The Early Development of Size-Exclusion

Chromatography: A Historical Perspective

This column installment traces the early development of SEC from its inception to its
acceptance as a modern analytical technique.

Separation by size-exclusion chromatography (SEC) is governed solely by the hydrodynamic

size and shape of macromolecules relative to the size and shape of the pores of the column
packing. Provided that the proper mobile phase is selected, the entire polymer sample will
be eluted from the column within a defined elution volume region dictated by the porosity of
the packing and the column dimensions. The elution order of a homologous series of
components is always large molecules first, followed by smaller-sized components, which
aids data interpretation.

SEC, when compared to high performance liquid chromatography (HPLC), requires no

gradient elution and, to a first approximation, no temperature control. Method development
is usually straightforward: for the analysis of biopolymers, an aqueous buffer is used, while
for synthetic polymers, an organic solvent is used for the mobile phase.

Table I: Applications of size-exclusion chromatography (SEC) with and without light

scattering (LS) or viscometric detection. See reference (24) for detailed explanation of
terms and procedures.

With this simple system, a wealth of information can be obtained about the properties and
characteristics of macromolecules (Tables I and II). Because of the usefulness of this
technique, especially for the analysis of complex samples with a wide molecular weight
range, most HPLC laboratories have a dedicated SEC instrument to aid in problem-solving.

Table II: Conventional and unconventional size exclusion chromatography applications

without column calibration

It took nearly 50 years from Tswett's momentous discovery in 1906 of column liquid
chromatography before SEC was first recognized, quite by accident, as a new separation
mode. After it was introduced to the scientific community, it became an essential technique
for the life sciences and polymer science.

Table III: The early milestones of aqueous size-exclusion chromatography (gel filtration
chromatography)

In this column installment, we will trace the early development of SEC from its inception to
its acceptance as a modern analytical technique for biopolymer and synthetic polymer
characterization. Some of the significant SEC milestones that will be covered are
summarized in Tables III and IV.

Table IV: The early milestones of nonaqueous size exclusion chromatography (gel
permeation chromatography)

In the Beginning

Gel "Filtration" Chromatography: In the early 1950s, investigators (1,2) had noted that
neutral small molecules and oligomers were eluted on the basis of decreasing molecular
weight from columns packed with cross-linked polystyrene ion-exchange resins. Intrigued
by this separation mode, Deuel (3) and Lindquist (4) reported similar results for the elution
of low-molecularweight solutes using polysaccharide packings, that is, cross-linked
galactomannan and starch granules.

In 1955–1956, two British biochemists, Lathe and Ruthven (5,6), used conventional open
columns packed with swollen starch granules to study the elution properties of low-and
highmolecular-weight polysaccharides and proteins. To their surprise, higher-molecular-
weight compounds were eluted ahead of lower-molecular-weight components, in
agreement with earlier work (1–4). Because of Lathe and Ruthven's more thorough
investigations, they are credited with performing the first biopolymer size separation (see
Figure 1). Although they correctly described the separation as being caused by "partial or
size-restricted penetration of solute molecules into the swollen gel particles," Lathe and
Ruthven incorrectly called the process "gel filtration," which implies that the high-molecular-
weight components are physically retained on the column.

Figure 1: First published SEC composite chromatogram of polysaccharides,

oligosaccharides, carbohydrates, and vitamin B12 using swollen potato starch (75 g filled to
56 cm) and a buffer for the mobile phase at 4 mL/h. Fractions were collected and analyzed
via UV–vis spectrophotometry using postcolumn derivatization. The following elution
volumes are estimated from the data provided: V0 ≈ 28 mL; Vi ≈ 46 mL; Vt ≈ 74 mL;
Vi /V0 ≈ 1.6 which is satisfactory. Because N ≈ 1000 plates (glucose) and H ≈ 600
µm, column efficiency is at least 30× lower than a typical HPLC column, which is a
reasonable value considering this is a conventional open-column separation. Total elution
time for this system is about 20 h. Adapted and reproduced with permission from reference
6.

The next milestone occurred in 1959, when Per Flodin, a biochemist with Pharmacia
Pharmaceutical Company, in collaboration with Jerker Porath, synthesized a series of
cross-linked dextran packings with different pore sizes and successfully demonstrated size
separation of peptides and oligosaccharides (7–9). Because Pharmacia had considerable
experience with the production and chemistry of dextran, which they used as a blood
plasma substitute for transfusions, the company was able to quickly produce and market
crosslinked packings of different porosities in the early 1960s. The packing was
trademarked Sephadex, an acronym taken from the first few letters of separations
Pharmacia dextran. Sephadex sales soared, and soon most biochemistry laboratories used
gel filtratrion chromatography (GFC). Within a short time, other types of cross-linked gels
were released by Pharmacia.

Apart from being used for estimating molecular weights of biological components from
elution time comparisons to molecular weight standards, Sephadex was primarily used in
place of dialysis for desalting or exchanging electrolytes in biopolymer preparations, or for
isolating and purifying biopolymers and other biologicals. (It seemed that most biochemical
laboratories at the time were situated next to slaughterhouses to ensure a steady stream of
starting material.)

Gel Permeation Chromatography: Motivated by the early success of gel filtration

chromatography, polymer scientists in the late 1950s and early 1960s focused their efforts
on the application of hydrophobic packings for the SEC of synthetic polymers, at the time
referred to as gel permeation chromatography (GPC). The concept of molecular weight
separation of nonpolar molecules was first demonstrated when Brewer (10,11) used
swollen rubber granules for the separation of a homologous series of hydrocarbons.
Determann and colleagues (12) evaluated cross-linked poly(methyl methacrylate) for SEC of
oligostyrenes.

In 1962, John Moore (13–15) of Dow Chemical Company produced a series of cross-linked
polystyrene resins of known porosities and particle sizes for the SEC of synthetic polymers.
For the first time, the molecular weight distribution of any polymer soluble in toluene could
be obtained within several hours compared to weeks or months with conventional
fractionation and procedures. Soon after the method was published, polymer laboratories
around the world clamored for these packings and associated GPC instrumentation.

Dow Chemical recognized the practicality and importance of GPC to their polymer business
and licensed the packing synthesis and instrumentation to Waters Associates, a consulting
independent laboratory located near Boston, Massachusetts. James Waters, its founder,
had rented the basement of a former police station for this new business venture and had
set up polymerizers on the first floor in an area that was formerly a women's jail. Waters
soon supplied packed columns and complete GPC systems for room temperature use; high-
temperature (135 °C) SEC for polyolefins; and largescale preparative separations. Waters
Associates became the GPC counterpart of Pharmacia's GFC venture. Waters Associates
was the only source of GPC packings and instrumentation for many years.

Packings

Initially SEC packings consisted of either cross-linked polysaccharides for aqueous SEC or
cross-linked polystyrene for nonaqueous SEC separations, as listed in Tables III and IV.
Packings for GFC were developed by Pharmacia, Bio-Rad Laboratories, LKB Produkter, and
E. Merck. Waters Associates (now Waters Corp.), which licensed the polystyrene
polymerization technology from Dow, and held the "GPC" name as their trademark, was the
sole producer of Styragel packings.

In the early 1970s, reduced-particle-size, porous silica was shown to have extremely high
plate counts for HPLC separations, including SEC, because of short diffusion paths traveled
by solutes. Over time, biochemists switched from large-diameter crosslinked
polysaccharide packings to high-performance silica-based packings and then on to
hydrophilic polymer packings for aqueous SEC as they became available in later years.
Small diameter cross-linked polystyrene packings, which were made rigid and
noncompressible, became available in the mid-1980s.

Companies that helped launch and promote the early development of high-performance
SEC packings were: Varian Inc., BioRad Inc., Pharmacia, Waters Associates, DuPont
Instruments, Toyo Soda (now Tosoh Bioscience), Showa Denko, Co., Synchrom Inc., and E.
Merck. Although controlledpore glass (CPG) was not a highperformance packing, it was
available in very large pore sizes with narrow pore-size distributions. Of these companies,
Waters, Tosoh Bioscience, and Showa Denko remain, and continue to advance, the
technique. Other major SEC column suppliers include Agilent Technologies, Polymer
Laboratories, PSS Polymer Standards Service, Phenomenex, Jordi Associates, BioChrom
Labs, Eprogen, Sepax Technologies, and YMC.

Instrumentation

Pumps: Because of the nature of SEC calibration, in which log M is plotted against elution
volume, small flow rate deviations cause large molecular weight errors, especially when
calculating average molecular weights of polydisperse samples. However, for proteins and
nucleic acids, which are mostly monodisperse, biochemists used SEC for determining
molecular weights at peak maxima, which was less dependent on flow rate variations. It is
of interest to note that the most popular uses of GFC were desalting, electrolyte exchange,
or biopolymer purification, which do not necessarily require highprecision flow rate.

The most common pumping systems for GFC were peristaltic pumps or motor-driven glass
syringes, which are still in use. Gravityinduced flow with open-column chromatography was
widely used. To help control flow with gravity feed, a simple device called the Mariotte flask
is often used. The Mariotte flask, which serves as the mobile-phase reservoir, has a tube
inserted for draining mobile phase onto the top layer of the packed column. The flask also
has a capillary tube with one end kept below mobile-phase level and the other end
connected to the outside for admitting air into the flask via a steady stream of bubbles. The
mobile-phase flow rate is controlled by adjusting the stream of air bubbles, and the flow rate
is determined by measuring mobilephase depletion in the flask.

When determining molecular weight averages of polydisperse samples, as in the case of

GPC, precise flow rate control is required. Most pumps at the time were industrial single- or
dualpiston pumps with modified seals for chemical inertness; metering pumps from Milton
Roy were commonly used with conventional column LC.

To monitor flow rate throughout a GPC run, a custom-made siphon "dump" was used up
until the early 1980s. This device was placed at the end of the column. The siphon, which
had a capacity of 1-, 2-, or 5-mL, would fill to volume and then dump its contents, producing
a signal at each volume increment. The average flow rate throughout the run could be
computed.
Detectors: When GFC was introduced in the 1950s, flow-through detectors were not
available. Instead, manual or automated fraction collection was accomplished with
subsequent UV measurements. For monitoring oligosaccharides, polysaccharides, or lipids,
fraction collection with postcolumn derivatization and subsequent UV–vis spectrometry
was required. Obviously, only a few data points across a peak could be taken, and a great
deal of peak broadening was introduced, as seen in Figure 1. For colored solutes, migration
through glass columns could be followed visually.

Data Acquisition and Processing: Although it is difficult to imagine a time before

dedicated computers, it did exist! Because cross-linked polysaccharide gels were soft and
compressible, low pressures or flow rates were required. As such, a single GFC run would
take approximately 12 h, and an additional one or two days to analyze collected fractions
and plot the results. The total time for a single GFC analysis would be approximately 1.5–
2.5 days, or close to a week for a duplicate run.

Cross-linked polystyrene packings for GPC were more robust, so higher flow rates could be
used. Depending on the number of Styragel columns that were in series, a single run could
take about 1.5 h, with an additional 2.5 h to manually measure peak heights off a
chromatogram, tabulate the data, and then calculate average molecular weights and graph
cumulative and differential molecular weight distributions. Total time would be roughly 4 h
per sample. If a large-frame computer were available with data entry using punched tape or
cards, total analysis time could be reduced to about 2.5 h per sample. Tedious, yes, but SEC
was much faster than the weeks required for classical fractionation with subsequent
osmometry and light-scattering measurements of every fraction collected.

SEC Elution Behavior

In the early history of SEC, experiments had confirmed that log M vs. elution volume has a
negative, nearly linear slope. Each polymer type was found to have a characteristic
calibration slope and intercept. Based on these observations, the SEC elution behavior of
macromolecules is

where VR is the elution volume of a given component, Vo is the interstitial volume of the
packed column, Vi is the total pore volume of the packed column, and KSEC is the distribution
coefficient of the SEC separation process. The maximum or total elution volume Vt of a
packed column is
and the distribution coefficient (or partition coefficient at ideal conditions) of a solute is

where c1 and c2 are the average solute concentrations in the pore volume and in the
interstitial volume, respectively. The distribution coefficient ranges from zero to unity.
Macromolecules too large to access the pore volume will be eluted at Vo. Small molecules
that can freely diffuse into the pores of the packing will have an elution volume of Vt.

Thermodynamics of SEC

It is instructive to examine the thermodynamics of a separation to provide additional insight

into the nature of the process. The distribution coefficient, Kt, of a system at equilibrium and
infinite dilution is

where ΔH is the change in enthalpy when a solute is transferred from phase 2 to 1, ΔS is
the entropy change when the solute diffuses from phase 2 to 1, R is the gas constant,
and T is the absolute temperature of the system. In SEC, it is critical that macromolecules
do not interact energetically with the outer surface or pore walls of the packing, thus