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Home , Affinity chromatography

ADVANCE SEPARATION METHODS

CHE 823

BY ADEWUYI Adewale

Chemical Sciences Redeemer’s University

This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.

Chromatography

A technique for analysis of chemical substances. The term literally means color writing, and denotes a method by which the substance to be analyzed is poured into a vertical glass tube containing an adsorbent, the various components of the substance moving through the adsorbent at different rates of speed, according to their degree of attraction to it, and producing bands of color at different levels of the column. The term has been extended to include other methods utilizing the same principle, although no colors are produced in the column.

The mobile phase of chromatography refers to the fluid that carries the mixture of substances in the sample through the adsorptive material. The stationary or adsorbent phase refers to the solid material that takes up the particles of the substance passing through it. Kaolin, alumina, silica, and activated charcoal have been used as adsorbing substances or stationary phases.

Classification

Classification of chromatographic techniques tends to be confusing because it may be based on the type of stationary phase, the nature of the adsorptive force, the nature of the mobile phase, or the method by which the mobile phase is introduced.

Conventionally, chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture mainly purification. Analytical chromatography is done normally with smaller amounts of material and is for establishing the presence or measuring the relative proportions of analytes in a mixture. Preparative chromatography is used to purify sufficient quantities of a substance for further use, rather than analysis.

A – According to mechanism of separation: The mechanism of separation depends mainly on the nature of the stationary phase. Based on separation mechanisms chromatography can be classified into: Adsorption Chromatography: It is the oldest and most common type of chromatography. The stationary phase is a solid with adsorption power. Mixture components will be adsorbed on the surface of the stationary phase with different powers and that account for separation. Silica gel is the most common stationary phase in adsorption chromatography Partition Chromatography : The stationary phase is a liquid forming a thin film on an inert solid acts as support. The stationary liquid is usually more polar than the mobile liquid. The two liquids must be immiscible with each other. Cellulose powder and wet silica gel are examples of supports in partition chromatography that carry film of water act as stationary phase. Partition chromatography is preferable over adsorption when dealing with polar compounds Ion Exchange Chromatography: It is used for separation of charged molecules. The stationary phase is an ion exchange resin to which a cationic or anionic groups are covalently bonded. Ions of opposite charges (counter ions) in the mobile phase will be attracted to the resin and compete with the components of the mixture for the charged group on the resin. Both the mixture components and the mobile phase must be changed. Mixture of Alkaloids (compounds with positive charges) can be separated on anionic exchanger, while mixture of organic acids (negative charges) can be separated using cationic exchanger. Both types are used for desalination of water. Molecular Exclusion (Size Exclusion) Chromatography: Stationary phase has pores of defined diameter. Very large molecules cant enter into the pores and so elute first, Large molecule enter with difficulties and so elute second. Small molecules enter all pores and elute as last ones Affinity Chromatography: It uses the affinity of to specific ligands such as . The is attached to suitable polysaccharide such as cellulose - – dextran. Chiral Chromatography: In this type we can separate enantiomers – we used chiral stationary phase that react with one enantiomer more then the other so separation takes place.

B - According to the mobile phase In this regard chromatography is classified into: Liquid Chromatography (LC):The mobile phase is liquid. In case of separation by adsorption the stationary phase is solid so it is called: Liquid-Solid Chromatography (LSC). If separation occurs through partition the stationary phase is liquid so it is called: Liquid - Liquid Chromatography (LLC). (GC): Where the mobile phase is inert gas nitrogen or helium. Again if the stationary phase is solid it is called: Gas–Solid Chromatography (GSC). When stationary phase is liquid it is called: Gas-Liquid Chromatography (GLC).

C- According to the technique applied (methods of holding the stationary phase) 1- Planar or Plane Chromatography: In this type of chromatography the stationary phase is used in the form of layer. Plane chromatography is further classified into: a- Thin Layer Chromatography (TLC): The stationary phase in the form of fine powder is spread on glass or plastic or aluminum sheets. b- (PC): A specific type of papers is used as stationary phase in the form of sheets. 2- Columnar or (CC): The stationary phase is held in to a tube made of glass or metal.

D- According to purpose of use Chromatography can be used for analytical work and also to obtain pure materials from mixtures. 1- Analytical Chromatography: a- Qualitative Chromatography: In this case Chromatography can be used to: 1- Confirm the absence or probable presence of certain constituent in the sample under investigation: 2- Give an idea about the complexity of the mixture and the least number of compounds present. 3- Check purity and identity of any compound. 4- Establish a (finger print ) pattern for extracts , volatile oils or pharmaceutical preparations. These finger prints can be then used to check the identity and purity in the future. 5- Monitor both column chromatography and organic chemical reactions.

b- Quantitative Chromatography: The development of modern instruments enable the use of chromatography to determine the amount of any component in a mixture as absolute amount or relative to another component HPLC/ GC/ HPTLC can be used for there applications. . 2- Preparative and Industrial scale Chromatography : This was the first and is the main application of chromatography. The technique was developed primarily for this purpose. Chromatography is used to obtain reasonable quantities of pure compounds from mixtures.

HPLC is distinguished from traditional ("low pressure") liquid chromatography because operational pressures are significantly higher (50–350 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. Due to the small sample amount separated in analytical HPLC, typical column dimensions are 2.1–4.6 mm diameter, and 30–250 mm length. Also HPLC columns are made with smaller sorbent particles (2–50 μm in average particle size). This gives HPLC superior resolving power (the ability to distinguish between compounds) when separating mixtures, which makes it a popular chromatographic technique.

Isocratic and gradient

A separation in which the mobile phase composition remains constant throughout the procedure is termed isocratic (meaning constant composition). The mobile phase composition does not have to remain constant. A separation in which the mobile phase composition is changed during the is described as a gradient elution. One example is a gradient starting at 10% methanol and ending at 90% methanol after 20 minutes. The two components of the mobile phase are typically termed "A" and "B"; A is the "weak" solvent which allows the solute to elute only slowly, while B is the "strong" solvent which rapidly elutes the solutes from the column. In reversed-phase chromatography, solvent A is often water or an aqueous buffer, while B is an organic solvent miscible with water, such as acetonitrile, methanol, THF, or isopropanol. In isocratic elution, peak width increases with retention time linearly according to the equation for N, the number of theoretical plates. This leads to the disadvantage that late-eluting peaks get very flat and broad. Their shape and width may keep them from being recognized as peaks.

Gradient elution decreases the retention of the later-eluting components so that they elute faster, giving narrower (and taller) peaks for most components. This also improves the peak shape for tailed peaks, as the increasing concentration of the organic eluent pushes the tailing part of a peak forward. This also increases the peak height (the peak looks "sharper"), which is important in trace analysis. The gradient program may include sudden "step" increases in the percentage of the organic component, or different slopes at different times – all according to the desire for optimum separation in minimum time. In isocratic elution, the selectivity does not change if the column dimensions (length and inner diameter) change – that is, the peaks elute in the same order. In gradient elution, the elution order may change as the dimensions or flow rate change.

The driving force in reversed phase chromatography originates in the high order of the water structure. The role of the organic component of the mobile phase is to reduce this high order and thus reduce the retarding strength of the aqueous component.

Normal–phase chromatography Normal–phase chromatography was one of the first kinds of HPLC that chemists developed. Also known as normal-phase HPLC (NP-HPLC) this method separates analytes based on their affinity for a polar stationary surface such as silica, hence it is based on analyte ability to engage in polar interactions (such as hydrogen-bonding or dipole-dipole type of interactions) with the sorbent surface. NP-HPLC uses a non-polar, non-aqueous mobile phase (e.g. Chloroform), and works effectively for separating analytes readily soluble in non-polar solvents. The analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity. The interaction strength depends not only on the functional groups present in the structure of the analyte molecule, but also on steric factors. The effect of steric hindrance on interaction strength allows this method to resolve (separate) structural isomers. The use of more polar solvents in the mobile phase will decrease the retention time of analytes, whereas more hydrophobic solvents tend to induce slower elution (increased retention times). Very polar solvents such as traces of water in the mobile phase tend to adsorb to the solid surface of the stationary phase forming a stationary bound (water) layer which is considered to play an active role in retention. Reversed-phase chromatography (RPC) Reversed phase HPLC (RP-HPLC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is a silica which has been surface-modified with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. With such stationary phases, retention time is longer for molecules which are less polar, while polar molecules elute more readily (early in the analysis). An investigator can increase retention times by adding more water to the mobile phase; thereby making the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase. Similarly, an investigator can decrease retention time by adding more organic solvent to the eluent. RP-HPLC is so commonly used that it is often incorrectly referred to as "HPLC" without further specification. The pharmaceutical industry regularly employs RP-HPLC to qualify drugs before their release.

1. Clinical diagnosis of diseases, disorders. 2. In scientific research for discovery. 3. In pharmaceutical labs for analysis. 4. In food industry for quality control. 5. For standards control by government. 6. For separation of similar molecules. Foods

High Performance Liquid Chromatography has brought desirable advantages in the field of food analysis. Food matrices are generally complex and extraction of analytes is not an easy task. To further complicate matters both desirable and undesirable components are often found in trace levels and classical extraction and analysis does not provide the required levels of accuracy and precision. HPLC offers viable solutions due to vast choice of stationary phases and mobile phase options. Common applications in foods are : Fat soluble vitamins (A,D,E and K)

• Water soluble vitamins (B-complex vitamins such as B1, B2, B3, B6, Folic acid, Pantothenic acid, B12, VitaminC) • Residual pesticides such as 2, 4-D and Monochrotophos. • Antioxidants such as TBHQ, BHA and BHT. • Sugars: Glucose, Fructose, Maltose and other saccharides. • Cholesterol and sterols • Dyes and synthetic colours. • Mycotoxins such as Aflatoxins B1,B2,G1,G2,M1,M2and ochratoxin • Amino acids • Residual antibiotics • Steroids and flavanoids • Aspartame and other artificial sweeteners. • Active ingredients of farm produce such as allin in garlic and catachin in tea extracts. Analysis of Vitamins

Vitamins are a well-known group of compounds that are essential for human health and are classified into two main groups, water-soluble and fat-soluble. Water-soluble vitamins include B group vitamins (thiamine/B1, riboflavin/B2, nicotinamide/nicotinic acid/B3, pantothenic acid/B5, pyridoxine/pyridoxal hydrochloride/B6, folic acid/B9, and cyanocobalamine/B12) and ascorbic acid (vitamin C). Fat-soluble vitamins include mainly retinol (vitamin A), tocopherol (vitamin E), radiostol (vitamin D), and antihemorrhagic vitamins (vitamin K).

HPLC Methods for Water- and Fat-Soluble

Vitamin Analysis Traditional HPLC Method

Reversed-phase HPLC is a well-suited technique for vitamin analysis. In typical regulated HPLC methods and commonly reported HPLC methods, water-soluble vitamins are determined using an aqueous mobile phase with low-organic solvent content, whereas fat-soluble vitamins are determined using organic solvent mobile phases. This is due to their different solubility and reversed-phase retention properties. Commonly used buffers for the separation of water- soluble vitamins are phosphate, formic acid, and acetic acid. Non-aqueous reversed-phase (NARP) retention is commonly used for fat-soluble vitamins so that the vitamins are soluble throughout the analysis. A typical NARP mobile phase consists of a polar solvent (acetonitrile), a solvent with lower polarity (e.g., dichloromethane) to act as a solubilizer and to control retention by adjusting the solvent strength, and a third solvent with hydrogen bonding capacity (e.g., methanol) to optimize selectivity. Sample preparation Vit C

AA can be mostly found in fruits and vegetables. The main sources of AA are citrus fruits, hips, strawberries, peppers, tomatoes, cabbage, spinach and others. If one wants to uptake AA from animal sources, liver and kidney are the tissues with highest contents of this molecule, but in comparison with plant sources the amount of AA is very low .

*The pharmaceutical preparation (a tablet) should be ground in a mortar (n = 5). Then, ground powder (about 1 mg) dissolved in ACS water (1 mL). Oranges and apples can be used. The pericarps of the fruits were removed, and then the fruits (app. 0.25 g) were homogenized using a mortar. The extracts obtained were filtered through filter paper, transferred into a volumetric flask and diluted with ACS water. Measurements of the samples were carried out immediately after preparation steps. The same for human blood serum samples. Human sera were frozen at –20 °C immediately after collection. The samples were 100 × diluted with ACS water and filtered through 0.45 µm Teflon membrane filter prior to measurement. *Calibration curve (internal standard) *Develop a suitable mobile phase and programming. *Method blank: Describe – Blank spike and Matrix spike [important]

*Chemical Structures, UV Spectra, and Detection Wavelengths The UV spectra of water-and fat-soluble vitamins vary significantly due to their multiple structures (Figure below) and therefore, multiwavelength detection is required for achieving the best sensitivity. Usually, the maximum absorbance is the best choice, but the wavelength selected can be different because at certain wavelengths, impurities may interfere with analyte detection. Method Blank The Method Blank will show contamination that may have occurred during the preparation step. If the level of the analyte in the blank is greater than 10% of the level of the analyte in the sample, the sample will be flagged. This will alert the data user that a high bias should be taken into account. The flagged data will only be reported when the analytes in question are significantly lower than the regulatory limits or insufficient volume was received to perform a re-analysis. Levels of analyte in the blank below 10% of the level of the analyte in the sample would not be considered significant, should not be flagged and would not affect data usability.

Blank Spike The Blank Spike is a measure of the accuracy of the test procedure. If an analyte for any Blank spikes is outside of criteria, that particular analyte needs to be evaluated. If Blank Spikes are above criteria, there is possibility of a high bias; below criteria, there is a possibility of a low bias for the analytes being evaluated. High bias would not be of concern for samples under a regulatory limit. Low bias would be of concern for samples that are under a regulatory limit depending on proximity to the limit. Extrapolation based on percent recovery would be advisable, however extremely low recoveries would affect data usability.

Matrix Spike The Matrix Spike is a measure of accuracy with regards to matrix effect. It is evaluated in the same way as the Blank Spike. The difference being that analytes that are outside of criteria in the Matrix Spike and not in the Blank Spike are showing matrix effects. Again extrapolation can be used however extremely low recoveries affect data usability. Vit C continue………….. Its maximum absorption is at approximately 245 nm; however, a large amount of vitamin C is usually added to some functional waters (e.g., sports drinks), which may result in the concentration being outside the linear range of calibration. Therefore, detection at other wavelengths (i.e., 254 or 265 nm) may place its concentration in a linear calibration range. Table 2 lists some reported detection wavelengths for water- and fat-soluble vitamins and the detection wavelengths used in the analysis presented here.

Analysis of Steroids in blood serum

100 µL of serum or plasma samples, calibrators and quality controls were spiked with 20 µL of internal standards (final concentrations specified ) followed by 400 µL of water.

Thus, calibration curves went through identical sample preparation and were derived in each analytical run.

Sample processing was performed. Briefly, each sample was applied to a 1 mL Bond Elut® C18 SPE cartridge previously conditioned with methanol and water. The sample loading rate was 0.1 mL/min, and the samples were washed with 1 mL of water followed by 1 mL of hexane at a flow rate of 1 mL/min. The steroid analytes were eluted with 1 mL of ethyl acetate at a flow rate of 0.1 mL/min. Solvents were evaporated to dryness under a stream of high purity nitrogen at 45°C using a sample concentrator. The dry extracts were reconstituted in 100 µL of 50∶50 methanol/water, and 40 µL was injected into the LC-MS/MS. Supercritical Fluid Chromatography (SFC) is a form of normal phase chromatography that is used for the analysis and purification of low to moderate molecule molecular weight, thermally labile molecules. It can also be used for the separation of chiral compounds. Principles are similar to those of HPLC, however SFC typically utilizes carbon dioxide as the mobile phase; therefore the entire chromatographic flow path must be pressurized. Since the supercritical phase represents a state in which liquid and gas properties converge, supercritical fluid chromatography is sometimes called "convergence chromatography." The mobile phase is composed primarily of supercritical carbon dioxide, but since CO2 on its own is too non-polar to effectively elute many analytes, cosolvents are added to modify the mobile phase polarity. Cosolvents are typically simple alcohols like methanol, ethanol, or isopropyl alcohol. Other solvents such as acetonitrile, chloroform, or ethyl acetate can be used as modifiers. For food-grade materials, the selected cosolvent is often ethanol or ethyl acetate, both of which are generally recognized as safe (GRAS). The solvent limitations are system and column based.

Limitations There have been a few technical issues that have limited adoption of SFC technology, first of which is the high pressure operating conditions. High-pressure vessels are expensive and bulky, and special materials are often needed to avoid dissolving gaskets and O-rings in the supercritical fluid. A second drawback is difficulty in maintaining pressure (backpressure regulation). Whereas liquids are nearly incompressible, so their densities are constant regardless of pressure, supercritical fluids are highly compressible and their physical properties change with pressure - such as the pressure drop across a packed-bed column. Currently, automated backpressure regulators can maintain a constant pressure in the column even if flow rate varies, mitigating this problem. A third drawback is difficulty in gas/liquid separation during collection of product. Upon depressurization, the CO2 rapidly turns into gas and aerosolizes any dissolved analyte in the process. Cyclone separators have lessened difficulties in gas/liquid separations.

Membrane Separation Techniques Membrane separation processes operate without heating and therefore use less energy than conventional thermal separation processes such as , sublimation or . The separation process is purely physical and both fractions (permeate and retentate) can be used.

Uses Cold separation using membrane technology is widely used in the food technology, biotechnology and pharmaceutical industries.

It enables separations to take place that would be impossible using thermal separation methods. For example, it is impossible to separate the constituents of azeotropic liquids or solutes which form isomorphic crystals by distillation or recrystallization but such separations can be achieved using membrane technology.

In wastewater treatment, membrane technology is becoming increasingly important. With the help of ultra/ it is possible to remove microorganisms, particles, colloids and macromolecules, so that waste-water can be disinfected in this way.

Principle Via Mass transfer: Two basic models can be distinguished for mass transfer through the membrane:  solution-diffusion model and  hydrodynamic model. Solution-diffusion model

In the solution-diffusion model, transport occurs only by diffusion. The component that needs to be transported must first be dissolved in the membrane. The general approach of the solution-diffusion model is to assume that the chemical potential of the feed and permeate fluids are in equilibrium with the adjacent membrane surfaces such that appropriate expressions for the chemical potential in the fluid and membrane phases can be equated at the solution-membrane interface. This principle is more important for dense membranes without natural pores such as those used for and in fuel cells. During the process a boundary layer forms on the membrane. This concentration gradient is created by molecules which cannot pass through the membrane. The effect is referred as concentration polarization and, occurring during the filtration, leads to a reduced trans-membrane flow (flux). Concentration polarization is, in principle, reversible by cleaning the membrane which results in the initial flux being almost totally restored. Using a tangential flow to the membrane (cross- flow filtration) can also minimize concentration polarization.

Hydrodynamic model Transport through pores – in the simplest case – is done convectively. This requires the size of the pores to be smaller than the diameter of the two separate components. Membranes which function according to this principle are used mainly in micro- and . They are used to separate macromolecules from solutions, colloids from a dispersion or remove bacteria. During this process the retained particles or molecules form a pulpy mass (filter cake) on the membrane, and this blockage of the membrane hampers the filtration. This blockage can be reduced by the use of the cross-flow method (cross-flow filtration). Here, the liquid to be filtered flows along the front of the membrane and is separated by the pressure difference between the front and back of the membrane into retentate (the flowing concentrate) on the front and permeate (filtrate) on the back. The tangential flow on the front creates a shear stress that cracks the filter cake and reduces the fouling.

Membrane separation processes Membrane separation processes differ based on separation mechanisms and size of the separated particles. The widely used membrane processes include: microfiltration, ultrafiltration, nanofiltration, reverse osmosis, electrolysis, dialysis, electrodialysis, gas separation, vapor permeation, pervaporation, membrane distillation, and membrane contactors. All processes except for pervaporation involve no phase change. All processes except (electro)dialysis are pressure driven. Microfltration and ultrafiltration is widely used in food and beverage processing (beer microfiltration, apple juice ultrafiltration), biotechnological applications and pharmaceutical industry (antibiotic production, purification), water purification and wastewater treatment, the microelectronics industry, and others. Nanofiltration and reverse osmosis membranes are mainly used for water purification purposes.

Dense membranes are utilized for gas separations (removal of CO2 from natural gas, separating N2 from air, organic vapor removal from air or a nitrogen stream) and sometimes in membrane distillation. The later process helps in the separation of azeotropic compositions reducing the costs of distillation processes.

Pore size and selectivity

The pore sizes of technical membranes are specified differently depending on the manufacturer. One common distinction is by nominal pore size. It describes the maximum pore size distribution and gives only vague information about the retention capacity of a membrane. The exclusion limit or "cut-off" of the membrane is usually specified in the form of NMWC (nominal molecular weight cut-off, or MWCO, molecular weight cut off, with units in Dalton). It is defined as the minimum molecular weight of a globular molecule that is retained to 90% by the membrane.

The cut-off, depending on the method, can by converted to so-called D90, which is then expressed in a metric unit. In practice the MWCO of the membrane should be at least 20% lower than the molecular weight of the molecule that is to be separated.

Filter membranes are divided into four classes according to pore size: (kDa =kilodalton)

Pore size Molecular mass Process Filtration Removal of > 10 "Classic" filter > 0.1 µm > 5000 kDa microfiltration < 2 bar larger bacteria, yeast, particles bacteria, macromolecules, proteins, 100-2 nm 5-5000 kDa ultrafiltration 1-10 bar larger viruses 2-1 nm 0.1-5 kDa nanofiltration 3-20 bar viruses, 2- valent ions < 1 nm < 100 Da reverse osmosis 10-80 bar salts, small organic molecules

The form and shape of the membrane pores are highly dependent on the manufacturing process and are often difficult to specify. Therefore, for characterization, test are carried out and the pore diameter refers to the diameter of the smallest particles which could not pass through the membrane. Microfiltration The process Microfiltration, ultrafitration and reverse osmosis are related membrane processes differing in the size of the material retained by the membrane. As shown in Figure 6, reverse osmosis membranes can generally separate dissolved microsolutes with a molecular weight below 500 by a solution-diffusion mechanism. When the molecular weight of the solute exceeds 500, the separation mechanism of the membrane is molecular filtration, in which separation characteristics are determined by the size of the particles in the mixture and the diameter of the pores in the membrane. By convention, membranes having pore sizes up to approximately 0.1 µm in diameter are considered to be ultrafiltration membranes. Microfiltration membranes are those with pore diameters in the range of 0.1 to 10 µm. Above 10 µm the separation medium is considered to be a conventional filter. Ultrafiltration/microfiltration membranes fall into two broad categories: screen membrane and depth membranefilters, as shown in Figure 7. Screenfilters are anisotropic with small surface pores on a more open substructure. The surface pores in screen membranefilters are uniform and show a sharp cutoff between material that is completely retained by the membrane and material that penetrates the membrane. Retained material accumulates on the membrane surface. Depth membranefilters have a much wider distribution of pore sizes and usually have a more diffuse cutoff than screen membranefilters. Very large particulates are retained on the surface of the membrane, but smaller particulates entering the membrane are trapped at constrictions or adsorbed onto the membrane surface. Screenfilters are usually used in ultrafiltration applications (see next section). The membrane pores are normally very small, on the order of 5-50 nm in diameter. Particulates and colloidal matter retained at the membrane surface are removed by a tangential flow of the feed solution. In this type of process, 80-90 vol% of the feed solution permeates the membrane as a clean filtrate. The remaining solution containing the rejected material is collected as a concentrated residue.

Depthfilters are usually used in microfiltration applications. The surface membrane pores can be quite large, on the order of 1-10 µm in diameter, but many smaller restrictions occur in the interior of the membrane. This means that bacteria or virus particles as small as 0.2 µm in diameter are completely prevented from penetrating the membrane. Microfiltration membranes are usually used as an in-line filter. All of the feed solution is forced through the membrane by an applied pressure. Retained particles are collected on or in the membrane. The lifetime of microfiltration membranes is often improved by using a more open prefilter membrane. directly before the final membrane. Prefilters are not absolute filters, but trap most of the very large particulates and many of the smaller ones before the feed solution reaches the finer membrane filter. This reduces the particle load that the finer membrane must handle, and thus increases its useful life.

Applications The primary market for microfiltration membranes is disposable cartridges for sterile filtration of water for the pharmaceutical industry and final point-of-use polishing of ultrapure water for the electronics industry. The cost of microfiltration compared with the value of the products is small. Cold sterilization of beer, wine and other beverages is another emerging market area. In these processes the microfiltration cartridge removes all yeast and bacteria from the filtrate. This process was introduced on a commercial scale in the 1960s. Although not generally accepted at that time, the process has become common in recent years.