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匀浆的转子或分散头的选择
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The field of homogenizing encompasses
a very broad area. The word homogenize means "to make
or render homogeneous" while homogeneous means "having
the same composition, structure, or character throughout".
Homogenizing is what is called an umbrella word - a
word which covers a very large area. When someone says
that they are homogenizing, they may mean that they
are actually doing one or more of the following, blending,
mixing, disrupting, emulsifying, dispersing, stirring
etc. Therefore during this writing when the word homogenizing
is used it may mean any one or more of the above mentioned
processes. |
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The current processes or methods of homogenizing can
be broken down into three (3) major categories, ultrasonic,
pressure, and mechanical. |
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ULTRASONIC HOMOGENIZING
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One widely used method to disrupt cells
is ultrasonic disruption. These devices work by generating
intense sonic pressure waves in a liquid media. The
pressure waves cause streaming in the liquid and, under
the right conditions, rapid formation of micro-bubbles
which grow and coalesce until they reach their
resonant size, vibrate violently, and eventually collapse.
This phenomenon is called cavitation. The implosion
of the vapor phase bubbles generates a shock wave with
sufficient energy to break covalent bonds. Shear from
the imploding cavitation bubbles as well as from eddying
induced by the vibrating sonic transducer disrupt cells.
There are several external variables which must be optimized
to achieve efficient cell disruption. These variables
are as follows: |
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Tip amplitude and intensity |
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Temperature |
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Cell concentration |
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Pressure |
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Vessel capacity and shape |
| Modem ultrasonic processors use piezoelectric
generators made of lead zirconate titanate crystals.
The vibrations are transmitted down a titanium metal
horn or probe tuned to make the processor unit resonate
at 15-25 kHz. The rated power of ultrasonic processors
vary from 10 to 375 Watts. low power output does not
necessarily mean that the cell disintegrator is less
powerful because lower power transducers are generally
matched to probes having smaller tips. It is the power
density at the tip that counts. Higher output power
is required to maintain the desired amplitude and intensity
under conditions of increased load such as high viscosity
or pressure. The larger the horn, the more power is
required to drive it and the larger the volume of sample
that can he processed. On the other hand, larger ultrasonic
disintegrators generate considerable heat during operation
and will necessitate aggressive external cooling of
the sample. Typical maximum tip amplitudes are 30-250
urn and resultant output intensities are in the range
of 200-2000 W/square cm. |
| The temperature of the sample suspension should be as
low as possible. In addition to addressing the usual
concerns about temperature lability of proteins, low
media temperatures promote high-intensity shock front
propagation. So ideally, the temperature of the ultrasonicated
fluid should be kept just above its freezing point.
The ultrasonic disintegrator generates considerable
heat during processing and this complicates matters.
Disruption can also be enhanced by increased hydrostatic
pressure (typically 15-60 psi) and increased viscosity,
providing the ultrasonic processor has sufficient power
to overcome the increased load demand and the associated
sample heating problems can be solved. For microorganisms
the addition of glass beads in the 0.05 to 0.5mm size
range enhances cell disruption by focusing energy released
by the bubble implosions and by physical crushing. Beads
are almost essential for disruption of spores and yeast.
A good ratio is one volume of beads to two volumes of
liquid. Tough tissues such as skin and muscle should
be macerated first in a blender or the like and confined
to a small vessel during ultrasonic treatment. The tip
should not be placed so shallow in the vessel as to
allow foaming. Antifoaming agents or other materials
which lower surface tension should be avoided. Finally,
one must keep in mind that free radicals are formed
in ultrasonic processes and that they are capable of
reading with biological material such as proteins, polysaccharides,
or nucleic acids. Damage by oxidatire free radicals
can be minimized by including scavengers like cysteine,
dithiothreitol, or other SH compounds in the media or
by saturating the sample with a protective atmosphere
of helium or hydrogen gas. |
| For practical reasons, the tip diameter of ultrasonic
horns cannot exceed about 3 inches. This sets a limit
on the scale-up of these devices. While standard sized
ultrasonic disrupters have been adapted to continuous
operation by placing the probe tip in a chamber through
which a stream of cells flow, cooling and free radical
release present problems. |
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PRESSURE HOMOGENIZING
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High-pressure homogenizers have been
used to disrupt microbial cells for many years. With
the exception of highly filamentous microorganisms,
the method has been found to be generally suitable for
a variety of bacteria, yeast and mycelia. |
| This type of homogenizer works by forcing
cell suspensions through a very narrow channel or orifice
under pressure. Subsequently, and depending on the type
of high-pressure homogenizer, they may or may not impinge
at high velocity on a hard-impact ring or against another
high-velocity stream of cells coming from the opposite
direction. Machines which include the impingement design
are more effective than those which do not. Disruption
of the cell wall occurs by a combination of the large
pressure drop, highly focused turbulent eddies, and
strong shearing forces. The rate of cell disruption
is proportional to approximately the third power of
the turbulent velocity of the product flowing through
the homogenizer channel, which in turn is directly proportional
to the applied pressure. Thus, the higher the pressure,
the higher the efficiency of disruption per pass through
the machine. The operating parameters which effect the
efficiency of high-pressure homogenizers are as follows: |
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Pressure |
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Temperature |
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Number of passes |
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Valve and impingement design |
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Flow rate |
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High-pressure homogenizers have long been the best
available means to mechanically disrupt nonfilamentous
microorganisms on a large scale. Animal tissue also
can be processed but the tissue must be pretreated with
a blade blender, rotor-stator homogenizer, or paddle
blender. The supremacy of high-pressure homogenizers
for disruption of microorganisms is now being challenged
by bead mill homogenizers. Still, in terms of throughput,
the largest industrial models of high-pressure homogenizers
outperform bead mills. The maximum volume of microbial
suspension per hour that can be treated by the larger
commercial machines is 4,500 liters for high-pressure
homogenizers versus about 1,200 liters for bead mills.
Even larger capacity high-pressure homogenizers are
available but their efficiency in disrupting microbial
cells has not been documented. This throughput advantage
is diminished somewhat by the fact that most high-pressure
homogenizers require several passes of the cell suspension
to achieve high levels of cell disruption whereas bead
mills frequently need only one.
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A familiar commercial high-pressure homogenizer for
the laboratory is the French press which uses a motor-driven
piston inside a steel cylinder to develop pressures
up to 40,000 psi. Pressurized sample suspensions up
to 35m1 are bled through a needle valve at a rate of
about 1 ml/min. Because the process generates heat,
the sample, piston and cylinder are usually pre-cooled.
Typical pressures used to disrupt yeast are 8,000 to
10,000 psi and several passes through the press may
be required for high efficiency of disruption. Generally,
the higher the pressure, the fewer the passes. Pressure
cells rated at 20,000 psi maximum come in capacities
of 3.7 and 35m1 and there is also a 35m1 capacity cell
rated at 40,000 psi.
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Most high-pressure homogenizers used for homogenization
were adapted from commercial equipment designed to produce
emulsions and homogenates in the food and pharmaceutical
industries. They combine high pressure with an impingement
valve. Those with a maximum pressure rating of 10,000
psi rupture about 40% of the cells on a single pass,
60% on the second pass, and 85% after four passes. Capacities
of continuous homogenizers vary from 55 to 4,500 liters/hr
at 10-17% w/v cell concentrations. With the larger capacity
machines several passes are needed to achieve high yields
of disruption. Considerable heat can be generated during
operation of these homogenizers and therefore a heat
exchanger attached to the outlet port is essential.
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MECHANICAL HOMOGENIZERS-ROTOR-STATOR HOMOGENIZERS
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Mechanical homogenizers can be broken
down into two (2) separate categories, rotor-stator
homogenizers and blade type homogenizers. |
| Rotor-stator homogenizers (also called
colloid mills or Willems homogenizers) generally outperform
cutting blade-type blenders and are well suited for
plant and animal tissue. Combined with glass beads,
the rotor-stator homogenizer has been successfully used
to disrupt microorganisms. However, the homogenized
sample is contaminated with minute glass and stainless
steel particles and the abrasive wear to the rotor-stator
homogenizer is unacceptably high. Cell disruption with
the rotor-stator homogenizer involves hydraulic and
mechanical shear as well as cavitation. Some people
in the homogenizing field also claim that there is to
a lesser extent high-energy sonic and ultrasonic pressure
gradients involved. |
| I personally do not believe in the theory
that high-energy sonic and ultrasonic pressure gradients
are involved with mechanical homogenizers. The only
thing that ultrasonic and mechanical (rotor-stator)
homogenizing have in common is that both methods generate
and use to some degree cavitation. Cavitation is defined
as the formation and collapse of low-pressure vapor
cavities in a flowing liquid. Cavitation is generated
as you move a solid object through a liquid at a high
rate of speed. In ultrasonics the object being moved
is the probe which is being vibrated at a very high
rate of speed generating cavitation. In mechanical homogenizing
(rotor-stator) the blade (rotor) is being moved through
the liquid at a high rate of speed generating cavitation. |
| The rotor-stator generator type homogenizer
was first developed to make dispersions and emulsions,
and most biological tissues are quickly and thoroughly
homogenized with this apparatus. Appropriately sized
cellular material is drawn up into the apparatus by
a rapidly rotating rotor (blade) positioned within a
static head or tube (stator) containing slots or holes.
There the material is centrifugally thrown outward in
a pump like fashion to exit through the slots or holes.
Because the rotor (blade) turns at a very high rpm,
the tissue is rapidly reduced in size by a combination
of extreme turbulence, cavitation and scissor like mechanical
shearing occurring within the narrow gap between the
rotor and the stator. Since most rotor-stator homogenizers
have an open configuration, the product is repeatedly
recirculated. The process is fast and depending on the
toughness of the tissue sample, desired results will
usually be obtained in 15-120 seconds. For the recovery
of intracellular organelles or receptor site complexes,
shorter times are used and the rotor speed is reduced.
The variables to be optimized for maximum efficiency
are as follows: |
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Design and size of rotor-stator (generator) |
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Rotor tip speed |
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Initial size of sample |
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Viscosity of medium |
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Time of processing or flow rate |
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Volume of medium and concentration of
sample |
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Shape of vessel and positioning of rotor-stator |
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PRO Scientific公司提供的精密匀浆器包含了多种速度的转子-定子分散头：
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| The size of the rotor-stator probe (also
called generator) can vary from the diameter of a pencil
for 0.01-10mI sample volumes to much larger units having
batch capacities up to 19,000 liters or, for on-line
units, capabilities of 68,000 liters/hr. Rotor speeds
vary from 3,000 rpm for large units to 8,000-60,000
rpm for the smaller units. In principle, the rotor speed
of the homogenizer should be doubled for each halving
of the rotor diameter. It is not the rpm's of the motor
but the tip velocity of the rotor that is the important
operating parameter. Other factors such as rotor-stator
design, which there are many, materials used in construction,
and ease of leaning are also important factors to consider
in selecting a rotor-stator homogenizer. |
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| Laboratory size rotor-stator homogenizers
process liquid samples in the 0.01 ml to 20 liter range.
The capacity of the rotor-stator should be matched to
the viscosity and volume of the medium and with the
type and amount of plant and animal tissue to be processed.
The speed and efficiency of homogenization is greatly
degraded by using too small a homogenizer, and the volume
range over which a given homogenizer rotor-stator size
will function efficiently is only about 10 fold. Also,
most of the laboratory-sized homogenizers function properly
only with liquid samples in the low to medium viscosity
range (<10,000 cps). This must be balanced against the
practical observation that concentrated samples, by
colliding more frequently, are broken up more rapidly.
Higher viscosity samples can be processed but require
specially shaped homogenization vessels or unique rotor-stator
configurations. The size of the sample prior to processing
with the homogenizer must be small enough to be drawn
inside the stator. Therefore, samples often must be
pre-chopped, cut or fragmented. |
| Foaming and aerosols can be a problem
with rotor-stator homogenizers. Keeping the tip of the
homogenizer well submerged within the media and the
use of properly sized vessels helps with the first problem.
Square-shaped or fluted vessels give better results
than round vessels and it is also beneficial to hold
the immersed tip off center. Aerosols can be minimized
by using covered vessels. Pro Scientific offers a complete
line of Safety Sealed chambers which eliminate the aerosoling
problem. The most widely used Safety Sealed chambers
are those of the ST series. The ST series uses four
different size standard laboratory test tubes and incases
them within a sealed cage. The four units currently
in the ST series are as follows: |
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ST-5 5mm Diameter Generator 12 x 75 Tube 5ml |
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ST-10 7mm Diameter Generator 17 x 100 Tube 16m1 |
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ST-15 7mm Diameter Generator 16 x 125 Tube 19m1 |
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ST-50 10mm Diameter Generator 50m1 Tube |
| There are no aerosols with in-line homogenizers.
Even though a number of the laboratory rotor-stator
homogenizers use sealed motors, none of them are truly
explosion-proof. Due caution should be followed when
using flammable organic solvents by conducting the homogenization
in a well ventilated hood. On the positive side, rotor-stator
homogenizers generate minimal heat during operation
and this can be easily dissipated by cooling the homogenization
vessel in ice water during processing. |
| The larger rotor-stator homogenizers
are either scaled up versions of the laboratory models
or in-line homogenizers. The latter contain teeth on
the edge of a horizontally oriented, multi-bladed, high-speed
impeller aligned in close tolerance to matching teeth
in a static liner. |
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MECHANICAL HOMOGENIZERS-BLADE TYPE HOMOGENIZERS
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Although less efficient than rotor-stator
homogenizers, blade homogenizers (also called blenders)
have been used for many years to produce fine brie and
extracts from plant and animal tissue. The cutting blades
on this class of homogenizer are either bottom or top
driven and rotate at speeds of 6,000 to 50,000 rpm.
Blenders are not suitable for disruption of microorganisms
unless glass beads or other abrasives are added to the
media and then one encounters the same problems as were
mentioned above for rotor-stator homogenizers. Many
plant tissue homogenizers undergo enzymatic browning
which is a biochemical oxidation process which can complicate
subsequent separation procedures. Enzymatic browning
is minimized by carrying out the extraction in the absence
of oxygen or in the presence of thiol compounds such
as mercaptoethanol. Sometimes addition of polyethylene
imine, metal chelators, or detergents such as Triton
X-100 or Tween-80 also helps. |
| Blade homogenizers are available for
a range of liquid sample sizes from 0.01 ml to multi
gallons. Some of the higher rpm homogenizers can reduce
tissue samples to a consistent particulate size with
distributions as small as 4um as determined by flow
cytometric analysis. Accessories for some blenders include
cooling jackets to control temperature and closed containers
to minimize aerosol formation and entrainment of air.
PRO Scientific has a complete line of Safety Sealed
chambers which eliminates the aerosoling problem as
well as the problem of introducing air into the sample. |
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