Polymer HPLC eluent molecules

The instruments for polymer HPLC except for the columns (Section 16.8.1) and for some detectors are in principle the same as for the HPLC of small molecules. Due to sensitivity of particular detectors to the pressure variations (Section 16.9.1) the pumping systems should be equipped with the efficient dampeners to suppress the rest pulsation of pressure and flow rate of mobile phase. In most methods of polymer HPLC, and especially in SEC, the retention volume of sample (fraction) is the parameter of the same importance as the sample concentration. The conventional volumeters— siphons, drop counters, heat pulse counters—do not exhibit necessary robustness and precision [270]. Therefore the timescale is utilized and the eluent flow rate has to be very constant even when rather viscous samples are introduced into column. The problems with the constant eluent flow rate may be caused by the poor resettability of some pumping systems. Therefore, it is advisable to carefully check the actual flow rate after each restarting of instrument and in the course of the long-time experiments. A continuous operation— 24h a day and 7 days a week—is advisable for the high-precision SEC measurements. THE or other eluent is continuously distilled and recycled. 

Separation of distinct rrracromolecules in polymer HPLC results from then-different retention within colirmn Retention mechanism of analyte is the general term recommended by lUPAC. It denotes the mutrral difference of elution rate of distinct macromolecirles. Similar to HPLC of low-molecirlar substances, elution rate of macromolecirles and molecules of mobile phase differ also in polymer HPLC. As a rule, in HPLC of low-molecular substances, the separated species elute with lower velocity than molecules of their original solvent, they are retained, decelerated. With few exceptions, elution rate of macromolecules is the same or slower than elution rate of eluent molecules also in various the coupled methods of polymer HPLC. 

Stated that depending on the thermodynamic quality of solvent, the same macromolecule can assnme various conformations and consequently also different sizes in solution Similar thoughts are to be applied also considering role of solvents in liquid chromatography systems, in which macromolecules necessarily interact with eluent molecules. However, the third component has to be taken into account in polymer HPLC, namely the column packing and its interactions not only with the macromolecules of sample but also with the eluent moleeules. 

Multicomponent eluents are very frequently apphed in coupled methods of polymer HPLC with the aim to control interaction between sample and column packing (see section 11.8) They are employed less commoidy in SEC, except when they should ensure sample solubility and its stability in solution. Inevitably, mixed eluents cause problems resulting from the phenomena of preferential solvation of sample molecules (see sections 11.2.4 and 11.6.1.4), preferential sorption on the column packing, and preferential evaporation from the eluent container. The latter occurences often give rise to the base hne perturbances including the appearance of system peaks. Depending on the particular case, the mixed mobile phases may either enhance or impair the sample detectability. 

Some authors have suggested the use of fluorene polymers for this kind of chromatography. Fluorinated polymers have attracted attention due to their unique adsorption properties. Polytetrafluoroethylene (PTFE) is antiadhesive, thus adsorption of hydrophobic as well as hydrophilic molecules is low. Such adsorbents possess extremely low adsorption activity and nonspecific sorption towards many compounds [109 111]. Fluorene polymers as sorbents were first suggested by Hjerten [112] in 1978 and were tested by desalting and concentration of tRN A [113]. Recently Williams et al. [114] presented a new fluorocarbon sorbent (Poly F Column, Du Pont, USA) for reversed-phase HPLC of peptides and proteins. The sorbent has 20 pm in diameter particles (pore size 30 nm, specific surface area 5 m2/g) and withstands pressure of eluent up to 135 bar. There is no limitation of pH range, however, low specific area and capacity (1.1 mg tRNA/g) and relatively low limits of working pressure do not allow the use of this sorbent for preparative chromatography. 

---