science

Mixed-mode chromatography

Mixed-mode chromatography is a science topic covered in the lgStudy science library. This page brings together a partial reference excerpt, illustrations, worked examples, real-world applications and a short study plan, so you can understand Mixed-mode chromatography rather than just read about it. In short: Mixed-mode chromatography (MMC), or multimodal chromatography, refers to chromatographic methods that utilize more than one form of interaction between the stationary phase and analytes in order to achieve their separation. What is distinct from conventional single-mode chromatography is that the secondary interactions in MMC cannot be too weak, and thus they also contribute to the retention of the solutes.

Key takeaways

Mixed-mode chromatography belongs to science; place it in that map before memorising details.
Learn the definition first, then one example that makes the definition concrete.
Connect Mixed-mode chromatography to a quantity you can measure, compute or draw — that is where exam questions come from.
Reproduce the core statement of Mixed-mode chromatography from memory before moving on to harder problems.

Reference excerpt

Mixed-mode chromatography (MMC), or multimodal chromatography, refers to chromatographic methods that utilize more than one form of interaction between the stationary phase and analytes in order to achieve their separation. What is distinct from conventional single-mode chromatography is that the secondary interactions in MMC cannot be too weak, and thus they also contribute to the retention of the solutes.

History Before MMC was considered as a chromatographic approach, secondary interactions were generally believed to be the main cause of peak tailing.

However, it was discovered afterwards that secondary interactions can be applied for improving separation power. In 1986, Regnier’s group synthesized a stationary phase that had characteristics of anion exchange chromatography (AEX) and hydrophobic interaction chromatography (HIC) on protein separation. In 1998, a new form of MMC, hydrophobic charge induction chromatography (HCIC), was proposed by Burton and Harding. In the same year, conjoint liquid chromatography (CLC), which combines different types of monolithic convective interaction media (CIM) disks in the same housing, was introduced by Štrancar et al. In 1999, Yates’ group [11] loaded strong-cation exchange (SCX) and reversed phase liquid chromatography (RPLC) stationary phases sequentially into a capillary column coupled with tandem mass spectrometry (MS/MS) in the analysis of peptides, which became one of the most efficient technique in proteomics afterwards. In 2009, Geng’s group first achieved online two-dimensional (2D) separation of intact proteins using a single column possessing separation features of weak-cation exchange chromatography (WCX) and HIC (termed as two-dimensional liquid chromatography using a single column, (2D-LC-1C).

Advantages Higher selectivity: for example, positive, negative and neutral substances could be separated by a reversed phase (RP)/anion-cation exchange (ACE) column in a single run. Higher loading capacity,

for example, loading capacity of ACE/ hydrophilic interaction chromatography (HILIC) increased 10-100 times when compared with RPLC,

which offered a new selection and idea for developing semi-preparative and preparative chromatography.

One mixed-mode column can replace two or even more single mode columns, which is economic and eco-friendly for employing the stationary phase more sufficiently and reducing the consuming and ‘waste’ of raw materials. Single mixed-mode column can be applied for on-line two-dimensional (2D) analysis in a sealed system via establishing corresponding chromatographic system or off-line 2D analysis as two columns.

Classification of MMC MMC can be classified into physical MMC and chemical MMC. In the former method, the stationary phase is constructed of two or more types of packing materials. In the chemical method, just one type of packing material containing two or more functionalities is used.

Physical methods The simplest approach is to connect two commercial columns in series, which is termed a “tandem column”. Another approach is “biphasic column”, by packing two stationary phases separately in two ends of the same column. The third approach is to homogenize two or more different types of stationary phases in a single column, which is termed a “hybrid column” or “mixed-bed column”.

Chemical methods

IEC/HIC Since IEC and HIC conditions are the closest ones to physiological conditions which are fit for maintaining biological activity, the combinations of them are widely used in the separation of biological products. IEC/HIC MMC has improved separation power and selectivity on the grounds that it applies both electrostatic and hydrophobic interactions.

IEC/RPLC IEC/RP MMC combines the advantages of RPLC and IEC. For example, WAX/RP has increased separation power and degree of freedom in adjusting the separation selectivity when compared with single WAX or RPLC.

HILIC/RPLC Liu et al. synthesized a HILIC/RP stationary phase which could show RPLC or HILIC retention by adjusting the organic phase in mobile phase.

HILIC/IEC Mant et al. reported that HILIC/CEX offered unique selectivity, stronger separation power and wider range of applications compared to RPLC for peptide separations.

SEC/IEC Hydrophobic interactions in protein SEC are relatively weak at low ionic strength, electrostatic effects may contribute significantly to retention, and this allows us to use an SEC column as a weak ion exchanger.

References

Worked examples

Example 1 — a first encounter with Mixed-mode chromatography

Start with the simplest possible case. Write down what Mixed-mode chromatography claims or describes in one sentence, then invent the smallest concrete situation in which that sentence is true. In science, the smallest case is usually a single object, a single equation or a single measurement. Check that every symbol or term in your sentence has a meaning in that case.

Example 2 — changing one variable

Take the situation from Example 1 and change exactly one quantity: double it, halve it, or set it to zero. Predict what should happen to Mixed-mode chromatography before you calculate. Comparing your prediction with the result is the fastest way to find out whether you understand the idea or only the words.

Example 3 — an exam-style question

Typical questions about Mixed-mode chromatography ask you to (a) state it precisely, (b) apply it to given data, and (c) explain a limitation. Practise writing all three answers in under five minutes; the third part is what separates a full-mark answer from an average one.

Applications of Mixed-mode chromatography

In research: Mixed-mode chromatography appears in science research whenever the underlying quantities have to be modelled precisely. Papers usually cite it as a starting assumption and then explore where it breaks down.
In technology and industry: Engineering practice reuses Mixed-mode chromatography in design rules, simulations and safety margins. Knowing the idea lets you read a specification sheet and understand why the numbers look the way they do.
In the classroom: Mixed-mode chromatography is common in secondary-school and first-year university syllabi. It links to neighbouring topics Chromatography, so understanding it makes those chapters shorter.
In everyday life: Look for Mixed-mode chromatography outside the textbook — in sport, cooking, traffic, electronics or the sky above you. An example you found yourself is remembered far longer than one you were given.

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How to study Mixed-mode chromatography in 20 minutes

Read the reference excerpt below once, without taking notes.
Close the page and write down what Mixed-mode chromatography means in your own words.
Compare your version with the excerpt and mark what you missed.
Work through the three examples above with pen and paper.
Explain Mixed-mode chromatography out loud to somebody else — or to Teacher Smith in the lgStudy chat.

Frequently asked questions

What is Mixed-mode chromatography in simple terms?

Why does Mixed-mode chromatography matter?

Because it connects several science ideas at once: it gives you a definition you can apply, a quantity you can calculate, and a way to check whether a result is plausible.

How should I study Mixed-mode chromatography?

Read the excerpt, restate it from memory, then work through the examples and applications listed on this page. The five-step study plan above takes about twenty minutes.

What does this page cover?

It gives you a compact reference excerpt plus original lgStudy explanations, examples, applications and study material on Mixed-mode chromatography.