Stationary phases reproducibility

FIGURE 5.7 Reaction schemes for the synthesis of a poly(ethylene co-acrylic acid)-glycidoxypropyl-silica (PEAGlyP) stationary-phase. (Reproduced from Wegmann, J., et al., Anal. Chem., 73, 1814, 2001. With permission.)... 

Figure 7.5—Determination of molecular mass. The use of a calibration curve made with standards of known molecular masses. It should be noted that the calibration curve is linear over a wide range of masses due to the use of a mixture of stationary phases. (Reproduced by permission of Polymer Lab.) The bottom right figure shows the geometry assumed by a linear polymer in solution (figure from PSS).

MIXED STATIONARY PHASES. REPRODUCED WITH PERMISSION FROM 4. ... 

Figure 7.5 Monolithic stationary phase (reproduced by permission of Merck). Top Separation of Gamonil and byproducts. Conditions column, 8.3cm x 7.2 mm i.d. stationary phase, SilicaROD RP 18 e mobile phase, water with 20 mM phosphoric acid-acetonitrile, combined solvent and flow gradient, 10-50% acetonitrile and 3-9 ml min UV detector, 256 nm. Bottom Scanning electron micrograph ofthe stationary phase.

Figure 22.3 Separation of norephedrine enantiomers with (+ )-dibutyl tartrate as the liquid stationary phase [reproduced with permission from C. Pettersson and H.W. Stuurman, J. Chromatogr. Sci., 22, 441 (1984)]. Conditions column, 15cm X 4.6mm i.d. stationary phase, ( + )-dibutyl tartrate on 5 j,m Phenyl Hypersil mobile phase, phosphate buffer (pH 6) containing hexafluorophosphate, saturated with dibutyl tatrate UV detector, 254nm.

Fig. 3.8. Chromatographic resolution of Sa//3-androstan-3a/jS-ols on a liquid crystalline stationary phase. Reproduced from [87] with permission of the American Chemical Society.

Fig. 3.14. Capillary GC separation of a racemic mixture of 19 protein amino acids on an optically active stationary phase. Reproduced from [23].

OV-11, OV-17, OV-101, QF-1, SE-54, SP-2250, EGA, Dexsil-300, and others) have been used successfully in packed columns. Extensive reviews on detectors, column supports, stationary phases, reproducibility, and separation efficiency of TAB, HFB, and TMS derivatives have been published (Husek and Macek, 1975 Blackburn, 1978) and require no further elaboration. However, the capillary columns deserve further mention. The use of various stationary phases, especially SE-30, SE-54, SE-2100, OV-1, OV-17, OV-101, OV-210, EGA, and Carbowax 20M, have been utilized OV-101 (Chauhan et al., 1982 Chauhan and Darbre, 1982 Moodie, 1981 Husek, 1982 Desgres et al., 1979), SE— 54 (Gajewski et al., 1982), and SE-30 (Poole and Verzele, 1978) are apparently superior m terms of separability, low background noise, and low column bleed... 

Fig. 18.16 Separation of proteins on a mixed stationary phase. (Reproduced by permission of Elsevier Science Publishers BV from Z. el Rassi and C. Horvath, J. Chromatogr., 359, 255 (1986). Stationary phases (A) strong anion exchanger (Zorbax SAX-300) (B) strong cation exchanger (Zorbax SCX-300) (C) 1 1 mixture of A and B. Particle size, 7pm. Column, 8cm x 6.2 mm i.d. (A and B), 10cm X 4.6mm i.d. (C) mobile phase, 20mM tris-HCI (pH 7.0), gradient from 0 to 0.3 M sodium chloride in 40 min, 1.5 ml min (A and B), 1 ml min (C) UV detector, 280 nm. RNase = ribonuclease A CYT = cytochrome c CHY = a-chymotrypsinogen A LYS = lysozyme Hb = haemoglobin CON = conalbumin LAC A = j5-lactoglobulin A.

Figure 9 (a) A polymeric crown ether stationary phase. (Repro-dnced from Ref. 56. lUPAC, 1982.) (b) structure of the polyether modified silica stationary phase. (Reproduced from Ref. 57. American Chemical Society, 1983.)... 

Figure 13 The monomCT of cryptand [2,2,2] used to graft to an ion chromatographic stationary phase. (Reproduced from Ref. 63. Elsevier, 2002.)...

In this section, we explain the principles that influence the selectivity of a reversed-phase column. The first parameter is the hydrophobicity of the stationary phase, which can be measured with purely hydrophobic probes. The second value describes the silanol activity, which is of special importance for basic analytes. Naturally, the silanol activity is best measured using a basic compound, using a correction for the hydrophobic contribution of the structure of the analyte to its retention. The third value is the polar selectivity, which measures the formation of hydrogen bridges between analytes and the stationary phase. With these values, one can create selectivity charts that can help in the selection of the best stationary phase for a particular separation problem. These charts help in method development, whether one would like to find a stationary phase that is drastically different or one that is rather similar to one that is available or in use. At the end of the chapter, we briefly touch on the subject of stationary phase reproducibility. 

SiHca, alumina, and other metal oxides and salts have been used as the stationary phase in gas—soHd chromatographic systems. The appHcabiHty of these materials is limited by the difficulty of producing a consistent, resiHent, reproducible material. 

In the first version with a mobile phase of constant composition and with single developments of the bilayer in both dimensions, a 2-D TLC separation might be achieved which is the opposite of classical 2-D TLC on the same monolayer stationary phase with two mobile phases of different composition. Unfortunately, the use of RP-18 and silica as the bilayer is rather complicated, because the solvent used in the first development modifies the stationary phase, and unless it can be easily and quantitatively removed during the intermediate drying step or, alternatively, the modification can be performed reproducibly, this can result in inadequate reproducibility of the separation system from sample to sample. It is therefore suggested instead that two single plates be used. After the reversed-phase (RP) separation and drying of the plate, the second, normal-phase, plate can be coupled to the first (see Section 8.10 below). 

Enantiomeric separations have become increasingly important, especially in the pharmaceutical and agricultural industries as optical isomers often possess different biological properties. The analysis and preparation of a pure enantiomer usually involves its resolution from the antipode. Among all the chiral separation techniques, HPLC has proven to be the most convenient, reproducible and widely applicable method. Most of the HPLC methods employ a chiral selector as the chiral stationary phase (CSP). 

The support materials for the stationary phase can be relatively inactive supports, e.g. glass beads, or adsorbents similar to those used in LSC. It is important, however, that the support surface should not interact with the solute, as this can result in a mixed mechanism (partition and adsorption) rather than true partition. This complicates the chromatographic process and may give non-reproducible separations. For this reason, high loadings of liquid phase are required to cover the active sites when using high surface area porous adsorbents. 

This means, in practice, that when employing a polar solvent with n-heptane (or any other paraffin for that matter) to reduce the retention, there will be a dramatic reduction in retention over the concentration range of about 0-2%w/v. However, subsequent changes in solute retention with polar solvent concentration will be relatively small. This will be true for any polar solute and was experimentally verified by Scott and Kucera for solutions of ethyl acetate, tetrahydrofuran and n-propanol in n-heptane. The very sensitive relationship between solvent concentration and retention at very low concentrations makes the phase system very difficult to make reproducible. This problem is one of the factors that deter analysts from using silica gel as a stationary phase for the separation of polar solutes. It is very satisfactory, however, for the separation of polarizable and weakly polar substances that can be eluted by paraffin/methylene dichloride or similar types of solvent mixtures. 

In HPLC, the mobile phase is a liquid delivered under high pressure (up to 400 bar (4 x 10 Pa)) to ensure a constant flow rate, and thus reproducible chromatography, while the stationary phase is packed into a column capable of withstanding the high pressures which are necessary. 

Table 2.1 HPLC capacity factors for secbuto-barbitone and vinbarbitone with an octadecyl silyl stationary phase and mobile phases of methanoiyO.l M sodium dihydrogen phosphate (40 60) at (a) pH 3.5, and (b) pH 8.5. From Moffat, A.C. (Ed.), Clarke s Isolation and Identification of Drugs, 2nd Edn, The Pharmaceutical Press, London, 1986. Reproduced by permission of The Royal Pharmaceutical Society...

The precision in retention from injection to injection will often be better than 1%. Over longer periods of time such precision requires the following (a) good flow control from the pump (b) constant mobile and stationary phases and (c) temperature control of the column. The critical question of reproducibility from column to column is still a matter of concern, especially when dealing with the more sophisticated packing materials, e.g. small porous particles, bonded phases While frequently this reproducibility is quite good, workers should recognize that care must be exercised to achieve and/or maintain reproducible columns. Undoubtedly, with experience, this need not be a severe problem. 

Advances in understanding solute interachons in liquid-liquid systems in a nonequilibrium environment brought reversed-phase (RP)-HPLC into the forefront of lipophilicity determinahon. The development and manufacturing of rigid, reproducible and well-characterized stationary phases and columns, as well as the accessibility and high level of automation of modern HPLC systems, have made RP-HPLC the method of choice for many laboratories. 

Figure 6.2 Influence of colunn internal diaeeter and stationary phase filn thickness on efficiency for open tubular colunns operated at 10 u with supercritical carbon dioxide under low density conditions as the nobile phase. (Reproduced with pemission from ref. 46 and 47. Copyright Dr Alfred Huethig Publishers).

Figure 2.12 Plot of the logaritlm of the specific retention volune and colunn efficiency (plates per aeter) as a function of column tM erature for benzaldehyde (BZA) and n-tridecane (C ) on the stationary phase tetra-n-butylanmonium picrate. (Reproduced with permission from ref. 58. Copyright Blsevier Scientific Publishing Co.)...

Trying to determine which column is ideal for a specific analysis can be difficult with over 1000 different columns on the market [74]. A proper choice implies a definition of parameters such as column material, stationary phase (polarity), i.d., film thickness and column length. Guides to column selection are available [74,75]. The most important consideration is the stationary phase. When selecting an i.d., sample concentration and instrumentation must be considered. If the concentration of the sample exceeds the column s capacity, then loss of resolution, poor reproducibility and peak distortion will result. Film thickness has a direct effect on retention and the elution temperature for each sample compound. Longer columns provide more resolving probe, increase analysis times and cost. 

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