Chromatography Ion-Pair

Ion-pair chromatography represents an alternative to ion-exchange chromatography. Many problems can be solved by either method but ion-exchange is not as good for separating mixtures of acids, bases and neutral products under certain circumstances, and this is where ion-pair chromatography then comes into its own. The fact that the reversed phases described in Chapter 10 can be used as stationary phases has added to its popularity. 

Ionic samples may be separated by reversed-phase chromatography, provided that they contain only weak acids or only weak bases (in addition to neutral compounds) present in undissociated form, as determined by the chosen pH this is known as ion suppression . Ion-pair chromatography is an extension of this principle. An organic ionic substance is added to the mobile phase and forms an ion pair with a sample component of opposite charge. This is, in fact, a salt but its chromatographic behaviour is that of a non-ionic organic molecule  

Reversed-phase chromatography can be used in this instance. E.g., an alkylsulphonate is added to cationic samples and tetrabutylammonium phosphate to anionic samples. A sample containing both anionic and cationic components has one type masked by a counter ion and the other suppressed by a suitable pH level. 

The advantages of ion-pair chromatography for separating ionic samples may be summarized as follows  

Practical High-Performance Liquid Chromatography, Fourth edition Veronika R. Meyer 2004 John Wiley Sons, Ltd ISBN 0-470-09377-3 (Hardback) 0-470-09378-1 (Paperback) 

The process cannot be used tor strong acids or bases because these are dissociated over a wide pH range and extreme pH levels would be required for ion suppression. Ion chromatography as described in Chapter 14 was developed specifically for cases such as this. 

Practical High-Performance Liquid Chromatography, Fifth edition Veronika R. Meyer 

A separation using ion-pair chromatography is based upon the resolution of solute molecules undergoing dynamic complexation with an added counter ion. Either a normal phase (90, 333) (silica gel, alumina, or cellulose, for example) or, more frequently, a reversed phase (41, 42, 89, 219) (e.g., C-18 or C-8) may be used. In general, the counter ion is initially adsorbed onto the stationary phase as well as being present in low concentration in the eluent. For reversed-phase ion-pair chromatography, the counter ion is usually relatively hydrophobic, whereas a normal-phase support such as silica gel requires adsorption of an aqueous solution of the counter ion, which gives rise to liquid-liquid partition characteristics for the separation. 

While the precise nature of the mechanism of separation of compounds by ion-pair chromatography has been thoroughly examined and is still the subject of considerable debate (146, 362, 369), the basic features can be summarized by a series of equilibria representing the partitioning and/or adsorption of the ions and the complex between the mobile and stationary phases. The equilibria pertinent to reversed phases (Fig. 2.22) account for the possibility of ion-pair formation in either the mobile or stationary phases as well as the partitioning of the ions and the complex between the two phases. When normal phases are considered, equilibria accounting for appropriate interactions with the normal-phase solid support as well as with the stationary aqueous phase bound to the solid support must also be included. The relative influence of these dynamic processes depends upon the nature of the solute, counter ion, mobile phase, and stationary 

The fundamental characteristic of ion-pair chromatography is that the addition of the counter ion enhances the retention of the solute, without which the solute would either move with the solvent, in the case of reversed-phase support, or be completely retained, in the case of normal-phase support (or at least experience severe tailing and poor resolution). The enhanced retention is a consequence of the partitioning of the ion pair into the stationary phase subsequent to partitioning of the ions into the stationary phase. Thus, the equilibrium constants defined in Fig. 2.22, which are unique for each particular solute, counter ion, and stationary and mobile phase, are the factors that define the retention of a particular solute, the column efficiency, and hence the efficiency of the separation. 

Parameters optimized for a specific separation problem include the pH of the aqueous phase, the addition of organic modifiers to the mobile solution, the concentration of the counter ion, and, to a lesser extent, the ionic strength of the aqueous phase. Resolution may be very sensitive to the pH of the aqueous phase, especially when solutes or counter ions with pKa s between 2 and 10 are to be separated. Under such conditions, protonation-deprotonation equilibria are accessible within the pH limitations of most HPLC columns, including reversed phases. Such equilibria provide another solute-differentiation mechanism that can be exploited for enhancement of resolution. Under such conditions, the dynamic equilibria, which define the interactions of the solute with both the stationary phase and the counter ion, must include the behavior of both the protonated and unprotonated solute. 

The capacity ratio of a solute is dependent upon the concentration of counter ion adsorbed on the stationary phase as well as on the concentration in the mobile phase when the stationary phase is saturated with adsorbed counter ion. As it is necessary to maintain a steady-state concentration of counter ion in the mobile phase, especially when the counter ion is UV-absorbing, the stationary phase is saturated with counter ion prior to application of the solute mixture. The concen- 

Equilibration is the first step in a separation. A mobile phase containing a pairing ion and a co-ion in an aqueous-organic solution is pumped through the column until equilibration is complete and a steady baseline is obtained. Suppose a tetrabutylammonium salt, Bu N Cl , is to be used in the mobile phase for separation of the sample anion. The larger cation equilibrates between the two phases  

After reconditioning, the sample is introduced as the mobile phase continues to be pumped through the column. Analyte retention has been suggested to follow a double-layer model in which the organic pairing ion occupies a primary layer on the stationary phase and the other ions in the system complete for the secondary layer [1]. Sample anion or cations are separated by differences in their affinity for the pairing ion sites on the stationary phase. 

An alternative mechanism is possible when organic sample ions or polarizable inorganic ions, such as iodide or thiocyanate, are to be separated. In this case ion pairs are partially formed in the liquid mobile phase and the pairs can then equilibrate with the stationary phase. 

Retention factors of sample ions can be adjusted by changing the chemical nature and concentration of the pairing ion and by altering the proportion of organic solvent in the mobile phase. As an example, larger k values are obtained for anions with R4N as the pairing ion in the series R = ethyl propyl butyl pen- 

Ion-pair chromatography is actually quite broad in scope. Chromatographic behavior can be distinctly different when organic ions, rather than inorganic, are to be separated. The precise mechanism has been hotly debated (see Section 9.3), and several names have been applied to this type of separation ion-interaction chromatography (IIC), mobile-phase ion chromatography (MPIC), as well as ion-pair (IPC) chromatography. We find the last name to be simple and descriptive. 

---

Two mechanisms for chiral separations using chiral mobile-phase additives, analogous to models developed for ion-pair chromatography, have been... 

K. Yamashita, M. Motohaslii and T. Yashiki, High-performance liquid cliromatograpliic determination of phenylpropanolamine in human plasma and urine, using column switching combined with ion-pair chromatography , J. Chromatogr. 527 103-114 (1990). 

EC = ion exchange chromatography IPC = ion pair chromatography LSC = liquid—solid chromatography... 

Separation of ionic and nonionic compounds of alkyl ether carboxylates can be done by reverse phase ion pair chromatography [241]. 

The ionic or polar substances can be seperated without any reaction on specially treated chromatographic columns and detected refractometrically. This is necessary because alkyl sulfosuccinates show only small absorption in the UV-visible region no sensitive photometric detection can be obtained. Separation problems can arise when common steel columns filled with reverse phase material (or sometimes silica gel) are used. This problem can be solved by adding a suitable counterion (e.g., tetrabutylammonium) to the mobile phase ( ion pair chromatography ). This way it is possible to get good separation performance. For an explanation of separation mechanism see Ref. 65-67. A broad review of the whole method and its possibilities in use is given in an excellent monograph [68]. 

Simple mixtures—like in alkyl sulfosuccinates—can be run using only one solvent. For more complex systems (e.g., ethoxylated fatty alcohol sulfosuccinates) a gradient technique is strongly recommended Technical mixtures of disodium laureth sulfosuccinate could be separated [68]. The separation was so effective that resolution of single homologs of ethoxylates was possible. The detection limit of this method lies at around 0.5 pg. Therefore reverse phase ion pair chromatography seems to be an excellent tool to analyze sulfosuccinates directly without the use of any kind of manipulation. 

There are very few examples of asymmetric synthesis using optically pure ions as chiral-inducing agents for the control of the configuration at the metal center. Chiral anions for such an apphcation have recently been reviewed by Lacour [19]. For example, the chiral enantiomerically pure Trisphat anion was successfully used for the stereoselective synthesis of tris-diimine-Fe(ll) complex, made configurationally stable because of the presence of a tetradentate bis(l,10-phenanthroline) ligand (Fig. 9) [29]. Excellent diastereoselectivity (>20 1) was demonstrated as a consequence of the preferred homochiral association of the anion and the iron(ll) complex and evidence for a thermodynamic control of the selectivity was obtained. The two diastereoisomers can be efficiently separated by ion-pair chromatography on silica gel plates with excellent yields. 

In addition to chromatography based on adsorption, ion pair chromatography (IP-HPLC) and capillary electrophoresis (CE) or capillary zone electrophoresis (CZE) are new methods that became popular and are sufficiently accurate for these types of investigations. Other methods involving electrochemical responses include differential pulse polarography, adsorptive and derived voltammetry, and more recently, electrochemical sensors. 

Hapten density, and also the common positions where haptens are bound, can also be estimated by cyanogen bromide or enzymatic cleavage of the protein and either MALDI-MS or separation of the components by reversed-phase ion-pair chromatography and electrospray or electrospray time-of-flight (TOF) analysis. 

Ion-pair chromatography (IPC) is a further example of the use of secondary chemical equilibria to control retention and... 

Figure 4.27 Flow chart for coluwi selection based on sample type (m - molecular weight). PLC precipitation-liquid chromatography SEC = size-exclusion chromatography lEC - ion-exchange chromatography HIC hydrophobic interaction chromatography LSC liquid-solid chromatography RPC - reversed-phase liquid chromatography BPC (polar) bonded-phase chromatography and IPC - ion-pair chromatography.

Typical ion-pairing reagents are, for cations, alkyl sulphonic acids, eg pentane, hexane, heptane or octane sulphonic acid, and for anions, tetrabutylammonium or dibutylamine ammonium salts. In ion-pair chromatography the retention of solutes can be controlled in a number of ways ... 

Steinbrech, B., Neugebauer, D., Zulauf, G. (1986). Determination of surfactants by liquid chromatography (HPLC). Reversed phase ion-pair chromatography of alkyl sulfates and alkyl sulfosuccinates. Analytische Chemie 324(2), 154—157. 

Ionic surfactants such as sodium dodecyl sulfate can also be detected by ESI. Figure 30 shows an overlay of sub ppm concentrations detected using ion pair chromatography with specific ion LC-MS detection (positive ESI at m/z 265). Gradient elution from 50.0% water containing 5 mM acetic acid triethylamine) to 50.0% 80/20 acetonitrile/water (5mM acetic acid triethylamine) was employed. 

Shea and MacCrehan [322] and Duane and Stock [323] determined hydrophilic thiols in marine sediment pore waters using ion-pair chromatography coupled to electrochemical detection. 

---