Eluents preparation

The retention indices, measured on the alkyl aryl ketone scale, of a set of column test compounds (toluene, nitrobenzene, p-cresol, 2-phenyl ethanol, and IV-methylaniline) were used to determine the changes in selectivity of a series of ternary eluents prepared from methanol/0.02M phosphate buffer pH 7 (60 40), acetonitrile/0.02 M phosphate buffer pH 7 (50 50) and tetrahydrofuran/0.02 M phosphate buffer pH 7 (25 65). The analyses were carried out on a Spherisorb ODS reversed-phase column. The selectivity changes were often nonlinear between the binary composition [83]. 

Figure 8 shows the reconstructed histogram from an AX purification of DMT-off crude ISIS 2302, a 20 mer phosphorothioate oligonucleotide. A strong AX resin, in this case POROS HQ/50 (PE BioSystems), was used. Sample loading in related experiments varied from 5 to 10 g. High pH eluents prepared with NaOH were used to minimize secondary structure... 

Recent studies on separation optimization showed that accurate control of mobile phase pH was essential to successfully resolve a number of important non-hydrolysate amino acids. With good control of a complex gradient profile the system could resolve a mixture of amino acids including, Asn, Gin, cysteine derivatives carboxymethyl cysteine and pyridylethyl cysteine, and the hydroxylated amino acids hydroxyproline (Hyp) and hydroxylysine (Hyl) as well as the hydrolysate amino acids (4). However, the required precision in the control of eluent pH unnecessarily complicated transfer of the method between laboratories. The method also lacked the ability to separate Orn from the hydrolysate amino acids. The current study demonstrates the utility of quaternary HPLC gradient systems for facilitating methods development and simplifying routine eluent preparation with excellent pH control. 

Solvent contamination Contamination of the solvent used for the eluent preparation could disturb the separation. This could for example happen with a recycled solvent this is particularly critical when the crude contains volatile... 

The author of this book does not presume to be able to look into the future, but with a little bit of fantasy one can imagine an ion chromatographic system that does not require manual eluent preparation. At first sight, this might appear strange, but in suppressor technology, evolution has already culminated in the development of a continuous, electrochemically regenerated membrane-based device that is extremely easy to use and, therefore, practically invisible. ... 

One of trends of development of thin-layer chromatography implies that replacement of aqueous-organic eluents by micellar surfactants solution. This is reduces the toxicity, flammability, environmental contamination and cost of the mobile phases, reduce sample prepar ation in some cases. 

FIGURE 7.13 Preparative separation of various proteins on Fractogel EMD BioSEC (S). The length of the column was 1000 mm and the inner diameter 100 mm. The flow rate was 6.2 ml/min with 20 sodium phosphate buffer (pH 7.2) containing 0.3 M NaCI as the eluent. The injected standard proteins can be used to create a calibration curve. 

FIGURE 7.18 Preparative separation of abnegates from monomer of human IgG on a Fractogel EMD BioSEC (S) column (1000 X 50 mm column volume 1.88 liter). Five milliliters of a sample consisting of IgG (10 mg/ml) was applied at a flow rate of 10 ml/min (21.4 cm/hr) to remove aggregates (eluent 20 mM sodium phosphate, 0.1 At sodium chloride, pH 7.2). 

The latest trend is to smaller beads in smaller columns, as this saves eluent and shortens the time for a chromatographic analysis. This argument can be correct if only one suitable detector is used. However, these modern small columns are not optimal for a combination of detectors. So-called multiple detection is a combination of some detectors with different measurement principles (differential refractometer, spectral photometer, light-scattering detector, on-line viscometer) behind the last column, mostly in series, seldom in a branched ( parallel ) order. In this way, the tedious preparative fractionation of a polymer sample can often be avoided. 

Stock of solvents were prepared so that their properties changed only little in the course of experiments. All mixed eluent compositions are given in weight %. 

FIGURE 16.1 Preparative SEC of short-chain (scb) branched glucans of small" (<3S /u.m) starch granules of potato species Ostara separated on Sephacryl S-1000 (88 X 2.6 cm) eluent 0.005 M NaOH the normalized chromatogram (area = 1.0) was constructed from an off-line determined carbohydrate content of succeeding 5-ml fractions flow rate 0.67 ml/mln V d = 185 ml, V, = 460 ml fraction I high dp fraction fraction 2 low dp fraction. 

A new brush-type CSP, the Whelk-0 1, was used by Blum et al. for the analytical and preparative-scale separations of racemic pharmaceutical compounds, including verapamil and ketoprofen. A comparison of LC and SFC revealed the superiority of SFC in terms of efficiency and speed of method development [50]. The Whelk-0 1 selector and its homologues have also been incorporated into polysiloxanes. The resulting polymers were coated on silica and thermally immobilized. Higher efficiencies were observed when these CSPs were used with sub- and supercritical fluids as eluents, and a greater number of compounds were resolved in SFC compared to LC. Compounds such as flurbiprofen, warfarin, and benzoin were enantioresolved with a modified CO, eluent [37]. 

The drawback of the described adsorbents is the leakage of the bonded phase that may occur after the change of eluent or temperature of operation when the equilibrium of the polymer adsorption is disturbed. In order to prepare a more stable support Dulout et al. [31] introduced the treatment of porous silica with PEO, poly-lV-vinylpyrrolidone or polyvinylalcohol solution followed by a second treatment with an aqueous solution of a protein whose molecular weight was lower than that of the proteins to be separated. Possibly, displacement of the weakly adsorbed coils by the stronger interacting proteins produce an additional shrouding of the polymer-coated supports. After the weakly adsorbed portion was replaced, the stability of the mixed adsorption layer was higher. 

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. 

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