Void markers

Fig. 6.5 A d iagram of the modified staircase approach. In this procedure, ligand and void marker are infused at increasingly higher concentrations. It differs from the normal procedure in that column washing between infusions is not performed. This is the simplest of FAC techniques for measuring ligand K. ...

Fig. 6.15 FAC-MS chromatograms of dual indicators for protein kinase Ca [32]. (a) In the chromatograms, the red lines correspond to a void marker, the blue lines correspond to the substrate-site indicator chelerythrine chloride and the magenta lines correspond to the ATP-site indicator PDl53035. Arrows...

Other Experimental Methods. It is probably suitable to discuss here column porous structure. Porous space of a conventional packed column consists of the interparticle volume (Vip—space around particles of packing) and pore volume (Vp— space inside porous particles). The sum of those two constitutes the column void volume. The void volume marker ( unretained ) should be able to evenly distribute itself in these volumes while moving through the column. Only in this case the statistical center mass of its peak will represent the true volume of the Uquid phase in the column. In other words, its chromatographic behavior should be similar to that of the eluent molecules in a monocomponent eluent. If a chosen void volume marker compound has some preferential interaction with the stationary phase compared to that of the eluent molecules, it will show positive retention and could not be used as void marker. If on the other hand it has weaker interaction, it will be excluded from the adsorbent surface and will elute faster than the real void time, meaning that it also could not be used. For any analytical applications (when no thermodynamic dependences are not extracted from experimental data), 10% or 15% error in the determination of the void volume are acceptable. It is generally recommended to avoid elution of the component of interest with a retention factor lower than 1.5. Accurate methods for the determination of the column void volume are discussed in Chapter 2. 

Figure 19-21. FAC-MS profiles of compounds eluting from the SDH column as measured using single ion monitoring of molecular ions. A nonbinding compound elutes first as the void marker because it shows no affinity to the target. The elution order of eight components analyzed as a mixture reflects their relative binding strengths, as confirmed by IC50 and values. (Reprinted from reference 111, with permission of the American Chemical Society.)...

Fig. 5.18. Effect of polymerisation and elution temperature on the enantiomer separation factor (a) in the separation of D- and L-PA on L-PA imprinted polymers. Polymers were prepared by thermochemical initiation at either 60 or 40°C using AIBN or ABDV respectively as initiators. The samples consisted of ca. 20 nmol of each of D- and L-PA and BOC-L-PA as void marker. Flow rate 0.5 mL/min. Mobile phase MeCN/acetic acid 95/5 (v/v). The columns were thermostatted by immersing them in a circulating water bath at the indicated temperature. From O Shannessy et al. [8].

Fig. 20.2. Structures and separation values (a) of compounds examined in linear dichroism study. The capacity factor k is calculated as (ts-O/tv, where these terms refer to the retention times of the sample and the void marker, respectively. The separation factor a is defined as k Jk y. the ratio of capacity factors for any two substances on the same column.

Here Bt is the number of binding sites on the column, V the elution volume of the analyte for which Kd is being determined, Vq is the elution volume of a void marker, which does not interact with the immobilized protein, and [L] the ligand concentration in the sample. Needless to say, a FAC-MS assay can be calibrated using SM with known interaction parameters. 

A typical HPLC chart is schematically illustrated in Fig. 4.1. The major of the two peaks corresponds to our target guest. Its retention time is tg. The minor peak (the retention time = t0) is that for the void marker (a standard), which is poorly bound by the polymer (e.g., acetic acid, acetone, or acetonitrile). An index of binding activity of the imprinted polymer toward our target guest, the capacity factor k, is defined by Eq. (1). 

Fig. 4-1 Schematic illustration of the HPLC chart of substrate (tg) and void marker (t0)...

The above-described column is used for the evaluation of imprinting efficiency (chromatographic analysis). Atrazine and an appropriate void marker are injected into the column, and the retention time for each is measured. The retention factor of atrazine is also compared with those of related compounds. The imprinting is sufficiently efficient, as confirmed by the fact that the retention factor of atrazine is selectively prolonged (see Table 6.1). 

Figure 11 Chromatogram of the imprinted stationary phase. This imprinted polymer does work in aqueous solvents and chromatographic characterizations are performed using an aqueous mobile phase (25 mM sodium citrate, pH 3.0) containing 10% MeCN at a flow rate of 1 mL/min and chromatograms, run in isocratic mode and recorded at 280 nm. Injections of 20 luL of 2 mM racemic isoproterenol HCl (40 pmol) dissolved in the mobile phase are done in order to evaluate the enantioselectivity towards its imprinted print molecule isoproterenol. Eluent used was a sodium citrate buffer (pH 3.0, 25 mM citrate, 10% MeCN) flow rate 1 ml/min, peak detection at 280 nm, injection of 20 pL of a racemic isoproterenol hydrochloride solution (2 mM), acetone was used as void marker. The structures of + and - isoproterenol are given in Fig. 8.

Polymer particles are suspended in water (25% MeCN) and then slurry-packed into stainless steel columns (250 mm x 4.6 mm i.d.) using an air-driven fluid pump (Haskel, Burbank, CA, USA) and water (25% MeCN) as the packing solvent. The packed columns are washed on-line on a Beckman HPLC system (comprising a solvent module 126 and diode array detector 168) using MeCN (20% acetic acid) to remove the print molecule until a stable base line is obtained. The mobile phase is then changed to a citrate buflfer (pH 3.0, 25 mM citrate) containing 10% MeCN (v/v) at a flow rate of 1 ml. min . For a test of chiral resolution, a racemic mixture of (+)- and ( )-isoproterenol (20 pL at 2 mM in the mobile phase) is injected, and the elution monitored at 280 nm. Acetone can be used as a void marker for the calculation of capacitor factor (k ) and separation factor (oc). 

Grossfeld GD, Wolf JS, Litwin MS etal. (2001) Evaluation of asymptomatic microscopic hematuria in adults the American Urologic Association vest practice policy recommendations. Part II patient evalimtion, cytology, voided markers, imaging, cystoscopy, nephrology evaluation, and follow-up. Urology 57 604-610... 

Ohring, M. Sun, P.H. Void marker motion during electromigration in Sn In thin films. Thin Solid... 

---