HPLC SOLVENT COMPRESSIBILITY - REVISITED

 Twelve years ago I published a short article here (HPLC PUMP SOLVENT COMPRESSIBILITY VALUES) which described the importance of setting the correct solvent compressibility values in the HPLC pump's table. Developing HPLC methods which exhibit smooth, stable baselines, with little measurable signal artifacts (e.g. spikes, noise, oscillation) and minimal pressure fluctuations help insure reliable, repeatable methods. Taking steps to insure that the LC pump operates is setup properly for the method are part of following good chromatography fundamentals

Over the past month I consulted for three different clients who needed help in troubleshooting various "pump stability problems". In all three cases, each HPLC system showed extreme pump pressure cycling, cavitation, noise and instability over time. Pressure fluctuations of 10% (or in one case, 10-30% Ripple values) were observed in several different HPLC methods that were used. One of the very first areas to check for problems with pump pressure instability is mobile phase degassing.  

Proper operation of the HPLC pump requires that efficient degassing of all mobile phases is performed before the liquids enter the pump head. 

Failure to properly degas liquids often results in pump cavitation, check valve sticking and baseline instability. An Inline vacuum degasser or continuous Helium sparing should be used to degas all mobile phase solution for use in HPLC (not sonication or vacuum filtration which perform poorly to solve degassing issues). 

In one of the three cases, the HPLC degasser was found to be broken and long overdue for service. Cleaning and servicing the degasser cured the problem and the method that once showed pressure ripple of >10% now shows no baseline disturbances and very low ripple of ~0.1% at ~ 70 bars system pressure. 

Before I was called in to assist each client, the clients had replaced numerous parts, including: pump seals, check valves, mixers, solvent frits and still had the same baseline instability issues (no change). As recommended by me, two of the clients had their very old degassers cleaned and serviced (as they were long overdue for service), but still had some baseline and pump instability (servicing the degassers improved the baselines, but the pump was not running as it should). In both cases, the cause for the remaining pump instability was quickly identified by me on-site (many problems can be quickly diagnosed on-site).

• The client had incompatible solvent compressibility values stored as part of their HPLC methods. This resulted in huge baseline disturbances, spikes, cavitation and occasional loss of prime. 

One of the clients normally ran methods containing high percentages of ACN (with some water) for their sample methods, but a few months earlier had switched to running with gradients containing high percentages of methanol. The solvent compressibility values stored in their system were appropriate for WATER, but they never updated them when they used the same method file to run samples in mostly methanol solutions (which need different compressibility values). Though they all had been using HPLC for many years, they had not received basic HPLC instrument training to know how to adjust and optimize these and other important instrument settings for EACH method (they were overwriting each new method, a common new user mistake, when making changes). Once we changed the method's solvent compressibility value to a more compatible one (in their case, for methanol), the baseline smoothed out in just a few minutes and all of the pressure instability issues went away (*they had replaced several thousand dollars worth of perfectly functioning parts trying to solve this issue before I arrived). Professional training in how to use and operate any HPLC instrument should always include how to set and optimize the compressibility value(s). Make sure you know how to incorporate the correct value in each new method that you create. Always spend up-front time to optimize each method for the application before you use it to analyze real samples. The initial time spent getting everything to run smoothly and reliably will improve overall accuracy plus save money and time.

• Note: In a low-pressure HPLC single-head pumping system with multi-position solvent selector valve (e.g. Most ternary or quaternary systems) one value is allowed, but in a true, dual-head binary pumping system each of the two pump-heads may have a separate field to input the solvent compressibility values.

The importance of inputting the correct and applicable solvent compressibility value(s) into the pump's settings, for each solvent used is one of many steps in creating an optimized HPLC method. There are no universal values, but the instrument manufacturer will have included a generic value in the pump's compressibility settings field. Should you use this generic value?  What are the chances that a randomly selected value used as a 'place holder' in the software is the correct value for your method?  Just as with flow rate, solvent composition, run time, stroke volume, wavelength etc., entering (and saving) the correct solvent compressibility value into EACH method helps to optimize the pumping performance. You will want to select an appropriate value FOR EACH AND EVERY HPLC METHOD YOU CREATE and use (and be sure to save the method with a unique name). Start by loading your HPLC method into the system, then look at the solvent compressibility value(s) used. Are they correct? Change the value(s) shown to values that are appropriate for your method. It is OK to experiment and try different values (we encourage it!). Monitor the S/N levels of the baseline noise for comparison. The instrument manufacturer should provide a table of suggestion solvent compressibility values for use with their system [For HP/Agilent systems, you can see an example table at the link I provided in the first paragraph of this article or review the operator's manual for more information].

• A related article to this one that you may find useful is; "Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)";
  

Cannabis, Cannabinoid (Hemp, THC, CBD, CBN, Marijuana) HPLC Analysis and Testing, Areas for Improvement [*Updated 8/2021]

Over the past few years we have observed an exponential growth in the number of state-level, legal businesses (in the USA) who offer Cannabis Analysis (e.g. Potency Analysis or Profiles) and/or related businesses such as Hemp Oil Extraction. Most related products which incorporate Marijuana, CBD, THC, Cannabinoids, Terpenes and/or other related compounds require formal analytical laboratory testing which should follow good laboratory practices. This article is targeted to help many of the people involved in this new analysis business (or anyone using chromatography as the analytical technique of choice for the same goals).

As a professional chromatography consultant, I have seen a large increase in the number of requests for my services to this new market. Most of these new businesses have questions about obtaining professional training, correct analysis procedures, improving reproducibility, documentation, optimizing method development, how to receive professional training in maintenance of the HPLC system(s) and need hands-on help to optimize the procedures used. Many users are not achieving acceptable results and need help finding out why. They want to know where they can take a class to learn HPLC method development and how to perform the required tests. 

These new businesses would benefit greatly from professional guidance EARLY in their setup and establishment, to improve the internal methods and procedures of analysis used. Time spent on the "front-end" of any process is always time spent wisely (in this context, knowledge and practical experience = confidence). A chromatography professional can quickly identify areas which may need improvement and/or suggest changes that can directly improve your company's accuracy, reproducibility, increase efficiency and of course, impact your bottom-line too. Focus is placed on the exact areas that will benefit you (rather than wasting time with non-targeted approaches, sales biased classes and trial-and-error approaches). 

• Please note that there are NO SCIENTIFICALLY LEGITIMATE ONE DAY, ONE WEEK or ONE MONTH LONG TRAINING CLASSES THAT CAN TEACH YOU HOW TO PERFORM HPLC METHOD DEVELOPMENT or ANALYSIS. NONE AT ALL. Most types of "Certification" offered are completely without scientific merit or value. The training needed takes many years of hands-on experience, in an industrial laboratory (not a school), to acquire just a basic level of proficiency (*Emphasis on 'Basic", not intermediate or expert). Be very cautious of anyone who claims to be able to provide you with all the training you need in a short time period.
  

Generating accurate and reproducible analytical data, esp. with HPLC, SFC or GC requires a great deal of knowledge, formal training and practical hands-on experience (not something which is taught at most university or school programs). These complex techniques require years of bench time and professional hands-on experience to learn). Shipping or selling products which contain unacceptable levels of impurities or which do not meet basic testing or regulatory qualifications could pose a health and/or liability risk. Hire people who have the needed training from industry before setting up the laboratory.

It has been my professional experience that some of the most common training areas that client's would benefit from are: GLP (Good Laboratory Practices/Procedures and SOPs) and additional instrument operational training to demonstrate proficiency in analytical chromatography. Address these areas early on and continuously update them to reduce errors and improve results. Training should continue on a regular basis to gain experience.
 
While each confidential consultation visit I have with a client may show different key issues which need to be addressed, many labs can start to improve their analytical results by addressing and improving how they address:

1. Documentation: Laboratory methods and sample analysis must be conducted using clearly written documentation. This should initially include having Standard Operating Procedures (SOPs) in place for all methods, procedures, qualification, verification and tests used. They should include SOPs, Document Control and Policy documents which also address: Training, Calibration, Maintenance, Frequency of the same, Mobile phase preparation, pH measurement, use of the balance and so on... I find that it is best to create an initial SOP Template to insure document uniformity (i.e. include such fields as: Business Name, SOP #, review/approval date(s)/names, Rev #, materials & tools needed, purpose, procedure steps, pass/fail definitions... plus any needed supporting documents).
2. Sample Preparation Methods: Be sure to document, test, review / standardize specific sample preparation methods, for each sample type. Variations in: temperature, extraction solvent or the solution(s) dissolved in, homogenization or grinding methods, mixing, times used, glass or plastic containers used may result in significant variation of the final reported results.
3. Correct Poor Reproducibility and/or Baseline Instability Issues: In chromatography analysis, if the method(s) used are not stable and reproducible, every time they are run, then little to no scientific value can be obtained from them. Methods used must follow basic good chromatography fundamentals and meet basic guidelines. Baseline noise or instability may directly impact integration results (which directly impacts reported results). Instrumentation must be operated in clean, climate controlled rooms. Failure to reproduce a result within acceptable limits (these will vary per method type) will invalidate the method used. Make sure that SOP's are followed, mobile phase solutions are made fresh each day (do not pre-mix solutions with acids and let them sit for several days before use; do not "top off" bottles), solutions should be degassed, HPLC columns are properly washed and re-equilibrated before each analysis, instruments are maintained (per a SOP) and serviced on a regular basis. 
4. Develop HPLC methods that follow good chromatography fundamentals: Retain, separate and resolve ALL peaks. Insure peak K primes are 2.0 or higher. If you have co-eluted peaks in your method, then method development is not finished. If you have ghost peaks or changing retention times, then you need to stop running samples and find out why. Be careful whose method(s) you use. A method that is "Validated" may not be scientifically valid method to use. Have the method checked by an experienced chromatographer.
   
5. Continuous Training is Required to become Proficient: To be proficient, at a basic level in chromatography, takes most chromatographers several years working in an industrial environment to gain practical hands-on time. That assumes that they have had professional training outside of college, in an industrial lab, and can demonstrate an understanding of the fundamentals of good chromatography. Note, that method development skills require a much higher level of understanding and hands-on training to acquire the needed skill set. Make sure your scientists have the needed level of training to operate, run analysis methods and troubleshoot any issues that come up (and issues will come-up, even under ideal conditions). Please do not make the mistake of thinking they will "figure it out" on their own. Hire people who already have several years of industrial chromatography experience, then provide them with additional training opportunities to advance their skills in the application areas that your business needs.  Get them help NOW, you will save money and time, plus get back on track moving forward with your project.
   

If you want to surpass your competitors and provide clients with the most accurate data, then investing in your employees professional knowledge and hands-on technical training is the fastest route to do so. This is an experience based technique where decades of practical knowledge are needed to improve your skill set. A professional can quickly provide you with practical information and show you techniques that will help you move forward. 1-2 days of on-site training often translates to nearly one years worth of knowledge. What is one-years worth of lost time worth to you?
  
Additional Resources:

• Modern HPLC Method Development Tips (PART I): 
• Modern HPLC Method Development Tips (PART II):
• EXPERT CANNABINOID HPLC Training and CONSULTING SERVICES: