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AIR LIFT
Surface Filtration Depth Filtration
particles are retained mostly on the surface of the a thicker media or multiple layers of media,
media, forming a layer of material that increases forming a torturous path to retain particles.
the efficiency or fineness of particles retained.
60% to 70% efficient at retaining the targeted This type of engineered media ideally retains larger
particle size and as the "cake layer" develops particles at the surface and progressively finer
eventually becoming nearly 100% efficient. particles through the thickness or layers
Screen material (nylon monofilament, at 95 to 99% efficiencies and therefore not reliant
monofilament metal) on a filter cake for their efficiency.
The advantage is that the monofilament structure the high temperature surface heat treatment, that is,
can be repeatedly washed, and the cost is relatively the instantaneous sintering technique can
low. effectively prevent the fiber from being affected by
filtration Flush high-speed fluid with the loss; felt
material is a disposable type (one-time use),
The disadvantage is that the surface filtration mode Felt material (acupuncture cloth, melt blown
can easily cause the surface of the filter bag to be nonwovens) is a common deep three-dimensional
clogged. filter material, which is characterized by loose
fibrous tissue, high porosity increases the amount
of impurity content, t
This type of product is most suitable for the coarse filtration accuracy 1-200μm
filtration with lower precision with the filtration
precision of 25-1200μm
Scientific & Technical Report WER 5300
Principles of Filtration
Scientific & Technical Report
Principles of Filtration
Introduction
Filters play an important role in any industrial dot that can be seen by the unaided eye is approx-
society. Filtration is the separation of particles imately 40 micrometers in diameter. For those
from a fluid (liquid or gas) by passage of that fluid who think in terms of the metric system, a microm-
through a permeable medium. 1 When the parti- eter is 1 / 1000 of a millimeter which is approxi-
cles represent a significant proportion of the fluid, mately one twenty-fifth of an inch. To translate
the process may be described as bulk solids col- directly into inches,
lection. When the particles represent only a very one micrometer = inch = 0.000039 inches.
small proportion of the total (0.01% or less), the 25,400
process is called fluid clarification.
“Micron”is the commonly used shortened form for
In most cases, Pall filters are used to remove par- micrometer, and its symbol is µm.
ticles ranging in size from fractions of a microm-
eter to 40 plus micrometers. The smallest pencil
See References a t end of pa per.
Filtration Processes
Suspended solids are separated from fluids via through a filter media, the liquid stream will take
three mechanisms: inertial impaction, diffusion- the path of least resistance to flow and will be
al interc eption and direc t interc eption. While diverted around the fiber. The particles, because
these mechanisms of filtration are operable, the rel- of their momentum, tend to travel in a straight
ative importance and role of each changes. Both line and, as a result, those particles located at or
inertial impaction and diffusional interception are near the c enter of the flow line will strike or
much less effective with liquids than with gases. impact upon the fiber and be removed. Figure 1
Since the density of a particle will typically be illustrates this process. The fluid stream, shown
closer to that of a liquid than to that of a gas, devi- as solid lines, flows around the filter fibers while
ation of a suspended particle from the liquid flow the particles continue along their path, shown as
line is much less, and thus impa ction on the struc-
ture of the medium is less likely. Moreover, Figure 1 Inertial Impaction
impac tion in many systems is not followed by
adhesion of the particles to the surface of the fil-
ter medium. Diffusional interception in liquids
oc c urs to only a very limited ex tent bec ause
Brownian Motion is not nearly as pronounced in
liquid suspensions as in gaseous suspensions.
1. Inertial Impaction
Particles in a fluid stream have mass and velocity
and, hence, have a momentum associated with
them. As the liquid and entrained particles pass
1
dashed lines, and strike the fibers. Generally, larg- role in liquid filtration. This results because of
er particles will more readily deviate from the flow the inherent nature of liquid flow which tends to
lines than will small ones. In practice, however, reduce the lateral movement or excursions of the
because the differential densities of the particles particle away from the fluid flow lines.
and fluids are very small, deviation from the liquid
flow line is much less and, hence, inertial impaction 3. Direct Interception
in liquid filtration plays a relatively small role. While inertial impac tion and diffusional inter-
ception are not as effective in liquid service as in
Figure 2
Diffusional gas service, direct interception is equally as effec-
Interception
tive in both and is the desired mechanism for sep-
arating particles from liquids. In a filter medium,
one observes not a single fiber, but rather an assem-
bly of a large number of such fibers. These fibers
define openings through which the fluid passes.
If the particles in the fluid are larger than the
pores or openings in the filter medium, they will
be removed as a result of direct interception by
the holes. Figure 3 portrays this removal mecha-
nism. Direct interception is easily understood in
Figure 3
Direct the case of a woven wire mesh filter with uni-
Interception form pores and no thickness or depth; once a par-
tic le passes through an opening, it proc eeds
unhindered downstream. Yet such a filter will
collect a very significant proportion of particles
whose diameter is smaller than the openings or
pores of the medium. Several factors that help
account for this collection are outlined below and
illustrated in Figure 4.
• In the real world most suspended particles,
even if quite small when viewed from some
2. Diffusional Interception directions, are irregular in shape and, hence,
For particles that are extremely small (i.e., those can “bridge” an opening.
with very little mass), separation can result from • A bridging effect can also occur if two or more
diffusional interception. In this process, particles particles strike an opening simultaneously.
are in collision with the liquid molecules. These • Once a particle has been stopped by a pore,
frequent collisions cause the suspended particles that pore is at least partially occluded and,
to move in a random fashion around the fluid subsequently, will be able to separate even
f lo w line s. Suc h mo ve me nt, whic h c an b e smaller particles from the liquid stream.
observed mic rosc opic ally, is c alled “Brownian • Specific surface interactions can cause a small
Motion.” Brownian Motion causes these smaller particle to adhere to the surface of the inter-
particles to deviate from the fluid flow lines and, nal pores of the medium. For example, a par-
hence, increase the likelihood of their striking the ticle considerably smaller than a pore is likely
fiber surface and being removed. Figure 2 shows to adhere to that pore provided the two sur-
the partic le flow c harac terized by Brownian faces are oppositely charged. Moreover, it is
Motion and impacting the filter fibers. As with iner- sometimes not necessary that they be oppo-
tial impaction, diffusional interception has a minor sitely charged. A very strong negatively charged
2
filter surface can cause a positive charge to be increased many fold, some of these smaller parti-
induced on a less strongly charged negative cles will probably be released downstream. To
particle. There c an also be other types of test for such release, “impulse” (rapidly varying)
interac tions suc h as “hydrogen bonding.” flow conditions can be deliberately set up, and par-
ticles so released collected downstream by a finer
Figure 4 filter for counting and inspection.
Removal
Mechanisms for
Particles Smaller Even if the buildup in pressure is gradual and not
Than the Pores of
the Medium that great, release of collected particles is likely to
occur if the structure of the medium is such that
the pore dimensions c an enlarge. Those c om-
mercially available filters prone to this type of
malfunction are the so-called “wound”and similar
fibrous types in which numerous poorly supported
fibers are present which move as pressure builds.
Direct interception can also obviously occur in fil- To summarize, every type of filter will collect par-
ters in which the pore openings are not uniform ticles finer than its absolute rating, and under
but instead vary in size (but within carefully con- extreme impulse conditions such finer particles
trolled limits) throughout the thickness of the fil- may release. Filters in which the structural por-
ter medium, resulting in a tortuous flow path. tions of the medium are free to move in response
Filters of this type include membranes, 2 Pall’s to increased pressure are particularly prone to
HDC® II medium, Pall’s Ultipor® glass fiber filters, this occurrence, and they may even release down-
and cellulosic paper filters. stream particles larger than their pore size.
Membrane filters like screen-type filters collect In a well designed filter, particles larger than the
particles smaller than their absolute removal rat- pore size do not pass downstream of the medium.
ing by the four means previously mentioned. The most successful approach to zero particle
However, the fact that the medium contains pores release is achieved by using a filter medium in
smaller than the absolute removal rating size fur- which the pores will not enlarge under pressure
ther enhances the possibility of particles smaller and in which the thickness is sufficient so that in
than this rating being removed from the fluid normal service substantially all the incident par-
stream by direct interception. ticles are collected in the first tenth to fifth of the
thickness, leaving the rest available to stop under-
4. Particle Release Under “Impulse” size particles which may be released from the
Conditions upper layer on impulse.
We have established that there are numerous meth-
ods by which a filter can collect particles smaller 5. Aids to Liquid Filtration
than its absolute removal rating. Under certain con- It is possible to supplement the three principal
ditions, a portion of these finer particles can be mechanisms of filtration, and to enhance a filter’s
released and pass downstream. For example, if a effectiveness in removing particles from a liquid.
wire mesh sc reen filter has c ollec ted particles Several methods will be discussed briefly.
smaller than its pore openings at a low, steady
rate of liquid flow, and the flow rate is then
3
A. Electrostatic Deposition B. Flocculation
Most particles carry a negative charge. The fibers Very fine size particles are difficult to filter. One
of the medium often carry charges that can affect way to enhance filterability is to cause them to
both particle removal efficiency and/or particle “clump”together to form larger particles that are
retention efficiency of the filter. It is possible, easier to filter and usually produce filter cakes
therefore, to enhance the particle capture mech- which result in less pressure drop and, in conse-
anisms of a filter by inducing a desired charge quence, an increase in throughput. Flocculation
(usually positive) on the fiber.3 Figure 5 illustrates of particles by the addition of polyelectrolytes
a typical particle-fiber interaction based upon dif- (long chain molecules with many positive and
ferences in zeta potential. The particle has a neg- negatively charged ionic points along the chain
ative surface charge associated with it resulting length) to the fluid system is a common practice.
from a double ionic layer formed on the particle Polyelectrolytes (e.g., soluble starches, gelatin, and
surface. The clear circles are positive ions while derivatives of polyacrylates) attach themselves to
the dark circles are negative ions. The filter media many oppositely charged particles in the liquid
has an induced positive zeta potential which can causing their agglomeration and increasing their
then promote particle adherence. settling rate. They should be selected empirical-
ly for each fluid process. Figure 6 summarizes
Figure 5 the basic flocculation process.
Zeta Potential Filter Media
Particle-Fiber Surface
Interaction (Positive Zeta) In everyday practice, the polyelectrolyte is dis-
solved and a very small quantity is added to a much
larger volume of the suspension of solids to be
flocculated. Agitation must be optimum for the sys-
tem – sufficient to disperse the polyelectrolyte but
Ionic Particle
Double (Negative Zeta) not too strong as to rupture the flocs. Continued
Layer
agitation can decrease the flocculation and increase
the amount of “haze” present. Air pressure, or a
non-shearing pump is preferred for moving the
flocculated material to the filter so as not to cause
The intensity of charge of both the particle and deflocculation. Recirculation of the suspension
the fiber is important. In general, as the charge should also be avoided for the same reason.
intensity inc reases and particle size dec reases,
capture efficiency will increase. The obvious effect Whether or not a polyelectrolyte can be used in
of a charged filter surface is the ability to remove a filter process depends on its performance, cost,
very fine sized particles by a medium having rel- and adulterating effect.
atively large pores, very often at low pressure
Figure 6 Flocculation Process
drops and high dirt holding capacity.
The Use of Polyelectrolytes, Large Molecules with
Multiple Ionic Sites of Different Charge Than the
One must use great caution when seeking to aid Zeta Potential of the Particle, Which Actually Binds
Particles Together to Form Aggregates.
the filtration of liquids through electrostatic depo-
TYPE STRUCTURE
sition, however. A charge is often unstable and [ -CH- ]n
CATIONIC
influenced by pH of the contacting fluid, the pas- N(R)3
sage of ionized gases, radiation, time, humidity, and [ -CH- ]n
ANIONIC C
the deposition of charged particles. Consequently,
predictability of particle capture efficiency is poor O O
Both Types of Charge
and should be determined empirically. NONIONIC With Overall Neutrality
4
C. Filter Aids this property which enables them to produce fil-
The ease with which fine size particles can be ter cakes of high permeability. Other filter aids
removed from a liquid stream can also be increased include perlite (an igneous rock formed by the
by the addition to the suspension of small amounts quenching of molten volcanic lava in water), car-
of filter aids. This is known as a “body feed” or bon, and cellulose.
“body aid,”and should not be confused with pre-
coat filtration where filter aid is first deposited Filter aid filtration is not common in fluid clarifi-
on a filter and the suspension then caused to flow cation but, when used, is often found upstream of
through. As in the case with flocculation, the pur- cartridge filtration. Cartridge filters are used as
pose of the filter aid is to achieve the desired cake “trap”filters downstream of precoat filters to cap-
permeability. ture any filter aid which may “bleed” past the fil-
ter medium.
Perhaps the most c ommonly used filter aid is
diatomaceous earth, which consists of the sedi-
mentary deposits of fossilized diatoms. The diatom
skeletons have a wide variety of shapes, and it is
Filter Types
In recent years it has become increasingly com- conditions these finer particles are more likely to
mon to classify filters and filter media as either be released by a filter whose pores can enlarge.
“depth type”or “surface type.” Unfortunately, filter
manufacturers have been unable to agree upon an This is always a problem with non-fixed random
“official”definition of the terms. As a result, much pore depth type media. Such media contain many
misunderstanding is encountered in the field on this tortuous passages, and there are many paths through
subject. The purpose of this section is to separate which fluid can flow. Naturally, the small passages
fact from fiction respecting this matter. become blocked first, resulting in more and more
flow being taken by the large passages. Since the
1. Non-Fixed Random Pore Depth Type structure of the medium is not an integral one,
Media increased pressure on the large passages can cause
Non-fixed random pore depth type media depend the medium to separate, thereby widening the
principally on the filtration mechanisms of inertial passages. It should be obvious that channeling of
impaction and/or diffusional interception to trap par- this nature adversely affects the performance of
ticles within the interstices or spaces of their inter- the filter.
nal structure. Examples of this type of media are
felts, woven yarns, asbestos pads, and packed fiber- Non-fixed random pore depth type filters depend
glass. Such filters are constructed of non-fixed not only on trapping but also on adsorption to
media of a thickness sufficient to trap particles in retain particles. As long as the dislodging force
a given size range on a finite statistical basis. exerted by the fluid is less than the force retaining
the particle, the particle will remain attached to
As noted earlier, release of collected particles is the medium. However, when such a filter has been
likely to occur if the structure of the medium is such onstream for a length of time and has collected a
that pore dimensions can enlarge as the pressure c ertain amount of partic ulate matter, a sudden
drop increases. Also, every type of filter will collect increase in flow and/or pressure can overcome
particles finer than its pore size but under impulse these retentive forces and cause the release down-
5
stream of some of the particles. This unloading Naturally, a surface or screen filter will stop all par-
will frequently occur after the filter has been in ticles larger than the largest pore (provided, of
use for some time and can give the false impression course, the structure of the medium is an integral
of long service life for the filter. one). While particles smaller than the largest pore
may be stopped because of factors previously dis-
Furthermore, most non-fixed random pore depth cussed (bridging, etc.), there is no guarantee that
type filters are subject to media migration. This such particles will not pass downstream. Woven
means that parts of the filter medium bec ome wire mesh filters are currently available with open-
detached and continue to pass downstream cont- ings down to 8 micrometers.
aminating the effluent (fluid that has passed through
the filter). Media migration is also sometimes incor- 4. Summary of Filter Types
rectly taken to include release of “built-in” conta- Upon reviewing the above information, it should
mination – for example, dust and fibers picked up become apparent that classifying filters as depth or
by the filter during its manufacture. surface is meaningless. Nearly all filters exhibit
“depth”when viewed under a microscope.
2. Fixed Random Pore Depth Type Media
Fixed random pore depth type filters consist of A more meaningful classification of filters is as
either layers of medium or a single layer of medi- follows:
um having depth, depend heavily on the mechanism • Non-fixed random pore depth type with pores
of direct interception to do their job, and are so con- whose dimensions increase at high pressure
structed that the structural portions of the medium (“wound,”low density felted).
cannot distort and that the flow path through the • Fixed random pore depth type with pores which
medium is tortuous. It is true that such filters retain do not increase in size at high pressures (most
some particles by adsorption as a result of inertial membrane filters, including Pall’s Nylon 66 and
impaction and diffusional interception. It is also true Ultipor® membranes, Pall HDC® II polypropy-
that they contain pores larger than their removal lene, Ultipor glass fiber, and Epocel® epoxy
rating. However, pore size is controlled in manu- impregnated cellulose filters).
facture so that quantitative removal of particles • Screen Type (woven cloth or screens).
larger than a given size can be assured. Moreover,
by providing a medium with sufficient thickness, The fixed random pore depth type filter is superi-
release of particles collected that are smaller than or for most purposes when compared with the
the removal rating can be minimized, even under screen type. It combines high dirt capacity per
impulse conditions. unit area with both absolute removal of particles
larger than a given size and minimum release of col-
3. Surface Type Media lected particles smaller than this rated size under
In the strict definition of the term, a surface or impulse conditions.
screen filter is one in which all pores rest on a sin-
gle plane, which therefore depends largely upon Non-fixed random pore depth type filters have no
direct interception to separate particles from a absolute ratings, are subject to media migration,
fluid. Only a few filters on the market today (for and unload partic les very badly on impulse.
example; woven wire mesh, woven cloth, and a Comparison of dirt capacity between fixed pore and
me mb rane filte r manufac ture d b y Whatman non-fixed pore depth types is meaningless because
Nuclepore) qualify as surface filters. the nominal removal ratings of non-fixed pore depth
type filters bear little relationship to their behavior
in service.
6
If it matters little whether a filter is surface type or probably unwise to use the filter. If not, then look
depth type, how should one judge filters? Only to the second question: Can the manufacturer guar-
two questions should concern the filter user. Is the antee that the filter will reliably remove all particles
filter subject to unloading, channeling, or media that should be removed from the fluid in question,
migration? If so, then for most applications it’s and is the filter safe to use?
Removal Ratings
Various rating systems have been evolved to uncommon for a filter with a Nominal Rating
desc ribe the filtration c apabilities of filter ele- of 10 µm to pass particles downstream rang-
ments. Unfortunately, there is at the moment no ing in size from 3 0 to over 1 0 0 mic rons.
genera lly a ccepted ra ting system and this tends C. Test data are often not reproducible, particu-
to confuse the filter user. Several of the systems larly among different testing laboratories.
now in use are described below. D. So me manufac ture rs do no t b ase th e ir
No minal Rating o n 9 8 % c o ntaminatio n
1. Nominal Rating removal by weight but instead on a contam-
Many filter manufacturers rely on a Nominal Filter ination removal effic ienc y of 95%, 90%, or
Rating whic h has been defined thusly by the even lower. Thus, it often happens that a Pall
National Fluid Power Association (NFPA): “An arbi- filter with an Absolute Rating of 10 µm is actu-
trary micron value assigned by the filter manually finer than a c ompetitive filter with a
facturer, based upon removal of some percentage Nominal Rating of 5 µm. Always check the cri-
of all particles of a given size or larger. It is rarely teria upon which a c ompetitor’s Nominal
well defined and not reproducible.” In practice, Rating is based.
a “contaminant”4 is introduced upstream of the fil- E. The very high upstream c ontaminant c on-
ter element and subsequently the effluent flow centrations used for such tests are not typical
(flow downstream of the filter) is analyzed micro- of normal system c onditions and produc e
scopically. A given Nominal Rating of a filter means misleading high efficiency values. It is com-
that 98% by weight of the contaminant above the mon for a wire mesh filter medium with a
specified size has been removed; 2% by weight of mean (average) pore size of 17 µm to pass a
the contaminant has passed downstream. Note 10 µm nominal spec ific ation. However, at
that this is a gravimetric test rather than a parti- normal system contamination concentrations,
cle count test. Counting particles upstream and this same filter medium will pass a lm ost a ll
downstream is a more meaningful way to measure 10 µm size particles.
filter effectiveness.
One cannot, therefore, assume that a filter with a
The various tests used to give non-fixed random Nominal Rating of 10 µm will retain all or most par-
pore depth type filters a Nominal Rating yield ticles 10 µm or larger. Yet some filter manufacturers
results that are nebulous if not misleading. Typical c ontinue to use o nly a Nominal Rating both
problems are as follows: because it makes their filters seem finer than they
A. The 98% contaminant removal by weight is actually are, and because it is impossible to place
determined by using a specific contaminant an Absolute Rating on a non-fixed random pore
at a given concentration and flow. If any one depth type filter. Figure 7 compares a 1 µm nom-
of the test conditions are changed, the test inal rated wound type cartridge to a Pall 30 µm
results could be altered significantly. absolute rated cartridge. The graph shows that the
B. The 2% of the contaminant passing through effluent stream from the 1 µm nominal cartridge
the filter is not defined by the test. It is not contains particles up to 50 µm in diameter while
7
the effluent stream from the 30 µm absolute rated minant” and before running any test an experi-
cartridge contains no particles larger than 30 µm. enced laboratory will have determined the amount
of such contamination in its test setup. A test will
be invalidated if the background contamination
Figure 7
Comparison of count is above established limits.
Filter Cartridge 56
Number of Particles in Effluent

Removal Ratings
1 µm Wound-Type
48 There are several recognized tests for establishing
Filter Cartridge
the Absolute Rating of a filter. What test is used
40
will depend on the manufacturer, on the type of
32 medium to be tested, or sometimes on the pro-
24 cessing industry. In all cases the filters have been
Pall 30 µm Absolute rated by a “challenge”system. A filter is challenged
16 Rated Filter by pumping through it a suspension of a readily
Cartridge
8 rec ognized c ontaminant (e.g., glass beads or a
bacterial suspension), and both the influent and
10 20 30 40 50 effluent examined for the presence of the test
Particle Size (µm) contaminant.
2. Absolute Rating Such challenge tests are destructive tests – i.e., the
The NFPA defines Absolute Rating as follows: “The challenged filter cannot be used thereafter in actu-
diameter of the largest hard spherical particle that al service. Consequently, integrity tests for for fil-
will pass through a filter under specified test con- t e r s h ave b e e n e st ab lish e d w h ic h are
ditions. It is an indication of the largest opening non-destructive and correlate with the destruc-
in the filter element.” Such a rating can be assigned tive qualification challenge test. In other words,
only to an integrally bonded medium (such as if the test filter was successfully integrity tested
Pall’s fixed pore depth media or sintered metal by the non-destructive test, that would mean it
media). wo uld pass the de struc tive c halle nge te st.
However, after passing the integrity test, the filter
The original te st and te rm for the Ab solute element can be placed in service and will pro-
Filtration Rating was proposed by Dr. Pall, founder vide the user with the results claimed by the fil-
of Pall Corporation, in the mid-1950s. It was con- ter manufacturer.
sidered by the Filter Panel of SAE Committee A-6
and with minor changes subsequently adopted. Figure 8 Schematic Representation of Glass Bead
Challenge Test Apparatus
One point that confuses users of absolute rated fil-

ters is that when measuring downstream conta-
Pressurized Container
mination, contaminants larger than the pore size for Contamination Suspension
indicated by the Absolute Rating are invariably

found. At first glance, this would seem to cast Test Filter
doubt on the very concept of an “absolute”rating.

However, one must realize that it is impossible to
take effluent samples, transfer them, run the test, Collection Vessel
or wash out a newly manufactured filter without

adding a quantity of contaminants. Even a new fil- Analysis Membrane for
Microscopic Examination
ter is contaminated when it is removed from its
packing! All of this is called “background conta-
8
3. Glass Bead Challenge Test the influent stream and effluent stream, a profile
One method of establishing the Absolute Rating of removal efficiency emerges for any given filter.
of a filter is the Glass Bead Challenge Test as depict- Figure 9 shows a single pass Beta test apparatus.
ed in Figure 8. A suspension of glass beads (all lying
within a specific size range) is passed through a fil- The Beta value is defined as:
ter and collected downstream on an analysis mem- β = Number of particles of a given size
brane. Glass beads are used because their spherical and larger in the influent
shape makes them easy to differentiate from back- Number of particles of a given size
ground contamination that may be generated dur- and larger in the effluent
ing the test. Those glass beads which pass the
filter are then examined under a microscope to Percent removal efficiency at a given particle size
determine the largest spherical glass bead passed. can be obtained directly from the β value and can
This will establish the filter’s Absolute Rating. be calculated as follows:
% Removal Efficiency = β -1 x 100
Figure 9
Schematic β
Representation of Agitated
Contaminant On Line Multichannel The relationship between β values and percent
Single Pass Beta Suspension Automatic Particle Counters
(β) Test Apparatus
removal efficiency is illustrated below.
Upstream Downstream
Metering
Pump
P1 P2 β % Removal
Constant Test
Flow Pump 0.5 lpm Filter 1 0
0.5 lpm
9.5 lpm 10 lpm 2 50
Clean-Up 10 90
Filter
9.5 lpm 100 99
Reservoir 1000 99.9
5000 99.98
10000 99.99
4. Beta (β) Rating System
100000 99.999
While absolute ratings are clearly more useful
than nominal ratings, a more recent system for
Usually a β = 5,000 can be used as an operational
expressing filtration rating is the assignment of
definition of an absolute rating.
Beta ratio values. Beta ratios are determined using
the Oklahoma State University, “OSU F-2 Filter
The β values allow the comparison of removal
Performance Test.” The test, originally developed
efficiencies at different particle sizes for different
for use on hydraulic and lubricating oil filters, has
cartridges in a meaningful manner. Figure 10 illus-
been adapted by Pall Corporation for rapid semi-
trates typical Beta curves for three different car-
automated testing of filters for service with aque-
tridges. Beta curves are part of all Pall published
ous liquids, oils, or other fluids.
literature.
The Beta rating system is simple in concept and Figure 10 Typical Beta (β) Curves
can be used to measure and predict the perfor- 104

104
mance of a wide variety of filter cartridges under
103
specified test conditions.
Filtration Ratio (βx)
103
102
102
The basis of the rating system is the concept of
10
titer reduction. If you measure the tota l pa rticle 10
counts a t severa l different pa rticle sizes, in both
0 2 4 6 8 10 0 10 20 30 40 50 60 70
Particle Size (µm)
9
Choosing the Proper Filter
Among the more important factors that must be a measure of the thickness or flowability of a
taken into consideration when choosing a filter for fluid. Water, ether, and alcohol have low viscosi-
a particular application are the size, shape, and ties; heavy oils and syrup have high viscosities.
hardness of the particles to be removed, the quan- Viscosity affects resistance directly. If all other
tity of those particles, the nature and volume of conditions remain constant, doubling the viscos-
fluid to be filtered, the rate at which the fluid ity in a filter system gives twice the original resis-
flows, whether flow is steady, variable and/or inter- tance to flow. Consequently, as viscosity increases,
mittent, system pressure and whether that pressure the pressure required to maintain the same flow
is steady or variable, available differential pres- rate increases. Centipoise is the unit of measure-
sure, compatibility of the medium with the fluid, ment comparing the viscosity of a fluid with that
fluid temperature, properties of the fluid, space of water which has a viscosity of 1 centipoise at
available for particle collection, and the degree 70°F/21°C.
of filtration required. Let us now examine how
some of these factors affect filter selection. 3. Temperature
The temperature at which filtration will occur
1. Nature of Fluid can affect both the viscosity of the fluid, the cor-
The materials from which the medium, the car- rosion rate of the housing, and filter medium com-
tridge hardware, and the housing are constructed patibility. Viscous fluids generally become less
must be compatible with the fluid being filtered. viscous as temperature increases. If a fluid is too
Fluids can corrode the metal core of a filter car- viscous, it may be advisable to preheat the fluid
tridge or a pressure vessel, and the corrosion will and to install heater bands in the filter. Thus, it is
in turn contaminate the fluid being filtered. Thus important to determine the viscosity of a fluid at
it is essential to determine whether a fluid is acid, the temperature at which filtration will oc c ur.
alkali, aqueous, oil or solvent based, etc.
High temperature also tends to accelerate corro-
2. Flow Rate sion and to weaken the gaskets and seals of filter
Flow rate (the units of measure of flow rate are housings. Very often disposable filter media cannot
given as volume per unit time – e.g., ml/min., or withstand high temperatures, particularly for pro-
liters/hr., or gallons/min.) is dependent on two longed periods of time. It is for this reason that one
general parameters, pressure [P] and resistance must often choose porous metal cleanable filters.
[R]. Flow rate depends directly on pressure and
inversely on resistance. Thus, for a constant [R], 4. Pressure Drop
the greater the pressure, the greater the flow. For Everything a fluid passes through or by contributes
a constant [P], the lower the resistance, the greater resistanc e to the flow of that fluid in an additive
the flow. fashion. The pressure losses due to flow of the
fluid through the tubing, piping, etc ., c ouples
Pressure can come from any number of sources with the pressure loss through the filter to pro-
and is usually expressed as pounds per square duc e resistanc e.
inch (psi). All other factors being equal, if the
pressure on a fluid is increased, then the flow Resistance to flow through a clean filter will be
rate of that fluid will increase. caused by the filter housing, cartridge hardware,
and the filter medium. For a fluid of given viscosity,
Viscosity is the resistance of a fluid to the motion the smaller the diameter of the pores or passages
of its molecules among themselves; in other words, in the medium, the greater the resistance to flow.
10
When a fluid meets resistance in the form of a fil- When this occurs, the proper solution is usually
ter, the result is a drop in pressure downstream of to increase pump capacity or gravity head or, as
that filter, and the measurement of the pressure an alternative, to reduce clean pressure drop by
drop across the filter is called the differential pres- increasing filter size.
sure or ∆P. Thus, for all practical purposes the
terms pressure drop, differential pressure, and ∆P Filter cartridges exhibit an exponentially increas-
are synonymous. ing pressure drop vs. dirt capacity curve as shown
in Figure 11. Usually, the capacity of the filter is
The more resistance a filter medium offers to fluid mostly consumed before the sharp increase in
flow, the greater the differential pressure at con- pressure drop. Consequently, the available sys-
stant flow. Since flow is always in the direction tem pressure source should be at least sufficient
of the lower pressure, the differential pressure to overcome the pressure drop ( ∆P) at the knee
will cause fluid to flow. Thus, it is differential of the curve so as to utilize most of the dirt hold-
pressure that moves the fluid through the filter ing capacity of the filter medium.
assembly and overcomes resistance to flow.
Maximum allowable cartridge pressure drop is
In the preceding discussion it was tacitly assumed the limit beyond which the filter might fail struc-
that the fluid was completely free of particulate turally should additional system pressure be applied
contamination. But in reality there will always be to maintain adequate flow. This limit is always
some particles present in a system. As the filter specified by the filter manufacturer.
does its job, particles will be stopped by and par-
tially occlude or block the pores or holes of the In choosing a pressure source, one must take into
filter medium, thereby increasing resistance to consideration the resistance to flow of the filter
flow and ∆P. – both c onstant resistanc e c omponents (filter
housing and element hardware), and the variable
In choosing a filter, therefore, one must provide for resistance components (filter cake and medium).
sufficient pressure source not only to overcome the As filtration proceeds at constant flow, there will
resistance of the filter, but also to permit flow to be an increase in pressure drop made up of a con-
continue at an acceptable rate as the medium plugs stant component and an increasing variable com-
so as to use fully the effective dirt holding capaci- ponent. Eventually, the increasing pressure drop
ty of the filter. If the ra tio of initia l clea n pressure component becomes so large as to either clog
drop through the filter to tota l ava ilable pressure the filter and stop flow or to structurally damage
is disproportiona lly high, una ccepta ble flow will the filter. Enough pressure drop should be avail-
quickly result even though the medium’s ca pa ci- able to satisfy both components at least to filter
ty fo r co llecting dirt ha s no t b een exha usted. clogging.
Figure 11
Cartridge Pressure
Dirt Capacity Typical
If a pressure head exists downstream, as for exam-
Drop vs. Dirt
Capacity ple in an elevated receiver, this must be overcome
without limiting the available pressure drop for the
filter. In suc h c ases a c hec k valve should be
∆P Element Only
installed downstream of the filter to prevent

reverse pressure from damaging the c artridge.
As noted above, the pressure drop across the fil-

ter assembly can be reduced by increasing the
AC Fine Dust (GMS) size of the assembly. This is usually an economi-
11
cal approach for continuous processing since the have reached 75 psi (there are 6 times as many
increase in total throughput is often more than lin- pores to plug: 30/5 = 6). Indeed, since flow rate
ear with respect to the cost of the larger number per sq. ft. of filter area is in the ratio of 5/30, the
of filter cartridges in the larger assembly (see below). pressure drop across the 30 sq. ft. filter will be 5/30
x 75 psi or 12.5 psi. If the 30 sq. ft. filter has a pres-
5. Surface Area sure drop of 12.5 psi in 144 hours, then it will have
It should be clear from the above discussion of a pressure drop of 75 psi in x hours.
pressure drop that the useful life of a filter relates Thus:
to its dirt capacity, defined in the NFPA glossary 12.5 = 75
as “The weight of a specified artificial contaminant 144 x
which must be added to the influent to produce
a given differential pressure across a filter at spec- 12.5x = 75 x 144
ified conditions.” While dirt capacity can be mea- 12.5x = 10,800
sure d using any c o nsiste ntly re p ro duc ib le x = 864 hours
contaminant, ACFTD is most often employed for
this purpose. 5 The life of the 30 sq. ft. filter is therefore 36 times
that of the 5 sq. ft. filter (864/24). If one calculates
The life of most screen and fixed pore depth type the square of the area ratio (30/5)2 the answer is 36!6
filters is greatly increased as their surface areas are
increased; in fact, the ratio ca n be as much as the The benefit of opting for a filter assembly with a
square of the area ratio! To understand why this large surface area can be expressed as follows:
is so, let us look at two filters of identica l m edi- Let T = Throughput (gallons) for a filter with area,
um (thus subject to the same pressure drop limit) A, (sq. ft.)
which pass the same fluid at the same flow rate.
Then T1 = T2 n
The first filter has a surface area of 5 sq. ft. and col-
( AA ) 1
2
lects a filter cake .005 ″ thick (128 µm) in a 24-hour where n is equal to or greater than 1 and less
period. After 24 hours most of the pores are than or equal to 2.
plugged; the pressure drop is 75 psi; and the use-
ful life of the filter has been exhausted. This relationship is expressed graphically in Figure
12. The curve shows that as the flow density (gal-
Let us then increase the surface area of the filter lons per minute per square foot) decreases, the
to 30 sq. ft. and calculate life. If a filter with a sur- total throughput increases. If one assumes a con-
face area of 5 sq. ft. collects a filter cake of .005 ″
in 24 hours, then at the same flow rate a filter of Figure 12 Filter Life vs. Flow Density
30 sq. ft. will collect that same filter cake in x

hours. Thus: n
T2
5 = 30 T1
= ( JJ )
1
2
24 x
1≤n≤2
Throughput (T)
Gallons
5x = 30 x 24
5x = 720
x = 144
While the 30 sq. ft. filter has collected a filter cake

of .005 ″ in 144 hours, its useful life will not be
Flow Density (J)
ex hausted bec ause the pressure drop will not Gallons Per Minute Per Square Foot
12
stant flow rate (gallons per minute), then the ratio From the foregoing it is apparent that an increase
of the flow densities simplifies to the ratio of the in surface area will yield at least a proportional
areas raised to the nth power which is exactly increase in service life. Under favorable circum-
the relationship previously discussed. stances, the ratio of service life may approach the
square of the area ratio. In many, if not most cases,
The life extension factor (n) will approach two pro- a filter user will save money in the long run by pay-
vided that: ing the higher initial cost of a larger filter assembly!
• The filter cake is not compressible. If the fil-
ter cake is compressible, n will tend to be For example, let’s assume an arbitrary cartridge cost
nearer to one. of $1.00/square foot of effective cartridge surface
• The collected cake does not become a finer area. Then, from the previous example, a 5 sq. ft.
filter than the medium itself (i.e., collect finer filter c osts $5.00 while a 30 sq. ft. filter c osts
solids as it builds up). To the extent that the $30.00. Further, as calculated, the 5 sq. ft. car-
filter cake acts as a finer filter than the medi- tridge lasts 24 hours while the 30 sq. ft. cartridge
um itself, n will tend to approach one. has a useful life of 864 hours. Therefore, the for-
• The solids collected are relatively uniform in mer requires 36 changeouts (864/24) as compared
particle diameter. to the latter. In terms of costs, the filter user will
be outlaying $180.00 (36 x $5.00) in cartridge
Figure 13 costs as opposed to $30.00 for the 30 sq. ft. car-
Schematic
Representation of
~ 2 3/ 4"
D= ~ 2 3/ 4"
D= tridge. This cost differential is exacerbated if one
Pleated Structure includes labor as well as other costs such as lost
Cartridge Design
production time.
~
=10" ~
=10"
~ 0.6 ft2
A1 = A2 ~
= 3-8 ft2 As the surface area is increased, a larger housing
(container or pressure vessel) is required. There
A = Filter Area
T = Thickness of is, of c ourse, a prac tic al limit to housing size.
Filter Medium
T2
It is for this reason that Pall Corporation uses con-

T1
voluted or pleated structures to provide large sur-
fac e areas in small envelopes, thereby keeping
housing size and cost to a minimum. Figure 13 is a
Figure 14 schematic representation of a pleated structure car-
Void Volume vs.
tridge design. The figure shows that for identical
Fiber Diameter at
Constant Pore Size envelope dimensions (2-3/4 ″ x 10 ″), the pleated
design provides for over 13 times the surface area.
6. Void Volume
Void volume, 7 or the open area of a medium, is
always of great importance. All other factors being
equal, the medium with the greatest void volume
is most desirable because it will yield longest life
and lowest initial clean pressure drop per unit
thickness. Figure 14 illustrates the relationship
between void volume and fiber diameter. As the
fiber diameter decreases, the void volume increas-
es, assuming constant pore size. Other factors,
13
however, such as strength, compressibility as pres- sufficient to justify prefiltration; overall cost reduc-
sure is applied (which reduces void volume), com- tion is usually the principal consideration.
patibility of the medium with the fluid being
filtered, cost of medium, cost of constructing that During 50 years experience with requests for pre-
medium into a useable filter, etc., must all be con- filtration, it has been Pall’s experience that most
sidered when designing a filter for a particular applications are better served by increasing final
application. filter area rather than by providing a prefilter. This
is because (as was shown in the previous section)
7. Degree of Filtration increasing final filter area a lwa ys yields a longer
Naturally, the filter chosen for a given application cycle and lower operating costs. Doubling the
must be able to remove contamination from the area of the final filter will result in two to four times
fluid stream to the degree required by the process the life. Since one-half to two-fold life increase due
involved. Once the size of the contaminants to be to prefiltration is typic al, and four times very
removed has been determined, it is possible to unusual, it may be seen that increasing the filter
choose a filter with the particle removal charac- area usually yields better results. This approach also
teristics needed to do the job. Choosing a filter results in lower costs, requires less labor input, and
with a pore size finer than required can be a cost- permits operation with less power at lower pres-
ly mistake. Remember, the finer the filtration, the sure drop. Cost is less because one housing is
more rapid the clogging and the higher the cost! used instead of two, and cost is further reduced
because the larger filter installation has life in ser-
Remember also that the filter selected must be able vic e whic h is proportionally larger than the
to retain particles removed from the subject fluid. increase in area. Clean pressure drop is reduced
As noted above, depth type filters of the type because of larger area, whereas a prefilter always
whose pores can increase in size as pressure is increases pressure drop. Power required is reduced
increased are subject to unloading. With surface because pressure drop through most of the fil-
type filters, or fixed random pore depth type filtration cycle is lower.
ters, one selects a medium which will not change
its structure under system-produced stress. For In addition, increased final filter area always increas-
example, as system pressure rises to accommo- es life at least in proportion, and often exponen-
date flow as filter cake builds, strands of woven tially, while prefiltration often involves much cut
wire mesh must not separate to produce a larger and try experimentation, which is not a lwa ys suc-
pore. Nor should the mesh rupture under the cessful in the end. Even when the prefilter tests
pressures possible in the system in question. are successful, that success can be lost because the
nature of the contaminants to be filtered changes
When it is necessary to support thin, membrane- with time.
type filters, attention must be given to the char-
acteristics of the support material chosen. Support Staged prefiltration, in which coarser but equal area
materials can react with the fluid, add significant absolute rated filters are arbitrarily selected to
resistance to flow, and cause poorer life. precede the final filter ca n also work – but in fact
does so only rarely. This is not a recommended
8. Prefiltration approach. However, if for some reason a final fil-
The purpose of a prefilter is to reduce overall ter area increase is not feasible, the various Pall car-
operating cost by extending the life of the final fil- tridges designated as prefilters can be tried using
ter. Extending final filter life may not in itself be the grade of prefilter recommended by our liter-
ature.
14
Don’t fall into the trap of always assuming that a are left onstream until clogged, then using two
final filter should always be preceded by a prefilter cartridges instead of one will increase life by more
with an equal number of cartridges. Using a larg- than two times, and possibly four to five times; this
er number of cartridges in a prefilter often results then results in lower operating cost, since more
in substantially lower overall costs. For example, fluid is processed per cartridge.
if the mode of operation is such that the prefilters
Summary
This paper discussed the principles of filtration. depth type, and surface type media and explained
The three c apture mechanisms by which sus- why these classific ations are more meaningful
pended solids are separated from fluids were than simply a depth or surface classification. After
examined and the conclusion reached that direct characterizing the various types of filters that exist
interception is the desired mechanism for sepa- in the field today, it is necessary to examine their
rating partic le s fro m liquids, while ine rtial removal efficiencies. Three removal rating sys-
impaction and diffusional interception are more tems were analyzed inc luding nominal rating,
effective in gas filtration. In order to enhance the absolute rating, and Beta ratings. The paper con-
filter’s separation effectiveness, it is possible to cludes by considering the most important factors
manipulate the particle/liquid/filter media system. that a filter user must take into account when
Three such techniques were described. Because making a filter selection.
of the confusion over filter media classification, the
paper compared and contrasted non-fixed ran-
dom pore depth type media, fixed random pore
15
Footnotes
1 permeable medium – a material containing inter- 6 The total pressure drop through a cartridge fil-
connected holes or pores, the presence of which ter assembly is the sum of the pressure drops of
permits passage of fluids. the housing, the medium and the filter core. For
simplification, this example does not take into
2 except Whatman Nuclepore, which consist of account pressure drops through the housing or
straight through irregular and circular holes. the filter core (the constant resistance compo-
nents), and deals only with the variable resis-
3 Filter Zeta Potential – a measurement of the elec- tance components. The proportion of the total
trical potential difference between a filter surface pressure drop due to the constant resistance
and the contacting liquid. components will decrease as the size of the unit
is increased, but this is a minor consideration.
4 Standardized test dusts are classified from natural
Arizona dust and are generally referred to as A.C. 7 Void volume is often confused with the term
Fine and A.C. Coarse Test Dusts. A.C. Fine Test “porosity.” Since porosity has been used in the
Dust (ACFTD) is commonly used to establish industry to mean both percentage void volume
the Nominal Rating of filters less than 50 µm. For and also pore size, we avoid use of the term.
coarse filters (i.e., those greater than 50 µm)
A.C. Coarse Test Dust is used.
5 Laboratory dirt capacity tests using ACFTD, or any

other test contaminant, very often do not cor-
relate with actual operating conditions in the
field, and at best offer a rough guide to anticipated
filter life. Also, dirt capacity tests between dif-
ferent types of media (e.g., wound or pleated
paper) are not meaningful as they do not cor-
relate to comparative actual field test results.
No laboratory dirt capacity test now in use, or
proposed for use, provides a meaningful index
of filter life in service because life in service
varies enormously depending on the nature of
the actual contaminant, its state of suspension in
the liquid, its size distribution, etc. Results also
vary greatly depending on the viscosity and flow
rate of the liquid. Meaningful data is obtained
only from in-system tests.
16
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trademark registered in the USA. is a service mark of Pall Corporation.
MEASUREMENT OF MICROBIAL GROWTH
Microbial growth to determine growth rates and generation times can be measured by different methods.
Since growth leads to increase both the number and the mass of the populations, either of the two may
be followed. It is necessary to make it clear that no single technique is always best; the most appropriate
approach depends upon the experimental situation
Measurement of Cell Numbers
Direct microscopic counts using a counting chamber can be used but this technique has limitations as
the dead cells cannot be distinguished from living cells. Electronic counting chambers count numbers
and measure size distribution of cells. These are more often used to count eucaryotic cells like blood
cells. Indirect viable cell counts also called plate counts may be used. This involves plating out dilutions
of a culture on nutrient agar. Each viable unit will form a colony and each colony that can be counted is
called a colony forming unit (cfu) and the number of cfus is related to the viable count in the sample.
1. Breed Method : A known volume of microbial cell suspension (0.01 ml) is spread uniformly over a
glass slide covering a specific area (1 sq. cm). The smear is then fixed by heating, stained and examined
under oil immersion lens, and the cells are counted. Customarily, cells in a few microscopic fields are
counted because it is not possible to scan the entire area of smear. The counting of total number of cells
is determined by calculating the total number of microscopic fields per one square cm. area of the smear.
The total number of cells can be counted with the help of following calculations:
(a) Area of microscopic field = πr2

r (oil immersion lens) = 0.08 mm.
Area of the microscopic field under the oil immersion lens
= πr = 3.14 x (0.08 mm)2 = 0.02 sq. mm.
2
(b) Area of the smear one sq. cm. = 100 sq. mm. Then, the no. of microscopic fields = 100 / 0.02= 5000
(c) No. of cells 1 sq. cm. (or per 0.01 ml microbial cell suspension) = Average no. of microbes per
microscopic field x 5000
Counting Chamber Technique: The number of cells in a population can be measured by taking direct
microscopic count using Petroff-Hausser counting chamber (for prokaryotic microorganisms) or hemo-
cytometers (to larger eukaryotic microorganisms). Prokaryotic microorganisms are more easily counted
if they are stained or if phase I contrast of florescence microscope is employed. These are specially
designed slides that have chambers of I known depth with an etched grid on the chamber bottom. , Each
square on the grid has definite depth and volume. Total number of microorganisms in a sample can be
calculated taking the count of number of bacteria per unit area of grid and multiplying it by a conversion
factor (depending on chamber volume and sample dilution used).
1
The direct microscopic method is easy, inexpensive and relatively quick to count microbial cell number.
However, using these method dead cells is not distinguished from living cells and also very small cells
are usually missed.
Viable Count: The bacterial culture need not contain all living cells. There might be few dead as well.
Only living cells will form colony when grown in proper solid medium and under standard set or growth
conditions. This fact is used to estimate number of living or dead bacterial cells (viable count) in the
given culture. Estimates thus obtained are expressed as a colony f9rming unit (CFU).
Viable count technique is very much useful in the dairy industry and the food industry for quantitative
analysis of milk and spoilage of food products. For convenience, to obtain a colony count for bacteria in
milk, 1 ml of well mixed milk is placed in 99 ml of sterile dilute solution (may be water or nutrient broth
or saline solution).
This results in s dilution of 1: 100 or 1 × 10-2. To the petri dish containing pre solidified medium 1 ml
of 1: 100 dilution is transferred and incubated at desired is repeated for the preparation of further
dilution as 1 : 1000 or 1 : 10, 0000 of bacteria per ml in original sample can be found by multiplying
bacterial colony count by the reciprocal of he dilution and of the volume used.
For Example, CFU = 50 for 1: 10, 000 if volume used is 1 ml then,
CFU = 50 × 10, 000 × 1
CFU = 5 × 105
Coulter Counter: Coulter counter is an electronic used to count number of microbes preferably
protozoa microalgae and yeasts. In This method, the sample of microbes is forced through a small
orifice (small hole). On the both sides of the orifice, electrodes are present measure the electric
resistance or conductivity when electric current is passed through the orifice. Every time a
microorganism passes through the orifice, electrical resistance increases or the conductivity drops and
the cell is counted. The Coulter counter gives accurate results with larger cells. The precaution to be
taken in this method is that the suspension of samples should be free of any cell debris or other
extraneous matter.
Coulter Counter
1. Electrode 2. Flow of Suspending Fluid 3. Bacterial Cell 4. Orifice

5. Electrode 6. Particle Location 7. Measurement of Voltage
Membrane-Filter Technique: Microbial cell numbers are frequently determined using special
membrane filters possessing millipores small enough to trap bacteria. In this technique a water sample
containing microbial cells passed through the filter. The filter is then placed on solid agar medium or on
a pad soaked with nutrient broth (liquid medium) and incubated until each cell develops into a separate
colony. Membranes with different pore sizes are used to trap different microorganisms. Incubation times
2
for membranes also vary with medium and the microorganism. A colony count gives the number of
microorganisms in the filtered sample, and specific media can be used to select for specific
microorganisms. This technique is especially useful in analyzing aquatic samples
Steps of Membrane Filter Technique
Measurement of Cell Mass
1. Dry Weight Technique
The cell mass of a very dense cell suspension can be determined by this technique. In this technique, the
microorganisms are removed from the medium by filtration and the microorganisms on filters are
washed to remove all extraneous matter, and dried in dessicator by putting in weighing bottle
(previously weighted). The dried microbial content is then weighted accurately. This technique is
especially useful for measuring the growth of microfungi. It is time consuming and not very sensitive.
Since bacteria weigh so little, it becomes necessary to centrifuge several hundred millions of culture to
find out a sufficient quantity to weigh.
Measurement of nitrogen content
As the microbes (bacteria) grow, there is an increase in the protein concentration (i.e. nitrogen
concentration) in the cell. Thus, cell mass can be subjected to quantitative chemical analysis methods to
determine total nitrogen that can be correlated with growth. This method is useful in determining the
effect of nutrients or antimetabolites upon the protein synthesis of growing culture
Measurement of Turbidity (Turbidometry)
Rapid cell mass determination is possible using turbidometry method. Turbidometry is based on the fact
that microbial cells scatter light striking them. Since the microbial cells in a population are of roughly
constant size, the amount of scattering is directly proportional to the biomass of cells present and
indirectly related to cell number. One visible characteristic of growing bacterial culture is the increase in
cloudiness of the medium (turbidity). When the concentration of bacteria reaches about 10 million cells
(107) per ml, the medium appears slightly cloudy or turbid. Further increase in concentration results in
greater turbidity. When a beam of light is passed through a turbid culture, the amount of light
transmitted is measured, Greater the turbidity, lesser would be the transmission of light through medium.
Thus, light will be transmitted in inverse proportion to the number of bacteria. Turbidity can be
measured using instruments like spectrophotometer and nephlometer
3
Determination of cell mass using turbidimetry method
1. Source of Light of a single wave-length (monochromatic) 2. Filter

3. Tube with cell Free medium 4. Tube with suspension of 5. Photocell or
microorganisms Detector
McFarland standards: In microbiology, McFarland standards are used as a reference to adjust

the turbidity of bacterial suspensions so that the number of bacteria will be within a given range.
Original McFarland standards were made by mixing specified amounts of barium chloride and sulfuric
acid together. Mixing the two compounds forms a barium sulfate precipitate, which causes turbidity in
the solution. A 0.5 McFarland standard is prepared by mixing 0.05 mL of 1.175% barium chloride
dihydrate (BaCl2•2H2O), with 9.95 mL of 1% sulfuric acid (H2SO4).Now there are McFarland standards
prepared from suspensions of latex particles, which lengthens the shelf life and stability of the
suspensions. The standard can be compared visually to a suspension of bacteria in sterile saline or
nutrient broth. If the bacterial suspension is too turbid, it can be diluted with more diluent. If the
suspension is not turbid enough, more bacteria can be added.
McFarland Nephelometer Standards:
McFarland Standard No. 0.5 1 2 3 4

1.0% Barium chloride (ml) 0.05 0.1 0.2 0.3 0.4
1.0% Sulfuric acid (ml) 9.95 9.9 9.8 9.7 9.6
Approx. cell density (1X10^8 CFU/mL) 1.5 3.0 6.0 9.0 12.0
% Transmittance* 74.3 55.6 35.6 26.4 21.5
Absorbance**at wavelength of 600 nm 0.132 0.257 0.451 0.582 0.669
spectrophotometer to estimate absorbance of cell suspensions. The absorbance at a particular

wavelength is proportional to the cell concentration. By plotting a standard curve of absorbance versus
cell concentration, the cell concentration of an unknown sample can be calculated.
4
A plate count may be done on plates by either the pour plate method or the spread plate method.
5
Bacterial growth rates during the phase of exponential growth, under standard nutritional conditions
(culture medium, temperature, pH etc.) define the bacterium’s generation time.
Generation times for bacteria vary from about 12 minutes to 24 hours. The generation time for E. coli in
the laboratory is 15-20 min. Symbionts such as Rhizobium tend to have a longer generation time. Some
pathogenic bacteria, e.g., Mycobacterium tuberculosis have especially long generation times and this is
thought to be an advantage to their virulence.
When growing exponentially by binary fission, the increase in a bacterial population is by geometric
progression. The generation time is the time interval required for cells (or population) to divide:
G =t/n
Where G is generation time, n is number of generations and t is time in min/hours
The equation for growth by binary fission is:
b = B x 2n
where b is number of bacteria at end of a time interval, B is number of bacteria at beginning of a time
interval, n is the number of generations (number of times the population doubles in the time interval).
logb = logB + nlog2
n = logb-logB n = logb-logB
log2 .301
n = 3.3logb/B
G= t
3.3logb/B
2.5.2 GROWTH KINETICS IN BATCH CULTURE

Batch culture occurs in a closed system that contains an initial limited amount of substrate. The
inoculated microorganism will pass through a number of growth phases (Fig. 2.2). During the log phase,
cell numbers increase exponentially at a constant maximum rate. In mathematical
terms, we can write: dx = μx
dt
where x is the concentration of microbial biomass, t is the time in hours and μ is the specific growth rate
in hours-1. If we integrate between time t0 and time t1 when the concentrations of the cells are X0 and
X1 we obtain:
xt = x0e μt
where x0 is the original biomass concentration, xt is the biomass concentration after a time interval t
hours and e is the base of the natural logarithm. On taking natural logarithms, the equation becomes:
ln xt = ln x0 + μt
Using this equation, a plot of the natural log of biomass
concentration versus time should yield a straight line, the slope

ln x
of which will equal the specific growth rate (μ).
During the exponential phase, nutrients are in excess and the microorganism is growing at maximum
specific growth rate μmax for the prevailing conditions.
6
The major problem of the exponential growth equation is that it does not predict an end to growth in a
batch environment. According to this model, not only the whole earth, but also the whole solar system
could become quickly covered by bacteria. However, growth results in the consumption of nutrients and
the excretion of microbial products. After a time, the growth rate of the culture ceases. The cessation of
growth may be due to the depletion of some essential nutrient in the medium (substrate limitation), the
accumulation of some autotoxic product in the medium or a combination of the two.
The nature of the limitation of growth can be explored by growing the microorganisms in a range of
substrate concentrations and plotting the biomass concentration in the stationary phase against the initial
substrate concentration, the nature of growth limitation may be explored (Fig. 2.4).
D
C
Biomass
B
concentration at
stationary phase
Initial substrate concentration
Fig.2.4: The effect of initial substrate concentration on the biomass concentration at the onset of
stationary phase in batch culture (Stanbury et al. 1995).
A proportional increase in biomass is observed to increasing initial substrate concentration in the area
between A and B which can be defined as:
x = Y (SR – S)
Where x is the concentration of biomass produced, Y is the yield factor (g biomass produced per g
substrate utilized), SR is the initial substrate concentration and S is the residual substrate concentration.
In the area between A and B, S is zero and therefore the equation above could be used to predict the
biomass that could be formed from a certain amount of substrate. Between B and C although biomass
increases with increasing substrate concentration, there is a diminished effect due to accumulation of
toxic products or reduced availability of some other substrate. In the region between C and D, there is no
change in biomass with increasing substrate concentration which may be attributed to increasing levels
of toxic products or the exhaustion of some other substrate.
Y, the yield factor is the measure of efficiency of conversion of any one substrate to biomass. Although
Y is not a constant and varies according to growth rate, pH, temperature, the limiting substrate and
concentration of the substrate in excess, it can be used to predict the substrate concentration required to
produce a certain biomass concentration. In the 1930s, Jacques Monod performed a number of initial
rate experiments and plotted the specific growth rate against the concentration of growth-limiting
substrate (Fig. 2.5). The result was a Langmuir type graph that appeared similar to enzymatic rate-
substrate relationships described by Michaelis-Menton’s model. Monod’s model describing the
relationship between the specific growth rate and the growth limiting substrate concentration is:
7
C A B
Fig.2.5: The effect of residual limiting substrate concentration on specific growth rate of a
hypothetical bacterium (Stanbury et al. 1995, adapted).
Where μm is the maximum specific growth rate, S is the residual substrate concentration and Ks is the
substrate utilization constant, numerically equal to substrate concentration when μ is half μm μm and is
a measure of the affinity of the organism for its substrate. Zone A to B in Fig. 2.5 represents the
exponential phase of growth in batch culture where substrate concentration is in excess and growth is at
μm. Zone A to C is the deceleration phase, substrate concentration becomes limiting and cannot support
growth at μm. An organism with a high affinity for the limiting substrate (low Ks) will have a short
deceleration phase as the growth rate will only be affected when the substrate concentration is very low.
Conversely, a microorganism with a low affinity for the substrate will have a very long deceleration
phase (growth slows down at high substrate concentrations). The point when growth rate has declined to
zero represents the stationary phase. This is a misnomer as many organisms are still metabolically active
and are producing products called secondary metabolites during this phase.
Monod’s model is widely used to describe the growth of many microorganisms. The equation
adequately describes fermentation kinetics and can be used to describe complex fermentation systems.
The equation adequately describes fermentation kinetics and can be used to describe complex
fermentation systems, e.g., a commonly used expression to describe product inhibition is:
Using the Monod model, a simple model microbial growth can be written as:
where Yxs is the biomass yield coefficient. The biomass yield coefficient is the efficiency of conversion
of substrate to biomass and is calculated as:
Biomass = Dry weight of biomass produced
Weight of substrate used
The kinetics of product formation may be described as growth-linked products and non-growth linked
products. In the first instance – these could relate to primary metabolites synthesized by growing cells
8
and the non-growth-linked products would be secondary metabolites. Formation of growth-linked
products can be defined by the following:
dp/dt = qpx
where p is the concentration of product, qp is the specific rate of product formation (mg product /g
biomass/h).
Product formation can also be expressed in relation to biomass as:
dp/dx = Yp/x
where Yp/x is the yield of product in terms of biomass (g product/g biomass). Combining these
equations:
qp = Yp/x .µ
Thus when product formation is linked to growth, the specific rate of product formation increases with
specific growth rate and will be highest at µm. In this instance improved output will be obtained by
increasing both biomass and µ. Non-growth linked product formation is related to biomass
concentration. As these products are produced only under certain physiological conditions (usually
limitation of a certain substrate), the biomass needs to be in the correct physiological state before
secondary metabolites are produced.
Batch fermentations may be used to produce biomass and primary and secondary metabolites. For (i)
biomass production: conditions supporting fastest growth rate and maximum cell concentration;
For (ii) primary metabolites: conditions to extend exponential phase accompanied by product excretion;
For (iii) secondary metabolites: conditions providing a short exponential phase and extended production
phase or conditions giving decreased growth rate in the log phase resulting in earlier secondary
metabolite production.
GROWTH KINETICS IN CONTINUOUS CULTURE

Exponential growth in batch culture may be prolonged by the addition of fresh medium, provided that
the medium is designed to be substrate-limiting. If the vessel is designed with an overflow mechanism,
such that the added medium displaced an equal volume of spent medium, then continuous culture of
cells can be achieved. A steady state will be achieved if the medium is fed continuously at a suitable
rate, i.e., formation of new biomass by the culture is balanced by the loss of biomass from the vessel.
The flow of medium is related to the volume of the vessel by the dilution rate (D) as follows:
D = F/V
Where F is the flow rate (l/h) and V is the volume (l).
The net change in cell concentration over time may be expressed as:
dx/dt = growth – output or dx/dt = µx – Dx

Under steady state conditions, the cell concentration remains constant, therefore
dx/dt = 0 and µx = dx and µ = D
A continuous culture may be operated at dilution rates below the maximum specific growth rate and so
within certain limits, the dilution rate may be used to control the growth rate of the culture. Cell growth
in such a continuous culture is controlled by the availability of the growth limiting substrate and the
system is referred to as a chemostat.
The mechanism underlying the controlling effect of the dilution rate is expressed in the Monod equation:
9
µ = µms/(Ks + s)
AT steady state, µ =D
Therefore, D = µm /(Ks + )
where is the steady state concentration of substrate in the chemostat.
Rearranging the equation:

KsD/ (µm – D)
which predicts that the substrate concentration is determined by the dilution rate. This occurs by the
growth of the cells depleting the substrate to a concentration that supports the growth rate equal to the
dilution rate. If the substrate becomes depleted below the level that supports the growth rate dictated by
the dilution rate, the following would occur:
(i) The growth rate of the cells will be less than the dilution rate and they will be washed out of the
vessel at a rate greater than they are being produced resulting in a decrease in biomass concentration.
(ii) The substrate concentration in the vessel will rise because fewer cells are left in the vessel to
consume it.
(iii)The increased substrate concentration in the vessel will result in the cells growing at a rate greater
than the dilution rate and biomass concentration will increase.
(iv) The steady state will be re-established.
Therefore a chemostat is a nutrient-limited, self-balancing culture system which may be maintained in a

steady state over a wide range of sub-maximum specific rates.
The cell concentration in a chemostat is defined by:

= Y(SR -
Where is the steady state cell concentration.
By combining equation of steady state substrate and biomass concentrations:

= Y[SR – {KsD/(μm – D)}]
Therefore biomass concentration at steady state is defined by operational variables SR and D. If SR is

increased, increases but remains the same.
If D is increased, μ will increase (μ = D), at the new steady state would have increased to support
the elevated growth rate and less substrate will be available to be converted to biomass resulting in
a lower
An alternative type of continuous culture to a chemostat is a turbidostat. Here the concentration of the
cells in the vessel is kept constant by controlling the flow of medium such that the turbidity of the
culture is kept within certain narrow limits. To achieve this, biomass is monitored using a photoelectric
cell, signals are sent to a pump controlling medium flow into the vessel. If the biomass exceeds a set
point, the pump is switched on and if the biomass falls below the set point it is switched off. Other
biomass measurement systems include CO2 concentration or pH - biostat. However, the chemostat is the
more commonly used system as it has the advantage over the biostat of not requiring complex control
systems to maintain steady state.
10
BIO SENSOR
The Biosensor is used to detect the analyte so the Biosensor is an analytical device and it gathers
the biological components with a Physicochemical detector. The sensing biological elements are
biometric components interact with the recognize and analyze the study and the components like
tissue, microorganisms, antibodies, nucleic acids and etc. The sensitive elements of biological
can also generate by the biological engineering. The detector elements transform the signals from
the interface of analyte with the biochemical elements into other signals like transducer and it
can be measured more easily and qualified. The Biosensor devices are associated with the
electronics and the signal processors and they are generally responsible for the display of the
results and they are user-friendly. The Biosensor research has a significant role in the
development of modern electronics. This article discusses about different types of
Biosensors working and applications. A Biosensor is an analytical device. The
sensor which integrates the biological
elements with the Physiochemical
transducer to produce an electronic signal
is proportional to a single analyte and
which is fetched into a detector.
Biosensor
A biosensor is a device for the detection of an analyte that combines a biological component
with a physicochemical detector component.
An analytical device which functions to analyse a sample for the presence of a specific
compound is known as sensor. A sensor which utilizes biological material to specifically interact
with an analyte is known as biosensor. An analyte refers to the compound which has to be
‘sensed’ or the presence of which has to be determined. The interaction of analyte and biosensor
is measured and converted to signals, which are again amplified and displayed. A biosensor thus
involves converting a chemical flow of information into electrical signals. The biological
materials used in biosensors are mostly enzymes, antibodies, nucleic acids, lectins, a cell as a
whole etc.
According to the mode of interaction biosensors are of two types:

Catalytic biosensor: The interaction of biological material in the biosensor and the analyte result
in modification of analyte into new chemical molecule. The biological material used is mainly
enzymes.
Affinity biosensor: Here, upon interaction, the analyte binds to the biomolecule on the biosensor.
These are mainly composed of antibodies, nucleic acids etc.
Essential properties of a biosensor:

(i) Specificity: a biosensor should be specific to the analyte which it interact.
(ii) Durability: it should withstand repeated usage.
(iii) Independent nature: It should not be affected by variations in the environment like
temperature, pH etc.
(iv) Stability in results: the results produced by interaction should be corresponding to the
concentration of analyte.
(v) Ease of use and transport: it should be small in size so that it can be easily carried and used.
Components and mechanism of a biosensor:

A biosensor mainly consists of two parts
(i) a biological part: this constitutes of enzymes antibodies etc., which mainly interacts with the
analyte particles and induce a physical change in these particles.
(ii) a transducer part: which collects information from the biological part, converts, amplifies and
display them. In order to form a biosensor, the biological particles are immobilized on the
transducer surface which acts as a point of contact between the transducer and analyte.
When a biosensor is used to analyse a sample, the biological part specific to the analyte
molecules, interacts specifically and efficiently. This produces a physicochemical change of the
transducer surface. This change is picked up by the transducer and gets converted into electric
signals. These then undergo amplification, interpretation and finally display of these electric
units accounting to the amount of analyte present in the sample.
Working of Biosensors
The preferred biological material like enzyme is
preferred for conventional methods like physical or
membrane entrapment and non covalent or covalent
binding. The preferred biological material is in contact
with the transducer. To produce a bound analyte through
the analyte binds to the biological material which
produces the electrical response to be measured. In
some cases the analyte changed to a product and have
some probability to associate with the release of heat,
gases like oxygen, electrons or hydrogen ions.
Types of biosensors:
(i) Calorimetric biosensor: some enzyme- analyte reactions are exothermic and releases heat into
the sample. This change in temperature is detected by the transducer. The amount of heat
generated is proportional to the analyte concentration present and is processed likewise.
(ii) Potentiometric biosensor: an electric potential is produced as a result of interaction which is
detected by the transducer
(iii) Amperometric biosensor: analyte when comes in contact with biological material induces a
redox reaction. This results in movement of electrons which is picked up by transducer.
(iv) Optical biosensors: in this, a biosensor reacts with analyte to absorb or release light which is
identified by the transducer and interpreted.
(v) Acoustic wave biosensors: biological component of biosensor undergoes a biomass change
ascertained by transducer.
The advantages of biosensors include accuracy in results, minute detection capability, ease of
use, versatile and continuous monitoring available.
1 Electrochemical Biosensor
Electrochemical Biosensor
Electrochemical Biosensors is a
simple device. It measures the
measurement of electronic current,
ionic or by conductance changes
carried by bio-electrodes.
Electrochemical Biosensor
2 Amperometric Biosensor
The Biosensors are based on the electrons

movement, i.e. electronic current
determination as a reaction of enzyme-
catalyzed redox reaction. Generally a normal
contact voltage passes through the
electrodes to analyze. In the enzymatic
reaction which produces the substrate or
product can transfer the electrons with the
surface of electrodes to be reduced.
Amperometric Biosensor
As a result an alternate current flow can be measured. The substrate concentration is directly
proportional to the magnitude of the current. The reduction of oxygen is acquired through the
oxygen electrodes and it is a simple way to from an Amperometric biosensor. The example is the
determination of glucose by glucose.
The above description is about the first generation of Amperometric biosensor and it has a direct
transfer of electrons which are released from the electrodes are having some difficulties. The
second generation Amperometric biosensors are developed in a mediator takes the electrons and
transfer to the electrodes.
Blood Glucose Biosensor

The blood glucose Biosensors are used
widely throughout the world for diabetic
patients. It has a single use disposable
electrodes with glucose oxide and derivatives
of a mediator (Ferrocence) and the shape of
the blood glucose Biosensor looks like a
watch pen. With the help of hydrophilic mesh
electrodes are converted. The Blood glucose
Biosensor is a good example of
Amperometric Biosensor.
3 Potentiometric Biosensor
In this type of Biosensors changes the

concentration of ionic is determined by the ion-
selective electrodes in this pH electrodes are used
most commonly. Hence a large amount of
enzymatic reactions is involved in the release of
hydrogen ions. Ammonia-selective and
Corbondioxide selective electrodes are some
other important electrodes
The Potentiometric electrode and the reference electrode can be measured with the help of
potential difference and it is directly proportional to the substrate concentration. The
Potentiometric Biosensors is the sensitivity of enzymes to ionic concentration like H+ and NH+4
The ion- selective field effect transistors are lower price devices. It can be used in the
miniaturization of Potentiometric Biosensors. The example of the ISFET Biosensor is to monitor
intra-myocardial for open heart surgery.
4 Conduct Metric Biosensor: In the biological system there are several reactions that change the
ionic species. The electronic conductivity can be measured with the help of an ionic species. The
example of the conduct metric Biosensor is the urea Biosensor which utilizing the immobilized
areas. The following reactions show the urea catalyses.
The given reaction is associated with the drastic alteration in ionic concentration and they are
used for the monitoring urea concentration. In generally during the dialysis and renal surgery the
urea Biosensor is very useful.
5 Thermometric Biosensor: There are many more biological reactions are connected with the
production of heat and it forms the basis of thermometric Biosensors. The diagram shows the
representation of a thermal Biosensor. The diagram consists of a heat insulated box fixed with
heat exchange.
6 Optical Biosensors
The optical Biosensor is a device, it utilizes the principle of optical measurements like
fluorescence, absorbance and etc. They used in fiber optics and Optolelectronic transducers. The
optical Biosensors are safe for non electrical remote sensing of materials. In the transducer
elements primarily optical Biosensors involves in the enzymes and antibodies. Usually the
Biosensors is not required any reference sensors and the comparative signals are generated by
using the sampling sensor. The important Biosensors is described briefly.
Fiber Optic Lactate Biosensor
The working of the fiber optic lactate
Biosensor is based on the measurement of
change in oxygen concentration, molecular
by identifying the effects of oxygen in
fluorescent dye. The following reaction is
reduced by the enzyme lactate mono-
oxygenase.
The oxygen depends on the amount of fluorescence generated by the dyed film this is because of
oxygen has a reducing effect on the fluorescence. In the reaction mixture the concentration of
lactate is increased, oxygen is utilized and as a result, there is a proportional decrease in the
quenching effect. Hence there is an increase in the fluorescence output can be measured
Optical Biosensors for Blood Glucose

For the diabetes patients the blood glucose is more important to monitor. In this simple technique
is used, i.e. Paper strips saturated with the reagents it contains glucose oxide, Horseradish
Peroxidase and a Chrmogen. The following reactions take place. Using the portable reflectance
meter it can measure the intensity of the colour of the dye. In the world wide the glucose strip
industry is very high. The calorimetric test strips of cellulose covered with the suitable enzymes
and reagents are in use for the view of more blood and the urine parameters. The other optical
fiber Biosensors are used in the devices of optical Biosensing it measures the p CO2 and in
critical care and in surgical monitoring.
7 Piezoelectric Biosensors
The principle of piezoelectric Biosensor is used in
sound vibrations, hence it is called acoustic Biosensors.
The basics of the Biosensors are formed by the
piezoelectric crystals and the characteristic frequencies
are trembling with the crystals of positive and negative
charge. By using the electronic devices we can measure
the certain molecules on the crystal surface and alters
the response frequencies using these crystals we can
attaché the inhibitors. The Biosensors for cocaine in the
gas phase has been developed by attaching the
antibodies cocaine to the surface of the crystal
8 Immuno–Biosensors
 The immobilized antibody can directly combine through the antigen

 The immobilized antigen can combine with the antibody which can twist to a second free
antigen.
 The immobilized antibody combined with the free antigens and enzyme labeled antigen
in opposition.
The immune Biosensors works on the

principle of immunological specificity
and mostly coupled with measurement
on the Potentiometric Biosensors.
There are different configuration of
probabilities for immune Biosensors
some of them are given below and the
figure shows the description
Applications of Biosensors
The applications of different types of biosensors are
 Food analysis
 Study of Biomolecules and their interactions
 Drug development, crime detection
 Medical diagnosis
 Environmental field monitoring
 Industrial process control
 Manufacturing of pharmaceuticals and replacement of organs
A biosensor has a wide range of applications in different fields.
Medicinal Application: biosensors have been used in various diagnostic procedures to determine
various tests.
Industrial application: various manufacturing processes can be monitored by biosensors to
provide assistance with regard to increase the quality and quantity of product obtained.
Environmental application: it helps in measuring the toxicity of water bodies, microbial
contamination of natural resources helping in developing steps towards a cleaner environment.
Military application: it helps to detect explosives, drugs etc., aiding in defence of the people.
Another breakthrough in the field of biosensors was the production of a product called ‘smart
skin’. It is a kind of biosensor which detects any chemical or biological attack nearby and warns
the person using the same.
Drug development: a biosensor called ‘nano sensors’ has been developed which detects and
analyse the binding of proteins to its targets which has proved very useful in drug designing.
This also helps to monitor certain side effects caused by some medicines.
Components of a Biosensor
Detector
“Biosensor” – Any device that uses specific biochemical reactions to

detect chemical compounds in biological samples.
Chiral : Obtaining enantiomerically pure medicinal drugs
One obvious way to obtain enantiomerically pure medicinal drugs is to isolate natural products from the
chiral pool of nature and, if necessary, to derivatize them. Yet, another possibility is a racemic
synthesis and the subsequent separation of the enantiomers (resolution of racemates). However, half of this
process's result would be undesired side product. As more and more enantiomerically pure active agents are
required today, selective synthesis is the subject of many research projects. The selective synthesis of
enantiomers is called asymmetric synthesis, while the corresponding technology is chiral technology.
Separation of enantiomers
RESOLUTION OF RACEMATES
The resolution of racemates is the separation of an equimolar mixture of enantiomers (racemate)

by physical or chemical methods. Usually, the separation is carried out after a preceding conversion
of the enantiomers into diastereomers, because, as a result of their practically identical chemical and
physical properties, entantiomers cannot be separated directly. Certainly, the methods of the
resolution of racemates can also be applied to non-equimolar mixtures of enantiomers that are
usually obtained by asymmetric synthesis, since asymmetric synthesis can never have a
stereoselectivity of 100 %.
Tab.1
Methods of resolution of racemates
Methods Description
In the crystallization process of enantiomers, derived a racemic mixture, the
Manual separation of enantiomers crystallize separately and form two macroscopically different kinds
enantiomers of crystals with a mirror-image relationship. These crystals can be separated
manually with a pair of tweezers.
By salt formation with a chiral acid or base, enantiomers can be converted into
diastereomers, which can then be separated as a result of their different physical
and chemical properties. Subsequently, the acid or base is released and the pure
enantiomers are recovered.Covalent derivatization of enantiomers with chiral
reagents also yields diastereomers. The diastereomers are separated by common
Resolution of
separation techniques, as they possess different physical and chemical properties.
racemates after
Afterwards, separated derivatives are returned to the initial chiral reagent and the
conversion into
desired pure enantiomers.In addition, the separation of enantiomers can be
diastereomers
carried out by chromatographic methods, such as gas chromatography (GC),
high-pressure liquid chromatography (HPLC), and thin-layer chromatography
(TLC) in association with a chiral stationary phase, which retards one enantiomer
relatively more than the other one by stereoselectively constructing of diversely
stable, chiral complexes.
Most enzymes convert their substrate in an enantioselective way. Therefore,
microorganisms can be used to metabolize only one of the enantiomers of a
Biochemical resolution
racemate. If the microorganism is wisely chosen, the desired enantiomer is the
of racemates
only thing that remains and can then be separated from the mixture by common
separation techniques.
In the reaction with chiral reaction partners or chiral catalysts, enantiomers
display different reaction rates. If the difference is large enough, the enantiomers
can be separated by stereoselectively converting only one of the enantiomers,
Kinetic resolution of
while the desired enantiomer remains. Since this is based on different reaction
enantiomers
rates, the separation technique is called kinetic resolution. A special case of
kinetic resolution is the biochemical resolution of racemates by stereoselective
enzymic conversion of one enantiomer.
Synthesis of enantiomerically pure compounds
ASYMMETRIC SYNTHESIS
Asymmetric synthesis is a reaction or reaction sequence in which one configuration of one or more
new stereogenic elements is selectively formed. In an asymmetric synthesis, an achiral molecule is
enantioselectively converted into a chiral molecule or a chiral molecule is diastereoselectively
converted into a new chiral molecule that contains at least one more chirality element.Summary: In
an asymmetric synthesis the enantiomers (or diastereomers) of a chiral product are formed in
different yields.
In effect, a synthesis can be stereoselective if it displays a diastereomeric transition state, because the
transition states for the formation of the different stereoisomeric products then usually contain different
energies. As a result, the activation energies differ from each other. Consequently, the stereoisomer whose
formation requires the lower activation energy is preferably formed. The stereoselectivity increases with
growing energy difference of the transition states. An unconditional prerequisite for a diastereomeric
transition state is the presence of at least one chiral reactant. This may be either the substrate, a reagent, or a
catalyst.
Tab.2
Methods of asymmetric synthesis
Stoichiometric
Description
methods
In an asymmetric synthesis, if an achiral molecule (substrate) should be
stereoselectively converted into a chiral molecule by reaction with an achiral reagent,
one of the reactants must previously be made chiral. This is achieved by covalently
attaching a chiral molecule to a substrate that can be removed and intactly recovered
after the asymmetric synthesis. Such a chiral molecule is called chiral auxiliary. The
stereoselectivity of the asymmetric synthesis is then controlled by the stereochemistry
of the chiral auxiliary. Certainly, in order to successfuly achieve a diastereomeric
Controlled by transition state, a chiral reagent that will not be recovered after the synthesis can also
reagent be applied. The stereochemistry of the chiral auxiliary, or the chiral reagent,
respectively, controls the stereoselectivity of the asymmetric synthesis. In contrast to
a chiral catalyst, the chiral auxiliary, as well as the chiral reagent, must be applied in
appropiate amounts, according to the stoichiometry of the reaction. Therefore, that
kind of asymmetric synthesis is a stoichiometric, reagent-controlled method. A well-
known example of such an asymmetric synthesis is the hydration of alkenes by the
reaction sequence of hydroboration, oxidation, and hydrolysis in association with a
chiral borane reagent.
If the substrate is chiral, the reagent does not necessarily have to be chiral for an
asymmetric synthesis, too. In this case, the stereoselectivity is controlled by the
stereochemistry of the substrate. The substrate is obviously applied in stoichiometric
amounts. Therefore, the asymmetric synthesis is a stoichiometric, substrate-controlled
method.The chiral substrate of such asymmetric syntheses frequently derives from the
large pool of chiral natural products. These starting products are often easily available
Controlled by in an enantiomerically (or diastereomerically) pure form and are, therefore, cheaper
substrate than chemically synthesized enantiomerically (or diastereomerically) pure starting
products. Examples of such chiral natural products are carbohydrates, optically active
carbon acids, terpenes, and sesquiterpenes.However, for many asymmetric syntheses,
no suitable chiral substrate can be found in the chiral pool of natural products. In such
a case, the advantages of reagent- or catalyst-controlled asymmetric syntheses are
obvious: they are broadly applicable and contain a high flexibility with respect to the
range of starting products and products.
An asymmetric synthesis can also be achieved by applying a chiral catalyst. The catalyst can be an enzyme,
or a synthetic catalyst, usually one such as a chiral transition-metal catalyst. Catalytic asymmetric
syntheses are usually more cost-effective, though the enzymes and chiral transition-metal catalysts are often
more expensive than chiral auxiliaries or reagents, because they have to be applied in small, catalytic
quantities instead of stoichiometric amounts. Therefore, an additional advantage of catalytic processes is that
the disposal of byproducts poses less of an environmental impact. However, a disadvantage is the toxicity of
transition metals.
Tab.3
Methods of asymmetric synthesis
Catalytic methods Description

In industrial asymmetric syntheses, more and more transition-metal catalysts with
chiral ligands are applied. A well-known example of an asymmetric synthesis used in
a large-scale production is the Monsanto process for the manufacture of L-DOPA (L-
Synthetic catalysts
dihydroxyphenylalanine). This proved useful in the treatment of Parkinson's disease.
In one catalytical step of this asymmetric synthesis, a chiral rhodium catalyst is
applied to a stereoselective hydrogenation of a double bond.
Enzymes show a high enantioselectivity (or diastereoselectivity) in asymmetric
syntheses. However, an enzyme can often be applied only to the conversion of a very
Enzymes
small group of substrates and, generally, not to a special process. In addition, they
usually need physiologic reaction conditions.
Dynamic kinetic resolution (DKR)

Dynamic kinetic resolution (DKR) is the most widely used technique for deracemisation. It is a combination of
‘classic’ kinetic resolution, in which a racemate (SS and SR) is resolved by chemical transformation of one single
enantiomer into a product (PR), with in situ racemisation of the ‘unwanted’ enantiomer (SS) of the starting material
(Scheme 1). Many examples have been revealed combining metal-catalysed racemisation with enzymatic kinetic
resolution. Since such chemoenzymatic DKRs have already been extensively reviewed,2,3 in this review, we will
focus on DKR processes employing either organocatalysis, transition metal catalysis or enzymatic
transformations. DKR of atropoisomers and so-called attrition-enhanced deracemisation will be also discussed.
Enzymatic DKR In addition to enantiopure organocatalysts and transition metal complexes, enzymes can be
powerful catalysts to induce enantioselective reactions due to their high substrate selectivity. In order to overcome
the 50% yield barrier in enzymatic resolutions of racemic mixtures, the DKR principle has also been recently
applied to a variety of enzymatic reactions.
In 2011 the group of Asano reported enzyme-promoted DKR of α-aminonitriles leading to optically pure α-
amino acids.16 This process consisted of three steps: (i) hydrolysis of racemic α-aminonitrile by a non-selective
nitrile hydratase (NHase), (ii) stereoselective hydrolysis of the resulting α-amino acid amide by an amino acid
amide hydrolase, and (iii) simultaneous racemisation of the α-amino acid amide by ACL racemase (Scheme 15).
Scheme 15 DKR of α-aminonitriles to form chiral α-amino acids.
Several substrates were tested using this system and both (R)- and (S)-amino acids could be obtained in
excellent yields and ee's (>99%). Such an approach, however, will always be dependent on the availability of
different enzymes that are able to convert both enantiomers.
The group of Ramström used a lipase from Burkholderia cepacia (a group of Gram-negative bacteria) to
catalyse the reaction between an acyl donor (phenyl acetate) and an amine to give the acetylated amine.17 In initial
experiments KR of α-aminonitrile 38 was investigated (Scheme 16). The conversion into product (R)-39 was
recorded at 60% with an enantiomeric purity of 88% ee. The conversion exceeding the theoretical 50% maximum
of KR and the observation that the remaining starting material 38 was racemic, clearly indicated that a DKR-type
process took place. Further mechanistic studies led to the conclusion that racemisation of the α-aminonitrile was
catalysed by lipase via a retro-Strecker mechanism through the same active site that catalyses transacylation.
Scheme 16 KR of α-aminonitriles promoted by lipase.

Racemisation most likely occurs when a His-residue abstracts a proton from the amino group, followed by
elimination of the cyanide ion (Scheme 17).
Scheme 17 Transition state for lipase-catalysed racemisation.

This method was demonstrated to give good yields (87–90%) and ee's (85–88%), but only after prolonged
reaction times (7–13 days).
In 2010 Kroutil et al. published a DKR of racemic mandelic acid 40 to yield the corresponding enantiopure α-
amino acid L-phenylglycine (42).18 The α-hydroxy acid was first oxidised to the α-ketoacid 41 using D-mandelate
dehydrogenase (D-MDH), after which it was converted into the α-amino acid via asymmetric reductive amination
with L-amino acid dehydrogenase (L-AADH) (Scheme 18).
Scheme 18 DKR of mandelic acid 40.
Because oxidation and reduction took place simultaneously, the NADH/NAD+ cofactor was recycled in this
system. In the screening process, 10 out of 24 tested AADHs gave excellent results (>97% conversion, >97% ee),
all favouring L-42.
D'Arrigo et al. reported in 2011 an enzymatic DKR of N-Boc-amino acid thioesters.19 In this process, the
authors take advantage of the fact that thioesters, as opposed to the resulting amides, are prone to racemisation
under basic conditions. The formation of the amide bond can be catalysed in an enantioselective manner by
proteases. Subtilisin, commercially available under the name alcalase, is known to be stable in organic solvents (an
aqueous reaction will lead to hydrolysis of the thioester) and in the presence of bases. In this system, ammonia or
amines can be used as nucleophiles (Scheme 19).
Scheme 19 DKR of N-Boc-amino acid thioesters.

Enzymes in pharmaceuticle industries
 Diagnostic procedures
 Extraction of medicinally important compounds
 Manufacture of chemical pharmaceuticals
 Molecular biology
 Supplements for deficiencies
ENZYME ENGINEERING
Improvement in the activity and usefulness of an existing enzyme or creation of a new enzyme activity by
making suitable changes in its amino acid sequence is called Enzyme Engineering. When this approach is
used to modify the properties of any protein, whether enzyme or nonenzyme, it is termed as protein
engineering. Since enzymes are proteins, enzyme engineering is a part of the larger activity of protein
engineering. Enzyme engineering utilizes r-DNA technology to introduce the desired changes in amino acid
sequences of enzymes. In addition, the level of production of an enzyme may be increased by introducing
more copies of the gene into the concerned organism.
OBJECTIVES OF ENZYME ENGINEERING
The chief objective of enzyme engineering is to produce an enzyme that is more useful for industrial and
other applications. The various properties of an enzyme that may be modified to achieve this objective are as
follows :-
1. Improved kinetic properties.

2. Elimination of allosteric regulation.
3. Enhanced substrate and reaction specificity.
4. Increased thermostability.
5. Alteration in optimal pH.
6. Suitability for use in organic solvents.
7. Increased/decreased optimal temperature.
PRINCIPLES OF ENZYME ENGINEERING
The structure and function of an enzyme molecule,

for that matter of any protein molecule, are chiefly determined by its amino acid sequence, i.e, its primary
structure. Therefore, any change in the properties of an enzyme is always reflected in its primary structure.
Conversely, a change in the amino acid

sequence should alter the properties of the
enzyme. But, this is not always the case
because the enzymatic properties, etc. are
changed only when amino acid changes
are introduced in certain critical regions
of the protein. Therefore, it is of great
importance to know the critical regions
for the various functions of an enzyme,
and to be able to predict the effect of
specific amino acid changes in these areas
on the various functions.
The present knowledge of the relationships between amino acid sequences, 3D structure of protein and
properties of enzymes, obtained from a large database is only partially operative. It allows an explanation of
the changes in structure and function on the basis of the changes in amino acid sequence, but it does not
allow a dependable prediction of the influences of specific amino acid changes on the structure and function
of the enzymes. As more elaborate databases and improved softwares become available, it should become
possible to predict with a far greater confidence the structural and functional changes in enzymes produced
by the specified changes in their amino acid sequences. The effectiveness of enzyme engineering will be
greatly enhanced then, and this activity may have a tremendous influence on enzyme technology.
STEPS IN ENZYME ENGINEERING
The strategies for enzyme engineering and their theoretical considerations are quite involved. The steps
involved in enzyme engineering are briefly described in simple terms :-
2. The first step consists of isolation
of the concerned enzyme and
determination of its structure and
properties. Both amino acid sequence and
the 3D structure are usually obtained from
X-ray diffraction, nuclear magnetic
resonance (NMR), etc.
2. The data so obtained are analyzed together with the database of known and putative structural
effects of amino acid substitutions on enzyme structure and function. Molecular modelling is
performed to determine a possible change in amino acid sequence for the desired improvement in the
structure/function of an enzyme.
3. The next step consists of constructing a gene that will encode the amino acid sequence
specified at the end of step 2. This is best achieved by isolation and cloning of the endogenous gene
encoding the concerned enzyme, and using this gene for site directed mutagenesis.
4. Once the appropriated gene is constructed, it is introduced and expressed in a suitable host,
e.g E.coli.
5. The recombinant or mutant enzyme so produced is isolated, purified and used for
determination of its structure and properties. The information so obtained is added to the database. If
the enzyme structure and function are not altered as desired, the next cycle of experimentation
(step 2-5) is undertaken.
Enzyme Technology
A most exciting development over the last few years is the application genetic engineering techniques
to enzyme technology. A full description this burgeoning science is beyond the scope of this text but
some suitable references are given at the end of this chapter. There are a number of properties which
may be improved or altered by genetic engineering including the yield and kinetics of the enzyme, the
ease of downstream processing and various safety aspects. Enzymes from dangerous or unapproved
microorganisms and from slow growing or limited plant or animal tissue may be cloned into safe high-
production microorganisms. In the future, enzymes may be redesigned to fit more appropriately into
industrial processes; for example, making glucose isomerase less susceptible to inhibition by the
Ca2+present in the starch saccharification processing stream.
The amount of enzyme produced by a microorganism may be increased by increasing the number of
gene copies that code for it. This principle has been used to increase the activity of penicillin-G-
amidase in Escherichia coli. The cellular DNA from a producing strain is selectively cleaved by the
restriction endonuclease HindIII. This hydrolyses the DNA at relatively rare sites containing the 5'-
AAGCTT-3' base sequence to give identical 'staggered' ends.
[8.4]
intact DNA cleaved DNA
The total DNA is cleaved into about 10000 fragments, only one of which contains the required genetic
information. These fragments are individual cloned into a cosmid vector and thereby returned to E.
coli. These colonies containing the active gene are identified by their inhibition of a 6-amino-
penicillanic acid-sensitive organism. Such colonies are isolated and the penicillin-G-amidase gene
transferred on to pBR322 plasmids and recloned back into E. coli. The engineered cells, aided by the
plasmid amplification at around 50 copies per cell, produce penicillin-G-amidase constitutively and in
considerably higher quantities than does the fully induced parental strain. Such increased yields are
economically relevant not just for the increased volumetric productivity but also because of reduced
downstream processing costs, the resulting crude enzyme being that much purer.
Figure 8.1. The protein engineering cycle. The
process starts with the isolation and
characterisation of the required enzyme. This
information is analysed together with the
database of known and putative structural
effects of amino acid substitutions to produce a
possible improved structure. This factitious
enzyme is constructed by site-directed
mutagenesis, isolated and characterised. The
results, successful or unsuccessful, are added to
the database, and the process repeated until the
required result is obtained.
Another extremely promising area of genetic engineering is protein engineering. New enzyme
structures may be designed and produced in order to improve on existing enzymes or create new
activities. An outline of the process of protein engineering is shown in Figure 8.1. Such factitious
enzymes are produced by site-directed mutagenesis (Figure 8.2). Unfortunately from a practical point
of view, much of the research effort in protein engineering has gone into studies concerning the
structure and activity of enzymes chosen for their theoretical importance or ease of preparation rather
than industrial relevance. This emphasis is likely to change in the future.
As indicated by the method used for site-directed mutagenesis (Figure 8.2), the preferred pathway for
creating new enzymes is by the stepwise substitution of only one or two amino acid residues out of the
total protein structure. Although a large database of sequence-structure correlations is available, and
growing rapidly together with the necessary software, it is presently insufficient accurately to predict
three-dimensional changes as a result of such substitutions. The main problem is assessing the long-
range effects, including solvent interactions, on the new structure. As the many reported results would
attest, the science is at a stage where it can explain the structural consequences of amino acid
substitutions after they have been determined but cannot accurately predict them. Protein engineering,
therefore, is presently rather a hit or miss process which may be used with only little realistic
likelihood of immediate success. Apparently quite small sequence changes may give rise to large
conformational alterations and even affect the rate-determining step in the enzymic catalysis. However
it is reasonable to suppose that, given a sufficiently detailed database plus suitable software, the
relative probability of success will increase over the coming years and the products of protein
engineering will make a major impact on enzyme technology.
Much protein engineering has been directed at subtilisin (from Bacillus amyloliquefaciens), the
principal enzyme in the detergent enzyme preparation, Alcalase. This has been aimed at the
improvement of its activity in detergents by stabilising it at even higher temperatures, pH and oxidant
strength. Most of the attempted improvements have concerned alterations to:
1. the P1 cleft, which holds the amino acid on the carbonyl side of the targeted peptide bond;
2. the oxyanion hole (principally Asn155), which stabilises the tetrahedral intermediate;
3. the neighbourhood of the catalytic histidyl residue (His64), which has a general base role; and
4. the methionine residue (Met222) which causes subtilisin's lability to oxidation.
It has been found that the effect of a substitution in the P1 cleft on the relative specific activity
between substrates may be fairly accurately predicted even though predictions of the absolute effects
of such changes are less successful. Many substitutions, particularly for the glycine residue at the
bottom of the P1cleft (Gly166), have been found to increase the specificity of the enzyme for particular
peptide links whilst reducing it for others. These effects are achieved mainly by corresponding
changes in the Km rather than the Vmax. Increases in relative specificity may be useful for some
applications. They should not be thought of as the usual result of engineering enzymes, however, as
native subtilisin is unusual in being fairly non-specific in its actions, possessing a large hydrophobic
binding site which may be made more specific relatively easily (e.g. by reducing its size). The
inactivation of subtilisin in bleaching solutions coincides with the conversion of Met222 to its
sulfoxide, the consequential increase in volume occluding the oxyanion hole. Substitution of this
methionine by serine or alanine produces mutants that are relatively stable but with reduced activity.
Figure 8.2. An outline of the process of site-directed mutagenesis, using a hypothetical example. (a) The primary structure of
the enzyme is derived from the DNA sequence. A putative enzyme primary structure is proposed with an asparagine residue
replacing the serine present in the native enzyme. A short piece of DNA (the primer), complementary to a section of the gene
apart from the base mismatch, is synthesised. (b) The oligonucleotide primer is annealed to a single-stranded copy of the
gene and is extended with enzymes and nucleotide triphosphates to give a double-stranded gene. On reproduction, the gene
gives rise to both mutant and wild-type clones. The mutant DNA may be identified by hybridisation with radioactively
labelled oligonucleotides of complementary structure.
An example of the unpredictable nature of protein engineering is given by trypsin, which has an active
site closely related to that of subtilisin. Substitution of the negatively charged aspartic acid residue at the
bottom of its P1 cleft (Asp189), which is used for binding the basic side-chains of lysine or arginine, by
positively charged lysine gives the predictable result of abolishing the activity against its normal
substrates but unpredictably also gives no activity against substrates where these basic residues are
replaced by aspartic acid or glutamic acid.
Considerable effort has been spent on engineering more thermophilic enzymes. It has been found that
thermophilic enzymes are generally only 20-30 kJ more stable than their mesophilic counterparts. This
may be achieved by the addition of just a few extra hydrogen bonds, an internal salt link or extra internal
hydrophobic residues, giving a slightly more hydrophobic core. All of these changes are small enough to
be achieved by protein engineering. To ensure a more predictable outcome, the secondary structure of
the enzyme must be conserved and this generally restricts changes in the exterior surface of the enzyme.
Suitable for exterior substitutions for increasing thermostability have been found to be
aspartate glutamate, lysine glutamine, valine threonine, serine asparagine, isoleucine threonine,
asparagine � aspartate and lysine � arginine. Small increases in the interior hydrophobicity may also
increase the thermostability. Making an enzyme more thermostable reduces its overall flexibility and,
hence, it is probable that the factitious enzyme produced will have reduced catalytic efficiency.
Enzyme catalysis in organic synthesis
–is widely recognized as a first choice opportunity in the preparation of a wide range of chemical
compounds. For numerous molecules the synthetic routes based on enzyme catalysis have turned out to be
competitive (and often superior!) compared with classic chemical as well as chemocatalytic synthetic
approaches. Thus, enzymatic catalysis is increasingly recognized by organic chemists in both academia and
industry as an attractive synthetic tool besides the traditional organic disciplines such as .classic. synthesis,
metal catalysis, and organocatalysis.
By means of enzymes a broad range of transformations relevant in organic chemistry can be
catalyzed, including, for example, redox reactions, carbon–carbon bond forming reactions, and hydrolytic
reactions. Organic chemists did not use enzymes as catalysts for their envisioned syntheses because of
observed (or assumed) disadvantages such as
 narrow substrate range,
 limited stability of enzymes under organic reaction conditions,
 low efficiency when using wild-type strains,
 diluted substrate and product solutions, thus leading to non-satisfactory volumetric productivities.
However, due to tremendous progress in enzyme discovery, enzyme engineering, and process
development, in recent years numerous examples of organic syntheses with (tailor-made) enzymes have
been developed that avoid these disadvantages.
Enzyme Catalysts: Three-Dimensional Structure and General Properties The unique functions of
enzymes as catalytically active proteins are a result of their complex three-dimensional structures and the
active site integrated therein. This enables a highly specific recognition of specific substrates, leading to
excellent selectivities.
Besides chemoselectivity, the stereoselectivity of enzymes is also in general high to excellent and,
furthermore, this is typically true for regio-, diastereo-, as well as enantioselectivity.
In addition to the substrate the reaction conditions also play a very important role in enzyme catalysis. it is
evident that enzyme catalysis requires specific suitable reaction conditions such as pH, temperature, and
solvent, which have to be considered in (bio-)process development. In the following, some selected reaction
parameters of specific importance for enzymatic reactions are briefly discussed. For enzymes pH and
temperature are certainly highly important reaction parameters in terms of both activity and stability.
Typically, enzymes operate in a more or less neutral or weakly basic/acidic pH range, usually between pH 5
and 10, although exceptions are known. The natural reaction environment for enzymes is water.
Interestingly, however, water as a reaction medium is not necessarily required and many enzymatic
transformations (including industrial processes) are run in organic reaction medium.
1
2
3
4
5
6
7
Molecular imprinting
is the technology of creating artificial recognition sites in polymeric matrices which are complementary to
the template in their size, shape and spatial arrangement of the functional groups. The interactions between
molecules that are important in processes such as affinity separation and enzymatic catalysis relay on
molecular recognition. Through molecular imprinting, it is possible to develop tailor made polymers that are
selective for different compounds.
Molecularly imprinted polymers (MIPs) and their incorporation with various transducer platforms are
among the most promising approaches for detection of several analytes. Recently, molecular imprinting
technology has been used for the creation of biorecognition surfaces on biosensors.
Molecularly imprinted polymers (MIPs) are prepared by co-polymerization of cross-linkingmonomer and a
complex which is pre-formed between the template molecule and functional monomers using covalent, non-
covalent or semi-covalent interactions. When the template molecule is removed from the imprinted material
after polymerization, it leaves behind specific cavities that are complementary to the template in size, shape
and chemical functionality .
MIPs have been used successfully in many applications, including purification , isolation , chiral separation ,
catalysis and in biosensors
There are different types of molecular imprinting techniques, including

bulk imprinting, surface imprinting and epitope imprinting.
Bulk Imprinting
a template molecule is imprinted as a whole in the polymer matrix and after polymerization it needs to be
wholly removed from the molecularly imprinted material. In the next step, to form small particles from
these bulk polymers, the bulk polymer is crushed mechanically and the formed particles are fractionated.
By this way, readily accessible, template-specific 3D interaction sites are obtained within the selective,
imprinted polymeric material. Bulk imprinting is generally preferred for the imprinting of small molecules
since, at least in theory, the adsorption and release of the template molecule are faster and reversible which
is an advantage for the re-usability of the imprinted support for several rounds of analyses.
Surface Imprinting
High affinity recognition sites are formed at the surface of a substrate in surface imprinting. Due to this the
recognition sites are more easily accessible with favorable binding kinetics. In other words,
template/polymer interactions are not diffusion limited to the same extent as was a general problem
encountered in bulk imprinting. Therefore, the technique is popular and most applicable especially for
1
imprinting of biomolecules including proteins. In surface imprinting, less template molecules are used as
compared to what is used in conventional imprinting techniques because template is only used in the
surface coating step in the technique. The main drawback of the method in sensor design is the possibility
of lower sensitivity as compared to bulk imprinting owing to the reduced number of imprinted sites.
Surface imprinted polymers have been widely used for different types of analytes, including proteins,
microorganisms and cells
types of surface imm: soft lithography, template immobilization, grafting, emulsion polymerization
In the soft lithography : general procedure of the method, micro- or nano-scaled patterns ranging from 30
nm to 100 _m are formed on the solid substrates by using a soft polymeric stamp [23]. A pre-polymerized
layer is coated on a transducer surface and the template stamp is pressed over this surface for a certain
period of time. Self-assembling of template structures including proteins, microorganisms onto a smooth
support are used to produce the template stamps which are complementary to the template in their structural,
geometrical and also chemical features.
Figure 1. Schematic representation of
fabrication process of patterned MIP
films via soft lithography and UV
polymerization. (1–2) Imprinting solution
containing the template molecule,
functional and cross-linking monomers
and initiator was dropped on the
substrate. (3) The patterned PDMS
mold was placed on the droplet of
solution under certain pressure to ensure
enough physical contact between the
PDMS and substrate. Then, UV
polymerization was initiated and
continued for 7–10 min. (4) Following de-
molding, the patterned MIP films were
dried at 60 _C. (5) In the last step,
template molecule (atrazine) was
removed from the surface (reproduced
from [29] with permission).
2
Emulsion Polymerization
Another surface imprinting strategy is emulsion polymerization. In the general procedure of the technique,
core particles are synthesized before they are covered with imprinted shells. Tan et al. [59] used surface
3
imprinting strategy based on covalently immobilized templates to prepare BSA-imprinted sub-micrometer
particles via two-stage core shellmini-emulsion polymerization. Through the encapsulation of Fe3O4
nanoparticles, the MIP particles gained magnetic properties which increases their potential applications in
various fields including magnetic bioseparation, cell labeling and bioimaging. Imprinted particles with non-
immobilized (free) template molecules (fMIP) did not display the expected template recognition and this
illustrates the importance of the template immobilization for a successful surface imprinting. The MIP
prepared according to this strategy displayed high specific recognition for BSA without any adsorption of
the competitive proteins. Therefore the technique has potential to be used for the preparation of highly
selective biosensor surfaces in further applications.
The general procedure of the technique involves three steps:

(1) In the first step, negatively charged bacteria were assembled with a positively charged pre-polymer
which contained vinyl groups,
(2) In the next step, bacteria-pre polymer complexes were used as the particle stabilizer to form emulsion of
a cross-linking monomer (the oil phase) in water. The cross-linking monomer was polymerized by free
radical polymerization and by this way the pre-polymer was covalently fixed to the core of the polymer
beads.
(3) After template removal, bacteria-imprinted sites were obtained on the surface of the polymer beads.
Factors influencing the characteristics of the imprinted polymers The structure of the polymer matrix is
crucial in molecular imprinting. The specific structure of the cavity is determined not only by the low
molecular weight template molecules but also by the fixed arrangement of the polymer chains. The
characteristics of the polymer matrix like the matrix structure, matrix configuration, matrix nature, template
used and the extent of crosslinking has a significant effect on rebinding.
(a) Rigidity/ Flexibility of the polymer support The stiffness of the polymer structure enables the cavities to
retain their shape even after the removal of the template, thus giving higher selectivity. This can be
achieved by high degree of crosslinking. The binding of the template into the polymer during
polymerisation, directs the positioning of the imprinted substrate within the cavities of the crosslinked
polymer and it is assumed that the polymers with optimum selectivity and specificity characteristics were
obtained by suitable selection of monomers and crosslinking agents.
(b) Functional and crosslinking monomers Majority of reports on molecular imprinting describe organic
polymers synthesised by radical polymerisation of functional and crosslinking monomers having vinyl or
acrylic groups. This can be attributed to the fairly straightforward synthesis of these materials and to the
vast choice of available monomers. Other approaches are the polystyrene-based and polysiloxane-based
systems, but to a lesser extent. The functional monomers can be basic, acidic, permanently charged,
hydrogen bonding, hydrophobic and others.
(c) Monomer–template ratio The molar relationship between the functional monomer and template (M/T)
has been found to be important with respect to the number and quality of MIP recognition sites. Low M/T
ratios result in less than optimal complexation on account of insufficient functional monomers. Most of the
functional monomer will be complexed and only a limited number of functional monomer will be located
outside the imprinted cavities scattered on the surface of the polymer particles. During rebinding, the
imprinted binding sites may compete successfully with the residual monomers for a limited number of
guests, thus increasing selectivity. But when the ratio (M/T) is high, only a limited number of functional
monomer is complexed with the template while those located outside the cavities, remain scattered in the
bulk of the polymer particles. Thus too high ratios yield non-selective binding – the extreme case being the
4
non-imprinted polymer. The parameter “imprinting effect” gives an estimation of how well the polymer
was imprinted by the template.
(d) Porogen / Rebinding solvent Solvent is used in the imprinting studies both as the porogen during
polymerisation and as rebinding medium. The trivial role of solvents is to dissolve the agents for
polymerisation. However they have more crucial roles. It provides porosity within the network, which
facilitates mass transfer of the analyte to and from the binding sites. In the polymerisation, solvent
molecules are incorporated inside the polymers and are removed in the post-treatment. Then the space
originally occupied by the solvent molecules is left as pores in the polymers. Another role of solvents is to
disperse the heat of reaction generated on polymerisation. Otherwise, the temperature of reaction mixture is
locally elevated, and undesired side-reaction occur
(e) Polymerisation temperature The position of equilibrium between free template - monomer(s) and their
corresponding complexes is a product of both temperature and pressure65. Sellergren group showed that
high pressure (1000 bar) polymerisation could be used to enhance the selectivity of the resultant imprinted
polymers66. Previous studies found that lower temperature of polymerisation is favourable for the
preparation of MIPs based on electrostatic interaction due to the greater strength of electrostatic interaction
at lower temperature67, 68. At low temperature the polymerisation process is slow and the chain formation
does not interfere with the template - monomer interactions. An optimal condition of temperature should be
found for each combination of template and monomer55. pH of the rebinding medium also induces a
significant role in molecular imprinting, especially in covalent imprinting69.

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sage of ionized gases, radiation, time, humidity, and [ -CH- ]n

Number of Particles in Effluent

One point that confuses users of absolute rated fil-

indicated by the Absolute Rating are invariably

doubt on the very concept of an “absolute”rating.

or wash out a newly manufactured filter without

can be used to measure and predict the perfor- 104

installed downstream of the filter to prevent

As noted above, the pressure drop across the fil-

30 sq. ft. will collect that same filter cake in x

While the 30 sq. ft. filter has collected a filter cake

It is for this reason that Pall Corporation uses con-

5 Laboratory dirt capacity tests using ACFTD, or any

023 9230 3303 phone

Measurement of Cell Numbers

(a) Area of microscopic field = πr2

1. Electrode 2. Flow of Suspending Fluid 3. Bacterial Cell 4. Orifice

Steps of Membrane Filter Technique

Measurement of Cell Mass

1. Dry Weight Technique

Measurement of nitrogen content

Measurement of Turbidity (Turbidometry)

1. Source of Light of a single wave-length (monochromatic) 2. Filter

McFarland standards: In microbiology, McFarland standards are used as a reference to adjust

McFarland Nephelometer Standards:

McFarland Standard No. 0.5 1 2 3 4

spectrophotometer to estimate absorbance of cell suspensions. The absorbance at a particular

2.5.2 GROWTH KINETICS IN BATCH CULTURE

Using this equation, a plot of the natural log of biomass

concentration versus time should yield a straight line, the slope

Initial substrate concentration

GROWTH KINETICS IN CONTINUOUS CULTURE

Where F is the flow rate (l/h) and V is the volume (l).

dx/dt = growth – output or dx/dt = µx – Dx

dx/dt = 0 and µx = dx and µ = D

where is the steady state concentration of substrate in the chemostat.

Rearranging the equation:

Therefore a chemostat is a nutrient-limited, self-balancing culture system which may be maintained in a

The cell concentration in a chemostat is defined by:

By combining equation of steady state substrate and biomass concentrations:

Therefore biomass concentration at steady state is defined by operational variables SR and D. If SR is

According to the mode of interaction biosensors are of two types:

Essential properties of a biosensor:

Components and mechanism of a biosensor:

The Biosensors are based on the electrons

Blood Glucose Biosensor

In this type of Biosensors changes the

Optical Biosensors for Blood Glucose

 The immobilized antibody can directly combine through the antigen

The immune Biosensors works on the

“Biosensor” – Any device that uses specific biochemical reactions to

The resolution of racemates is the separation of an equimolar mixture of enantiomers (racemate)

Catalytic methods Description

Dynamic kinetic resolution (DKR)

Scheme 16 KR of α-aminonitriles promoted by lipase.

Scheme 17 Transition state for lipase-catalysed racemisation.

Scheme 19 DKR of N-Boc-amino acid thioesters.

OBJECTIVES OF ENZYME ENGINEERING