Acoustic standing wave technology combined with microtechnology opens up new areas for the development of advanced particle and cell separating microfluidic systems. This tutorial review outlines the fundamental work performed on continuous flow acoustic standing wave separation of particles in macro scale systems. The transition to the microchip format is further surveyed, where both fabrication and design issues are discussed. The acoustic technology offers attractive features, such as reasonable throughput and ability to separate particles in a size domain of about tenths of micrometers to tens of micrometers. Examples of different particle separation modes enabled in microfluidic chips, utilizing standing wave technology, are described along a discussion of several potential applications in life science research and in the medical clinic. Chip integrated acoustic standing wave separation technology is still in its infancy and it can be anticipated that new laboratory standards very well may emerge from the current research.

A novel method, free flow acoustophoresis (FFA), capable of continuous separation of mixed particle suspensions into multiple outlet fractions is presented. Acoustic forces are utilized to separate particles based on their size and density. The method is shown to be suitable for both biological and nonbiological suspended particles. The microfluidic separation chips were fabricated using conventional microfabrication methods. Particle separation was accomplished by combining laminar flow with the axial acoustic primary radiation force in an ultrasonic standing wave field. Dissimilar suspended particles flowing through the 350-microm-wide channel were thereby laterally translated to different regions of the laminar flow profile, which was split into multiple outlets for continuous fraction collection. Using four outlets, a mixture of 2-, 5-, 8-, and 10-microm polystyrene particles was separated with between 62 and 94% of each particle size ending up in separate fractions. Using three outlets and three particle sizes (3, 7, and 10 microm) the corresponding results ranged between 76 and 96%. It was also proven possible to separate normally acoustically inseparable particle types by manipulating the density of the suspending medium with cesium chloride. The medium manipulation, in combination with FFA, was further used to enable the fractionation of red cells, platelets, and leukocytes. The results show that free flow acoustophoresis can be used to perform complex separation tasks, thereby offering an alternative to expensive and time-consuming methods currently in use.

A method to separate suspended particles from their medium in a continuous mode at microchip level is described. The method combines an ultrasonic standing wave field with the extreme laminar flow properties obtained in a silicon micro channel. The channel was 750 microm wide and 250 microm deep with vertical side walls defined by anisotropic wet etching. The suspension comprised "Orgasol 5 microm" polyamide spheres and distilled water. The channel was perfused by applying an under pressure (suction) to the outlets. The channel was ultrasonically actuated from the back side of the chip by a piezoceramic plate. When operating the acoustic separator at the fundamental resonance frequency the acoustic forces were not strong enough to focus the particles into a well defined single band in the centre of the channel. The frequency was therefore changed to about 2 MHz, the first harmonic with two pressure nodes in the standing wave, and consequently two lines of particles were formed which were collected via the side outlets. Two different microchip separator designs were investigated with exit channels branching off from the separation channel at angles of 90 degrees and 45 degrees respectively. The 45 degrees separator displayed the most optimal fluid dynamic properties and 90% of the particles were gathered in 2/3 of the original fluid volume.

Improved continuous acoustic particle separation (separation efficiency close to 100%) and separation of erythrocytes (red blood cells) from lipid microemboli in whole blood is reported.

A method to continuously separate different particle types in a suspension is reported. Acoustic forces in a standing wave field were utilized to discriminate lipid particles from erythrocytes in whole blood. The presented technology proposes a new method of cleaning, i.e. removing lipid emboli from, shed blood recovered during cardiac surgery. Blood contaminated with lipid particles enter a laminar flow micro channel. Erythrocytes and lipid particles suspended in blood plasma are exposed to a half wavelength standing wave field orthogonal to the direction of flow as they pass through the channel. Because of differences in compressibility and density the two particle types move in different directions, the erythrocytes towards the centre of the channel and the lipid particles towards the side walls. The end of the channel is split into three outlet channels conducting the erythrocytes to the centre outlet and the lipid particles to the side outlets due to the laminar flow profile. The separation channel was evaluated in vitro using polyamide spheres suspended in water, showing separation efficiencies approaching 100%. The system was also evaluated on whole blood using tritium labelled lipid particles added to bovine blood. More than 80% of the lipid particles could be removed while approximately 70% of the erythrocytes were collected in one third of the original fluid volume. The study showed that the further reduced micro channel dimensions provided improved performance with respect to; (i) separation efficiency, (ii) actuation voltage, and (iii) volumetric throughput as compared to earlier work.