Subtraction methods are widely used in chromatographic practice, mainly for identify- ing chromatographic zones in the analysis of unknown mixtures. This is a very important, but not the only, problem that can be solved by use of this method. Subtraction methods can also be successfully employed for solving the following problems: (1) demasking of impurity zones, which are often masked by the zone of the principal components; (2) concentrating impurities with the use of selective absorbers; and (3) determination of the content of components in the overall unseparated zone.
Simplicity and flexibility are the attractive features of the subtraction method. Despite the utilization of this method in practice it has not been developed thoroughly enough, even for identification purposes. Numerous publications on this method mainly contain, with rare exceptions, a description of its application to several classes of organic com- pounds, although its practical value essentially depends on how comprehensively the reagent used has been studied (selectivity, interaction with many classes of organic com- pounds, volatility, maximum temperature of utilization, reaction rate, completeness of
reaction under different conditions, side-reactions, etc.).
The development of the subtraction method requires the elaboration of a systematic method of analysis of organic compounds and the development of techniques for its utilization in capillary chromatography.
An especially promising modification of the subtraction method, which should, in our opinion, be given special attention, is the two-stage version, the first stage being subtrac- tion and the second being isolation (inverse subtraction) and chromatographic analysis of the components removed in the first stage. The selectivity, accuracy and reliability are much higher in this version than in the conventional one.
The method can be substantially simplified by using selective volatile reagents. This aspect is also extremely promising; in particular, its utilization makes the two-stage ver- sion easier to achieve. Further development of the method for concentrating impurities, including those in the environment, is also of interest.
The examples considered in this chapter indicate that further development of the method will be of practical value.
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Chapter 6
Chemically selective stationary phases
The main aim of a chromatographic investigation is usually the separation of com- pounds with similar properties. The separation coefficient, R , of two compounds j and i between chromatographic zones is described (e.g., refs. 1 and 143) by the equation
where tRi are the t R i retention times of j and i, and uti is the standard deviation (in time units) of the component peak. The value of Rji is dependent on the characteristics of the chromatographic process:
where ki = Ci&, * V,/V, is the capacity ratio of component i, Cis and Cim are the molar concentrations of component i in the stationary (s) and mobile (m) phases, V, and Vm are the volumes of the stationary and mobile phases, respectively, in the column, ajj = k j / k i is the relative retention (selectivity coefficient, separation factor) andNi is the number of theoretical plates.
The separation is mainly conditioned by the value of the selectivity coefficient, aji [2], as follows from the analysis of eqn. 6.2. A small increase in the selectivity coef- ficient, especially in the region where it differs insignificantly from unity, causes a sharp decrease in the number of theoretical plates required for the separation. As the necessary number of theoretical plates is approximately proportional to the retention time, an increase in aji causes a sharp decrease in the duration of the analysis [3]. This is why the development of selective stationary phases has attracts the attention of many investi- gators, in the last 25 years. For example, ca. 33% of the papers on gas chromatography published in the Journal of Chromatography in 1981 was devoted to problems of stationary phases.
The most striking example of specially selective stationary phases is those based on complexing reactions of volatile compounds to be separated with an involatile active component of the stationary phase. The selectivity of this type of stationary phase is so high in some instances, that it is sufficient for the separation of organic isomers, includ- ing nuclear isomers, the molecules of which differ, for example, by presence of atoms of hydrogen-1, -2 and -3 [4] .
In this chapter stationary phases whose interaction with compounds to be separated is significant are examined; interactions based on the formation of hydrogen bonds are not examined here, although it is in principle a particular case of chemical interactions.
Separations on stationary phases with the formation of hydrogen bonds were examined by Iogansen and Kurkchi [ 51 . The separation of diastereoisomers of a number of organic substances is based on the selective formation of intermolecular hydrogen bonds [6] .
Although different kinds of complexes of organic compounds are known, complexes of metals are most widely used in gas chromatography (GC).
The general properties of complexes of this type have been examined in a number of books and reviews (see, for example, refs. 7-1 1). Donor-acceptor complexes of metals (electron acceptors) and of organic compounds (electron donors) are often not stable (the equilibrium constant is low) and exist in equilibrium with the free components; the rate of formation o f these complexes is usually sufficiently great. These properties of the com- plexes combined with high chemical selectivity are requirements for the utilization of the complexes in chromatographic separations. It was shown for the first time by Bradford et al. [ 121 in 1955 for the complete separation of ethane and ethylene using a solution of silver nitrate in ethylene glycol as the liquid stationary phase. It should also be noted that GC is an effective method for the investigation of complex formation, as was shown for the first time by Du Plessis and Spong [ 131 . GC methods have several advantages, e g , ( I ) rapidity; (2) high accuracy and; (3) micro-scale operation. In a number of applications no alternative methods exist. For example, the spectra of cis- and trans-isomers of chromium(II1) trifluoroacetylacetonate are very close in the visible, infrared and ultra- violet regions [ 141 and nuclear magnetic resonance spectroscopy is also unsuitable. Gas chromatography allows the separation of these isomers [ 151. Gil-Av and Herling [ 161 made a great contribution to the development of quantitative methods for the study of complex formation.
The complete theory of the retention of volatile compounds on liquid stationary phase (LSPs), which form a complex with a chromatographated substance, was developed by Purnell [I71 and Martire and Riedl [18]. Let us examine the method for determi- nation of the character of complex formation according to Cadogan and Purnell [ 191 .
A ligand (B) is dissolved in an LSP. A volatile substance A, which reacts with the ligand B, is introduced into the column and is distributed between the gas and liquid phases :
+ A(1) (6.3)
The equilibrium 6.3 is characterized by a constant K i :
where V g is the retention volume of the substance A on the LSP which does not contain ligand B (when defining the value of if: corrections for non-ideality of the gas phase, adsorption effects, compressibility of the gas phase, etc., are introduced) and V& is the volume of the LSP in the column. Compound A, dissolved in the LSP, reacts with the ligand B to form a complex AB:
A(1) -t B(1) + AB(1) (6 -5)
The formation constant of this complex can be expressed by the following equation:
where aA , aB and aAB are the activities of compounds A, B and AB, respectively; cA, cB and CAB are the concentrations of compounds A, B and AB, respectively; and y A , yB and TAB are the activity coefficients of compounds A, B and AB, respectively.
The coefficient of distribution of the substance A in the system composed of the LSP containing the ligand and the gas phase can be defined with the help of the equation
where V, is the retention volume of substance A on the LSP containing Iigand B and VLsp is the volume of LSP in the column. From the above equations, we can write KR = K i ( 1 + KlaB)
KR = K g ( 1 + K ~ c B )
(6.8)
(6 -9) With an ideal solution
To define the value of K 1 , the experimental function K R = f(cB) is defined and then K g and K g K I can be found graphically. This method is also used to characterize com- plexes with hydrogen bonds [19] and complexes with the transfer of electric charge between an aromatic hydrocarbon and di-n-propyl tetrachlorophthalate [20] , between dienes, aromatic hydrocarbons and 2,4,7-trinitrofluorene [21] .
Stationary phases containing a solution of a silver salt in an organic solvent are most often used in GC. The interaction of olefins with a silver ion was studied in detail by De Ligny and co-workers [22, 2 3 1 . As follows from the above equations, K g must change with changing ionic strength of the solution. Hence solutions of an unreactive salt in the same involatile liquid were used as stationary phases for exact measurements carried out with a comparison column. For example, columns containing 0.25, 1 .O and 2.3M lithium nitrate in ethylene glycol were used as comparison columns for columns containing 0 . 2 5 , 1 .O and 3 M silver nitrate in ethylene glycol, respectively. To avoid the influence of adsorption effects, a column containing 40% of the LSP was used. It is worth noting that an increase in the level of the LSP to 40% does not always allow the contri- bution of adsorption phenomena to the retention value to be neglected. These questions have been considered in more detail elsewhere [24,25] .
Detailed surveys of the theory and experimental determination of the stability con- stants of complexes were published by Locke [26] and De Ligny [27]. To interpret experimental results on the formation of complexes of ogranic compounds with metal ions it is necessary to use not only the complex formation constants but also the entropy and heat of formation of the complexes [ 7 ] . Silver-containing sorbents are mainly used to separate unsaturated compounds. The stability of complexes of silver ion with organic compounds and therefore their retention depend on the number of double bonds, their type and the molecular structure of the unsaturated compound. The following character- istics of retention of unsaturated compounds on stationary phases containing silver ion (see, e.g., refs. 12, 22, 23, 27, 28 and 3 8 ) were formulated in surveys by Guha and Janak [4].
( 1 ) Substituents with a double bond decrease the retention volume of unsaturated compounds.
188
( 2 ) Retention increases in the series 1 -alkyl < 3-alkyl< 4-alkyl compounds.
(3) Olefins with substituents in the 3-position have higher retention volumes than (4) Cyclobutanes are characterized by lower retention volumes than five- and six- ( 5 ) Cyclopentene derivatives are retained more strongly than isomeric cyclohexenes.
(6) In solutions of silver salts in ethylene glycol dienes with independent double bonds form stronger complexes than in aqueous solutions.
After the first work by Bradford et al. [12] on the application of solutions of silver salts in ethylene glycol for the separation of isomeric unsaturated gases, the high selectivity of silver salts for separation of isomeric unsaturated compounds was illustrated by many workers (e.g., refs. 30-34). The study by Smith and Ohlson [33,34] should be particularly noted; they published retention data on 83 hydrocarbons on stationary phases consisting of silver nitrate solution in ethylene glycol. However, silver-organic stationary phases have definite disadvantages, the main ones being low inter-laboratory and inter-column reproducibility and low temperature limits. Also, solutions of silver salts in organic solvents are not sufficiently stable. For example, Keulemans [35] found that a stationary phase consisting o f silver nitrate solution in ethylene glycol becomes unstable and the selectivity decreases at temperatures above 40°C. Therefore, many workers sought stationary phases in which the disscilved silver salt is stable. For example, the chromatographic properties of silver nitrate solutions were investigated in polyethylene glycol and glycerine [36] , triethylene glycol [37]. benzyl cyanide [38-401 and diethylene glycol [41]. It was shown that stationary phases containing silver nitrate can be used at 6 5 ° C ; with a further increase in temperature the stationary phase loses its activity as a result of silvcr ion reduction. Silver nittate solutions in benzyl cyanide are fairly stable but the selectivity is lower than that for silver nitrate in alcohol stationary phases, which can be explained by the lower solubility of the silver salt. Also, the use of benzyl cyanide as a stationary phase is limited by its volatility.
To eliminate the column “noise” caused by volatilization of the stationary phase it is advisable to insert an extra column (length 3.6m, filled with 8% DC-550 silicone oil on a solid support) between the column of silver nitrate solution in benzylcyanide and the detector [ 4 2 ] .
The above disadvantages of the silver-containing stationary phases are partially com- pensated for. however, by their unique selectivity. For example, it was shown [41] that on a squalane capillary column (45 m) a t 25°C a poor separation of methylcycloheptene isomers is observed, whereas on a packed column ( 3 m x 6 mm I.D.) of silver nitrate solution in triethylene glycol the separation was complete at 40°C.
To widen the field of application of these stationary phases an attempt was made to use high-boiling solvents for the silver nitrate, but this did not give any appreciable advantages 1431. Zlatkis [ 4 4 ] , who had used these stationary phases in capillary chroma- tography, demonstrated the extension of the range of compounds that could be analysed and an increase i n the separation efficiency.
Wasic and Tsang [45] showed that the use of aqueous solutions ofconiplexing metal ions as stationary phases is promising. Accordingly to them, water can be used as a stationary phase without any difficulties [46] .
their isomers with substituents in the 4-position.
membered cycloolefins with the same number of carbon atoms.