Column Stationary Phase

In the development of a SE-HPLC method the variables that may be manipulated and optimized are the column (matrix type, particle and pore size, and physical dimension), buffer system (type and ionic strength), pH, and solubility additives (e.g., organic solvents, detergents). Once a column and mobile phase system have been selected the system parameters of protein load (amount of material and volume) and flow rate should also be optimized. A beneficial approach to the development of a SE-HPLC method is to optimize the multiple variables by the use of statistical experimental design. Also, information about the physical and chemical properties such as pH or ionic strength, solubility, and especially conditions that promote aggregation can be applied to the development of a SE-HPLC assay. Typical problems encountered during the development of a SE-HPLC assay are protein insolubility and column stationary phase... 

For the GPC separation mechanism to strictly apply, there must be no adsorption of the polymer onto the stationary phase. Such adsorption would delay elution of the polymer, thereby resulting in the calculation of too low a molecular weight for the polymer. The considerable variety of undesirable interactions between polymers and column stationary phases has been well reviewed for GPC by Barth (1) and this useful reference is recommended to the reader. Thus, the primary requirement for ideal GPC is that the solvent-polymer interaction be strongly thermodynamically favored over the polymerstationary phase interaction. 

The particle diameter of the GPC column stationary phase plays a role not just in determining the resolution of the column, but also in determining how well the column elutes insolubles or microgels that may be present. It is usually advisable to avoid the injection of insolubles or microgels that might block the frits or interstices of any GPC column, but in some instances the analysis of these materials by GPC is possible and even desirable. 

Current trends in GC relate to miniaturisation, fast-GC, improved selectivity (mainly for short columns), stability of column stationary phases (reduction of bleeding) and increasing use of MS detection [117]. Finally, GC can be readily hyphenated with spectroscopic techniques without using involved interfaces and thus can easily provide unambiguous solute identification. 

Many of the possible column combinations that are useful in 2DLC are listed in Chapter 5. Besides the actual types of column stationary phases, for example, anion-exchange chromatography (AEC), size exclusion chromatography (SEC), and RPLC, many other column variables must be determined for the successful operation of a 2DLC instrument. The attributes that comprise the basic 2DLC experiment are listed in Table 6.1. 

FIGURE 17.10 Gradients for the separation of the FAE mixture on a monolithic column, stationary phase Chromolith C18, 10 x 0.46 cm i.d., mobile phase MeOH H20. 

Adequate resolution of the components of a mixture in the shortest possible time is nearly always a principal goal. Establishing the optimum conditions by trial and error is inefficient and relies heavily on the expertise of the analyst. The development of computer-controlled HPLC systems has enabled systematic automated optimization techniques, based on statistical experimental design and mathematical resolution functions, to be exploited. The basic choices of column (stationary phase) and detector are made first followed by an investigation of the mobile phase composition and possibly other parameters. This can be done manually but computer-controlled optimization has the advantage of releasing the analyst for other... 

Recent advances in column stationary phases are remarkable. High performance silica-based reversed-phase 3 to 5 jxm packing materials have been developed for biological sample separations... 

Gas-Liquid Chromatography. In gas-liquid chromatography (GLC) the stationary phase is a liquid. GLC capillary columns are coated internally with a liquid (WCOT columns) stationary phase. As discussed above, in GC the interaction of the sample molecules with the mobile phase is very weak. Therefore, the primary means of creating differential adsorption is through the choice of the particular liquid stationary phase to be used. The basic principle is that analytes selectively interact with stationary phases of similar chemical nature. For example, a mixture of nonpolar components of the same chemical type, such as hydrocarbons in most petroleum fractions, often separates well on a column with a nonpolar stationary phase, while samples with polar or polarizable compounds often resolve well on the more polar and/or polarizable stationary phases. Reference 7 is a metabolomics example of capillary GC-MS. 

The most widely used support substance for the manufacture of packing materials in analytical HPLC columns is silica. Silica can be treated with organochlorosilanes or similar reagents to produce siloxane linkages of any derived polarity similar to what is done for GC columns (stationary phases). The most popular materials are octadecyl silane (ODS), which contains a carbon loading of CIS groups and octyl, which contains C8 groups materials such C2, C6, and C22 are also available. 

System (7) is designed to separate hydrocortisone, cortisone and aldosterone [155]. The column stationary phase was Sil-X (R-P) with octadecyltrichlorosilane, and the mobile phase was either 100 4 or 100 5 chloroform 1,4-dioxane. Detection was based on the UV absorbance of the analytes. 

Column Kontron steel column Stationary phase ODS II, 5 pm, 250 x 4.6 mm... 

Gaseous samples were determined by GC (column stationary phase, carbosieve detector, catharometer, programmed temperature 30 to 230°C, 4°C/min.). 

Fig. 7 Comparative HPLC separations of a standard solution of six vitamins using (A) 250 X 4.0-mm-ID standard-bore and (B) 250 X 2.0-mm-ID narrow-bore columns. Stationary phase (both columns), 5-/tm Nu-cleosil-120-5 C8 (octyl) mobile phase, methanol/water (92 8). Flow rate (A) 0.7 ml/min, (B) 0.2 ml/min. Injection volume, 1 fi 1. Wavelength-programmed absorbance detection. Peaks (1) retinol (2) retinyl acetate (3) vitamin D3 (4) a-tocopherol (5) a-tocopheryl acetate (6) retinyl palmitate. (From Ref. 108.)...

In this chapter we attempt to present an organized approach to method development in SFC, with most of our emphasis on step 6, the optimization of the separation. Although a detailed discussion of sample characterization (step 1) and system selection (steps 2-4) is beyond our present scope, we have included for the novice a brief summary of the choices available for columns, stationary phases, mobile phases, sample introduction, and detection at the beginning of this chapter. We assume the reader is familiar with the basics of SFC if not, numerous reviews and monographs are available (1-5). 

The reverse is true for the line drawn in figure 3.7 with a negative slope (Ss < Sm), and for this reason this system is called a reversed phase (RP) system. The particular line in figure 3-7 connects a typical non-polar (alkane-like) phase with Ss=7 to a polar mobile phase with Sm = 18. Such a mobile phase could for instance be created by mixing methanol (5 16) with water (5w 25) in the correct proportions. Since a very wide range of mobile phase polarities can be covered with mixtures of methanol and water, or even tetrahydro-furan (THF Sx 10) and water, the reversed phase system is a very flexible one. Without changing the column (stationary phase), it can be applied for the elution of a wide variety of solutes. 

Unfortunately, we cannot just use any combination of phases that would constitute a very selective phase system. For example, we might want to opt for the combination of an RPLC column with typically 5, 7, with pure water (8m=25.5) asj he mobile phase, which would result in a selectivity (V) of about 18. However, in this particular phase system only a very polar solute with 5t. 16 would satisfy eqn.(3.30). It will come as no surprise that in this particular (highly selective) phase system all but the very polar solutes will have extremely high capacity factors. In fact, for a given solute, once a given column (stationary phase) has been selected, the appropriate mobile phase can readily be obtained from eqn.(3.30) (or graphically from figure 3.7). From a substitution of eqn.(3.30) in eqn.(3.32) we find... 

All analytics should be performed on LC/MS systems in industrial labs very often ion traps or quadrupole systems are used. Single ion monitoring in either positive or negative mode (compound dependent) is used in HTS conditions. Local LC conditions (injection volume, mobile phase gradients) should be applied to accommodate variations in LC equipment. The selected LC column stationary phase can be used routinely in different labs (for example, C-18 phase selection is compound dependent). 

As with any form of chromatography, the separating efficiency of capillary columns in gas chromatography is strongly dependent on the column stationary phase, carrier gas flow rate, and temperature. Because of the high separation efficiency of capillary columns, only a limited number of stationary phases can be substituted for the numerous phases used in most packed column applications. The choice of a stationary phase is commonly dictated by experience. A phase that has been successfully used by others is usually a good choice. Fre-... 

Basic equipment and components An HPLC system comprises four key components, namely (1) solvent pump(s) (delivery of mobile phase) (2) sample injector (3) HPLC column (stationary phase) and (4) detector linked to recording device. The basic components comprising an HPLC system are illustrated in Figure 7.8. 

Question 21 Solvent pump (delivery of mobile phase) sample injector HPLC column (stationary phase) and detector linked to recording device. 

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