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CHROMATOGRAHY
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Classification of chromatography
A. Mode of Separation Adsorption (NPC, LSC) separates molecules based on polarity, least polar eluting first Partition - Separates molecules based on combination of solubility parameters, partition coefficients, and polarity, most polar eluting first. Ion exchange Separates molecules on basis of molecular charge Size exclusion (GPC) separation based on molecular size, largest eluting first Affinity based on affinity with ligand
B. Basis of Mobile Phase Liquid Chromatography LLC, LSC Gas chromatography GLC, GSC
Two common approaches are used to bring the mobile phase and stationary phase into contact. In column chromatography, the stationary phase is placed in a narrow column through which the mobile phase moves under the influence of gravity or pressure. The stationary phase is either a solid or a thin, liquid film coating on a solid particulate packing material or the columns walls. In planar chromatography the stationary phase coats a flat glass, metal, or plastic plate and is placed in a developing chamber. A reservoir containing the mobile phase is placed in contact with the stationary phase, and the mobile phase moves by capillary action.
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solvent front
component B
dS dB
component A dA origin
A given co ompound will always a travel a fixed distanc ce relative to th he distance the e solvent travel ls; this ratio is called the Rf (Ret tentension fac ctor) Rf of comp ponent A = dA / dS or distan nce travelled by b analyte fro om origin / distance travell led by solvent t from origin ponent B = dB / dS Rf of comp The Rf of a compound cant c be great ter than 1 but it can be 1 in some cases lik ke if analyte is having more affinity a for the sol lvent or it is co ompletely not absorbed on the t stationary phase p as the an nalyte is highl ly non polar an nd the stationary phase p in TLC is i polar. So both the solvent and a the analyte e will move to the same dista ance on TLC pl late. The efficie ency of thin la ayer plate is ex xpressed by its s number of th heoretical plat tes (N) and pl late height (H) ). N = 16 (dA / w) 2 dA is the t distance tra avelled by the analyte and w is width of the e spot. H = dA / N here N is nu umber of theore etical plates K ` = 1 Rf Rf or dS dA dA K ` is capacity fa actor for the an nalyte, dS is the e distance trave elled by solven nt.
Thin layer r plate prepar ration Slurry of SP S phase is usu ually prepared in water or ch hloroform is ap pplied to a glas ss, plastic or fo oil plate genera ally 20 cm square e with the help p of the plate e spreader wh hich gives a un niform thin lay yer. The thickn ness of the slu urry is important as a for the analytical separa ations the thick kness of the SP S must be 0.2 25 mm while for the prepa arative separation ns it may be up u upto 2 mm m. Binding age ent calcium su ulphate (gyps sum, 10-15%) is incorporate ed into the slurry in i order to faci ilitate the adhe esion of adsorb bent on plate. So S once the slu urry layer has been b prepared then t it is dried to o 1100C for 30 0 mins in oven n and this pro ocess is called d as activation n of the adsor rbent as water in the
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slurry has to be removed otherwise it will block the adsorption (e.g. in silica gel when used as SP, the active part for adsorption are silanols or Si- OH grp which undergoes reversible H- bonding with the analyte become deactivated in presence of water). So the heating of SP activate the adsorbent by evaporation of water molecules. Sometimes silica become highly active and the polar compounds get strongly retained on it, so then we have to deactivate the silica. So water is playing an important role for activation and deactivation of adsorbent in SP. After the plate has been dried than sample is applied to the plate 2-2.5 cm above from the edge of plate either with the aid of capillary or micropipette or microsyringe as a uniform spot. A TLC developing chamber or jar contains a filter paper wrapped over its inside wall boundary, a covering lid and solvent mixture or mobile phase. When filter paper is completed wetted with mobile phase (MP move into the filter paper due to capillary action) indicate that chamber equilibrated with the solvent. After the chamber is equilibrated with MP then lid is taken off and the TLC plates are placed vertically in the TLC chamber and again lid is placed. (Applied analyte spots must be above from the level of the MP in the TLC chamber, otherwise the analyte may dissolve in the MP resulting in sample loss) The MP move due to capillary action in SP which is porous in nature and separates the compounds on the basis of polarity. Now the analyte which have stronger affinity for SP i.e. polar retained while nonpolar analyte move along with the nonpolar mobile phase and there retention factor is calculated with respect to distance travelled by the mobile phase. Generally MP for TLC is hexane & ethyl acetate. We start with higher % of nonpolar hexane (say 95%) and lower % of polar ethyl acetate (say 5%). In this concentration range mostly nonpolar analytes goes along with the hexane and occur at the top of the TLC plate (most nonpolar) but polar compounds retained on SP and they are separated by increasing % of ethyl acetate from 56%, 6- 7%, 7- 20 % etc for polar compounds as they get retained on TLC plate and not showing any distance travelled from the origin line. As the % of the polar mobile phase (ethyl acetate) is increased polar compounds starts to move from the origin line or sample application line and attained a particular distance with particular concentration of the solvent. So the distance travelled by the analyte is dependent upon the % of nonpolar and polar MP in the solvent mixture. Nowadays precoated silica plates in which silica is absorbed on aluminium plate are available supplied by Merck. Detection of compounds on TLC plates: 1. 2. Ultraviolet Light some organic compounds illuminate or fluoresce under short-wave UV light Iodine Vapor forms brown/ yellow complexes with organic unsaturated compounds and get added to double bonds 3. Fluorescent compounds fluoresce when placed under UV light. Other can be thin layer adsorbents which contain a fluorescent dye so that when the plate is placed under UV light, the separated compounds show blue, black , green areas against fluorescent background. E.g. zinc silicate
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4. 5. Silver Nitrate Spray (for Alkyl Halides)dark spots form upon exposure to light Sulfuric Acid Spray (50%) + Heat (1100 C)permanent charred spots and showing brown spots (Universal method of detection) 6. 7. Ninhydrin reagent - for detection of amino acids and peptides Autoradiography if the compound is radiolabelled
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A. Column C pac cking can be done d as Slurry y packing and Dry packing. A column is prepared by y placing a sma all plug of glas ss wool in the bottom b of the cylindrical gla ass column, fol llowed by a small layer of sand. The column is i then packed with the solid adsorbent pha ase (silica gel). . Slurry of adsorbent olvent to be late er used in sepa aration. The slu urry is carefull ly and slowly poured p in solvent is prepared with the same so olumn after it is partially fill led with solve ent in order to prevent distur rbance of the sand. The solv vent is into the co allowed to drain as the si ilica packs tigh htly. Once the solvent s just bar rely becomes le evel with the silica s (without drying d er small layer of o sand is appli ied carefully without w disturbin ng the silica. it!), anothe B. Sample S app plication 1. 2. 3. Dissolv ved in mobile phase (~ 25% % solution for viscous v sample) Dry loa ad - Adsorptio on on silica (1:2 ratio) Neat - non viscous sa ample
The compo ound mixture is dissolved in n a minimum amount a of solv vent (same as in the column n) and very car refully added to th he top of the column using a Pasteur pipe ette. After allo owing the com mpound to adso orb into the co olumn, solvent is continually c add ded to the top of o the column until u each band d resolves and is carefully collected. With colo ored substanc ces, the bands s may be dire ectly observed and collect ted as they ru un off the co olumn. However, with colorless s compounds, , the developm ment can be observed by co ollecting many y small fractio ons of g solvent and testing t each by b thin layer ch hromatograph hy under UV lamp. l the eluting
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C. Adsorbents Stationary phases used in order of polarity includes Alumina (highest Polarity), Magnesium oxide, Carbon, Silica, Starch, Cellulose (lowest Polarity). The most commonly used adsorbent in column chromatography is silica. It is available in different particle size and shapes. Greater the particle size, lower is the surface area, lower the number of theoretical plates, lower is the efficiency of the column. Lower the particle size, higher is the efficiency of column. Generally the particle size of silica for column chromatography: 60-200 m For Thin Layer chromatography (TLC) pre coated plates: 10 m For High Performance Liquid Chromatography (HPLC): 3-10 m Stationary phases (adsorbents) may be acidic or basic. Silica is acidic has preferential ability to retain amines and other basic compounds. Alumina and magnesia are basic they preferentially retain acidic compounds. Adsorption a boundary reaction between dissolved substance and solid substance, weak and reversible interactions (weak bonds) between two phases. The interactions includes Dipole Interactions (Bonding between two atoms of different electronegativity), Hydrogen bridge bonds (Electrostatic bonds between hydrogen of one molecule and strongly electronegative element of the other molecule (F,O, N,S), Pi-Complex (Adduct of electrophilic species with C-C double bond). D. Activity of Stationary Phase Adsorbents require activation before use. Activity of silica is due to free silanol groups Si OH grp. Addition of water results in loss of activity and too much heating results in loss of activity. Exposure to moisture increases physically adsorbed water on surface and decreases interaction between solute and silanol. So silica activated by heating at 110oC for 30 min as at this temp as adsorbed water present on surface is removed and max number of silanols are present. Heating silicas above 2000C leads to conversion of silanol groups to siloxane groups (Si-OSi). This decreases the ability of surface to retain polar materials. Alumina heat at 200oC for 4h (active), too long heating at high temperature (1000oC) results in total loss of activity. If fresh silica or alumina is too active, deactivation is done by adding water. Brockman Activity Scale for Alumina 1. 2. 3. Grade I most active, heating at 350oC for few hrs Grade II 2-3 % water added Grade III 5-7 % water added
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The activity can be tested by relative adsorbtivity of azodyes Azobenzene least adsorbed p-phenylazophenol most strongly adsorbed
Silica gel Silica gel is totally Porous, narrow pore size distribution, wide choice of pore size and particle size. Silica gel is manufactured by releasing silicic acid from a strong solution of sodium silicate by hydrochloric acid. Na2SiO3 +H2O + 2HCl Si (OH)4 + 2NaCl
The matrix of the primary silica gel particle consists of a core of silicon atoms joined together with oxygen atoms by siloxane bonds (silicon-oxygen-silicon bonds). Sodium silicate is prepared by heating sand at high temperature in contact with sodium carbonate, initially silicic acid is released which condenses with itself with elimination of water to form dimers, trimers and eventually polymeric silica. The polymer grows, forms polymer aggregates and then polymer spheres (primary silica particles) of few Angstrom in dia. The primary particles continue to grow until surface silanols on adjacent primary particles condense with elimination of water; this condensation causes silica to gel. The structure of Silica gel The matrix of primary silica particles consist of core of silicon atoms joined together with oxygen atoms by siloxane bonds. On the surface of each primary particle some residual, uncondensed hydroxyl groups from the original polymeric silicic acid remain. These residual hydroxyl groups confer upon silica gel its polar properties. These hydroxyl groups react with the silane reagents to form bonded phases. The silica surface is quite complex and contains more than one type of hydroxyl group, and strongly bound or chemically adsorbed water and loosely bound or physically adsorbed water.
OH Si O O Si O O O O O Si O O O O Si O O Si O O OH Si O O Si O O OH Si O O Si O O
OH Si O O O Si
OH Si
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Three types of silanols Free silanols low concentration, strong binding to basic solutes, gives broad and tailing peaks Fully hydroxylated high concentration of geminal and Associated silanols most favored (less acidic) The disadvantage of silica gel includes it start to dissolves at pH 8 due to breakage of siloxane bond. Purity of silica gel is important. Metal contaminants can complex with chelating solutes lead to tailing peaks or even complete retention Alumina Alumina is a network of aluminium and oxygen atoms. Active points are both the Al3+ centers and the connecting O2- atoms. Natural alumina is basic, however it is also available in basic or neutral forms. Chemisorption (Irreversible binding) more probable in alumina mainly when analyte contain COOH grp, so used only for specific purposes.
Elution pattern in normal phase column i.e. stationary phase is polar and MP is nonpolar Normal Phase Alkanes olefins nitro compounds sulfoxides amides aromatics organic halogen compounds alcohols/amines sulfides ethers sulfones
In NP Chromatography polarity of the mobile phase determines the elution time. Solvents in order of increasing polarity Eluotropic Series Pentane/hexane <pet ether <cyclohexane <xylene <toluene <diethyl ether <chloroform <dichloromethane <THF <acetone <dioxane <acetonitrile <methanol <water
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A: Reaction of surface silanol with chlorodimethylsilane B: Reaction of surface silanol with trifunctional silane C: Reaction of surface silanol with trifunctional alkoxysilane
Reacting silanol with chlorodimethylalkyl silanes or chloroalkoxy silanes transform polar stationary phase to non polar stationary phase, chain length varying.
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An HPLC typically includes two columns: an analytical column responsible for the separation and a guard column. The guard column is placed before the analytical column, protecting it from contamination. Guard Columns Two problems tend to shorten the lifetime of an analytical column. First, solutes binding irreversibly to the stationary phase degrade the columns performance by decreasing the available stationary phase. Second, particulate material injected with the sample may clog the analytical column. To minimize these problems, a guard column is placed before the analytical column. Guard columns usually contain the same particulate packing material and stationary phase as the analytical column, but are significantly shorter and less expensive; a length of 7.5 mm and a cost one-tenth of that for the corresponding analytical column is typical. Because they are intended to be sacrificial, guard columns are replaced regularly. Detectors used in HPLC 1. 2. 3. 4. 5. Ultraviolet visible spectrophotometer (most common) Fluorescence detectors Electrochemical detectors (amperometric and coulometric) HPLC-Mass spectrophotometer Refractive index detectors
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Chromatographic terms: t
Poor resolution
t0 = colu umn dead time = time e an unretarde ed compound needs to pass s the column, tR = retention n time
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If
th he
substance
is
not
retained
by
t the
stati ionary
p phase,
Resolution:
Plate nu umber and plate heigh ht The numb ber of theoretical plates is pr roportional to the t column len ngth L. The lon nger a column, the more theo oretical plates.
emter Curv ve reflects the relationship be etween theoreti ical plate heigh ht H and the lin near velocity u of the Van Dee mobile pha ase. Since the plate p height H is a measure for fo the band bro oadening of the e injection pea ak of a substanc ce, the path of th he H(u) curve of a separation n column is of f great interest t for determinin ng the chromatographic cond ditions for practica al applications.
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Eddy diffu usion (A) The band broadeni ing as a result of o the Eddy di iffusion is crea ated by the va arious flow pa aths of individual l sample mole ecules through h the column. In the ideal case, c the sampl le molecules sh hould travel th hrough the column ns as a group or o band. But si ince several molecules m are de elayed by obst tacles or deviat te from the path, the band broad dens during Ed ddy diffusion, independent of o the flow spe eed u of the mobile m phase. We W receive a parallel p straight lin ne to the u-axis for term A. Longitudinal diffusion (B) Length diffusion effects s a dilution of f sample mole ecules in direc ction of the co olumn a very low flo ow speed of th he mobile phase. In the rang ge of the usual l flow speeds for chromatog graphic axis only at separations s length diffusi ion seldom pla ays an importan nt part. Mass tran nsfer into por res in stationa ary phase (C) The dominan nt factor of ban nd broadening is the movem ment of sample mo olecules betwee en mobile phas se and stationa ary phase. it Re elates to the kin netics of the mass m transfer be etween mobile and d stationary ph hase. Molecule es which are completely c in the mobile ph hase always ar re slightly ahe ead of the peak center. c
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Gas chromatography
The compound separated with gas chromatography must be volatile or can be derivatized so that become volatile and it must be thermally stable. Carrier gas either Nitrogen, Helium, hydrogen are used as mobile phase. It can be Gas solid chromatography (GSC) or Gas liquid chromatography (GLC). In GLC mobile phase is a gas and the stationary phase is a liquid coated either on a solid packing material or on the columns walls. It is most valuable for the compounds of relatively low polarity or nonpolar. The polar compounds which contain carboxylic acids, alcohols or primary amines as functional grps badly tailed due to interactions of these functional grps with stationary phase. To overcome this problem pyrolysis GC or photolysis is done. In pyrolysis GC the sample for analysis is heated to very high temp. (8000C) for an extremely short time then the thermally destroyed samples are swept into gas chromatograph. A stationary phase of high boiling point liquid like silicone grease is supported on an inert granular solid like diatomaceous earth or celite. This celite or diatomaceous earth or celite has one problem as it also have free hydroxyl grps which can cause support sample interaction. So these hydroxyl grp must be modified by silanization agent like trichlorosilane. More recently, solid supports made from glass beads or fluorocarbon polymers have been introduced. These supports have the advantage of being more inert than diatomaceous earth. The main criteria for selecting a stationary phase are that it should be chemically inert, thermally stable, of low volatility, and of an appropriate polarity for the solutes being separated. The stationary phase is often high boiling point organic compounds which includes polyethylene glycols, methyl phenyl siloxane, squalene and esters of adipic acid, succinic and pthalic acid. Another important characteristic of a gas chromatographic column is the thickness of the stationary phase. Separation efficiency improves with thinner films. The most common film thickness is 0.25 m. Nonvolatile analytes must be chemically converted to a volatile derivative before analysis. For example, amino acids are not sufficiently volatile to analyze directly by gas chromatography. Reacting an amino acid with 1butanol and acetyl chloride produces an esterfied amino acid. Subsequent treatment with trifluoroacetic acid gives the amino acids volatile N-trifluoroacetyl-n-butyl ester derivative. The steps involved in GC 1. 2. 3. A vaporized sample is injected onto the chromatographic column. Second, the sample moves through the column through the flow of inert gas. Separation occurs as a result of unique equilibrium established between the solutes and the stationary phase (the GC column). The components are recorded as a sequence of peaks as they leave the column.
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Gas chr romatograp phy columns 1. Packed Column ns are made up p of glass, nick kel or stainless steel. They ar re typically 1.5 5-10 m long an nd 2-4 m in diameter. Contain iner mm rt support mat terials like dia atomaceous ea arth for liquid d stationary ph hase. 2. Capillary C (Ope en Tubular) Columns are ty ypically 10-50 m long and 0.3-0.5 mm in diameter. d Cap pillary or r open tubular columns are e constructed from fused sil lica coated wit th a protective polymer. Th hey are m more efficient th han packed col lumns. They ar re composed of two major pa arts: tubing and d stationary ph hase. A th hin film (0.1-10 0.0 micro mete ers) of high mo olecular weigh ht, thermally stable s polyme er is coated on nto the in nner wall of sm mall diameter tubing. I. Suppo ort coated ope en tubular (S SCOT) a thin layer l of a solid support, suc ch as a diatoma aceous earth, coated c with a liquid stationa ary phase is att tached to the ca apillarys inner r wall. II. Porous layer open tubular (PLOT) ( Capill lary GC colum mns in which th he stationary phase p is based on an ad dsorbent or a porous polym mer III. W Wall-coated op pen tubular (WCOT) ( con nsist of a capi illary tube wh hose walls are e coated with liquid st tationary phase e Oven Te emperature e Columns are a kept in a heated oven. Co olumn tempera ature relies on the boiling po oint of the sam mple being ana alyzed, and should d be slightly higher h than its s boiling point t. Temperature e must be stric ctly controlled d within fractio ons of degrees.
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Temperature programming Start at low temperature and gradually ramp to higher temperature. Detector There are several types of detectors with each responding to different compounds or properties 1. Flame Ionization Detectors (FID) is only used for organic compounds
Combustion of an organic compound in an H2/air flame results in a flame rich in electrons and ions. If a potential of approximately 300 V is applied across the flame. Most carbon atoms, except those in carbonyl and carboxylic groups, generate a signal, making the FID an almost universal detector for organic compounds. Most inorganic compounds and many gases, such as H2O and CO2, cannot be detected, making the FID detector ideal for the analysis of atmospheric and aqueous environmental samples.
2.
Electron Capture Detectors (ECD) used for pesticide determination or halogenated compounds
The detector consists of a beta emitter (a beta particle is an electron) such as 63Ni. The emitted electrons ionize the mobile phase, which is usually N2, resulting in the production of additional electrons that give rise to an electric current between a pair of electrodes. When a solute capture of electrons elutes from the column, the electric current decreases. This decrease in electric current serves as the signal. The ECD is highly selective toward solutes with electronegative functional groups, such as halogens, and nitro groups and is relatively insensitive to amines, alcohols, and hydrocarbons.
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3.
As the mobile phase exits the column, it passes over a tungstenrhenium wire filament. The filaments electrical resistance depends on its temperature, which, in turn, depends on the thermal conductivity of the mobile phase. Because of its high thermal conductivity, helium is the mobile phase of choice when using a thermal conductivity detector (TCD). When a solute elutes from the column, the thermal conductivity of the mobile phase decreases and the temperature of the wire filament, and thus its resistance, increases. A reference cell, through which only the mobile phase passes, corrects for any time-dependent variations in flow rate, pressure, or electrical power, all of which may lead to a change in the filaments resistance. A TCD detector has the advantage of universality, since it gives a signal for any solute whose thermal conductivity differs from that of helium.
4.
Other detectors
Two common detectors, which also are independent instruments, are Fourier transform infrared spectrophotometers (GC-FTIR) and mass spectrometers (GC-MS). In GCMS effluent from the column is introduced directly into the mass spectrometers ionization chamber in a manner that eliminates the majority of the carrier gas. In the ionization chamber all molecules (remaining carrier gas, solvent, and solutes) are ionized, and the ions are separated by their mass-to-charge ratio. Because each solute undergoes a characteristic fragmentation into smaller ions, its mass spectrum of ion intensity as a function of mass-to-charge ratio provides qualitative information that can be used to identify the solute.
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Ion-Exchange Chromatography
In ion-exchange chromatography (IEC) the stationary phase is a cross-linked polymer resin, usually divinylbenzene cross-linked polystyrene, with covalently attached ionic functional groups. The counter ions to these fixed charges are mobile and can be displaced by ions (present in mobile phase) that compete more favorably for the exchange sites. Ion-exchange resins are divided into four categories: strong acid cation exchangers; weak acid cation exchangers; strong base anion exchangers; and weak base anion exchangers.
The ion exchange sites, indicated by R, are mostly in the para position and are not necessarily bound to all styrene units.
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1. 2. 3. Inorganic polymers Natural Synthetic Aluminosilicates Zeolites copolymers of styrene & DVB OR methacrylic acid Strong acid cation exchanger Weak acid cation exchanger Strong base anion exchanger Weak base anion exchanger Sulfonic acid Carboxylic acid Quaternary amine Amine SO3-, CH2CH2SO3COO, CH2COO CH2N+ (CH3)3 N+H3, CH2CH2N+H (CH2CH3)2
The ion-exchange reaction of a monovalent cation, M+, at a strong acid exchange site is SO3 H+(s) + M+(aq) SO3 M+(s) + H+(aq)
Strong acid cation exchangers include a sulfonic acid functional group that retains its anionic form (pH 1-14), and thus its capacity for ion-exchange, in strongly acidic solutions. The functional groups for a weak acid cation exchanger, however, are fully protonated at pH levels less than 4, thereby losing their exchange capacity. The strong base anion exchangers are fashioned using a quaternary amine, therefore retaining a positive charge even in strongly basic solutions. Weak base anion exchangers, however, remain protonated only at pH levels that are moderately basic. Under more basic conditions, a weak base anion exchanger loses its positive charge and, therefore, its exchange capacity. Generally, for weak acids or base which are only ionized at either low pH (weak bases) or high pH (weak acids) strong anion exchangers are used. And if compounds are strong acid or base then weak anion exchangers are used. The equilibrium constant for this ion-exchange reaction, which is also called the selectivity coefficient, is
Highly charged ions bind more strongly than ions of lower charge
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IEX is mostly employed for the purification of the proteins. IEX separates proteins with differences in charge to give a very high resolution separation with high sample loading capacity. The separation is based on the reversible interaction between a charged protein and an oppositely charged chromatographic medium. Proteins bind as they are loaded onto a column. Conditions are then altered so that bound substances are eluted differentially. This elution is usually performed by increases in salt concentration or changes in pH. The net surface charge of proteins varies according to the surrounding pH. When above its isoelectric point (pI) a protein will bind to an anion exchanger, when below its pI a protein will behind to a cation exchanger. Typically IEX is used to bind the target molecule, but it can also be used to bind impurities if required. IEX can be repeated at different pH values to separate several proteins which have distinctly different charge properties.
The mobile phase in IEC is usually an aqueous buffer, the pH and ionic composition of which determines a solutes retention time. Gradient elutions are possible in which the ionic strength or pH of the mobile phase is changed with time. For example, an IEC separation of cations might use a dilute solution of HCl as the mobile phase. Increasing the concentration of HCl speeds the elution rate for more strongly retained cations, since the higher concentration of H+ allows it to compete more successfully for the ion-exchange sites. The most common detector measures the conductivity of the mobile phase as it elutes from the column. The high concentration of electrolyte in the mobile phase is a problem, however, because the mobile-phase ions dominate the conductivity. For example, if a dilute solution of HCl is used as the mobile phase, the presence of large concentrations of H3O+ and Cl produces a background conductivity that may prevent the detection of analytes eluting from the column. To minimize the mobile phases contribution to conductivity, an ion-suppressor column is placed between the analytical column and the detector. This column selectively removes mobile-phase electrolyte ions without removing solute ions. For example, in cation ion-exchange chromatography using a dilute solution of HCl as the mobile phase, the suppressor column contains an anion-exchange resin. The exchange reaction replaces the ionic HCl with H2O. H+(aq) + Cl(aq) + Resin+ OH Resin+Cl + H2O(l)
Analyte cations elute as hydroxide salts instead of as chloride salts. A similar process is used in anion ion-exchange chromatography in which a cation ion-exchange resin is placed in the suppressor column. If the mobile phase contains Na2CO3, the exchange reaction replaces a strong electrolyte, Na2CO3, with a weak electrolyte, H2CO3. 2Na+(aq) + CO3 2(aq) + 2ResinH+ 2ResinNa+ + H2CO3(aq)
Ion suppression is necessary when using a mobile phase containing a high concentration of ions.
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Gel permeat tion chrom matograp phy or siz ze exclusi ion chrom matography
Gel-Perme eation Chrom matography is a mechanical l sorting of molecules m based on the size of the molecu ules in solution. Small molecu ules are able to permeate more m pores and a are, there efore, retained d longer than large molecules. .
v large mol lecule such as thyroglobulin to determine the void volume which doe es not A sample containing a very goes into the pores. Th he elution volum mes of the sta andards are div vided by the el lution volume of the thyroglobulin nd plotted against the log of the t standards' molecular m weig ghts. (Ve/Vo) an
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KD KD Ve Vo Vi = = = = = (Ve-Vo) / Vi Partition coefficient where stationary phase is interstitial spaces of the polymer bead Elution vol. of solute Void vol. the elution of comps that are completely excluded from the gel pores Vol. of liquid inside the gel pores available to very smallest molecules
Affinity Chromatography
Affinity chromatography depends upon the specific interactions of biological molecules like 1. 2. 3. 4. 5. Antibody-antigen interaction, Enzyme inhibitor interaction, DNA-DNA binding, DNA-protein interaction or Receptor agonist / antagonist interactions.
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SPECTROSCOPY
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Proton NM MR differs fro om 13C NMR in a number of o ways. 1H is the major isotope of hydrogen (9 99.985% natura al abundance), , while 13C is only o a minor is sotope (1.1%) 1H NMR is quantitative e: the area unde er the peak tell ls us the numbe er of hydrogen n nuclei, while 13C NMR ma ay give w peaks from m the same nu umber of 13C nu uclei strong or weak Protons interact magn netically (coup ple) to reveal l the connectiv vity of the str ructure, while clei to be seen coupling between C nuc
13 13
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INSTRUMENTATION
1. UV/Vis spectroscopy: Electronic transitions Source: Tungsten lamp: for visible region i.e. 400-800nm Deuterium lamp: For ultraviolet region i.e. 190-400 nm Sample cell is made up of Quartz Solvent generally used are 95 % Ethanol, 1,4-Dioxane Lambert-beers law: A= bc Graph is plotted between concentration vs optical density (Absorbance) o
Detector: Photomultiplier tubes 2. IR spectroscopy: Viberational changes for compounds having fluctuating dipole moment Source: Globar source (silicon carbide rod) Nerst glower (Ytterinium etc ) Incandescent lamp Detectors: Bolometers, Thermocouples, Golay cells (xenon gas), Pyroelectric (Triglycerine sulphate), Photo cells Cells are constructed of metal halides like NaCl or KBr
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For examination of solid samples either by KBr pellet or mull techniques uses Nujol (high boiling point petroleum oil or mineral oil) is used but when hydrocarbon in the sample interfere with it than hexachlorobutadiene is used.
3. NMR: Spin changes Radio waves oscillator and magnetic field (1Tesla = 104 Gauss) Chemical shift is measured in ppm and it is independent on magnetic field. Coupling constant is measured in Hz. 4. ESR: Rotational changes Microwaves (Klystron oscillator) and electromagnets Internal standard: DPPH 5. Mass spectroscopy: No electromagnetic radiations are used Source: Electron impact Chemical ionization (carrier gas Methane and ammonia) Fast atom bombardment (carrier gas Argon) Laser desorption (Matrix Assisted Laser Desorption Ionization) is Soft technique used for protein samples Electron spray Ionization Mass analyzers: Quadra pole Magnetic sector Electronic sector Detector: Electron multiplier tubes
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Faraday Cup
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