BIOSEPARATIONS AND DOWNSTREAM TECHNOLOGY

PRESSURE LEAF FILTER

The Pressure Leaf Filter is a MS/SS Vertical Vessel with Filter Leaves inside. The leaves are mounted vertically on a common manifold pipe, through which the filtered liquid flows out. On the top, the leaves are held by a vibrating shaft . A mechanical vibrator driven by electric motor/pneumatic vibrator is provided for vibrating the leaf shaft for cake discharge. Jacket for hot filtration can be offered if desired Over flow, vent/steam/air charging, pressure gauge & safety valve are provided on the top.

The top cover is provided with devit arm mechanism for raising the lid for cleaning/ removing the leaves. “I” bolts are provided for quick opening and closing of top lid. A mechanical jack is provided to lift the top for cleaning/removing the leaves. Lugs are provided for mounting the PLF.

The Filter leaves filter on both sides and hence a large filtration area is obtained in a relatively small vessel.

 

Working Of The Pressure Leaf Filter
Unfiltered liquid is pumped into the filter vessel. Initially the filter aid starts forming a precoat layer on both sides of the filter leaves, until then cloudy material comes out of the Filter. Once the layer is formed , Pressure starts developing, restricting the impurities. Clear liquid flows from both sides into the leaves ( filter elements), flows along the tubular channel & gets discharged from Bottom of the leaf. All the leaves are mounted on a common manifold. The leaves start getting choked on both the sides by impurities forming cake, which is in wet form. Once the leaves get chocked completely, the pressure rises to 3-4 Kg/cm² & the output flow almost stops.

The Pump is stopped & steam /air pressure is applied from the top ( without dropping the filter pressure) to filter the material around the leaves held up in the tank & to squeeze the cake further & reduce the liquid retention in it . The hold up unfiltered liquid in the conical portion is taken back.

In PLF the cake can be dried by steam /hot air & then discharged from the bottom with the help of mechanical/pneumatic leaves vibrator. The whole operation of cake drying takes 30-45 min, & cake discharge takes 5-10 min.

For wet cake discharge water spray nozzles are provided.

The Pressure Leaf Filter is used in

• EDIBLE OIL INDUSTRY :- Bleached ,winterized , Deodorized , Hydrogenated Fractionzed oils, Dewaxing, Catalyst, Mineral oil, Sulphur.
• BEVERAGE INDUSTRY :- For Glucose, fruit Juices, cold drinks, sugar, vinegar.
• CHEMICAL INDUSTRY : - For Organic & Inorganic salts , dyes, chemicals, plastizers
• PHARMA INDUSTRY :- For Pharmaceutical intermediates, syrup, bulk drugs, antibiotics, intravenous solutions.
• PETROCHEMICAL INDUSTRY :- Crude oil, LPG, lubricating oil, sulphur.
• Interesterification
• Herbal Extraction Plants
• Micronutrient Recovery
• Essential Oil Distillation
• High Vacuum Separation by Molecular Distillation
• Lube Oil Rerefining

Advantages

·         The cloth or woven mesh screens that cover the leaves of horizontal tanks may be accessed easily once the stack is pulled out of the vessel.

·         Mechanically simple since there are no complex sealing glands or bearings.

Disadvantages

• High headroom is required for dismantling the leaves on vertical vessels.
• Large floor space is required for discharging the cake on horizontal vessels.
• The emptying of the vessel in between cake filtration, washing and drying requires close monitoring of the pressure inside the vessel to ensure that the cake holds on to the candles.

Adsorption

Adsorption, the binding of molecules or particles to a surface, must be distinguished from absorption, the filling of pores in a solid. The binding to the surface is usually weak and reversible.

The most common industrial adsorbents are activated carbon, silica gel, and alumina, because they present enormous surface areas per unit weight. Activated carbon is produced by roasting organic material to decompose it to granules of carbon - coconut shell, wood, and bone are common sources. Silica gel is a matrix of hydrated silicon dioxide. Alumina is mined or precipitated aluminum oxide and hydroxide. Although activated carbon is a magnificent material for adsorption, its black color persists and adds a grey tinge if even trace amounts are left after treatment; however filter materials with fine pores remove carbon quite well.

A surface already heavily contaminated by adsorbates is not likely to have much capacity for additional binding. Freshly prepared activated carbon has a clean surface. Charcoal made from roasting wood differs from activated carbon in that its surface is contaminated by other products, but further heating will drive off these compounds to produce a surface with high adsorptive capacity. Although the carbon atoms and linked carbons are most important for adsorption, the mineral structure contributes to shape and to mechanical strength. Spent activated carbon is regenerated by roasting, but the thermal expansion and contraction eventually disintegrate the structure so some carbon is lost or oxidized.

Temperature effects on adsorption are profound, and measurements are usually at a constant temperature. Graphs of the data are called isotherms, that is, the amount of adsorbate on the adsorbent as a function of its pressure (if gas) or concentration (if liquid) at constant temperature.

In the process of adsorption, adsorbate gets adsorbed on adsorbent.

Freundlich Adsorption Isotherm:

Though Freundlich Isotherm correctly established the relationship of adsorption with pressure at lower values, it failed to predict value of adsorption at higher pressure. This relation is called as the freundlich adsorption isotherm.

Freundlich Isotherm is empirical one but desribe the adsorption of a wide variety of antibiotics, steroids, harmones.

Langmuir Adsorption Isotherm:

It is a semi-empirical isotherm derived from a proposed kinetic mechanism. It is used to describe the adsorption of proteins.

It is based on four assumptions:

1.    The surface of the adsorbent is uniform, that is, all the adsorption sites are equivalent.

2.    Adsorbed molecules do not interact.

3.    All adsorption occurs through the same mechanism.

4.    At the maximum adsorption, only a monolayer is formed: molecules of adsorbate do not deposit on other, already adsorbed, molecules of adsorbate, only on the free surface of the adsorbent.

Protein adsorption of biomaterials

Protein adsorption is a process that has a fundamental role in the field of biomaterials. Indeed, biomaterial surfaces in contact with biological media, such as blood or serum, are immediately coated by proteins. Therefore, living cells do not interact directly with the biomaterial surface, but with the adsorbed proteins layer. This protein layer mediates the interaction between biomaterials and cells, translating biomaterial physical and chemical properties into a "biological language".^[8] In fact, cell membrane receptors bind to protein layer bioactive sites and these receptor-protein binding events are transduced, through the cell membrane, in a manner that stimulates specific intracellular processes that then determine cell adhesion, shape, growth and differentiation. Protein adsorption is influenced by many surface properties such as surface wettability, surface chemical composition ^[9] and surface nanometre-scale morphology.

Microfiltration

Microfiltration (commonly abbreviated to MF) is a type of physical filtration process where a contaminated fluid is passed through a special pore-sized membrane to separate microorganisms and suspended particles from process liquid. It is commonly used in conjunction with various other separation processes such as ultrafiltration and reverse osmosis to provide a product stream which is free of undesired contaminants.

Microfiltration usually serves as a pre-treatment for other separation processes such as ultrafiltration, and a post-treatment for granular media filtration. The typical particle size used for microfiltration ranges from about 0.1 to 10 µm. The filters used in the microfiltration process are specially designed to prevent particles such as, sediment, algae, protozoa or large bacteria from passing through a specially designed filter. More microscopic, atomic or ionic materials such as water (H2O), monovalent species such as Sodium (Na+) or Chloride (Cl-) ions, dissolved or natural organic matter, and small colloids and viruses will still be able to pass through the filter.^[3]

A pump is commonly fitted onto the processing equipment to allow the liquid to pass through the membrane filter. There are also two pump configurations, either pressure driven or vacuum. A differential or regular pressure gauge is commonly attached to measure the pressure drop between the outlet and inlet streams. Applications:

• Water Treatment
• Sterilization
• Petroleum Refining
• Dairy Processing

 

           Micro filtration                      Ultra filtration                                  RO

Ultrafiltration

Ultrafiltration (UF) is a variety of membrane filtration in which forces like pressure or concentration gradients lead to a separation through a semipermeable membrane. Suspended solids and solutes of high molecular weight are retained in the so-called retentate, while water and low molecular weight solutes pass through the membrane in the permeate. This separation process is used in industry and research for purifying and concentrating macromolecular (10³ - 10⁶ Da) solutions, especially protein solutions. Ultrafiltration is not fundamentally different from microfiltration. Both of these separate based on size exclusion or particle capture. It is fundamentally different from membrane gas separation, which separate based on different amounts of absorption and different rates of diffusion. Ultrafiltration is applied in cross-flow or dead-end mode.

Principles

The basic operating principle of ultrafiltration uses a pressure induced separation of solutes from a solvent through a semi permeable membrane. The relationship between the applied pressure on the solution to be separated and the flux through the membrane is most commonly described by the Darcy equation:

where J is the flux (flow rate per membrane area),TMP is the transmembrane pressure (pressure difference between feed and permeate stream), μ is solvent viscosity, R_t is the total resistance (sum of membrane and fouling resistance).

Applications

Drinking Water

Protein Concentration

Filtration of effluent from paper pulp mill

Cheese manufacture, see ultrafiltered milk

Removal of pathogens from milk

Process and waste water treatment

Enzyme recovery

Fruit juice concentration and clarification

Dialysis and other blood treatments

Desalting and solvent-exchange of proteins (via diafiltration)

Laboratory grade manufacturing

 

 

 

Reverse osmosis

Reverse osmosis (RO) is a water purification technology that uses a semipermeable membrane. This membrane technology is not properly a filtration method. Reverse osmosis can remove many types of molecules and ions from solutions, and is used in both industrial processes and the production of potable water. The result is that the solute is retained on the pressurized side of the membrane and the pure solvent is allowed to pass to the other side. To be "selective", this membrane should not allow large molecules or ions through the pores (holes), but should allow smaller components of the solution (such as the solvent) to pass freely.

Reverse osmosis is most commonly known for its use in drinking water purification from seawater, removing the salt and other effluent materials from the water molecules.

 

Process

Osmosis is a natural process. When two liquids with different concentrations of a solute are separated by a semipermeable membrane, the fluid has a tendency to move from low to high solute concentrations for chemical potential equilibrium.

Formally, reverse osmosis is the process of forcing a solvent from a region of high solute concentration through a semipermeable membrane to a region of low solute concentration by applying a pressure in excess of the osmotic pressure. The largest and most important application of reverse osmosis is the separation of pure water from seawater and brackish waters; seawater or brackish water is pressurized against one surface of the membrane, causing transport of salt-depleted water across the membrane and emergence of potable drinking water from the low-pressure side.

The membranes used for reverse osmosis have a dense layer in the polymer matrix—either the skin of an asymmetric membrane or an inter facially polymerized layer within a thin-film-composite membrane—where the separation occurs. In most cases, the membrane is designed to allow only water to pass through this dense layer, while preventing the passage of solutes (such as salt ions). This process requires that a high pressure be exerted on the high concentration side of the membrane, usually 2–17 bar (30–250 psi) for fresh and brackish water, and 40–82 bar (600–1200 psi) for seawater, which has around 27 bar (390 psi)^[4] natural osmotic pressure that must be overcome. This process is best known for its use in desalination (removing the salt and other minerals from sea water to get fresh water), but since the early 1970s, it has also been used to purify fresh water for medical, industrial, and domestic applications.

Applications:

 

Drinking water purification

Food industry

Maple syrup production

Hydrogen production

Reef aquariums

Window cleaning

 

Electrodialysis

Electrodialysis (ED) is used to transport salt ions from one solution through ion-exchange membranes to another solution under the influence of an applied electric potential difference. This is done in a configuration called an electrodialysis cell. The cell consists of a feed (diluate) compartment and a concentrate (brine) compartment formed by an anion exchange membrane and a cation exchange membrane placed between two electrodes. In almost all practical electrodialysis processes, multiple electrodialysis cells are arranged into a configuration called an electrodialysis stack, with alternating anion and cation exchange membranes forming the multiple electrodialysis cells. Electrodialysis processes are different compared to distillation techniques and other membrane based processes (such as reverse osmosis) in that dissolved species are moved away from the feed stream rather than the reverse. Because the quantity of dissolved species in the feed stream is far less than that of the fluid, electrodialysis offers the practical advantage of much higher feed recovery in many applications.

Method

In an electrodialysis stack, the diluate (D) feed stream, brine or concentrate (C) stream, and electrode (E) stream are allowed to flow through the appropriate cell compartments formed by the ion exchange membranes. Under the influence of an electrical potential difference, the negatively charged ions (e.g., chloride) in the diluate stream migrate toward the positively charged anode. These ions pass through the positively charged anion exchange membrane, but are prevented from further migration toward the anode by the negatively charged cation exchange membrane and therefore stay in the C stream, which becomes concentrated with the anions. The positively charged species (e.g., sodium) in the D stream migrate toward the negatively charged cathode and pass through the negatively charged cation exchange membrane. These cations also stay in the C stream, prevented from further migration toward the cathode by the positively charged anion exchange membrane.^[7] As a result of the anion and cation migration, electric current flows between the cathode and anode. Only an equal number of anion and cation charge equivalents are transferred from the D stream into the C stream and so the charge balance is maintained in each stream. The overall result of the electrodialysis process is an ion concentration increase in the concentrate stream with a depletion of ions in the diluate solution feed stream.

Applications

·         Large scale brackish and seawater desalination and salt production.

·         Small and medium scale drinking water production (e.g., towns & villages, construction & military camps, nitrate reduction, hotels & hospitals)

·         Water reuse (e.g., industrial laundry wastewater, produced water from oil/gas production, cooling tower makeup & blowdown, metals industry fluids, wash-rack water)

·         Pre-demineralization (e.g., boiler makeup & pretreatment, ultrapure water pretreatment, process water desalination, power generation, semiconductor, chemical manufacturing, food and beverage)

·         Food processing

·         Agricultural water (e.g., water for greenhouses, hydroponics, irrigation, livestock)

·         Glycol desalting (e.g., antifreeze / engine-coolants, capacitor electrolyte fluids, oil and gas dehydration, conditioning and processing solutions, industrial heat transfer fluids, secondary coolants from heating, venting, and air conditioning (HVAC))

·         Glycerin Purification

Retention time

The time taken for a particular compound to travel through the column to the detector is known as its retention time. This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound.

Different compounds have different retention times. For a particular compound, the retention time will vary depending on:

·         the pressure used (because that affects the flow rate of the solvent)

·         the nature of the stationary phase (not only what material it is made of, but also particle size)

·         the exact composition of the solvent

·         the temperature of the column

Retention volume

The product of retention time and eluent flow rate, so called "retention volume", is more of a global retention parameter. Retention volume, V_R represent the volume of the eluent passed through the column while eluting a particular component.

Component retention volume V_R could be split into two parts:

1. Reduced retention volume is the volume of the eluent that passed through the column while the component was sitting on the surface.
2. Dead volume is the volume of the eluent that passed through the column while the component was moving with the liquid phase.

Retention volume is independent of the flow parameters for the particular run, but it depend on the geometrical parameters of the column. V_R will be different for the same compound eluted on the different columns packed with the same type of adsorbent.

The more universal and fundamental retention parameter is the ratio of the retention volume and dead volume (k).

k = V_R/V_o

Affinity chromatography

Affinity chromatography is a method of separating biochemical mixtures based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.

Principle

The stationary phase is typically a gel matrix, often of agarose; a linear sugar molecule derived from algae. Usually the starting point is an undefined heterogeneous group of molecules in solution, such as a cell lysate, growth medium or blood serum. The molecule of interest will have a well known and defined property, and can be exploited during the affinity purification process. The process itself can be thought of as an entrapment, with the target molecule becoming trapped on a solid or stationary phase or medium. The other molecules in the mobile phase will not become trapped as they do not possess this property. The stationary phase can then be removed from the mixture, washed and the target molecule released from the entrapment in a process known as elution. Possibly the most common use of affinity chromatography is for the purification of recombinant proteins.

Uses

Affinity chromatography can be used to:

• Purify and concentrate a substance from a mixture into a buffering solution
• Reduce the amount of a substance in a mixture
• Discern what biological compounds bind to a particular substance
• Purify and concentrate an enzyme solution.

What is HPLC?

High-performance liquid chromatography or high-pressure liquid chromatography (HPLC) is a chromatographic method that is used to separate a mixture of compounds in analytical chemistry and biochemistry so as to identify, quantify or purify the individual components of the mixture.

Benefits of HPLC

The key benefits of HPLC systems are as follows:

• Controls and automates chromatography instrumentation
• Provides data management, security features, and reporting and instrument validation.
• Powerful and adaptable
• Increases productivity by managing all the areas of analysis - from sample to instrument, and from separation to reporting results.
• Affordable

Applications of HPLC

HPLCs can be used in the following applications:

• Water purification
• Preconcentration of trace components
• Ligand-exchange chromatography
• Ion-exchange chromatography of proteins
• High-pH anion-exchange chromatography of carbohydrates and oligosaccharides

Size-exclusion chromatography

Size-exclusion chromatography (SEC) is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight.^[1] It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography.

Gas chromatography

Gas chromatography (GC), is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture (the relative amounts of such components can also be determined). In some situations, GC may help in identifying a compound. In preparative chromatography, GC can be used to prepare pure compounds from a mixture.^[1][2]

In gas chromatography, the mobile phase (or "moving phase") is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen. The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called a column (an homage to the fractionating column used in distillation). The instrument used to perform gas chromatography is called a gas chromatograph (or "aerograph", "gas separator").

Instrumental components

Carrier gas

The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is often dependant upon the type of detector which is used. The carrier gas system also contains a molecular sieve to remove water and other impurities.

 

Sample injection port

For optimum column efficiency, the sample should not be too large, and should be introduced onto the column as a "plug" of vapour - slow injection of large samples causes band broadening and loss of resolution. The most common injection method is where a microsyringe is used to inject sample through a rubber septum into a flash vapouriser port at the head of the column. The temperature of the sample port is usually about 50°C higher than the boiling point of the least volatile component of the sample.

Columns

There are two general types of column, packed and capillary (also known as open tubular). Packed columns contain a finely divided, inert, solid support material (commonly based on diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm.

Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of two types; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT).

Both types of capillary column are more efficient than packed columns.

Column temperature

For precise work, column temperature must be controlled to within tenths of a degree. The optimum column temperature is dependant upon the boiling point of the sample. As a rule of thumb, a temperature slightly above the average boiling point of the sample results in an elution time of 2 - 30 minutes. Minimal temperatures give good resolution, but increase elution times. If a sample has a wide boiling range, then temperature programming can be useful. The column temperature is increased (either continuously or in steps) as separation proceeds.

Detectors

There are many detectors which can be used in gas chromatography. Different detectors will give different types of selectivity. A non-selective detector responds to all compounds except the carrier gas, a selective detector responds to a range of compounds with a common physical or chemical property and a specific detector responds to a single chemical compound.

Detector	Type	Support gases	Selectivity
Flame ionization (FID)	Mass flow	Hydrogen and air	Most organic cpds.
Thermal conductivity (TCD)	Concentration	Reference	Universal
Electron capture (ECD)	Concentration	Make-up	Halides, nitrates, nitriles, peroxides, anhydrides, organometallics
Photo-ionization (PID)	Concentration	Make-up	Aliphatics, aromatics, ketones, esters, aldehydes, amines, heterocyclics, organosulphurs, some organometallics

 

The effluent from the column is mixed with hydrogen and air, and ignited. Organic compounds burning in the flame produce ions and electrons which can conduct electricity through the flame. A large electrical potential is applied at the burner tip, and a collector electrode is located above the flame. The current resulting from the pyrolysis of any organic compounds is measured. FIDs are mass sensitive rather than concentration sensitive; this gives the advantage that changes in mobile phase flow rate do not affect the detector's response. The FID is a useful general detector for the analysis of organic compounds; it has high sensitivity, a large linear response range, and low noise. It is also robust and easy to use, but unfortunately, it destroys the sample.

Applications

Gas chromatography is a physical separation method in where volatile mixtures are separated. It can be used in many different fields such as pharmaceuticals, cosmetics and even environmental toxins. Since the samples have to be volatile, human breathe, blood, saliva and other secretions containing large amounts of organic volatiles can be easily analyzed using GC. Knowing the amount of which compound is in a given sample gives a huge advantage in studying the effects of human health and of the environment as well. 

Air samples can be analyzed using GC. Most of the time, air quality control units use GC coupled with FID in order to determine the components of a given air sample. Although other detectors are useful as well, FID is the most appropriate because of its sensitivity and resolution and also because it can detect very small molecules as well.

GC/MS is also another useful method which can determine the components of a given mixture using the retention times and the abundance of the samples. This method be applied to many pharmaceutical applications such as identifying the amount of chemicals in drugs. Moreover, cosmetic manufacturers also use this method to effectively measure how much of each chemical is used for their products. 

GAS-LIQUID CHROMATOGRAPHY

All forms of chromatography involve a stationary phase and a mobile phase. In all the other forms of chromatography you will meet at this level, the mobile phase is a liquid. In gas-liquid chromatography, the mobile phase is a gas such as helium and the stationary phase is a high boiling point liquid absorbed onto a solid.

How fast a particular compound travels through the machine will depend on how much of its time is spent moving with the gas as opposed to being attached to the liquid in some way.



Injection of the sample

Very small quantities of the sample that you are trying to analyse are injected into the machine using a small syringe. The syringe needle passes through a thick rubber disc (known as a septum) which reseals itself again when the syringe is pulled out.

The injector is contained in an oven whose temperature can be controlled. It is hot enough so that all the sample boils and is carried into the column as a gas by the helium (or other carrier gas).

 How the column works

 The packing material

There are two main types of column in gas-liquid chromatography. One of these is a long thin tube packed with the stationary phase; the other is even thinner and has the stationary phase bonded to its inner surface.

The column is typically made of stainless steel and is between 1 and 4 metres long with an internal diameter of up to 4 mm. It is coiled up so that it will fit into a thermostatically controlled oven. 

The column temperature

The temperature of the column can be varied from about 50°C to 250°C. It is cooler than the injector oven, so that some components of the mixture may condense at the beginning of the column.

How separation works on the column

One of three things might happen to a particular molecule in the mixture injected into the column:

·         It may condense on the stationary phase.

·         It may dissolve in the liquid on the surface of the stationary phase.

·         It may remain in the gas phase.

None of these things is necessarily permanent.

A compound with a boiling point higher than the temperature of the column will obviously tend to condense at the start of the column. However, some of it will evaporate again in the same way that water evaporates on a warm day - even though the temperature is well below 100°C. The chances are that it will then condense again a little further along the column.

Retention time

The time taken for a particular compound to travel through the column to the detector is known as its retention time. This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound.

Different compounds have different retention times. For a particular compound, the retention time will vary depending on:

·         the boiling point of the compound

·         the solubility in the liquid phase.

·         the temperature of the column

The detector

There are several different types of detector in use. The flame ionisation detector described below is commonly used and is easier to describe and explain than the alternatives.

 A flame ionisation detector

In terms of reaction mechanisms, the burning of an organic compound is very complicated. During the process, small amounts of ions and electrons are produced in the flame. The presence of these can be detected.

The whole detector is enclosed in its own oven which is hotter than the column temperature. That stops anything condensing in the detector.

As it burns, it will produce small amounts of ions and electrons in the flame. The positive ions will be attracted to the cylindrical cathode. Negative ions and electrons will be attracted towards the jet itself which is the anode.

At the cathode, the positive ions will pick up electrons from the cathode and be neutralised. At the anode, any electrons in the flame will transfer to the positive electrode; and negative ions will give their electrons to the electrode and be neutralised.

This loss of electrons from one electrode and gain at the other will result in a flow of electrons in the external circuit from the anode to the cathode. In other words, you get an electric current.

The current won't be very big, but it can be amplified. The more of the organic compound there is in the flame, the more ions will be produced, and so the higher the current will be. As a reasonable approximation, especially if you are talking about similar compounds, the current you measure is proportional to the amount of compound in the flame.

Interpreting the output from the detector

The output will be recorded as a series of peaks - each one representing a compound in the mixture passing through the detector.

Column chromatography

Column chromatography in chemistry is a method used to purify individual chemical compounds from mixtures of compounds. It is often used for preparative applications on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process.

 

Column chromatography is one of the most useful methods for the separation and purification of both solids and liquids. This is a solid - liquid technique in which the stationary phase is a solid & mobile phase is a liquid. The principle of column chromatography is based on differential adsorption of substance by the adsorbent. 

The usual adsorbents employed in column chromatography are silica, alumina, calcium carbonate, calcium phosphate, magnesia, starch, etc., selection of solvent is based on the nature of both the solvent and the adsorbent. The rate at which the components of a mixture are separated depends on the activity of the adsorbent and polarity of the solvent. If the activity of the adsorbent is very high and polarity of the solvent is very low, then the separation is very slow but gives a good separation. On the other hand, if the activity of adsorbent is low and polarity of the solvent is high the separation is rapid but gives only a poor separation, i.e., the components separated are not 100% pure.

The adsorbent is made into slurry with a suitable liquid and placed in a cylindrical tube that is plugged at the bottom by a piece of glass wool or porous disc. The mixture to be separated is dissolved in a suitable solvent and introduced at the top of the column and is allowed to pass through the column. As the mixture moves down through the column, the components are adsorbed at different regions depending on their ability for adsorption. The component with greater adsorption power will be adsorbed at the top and the other will be adsorbed at the bottom. The different components can be desorbed and collected separately by adding more solvent at the top and this process is known as elution. That is, the process of dissolving out of the components from the adsorbent is called elution and the solvent is called is called eluent. The weakly adsorbed component will be eluted more rapidly than the other. The different fractions are collected separately. Distillation or evaporation of the solvent from the different fractions gives the pure components.

Intermolecular forces, which vary in strength according to their type, make organic molecules to bind to the stationary phase. The stronger the intermolecular force, the stronger the binding to the stationary phase, therefore the longer the compound takes to go through the column.

Ion exchangers

Ion exchange is an exchange of ions between two electrolytes or between an electrolyte solution and a complex. In most cases the term is used to denote the processes of purification, separation, and decontamination of aqueous and other ion-containing solutions with solid polymeric or mineralic 'ion exchangers'.

Typical ion exchangers are ion exchange resins (functionalized porous or gel polymer), zeolites, montmorillonite, clay, and soil humus. Ion exchangers are either cation exchangers that exchange positively charged ions (cations) or anion exchangers that exchange negatively charged ions (anions).

However, the simultaneous exchange of cations and anions can be more efficiently performed in mixed beds that contain a mixture of anion and cation exchange resins, or passing the treated solution through several different ion exchange materials.

Ion exchangers can be unselective or have binding preferences for certain ions or classes of ions, depending on their chemical structure. This can be dependent on the size of the ions, their charge, or their structure.

Applications of Ion Exchange:

Water and Waste Treatment

·  Softening (removal of hardness), demineralizing, silica removal, and alkalinity reduction

·  Removal of cations and anions from boiler feeds

·  Deionizing water

·  Treatment of trade effluents.

Purification

·  Recovery of organic and inorganic substances

·  Separation of ion mixtures

What is SDS-PAGE electrophoresis?

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a technique for separating proteins based on their ability to move within an electrical current, which is a function of the length of their polypeptide chains or of their molecular weight. This is achieved by adding SDS detergent to remove secondary and tertiary protein structures and to maintain the proteins as polypeptide chains. The SDS coats the proteins, mostly proportional to their molecular weight, and confers the same negative electrical charge across all proteins in the sample.

SDS-PAGE workflow

Sample preparation		SDS-PAGE gel electrophoresis		Protein blotting		Protein detection

 

Affinity protein isolation

Convenient protein separation

Efficient, 7-minute western blotting

Accurate protein identification

 

Freeze-drying

Freeze-drying, also known as lyophilisation, lyophilization, or cryodesiccation, is a dehydration process typically used to preserve a perishable material or make the material more convenient for transport. Freeze-drying works by freezing the material and then reducing the surrounding pressure to allow the frozen water in the material to sublimate directly from the solid phase to the gas phase.

The freeze-drying stages

There are four stages in the complete drying process: pretreatment, freezing, primary drying, and secondary drying.

Pretreatment

Pretreatment includes any method of treating the product prior to freezing. This may include concentrating the product, formulation revision (i.e., addition of components to increase stability and/or improve processing), decreasing a high vapor pressure solvent or increasing the surface area. Methods of pretreatment include: freeze concentration, solution phase concentration, formulation to preserve product appearance, formulation to stabilize reactive products, formulation to increase the surface area, and decreasing high vapor pressure solvents.

Freezing

In a lab, this is often done by placing the material in a freeze-drying flask and rotating the flask in a bath, called a shell freezer, which is cooled by mechanical refrigeration, dry ice and methanol, or liquid nitrogen. On a larger scale, freezing is usually done using a freeze-drying machine. In this step, it is important to cool the material below its triple point, the lowest temperature at which the solid and liquid phases of the material can coexist. This ensures that sublimation rather than melting will occur in the following steps. Larger crystals are easier to freeze-dry.

Primary drying

During the primary drying phase, the pressure is lowered (to the range of a few millibars), and enough heat is supplied to the material for the water to sublime. The amount of heat necessary can be calculated using the sublimating molecules’ latent heat of sublimation. In this initial drying phase, about 95% of the water in the material is sublimated. This phase may be slow (can be several days in the industry), because, if too much heat is added, the material’s structure could be altered.

Secondary drying

The secondary drying phase aims to remove unfrozen water molecules, since the ice was removed in the primary drying phase. This part of the freeze-drying process is governed by the material’s adsorption isotherms. In this phase, the temperature is raised higher than in the primary drying phase, and can even be above 0 °C, to break any physico-chemical interactions that have formed between the water molecules and the frozen material. Usually the pressure is also lowered in this stage to encourage desorption (typically in the range of microbars, or fractions of a pascal). However, there are products that benefit from increased pressure as well.

After the freeze-drying process is complete, the vacuum is usually broken with an inert gas, such as nitrogen, before the material is sealed.

Applications of freeze-drying

Pharmaceutical and biotechnology

Pharmaceutical companies often use freeze-drying to increase the shelf life of the products, such as vaccines and other injectables. By removing the water from the material and sealing the material in a vial, the material can be easily stored, shipped, and later reconstituted to its original form for injection.

Food and Agro industry

Although Freeze-drying is used to preserve food, the resulting product being very light weight, its early use was initiated processing of crops such as peanuts/groundnuts . The process has been popularized in the forms of freeze-dried ice cream, an example of astronaut food. It is also widely used to produce essences or flavourings to add to food. Instant coffee is sometimes freeze-dried, despite the high costs of the freeze-driers used.

Technological industry

In chemical synthesis, products are often freeze-dried to make them more stable, or easier to dissolve in water for subsequent use.

In bioseparations, freeze-drying can be used also as a late-stage purification procedure, because it can effectively remove solvents.

Other uses

In bacteriology freeze-drying is used to conserve special strains.

In high-altitude environments, the low temperatures and pressures can sometimes produce natural mummies by a process of freeze-drying.

Bioleaching

Bioleaching is leaching where the extraction of metal from solid minerals into a solution is facilitated by the metabolism of certain microbes - bioleaching microbes. Bioleaching is a process described as "the use of microorganisms to transform elements so that the elements can be extracted from a material when water is filtered trough it".

Methods

Heap leaching is the most common method for bioleaching and is mainly used for secondary copper ores. Stirred tank leaching is used for refractory gold concentrates where gold is locked into the pyrite/arsenopyrite matrix.

As the microbes do not necessarily need to contact the valuable metal-bearing material that is bioleached, they can be physically separated from it:

• Direct bioleaching. The microbes are kept together with the valuable metal-bearing material
• Indirect bioleaching. The microbes are kept in a pond external to the valuable metal-bearing material and provide the leaching chemicals at a distance.

The process

The extraction of iron can involve numerous ferrous and sulfur oxidizing bacteria, including Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans (formerly known as Thiobacillus). For example, bacteria catalyse the breakdown of the mineral arsenopyrite (FeAsS) by oxidising the sulfur and metal (in this case arsenic ions) to higher oxidation states whilst reducing dioxygen by H₂ and Fe³⁺. This allows the soluble products to dissolve.

The electrons pass into the cells and are used in biochemical processes to produce energy for the bacteria to reduce oxygen molecules to water.

In stage 2, bacteria oxidise Fe²⁺ to Fe³⁺ (whilst reducing O₂).

They then oxidise the metal to a higher positive oxidation state. With the electrons gained, they reduce Fe³⁺ to Fe²⁺ to continue the cycle.

The iron is now separated from the ore and in solution.

The process for copper is very similar however the efficiency and kinetics depend on the copper mineralogy. The most efficient minerals are supergene minerals such as chalcocite, Cu₂S and Covellite, CuS. The main copper mineral chalcopyrite (CuFeS₂) is not leached very efficiently which is why the dominant copper producing technology remains flotation followed by smelting and refining. The leaching of CuFeS2 follows the two stages of being dissolved and then further oxidised, with Cu²⁺ ions being left.

Advantages of bioleaching

• economical: bioleaching is in general simpler and, therefore, cheaper to operate and maintain than traditional processes, since fewer specialists are needed to operate complex chemical plants.
• environmental: The process is more environmentally friendly than traditional extraction methods. For the company this can translate into profit, since the necessary limiting of sulfur dioxide emissions during smelting is expensive. Less landscape damage occurs, since the bacteria involved grow naturally, and the mine and surrounding area can be left relatively untouched. As the bacteria breed in the conditions of the mine, they are easily cultivated and recycled.
• Ore concentration: Bioleaching can be used extract metals from ores that are too poor for other technologies. It can be used to partially replace the extensive crushing and grinding that translates to prohibitive cost and energy consumption in a conventional process.

Applications

Bioleaching is a preparatory step to metal recovery. In subsequent processes, different from bioleaching, the metal is recovered from the leachate.

Bacterial oxidation such as bioleaching or biooxidation on a commercial scale has been done on sulfide metal bearing materials such as arsenopyrite, pyrite, pyrrhotite, covellite and chalcocite ores and concentrates, the one exception to this processing being the oxidation of chalcopyrite ores and concentrates.

Gold and copper are the dominating valuable metals that are commercially extracted:

• Copper from low-grade secondary copper sulfide ores by heap bioleaching.
• Gold from refractory gold concentrates by stirred tank leaching as a pretreatment step. The biooxidation residue is further treated by cyanide leaching to recover gold.

Cell separation techniques

A large variety of cell separation methods are currently commercially available, these are predominantly based on three methodologies: adherence, density and antibody binding. New techniques are being developed that utilize micro fluidic technologies and take advantage of a variety of cellular properties such as elasticity in response to acoustic waves and membrane polarization in a non-uniform electric field. However, these techniques are mostly still experimental and not yet available commercially for research. The choice of separation method depends upon a variety of factors, and each methodology has benefits and drawbacks that affect its applicability in a given situation. In this section, we will briefly outline the three overall methodologies with specific examples of each.

Adherence

Techniques that utilise cellular adherence are some of the most simple methods used for cell separation and are routinely used when isolating cells from digested or explanted primary tissues. Adherence can also take time leading to some uncertainty as to the success of a separation. Recently, techniques based on cell adherence, such as differential binding of cells to polymer brushes of varying lengths, grafted to glass surfaces, have been developed and these are currently being refined. However, despite this progress, current uses of adherence sorting are mostly only applicable when cell purity is not of concern and isolation of various subpopulations is not required.

Density

Density-based techniques are now mostly based on the use of centrifugation, although historically sedimentation-based methods have been employed.¹⁸ Techniques based on centrifugation are commonly used in many laboratories and are also routinely used clinically. The ability to sort large numbers of cells based on their density, relative to a graduated separation medium (usually sugar based), makes these techniques particularly applicable for separations involving the use of blood. By using these techniques, it is possible to isolate different cell types from a complex mix, including disrupted solid tissues (Figure 3) such as mouse liver.²⁰ However, although technically feasible, this is still challenging to perform with high specificity.

Antibody binding

Antibody-binding methods generally refer to the commonly used techniques of fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS).Both technologies utilise the same cellular properties for separation, namely, cell surface antigens against which antibodies are raised. FACS separation relies on the conjugation of fluorescent labels to these antibodies, whereas MACS uses conjugation to iron oxide containing microbeads. FACS separation is achieved by laser excitation of the bound fluorophores, with excitation above a threshold level signalling the corresponding cell to be separated . MACS requires the cells to be placed in a magnetic field; unlabelled cells are eluted, and labelled cells are retained in the field until they are removed from the magnet, giving the separated populations.

Protein purification

Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins.

The methods used in protein purification can roughly be divided into analytical and preparative methods. The distinction is not exact, but the deciding factor is the amount of protein that can practically be purified with that method. Analytical methods aim to detect and identify a protein in a mixture, whereas preparative methods aim to produce large quantities of the protein for other purposes, such as structural biology or industrial use.

Preliminary Steps

Extraction

Depending on the source, the protein has to be brought into solution by breaking the tissue or cells containing it. There are several methods to achieve this: Repeated freezing and thawing, sonication, homogenization by high pressure, filtration, or permeabilization by organic solvents. The method of choice depends on how fragile the protein is and how sturdy the cells are. Usually for most of the conventional purposes, column chromatography is used to achieve purification. After this extraction process soluble proteins will be in the solvent, and can be separated from cell membranes, DNA etc. by centrifugation. The extraction process also extracts proteases, which will start digesting the proteins in the solution. If the protein is sensitive to proteolysis, it is usually desirable to proceed quickly, and keep the extract cooled, to slow down proteolysis.

Precipitation

In bulk protein purification, a common first step to isolate proteins is precipitation with ammonium sulfate (NH₄)₂SO₄. This is performed by adding increasing amounts of ammonium sulfate and collecting the different fractions of precipitate protein. Ammonium sulphate can be removed by dialysis.The hydrophobic groups on the proteins gets exposed to the atmosphere and it attracts other protein hydrophobic groups and gets aggregated. Protein precipitated will be large enough to be visible. One advantage of this method is that it can be performed inexpensively with very large volumes.

Ultracentrifugation

Centrifugation is a process that uses centrifugal force to separate mixtures of particles of varying masses or densities suspended in a liquid. When a vessel (typically a tube or bottle) containing a mixture of proteins or other particulate matter, such as bacterial cells, is rotated at high speeds, the inertia of each particle yields an outward force proportional to its mass. The tendency of a given particle to move through the liquid because of this force is offset by the resistance the liquid exerts on the particle

Purification Strategies

An analytical purification generally utilizes three properties to separate proteins. First, proteins may be purified according to their isoelectric points by running them through a pH graded gel or an ion exchange column. Second, proteins can be separated according to their size or molecular weight via size exclusion chromatography or by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis. Proteins are often purified by using 2D-PAGE and are then analysed by peptide mass fingerprinting to establish the protein identity. Thirdly, proteins may be separated by polarity/hydrophobicity via high performance liquid chromatography or reversed-phase chromatography

·         Size exclusion chromatography

·         Ion exchange chromatography

·         Affinity chromatography

·         HPLC

Concentration of the purified protein

A selectively permeable membrane can be mounted in a centrifuge tube. The buffer is forced through the membrane by centrifugation, leaving the protein in the upper chamber.

At the end of a protein purification, the protein often has to be concentrated. Different methods exist.

Lyophilization

If the solution doesn't contain any other soluble component than the protein in question the protein can be lyophilized (dried). This is commonly done after an HPLC run. This simply removes all volatile components, leaving the proteins behind.

Ultrafiltration

Ultrafiltration concentrates a protein solution using selective permeable membranes. The function of the membrane is to let the water and small molecules pass through while retaining the protein. The solution is forced against the membrane by mechanical pump, gas pressure, or centrifugation.

Evaluating purification yield

The most general method to monitor the purification process is by running a SDS-PAGE of the different steps. This method only gives a rough measure of the amounts of different proteins in the mixture, and it is not able to distinguish between proteins with similar apparent molecular weight.

If the protein has a distinguishing spectroscopic feature or an enzymatic activity, this property can be used to detect and quantify the specific protein, and thus to select the fractions of the separation, that contains the protein. If antibodies against the protein are available then western blotting and ELISA can specifically detect and quantify the amount of desired protein. Some proteins function as receptors and can be detected during purification steps by a ligand binding assay, often using a radioactive ligand.