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Introduction
     Chromatography is a 1906 Russian botanist Michael Tswett colored leaves of plants containing pigment and chalk solution through a column packed with adsorbent particles in an attempt to separate them from time to time discovered and named. Various pigments at different rates through the column, and thus separated from each other. Separated pigmentation different ribbons and easy to distinguish, thus named for chromatography (Chromatography), also known as chromatography. A major advance in 1941 followed by Martin and Synge found liquid - liquid (distribution) chromatography [Liquid-Lipuid (partition) Chromatography, abbreviated LIC]. And with the mobile phase immiscible fixative They cover the adsorbent surface instead of only the previous solid adsorbent. Sample components are dissolved in accordance with its distribution between the two phases. Martin and Synge because the work was awarded the 1952 Nobel Prize in Chemistry. In the early years of using column chromatography, reliable identification of small amounts of substances to be separated it is difficult, so the research and development of paper chromatography (Paper Chromatography, abbreviated PC). In this "flat" technology, the separation is mainly through the distribution of filter paper to achieve. Since then fully consider the advantages of planar chromatography and the development of thin-layer chromatography (Thin-Layer Chromatography, abbreviated TLC), in this method, a thin layer separation was coated on a glass plate or some hard materials carried on the adsorbent.
     After Stah-l classic work carried out in 1958 to standardize technology and materials,
     TLC party won a reputation. To help improve the paper chromatography or thin layer chromatography separation efficiency of the ionic compound, an electric field may be applied to the paper or board. This improved method are called paper electrophoresis or thin layer electrophoresis.
     Newly developed chromatography
     
     Gas chromatography is Martin and James first described in 1952, has become all the most advanced chromatography and a method most widely used, it is particularly suitable for gas or a mixture of volatile liquids and solids, even for very complex The mixture separation time is only a few minutes or so, it is already commonplace. High resolution, rapid and comprehensive analysis of detection sensitivity and so are several advantages of the gas phase chromatography became almost every chemical laboratory conventional method to be used. In recent years, because of new liquid chromatograph and a new column packing for chromatography theory development and better understanding, once again aroused interest in the sealed liquid chromatography column. HPLC (High-Performance Liquid Chromatography, abbreviated HPLC) is rapidly becoming as gas chromatography method widely used for the rapid separation of the sample of non-volatile or thermally unstable, the HPLC often It is preferable.
     Chromatography Categories
     There are many types of chromatography, there are a variety of classifications.
     
     (A) by the state in which the two-phase classification
     
     A liquid as the mobile phase, referred to as "liquid chromatography" (liquid chromatograp-hy); gas as a mobile phase, referred to as "GC" (gas
     chromatogr-aphy). There are two stationary phase state to the solid adsorbent as a stationary phase and in the solid-carrying liquid as the stationary phase, so chromatography by which the state can be divided into two phases:
     
     Liquid - solid chromatography (liquid-solid chromatography)
     Liquid - liquid chromatography (liquid-liquid chromatography)
     Gas - solid chromatography (gas-solid chromatography)
     Gas - liquid chromatography (gas-liquid chromatography)
     (B) the process of classification mechanism chromatography
     Adsorption chromatography (adsorption chromatography) using an adsorbent surface adsorption performance differences between different components, to achieve the purpose of separation and identification.
     
     Partition chromatography (partition chromatography) using different components between the mobile phase and the stationary phase partition coefficient (or solubility) different, leaving the method of separation.
     Ion exchange chromatography (ion-exchange chromatography) of different components with different affinity for the ion exchanger, the separation method is performed. Gel chromatography (gelchromatography) the use of certain gel due to different components of different molecular size differences and different blocking effect, separation techniques.
     
     (C) by operating in the form of different categories
     Column chromatography (colum chromatography) stationary phase packed in the column, the sample was moved in one direction to achieve separation. Paper chromatography (paper chrmatography) as the carrier liquid with filter paper (Tam body support), after spotting with the mobile phase to start in order to achieve the purpose of separation and identification. TLC (thin layper chromatography) will be paved with a thin layer of sorbent particle size appropriate to paper chromatography method similar separation and identification of substances.
     Adsorption chromatography
     Adsorption chromatography, often called the liquid - solid chromatography (Liquid-Solid
     Chromatography, referred LSC), which is based on the solute and used as a stationary solid adsorbent interactions between fixed active site. The adsorbent can be loaded in the column, the cover on the board, or immersed in a porous filter paper. Is active adsorbent having a large surface area of ​​the porous solid, such as silica gel, alumina, and activated carbon. Active point such as silica surface silanol compound generally be separated polar functional groups interact. Non-polar portion of the molecule (e.g., hydrocarbon), only a small influence on the separation, so liquid - solid chromatography is well suited for separating different kinds of compounds (e.g., alcohols and aromatics separation).
     Partition chromatography
     In partition chromatography (also known as body fluids - liquid chromatography), the solute molecules between two immiscible liquid that is between the stationary and mobile phases according to their relative solubility allocated. Stationary phase uniformly cover in an inert carrier ─ porous or non-porous solid particles or porous paper (paper spectrum). To avoid mixing the two phases, two dispensing liquid in polarity must be significantly different. If the stationary phase is polar (e.g., ethylene glycol), non-polar mobile phases (such as ethane), then the polar component will be relatively strong retention. This is the usual mode of operation. On the other hand, if the non-polar stationary phase (e.g., decane), the mobile phase is polar (e.g. water), then the polar component easily partitioned mobile phase to elute faster. The latter method (which have opposite polarity) ─ was referred to as reverse phase liquid chromatography. Due to slight differences in the effects of solubility, so liquid ─ liquid chromatography is suitable for separating homologues isomers. ─ liquid in liquid chromatography, the stationary phase are almost chemically bonded to the carrier material, rather than a mechanical covering its surface. This is called bonded phase chromatography, chromatography (Bonded-Phase Chromatography, abbreviated BPC). In this way the mechanism is not clear, the mechanism may be allocated, it may be adsorption mechanism, depending on the experimental conditions. High performance liquid chromatography, bonded phase chromatography applications far exceed all other modes.
     Ion exchange chromatography
     Sober and Peterson for the first time in 1956 in conjunction with ion-exchange group to the cellulose, ion exchange cellulose formed, successfully applied to the separation of proteins. Since biological macromolecules fractionation method has achieved rapid development. Ion exchange groups not only bound to the fibers can be bonded to the cross-linked dextran (S-ephadex) and agarose gel (Sepharose) on.
     In recent years, ion exchange chromatography has been widely used in the separation and purification of proteins, enzymes, nucleic acids, peptides, oligonucleotides, viruses, bacteriophages and polysaccharides. Their advantages are: ⑴ an open support frame, molecules can freely enter and spread rapidly, so adsorption capacity. ⑵ hyhilic, adsorption of macromolecules small firm with a mild elution conditions may not cause protein denaturation or inactivation of the enzyme. ⑶ porosity, surface area, large switching capacity, high recovery rate, can be used to isolate and process.
     First, the basic theory
     
     Ion exchangers is generally an insoluble polymer compound, such as resins, cellulose, dextran, sugar, alcohol, fat, its molecules containing dissociable group, the other of these genes can be in aqueous solution and cation or anion exchange interaction plays. Although the exchange reaction is an equilibrium reaction, but the chromatographic column, since the continuous addition of new switching solutions, balanced by positive direction constantly, until completely. The atomic ion can be ion exchanger on the whole eluted Similarly, when a certain amount of the solution through the exchange column, since the ions in solution are exchanged and continuously by reducing waviness, therefore, may all be exchanged and adsorbed on the resin. If there are two or more components are adsorbed on the exchanged ion exchanger, elution with an eluent, the eluted capacity is determined by the equilibrium constant of the reaction the respective wash. Ion exchange process has two phases ── protein adsorption and desorption. Protein adsorption on ion exchangers can be obtained by changing the pH of the protein adsorbed to lose charge and reach dissociated but more by increasing the ionic strength, the charge position to compete with the protein ions added on an ion exchanger, so that the adsorbed proteins and ion exchanger untied. Different number key, i.e., the size of affinity differences, thus simply the appropriate conditions for elution of components of the mixture can be eluted individually, to achieve the purpose of separation and purification of proteins formed between the ion exchanger.
     Second, the ion exchange of classified and common species
     (A) Categories
     Ion exchangers are divided into two categories, namely cation exchanger and anion exchanger. Various types of exchangers according to its dissociative size, can be divided into strong and weak two, namely a strong acid cation exchanger agent
     Weakly alkaline agents strong weak base anion exchanger type.
     1. The cation exchanger
     Cation exchanger in the acid dissociable genes (-SO3H), phosphoric acid (-PO3H2), carboxylic acid (COOH) and phenolic hydroxyl group (-OH) and other acidic groups.
     Some exchangers in exchange reactions are as follows:
     Strong acid: R-SO3 -H + + Na + R-SO3- Na + H +
     Weak acid: R-COOH + Na + R-COONa + H +
     Domestic resin acids 1 × 7 (Shanghai resin # 732) and foreign products Dowex 50, Zerolit 225 and so on strongly acidic ion exchanger.
     2. The anion exchanger
     The anion exchanger can be dissociated gene is a primary amine, (- NH2), secondary amine (-NHCH3), tertiary amines [N- (CH3) 2] and quaternary amine [-N (CH3) 2] and other alkaline base group. Some exchange reaction is as follows:
     Strong Alkaline: R-N + (CH3) 2 H · OH- + Cl R-N + (CH3) 2 Cl + OH-
     Weak alkaline: R-N + (CH3) 2 H · OH- + Cl R-N + (CH3) 2 HCl + OH-
     Strong alkaline No. # 201 domestic resin and abroad Dowex1, Dowex2, ZerolitFF belong to a strong base anion exchanger.
     (Ii) species
     1. The cellulose ion exchangers: cation exchanger has hydroxymethylcellulose (CM- cellulose), there is chloro anion exchanger triethylamine fiber yarn (DESE- cellulose).
     2. The cross-linked dextran ion exchangers: the exchange is made on the gene connected to a class of cross-linked dextran exchangers, thus both ion exchange, but also has the molecular sieve effect, is a class of widely used chromatography separation of substances. Commonly used Sephadex ion exchangers are also two types of anion and cation exchangers. There anion exchanger DEAE-Sephadex
     A-25, A-50 and QAE- Sephadex A25, A50; cation exchanger has a CM-Sephaetx
     C-50, C-50 and Sephadex
     C-25, C-50. Anion exchanger with English prefix A, cation exchangers of English words is C. English words behind the numbers indicate Sephadex models.
     3. Sepharose ion exchangers from: is DESE- or CM- group attached to Sepharose CL-6B
     Formed, DEAE-Sephades (anion) and CM-Sepharose (cation), has high hardness,
     Stable flow rate of gel after a good, strong separation ability and so on.
     Third, the experimental operation
     
     (A) processing exchanger, regeneration and transformation
     The new resin factory is dry resin, to be soaked with water so that the full swelling. It contains some impurities achievements, want to water, acid and alkali washing. General procedures are as follows: new factory of dry resin soaked in water 2
     Hours after the pumping pressure to reduce bubbles, decanted water, copious amount of deionized water until clear, after 4 times the amount of water was added to 2N HCl stirring shake four hours to remove the acid, washed with water to neutral, plus 4 times the amount of 2N
     NaOH stir shaking four hours, except alkaline liquid,
     Washed with water to neutral stand. The resin bring some desired ion operation is called transformation. If you wish cation resin with Na +, with four times the amount of NaOH is stirred soak for more than two hours; if they wish to resin with H +, available HCl. Anion resin transformation also, if you want to use with Cl- HCl, hope to bring OH- then use NaOH. The resin used to make restitution method called regeneration. Not always have to use another acid, alkaline washing liquid, often on the line as long as the transition process.
     (Two) column operation
     ⑴ exchanger packed easiest exchange chromatography can be used in place of alkaline burette. Treated resin into a beaker, stirring, pour a small amount waterside perpendicular chromatography tube, the resin is slow to settle. Exchanger in the column must be evenly distributed.
     On ⑵ sample poured into the sample liquid chromatography column
     ⑶ eluting collection
     Different different samples chosen eluent. Principle is to use a more active sorbent material than ions, the attraction was swapped out. Since the substance to be adsorbed is often not a single substance we are asking for, so in addition to the correct choice of mining method of controlling things, eluent flow rate and the distribution of the collected material to obtain a single desired.
     Gel chromatography
     GPC technology is developed in the early sixties, a rapid and simple separation minute technology, because the equipment is simple, easy to operate, no organic solvent, the polymer material has a high separation efficiency. It has now been biochemistry, molecular biology related fields, bioengineering, molecular immunology and medicine and other widely used, not only apply to the experimental study of science, and has been used in large scale industrial production.
     First, the basic theory
     (A) molecular sieve benefit
     When a sample solution containing various molecules slowly flows through the gel column, each molecule in the column at the same time two different movements: vertical downward movement and the diffusion of non-directional movement. Because macromolecules larger diameter, easy to enter the pores of the gel particles, but only the distribution between the particles, the downward movement in the eluted faster. After the addition of small molecules can diffuse in the gel particles in outer space, you can also enter the pores of the gel particles that enter the gel phase in the process of downward movement, from a gel diffusion into the particle gap then into another gel particles, and thus into the diffusion constant, speed down behind the small molecules are macromolecules, whereby large molecules in the sample to exit the column after flowing medium molecules, the smallest molecule final outflow, a phenomenon called molecular sieve effect. It is a molecular sieve having a porous gel. Various zeolite pore size distribution has a range of maximum limits and minimum limits. Molecular diameter than the maximum pore diameter of the gel, it will all be outside the exclusion gel particles, this situation is called full exclusion. Even the full exclusion of two molecules of different sizes, can not have separation. Gel pore diameter than the diameter of the smaller molecules can enter all of the pores of the gel. If the two molecules can enter all pores gel, even if their size difference, there will not be a good separation effect. Therefore, certain molecular sieves has its certain range. In summary, there are three cases in gel chromatography, one molecule is small, can enter all the pores of the molecular sieve; the second is a large molecule, can not enter any of the pores of the gel; the third is moderate molecular size , can enter the pores in the gel pore size appropriate section. Large, medium and small three molecules easier to separate from each other, but each molecule outside the gel separation range, without changing the gel kind of situation it is difficult to separate. For molecules of different sizes, but belong to a variety of molecules within the gel separation range, in the distribution of the gel bed is different: the larger molecules into only that part of the larger pore size gel pores, and the molecule may enter the larger gel particles, such larger molecules move shorter distances, smaller molecules to move a long distance in the gel bed. So the larger molecules to smaller molecules through the gel bed and after through the gel bed, thus separating the molecular sieve can use different substances. Further, the gel itself having a three-dimensional network structure, when the large molecules through the pores in the network structure of this greater resistance, the resistance is small by small molecules. In adopting the gel bed, according to molecular size "queue, the gel performance molecular sieve effect of a variety of different molecular weight components.
     Important parameters (b) column
     ⑴ column volume: refers gel column volume is packed from floor post to the deposition surface of the gel volume. Filled with gel in the column section called gel bed, because the lead column volume called "bed" volume, commonly Vt
     FIG.
     ⑵ void volume: gel particles within the column space, this part of the volume of said void volume, also known as interstitial volume, commonly expressed Vo.
     ⑶ inner volume of water: because the gel is a three-dimensional network structure, there is still room within the particles, liquid particles into the interior, which divide the sum of the volume of water within the gap, known as phase volume, Vi represents common.
     It does not include the volume of the solid support (Vg).
     ⑷ peak elution volume: refers to the substances to be separated by gel column eluent required to have volume, commonly Ve
     FIG. When little use when the volume of the sample, (and elution volume is relatively negligible), the figure in the elution from the sample to the peak position of the elution volume Ve.
     When the sample volume and elution volume comparison can not be ignored, the elution volume can be calculated from the volume of the sample to half the peak position. When the sample is large, the elution volume calculation can be applied sample from the beginning to the elution peak rising inflection point (or half-height).
     
     
     
     Second, the type and nature of the gel
     (A) cross-linked dextran gel (Sephadex)
     ⑴Sephadex
     G cross-linked dextran tradename Sephndex, different specifications of dextran with a letter G, said the number of the back of the G Arab gel was 10 times the value of water. For example, G-25 to 2.5 grams per gram of absorbent gel expansion, the same G-200 grams per gram of thousands of plastic water-absorbing 20 grams. Category Sephadex are G-10, G-15, G-25, G-50, G-75, G-100, G-150, and G-200. Thus, "G" reflects the degree of crosslinking gel swelling degree and segment range.
     ⑵Sephadex LH-20, is ─Sephadex G-25 carboxypropyl derivative, soluble in water and lipophilic solvents, for separating water-insoluble substances.
     (B) Agarose gel:
     Many trade name, a common, Sepharose (Sweden, pharmacia), Bio-Gel-A (American Bio-Rad) and the like. Agarose gel rely secondary chain such as hydrogen bonding between sugar chain to maintain the network structure, the density of the network structure relies agarose concentration. In general, its structure is stable, can be used (such as water, saline solution within the range pH4-9) under a number of conditions. Agarose gel begins to melt at above 40 ℃, nor autoclaving, chemical sterilization available live processing.
     (C) polyacrylamide gel:
     Is a synthetic gel is acrylamide as a unit,
     By A. bisacrylamide cross-linked into, the dried crushed or processed into granular shaped, controlled amount of crosslinking agent can be made of various types of gel. The more the crosslinking agent, the smaller the pores. Polyacrylamide gel of goods for the biological glue -P
     (Bio-Gel P), by the American Bio-Rod Factory, many models, from P-2 to P-300 total of 10 kinds, P
     Then by 1000 the latter figure is equivalent to the gel exclusion limit.
     (Iv) polystyrene gel commodity Styrogel, with a large mesh structure,
     Biological macromolecules can be used to separate the molecular weight of 1600 to 40,000,000, for organic polymers, molecular weight determination and fat-grade natural products, good mechanical strength of the gel, eluent available sulfoxide.
     Third, experimental techniques
     (A) column
     Gel chromatography column is the body, usually with a glass or plexiglass. The diameter of the column does not affect the separation, the sample volume is big, can increase the diameter of the column, usually prepared gel column, diameter greater than 2 cm, but the sample in the sample should be evenly distributed in the gel bed on. In addition,
     Diameter increased, the elution liquid volume increases, sample dilution degree. High degree of separation depends on the column, for the separation of the different components of the gel bed must have a suitable height, associated with a high degree of separation column square root, but because of soft gel too high extrusion blocked, usually not more than one meter . When the sub-family separated by short column, usually 20-30 cm long gel column, column height and diameter compare 5: 1─10: 1, gel bed volume of 4-10 times the volume of the sample solution.
     When fractionated line column height and diameter of 20: 1─100: 1, commonly used gel has 50 × 25 厘米, 10 × 25 cm. Dead volume of the column under the filter plate should be as small as possible, large dead volume if the filter plate under the palm branch, the possibility of separation between the components remixed big, with the result that affect the elution peak shape, appear trailing the like, lower differentiate force. When precise separation, the dead volume can not exceed the total bed volume 1/1000.
     (B) a gel ed according to the desired gel volume, the estimated amount of adhesive required. Usually dextran gel after water absorption gel volume is approximately 2 times the amount of water absorption, such as Sephadex
     The amount of water absorption of G-20 Sephadex 20,1 g
     G─200 absorbing gel volume after the formation of about 40ml. The particle size of the gel may also affect the chromatographic separation. Size cell separation is good, but resistance, slow flow. General laboratory separation of proteins using 100-200 mesh number of Sephadex
     G-200 is good, desalted using Sephadex G-25, G-50, with coarse, short column, flow speed.
     (C) Preparation of the gel
     Product was dried gel particles swell before use directly in the eluent used in the desire. In order to accelerate the expansion of available heating method, namely in the boiling water bath in the wet gel is gradually heated to near boiling, which can greatly speed expansion, usually can be completed in 1-2 hours. Especially in the use of soft,
     Natural expansion need 24 hours to several days, but with the heating method can be completed in a few hours. This method not only saves time, but also disinfection, remove the air inside the bacteria and remove gum gels pollution.
     (Iv) treating the sample solution if precipitation sample solution should be removed by filtration or centrifugation, such as lipid-containing high-speed centrifugation or by Sephadex
     G-15 short column removed. The viscosity of the sample is not large, protein-containing more than 4%, the viscosity of high-impact separation efficiency. Volume of the sample solution is determined according to the column bed volume of gel separation requirements. Volume isolated protein sample was 1-4% gel bed (generally about 0.5-2ml), proceeds with Family separate sample solution may be 10% gel bed when the protein solution desalting gel sample up 20-30% of the bed.
     Fractionated sample size should be small, the sample layer as narrow as possible, eluted peak shape better.
     (E) to prevent microbial contamination and cross-linked dextran polysaccharides are agarose, to prevent the growth of microorganisms, very important in the gel filtration chromatography, the commonly used antibacterial agents include:
     ⑴ stack of ammonia, sodium (NaN3) as long as the gel chromatography with 0.02% sodium azide has been sufficient to prevent the growth of microorganisms, a water-soluble sodium azide, at 20 ℃ is about 40%; it is not a protein or carbohydrate compound interaction, and therefore does not affect the antibody sodium azide vitality; it does not alter the protein and carbohydrate chromatography my characteristics. Sodium azide can interfere with fluorescently labeled proteins.
     ⑵ cola ketone [Cl3C-C (OH) (CH3) 2] used in the gel chromatography concentration of 0.01-0.02%. In acidic solution, its optimal bactericidal effect, easy to cause decomposition in strong alkaline solution or a temperature higher than 60 ℃ and failure.
     ⑶ ethylmercury-generation mercapto sodium salicylate gel chromatography as a bacteriostatic agent concentration of 0.05-0 01%. In acidic solution, the most effective. Heavy metal ions can sulfhydryl-ethyl-generation combination of substances, which contain hyhobic groups of proteins may reduce its antibacterial effect in varying degrees.
     ⑷ phenyl mercury salt substitutes
     Gel chromatography using a concentration of 0.001 to 0.01%. In slightly alkaline solution, suppressing the best for a long time with halogen, nitrate ions to produce precipitates upon standing; reducing agents may cause decomposition of this compound; with sparse groups of substances can decrease or inhibit its inhibitory effect .
     Affinity chromatography
     First, the basic theory
     (A) Principle
     In vivo, many macromolecules AKSJDHFKLSDFHKLSDJ have some corresponding specific molecule reversible binding characteristics. Such as antigens and antibodies, enzymes and substrates and coenzyme, hormones and receptors, RNA and its complementary DNA, etc., they have this feature. Between biomolecules such a specific binding ability is called affinity, according to the principle of biomolecular affinity adsorption and dissociation, established affinity chromatography, chromatography said. Affinity Chromatography two were specific binding molecules called each other as ligand. Such as antigens and antibodies, antigens can be considered as ligand antibodies, and vice versa antibodies can also be considered as the antigen ligand. The absence of a water-soluble ligand without hurting its biological function in conjunction with water-insoluble carrier called ligand immobilization. Affinity chromatography is the basic process:
     1. Ligand immobilization. It will be binding and purification of target specificity of substance, connected to the water-insoluble carrier, made after the affinity adsorbent packed (called affinity).
     2. affinity adsorption. The mixture containing purified by affinity column objects, objects adsorption purification column and other substances out of the column.
     3. desorption. With some kind of buffer or solution through an affinity column, the adsorption affinity column purified material eluted desire.
     (B) Application
     Affinity chromatography can be used for the following biological systems:
     Enzyme: substrate, inhibitor, coenzyme
     Antibody: antigen, virus, cell
     Lectins: polysaccharides, glycoproteins, cell surface receptors,
     Cellular nucleic acid: complementary base sequence, histone, nucleic acid polymerase, the binding protein
     Hormones and vitamins: receptor, a carrier protein
     Cell: cell surface specific protein, lectin
     Advantages affinity chromatography is simple operation, high efficiency, mild conditions, the disadvantage is the use of large limitations.
     (Iii) carrier ion principle
     
     
     Ideal carrier for affinity chromatography should have the following characteristics: ⑴ insoluble: insoluble in water; ⑵ permeability: loose network structure, to allow free passage of macromolecules; ⑶ a certain hardness, preferably uniform bead; ⑷ have a large number of available reactive chemical groups can be connected with a number of ligands covalently; ⑸ low non-specific adsorption capacity; ⑹ resistant microorganisms and enzymes erosion; ⑺ have good chemical stability; ⑻ hyhilic. Select specific ligands according to a comprehensive understanding of the purification of macromolecules.
     Choices ligands have two criteria:
     The first is to have a strong affinity between the protein and ligand dissociation constants in 5mM above is not a good ligand;
     Instead affinity too is harmful, because in the dissociation of protein ligand complexes ── conditions required going strong, this may denature proteins. For example using avidin as a ligand purification containing biotin carboxylase, biotin-avidin solution ── anti-complex dissociation constant of 10-15M, when required pH1.5,6M guanidine hydrochloride dissociation, In such a condition. Most carboxylase has been denatured.
     Select ligand second criterion is that the ligand must have the appropriate chemical group, such a group is not involved in the specific binding between the ligand and protein, but can be used to activate and support connected without affecting ligand affinity between proteins.
     Features (iv) common carrier
     Agarose gel: hyhilic strong, stable physical and chemical properties (trade name: Sepharose)
     Polyacrylamide gel: the physical and chemical properties of stability, resistance to organic solvents and detergents, antimicrobial ability, especially suitable for use with extracts affinity ligands weak material.
     Dextran: good chemical and physical properties, through a small hole.
     Cellulose: Sofitel easily adsorbed serious, cheap, easy to get.
     Second, the experimental method
     Affinity chromatography methods of separation of different substances with each have different. The general procedure is recommended as follows:
     1 Select ligands; 2 Select coupling gel; 3 conjugated ligand; 4 is loaded right column; 5 balance (2-3 times the volume of buffer); 6 Application sample
     7 washing unbound material;; article 8 eluting bound material → desalination; 9 regeneration
     HPLC
     Martin and
     Synge in 1941 to propose ideas chromatography efficient, but until the late 1960s due to the development of various technologies, high performance liquid chromatography was put into practice. This chromatographic technique has been known as high performance liquid chromatography (HighSpeed
     Liquid Chromatography), high pressure liquid chromatography (High Parss-ure Lipuid Chromatography), currently the most used name is HPLC (High Pe-rformance Liauid
     Chromatography, HPLC). HPLC has been widely used, it has become an indispensable technology. Its main advantage is that ⑴ resolution than other chromatography; ⑵ speed, ten minutes to tens of minutes to complete; ⑶ high repeatability; ⑷ efficient phase columns can be used repeatedly; ⑸ automation, analysis accuracy . Differences in the separation process according to the solute molecules interacting with the stationary phase, high performance liquid chromatography can be divided into four basic types, namely liquid - solid chromatography, liquid - liquid chromatography, ion exchange chromatography and size exclusion chromatography. Isolation and identification of high-performance liquid chromatography in the biological area is widely used in the following products: ⑴ amino acids and their derivatives; ⑵ organic acids; ⑶ steroids; ⑷ alkaloids; ⑸ antibiotics; ⑹ carbohydrate; ⑺ porphyrin; ⑻ nucleic acids and their degradation products; ⑼ proteins, enzymes and peptides; ⑽ fat
     
     First, the classification
     HPLC can be divided into four basic types: the liquid - solid chromatography, bonded phase chromatography, ion exchange chromatography and size exclusion chromatography.
     (A) liquid - solid chromatography
     Liquid - solid chromatography usually called adsorption chromatography, adsorbents have activated carbon, alumina and silica gel, in liquid - with a solid carrier is silica gel chromatography. Silica gel adsorption capacity of the solute molecules are not evenly distributed throughout the silicon off the surface of the silica surface in some areas and solute molecules interact strongly, these regions of the active sites, and solute molecules between silica primary role is hydrogen dipole force bonds and electrostatic interactions. More polar, and compound residence time in a silica gel column long. In the liquid - solid chromatography, relying on the mobile phase solvent molecules and solute molecules compete with each other active fixed position,
     So that the solute is eluted from the column. The ability to bind to the silica surface active position and strong solvent elution strong solute molecules, thus said the strong solvent, whereas a weak solvent. Characteristics of liquid chromatography ─ solid suitable for chromatographic separation of geometric isomers, can be used for quality liposoluble compounds such as phospholipids, steroids, fat-soluble vitamins, prostaglandins and the like.
     (B) bonded phase chromatography
     Bonded phase chromatography is a liquid - liquid chromatography, partition chromatography that is developed. Bonded phase chromatography stationary phase is covalently bound to the carrier particles, to overcome the partition chromatography stationary phases due to trace amounts of dissolved and flow in the flow phase column by mechanical impact during the stationary phase continued loss column gradually changed the nature of other shortcomings. Bonded phase chromatography can be divided into normal phase chromatography and reverse phase chromatography.
     1. normal phase chromatography
     In normal phase chromatography covalently bound to the carrier group are polar groups, such as primary amino, cyano, diol group, a dimethylamino group and the second group and the like. The mobile phase solvent adsorption chromatography with a mobile phase is very similar to a non-polar solvents such as heptane, hexane and iso-octane. Since the polarity of the stationary phase is, therefore, the stronger the flow of solvent polarity, the stronger elution ability, namely polar solvent is a strong solvent. This relationship stationary phase coincided with the mobile phase liquid - solid chromatography same, saying that chromatography is a normal phase chromatography. However, normal phase chromatography separation principle of the separation of different major phase distribution coefficient in the fixed and mobile phases based on compound, it is not suitable for the separation of geometric isomers.
     2. reverse phase chromatography
     In reverse phase chromatography stationary phase covalently bound to the carrier are some straight-chain hydrocarbons, such as n-octyl. Polar mobile phase is stronger than polar stationary phase. RP-HPLC chromatography in the most widely used.
     In reverse phase chromatography, retention solute main role is hyhobic interaction, in high-performance liquid chromatography is also known solvophobic role. The so-called hyhobic interaction i.e. when there is a non-polar solutes in water, the interaction between the interaction, the solute molecules and water molecules between solute molecules is much smaller than the interaction between water molecules,
     Therefore solute molecules from the water to be "squeezed" out. Visible reversed phase chromatography, the stronger the hyhobic compounds more easily go out of phase from the flow, residence time in the column is long, so different compounds obtained in reversed phase chromatography separated according to their hyhobic properties. Reverse phase chromatography suitable for separating compounds with different hyhobic group, a compound that is non-polar group. Moreover, reverse-phase chromatography can be used compounds with different polar groups separated. By changing the solvent and mobile phase composition and pH, in order to influence the interaction between the solute molecules and the mobile phase, change their residence behavior. In addition, the flow of water in the phase reversed phase chromatography accounted for a large proportion of scalability, can / from 0-100%, so that reverse phase chromatography can be used for water-soluble, fat-soluble compounds isolated. Reverse phase chromatography stationary phase is covalently bonded to a straight-chain saturated alkanes silica carrier, its chains of different length, the longest octadecyl, which is used most of the stationary phase. Straight-chain saturated hydrocarbons with the hyhobic character of the hydrocarbon chain length increases, the time due to the hyhobic interaction and solute retention will also be with the length of the hydrocarbon chain increases reversed phase column. In general, this means using a hydrocarbon chain length of reversed phase columns can get a better resolution, in most cases it is to rely repeatedly to the column. Since the reverse phase chromatography the stationary phase is hyhobic hydrocarbon effects, between the solute and the stationary phase of mainly non-polar interactions, or hyhobic interaction, and therefore the strength with polar solvents decreases. Water is the most polar solvents, but also the weakest reverse phase chromatography solvent. Often used in reversed phase chromatography and solvent base, to which different concentrations, can be water-miscible organic solvent, to give the flow of different intensity phase, these organic solvents called modifiers. Reversed phase chromatography is the most common organic solvents methanol and acetonitrile, in addition, ethanol, tetrahydrofuran, isopropanol and dioxane are also often used as a modifier.
     In the biochemical analysis, reversed phase chromatography very wide application. ① it can be used to analyze amino acids and peptides; separating ② protein; ③ base analysis, nucleic acid and nuclease; ⑤ as well as a few others such as separation of catechol amines, histamine, sugar and vitamins; analysis ④ Steroids .
     (C) ion-exchange chromatography
     Ion exchange chromatography stationary phase are some charged groups,
     These charged groups of opposite charge and ionic bonding by electrostatic interaction zone. If the mobile phase other oppositely charged ions, according to the law of mass action, these ions will be bound to the stationary phase counter-ions are exchanged. When stationary phase group with a positive charge, which is exchangeable ion is an anion, such ion exchange agent for the anion exchanger; stationary phase charged group with a negative charge, can be used with mobile ions are exchanged with cations, such ion exchangers is called the cation exchanger. Anion exchange column functional group is mainly -NH2, and -MH3 +: functional groups cation exchanger is mainly -SO3H and -COOH.