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)".

LC-MS Contamination? Another Possible Cause. Are your Mobile Phase Bottles and Pick up Filters Clean ?

One of the more common LC/MS problems I am asked to help solve deals with contaminated LC-MS or LC/MS/MS systems. Over time, many systems will become contaminated with a wide variety of plasticizers, detergents, salts, metals and ion pairing agents that routine source cleaning will not remove. Often, these compounds are introduced to the system through the tools used (e.g. pipettes) chemicals, solvents, mobile phase additives or even the samples themselves. "Dirty" samples sometimes persist inside the system long after the analysis work is complete, leaving material in poorly maintained injection valves but also through the use of poorly washed / contaminated and fouled HPLC columns. Even the modern inline HPLC vacuum degasser has proven to be a source of contamination. 

In addition to the above mentioned sources of contamination, another more obvious source of contamination should always be addressed early in the process of cleaning the system. Specifically, the glass mobile phase bottles and the associated solvent pickup tubing and solvent pickup filters used with them. Contamination in these areas may directly infuse the system with undesirable material. Good cleaning and maintenance practices must be maintained to reduce this source of potential contamination. 

As a general guideline, we shall not place our mobile phase reservoir bottles in any type of dishwasher or wash them using any dish soaps. These may leave a residue easily detected by even the weakest mass spectrometer. Avoid contamination by purchasing high quality glass bottles with vented caps to keep dust out. If rinsing with organic solvents (and/or freshly prepared and filtered high resistance water) does not clean them, you can try a Nitric Acid rinse (up to 30%) followed by a neutralizing wash in 2M Sodium hydroxide. Follow-up with many rinses of HPLC Grade water (or LC/MS grade), oven drying, then re-fill with an appropriate mobile phase. Don't forget to replace those solvent pickup filters too. While many 316 SS pickup filters can be cleaned, most of the sintered glass style filters are designed to be disposed of (not cleaned or put in an ultrasonic cleaner!). So periodically dispose of the glass types and install new filters and fresh mobile phase into those recently cleaned bottles (before you start looking for the source of contamination in the more expensive parts of the instrument, clean or replace the filters). - Please don't re-contaminate an expensive HPLC or LC/MS system and invalidate your methods and data because you skipped replacing a $10 part. Keep commonly used spare parts in-stock and always maintain a clean system.

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.