Direct comparison
HPLC vs FPLC: Pressure, Media and When to Use
HPLC vs FPLC compared on aim, analyte size, pressure, column media and wetted materials, with a decision row for analysis versus protein purification.
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How do HPLC, FPLC compare side by side?
The table below compares HPLC, FPLC across 10 procurement-relevant dimensions, from typical aim through source.
Side-by-side comparison
| Dimension | HPLC | FPLC |
|---|---|---|
| Typical aim | Identify and qualify analytes | Obtain as much pure, native product as possible |
| Typical analytes | Usually small compounds, from a few atoms up to approximately 3,000 Da | Large biomolecules of several kDa, such as proteins, nucleotides and peptides |
| Operating pressure | High pressure; columns are built to withstand it | Pressure is kept low because proteins are sensitive; set by the resin and column rating, so it varies by system (a Cytiva ÄKTA pure with the 25 pump is rated to 20 MPa / 200 bar, the 150 pump to 5 MPa / 50 bar) |
| Column media | Silica beads with very small particle sizes and high pressure resistance | Agarose or polymer media, with larger particle sizes for most methods; less pressure stable than silica and very sensitive to air bubbles |
| Column and wetted materials | Pressure-resistant stainless steel columns | Stainless steel avoided, because metal ions can interfere with the protein; biocompatible PEEK, ceramic or titanium, and transparent biocompatible glass columns that let you see air bubbles |
| Buffers | — | Isosmotic pH and salt concentrations similar to the cell environment |
| Temperature | — | Proteins are usually worked at 4 °C, so the system is often placed in a cold room |
| Monitoring features (example system) | — | ÄKTA pure offers an optional in-line pH monitoring valve and a pressure monitor that adjusts flow to avoid reaching a defined pressure limit |
| Pick it when | You need to identify or quantify small-molecule analytes | You need to purify a protein or other biomolecule and keep it native |
| Source | LCGC International, S. Runde, "FPLC versus Analytical HPLC" (22 Aug 2016), read 2026-10-29; Cytiva ÄKTA pure datasheet, read 2026-10-29 | Same two sources: https://www.chromatographyonline.com/fplc-versus-analytical-hplc-two-methods-one-origin-many-differences-0 ; https://cdn.cytivalifesciences.com/api/public/content/digi-16276-pdf |
Common questions
Common questions about HPLC vs FPLC
What is the main difference between HPLC and FPLC?
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The aim. HPLC is used to identify and qualify analytes, usually small compounds up to approximately 3,000 Da. FPLC purifies large biomolecules such as proteins, nucleotides and peptides, aiming for as much pure and native product as possible (LCGC International, 2016).
Does FPLC always run at lower pressure than HPLC?
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FPLC avoids high pressure because proteins are sensitive and its resins are less pressure stable than silica. But the working pressure depends on the system and column: the Cytiva ÄKTA pure datasheet rates the 25 pump to 20 MPa (200 bar) and the 150 pump to 5 MPa (50 bar).
Why does FPLC avoid stainless steel?
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The source states that metal ions from stainless steel can interfere with the protein and disturb its structure, so biocompatible materials such as PEEK, ceramic or titanium are used instead.
Why is an FPLC often in a cold room?
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The working temperature for proteins is usually 4 °C, so FPLC systems are often placed in a cold chamber or cold room.
Going deeper
