Different types of liquid chromatography explained

Liquid chromatography is one of the most widely used analytical techniques in pharmaceutical development.

Almost every modern drug substance including small molecules, peptides, oligonucleotides, and the conjugates built on them is characterized, purified, and released using some form of it.

But “liquid chromatography” is a broad label. Underneath it sit several distinct techniques, each with its own separation mechanism, ideal use cases, and limitations.

For a CDMO, choosing the right one, or, more often, the right combination of two, has a direct impact on what an analyst can see, what they can confirm, and what might slip through unnoticed.

Here we’ll walk through the different types of liquid chromatography used in peptide and oligonucleotide analysis, and explain how 2D liquid chromatography combines the strengths of two.

What is liquid chromatography?

Liquid chromatography is a technique for separating the components of a mixture.

A sample is dissolved in a liquid (the mobile phase) and pushed through a column packed with a solid material (the stationary phase). Different molecules interact with the stationary phase to varying degrees, so they travel through the column at different speeds.

By the time the sample exits the column, each component has been resolved into its own peak. That peak is then measured, most often with a UV detector, and increasingly with mass spectrometry where structural confirmation is needed.

The different types of liquid chromatography differ in how the stationary phase interacts with the sample. What mechanism drives the separation defines which kinds of molecules a method can resolve and which impurities it can detect.

The main types of liquid chromatography

Five separation mechanisms dominate the analysis of pharmaceutical molecules. Each one is suited to a particular kind of separation challenge.

1. Reversed-phase liquid chromatography (RPLC)

Reversed-phase liquid chromatography is one of the most widely used forms of liquid chromatography. The stationary phase is water-repellent (typically C18-modified silica) and the mobile phase is water-based. Molecules separate according to how strongly their water-repellent regions interact with the column: more hydrophobic compounds elute later.

RPLC is the workhorse method for small molecules, peptides, and many oligonucleotides. It’s robust, well-understood, and uses mobile phases that mass spectrometers can tolerate, making it the default choice for routine release testing.

2. Ion-pair reversed-phase chromatography (IP-RP)

Strongly charged molecules, including oligonucleotides, whose backbones carry repeating negative charges, do not retain well on a standard reversed-phase column on their own.

Ion-pair reversed-phase chromatography solves this by adding an ion-pairing reagent to the mobile phase. The reagent neutralizes the charge on the molecule so that it can interact with the water-repellent stationary phase in the usual way.

IP-RP is the gold-standard method for oligonucleotide purity analysis and is widely used as the primary release method for oligonucleotide APIs.

3. Ion-exchange chromatography (IEX)

Ion-exchange chromatography separates molecules based on charge.

The stationary phase carries fixed charges that are either positive (anion exchange) or negative (cation exchange), and the sample is eluted by gradually increasing the salt concentration in the mobile phase. Molecules with the opposite charge bind, then release as the salt level rises.

For oligonucleotides, strong anion exchange (SAX) is particularly valuable. Because its separation mechanism is completely different from the reversed-phase family, SAX often resolves impurities that IP-RP cannot. This makes it the most common orthogonal cross-check (an independent method that looks at the same sample through a different lens) in oligonucleotide purity analysis.

The limitation is that IEX methods use non-volatile salts, which means they cannot be coupled directly to a mass spectrometer.

4. Hydrophilic interaction chromatography (HILIC)

HILIC inverts the logic of reversed-phase chromatography. The stationary phase is water-loving, the mobile phase is largely organic, and polar molecules retain more strongly than nonpolar ones. This makes HILIC well-suited to molecules that are too polar to be retained by RPLC, including some metabolites, sugars, and modified peptides.

HILIC is MS-compatible, which makes it useful where RPLC simply cannot hold the analyte on the column long enough to separate it.

5. Size-exclusion chromatography (SEC)

Size-exclusion chromatography separates molecules by size rather than by chemical interaction.

The stationary phase is a porous gel. Smaller molecules diffuse into the pores and take longer to pass through; larger molecules pass around them and elute first.
SEC is commonly used for biologics. For example, SEC can be used to assess aggregation in monoclonal antibodies, but is also a useful screening tool for confirming the integrity of double-stranded oligonucleotides.

Two-dimensional liquid chromatography (2D-LC)

Each of the methods above describes a single separation mechanism. Two-dimensional liquid chromatography combines two of them in series.

How 2D-LC works

In 2D liquid chromatography, the sample is first separated using one mode, such as ion exchange. A specific peak (or the whole chromatogram) is then re-injected onto a second column running a different separation mode- in this example, an ion-pair reversed-phase.

Because the two modes separate the sample by different mechanisms, anything that co-eluted in the first dimension can be pulled apart in the second. The output is a much richer picture of what is actually in the sample than either method could give on its own.

Benefits of 2D-LC

The benefits of 2D-LC follow directly from that principle. It allows analysts to:

• Confirm whether a peak that looks pure on one method is genuinely pure, or whether impurities are co-eluting underneath. See this verified on a duplex siRNA API with our case study.
• Combine the orthogonal selectivity of one method (such as ion exchange) with the mass-spectrometry compatibility of another (such as IP-RP), so detection no longer constrains the choice of separation.
• Identify low-level impurities that would be invisible on a one-dimensional method
  Reduce reliance on reference standards, which often do not exist for the impurities of greatest interest

Essentially, 2D chromatography turns the limitations of any single technique into a problem the next dimension can solve.

When 2D-LC adds value

2D-LC is not the right tool for every analytical question.

For routine release testing, a well-developed one-dimensional method (typically IP-RP with UV detection) remains the standard, because it’s faster, simpler, and easier to validate.

2D-LC adds the most value during method development and verification, where the goal is not to release a batch but to confirm that a release method is actually doing its job. It’s particularly valuable when:

• The primary release method is suspected of hiding co-eluting impurities
• The orthogonal cross-check is not MS-compatible on its own
• Reference standards for likely impurities are not available
• The molecule is structurally complex — for example, modified or conjugated oligonucleotides and peptides

A real-world example: 2D-LC for oligonucleotide purity

The clearest place to see this play out is in oligonucleotide analysis.

The industry-standard workflow pairs IP-RP (MS-compatible, used for release) with SAX (orthogonal cross-check, not MS-compatible). The very thing that makes SAX valuable is its different separation mechanism. This is also what makes its main peak hardest to verify, because a clean-looking UV peak cannot prove that nothing is co-eluting underneath.

Bachem developed an in-house 2D SAX–IP-RP–MS method to close that gap. The first dimension runs the SAX separation as standard. The main peak is isolated and re-analyzed in the second dimension by IP-RP coupled to mass spectrometry. Any impurity sitting under the SAX main peak now has somewhere to go.

The full case study, applied to a modern duplex siRNA API, shows what this revealed about a peak that, by UV alone, would have been judged pure.

How to choose the right liquid chromatography method

No single mode of liquid chromatography is universally best. Choosing the right one (or the right combination) comes down to three questions.

What is the molecule?

Charge, polarity, size, and modifications all narrow the field. A small lipophilic drug points to RPLC. A modified oligonucleotide points to IP-RP for release and SAX as an orthogonal check. A biologic prone to aggregation points to SEC.

What separation are you trying to achieve?

Bulk purity, low-level impurities, isomer separation, and aggregation are different problems and need different selectivity. The separation question, rather than the instrument, should determine the method.

What detection do you need?

UV is universal but limited. Mass spectrometry confirms what UV cannot, but only works with MS-compatible methods. Where the answer requires both orthogonal selectivity and MS detection, 2D-LC is often the only way to get them at the same time.

The different types of liquid chromatography are not interchangeable. Each one is a tool with a specific job, and the most reliable analytical strategies use them in combination rather than relying on any one in isolation.

2D liquid chromatography is the clearest example. It is not a new separation mechanism, but a way to combine two existing ones so that no impurity can hide behind the limitations of a single technique.

For complex modalities like oligonucleotides and peptides, where impurities can differ from the main peak by only a single nucleotide, a single oxidation state, or a single stereocenter, that combination is increasingly what the analytical strategy demands.

How Bachem can help

Bachem’s analytical capabilities span the full range of liquid chromatography methods used in peptide and oligonucleotide development: from routine release methods to specialized 2D workflows that probe the limits of what any single mode can resolve.

We support customers with:

• Method development across reversed-phase, ion-pair, ion-exchange and other LC modes
• Orthogonal method strategies designed to catch what a single method might miss
• 2D-LC method development and verification, including the 2D SAX–IP-RP–MS workflow described above
• MS-based identification and characterization of impurities and degradation products
• Full release and stability analytics for peptide and oligonucleotide APIs

With more than fifty years of experience as a CDMO, we combine analytical depth with the chemistry and process understanding that make those methods meaningful.

Connect with our team to discuss how our analytical capabilities can support your molecule from early development to commercial launch.