Chapter 11 Column Liquid Chromatography

Transcription

1 Chapter 11 Column Liquid Chromatography

2 Types of Liquid Chromatography Partition chromatography Adsorption or liquid-solid chromatography Ion Exchange chromatography Size exclusion or gel chromatography

3 Adsorption Chromatography The stationary phase is solid. Separation is due to adsorption/desorption steps

4 Adsorbent can be packed in a column spread on a plate, or impregnated in a A porous paper Both solutes and solvents will be attracted to the stationary phase If the solutes have different degrees of attraction separation would be achieved

5 Partition chromatography Separation is based on solute partitioning between two liquid phases More highly retained species have greater affinity (solubility) for the stationary phase compared to the mobile phase (solvent) Separation of solutes is based on the difference in the relative solubilities

6 Modes of separation Normal phase partition chromatography Polar stationary phase and nonpolar solvent Reverse phase partition chromatography Nonpolar stationary phase and polar solvent Reverse phase is now more common

7 Changing the mode of separation will lead to a change In the elution order of the solutes that could be almost Reversed.

8 Ion exchange chromatography Stationary phase has charged surface opposite that of the eluents Separation is based on the affinity of ions in solution for oppositely charged ions on the stationary phase

9 Separation by ion exchange chromatography

10 Size exclusion chromatography Separation is based on molecular size. Stationary phase is a material of controlled pore size. It is also called Gel permeation chromatography

11 Size exclusion chromatography Columns are made to match the separation of specific size ranges Larger species will elute first. They cannot pass through many pores so their path is shorter Size exclusion liquid chromatog. Is useful for determining size, size range and molecular weights of polymers and proteins.

12 Separation by size exclusion chromatography

13 LC Solvents (Mobile phase) LC solvents depend upon the type of chromatographic mode used: * Normal Phase * Reversed phase

14 Solvent selection If the sample is water insoluble or nonpolar- normal phase mode is used If the sample is water soluble or not soluble but polar- use the reverse phase mode It is seldom to find a single solvent does the job. Thus mixtures of two or more solvents are used Two factors are considered: Solvent strength, ( o ) A measure of relative solvent polarity (ability to displace a solute). It is the adsorption energy per unit area of solvent. o for silica is about 0.8 of those on alumina Polarity index, (P ) Solvents that interact strongly with solutes are strong or polar solvents Polarity of solvents has been expressed by many terms, one of which is the polarity index. Thus, the P value measures the relative polarity of various solvents Used for reversed phase methods

15 Solvent strength and polarity index o P

16 Solvents used as mobile phase in liquid chromatography Snyder classified solvents based on acidity and basicity, dipole and chemical properties

17 Gradient elution

18 Advantages of gradient elution

19 Gradient elution

20 Gradient elution

21 Mobile Phases for adsorption chromatography Common mobile phases on alumina hexane; chloroform; 2-propanol Example: separation of amines Common mobile phases on Silica hexane; chloroform; 2-propanol Examples: separation of ethers, esters, prophyrins, vitamins

22 Mobile Phases for partition chromatography

23 Ion exchange phases

24 High Performance Liquid Chromatography

25 Column Liquid Chromatography LC techniques are: Classical LC and HPLC or HSLC (S = speed) Both techniques have same basic principle for separation but differ in apparatus and practice used HPLC gives high speed, high resolution, high sensitivity and convenient for quantitative Analysis.

26 HPLC originally refered to: High Pressure Liquid Chromatography currently refers to: High Precision Liquid Chromatography the high pressure allows using small particle size to allow proper separation at reasonable flow rates

27 Features of HPLC compared to Classical LC Particle size of the packing substance Classical LC utilises large porous particles that make it difficult to speed up the flow rate by pumping due to a decrease in resolution that results from the mass transfer limitation in the deep pores. These high capacity particles are good for preparative chromatography Since the mass transfer coefficient is a function of the square of d p (van deemter eq.) thus the HPLC was based on using pellicular and porous microparticles. Pellicular particles when packed into narrow columns will lead to an increase in column efficiency of 10 to 100 folds Pellicular particles have dense solid cores thus they are easily packed V s is significantly reduced and the sample capacity is reduced to 0.05 to 0.1 of the totally porous packing

28 Effect of particle packing size on column efficiency

29 Column length Efficiency is very high due to packing thus shorter columns are used (~ 20 cm) For difficult separations longer ( cm) are used with smallest available particles and high pressure solvent feed Effect of sample size on column efficiency Efficiency increases as the sample size decreases

30 Effect of sample mass on column efficiency

31 Columns LC columns could be made of stainless steel, glass and glass lined stainless steel that is used for extremely inert surfaces. In classical LC, elution takes place under gravity or low pressure by using small pumps Columns can be thermostated by placing in an oven or using a water jacket HPLC columns are mainly made of stainless steel packed with the microparticles When same amount is injected in the HPLC column, narrower and longer peaks are obtained that leads to greater detector sensitivity In HPLC solvent consumption is reduced HPLC columns facilitate coupling to MS that requires flow rates <50 L/min to avoid over pressuring the ion source in the MS

32 Components Solvent Reservoir and Degassing System Pumps Precolumns Sample Injection System Columns Temperature Control Detectors Readouts

33 Schematic diagram of a typical high performance liquid chromatograph

34 Schematic of Liquid Chromatograph

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37 Solvent Reservoir and Degassing System isocratic elution - single solvent separation technique gradient elution - 2 or more solvents, varied during separation

38 Improvement in separation efficiency by gradient elution. Column: 1m x 2.1mm id, precision-bore stainless. Sample: 5 L of chlorinated benzenes in isopropanol. Detector: UV photometer (254 nm). Conditions: temperature 60 o C, pressure, 1200 psi.

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40 Pumping systems Requirements for pumping system Generation of pressure up to 6000 psi Pulse free output Flow rates of 0.1 to 10 ml/min Flow control and flow reproducibility of 0.5% relative or better Corrosion resistance components Types of pumps: Direct pressure pumps (pneumatic pumps) Syringes (displacement pumps) Reciprocating pumps

41 They are limited to pressures less Than 2000 psi

42 They are of limited solvent capacity (about 250 ml) and inconvenience when the solvent is changed.

43 They have small internal volume ( L) High output pressure (10000 psi) Readily adoptable to gradient elution Constant flow rates that are independent of column back pressure and solvent viscosity Widely used pumping; motor driven piston that pumps the solvent into the chamber. The two ball check valves (f) Open and close alternatively to control the solvent into and out of the chamber. It has the disadvantage of producing pulsed flow which must be damped because its presence is manifested as baseline noise

44 Reciprocating pump

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51 Sample Loop

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55 Detectors Properties of good detectors

56 Types of Detectors Absorbance (UV with Filters, UV with Monochromators) IR Absorbance Fluorescence Refractive-Index Evaporative Light Scattering Detector (ELSD) Electrochemical Mass-Spectrometric Photo-Diode Array

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61 Ultraviolet detector

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64 Refractive-Index Detector Schematic of a differential refractive-index detector

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69 Mass spectrometric detectors LC and MS appear to be incompatible. HPLC Liquid phase operation o C operation MS Gas phase operation o C operation Almost no sample limitation same volatility desired Relatively inexpensive Uses inorganic buffers Conventional flow rate produce 550 ml/min gas at STP Expensive can t tolerate inorganic buffers Accepts 10 ml/min

70 Features of LS-MS Its sensitivity approaches sub nanograms The significant problem here is 1 ml of hexane, methanol, or water generates respectively, 180, 350 and 1250 ml/min gas Since MS operates under vacuum the sample vapor must be removed without removing a substantial amount of solute

71 LC-MS interface techniques Direct liquid inlet: small portion of LC effluent is directed into the ion source Moving belt method: LC effluent is deposited onto a moving belt, the solvent is evaporated, and the sample is volatilized from the belt into the ion source Thermospray ionization: ions are created when aqueous buffered mobile phase is passed through a heated stainless steel capillary creating a supersonic jet of vapor with subsequent evaporation of the mobile phase from charged liquid droplets

72 Applications of Liquid Chromatography

73 Applications Preparative HPLC -the process of isolation and purification of compounds. analytical HPLC-obtain information about the sample compound. identification, quantification, resolution of a compound. Chemical Separations - using HPLC certain compounds have different migration rates given a particular column and mobile phase. Purification - the process of separating or extracting the target compound from other (possibly structurally related) compounds or contaminants. Each compound should have a characteristic peak under certain chromatographic conditions. choose the conditions, such as the proper mobile phase, to allow adequate separation to collect or extract the desired compound as it elutes from the stationary phase. Identification of compounds by HPLC is a crucial part of any HPLC assay. accomplished by researching the literature and by trial and error. Identification of compounds can be assured by combining two or more detection methods. Quantification - the process of determining the unknown concentration of a compound in a known solution. inject a series of known concentrations of the standard compound solution chromatograph of these known concentrations peaks that correlate to the concentration of the compound injected

74 Adsorption chromatography

75 Adsorption chromatography It is LSC and oldest chromatographic method introduced by Tswett that became the HPLC technique Silica and alumina are mostly used as the stationary phases in thin layer or column How do adsorbents separate compounds? Consider the surface of the most widely adsorbent, silica gel. Silica gel is a stable porous solid terminated at the surface with silanol (Si-OH) or siloxane (Si-O-Si) bonds The slightly acidic silanol groups are of importance in separation however, siloxane bonds are of little or no influence

76 Most acidic Silanol groups

77 Silanol groups and interactions with solutes Silanol groups have varying degrees of acidity The most acidic ones are located at adjacent silicon atoms with intermolecular H-bonding. These lead to undesirable effects like chemisorption and peak tailing To avoid problems, polar modifier such as water is added in order to deactivate the strongest adsorption sites Interactions between adsorbent surface and solute vary from nonspecific (dispersion or vander Waal s forces) to specific ones (electrostatic interactions such as permanent dipoles or electron donor acceptor interactions such as hydrogen bonding) Retention on silica gel or alumina is governed mainly by interactions with the polar functional groups of the solute Compounds of different chemical types (hydrocarbons and alcohols) are easily separated by LSC

78 Homologous or other mixtures differing in the extent of aliphatic substitution (no change in polarity) cannot be differentiated LSC is unique in its ability to separate polyfunctional compounds especially positional isomers

79 With a few exceptions, the order of retention times on silica and alumina is: olefins < aromatic hydrocarbons < halides, sulfides < ethers < nitro compounds < esters ~ aldehydes ~ ketones < alcohols amines < sulfones < sulfoxides < amides < carboxylic acids

80 Intramolecular hydrogen bonding; i.e, less intermolecular interaction with the surface

81 Solvent Selection for Adsorption Chromatography In liquid-solid chromatography, the only variable available to optimize the retention factor and the selectivity factor is the composition of the mobile phase (in contrast to partition chromatography, where the column packing has a pronounced effect on the selectivity factor)

82 Influence of the Mobile Phase in LSC: Gradient Elution Interactions in LSC involve a competition between the solute molecules (X) and the molecules (S) of the mobile phase adsorption sites. This equilibrium is illustrated by

83 Thus, stronger adsorption of the mobile phase decreases adsorption of the solute. Solvents can be classified according to their strength of adsorption (solvent or eluent strength, o ). Such a quantitative classification is referred to as an eluotropic series. An eluotropic series can be used to find an optimum solvent strength for a particular separation. Using a solvent of constant composition is called isocratic elution.

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85 Solvent Strength The polarity index, P', used in partition chromatography can also serve as a rough guide to the strengths of solvents for adsorption chromatography. Solvent (Eluent) strength, which is the solvent adsorption energy per unit surface area is a much better index. This parameter depends upon the adsorbent. values for silica are about 0.8 of those on alumina. Note that solvent-to-solvent differences in roughly parallel those for P'. For a given isocratic elution, the initial solvent is selected by matching the relative polarity to that of sample components. Solvent is chosen to match the most polar functional group. Alcohols for OH group and amines for amino acids. If in an isocratic elution the k'-values for the solutes are too small (sample elutes rapidly) then a weaker (low, less polar) solvent is selected On the other hand, if the sample does not elute in a reasonable time because of high k values then a stronger (high, more polar) solvent would be selected.

86 Choice of Solvent Systems (binary mixture) Binary solvent mixtures may be used to find an optimum value of the solvent-strength parameter Two compatible solvents are chosen, one of which is too strong ( too large) and the other is too weak. A suitable value for k' is then obtained by varying the volume ratio of the two. a mixture of isooctane ( = 0.01) and methylene chloride ( = 0.42) can be matched with an isocratic solvent strength similar to that of carbon tetrachloride ( = 0.18). Unfortunately does not vary linearly (because of solvent-solvent and preferential solvent-surface interactions) with volume ratios. Thus, calculating an optimal mixture is more difficult.

87 General Elution Problem This problem appears with isocratic solvent systems and multicomponent samples with widely differing k'-values. If a strong isocratic mobile phase is selected that will adequately elute strongly retained compounds, then the weakly retained ones will be eluted too quickly and will be poorly separated Conversely, if a weak mobile phase is chosen, so that weakly retained sample components will be retained and separated, then very strongly retained solutes may not be eluted at all-or only very slowly The most common solution is using a technique called solvent programming or gradient elution. Here, elution is begun with a weak solvent and the solvent strength is increased with time. The changes are made either stepwise or continuously

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90 Applications of Adsorption Chromatography Adsorption chromatography is best suited for nonpolar compounds having molecular weights less than perhaps Although some overlap exists between adsorption and partition chromatography, the methods tend to be complementary. Generally, liquid-solid chromatography is best suited to samples that are soluble in nonpolar solvents and correspondingly have limited solubility in aqueous solvents such as those used in the reversed-phase partition procedure. A particular strength of adsorption chromatography, which is not shared by other methods, is its ability to differentiate among the components of isomeric mixtures.

91 Partition Chromatography (Liquid-liquid and Bonded phase Chromatography)

92 Features of Partition Chromatography Most of the applications have been to nonionic, polar compounds of low to moderate molecular weight (usually <3000). Recently, however, methods have been developed (derivatization and ion pairing) for separations to ionic compounds. Partition chromatography can be subdivided into liquid-liquid and bonded-phase chromatography. The difference in these techniques lies in the method by which the stationary phase is held on the support. With liquid-liquid, a liquid stationary phase is retained on the surface by physical adsorption. With bonded-phase, the stationary phase is bonded chemically to the support surfaces.

93 Liquid-liquid chromatography is limited to compounds with comparatively low values of K (or k'), because the stationary phase must be a good solvent for the sample but a poor solvent for the mobile phase. In practice, increasing solvent strength in order to elute compounds with high K- (or k'-) values will increase the solubility of the stationary phase and remove the stationary phase from its support. When the solvent strength is high enough to dissolve an appreciable amount of stationary phase, presaturation is made difficult. in conventional LLC solvent programming is ruled out. Even with its limitations, LLC is a very useful technique because it can resolve minute differences in the solubility of the solute. Many solvent pairs are available, and the choice of the proper ones allows great selectivity to be achieved. Both Paper chromatography and TLC are examples of LLC.

94 Liquid-liquid chromatography The stationary and mobile phases are selected so as to have little or no mutual solubility. Therefore, they generally are quite different in their solvent properties. For example one might choose water as the stationary phase and pentane as the mobile phase for normal LLC. However, water does have a finite (though very slight) solubility in pentane. Using pentane will slowly remove the water and change the nature of the separation mechanism. For this reason, the mobile phase must be presaturated with the stationary phase before it enters the column (or plate). Presaturation can be done by stirring the two phases together until equilibration takes place; but, in LC, it is more conveniently done by placing a precolumn before the injector and the chromatographic column. The precolumn should contain a high-surface-area packing, such as silica gel, coated with a high percentage (say 30 to 40% by weight) of the stationary phase used in the analytical column.

95 Drawbacks of LLC: finding immiscible solvent pairs, presaturing the mobile phase to avoid removal of coated stationary phase, the impossibility of using gradient elution to solve the general elution problem Solution Use of chemically bonded stationary phases. Bondedphase chromatography (BPC) now dominates in use all modes of HPLC. Microparticulate silica gel is the base material used for the synthesis of almost all chemically bonded phases.

96 Bonded-Phase Chromatography The supports are prepared from rigid silica, or silica-based, compositions. The surface of fully hydrolyzed silica (hydrolyzed by heat ing with 0.1 M HCl for a day or two) is made up of chemically reactive silanol groups. The most useful bonded-phase coatings are siloxanes formed by reaction of the hydrolyzed surface with an organochlorosilane

97 R : alkyl group or a substituted alkyl group. The unreacted SiOH groups, unfortunately, impart an undesirable polarity to the surface, which may lead to tailing of chromatographic peaks, particularly for basic solutes. To lessen this effect, siloxane packings are frequently capped by further reaction with chlorotrimethylsilane that, because of its smaller size, can bond many of the unreacted silanol groups.

98 The -Si-O-Si-C bond is stable under most conditions used in LC but is attacked by hydrolysis under basic conditions (ph > 7). Bonded phase stationary phase can be used with gradient elution. This is a major advantage of BPC. Two main techniques can be classified, based on the relative polarities of the stationary and mobile phases: (a) Normal phase BPC, and (b) reversed-phase BPC. Normal-phase BPC is used when the stationary phase (e.g., aminopropyl) is more polar (as evidenced by the predominant functional group) than the mobile phase (e.g., hexane). Reversed-phase BPC is used when the stationary phase is nonpolar (e.g., octadecylsilane) and the mobile phase is polar (e.g., water-methanol). Solute-elution order is often the reverse of that observed with normal-phase BPC. The technique is ideally suited to substances insoluble or only sparingly soluble in water but soluble in alcohols or other water-miscible organic solvents. Because many organic compounds show this solubility behavior, reversed-phase BPC is the most widely used mode of HPLC, accounting for about 60%of the published applications.

99 Reversed-Phase and Normal-Phase Bonding Phase Chromatography Based upon the relative polarities of the mobile and stationary phases, two types of partition chromatography are distinguishable. normal-phase BP chromatography Highly polar stationary phase such as water or triethyleneglycol supported on silica or alumina particles; a relatively nonpolar solvent such as hexane serves as the mobile phase. The least polar component is eluted first because in a relative sense, it is the most soluble in the mobile phase Increasing the polarity of the mobile phase has the effect of decreasing the elution time. Normal-phase BPC can replace LSC on silica gel in many applications

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101 Reversed-phase chromatography The stationary phase is nonpolar, often a hydrocarbon, and the mobile phase is relatively polar (such as water, methanol, or acetonitrile). The most polar component appears first, and increasing the mobile phase polarity increases the elution time. Perhaps three quarters of all high performance liquid chromatography is currently being carried out in columns with reversed-phase packings.

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103 Reasons for the wide usage of RP-BPC Nonionic, ionic, and ionizable compounds can often be separated, sometimes at the same time, using a single column and mobile phase. Bonded-phase columns are relatively stable provided certain precautions, especially ph control, are taken. The predominant mobile phase, water, is inexpensive and plentiful. The most frequently used organic modifier, methanol, can be obtained at a reasonable price and of sufficient purity in most places in the world. The elution order is often predictable because retention time usually increases as the hydrophobic character of the solute increases. Columns equilibrate rapidly, thereby permitting faster method development and sample turnaround after gradient elution.

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105 Relationship between polarity and elution times for normal phase and reversed phase chromatography

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107 Effect of chain length of the alkyl group of the bonded phase upon performance

108 Longer chains produce packings that are more retentive. In ad dition, longer chain lengths permit the use of larger samples. In most applications of reversed-phase chromatog raphy, elution is carried out with a highly polar mobile phase such as an aqueous solution containing various concentrations of such solvents as methanol, acetoni trile, or tetrahydrofuran. In this mode, care must be taken to avoid ph values greater than about 7.5 because hydrolysis of the siloxane takes place, which leads to degradation or destruction of the packing.

109 Applications of Partition Chromatography Reversed-phase bonded packings, when used in con junction with highly polar solvents (often aqueous), approach the ideal, universal system for liquid chro matography. Because of their wide range of applicability, their convenience, and the ease with which k' and a can be altered by manipulation of aqueous mobile phases, these packings are frequently applied before all others.

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112 Typical applications of bonded-phase chromatography. (a) Soft-drink addi tives. Column: 4.6 x 250 mm packed with polar (nitrile) bonded-phase packings. Isocratic solvent: 6% HOAC/94% HZO. Flow rate: 1.0 cm3/min. (b) Organophosphate insecticides. Column: 4.5 x 250 mm packed with 5-wm, C8, bonded-phase par ticles. Gradient: 67% CH30H/33% H20 to 80 CH3/20% H20. Flow rate 2 ml/min. Both used 254-nm UV detectors.

113 Derivative Formation In some instances, the components of a sample are converted to a derivative before, or sometimes after, chromatographic separation is undertaken for the following reasons: Reduce the polarity of the species so that partition rather than adsorption or ion-exchange columns can be used Increase the detector response and thus sensitivity, for all of the sample components Enhance the detector response to certain components of the sample.

114 Use of derivatives to reduce polarity and enhance sensitivity

115 Ion-Pair Chromatography Ion-pair (or paired-ion) chromatography is a type of reversed-phase partition chromatography that is used for the separation and determination of ionic species. The mobile phase in ion-pair chromatography consists of an aqueous buffer containing an organic solvent such as methanol or acetonitrile and an ionic compound containing a counter ion of opposite charge to the analyte. A counter ion is an ion that combines with the analyte ion to form an ion pair, which is a neutral species that is retained by a reversed-phase packing. Most of the counter ions contain alkyl groups to enhance retention of the resulting ion pair on the nonpolar stationary phase. Elution of the ion pairs is then accomplished with an aqueous solution of methanol or other water soluble organic solvent. Applications of ion-pair chromatography frequently overlap those of ion-exchange chromatography. An example of where the ion-pair method provides better separations is for analyzing mixtures of chlorate and nitrate ion. For this pair of solutes, selectivity with an ion-exchange packing is poor.

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117 ION-EXCHANGE CHROMATOGRAPHY Ion-exchange chromatography (IC), which is often shortened to ion chromatography refers to modern and efficient methods of separating and determining ions based upon ion-exchange resins. Ion chromatography was first developed in the mid-1970s when it was shown that anion or cation mixtures can be readily resolved on HPLC columns packed with anion-exchange or cation-exchange resins. At that time, detection was generally performed with conductivity measurements. Currently, other detectors are also available for ion chromatography. Ion chromatography was an outgrowth of ion-ex change chromatography,

118 Ion-Exchange Equilibria Ion-exchange processes are based upon exchange equilibria between ions in solution and ions of like sign on the surface of an essentially insoluble, high-molecular weight solid. Natural ion-exchangers, such as clays and zeolites, have been recognized and used for several decades. Synthetic ion-exchange resins were first produced in the mid-1930s for water softening, water deionization, and solution purification. The most common active sites for cation-exchange resins are the sulfonic acid group -SO - 3 H +, a strong acid, and the carboxylic acid group -COO - H +, a weak acid. Anionic exchangers contain tertiary amine groups -N(CH 3 ) 3 OH - or primary amine groups -NH 3+ OH - ; the former is a strong base and the latter a weak one. When a sulfonic acid ion-exchanger is brought in contact with an aqueous solvent containing a cation M x+ :

119 Cation exchanger Similarly a strong base exchanger interacts with the anion A x- as shown by the reaction Anion exchanger

120 Influences on Distribution Coefficients and Selectivity Ion-exchange chromatography involves more variables than other forms of chromatography. Distribution coefficients and selectivities are functions of: ph, solute charge and radius, resin porosity, ionic strength and type of buffer, type of solvent, temperature, and so forth. The number of experimental variables makes ion-exchange chromatography a very versatile technique, since each may be used to effect a better separation, but a difficult one because of the time needed to optimize a separation.

121 By selecting a common reference ion such as H +, distribution ratios for different ions on a given type of resin can be experimentally compared. Such experiments reveal that polyvalent ions are much more strongly held than singly charged species. Within a given charge group, however, differences appear that are related to the size of the hydrated ion as well as to other properties. Thus, for a typical sulfonated cation exchange resin, values for K ex decrease in the order For anions, K ex for a strong base resin decreases in the order

122 Ion Chromatography Ion chromatography (IC) is an ion-exchange technique that uses, most popularly, a low-capacity column combined with a conductivity detector Its most frequent practical application is the determination of trace anions in aqueous solution. The low-capacity column allows the use of a buffer with a low ionic strength. There are two forms of IC practiced today: (a) suppressed or dual-column IC, (b) nonsuppressed or single column IC.

123 Ion Chromatography with Eluent Suppressor Columns The widespread application of ion chromatography for the determination of inorganic species was inhibited by the lack of a good general detector. Conductivity detectors are an obvious choice for this task. They can be highly sensitive, they are universal for charged species, and, as a general rule, they respond in a predictable way to concentration changes. The only limitation arises from the high electrolyte concentration required to elute most analyte ions in a reasonable time.

124 As a consequence, the conductivity from the mobile-phase components tends to swamp that from analyte ions, thus greatly reducing the detector sensitivity. The problem of high eluent conductance was solved by the introduction of a so-called eluent suppressor column immediately following the analytical ion-exchange column. The suppressor column is packed with second ion-exchange resin that effectively converts the ions of the solvent to a molecular species of limited ionization without affecting the analyte ions.

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126 For example, when cations are being separated and determined, hydrochloric acid is often chosen as the eluting reagent, and the suppressor col umn is an anion-exchange resin in the hydroxide form. The product of the reaction in the suppressor is water. That is, H + (aq) + Cl - (aq) + Resin + OH - (s) ---> Resin + Cl - (s) + H 2 O The analyte cations are, of course, not retained by this second column. For anion separations, the suppressor packing is the acid form of a cation-exchange resin. Sodium bi carbonate or carbonate may serve as the eluting agent. The reaction in the suppressor is then Na + (aq) + HCO - 3 (aq) + Resin - H + (s) -> Resin - Na + (s) + H 2 CO 3 (aq) Here, the largely undissociated carbonic acid does not contribute significantly to the conductivity. An inconvenience associated with the original suppressor columns was the need to regenerate them periodically (typically, every 8 to 10 hr) in order to convert their packings back to the original acid or base form.

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128 Single-Column Ion Chrornatography Equipment has also become available commercially for ion chromatography in which no suppressor column is used. This approach depends upon the small differences in conductivity between the eluted sample ions and the prevailing eluent ions. To amplify these differences, low-capacity exchangers are used, which make possible elution with species having low equivalent conductances. Single-column chromatography tends to be somewhat less sensitive and to have a more limited range than ion chromatography with a suppressor column. An indirect photometric method that permits the separation and detection of nonabsorbing anions and cations without a suppressor column has recently been described. Here also, no suppressor column is used, but instead, anions or cations that absorb Uv or Vis radiation are used to displace the analyte ions from the column. When the analyte ions are displaced from the exchanger, their place is taken by an equal number of eluent ions (provided, of course, that the charge on the analyte and eluent ions is the same).

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132 Size-exclusion Chromatography Gel-permeation or gel-filtration chromatography applicable to high molecular weight species Packing is small silica or polymer particles containing a network of uniform pores into which solute and solvent molecules can diffuse. The average residence time of analyte molecules in the pores depends upon the effective size of these molecules Molecules that are larger than the average pore size will not be retained Molecules with sizes smaller than those of the pores will be retained Intermediate size molecules will penetrate according to their sizes. Thus fractionation occurs.

133 The process is almost always carried out in a column, but it also has been performed on a thin layer. Column packing materials with pores of different (controlled) sizes are generally used. The materials can be soft gels, semirigid gels, or rigid materials. The soft and semirigid gels can change their pore sizes, depending on the solvent used as a mobile phase. The soft gels, of the polydextran or agarose type, can swell to many times their dry volume, whereas the semirigid gels of the polyvinylacetate or polystyrene type swell to 1.1 to 1.8 times their dry volume. Rigid materials, such as porous glass or porous silica beads, have fixed pore sizes and do not swell at all.

134 Theory of Size-Exclusion Chromatography The total volume V t of a column packed with a porous polymer or silica gel is given by V t = V g + V i + V o V g is the volume occupied by the solid matrix of the gel, V i is the volume of solvent held in its pores, V o is the free volume outside the gel particles. Assuming no mixing or diffusion, V o also represents the theoretical volume of solvent required to transport through the column those components too large to enter the pores of the gel. In fact, however, some mixing and diffusion will occur, and as a consequence the nonretained components will appear in a Gaussian-shaped band with a concentration maximum at V o. For components small enough to enter freely into the pores of the gel, band maxima will appear at the end of the column at an eluent volume corresponding to (V i + V o ).

135 Molecules of intermediate size are able to transfer into some fraction K of the solvent held in the pores; the elution volume V e for these retained molecules is V e = V o + K C V i (1) Equation 1 applies to all of the solutes on the column. For molecules too large to enter the gel pores, K C = 0 and V e = V o ; for molecules that can enter the pores unhindered, K C = 1 and V e = (V o + V i ). In deriving Eq. 1, the assumption was made that no interaction, such as adsorption, occurs between the solute molecules and the gel surfaces. With adsorption, the amount of interstitially held solute will increase; with small molecules, K C will then be greater than unity.

136 Eq. 1 rearranges to K C = (V e - V o )/V i = C s lc m (2) where K C is the distribution constant for the solute. Values of K C range from zero for totally excluded large molecules to unity for small molecules.

137 The useful molecular weight range for a size exclusion packing is conveniently illustrated by means of a calibration curve such as that shown in the upper part of the Figure. Molecular weight, which is directly related to the size of solute molecules, is plotted against retention volume V R. Note that the ordinate scale is logarithmic. The exclusion limit defines the molecular weight of a species beyond which no retention occurs. All species having greater molecular weight than the exclusion limit are so large that they are not retained and elute together to give peak A in the chromatogram shown.

138 The permeation limit is the molecular weight below which the solute molecules can penetrate into the pores completely. All molecules below this molecular weight are so small that they elute as the single band labeled D. As molecular weights decrease from the exclusion limit, solute molecules spend more and more time, on the average, in the particle pores and thus move progressively more slowly. It is in the selective permeation region that fractionation occurs, yielding individual solute peaks such as B and C in the chromatogram.

139 No retention beyond this MW Vo Vi Limit below which solute molecules can penetrate completely into the pores Permeation limit Molecules below this MW are so small that they elute as the single band D A B C D Unretained large molecules Such calibration curves are supplied by manufacturers of packing materials

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