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Void volume (V0) is the volume of mobile phase inside the column — the space between and within the packing particles that liquid actually occupies. For a typical 4.6 × 150 mm fully porous column it is about 1.65 mL.
It sets the earliest time anything can elute, anchors every retention factor you calculate, and determines how long a gradient takes to reach the column. Get it wrong and your retention factors, gradient timing and method transfers are all wrong with it.
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1. What is Void Volume?
The void volume is the volume the mobile phase occupies inside the column. If 66% of the column’s internal volume is taken up by liquid and the rest by packing material, the void volume is 66% of the total column volume.
It appears in the literature under several names, and the overlap is the single biggest source of confusion in this topic:
| Term | Symbol | What it means |
|---|---|---|
| Void volume | V0 or VM | Mobile phase volume inside the column |
| Column volume | Vc | The full internal volume of the empty tube — packing included |
| Void time (dead time) | t0 | Time for unretained compound to elute: V0 ÷ flow rate |
| Extra-column volume | — | Volume outside the column: tubing, injector, detector cell |
2. Void volume vs dead volume: what is the difference?
In everyday lab conversation “void volume” and “dead volume” are used interchangeably, and most of the time nobody is misunderstood. Strictly, though, they are not the same thing:
- Void volume is the mobile phase volume inside the column. It is a property of the column.
- Dead volume, used precisely, means volume in the flow path that contributes nothing to separation — connecting tubing, the injector, the detector flow cell. This is outside the column and is a property of the instrument.
The practical consequence: void volume is what you use for retention calculations; extra-column dead volume is what you minimise to keep peaks sharp. When someone says “my dead volume is too high” and means peak broadening, they are talking about the plumbing, not the column.
The confusion arises because column manufacturers often print “column dead volume” on a datasheet when they mean V0. If a value is quoted for a specific column, it is almost certainly the void volume; if it is quoted for a system, it is extra-column volume.
3. How to Calculate Void Volume?
The geometric estimate multiplies the empty tube volume by the total porosity of the packing:
V₀ = π × r² × L × ε r = column internal radius (mm) L = column length (mm) ε = total porosity of the packing ε ≈ 0.66 fully porous particles ε ≈ 0.49 superficially porous (core–shell) particles The result is in mm³. Divide by 1000 for mL.
Worked example — 4.6 × 150 mm, fully porous:
r = 4.6 ÷ 2 = 2.3 mm V₀ = π × 2.3² × 150 × 0.66 = 1645 mm³ = 1.65 mL
Note that ε is the total porosity — the interstitial volume between particles plus the pore volume within them — not the pore volume alone. The two porosity figures above are typical values; a specific column may differ by a few percent, which is why the experimental method in the next section is more reliable.
4. Void volume chart for common HPLC columns
Calculated with the formula above. Vc is the empty-tube column volume; the two V0 columns give the void volume for fully porous and core–shell packings.
| Column (ID × length) | Vc (mL) | V0 fully porous (mL) | V0 core–shell (mL) |
|---|---|---|---|
| 4.6 × 250 mm | 4.15 | 2.74 | 2.04 |
| 4.6 × 150 mm | 2.49 | 1.65 | 1.22 |
| 4.6 × 100 mm | 1.66 | 1.10 | 0.81 |
| 4.6 × 50 mm | 0.83 | 0.55 | 0.41 |
| 3.0 × 150 mm | 1.06 | 0.70 | 0.52 |
| 3.0 × 100 mm | 0.71 | 0.47 | 0.35 |
| 3.0 × 50 mm | 0.35 | 0.23 | 0.17 |
| 2.1 × 150 mm | 0.52 | 0.34 | 0.25 |
| 2.1 × 100 mm | 0.35 | 0.23 | 0.17 |
| 2.1 × 50 mm | 0.17 | 0.11 | 0.08 |
| 2.1 × 30 mm | 0.10 | 0.07 | 0.05 |
Treat these as estimates to within a few percent. Notice how small V0 becomes on narrow-bore columns — a 2.1 × 50 mm core–shell column holds only about 85 µL. At that scale the extra-column volume of the instrument can rival the column itself, which is why UHPLC systems use such narrow tubing.
5. How to Experimentally Determine Void Volume?
The formulas above are only an estimate. To find the actual void volume of a particular column on a particular system, inject a compound that does not retain on the packing — uracil is the usual choice for reversed phase, and thiourea or potassium nitrate also work. Then:
V₀ = t₀ × F t₀ = retention time of the unretained peak (min) F = flow rate (mL/min)
Example: an unretained peak elutes at 2.32 min with the flow rate at 0.85 mL/min:
V₀ = 2.32 min × 0.85 mL/min V₀ = 1.97 mL
This measured value includes the extra-column volume of the system as well as the column itself, so it will read slightly higher than the geometric estimate. That is usually what you want, because retention factors calculated from it reflect the system you are actually running.
The reverse calculation is just as useful. Knowing V0, the void time at any flow rate is V0 ÷ F — for a 4.6 × 150 mm porous column at 1.0 mL/min, t0 is 1.65 min; at 1.5 mL/min it is 1.10 min.
6. Why void volume matters
Void volume turns up across HPLC, size exclusion chromatography (SEC) and gel filtration chromatography (GFC), and an incorrect value propagates into several places at once:
- Retention factors are wrong. k = (tR − t0) ÷ t0. Every k in the method depends on t0, so an error here shifts all of them.
- Gradient timing drifts. The gradient takes V0 (plus the system dwell volume) to reach the column outlet. Methods transferred between systems fail most often for exactly this reason.
- Method transfer breaks. Scaling a method between column dimensions means scaling the gradient in column volumes, which requires V0 for both columns.
- In SEC, it defines the calibration. V0 is the exclusion limit — molecules too large to enter the pores elute there, and everything is calibrated relative to it.
- You cannot tell a real peak from the void. An unretained peak at t0 is not a separation; knowing where t0 falls stops you integrating solvent front artefacts as analyte.
For related column troubleshooting, see HPLC peak shape problems — a void at the column head is a different fault from the column void volume, and the two are easy to confuse by name.
7. Frequently asked questions
What is void volume in chromatography?
The volume of mobile phase inside the column — the space between and within the packing particles. About 1.65 mL for a 4.6 × 150 mm fully porous column.
Is void volume the same as dead volume?
Loosely, yes; strictly, no. Void volume is mobile phase inside the column. Dead volume, used precisely, is non-separating volume outside it — tubing, injector, detector.
How do you calculate void volume?
V0 = πr2Lε, using ε ≈ 0.66 for fully porous and 0.49 for core–shell packing, with r and L in mm and the result divided by 1000 for mL.
What is the difference between column volume and void volume?
Column volume (Vc) is the whole internal volume of the empty tube. Void volume is only the fraction that mobile phase occupies once the tube is packed — roughly two-thirds of Vc for fully porous particles.
What is void time?
The time an unretained compound takes to pass through: t0 = V0 ÷ flow rate.
Which marker should I use to measure void volume?
Uracil is standard for reversed phase. Thiourea and potassium nitrate are also used. The requirement is simply that the compound has no retention on the packing.
Why is my measured void volume higher than the calculated one?
Because the measurement includes the extra-column volume of tubing, injector and detector, while the geometric formula covers only the column.
8. References
Total porosity values used here (ε ≈ 0.66 fully porous, ≈ 0.49 superficially porous) are typical figures for modern silica-based HPLC packings; consult the manufacturer’s datasheet for a specific column.