Void volume markers

Elution Volume of Proposed Void Volume Markers... 

In acidic eluents (Figure 3.9C and D), the retention of acidic compounds becomes stronger and that of basic compounds becomes weaker. In this system, uric acid (h) is not suitable as a void volume marker due to its longer retention time. In neutral and basic eluents, an ionized acid can be used as the marker because no other compounds are eluted more rapidly. Fructose (c) is a very... 

Figure 3.9 Elution volumes (ml) of alternative void volume markers. Column, 5 im octadecyl-bonded silica gel, 15 cm x 4.5 mm i.d. eluents A and B, 10-90% aqueous acetonitrile, eluents C and D, 10-90% aqueous acetonitrile containing 50 mMphosphoric acid flow rate, 1 ml min temperature, 30 °C detection, UV 210 nm and refractometer. Sample a, acetonitrile b, methanol c, fructose d, 2,4-dinitronaphthol e, sodium nitrate, f, tetrahydrofuran g, deuterium oxide, and h, uric acid.

Sander and Wise [88] reported a new standard reference material SRM 870, which contains the neutral compounds ethylbenzene and toluene, to monitor hydrophobicity and methylene selectivity uracil (void volume marker) quinizarin to measure activity toward metal chelators and amitriptyline to measure activity toward bases. The... 

In most analytical applications of HPLC, all these discrepancies are quietly and conveniently forgotten, and selection of some so-called nonretained component as a void volume marker is a common way for void volume measurement. In the majority of recent analytical publications, either thiourea or uracil were used as the void volume markers. As a disclaimer, we have to say here that for the purposes of analytical method development, qualitative or quantitative separation of complex mixtures which involves the use of a nonretained component as a void volume marker is acceptable insofar as there are no physicochemical generalization, thermodynamic development, or futher theoretical development performed upon the basis of these pseudo void volume determinations. 

In everyday method development practice, it is important to ensure the separation of target compounds, matrix components, and other impurities. The elution of the analyte at the void volume means that it did not interact with the stationary phase and thus could not be separated from other components that do not interact with the surface either. To ensure the analyte interaction with the stationary phase, it is usually recommended to choose chromatographic conditions when any component of interest elutes with at least 1.5 void volume values or even greater. The error of the void volume determination for these purposes could be 20% or even greater (insofar as these void volume values are not used for any calculations but just to estimate where the least retained analye elutes). The use of uracil, thiourea, or allantoin as analytical void volume markers is most common in practical analytical work. 

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. 

M where and ii are the retention times of the first- and second-eluted fractions, respectively, and to is the retention time of an unretained compound void volume marker), d) Resolution factor Rs = 2(t2 - ti)/ W] + Wi) where Wj and Are the band-widths of the first- and second-eluted fractions at the base-line level, respectively, e) Mono-MaNP ester. 

Sadek, P.C. Carr, P.W. Bowers, L.D. Evaluation of several void-volume markers for reversed-phase HPLC. LC, Liq. Chromatogr. HPLC Mag. 1985, 3, 590. 

Cytochrome c solution (20 mglrrd) Prepare in phosphate-buffered saline, store at +4°C, and use as a void volume marker. Both solutions 2 and 3 should be filtered (0.22 m) and can be stored at least for 3 months at -l-4°C. 

Band spreading is also related to an obstructive factor that is not a constant in a column bed. Both Kubin [6] and Pfannkoch et al. [11] have shown that plate height varies as a function of K. It appears that as its molecular size approaches the pore dimensions, a solute experiences diffusion limitations, decreasing its effective diffusion coefficient. This influence of restricted molecular movement on plate height (//) can be readily observed in SEC profiles. The first peak to elute after the void volume marker is frequently the broadest peak in the chromatogram. If one were able to obtain columns with different pore diameters but similar plate counts, pore volumes, and calibration curve slopes, it would be best to select a column on which would be 0.2 or greater for the solutes to minimize the effect of the obstructive factor. 

Measure the dead time by injecting 1,3,5-tri-i-butylbenzene, a void volume marker (13), onto the column. Iq equals 0.43 min. 

Pirkle, W. H. and Welch, C. J. (1991) A convenient void volume marker for several chiral HPLC columns. J. Liq. Chromatogr. 14,1-8. 

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