Cover story

In brief
By some estimates, this year
marks the 50th anniversary
of the method we now know
as high-performance liquid
chromatography, or HPLC.
The story of HPLC even from
the beginning has been one of
evolution rather than revolution.
Here, we revisit the early
days of HPLC and retrace the
incremental development of
the technique’s all-important
particle-packed columns (see
page 29). Then, we take a
look ahead, checking in with
companies and researchers
who are pushing the limits of
column-packing materials and
pressures to nudge HPLC toward
higher speeds and more complex
samples (see page 35).

28 C&EN | CEN.ACS.ORG | JUNE 13, 2016

 50 years
of HPLC
CELIA HENRY ARNAUD,
A look back at the history of high-performance
liquid chromatography and the column-
C&EN WASHINGTON packing materials that enabled its success

F
or some chromatographers, their first inkling that high-performance liquid chroma-
tography was going to be a big deal came in 1969. That year, they traveled to Las Vegas
to attend the fifth in a series of meetings organized by University of Houston chemist
Albert Zlatkis.
Earlier meetings in the “Advances in Chromatography” series had focused on gas chromatog-
raphy, which separates volatile compounds in a mixture as it passes through a gas-filled column.
So, many of the attendees at that meeting in 1969 were GC experts who hadn’t heard much
about the technique that was to become HPLC. They were in for an awakening. “That meeting
was pivotal in terms of really giving a jump start to HPLC,” says Ronald Majors, who was in at-
tendance. After hearing about the still-nascent technique, “everybody got really excited.”
GC was still the dominant separation technology at Zlatkis-organized meeting as a turning point for HPLC.
the time. But GC had its drawbacks. Most important, GC But even more, he remembers what happened when he
requires that compounds be volatile or be derivatized to came home. Together with other people who had attend-
become volatile. “But it’s such a pain doing derivatiza- ed the meeting, Snyder organized a half-day meeting at
tion,” Majors says. Therefore, many large or polar mole- California State University, Fullerton, to update people
cules couldn’t be analyzed by GC. who hadn’t been able to go.
But they could be by HPLC. In HPLC, a liquid mobile “We planned for maybe 15 or 20 people showing up,”
phase carries mixtures of compounds through a column Snyder says. “They announced the meeting and sched-
packed with particles. As a pump forces the mobile phase uled it for an ordinary classroom. As the response came
through the column, the compounds interact with the in, it progressively moved to larger rooms and finally the
stationary phase—the particles’ surface—to different auditorium. About 150 people showed up. I could see this
degrees and are separated in the process. was hot stuff.”
HPLC was poised to catch on with scientists at that According to some estimates, this year marks the 50th
fateful Las Vegas meeting because there were appli- anniversary of HPLC. Those estimates typically point
cations—drug discovery and biological separations, to Csaba Horváth and Seymour (Sandy) Lipsky of Yale
among others—where leading separation methods such University publishing a paper in 1966 on ion-exchange
CREDIT: SHUTTERSTOCK

as GC just weren’t getting the job done. Since those days,
HPLC has become a workhorse of the analytical chem-
istry lab. And applications—from chiral separations to
proteomics—continue to drive advances in HPLC today.
In 1969, though, HPLC was a relative unknown. Lloyd
Snyder, then a scientist at Union Oil, remembers the
separation of organic compounds (Nature 1966, DOI:
10.1038/211748a0). They followed up one year later with a
paper on fast liquid chromatography in which they used
a pump operating at higher pressures than in their previ-
ous work (Anal. Chem. 1967, DOI: 10.1021/ac60256a003).
They were among the pioneers at the landmark Las Ve-

JUNE 13, 2016 | CEN.ACS.ORG | C&EN 29

gas meeting who introduced the rest of the
chromatography community to what would
come to be called HPLC.
But pinning down an exact birthday for
HPLC isn’t easy. It was predicted long before
it was reduced to practice. In 1941, future No-
bel Laureates Archer J. P. Martin and Richard
L. M. Synge wrote that, for a method called
liquid-liquid partition chromatography, the
ability of a column to separate compounds
was dependent on the liquid-mobile-phase
flow rate and the square of the diameter of
the particles packed inside the column. They
talked in terms of theoretical plates, some-
thing chromatographers use to describe
how well a column can resolve mixtures.
The thinner the theoretical plate, the better.
“The smallest [theoretical plate] should be
obtainable by using very small particles and
a large pressure difference across the length
of the column,” they wrote (Biochem. J. 1941,
DOI: 10.1042/bj0351369).
So people knew as early as 1941 that ef-
the first published reports of
modern HPLC. But if you ask
chromatographers about the be-
ginnings of modern HPLC, they
are more likely to mention 1964.
That year, Joseph Jack Kirkland
visited Eindhoven University of
Technology.
“In one of the labs I visited,
there was this fellow, Josef F. K.
Huber, who was doing what I
now call HPLC,” Kirkland says.
“He had a very crude instrument
with a UV detector. He had
packed a column with particles.
They were actually GC particles,
but he had coated them with
liquids.”
Kirkland worked for DuPont Solving a problem for Robert Woodward helped
at the time. He had tried to use expand the market for HPLC. Shown here are Helmut
liquid chromatography a few Hamberger (from left), Josef Huber, James Waters,
years earlier, but the available and Woodward in a publicity photo taken in 1973.
equipment and knowledge

CREDIT: COURTESY OF JAMES WATERS

ficient liquid chromatography separations
would require a combination of high pres-
sure and small-particle packing materials.
But it took nearly 30 years to move from the-
ory to practice because there was still much
to be learned.
Horváth is generally credited with build-
ing the first HPLC instrument. And the
Nature and Analytical Chemistry papers he
published with Lipsky may indeed have been
weren’t yet up to the task. His
meeting with Huber encouraged him to try
again.
When Kirkland came home, he convinced tography. The first “liquid” in this method
managers at DuPont to let him work on
HPLC. “My work largely was in the pesticide
area,” Kirkland says. “We had a lot of com-
pounds that did not lend themselves to GC.
Liquid chromatography was something I
really had my eye on.”
Even before HPLC, chromatographers
were already doing liquid-liquid chroma-
refers to the mobile phase, and the second
one refers to the liquid coated onto the
packing material. The two liquids need to be
immiscible.
To make the technique work, Kirkland
says, you have to make sure the ratio of coat-

1900s 1940s 1950s 1960s

Chromatography Landmark paper

invented
1900 Russian botanist
Mikhail Tsvet invents
chromatography to
separate plant pigments.
The work is later
published in 1906.
1941 In their paper on partition chromatography, English Predecessor
chromatographers Archer J. P. Martin and Richard L. M. Synge predict 1963 Waters introduces its
that high pressure and small particle size will lead to efficient separations. GPC-100, an instrument for gel-
“The [theoretical plate] is proportional to the rate of flow of permeation chromatography.
liquid and to the square of the particle diameter. Thus the smallest
[theoretical plate] should be obtainable by using very small
particles and a high pressure difference across the length of the
column.”
Improving
performance
First report Commercialization 1972 The introduction
of 6,000-psi pumps,
1966 Csaba 1967 Waters Associates columns containing
Horváth and introduces ALC-100, a slight 10-µm particles, and
Seymour (Sandy) modification of the GPC- septumless injectors
Lipsky publish the 100 and considered by many marks the transition of
first report of HPLC, to be the first commercial HPLC from just “high
the ion-exchange HPLC instrument. pressure” to “high
separation of performance.”
nucleotides and
thyroid compounds.

CREDIT: WIKIMEDIA COMMONS (TSVET); WATERS (ALC-100 BROCHURE);

ANAL. CHEM (CHROMATOGRAM); JAMES JORGENSON (PARTICLES)
30 C&EN | CEN.ACS.ORG | JUNE 13, 2016
ing to mobile phase is just right so the mobile
phase doesn’t wash away the coating. He
adds: “It’s difficult to operate, but it’s a very
powerful technique.”
While Huber and Kirkland were working
on their instruments, Horváth was working
on an instrument to separate biological
molecules. In fact, he called his instrument a
nucleic acid analyzer.
Meanwhile, chromatography firm Waters
Associates, now known as Waters Corp.,
was pushing to roll out the first commercial
HPLC. Depending on which instrument you
want to count, it was in the HPLC market
even before Huber built his version.
In the early 1960s, Waters licensed tech-
nology for gel permeation chromatography
from Dow Chemical. That method separates
large molecules such as polymers on the
basis of their size by passing them through
a column packed with cross-linked polymer
beads. Waters introduced its GPC-100 in
1963. That instrument included a pump that
operated at 500 psi. There’s some disagree-
ment about whether that GPC instrument
should be considered the first commercial
HPLC or merely a precursor to it.
Not long after that, the company intro-
duced another instrument for liquid-liquid
chromatography, incorporating technology
licensed from Shell Development. That
instrument was less than successful. In fact
“it was a disaster,” company founder James
L. Waters remembers. “The liquid that was
supposed to be stationary on the packed par-
ticles wouldn’t stay on all the time. We built
three instruments, sold one, took it back,
and that was the end of that.”
But Waters didn’t give up on HPLC. The
company’s next foray into the field was
more successful. That instrument, called the
ALC-100, used a higher pressure pump than
its GPC predecessor and was intended as a
general-purpose analytical instrument. The
ALC-100, which was introduced in 1967, is
widely considered to be the first successful
commercial HPLC.
The market for HPLC opened up con-
siderably when Waters won over world-re-
nowned organic chemist Robert B. Wood-
ward, a Chemistry Nobel Laureate and
professor at Harvard University. One Friday
in 1972, Woodward’s postdoc Helmut Ham-
berger came to Waters with a problem. He
was trying to separate isomers of an inter-
mediate in the synthesis of vitamin B-12.
At the time, “I didn’t know who Wood-
ward was. He was just some chemist at Har-
vard,” James Waters says.
But Waters’s colleague James Little knew.
Waters remembers Little telling him: “If we
can solve a problem for him, we can sell a lot
of instruments.”
Waters had been thinking that organic
synthesis might become a major market for
HPLC, so he took the ALC-100 to Wood-
ward’s lab himself. “I had the problem pretty
well solved by the end of the second day
and separated small amounts of material
for him,” Waters says. To separate larger
amounts, Waters injected more material
and cycled the output back through the col-
umn. “I had to cycle it something like seven
times to get the other isomers separated so
I had the one Woodward wanted,” Waters
remembers.
Waters parlayed that success into a new
market. He used the American Chemical So-
ciety Directory of Graduate Research to as-
semble a list of organic chemists. He mailed
each one a brochure with a photo of Wood-
ward and a description of what they’d done.
In the following year, “we probably sold 40
instruments,” Waters says. “After that, we
owned the organic synthesis market because
we had the reputation and the experience.”
Since those early days, the development
of HPLC has been one of evolution rather
than revolution, which is fitting, given that
even HPLC’s beginnings involved incre-
mental changes. Although there have been
developments in the hardware—pumps,
detectors, and especially the introduction
of electrospray, an ionization technique
that allowed HPLC to be interfaced with
mass spectrometers—much of the history
of HPLC is the story of new column-packing
materials.
Horváth packed his early columns with

1970s 1980s 1990s 2000s

Shrinking
Chiral separations New particles
Expanding detector Mid-2000s
1979 to
markets early 1980s Mid- to late New systems are
1972 James Waters and Chromatographers 1980s John Fenn introduced that contain
James Little of Waters develop methods and colleagues particles smaller than
help Robert B. Woodward for separating at Yale University 2 µm. They require
and his postdoc Helmut enantiomers with develop an pressures of 15,000 to
Hamberger separate HPLC. At first they electrospray 20,000 psi to pump the
isomers of intermediates add modifiers to ionization mobile phase through
in the synthesis of the mobile phase. source for mass the chromatography
vitamin B-12. Their Later, they develop spectrometry, column.
success helps open stationary phases which allows mass
the organic synthesis that can separate spec to be used as
community as a market enantiomers. a detector for LC.
for HPLC.

First meeting
1973 First HPLC meeting is held in Interlaken,
Switzerland. For almost a decade thereafter, the
meeting is held biennially. In 1982, it becomes an
annual meeting. The 44th HPLC meeting is being
held in 2016 in San Francisco.
Proteins
Late 1970s to early 1980s Chromatographers
develop methods for separating peptides and proteins.
Those methods lay the groundwork for the proteomics
and biopharmaceutical analyses that are done today.
JUNE 13, 2016 | CEN.ACS.ORG | C&EN 31
material he called pellicular particles, which
that any silanol groups remain-
were impermeable spheres coated with a po- ing on the particle surface not
rous ion-exchange resin. But Horváth’s col- attached to C18 are chemically
umns were inefficient because compounds blocked. They can be mixed
couldn’t diffuse deeply into them. modes, meaning that something
Kirkland, collaborating with Ralph Iler other than C18 is also bonded
at DuPont, improved efficiency by making to the silica. “There are so many
spherical particles in which the solid silicavariations of how you can make
core was coated with a porous outer shell of a C18,” Majors says.
tiny beads. The resulting core-shell particles And the various C18 columns
had more surface area and capacity for in- don’t provide identical separa-
teracting with and separating compounds. tions. The underlying silica, the
Kirkland and Iler followed these superfi- surface area, and the pore sizes
cially porous particles with fully porous can all be different, creating
particles of different controlled sizes and different interactions with the Jack Kirkland 40 µm. Throughout the 1970s
porosities. compounds being separated. (left), with and 1980s, the particle size
Then came bonded phases. One of the In all, there are about 1,400 technician Glen decreased to 10 µm, then 7 µm,
biggest developments in the history of different stationary phases, Wallace, in front then 5 µm.
HPLC, the bonded phase consists of mol- Majors estimates. But many of of a homebuilt By the late 1980s, the field had
ecules covalently linked to silica particles.them are for niche applications. HPLC instrument settled on particle diameters of
Octadecylsilane, also called C18, was the “There are a lot of different sta- in 1967. about 3 µm and upper pressures
most common of these molecules, which act tionary phases, but they’re not of about 6,000 psi, which were
as the stationary phase. Majors, who wrote used that much. I would guess that probably needed to push the mobile phase through
about columns and sample prep for the the top five or six stationary phases would the tightly packed columns. As the particles
magazine LCGC for more than 30 years, esti- separate 90 or 95% of all samples that are got smaller, the columns became shorter and
mates that 800 different C18 columns have done now,” Kirkland says. the separations became faster, partially as a
been introduced over the years. Over the years, the particles have become way to avoid operating at the pressure limit.
One might think that with so many C18 progressively smaller and more uniform. “They were trying to keep the columns
stationary phases, they’re just duplicating Before the advent of HPLC, chromatog- operating around 2,000 or 3,000 psi at
each other. But that’s not the case, Majors raphy columns were packed with irregu- most,” says James W. Jorgenson, a chro-
says. The C18 phases can be monomeric or larly shaped particles larger than 100 µm. matographer at the University of North
polymeric. They can be endcapped, meaning Horvath’s pellicular particles were about Carolina, Chapel Hill. “Even though the
equipment can go to 6,000 psi, you don’t
want to run a column at the pressure limit.
Progressive improvements What kind of lifetime is it going to have
Smaller particles and shorter columns have led to faster separations over the years. before it starts plugging up and exceeds the
Each chromatogram here shows the separation of a mixture (in order of elution, lef pressure limit?”
to right) of 1-methylxanthine, 1,3-dimethyluric acid, 3,7-dimethylxanthine, and In the 1990s, Jorgenson started using
1,7-dimethylxanthine. The conditions are identical, except for the particle size, column even smaller particles (less than 2 µm) and
length, and injection volume (not listed). higher pressures (as high as 10,000 psi) to
carry out separations. “We were after very
high efficiency separations,” he says.
Waters Corp. began its own work with
higher pressures and smaller particles, but
its goal was speed. “That was a wise choice
on their part,” Jorgenson says, “because a
large chunk of the market—people who do
pharmaceutical separations—needs high
throughput.” If Waters could promise highly
resolved compounds and get it done in 10
minutes instead of 30 minutes, Jorgenson
adds, it was a huge selling point.
The move to sub-2-µm particles and
higher pressures—eventually dubbed
CREDIT: COURTESY OF JACK KIRKLAND

ultra-high-performance liquid chromatog-

raphy, or UHPLC—required a complete re-
design of the equipment around the column.
“You didn’t just change pumps and every-
body was happy,” says Peter Carr, a chroma-
tographer at the University of Minnesota,
Twin Cities. “You had to reengineer every
part of the system. The pumps, the injector,
the connections, the column itself, and the
Source: Agilent Technologies detector—all brand-new.”

32 C&EN | CEN.ACS.ORG | JUNE 13, 2016


Most important, the volumes of all those
parts needed to be reduced. “The more
efficient columns challenge the equipment
in terms of injector volumes, connecting
tubing volumes, and detector volumes,” Jor-
CREDIT: COURTESY OF RON MAJORS
genson says.
Even today, researchers continue de-
creasing the particle size, leading to the in-
evitable pressure increases. But some, such
as Kirkland, question the need to continue
pushing higher.
Pressures of about 15,000 psi are suffi-
cient for most applications, Kirkland says.
“People are beginning to understand better
the compromise between pressure and res-
Ron Majors working on a Varian
8500 system circa 1975.
olution. If you’ve got a difficult
sample with a lot of material in it
you want to separate, you actual-
ly have to use a very long column
with large particles. You use the
pressure needed for larger par-
ticles to be able to operate that
long column.”
And industrial chromatog-
raphers are likely to view pres-
sures much higher than the cur-
rent 15,000 to 20,000 psi with
suspicion. “In industry, there’s
going to be an evaluation of high pressure
from a safety perspective,” says Mary Ellen
McNally, a chromatographer at DuPont.
Most companies would probably require
instruments operating at exceptionally high
pressure to be isolated in special labs, she
says. That skepticism of high pressures has
led to a resurgence of core-shell particles
(see page 35).
Improvements in separation efficiency
are still needed as chromatographers turn
their attention to complicated biological
mixtures. One way they are achieving such
separations is by using two—or more—col-
umns in series. In the simplest form of this
multidimensional chromatography, each
column has different selectivity, so the com-
bination can tease apart more components
than any of the columns on its own.
“When you have 10,000 or 100,000
compounds, you can’t separate them with
one column,” Majors says. “You’ve got to go
beyond that.”
In two-dimensional HPLC, the output
from one column is injected onto a second
column. When two columns are used in this
way, the method effectively multiplies, rath-
er than adds, their capability for resolving
components.
Carr, who works in multidimensional
chromatography, is cautiously optimistic
about the method’s potential. He thinks it
could eventually account for as much as 40%
of liquid chromatography separations.
“If 2-D LC can change in a big way how
methods are developed, it will be really im-
portant,” Carr says. “If the way methods are
developed stays the same, then 2-D will be
important to certain fields, such as metab-
olomics, proteomics, and lipidomics, but it
won’t be a routine way to do business. The
next five years will tell that story.”
And so the evolution of HPLC continues.
And much as in the early days, the applica-
tions scientists want to use them for will
push the developments. ◾
JUNE 13, 2016 | CEN.ACS.ORG | C&EN 33