Webster's New Collegiate Dictionary defines a myth as "an ill-founded belief held uncritically, especially by an interested group." Could that group be misinformed
chromatographers? In the first of a two-part feature from Ron Majors, the top 10 high performance liquid chromatography (HPLC)
column myths are presented and attempts are made to demystify them by offering some evidence that they are untrue. This part
will feature myths 10 to six. Since ultrahigh-pressure liquid chromatography (UHPLC) has come about, new myths are popping
up and these shall also be dealt with here.
In any field there are often "misconceptions" or "myths" that are perpetuated and passed on to the next generation. These
myths are often driven by a lack of understanding by practitioners of the real issues, and can change as time moves on. Originally,
seven years ago, in a "Column Watch" instalment (1), the 10 most popular myths of the time around high performance liquid
chromatography (HPLC) column technology were demystified by discussing the issues at hand. Because HPLC is approaching its
50th year, many column myths have already been passed down to two generations of liquid chromatographers. Recently, ultrahigh-pressure
liquid chromatography (UHPLC) has come into its own and a new set of myths are arising. The purpose of this instalment of
"Column Watch" is to revisit and update readers on the most popular column myths of today and try to dispel some of these
myths before they get perpetuated. This column is an adaptation of an oral presentation at the HPLC2013 conference in Amsterdam,
the Netherlands (2). In keeping with the "countdown theme," I will start with number 10 and work my way up to the top myth.
Myth 10: Air Will Kill an HPLC Column
Figure 1: Chromatograms obtained using C18 bonded phases with the same base material but different chemistries: (a) Zorbax
Eclipse Plus C18 (different surface treatment for same base silica, double endcapping, same bonding chemistry as Eclipse XDB-C18);
(b) Zorbax StableBond SB-C18 (same base silica, sterically protected C18 phase, no endcapping); (c) Zorbax Eclipse XDB-C18
(same base silica, monomeric bonding chemistry, double endcapping); (d) Zorbax Extend-C18 (same base silica, bidentate bonding
chemistry, double endcapping). Column dimensions: 50 mm × 4.6 mm, 1.8-μm dp; mobile phase: 69:31 acetonitrile-water; flow rate: 1.5 mL/min; temperature: 30 °C; detection: single-quadrupole electrospray
ionization MS, positive mode scan. Peaks: 1 = anandamide, 2 = palmitoylethanolamide, 3 = 2-arachinoylglycerol, 4 = oleoylethanolamide.
False: HPLC and UHPLC columns are shipped with plugs of either stainless steel or polymeric construction installed at both end.
Users are told that a column should always be capped tightly after the column is disconnected from the instrument. The thought
is that large amounts of air can get inside the column, perhaps damaging the packing material, causing bubbles in the detector
flow cell when installed into the HPLC system in the future, and maybe disrupting the packed-bed morphology. One should first
realize that the tiny hole in the endfitting is less than 0.02 in. in diameter and therefore has an extremely small cross-sectional
area. If left open, the small amount of air that diffuses into the column could hardly cause irreparable damage. Depending
on the volatility of the solvent used to store the column, there could be some evaporation near the end of the column. But
large quantities of air would have a hard time diffusing through the microparticles in the packed bed seeing that we need
thousands of pounds per square inch of pressure to push liquid mobile phases through these micrometre-sized particles. The
small amount of air that could conceivably enter into the ends of the column would be immediately dissolved once the system
was pressurized or, at least be flushed out in the initial pressurization in a short time and should not cause any problems
with the chromatography later on. However, if you feel more secure by capping the endfittings, by all means do so.
Myth 9: All C18 (L1) Columns Are the Same
Figure 2: Separation of the same mixture on three reversed-phase columns under the same conditions: (a) Ace C8 (Advanced Chemical
Technologies); (b) Precision C8 (Mac-Mod); (c) Inertsil C8 (GL Sciences). Column dimensions: 15 cm × 4.6 mm; flow rate: 2.0
mL/min; temperature: 35 °C; mobile phase: 50:50 30 mM potassium phosphate buffer (pH 2.8)–acetonitrile. Peaks: 1 = N,N-diethylacetamide,
2 = nortriptyline, 3 = 5,5-diphenylhydantoin, 4 = benzonitrile, 5 = anisole, 6 = toluene, 7 = cis-chalcone, 8 = trans-chalcone,
9 = mefenamic acid. (Courtesy of Lloyd Snyder and John Dolan, LC Resources).
False: All of our HPLC column surveys have shown that C18 is, by far, the most popular bonded phase in existence (3). Because pharmaceutical
manufacturers were the earliest adopters of HPLC, the United States Pharmacopeial Convention (USP), not wanting to favour
any particular manufacturer of HPLC columns, developed a classification system that gave a generic description for each type
of bonded phase column that was submitted under a new drug application. For HPLC columns, an "L" designation was given, and
because C18 is used for a majority of submittals, its designation was "L1." As additional phases came along, they were given
their own "L" number such as C8 (L7), CN (L10), phenyl (L11), and so on. The implication with this system was that each C18
column that was submitted also designated as L1, was the same as the last L1 column. Unfortunately, this system proved to
be unreliable because columns from different manufacturers, produced from different base silicas and bonded with different
silane reagents using different synthetic routes, were not chromatographically the same and one could therefore not be substituted
for another. With more than 800 different L1 columns introduced into the marketplace, it has proven to be a confusing system.
Several approaches, including the use of the hydrophobic subtraction model (4,5) that gives a more detailed classification
of reversed-phase columns, have been proposed but to this day the "L" classification is still in widespread use. Thus, some
chromatographers who do not really understand the issues still believe that "all C18 columns are the same." Simple examples
that this is not the case are shown in Figures 1 and 2. In Figure 1, four different C18 silica bonded phase columns are shown
for the same separation under the same operating conditions; each phase provides a different chromatogram. To demonstrate
that the L system also doesn't hold for other bonded phases as well, Figure 2 provides an example of three different C8 (L7)
columns, one of which (Figure 2[b]) was very similar to the original chromatogram and could probably be substituted in an
HPLC method while the third column (Figure 2[c]) is quite different and might even be considered as orthogonal to the first
two columns. The Fs designation shown alongside each chromatogram is a numerical classification of how "close" of a fit columns are to one another
(4,5). Close Fs numbers are potentially replacement columns while large values of Fs imply that the column would not be a "drop-in" replacement in a particular HPLC method and, in fact, might be a useful column
when first performing method development because it offers a different selectivity to the other columns. So, the bottom line
is: All C18 (L1) and other reversed phase columns are not the same.
How useful do you find attending events such as HPLC 2013 for your research?
Not very useful
LCGC COLUMNISTS 2013
Column Watch | Ronald E. Majors: LCGC Columnist Ron Majors, established authority on new column technologies, keeps readers up-to-date with new sample preparation trends in all branches of chromatography and reviews developments in existing technology lines.
History of Chromatography | Industry Veterans: With each installment of this column, a different industry veteran covers an aspect of the evolution and continued development of the science of chromatography, from its birth to its eventual growth into the high-powered industry we see today.
MS — The Practical Art | Kate Yu:
Kate Yu is the editor of 'MS-The Practical Art' bringing her expertise in the field of mass spectrometry and hyphenated techniques to the pages of LCGC. In this column she examines the mass spectrometric side of coupled liquid and gas-phase systems. Troubleshooting-style articles provide readers with invaluable advice for getting the most from their mass spectrometers.
LC Troubleshooting | John Dolan: LC Troubleshooting sets about making HPLC methods easier to master. By covering the basics of liquid chromatography separations and instrumentation, John Dolan, Vice President of LC Resources and world renowned expert on HPLC, is able to highlight common problems and provide remedies for them.