Size exclusion chromatography (SEC) separates molecules based on size, with larger molecules eluting earlier than smaller ones. SEC is driven by entropy rather than adsorption, with no interaction between the stationary and mobile phases. Temperature has little impact on retention in SEC. Separation is achieved as molecules of different sizes access different portions of pores in the stationary phase packing material. The document provides examples of using SEC to analyze various proteins and polymers, demonstrating the impact of parameters like particle size, temperature, mobile phase composition, and column type on separation efficiency and resolution.

1. size exclusion chromatography

2. Illustrative description of
separation in SEC

4. HONG, Paula; KOZA, Stephan; BOUVIER, Edouard SP. A REVIEW SIZE-EXCLUSION
CHROMATOGRAPHY FOR THE ANALYSIS OF PROTEIN
BIOTHERAPEUTICS AND THEIR AGGREGATES. Journal of liquid
chromatography & related technologies, v. 35, n. 20, p. 2923-2950, 2012.
FEKETE, Szabolcs et al. Theory and practice of Size Exclusion
Chromatography for the analysis of protein aggregates. Journal of
Pharmaceutical and Biomedical Analysis, 2014.

6. The free energy change of a chromatographic process can be described by, where ΔG 0, ΔH 0,
and ΔS 0 are the standard free energy, enthalpy, and entropy differences, respectively; R is the
gas constant: T is absolute temperature, and k is the partition coefficient. For most
chromatographic modes of separation, the enthalpy of adsorption is the dominant contributor
to the overall change in free energy. SEC is unique in that partitioning is driven entirely by
entropic processes as there ideally is no adsorption, ΔH = 0. Thus the previous equation
becomes: where K D is the thermodynamic retention factor in SEC. Thus, in SEC separations,
temperature should have no impact on retention. In practice, temperature can indirectly impact
retention to a small degree by altering the conformation of the proteins, as well as by affecting
mobile phase viscosity and analyte diffusivity.

7. The thermodynamic SEC retention factor is the fraction of intraparticle pore
volume that is accessible to the analyte: where V R , V 0, and V i are the respective
retention volumes of the analyte of interest, the interstitial volume, and the intra-particle
volume. K D will range from a value of 0 where the analyte is fully excluded
from the pores of the stationary phase, to a value of 1 where the analyte fully
accesses the intraparticle pores.

8. Separation of (1) thyroglobulin, (2) IgG, (3) BSA, (4) Myoglobin, and (5) Uracil on a Waters
ACQUITY UPLC BEH200 SEC, 1.7 μ, 4.6 × 150 mm. Mobile phase: 100 mM sodium phosphate,
pH 6.8. Flow rate: 0.3 mL/min. Temperature: 30°C (black), 40°C (blue), 50°C (red). Reproduced
with permission from Waters Corporation, Milford, MA

9. Typical SEC calibration curve

11. Theoretically expected impact of the particle size and mobile phase temperature on column
performance. (For the calculations, a 50 kDa protein was assumed.).

12. Effect of linear velocity on plate height for (a) ribonuclease A (red) and (b) a monoclonal
antibody (blue) on two columns varying in particle size. The 4.6 × 150 mm columns were packed
with either 1.7 micron (solid line) or 2.6 micron particles (dashed line). Pore size of stationary
phase sorbent: 200 Å. Mobile phase consisted of 100 mM sodium phosphate, pH 6.8.
Reproduced with permission from Waters Corporation, Milford, MA.

13. Comparison of Columns: Effect of Particle Size on Efficiency and Resolution for a Reduced
Antibody
Theoretical Plates
[-17pt]
Columns
Dimension
s (m i.d. ×
mm length)
Particle
size (μm)
Pore sizes
(Å) HC LC Resolution
TSKgel
G3000SW
7.5 × 300 10 250 1980 3845 3
TSKgel
G3000SWxl
7.8 × 300 5 250 5060 10674 4
Shodex
KW-804
8.0 × 300 7 250 4952 8859 2
Protein-Pak
300SW
7.5 × 300 10 250 2078 4271 3
BioSuite
250
7.8 × 300 5 250 5149 9403 3

17. The drug aprotinin (Trasylol, previously Bayer and now Nordic Group
pharmaceuticals), is the small protein bovine pancreatic trypsin inhibitor,
or BPTI, which inhibits trypsin and related proteolytic enzymes. Under the
trade name Trasylol, aprotinin was used as a medication administered
by injection to reduce bleeding during complex surgery, such as heart and liver
surgery. Its main effect is the slowing down of fibrinolysis, the process that
leads to the breakdown of blood clots. The aim in its use was to decrease the
need for blood transfusions during surgery, as well as end-organ damage due
to hypotension (low blood pressure) as a result of marked blood loss. The drug
was temporarily withdrawn worldwide in 2007 after studies suggested that its
use increased the risk of complications or death

27. Schematic representation of some of the key steps in non-native aggregation

28. Plate heights (HETP) vs.
linear velocity (u0) plots
of Panitumumab (A),
chicken ovalbumin (B)
and β-lactoglobulin (C).
Columns: Acquity UPLC
BEH200 SEC 1.7 μm,
150 mm × 4.6 mm
(operated at 30, and
60 °C), YMC-Pack-Diol-
200 5 μm,
300 mm × 6 mm and
Phenomenex Yarra SEC-
3000 3 μm,
300 mm × 4.6 mm
(operated at 30 and
50 °C). Mobile phase:
20 mM disodium
hydrogen-phosphate
buffer of pH = 6.8.
Journal of
Pharmaceutical and
Biomedical Analysis 78–
79 (2013) 141– 149

29. Effect of pressure (A) and temperature (B)
on the observed aggregates. Column:
Acquity UPLC BEH200 SEC 1.7 μm,
150 mm × 4.6 mm. Mobile phase: 20 mM
disodium hydrogen-phosphate buffer of
pH = 6.8. Flow rate: 200 μl/min, detection:
FL (Ex: 280 nm, Em: 360 nm). The column
pressure (head pressure) was varied by
adding restrictor capillaries to the column
outlet (131, 271, 406 and 465 bar were
generated, including the column pressure)
on (A). Sample: heat stressed
panitumumab.

30. Representative chromatograms on the effect of column temperature (A) and pressure (B) on the
observed amount of antibody aggregates

31. Representative chromatograms on BEH 1.7 μm column (A) and on YMC diol 5 μm column (B)
obtained by injecting the same sample (native panitumumab). Mobile phase: 20 mM disodium
hydrogen-phosphate buffer of pH = 6.8. Flow rate: 500 μl/min, detection: FL (Ex: 280 nm, Em:
360 nm), mobile phase temperature: 30 °C. The generated pressure was 274 bar (A) and 73 bar
(B).

32. Fast separation of the aggregate and native form of β-lactoglobulin (A) and of chicken egg
ovalbumin (B). Column: Acquity UPLC BEH200 SEC 1.7 μm, 150 mm × 4.6 mm. Mobile phase:
20 mM disodium hydrogen-phosphate buffer of pH = 6.8. For β-lactoglobulin: flow rate:
700 μl/min, mobile phase temperature: 45 °C and for egg ovalbumin: flow rate: 850 μl/min,
mobile phase temperature: 60 °C. Detection: FL (Ex: 280 nm, Em: 360 nm) for both cases. Peaks:
1, 2 and 3: high molecular weight species.

33. C. Wong et al. / J. Chromatogr. A 1270 (2012) 153– 161
SE-HPLC chromatogram profile at 280 nm showing fronting shoulder on monomer of a drug
product at time zero (red) and 40 °C 2 months stability sample (black). Column TSKgel BioAsisst
G3SWXL

34. Representative chromatograms of formulated bulk separation using 0.25 M NaCl (black), 0.5 M
NaCl (blue), 0.75 M NaCl (green), 1.0 M NaCl (cyan), 1.25 M NaCl (magenta), 1.5 M NaCl
(purple), and 1.7 M NaCl (red) sodium chloride in 20 mM sodium phosphate, pH 7.0 mobile
phase, and Waters Acquity BHE200 4.6 mm × 300 mm column shown at 280 nm. (For
interpretation of the references to color in this figure legend, the reader is referred to the web
version of the article.)

35. Representative
chromatogram of (A)
formulated bulk material
(blue) and purified monomer
fraction (black), and (B) 40 °C,
9 months stability sample
(blue) and purified pre-peak
fraction (black) using the final
mixed mode UPLC method at
280 nm.
Mobile phase containing
20 mM sodium phosphate at
pH 7.0 and a flow rate of
0.15 mL/min showed the best
separation for the mixed mode
UPLC method. The pre-peak
and the monomer peak were
fractionated using the final
mixed mode UPLC method for
characterization

36. J. Sep. Sci. 2013, 36, 2718–2727
Chromatograms of PS standard (Mp ∼ 11 600 g/mol, -D- ∼ 1.03) obtained on XBridge(TM) C18
and ACQUITY R C18 columns (4.6 × 150 mm) packed with different size particles: 10, 5, 3.5, and
1.7 m. Mobile phase, THF; flow rate, 1 mL/min; detection, UV at 254 nm.

37. Stacked chromatograms of SEC separation of two proprietary polymers on BEH 45 unbonded
and BEH 45 TMS columns, 4.6 × 150mmin THF at 1 mL/min using UV detection at 254 nm. (A)
Polymer A on BEH 45 unbonded; (B) polymer B on BEH 45 unbonded; (C) polymer A on BEH
45 TMS; (D) polymer B on BEH 45 TMS

38. Chromatograms of PS
standards on (A) BEH 200
diol, 4.6 × 150 mm,
detection, UV at 254 nm;
(B) same as (A), but with
ELS detection. Five
replicate injections are
shown, demonstrating
repeatability; (C) 5 m
PLgel MiniMix D (4.6 ×
250 mm); detection, UV
at 254 nm. In all cases,
mobile phase was THF
and flow rate was 1
mL/min.

39. Chromatograms of (A) fourteencomponent mixture of PS standards obtained on two 4.6 × 150
mm columns connected in series. First column, 1.7 m BEH diol 200 A° ; second column, 1.7 m
BEH diol 450 A° . Mobile phase, THF; flow rate, 1 mL/min; detection, ELS, (B) twelve-component
PS standards obtained on three PL gel SEC columns (7.5 × 300 mm each) packed with 5 m
Psdivinylbenzene particles with pore sizes labeled as 10E2, 10E3, and 10E4 A° . Mobile phase,
THF; flow rate, 1 mL/min; detection, RI.

40. Chromatograms of PMMA standards and corresponding calibration curve (fifth-order fit). Mobile
phase, THF; flow rate, 0.4 mL/min; column, 4.6 × 150 mm, packed with 1.7 mdiol-bonded
particles with amean pore size of 200 A° ; detection, ELS.