 2.00 minutes and your flow rate was 1.000 ml/min. Your system dwell volume would be 2.000 mls. 

A more accurate system dwell volume value can be obtained by next running the same method with an injection of acetone (e.g. 1 ul) and noting the time at which the injection peak is first seen. That will give you the time it takes the sample (and therefore the volume needed) to go from the injector to the flow cell. If you subtract this time off the system dwell time you recorded in the last test, you will have the actual measured time from the pump head (or proportioning valve) to the head of the column (vs the flow cell). Normally the volume contained in this tubing and flow cell are very small relative to the volume in the rest of the system, so we can ignore them. However, when using some of the very low volume columns (e.g. 2.1 x 50 mm), the volume contained in these areas can become significant so when appropriate, we need to be aware of them.

Failure to take into account changes in HPLC system dwell volumes can result in methods which no longer work or provide different results. This is because the gradient rate change you program in your method may not allow enough time for the new mobile phase composition to reach and flow all the way through the column in the time that you have programmed. A common mistake we see is when users forget to adjust the gradient profile when changing column dimensions or program changes using too fast a time.

BTW: One common trick we use to improve compatibility between systems which have different dwell volumes is to include an initial (time 0.0)  isocratic hold-time into the start of each method. If all systems used have system delay volumes under 3 mls, then add a 3 minute isocratic hold time at the start of each method (if 1.000 ml/min flow rates are used), before any gradient starts. While not the best way to deal with the issue, this type of “cheat” can make it possible to quickly adapt a method for use on several different system types.

*Note: This is a generic method to determine the system dwell volume or gradient delay volume. Detector signal buffering and flow cell volume also adds to the delay and in some cases, must also be accounted for too. There are many other methods which can be used for this determination as well. This proposed example serves to illustrate the concept only.