Elution of small molecules

The development of rigorous theoretical treatments for the elution behavior of small molecules has been largely ignored due to the adequacy of predictive ability of log M, log effective molecular length (ref. 9) or log effective molecular volume models (ref. 10). However, the calculation of these parameters for macromolecular solutes with the same degree of confidence is not yet possible. 

The search for the best estimate of the effective hydrodynamic volume for biopolymers in SEC, which usually can be described as roughly spherical (proteins), long rods (viruses) or random-coil structures (polysaccharides and single stranded polyribonucleic acids), has led to the investigation of Rg, the solute radius of gyration in solution (ref. 11), and other related parameters. (See section 2.3). 

---

Typically, the elution of small molecules has been described as a linear function of log M and the elution volume, Vg. Molecular weight calibration curves obtained with members of a specific homologous series often give very good linearity with remaining members of the series, within the limit of the included volume of the column system used. An example of this application with oligosaccharides and several monosaccharides is shown in Figure 1. The predictive ability of such log M curves decreases as the solute studied... 

FIGURE 4.24 Adsorption chromatography of small molecules with a TSK-GEL G2500PWxl column. Column TSK-GEL G2500PWxl, 6 /tm, 7.8 mm X 30 cm. Sample (I) phenylacetic acid. (2) 3-phenylpropionic acid, (3) 4-phenylbutyric acid, (4) benzylamine, (5) 2-phenylethylamine, (6) 3-phenylpropylamine, (7) benzyl alcohol, (8) 2-phenylethanol, and (9) 3-phenyl-1 -propanol. Elution 0.1 M NaCIO, in water. Flow rate 2.0 ml/min. Temperature 65 C. Detection UV at 215 nm. 

As known, SEC separates molecules and particles according to their hydro-dynamic volume in solution. In an ideal case, the SEC separation is based solely on entropy changes and is not accompanied with any enthalpic processes. In real systems, however, enthalpic interactions among components of the chromatographic system often play a nonnegligible role and affect the corresponding retention volumes (Vr) of samples. This is clearly evident from the elution behavior of small molecules, which depends rather strongly on their chemical nature and on the properties of eluent used. This is the case even for... 

Varian (see Catalog, SPE products) Bond Elut Plexa Highly cross-linked polymer with hydroxylated surface Hydrophobic retention of small molecules and hydrophilic exclusion of proteins See catalog... 

Gel Filtration Separation is in accordance with size. Large molecules elute in void volumes and are eluted earlier. Small molecules penetrate pores of the matrix and elute later because of the increase in path length. 

A second approach to on-line SPE is to use an SPE extraction column that can be used for hundreds of samples. In the simplest of systems, two pumps (either HPLC systems or stand-alone pumps) are connected to an extraction column and an analytical column via 6 or 10 ports, and these are further linked to an MS system. The pump that is connected in-line with the autosampler loads the sample under high flow rate (3 to 5mL/min). The large molecules from the matrix are not retained by the SPE sorbent and are diverted to waste. The analytes of interest are retained by the sorbent. The valve then switches so that the second pump with the elution solvent is now in-line with the SPE column and elutes the analytes onto the analytical column for HPLC/MS analysis. This type of system has proved useful for the analysis of small molecules in a variety of sample matrices such as plasma and urine. While it is relatively straightforward to plumb this type of system with components already in the laboratory, commercial systems are available from such companies... 

We have recently reported (1 )) the use of this technique for characterization of various compounds. Experimental data obtained for a number of compounds are shown in Figure 2. We calculated the "size factors" for a number of small molecules and oligomers. This factor is a measure of the deviation of the elution volume of a given species from the calibration curve for n-alkanes which is assigned a size factor of 1. This size factor, F, is defined to be equal to A/M, where M is the molecular weight of the compound and A is the molecular weight of a real or hypothetical n-alkane which will elute at the same retention volume as the compound. Size factors for a number of... 

A characteristic of small-molecule liquid chromatography is the reversibility of their contacts with the stationary phase. The distribution equilibrium constant determines the duration of the stationary periods and, thus, the retention of the solute. With polymers, isocratic retention factors of normal degree (i.e., 1 gk 10) generally do not occur. A fractional alteration of elution conditions may cause transition from zero retention to infinity. As a rule of thumb, polymers either pass without retention or remain in the column. This off or on behavior produces the impression of irreversible fixation under the conditions of retention. 

Compared to LC/MS, GC/MS methods are applied to a much smaller extent, mainly because many pharmaceutical compounds cannot be eluted from a GC column without prior derivatization steps. However, GC/MS is still the method of choice for the analysis of small molecules that can be evaporated without decomposition due to its unmatched chromatographic resolution and highly reproducible, compound-characteristic EI-MS spectra [40]. Whereas compound-specific derivatization is time-consuming, the GC/MS analysis itself can be very fast, as a review by Leclercq and Cramers demonstrates [41 ]. 

Visser and coworkers combined LC, SPR, and MS together and immobilized cGMP molecules to an SPR chip to monitor the binding and dissociation of proteins from a human lysate by sequential elution steps and SPR [51]. The eluted proteins were thereafter identified by LC-MS/MS. The data indicate that SPR-based chemical proteomics is a viable alternative for quantitative extraction and identification of small-molecule-binding proteins from complex matrices. In fact, SPR-MS technological innovations have grown considerably in the last few years and have even sparked enough interest that leads to a book chapter devoted to the topic [52]. 

Chiral WCOT columns suitable for the enantiomeric separation of small molecules such as esters, ketones, alkanes and alcohols have recently been introduced. One example of a chiral column has a stationary phase film consisting of a non-bonded mid-polar, 35% phenyl 65% methyl siloxane modified by embedding in the film permethylated a- or /3-cyclodextrin. Elution characteristics are modified by the cyclodextrin content DEX 110 contains 10% cyclodextrin and DEX 120, 20% [127, 128]. 

Displacement chromatography offers an alternative to elution chromatography for preparative-scale separations under nonlinear conditions [10,66,82,83]. It has found limited success for the purification of biopolymers by reversed-phase [10,83,84] and ion-exchange chromatography [85-88], but is not widely used for the purification of small molecules [89-91]. It has the potential for greater use, but remains a minor technique compared with elution chromatography. 

The development of a theory of retention and band broadening for macro-molecular HPLC is intended primarily for improving such separations. We need to relate various separation goals to (1) experimental conditions, (2) the choice of column type and HPLC method (RPLC, SEC, etc.), and (3) the nature of the sample. The present model allows os to do this 1 prediction, rathor than by experiment Our main requirement is usuaOy to achieve adequate separation, or some minimum resolution R, betw adjacent bands of interest. In this section we will show that the separation of macro-molecular samples by either isocratic or gradient elution is understandable and controllable, using the same concepts that we use for optimizing the isocratic separation of small molecules. 

---