Size exclusion chromatography (SEC) — also called gel filtration chromatography (GFC) when the mobile phase is aqueous, or gel permeation chromatography (GPC) when it is organic-solvent-based — separates molecules by hydrodynamic size rather than by charge or affinity. Large molecules are excluded from the pores of the resin beads and travel through the spaces between them, so they elute first. Small molecules diffuse into the pores, take a longer path through the bed, and elute later. Because elution volume tracks size rather than mass directly, SEC is the standard method for estimating an unknown protein’s apparent molecular weight against a set of known standards, for checking oligomeric state and aggregation, and for buffer exchange or desalting when resolution isn’t the goal.
This guide covers the volumes and coefficients you need to run a calibration, works a full numeric example from raw elution volumes to an estimated molecular weight, and gives selection and troubleshooting tables for choosing and running a column.
The volumes and terms that define a run
| Symbol | Name | What it is | How it’s determined |
|---|---|---|---|
| V0 | Void volume | Volume of mobile phase between the resin beads; the elution volume of a molecule too large to enter any pore | Elution volume of a fully-excluded marker, typically blue dextran 2000 (~2,000 kDa) or a large aggregate/virus standard |
| Vt | Total (geometric) column volume | Cross-sectional area × bed height | Calculated from column dimensions, or measured as the elution volume of a small molecule that fully permeates the pores (e.g. acetone, DNP-aspartate) |
| Vi | Internal (pore) volume | Mobile phase volume inside the resin pores | Not directly measured in routine work; Vt − V0 approximates V0 + Vi + matrix volume together, which is why Kav (below) is used instead of the theoretically stricter Kd |
| Ve | Elution volume | Volume at which a given analyte’s peak maximum elutes | Read directly from the chromatogram for each standard and for the unknown |
| Kav | Available partition coefficient | Fraction of the internal (pore) volume accessible to a given molecule, normalized 0–1 | Kav = (Ve − V0) / (Vt − V0) |
Kav = 0 means a molecule is fully excluded (elutes at V0); Kav = 1 means it fully permeates every pore and elutes at Vt. Everything of practical interest falls between those two limits — a well-packed column with the right resin for the target’s size range should place your analytes and standards somewhere in the Kav 0.1–0.7 window, where the relationship between Kav and log(molecular weight) is closest to linear.
Worked calculation: from elution volumes to an estimated molecular weight
The illustrative values below are for demonstrating the arithmetic, not measurements from any specific experiment — substitute your own instrument’s readings. Assume a column with total volume Vt = 120 mL and a void volume V0 = 40 mL, determined by running blue dextran 2000 and reading its peak elution volume.
Step 1 — Calculate Kav for each protein standard
| Standard | MW (Da) | log10(MW) | Ve (mL) | Kav = (Ve − 40) / 80 |
|---|---|---|---|---|
| Thyroglobulin | 669,000 | 5.825 | 45 | 0.0625 |
| Ferritin | 440,000 | 5.643 | 58 | 0.225 |
| Aldolase | 158,000 | 5.199 | 72 | 0.400 |
| Conalbumin | 75,000 | 4.875 | 85 | 0.5625 |
| Ovalbumin | 44,000 | 4.643 | 95 | 0.6875 |
| Ribonuclease A | 13,700 | 4.137 | 108 | 0.850 |
Each Kav is computed the same way, e.g. for aldolase: Kav = (72 − 40) / (120 − 40) = 32 / 80 = 0.400.
Step 2 — Fit log10(MW) against Kav
Plot log10(MW) on the y-axis against Kav on the x-axis and fit a straight line through the standards (ordinary linear regression across all six points in practice; the two-point slope/intercept shown here is only to demonstrate the arithmetic). Using ferritin and ribonuclease A as the reference pair:
slope = (4.137 − 5.643) / (0.850 − 0.225) = −1.506 / 0.625 = −2.41
intercept = 5.643 − (−2.41 × 0.225) = 5.643 + 0.542 = 6.185
Calibration line: log10(MW) = 6.185 − 2.41 × Kav
Sanity-check against aldolase (Kav = 0.400, actual MW 158,000): predicted log10(MW) = 6.185 − (2.41 × 0.400) = 6.185 − 0.964 = 5.221 → MW ≈ 10^5.221 ≈ 166,000 Da, within the scatter expected from a two-point fit against a six-point curve that isn’t perfectly linear at the extremes.
Step 3 — Apply the line to an unknown
An unknown protein elutes at Ve = 90 mL. Kav = (90 − 40) / 80 = 0.625. log10(MW) = 6.185 − (2.41 × 0.625) = 6.185 − 1.506 = 4.679. MW = 10^4.679 ≈ 47,700 Da — reported as an apparent molecular weight, because the whole calculation assumes the unknown behaves as a compact, globular, monomeric protein with roughly the same shape as the standards. An elongated, glycosylated, intrinsically disordered, or oligomeric protein will elute earlier or later than its actual mass predicts, sometimes by a large margin, because SEC separates by hydrodynamic (Stokes) radius, not mass. When the true molecular weight matters — not just relative size — pair SEC with a MALS (multi-angle light scattering) detector; SEC-MALS measures absolute molecular weight from scattered light intensity and doesn’t depend on the column calibration or an assumption about shape.
Column and resin selection
The single most important selection decision is matching the resin’s fractionation range to where your analyte and any contaminants you need to resolve actually sit — a column whose range is too wide gives poor resolution because everything of interest is compressed into a narrow part of the Kav curve; too narrow and your analyte elutes outside the linear region entirely (at or near V0 or Vt, both useless for sizing). The ranges below are the manufacturer-published, typical figures for common resin lines; treat them as a starting point, not a substitute for the current datasheet for the exact product you’re using, since cross-linking density and bead chemistry are periodically revised.
| Resin / matrix | Approximate fractionation range (globular proteins) | Typical use |
|---|---|---|
| Sephadex G-25 | ~1–5 kDa | Desalting, buffer exchange, removing small molecules from proteins |
| Sephadex G-50 / G-75 | ~1.5–80 kDa | Group separation of small peptides from larger proteins |
| Superdex 75 | ~3–70 kDa | High-resolution polishing of small proteins and peptides |
| Sephacryl S-200 HR | ~5–250 kDa | Mid-range protein purification, preparative scale |
| Superdex 200 | ~10–600 kDa | General-purpose protein purification and MW estimation, the most common analytical/preparative default |
| Sephacryl S-300 / S-400 HR | ~10 kDa–2 MDa (approx.) | Large protein complexes, some viruses |
| Superose 6 / Sepharose CL-6B | ~5 kDa–5 MDa (approx.) | Very large complexes, ribosomal subunits, plasmids, aggregate/monomer separation |
Beyond fractionation range, three further parameters shape the choice:
- Particle/bead size and pore-size distribution uniformity. “HR” (high-resolution) and analytical-grade resins use smaller, more uniform beads for sharper peaks and better resolution, at the cost of higher back pressure and lower flow-rate tolerance than a “prep grade” resin built for throughput over resolution.
- Column length and bed height. Resolution in SEC scales with the square root of column length (more theoretical plates), so doubling column length gives a smaller resolution gain than the flow-time cost implies — long, narrow columns run at low flow rate are the standard configuration for high-resolution analytical SEC, not short, wide ones.
- Chemical compatibility. Dextran-based resins (Sephadex, Sephacryl) and agarose-based resins (Sepharose, Superose) tolerate different pH ranges, organic solvent content and pressure limits; silica-based SEC/GPC columns used for polymer characterization in organic solvents are a different chemistry class entirely from the biocompatible resins used for native protein work.
Operating parameters and what they trade off
| Parameter | Effect if too high | Effect if too low |
|---|---|---|
| Flow rate | Reduced resolution (less time for equilibration between mobile and pore phases); higher back pressure, risking bed compression | Longer run time, band broadening from diffusion, lower throughput |
| Sample volume | Peak broadening, loss of resolution — SEC has essentially no on-column concentrating effect, unlike ion-exchange or affinity modes | Detector sensitivity limits how dilute a sample can be and still give a usable peak |
| Mobile-phase ionic strength | Not typically a problem in itself, but very high salt can affect some detectors | Low-ionic-strength buffers allow non-specific ionic interactions between analyte and residual charged groups on the matrix, causing anomalously early or late elution unrelated to true size |
| Column temperature | Faster diffusion can slightly improve mass transfer but risks protein instability | Higher viscosity slows mass transfer and increases back pressure |
As a working rule, sample volume should stay at or below roughly 1–5% of the total column bed volume for analytical resolution runs; larger sample volumes are acceptable when the goal is group separation (e.g. desalting) rather than resolving closely-sized species. Mobile-phase choice should include enough ionic strength (commonly ~100–150 mM salt) to suppress non-specific electrostatic interaction between the analyte and the matrix, since an uncorrected ionic interaction is one of the most common causes of an SEC elution volume that doesn’t match the expected molecular weight.
Troubleshooting
| Symptom | Likely cause | Fix |
|---|---|---|
| Void volume peak shifts earlier over repeated runs | Bed shrinkage, channeling, or a cracked/settled resin bed | Re-pack or re-equilibrate the column; check for air bubbles or particulates introduced during sample loading |
| Broad, poorly resolved peaks across the whole run | Flow rate too high for the resin’s optimal linear velocity, sample volume too large, or a degraded/fouled bed | Reduce flow rate, reduce injected volume, re-evaluate column plate count with a small-molecule test sample |
| Analyte elutes earlier than its known MW predicts | Aggregation, or the protein is genuinely non-globular (elongated, multimeric) and has a larger hydrodynamic radius than a compact protein of the same mass | Check for aggregation by dynamic light scattering or a second SEC pass; if shape is the cause, use SEC-MALS for an absolute (shape-independent) MW instead of relying on the calibration curve |
| Analyte elutes later than its known MW predicts | Non-specific ionic or hydrophobic interaction with the resin matrix | Increase mobile-phase ionic strength; confirm the resin/buffer combination is recommended for the analyte class |
| Rising back pressure over time | Particulates or precipitated protein fouling the column inlet or frit | Filter and centrifuge samples before injection; add or replace an in-line guard filter; check the inlet frit for blockage |
| Poor reproducibility between runs | Inconsistent column equilibration, temperature drift, or degrading resin | Standardize equilibration volume (typically ≥1.5 column volumes), control room/column temperature, re-run standards periodically to confirm the calibration line is still valid |
| Ghost or unexpected extra peaks | Carryover from a previous run, or sample degradation/aggregation | Increase wash/equilibration between injections; check sample stability under running buffer conditions |
Frequently asked questions
Is size exclusion chromatography the same as gel filtration?
Yes, in practice — “gel filtration” is the traditional name for SEC run in an aqueous buffer, most often for proteins. “Gel permeation chromatography” (GPC) is the same physical separation principle applied with organic solvents, typically for synthetic polymer characterization. All three terms describe separation purely by hydrodynamic size, with no ion-exchange or affinity chemistry involved.
What is Kav and why not just use elution volume directly?
Kav normalizes elution volume to a 0–1 scale that’s independent of that specific column’s exact dimensions, so a calibration built on one column run can be compared conceptually against literature values or a different column of the same resin. Raw Ve alone depends on the specific column’s void and total volumes and isn’t directly comparable across setups.
Can SEC give an absolute molecular weight?
Not on its own — a standard SEC calibration curve gives an apparent MW that assumes the unknown has roughly the same shape (hydrodynamic radius per unit mass) as the standards used to build the curve. For a true, shape-independent absolute MW, SEC needs to be paired with a light-scattering detector (SEC-MALS) or another orthogonal method such as analytical ultracentrifugation.
How do I choose between Superdex 75 and Superdex 200 for a protein around 100 kDa?
Superdex 200’s ~10–600 kDa range comfortably brackets 100 kDa with room on both sides for resolving from smaller contaminants or from higher-order aggregates. Superdex 75’s ~3–70 kDa upper limit means a 100 kDa protein would elute near or past its exclusion behavior for the low end, giving compressed, poorly resolved data — for a target in the 70–600 kDa range, Superdex 200 (or an equivalent resin with a comparable range) is the right general-purpose choice.
Why does my protein elute earlier than its known monomer molecular weight predicts?
The two most common causes are aggregation (the protein is genuinely running as a larger species than the monomer) and non-globular shape (an elongated or heavily glycosylated protein has a larger hydrodynamic radius than a compact globular protein of the same mass, so it behaves like a “bigger” molecule on the column even though its actual mass is unchanged). Distinguishing the two usually requires an orthogonal technique — dynamic light scattering or SEC-MALS — rather than the calibration curve alone.
Related lab techniques
SEC is one of several chromatography and separation modes used in the same lab workflows. For separations based on other principles, see HPLC: columns, mobile phases and troubleshooting and column chromatography: packing, solvent selection and fractions. When SEC is coupled to mass spectrometry for identification rather than sizing, see LC-MS explained; for a simpler planar separation technique, see thin-layer chromatography: running a plate and calculating Rf.
For orthogonal ways to check size, shape and purity alongside or instead of SEC, see dynamic light scattering: Z-average, PDI and how to read a DLS report, surface plasmon resonance: running a binding assay and calculating KD, and agarose gel electrophoresis protocol basics. Accurate SEC results also depend on well-maintained supporting equipment — see analytical balance calibration and weighing technique and spectrophotometer calibration for detector- and sample-prep-adjacent instrumentation, and buffer and solution preparation plus molarity and solution calculations for the lab for preparing the mobile phase itself.
