The LCGC Blog: Looking for Love in all the Wrong Mobile Phases

Blog
Article
Image generated by Jonathan G. Shackman using Copilot

Image generated by Jonathan G. Shackman using Copilot

It’s Simple, Right?

A stationary phase and a mobile phase: the perfect pairing... when done right. Assuming the most common of ultrahigh- or high-performance liquid chromatography (U/HPLC) separations, achiral reversed-phase (RP) with UV detection, column selection can be as simple as pulling your favorite C18 out of the drawer or as complex as performing solute modeling to predict which of the hundreds of available phases will be best suited to your mixture (1). When adding in column geometry as a variable, the sheer number of possible column part numbers should far overshadow the selection of mobile phases (MPs). All we need is some water and an organic solvent. Flipping through my 3rd edition of “Introduction to Modern Liquid Chromatography,” (2), I see the authors rank RPLC solvents as:

Acetonitrile (preferred) > Methanol > Isopropanol >> Tetrahydrofuran (less useful)

Ah, simplicity! Unless, as they note, you live in a country that considers acetonitrile too toxic to use. Also, being published in 2010, the Great Acetonitrile Shortage of 2008 was long past, and hopefully not a recurring event (3). Aside from those issues, the ultraviolet (UV) cutoff, relatively low back pressure, and strong elution power appear to justify acetonitrile’s preferred status. Methanol, while typically more affordable and less toxic, does have higher back pressure, higher UV cutoff, and a pesky habit of being reactive, both with other mobile phase components and analytes themselves (4). Of course, methanol, either alone or as a ternary blend, can offer some beneficial selectivity changes, especially when dealing with π-π interactions (5). The other listed solvents seem much rarer in RPLC, finding more utility in normal phase methods. For tetrahydrofuran, this is probably for the best, to avoid novice chromatographers from storing it to the point of forming explosive peroxides, buying stabilized forms with now-useless UV cutoffs, or using it on LC separations with incompatible materials. On this last point, most vendors list solvent compatibility in their manuals or have it readily available upon request. Upgrade kits may even be available so that you can happily run tetrahydrofuran, acetone, and hexafluoroisopropanol all day without destroying your nonmetallic fluid lines. Should you desire a more exotic MP, references (2) and (6), and especially their appendices, offer a wealth of information on LC-relevant solvents.

Beyond Neutrality

RPLC analyses will frequently include analytes with ionizable groups. Hence, MP pH is a critical lever in selectivity, and controlling it is necessary to ensure a robust method. Even unmodified aqueous solvent systems can have quite variable pH ranges around neutrality due to reagent source (e.g., bottled vs. in-house generated), degassing technique (e.g., none, vacuum, sonication, sparging), age, and storage/environmental conditions. We used to think all we needed was phosphate with a dash of acetate to fill in the gap around pH 5 to satisfy all our separations, which were usually limited to pH 2–8. With the advent of advanced hybrid particles, zirconia, and polymeric resin supports, we now have a much broader pH range (e.g., 1–14) that can be leveraged. Additionally, simplified lower-cost mass spectrometry detectors have widely proliferated into measurement labs, typically in-line with UV detectors. Unfortunately, the best buffers with low UV cutoffs tend to be nonvolatile and unfriendly to MS; conversely, the least ion-suppressing volatile buffers for MS tend toward higher UV absorbance, impurity-containing reagents. At present, there appears to be no ideal universal buffer that perfectly pairs with both detectors, whilst maintaining suitable buffer capacity, stability, and reasonable solubility with organic solvents. Perhaps it’s a lost cause, and the only solution is on-line buffer exchange or two-dimensional liquid chromatography (2D-LC), but this would seem to be at odds with the requirements of robustness and simplicity of operation to be widely put into routine practice.

Chasing Ghosts

Even with the perfect MP recipe, the source and quality of the ingredients can be critical to achieving a successful and interference-free separation. Just as critical as the inputs can be the unit operations to generate the final compositions. Is distilled water from the grocery store good enough, or does it need to be certified UHPLC-UV-MS-Gradient grade? Does filtering remove more column-clogging particles than it contributes UV-absorbing leachables? Is it better to filter just the solvents, or should filtration occur after additives are mixed in, possibly changing the composition of volatile components? How much organic solvent should be premixed into MP A to prevent microbial growth (a surprisingly high amount, but then again, microbes can grow in volcanos and at Chernobyl)? What is my buffer solubility limit in MP B (also some surprising misconceptions are propagating out there [7])? Perhaps most frustrating is when you feel the optimal conditions are achieved, only to have utter failure set in when others attempt to run your method. I’ve run out of digits to count the number of horror stories from method transfers hamstrung by interference peaks. Chasing ghosts (8) can drive you mad, as there are so many potential sources of contamination, from the reagents to the glassware to the analysts themselves. To overcome some transfer issues quickly, in some instances, we have had to: acid-wash bottles to remove detergent peaks; specify white, dye-free cleanroom gloves for analysts; prohibit stir bars in favor of orbital shakers; and restrict the amount of pre-equilibration time in methods to prevent system peaks.

Quest for Perfection

Our criteria for the perfect (or at least, suitable for a majority of applications) RPLC mobile phase now includes:

  • Appropriate elution strength for a diverse range of analytes
  • Compatibility with a diverse set of columns
  • Low (< 195 nm) UV cutoff
  • MS compatibility with low signal suppression (preferably without creating adducts)
  • No impurity-generating system peaks
  • No particle contamination or component insolubilities
  • No microbial growth
  • Stability and inertness
  • Low viscosity
  • Common material compatibility (stainless steel, PEEK, glass, etc.)
  • Robust buffering from pH 1-14
  • Non-toxic / unrestricted usage
  • Green, sustainable and economical

I’ll leave open the debate if ion-pairing should be a beneficial attribute, and you may have additional considerations I missed. Clearly, we’re probably chasing a unicorn here to hit all these points. Indeed, even untargeted, generic methods, such as Dong’s example using acetonitrile and formate-based MPs (9), only meet a few of these criteria. With the potential of per- and polyfluoroalkyl substances (PFAS) bans coming, even my own department’s generic MP using water, acetonitrile, and 0.05% trifluoroacetic acid may soon need to be replaced (as well as additional possible impacts across the labs [10]). Yet research continues, and alternatives are being evaluated (11). Perhaps one day, the unicorn will be caught.

References

(1) Wen, Y.; Talebi, M.; Amos, R. I. J.; Szucs, R.; Dolan, J. W.; Pohl, C. A.; Haddad, P. R. Retention Prediction in Reversed Phase High Performance Liquid Chromatography Using Quantitative Structure-Retention Relationships Applied to the Hydrophobic Subtraction Model. J. Chromatogr. A 2018, 1541, 1–11. DOI: 10.1016/j.chroma.2018.01.053

(2) Snyder, L. R.; Kirkland, J. J.; Dolan, J. W. Introduction to Modern Liquid Chromatography, 3rd Ed.; John Wiley & Sons, Inc., Hoboken, 2010.

(3) Majors, R. E. The Continuing Acetonitrile Shortage: How to Combat It or Live with It. LC-GC North America 2009, 27 (6), 458–469.

(4) Metzger, B.; Shoykhet, K.; Buckenmaier, S.; Stoll, D. Chemistry in a Bottle: Ester Formation in Acidified Mobile-Phase Solvents. LCGC North America 2022, 40 (9), 411–416.

(5) Taylor, T. The LCGC Blog: Getting the Most from Phenyl Stationary Phases for HPLC. LCGC International 2016. https://www.chromatographyonline.com/view/getting-most-phenyl-stationary-phases-hplc (accessed 2024-12-23)

(6) Snyder, L. R.; Kirkland, J. J.; Glajch, J. L. Practical HPLC Method Development, 2nd Ed.; John Wiley & Sons, Inc., Hoboken, 1997.

(7) Taylor, T. The LCGC Blog: Ammonium Acetate Woes. LCGC International 2019. https://www.chromatographyonline.com/view/lcgc-blog-ammonium-acetate-woes (accessed 2024-12-23)

(8) Zhang, D. D.; Sadikin, S.; Redkar, S.; Inloes, R. Ghost Peak Investigation in a Reversed-Phase Gradient LC System. LCGC North America 2011, 29 (5), 394–400.

(9) Dong, M. W. A Universal Reversed-Phase HPLC Method for Pharmaceutical Analysis. LC-GC North America 2016, 34 (6), 408–416.

(10) Tyrrell, N. D. A Proposal That Would Ban Manufacture, Supply, and Use of All Fluoropolymers and Most Fluorinated Reagents Within the Entire EU. Org. Process Res. Dev. 2023,27 (8), 1422–1426.

(11) Yabré, M.; Ferey, L.; Somé, I. T.; Gaudin, K. Greening Reversed-Phase Liquid Chromatography Methods Using Alternative Solvents for Pharmaceutical Analysis. Molecules 2018, 23 (5), 1065.

About the Author

Jonathan G. Shackman is a Scientific Director in the Chemical Process Development department at Bristol Myers Squibb (BMS) and is based in New Jersey, USA. He earned his two B.S. degrees at the University of Arizona and his Ph.D. in Chemistry from the University of Michigan under the direction of Prof. Robert T. Kennedy. Prior to joining BMS, he held a National Research Council position at the National Institute of Standards and Technology (NIST) and was a professor of chemistry at Temple University in Philadelphia, PA. To date, he has authored more than 40 manuscripts and two book chapters. He has presented more than 40 oral or poster presentations and holds one patent in the field of separation science. Shackman has proudly served on the executive board of the ACS Subdivision on Chromatography and Separations Chemistry (SCSC) for three terms.

Jonathan G. Shackman is a Scientific Director in the Chemical Process Development department at Bristol Myers Squibb (BMS) and is based in New Jersey, USA. He earned his two B.S. degrees at the University of Arizona and his Ph.D. in Chemistry from the University of Michigan under the direction of Prof. Robert T. Kennedy. Prior to joining BMS, he held a National Research Council position at the National Institute of Standards and Technology (NIST) and was a professor of chemistry at Temple University in Philadelphia, PA. To date, he has authored more than 40 manuscripts and two book chapters. He has presented more than 40 oral or poster presentations and holds one patent in the field of separation science. Shackman has proudly served on the executive board of the ACS Subdivision on Chromatography and Separations Chemistry (SCSC) for three terms.

Recent Videos
Toby Astill | Image Credit: © Thermo Fisher Scientific
John McLean | Image Credit: © Aaron Acevedo
Related Content