Multitude of Methods for Extraction From Non-Liquid Samples
Homogenization seeks to create samples in which analytes of interest are dispersed uniformly throughout. Numerous technologies can achieve this, including pressure, mechanical (rotorstator or blade-type), and bead-beating. “Depending on the application, one approach may be more appropriate than others,” notes Holly Yacko Archibald, sales director at PRO Scientific (Oxford, CT). PRO Scientific specializes in blade and rotor-stator models that comprise 95 percent of the homogenizers they sell.
Mechanical homogenization is based principally on mechanical shear and the disruption of structures that include tissues from plants, animals, or humans. This mechanical breakdown is assisted to some degree by cavitational forces. Regardless, the operational variables for mechanical homogenization are time, rotor speed, and rotor type.
Ultrasonic homogenizers rely exclusively on cavitation, a phenomenon that has drawn a lot of attention for chemical synthesis. Cavitation involves sound waves traveling through fluids (typically water) that create microscopic bubbles that expand and then collapse violently, attacking and disrupting nearby cells and structures.
Laboratory workers tend to use equipment they are either familiar with or have read about in the literature. This has led to numerous fallacies about mechanical homogenization.
Mechanical homogenizers have the reputation for breaking samples down to nanometer-sized particles, and this is certainly true for long, high-speed runs. But as Ms. Archibald notes, breakdown into almost any particle size domain is possible. “It’s possible to gently mix samples by running for a few seconds at low speed, which will provide a broader particle size bandwidth.”
Another misconception involves the alleged inability to process very small sample sizes, an important consideration as samples become more valuable. Mechanical probes have no problem processing samples even in 0.5 mL tubes, assures Ms. Archibald.
Since mechanical homogenizers resemble kitchen blenders in many ways, some cost-conscious lab managers assume that a $12 appliance will perform as well as a homogenizer. While that may be true for some easily processed samples for which precision and reproducibility are nonissues, dedicated homogenizers hold several trumps over simple blenders.
Mechanical homogenizers use precision- crafted stainless steel rotors and probes that are easy to clean, chemically resistant, and autoclavable. “And their results are reproducible,” Ms. Archibald adds.
PRO Scientific has recently introduced the DSP-20, which incorporates both ultrasonic and mechanical homogenization modes and full automation homogenization for up to 20 samples.
The main advantage of dual-mode homogenization is the ability to reach submicron particle sizes rapidly and reproducibly while mitigating somewhat the drawbacks of both modes. Mechanical and ultrasonic homogenization occur sequentially: First, mechanical disruption handles the larger-sized particles, then the ultrasonic probe turns on to complete the process. Alternatively, users may use either mode alone.
Since ultrasound tends to heat samples through cavitational effects, the technique is not always suitable for heat-sensitive samples. Dual-mode homogenization minimizes sample contact with ultrasound to a minimum, thereby sparing labile analytes from long exposure to ultrasound.
So far, dual-mode homogenization has received attention from a wide range of end users, according to Ms. Archibald, including those involved with food testing, wastewater treatment, and plant pathology.
The smaller the sample, the more critical the homogenizer
Smaller sample volumes and high throughput are recurring trends in homogenization, not only in biology but also in food, materials, and environmental testing. “Until fairly recently, sample sizes ranged from about fifteen milliliters up to a few hundred milliliters, and samples were processed in glass,” notes Reed Barrickman, marketing communications manager at Omni International, Inc. (Kennesaw, GA). The widespread adoption of 2 mL plastic microtubes now enables homogenization and analysis of hundreds of samples per day. Homogenization has become so efficient, in fact, that downstream analysis has become a workflow bottleneck, which according to Mr. Barrickman presents serious workflow and throughput challenges to analytical laboratories.
Omni specializes in multi-sample systems that even in relatively low-volume formats (e.g., the six-sample Omni- Prep rotor-stator homogenizer and the 24-sample Omni Bead Ruptor 24 bead mill) provide a decent level of automation for medium-throughput labs. Omni’s LH-96 model, a high-throughput fully automated system, can rightly be termed the Cadillac of homogenizers. This model handles up to 192 samples in full walk-away mode. With the LH- 96, Omni has introduced a “smart” and familiar Microsoft Windows touchscreen- based interface and an option to interface directly with microtiter plates.
One of the more interesting recent developments in homogenization has been the Ultra Turrax Tube Drive (UTTD) from IKA Works (Wilmington, NC). The system consists of single- use, hermetically sealed tubes, each fitted with a reusable, sterilizable grinding element (a stirrer, glass or stainless steel balls, or a rotor/stator assembly). Special tube designs accommodate homogenization of drugs, plant or animal tissues, and sterile samples, with an option for piercable covers for easy sample withdrawal. Tubes are available in two capacities: 2-15 mL and 15-50 mL.
“The UTTD’s disposable tubes eliminate sample vial cleaning and practically reduce cross-contamination to zero,” says Tracy Christian, marketing coordinator at IKA.
The UTTD satisfies two goals of modern homogenization equipment: reproducibility and compressed workflows.
What to look for
“Users should consider what they are trying to homogenize, and how small they need their samples to be,” she tells Lab Manager Magazine. Achieving submicron particle sizes involves either ultrasonic disruption or bead milling. The former is faster but is noisy, tends to heat the sample, and in some instances can alter its chemical characteristics.
According to Ms. Christian, other desirables include:
- easy cleaning of product-contact surfaces
- a low motor noise, since homogenizers are usually located on workbenches in close proximity to operators
- ease of use
- rapid homogenization
- user control over homogenization parameters through a familiar digital display
- low heat generation, which is particularly important for labile tissue samples
- a programmable library of methods