Mixing and dissolving: vortex mixers, sonication and magnetic stirrers

Making up a buffer, a standard or an assay solution comes down to two steps: getting a solid into solution, and making sure the solution is the same all the way through. Labs have several tools for this, from a simple swirl to an ultrasonic bath. Each one puts a different amount of energy into the liquid, and more isn't always better. This guide explains how vortex mixers, magnetic stirrers and sonicators work, which suits which job, and how to avoid the common ways mixing goes wrong.

Terms are defined in the glossary.

Choosing a method

The general rule is to use the gentlest method that does the job.

Vortex mixers

A vortex mixer has a small rubber cup on top of a motor that moves in a tight circle, often at a few thousand revolutions per minute. Pressing a tube onto the cup makes the liquid inside spin into a funnel-shaped vortex, which mixes it within seconds.

• Touch mode runs only while a tube is pressed down. Continuous mode runs all the time, and can hold a rack or foam insert for several tubes.
• Speed control lets you start low and increase it. High speed in a full tube throws liquid onto the cap.
• Best for small volumes in microcentrifuge tubes and test tubes, such as mixing a dilution or resuspending a pellet after a spin. See centrifuges.

Vortexing is short and vigorous. It can whip air into solutions that contain proteins, peptides or detergents, which then foam. For those, a few gentle inversions or a brief, low-speed pulse is safer.

Magnetic stirrers

A magnetic stirrer is a flat plate with a rotating magnet underneath. A stir bar (or "flea"), a small magnet coated in PTFE, is dropped into the beaker or flask. As the magnet below turns, the bar spins with it and stirs the liquid.

• Stir bars come in many shapes. Plain cylinders suit flat-bottomed beakers. Oval or egg shapes suit round-bottomed flasks. Cross-shaped bars move more liquid.
• Hotplate stirrers combine stirring with heating. They are useful for dissolving salts that dissolve slowly when cold, but the plate can get much hotter than the liquid. Use a probe to control the liquid temperature.
• Decoupling happens when the speed is raised too quickly: the bar loses grip on the magnet and rattles. Start slow and build up.
• Overhead stirrers, with a paddle on a motor shaft, are used for thick, viscous liquids that a stir bar can't turn.

Magnetic stirring is the usual way to make up buffers, because it mixes steadily while you add reagents and adjust the pH. See buffers and pH and pH meters.

A stir bar spinning against a solid can grind it, and a fast vortex can draw air into the liquid. Moderate speed is enough for most solutions.

Sonication

What is sonication?

Sonication is the use of ultrasound, sound at frequencies above human hearing, to agitate a liquid, either in an ultrasonic bath or with a probe dipped into the sample.

How it works: cavitation

The sound waves create rapid cycles of high and low pressure. In the low-pressure phase, tiny bubbles form. In the high-pressure phase they collapse violently. This is cavitation. Each collapse produces a brief hot spot of roughly 5,000 °C and 500 atmospheres in a tiny region, along with shock waves and shear (Suslick, Science 1990). The bulk liquid stays far cooler, because each hot spot is microscopic and lasts only microseconds. That is what breaks up clumps, knocks particles off surfaces and drives out dissolved gas.

Ultrasonic baths

An ultrasonic bath is a steel tank of water with transducers fixed underneath, running at a fixed ultrasonic frequency, usually some tens of kilohertz. The sample sits in its own sealed tube or flask, standing in the water.

• Breaking up particles. A few minutes in a bath helps larger particles dissolve (Bachem). See peptide solubility for choosing a solvent first.
• Degassing solvents. Sonication drives dissolved air out of liquids such as HPLC mobile phases, which helps prevent bubbles forming in the pump and detector. See HPLC and LC-MS solvents.
• Cleaning. Ultrasonic baths are widely used to clean glassware and small parts.

The energy is spread through the whole tank, so a bath is fairly gentle. But the water warms steadily during use, and can get noticeably warm over a long run. Check the temperature, add ice to the bath if needed, and use short runs.

Where the tube sits matters. The energy is uneven across a tank, and the liquid level in the tube should be near the water level in the bath. Moving the tube around finds the most active spot.

Probe sonicators

A probe sonicator puts a titanium horn directly into the sample, usually at a lower frequency than a bath. It delivers far more energy to a small volume than a bath.

Probe sonicators are used to break open cells, shear DNA and disperse tough suspensions. They are too harsh for most small-molecule and peptide solutions:

• Heat builds up quickly in a small volume. Samples are kept on ice and sonicated in short pulses.
• Radicals. Cavitation splits water into hydroxyl radicals and hydrogen atoms, which can form hydrogen peroxide or react with what is dissolved (Riesz et al., Environ. Health Perspect. 1985). Sensitive groups such as methionine and cysteine can be oxidised. See oxidation and reduction.
• Foaming and aerosols. The probe can whip air into the liquid and throw fine droplets into the air.
• Contamination. The probe tip wears with use and can shed tiny metal particles into the sample.

Mixing without damaging a sample

• Start gentle. Swirl or invert first, then vortex, then sonicate in a bath only if needed.
• Avoid foam. Foam means air is being worked into the liquid. With peptides and proteins, the air–water surface can encourage clumping (aggregation).
• Control temperature. Watch for warming in baths and on hotplates. Gentle warming helps some solids dissolve, but keep it modest. The solubility guide suggests staying below about 40 °C.
• Mix after every addition. Concentration gradients form quickly when a solution is topped up, especially in a volumetric flask. Invert the flask several times after making up to the mark. See pipettes and volumetric glassware.
• Check it has really dissolved. Hold the solution against a dark background. A haze or visible particles mean a suspension, not a solution, and its concentration is unknown.

Related guides

For choosing a solvent, see peptide solubility. For making up solutions to a set concentration, see molarity, mg/mL and dilutions and the dilution calculator.