Size mobile phases

Figure 1 Chromatogram of a neutral compound (toluene) with watenacetonitrile mobile phase. Chromatographic conditions — column 30 cm x 3.9 mm p-Bondapak C18 (10-pm particle size) mobile phase watenacetonitrile (50 50) flow rate 1.5 ml/min column temperature ambient detector wavelength 254 nm.

FIGURE 14.8 Relationship of HETP and mobile phase linear velocity for column packing materials of 2, 3, 5, and 8 /an ROSIL C18 particle size. Mobile phase was 75 25 acetonitrile water. Sample test probe was pyrene at k = 6. 

Available particle size Mobile phase viscosity... 

In the development and optimization of a comprehensive LCxLC method, many parameters have to be taken in acconnt in order to accomplish snccessfnl separations. First of all, selectivity of the columns used in the two dimensions must be different to get maximum gain in peak capacity of the 2D system. For the experimental setup, column dimensions and stationary phases, particle sizes, mobile-phase compositions, flow rates, and second-dimension injection volumes should be carefully selected. The main challenges are related to the efficient coupling of columns and the preservation of mobile phase/column compatibility. 

Figure 11.6.3 Gradient HPLC separation of isoflavone standards (see Basic Protocol 3). Peaks 1, daidzin 2, glycitin 3, genistin 4, malonyldaidzin 5, malonylglycitin 6, acetyldaidzin 7, acetylglycitin 8, malonylgenistin 9, daidzein 10, glycitein 11, acetylgenistin 12, genistein. Conditions Waters Nova-Pak C18 reversed-phase column (150 x 3.9 mm 4-pm i.d. 60 A pore size) mobile phase 1% acetic acid in water (solvent A) and acetonitrile (solvent B) flow rate 0.60 ml/min UV detector 260 nm column temperature 25°C. The dotted line represents the gradient of solvent B.

HPLC column p-Bondapak C-18, 10 pm particle size mobile phase Na2HP04 (0.005 AT) in methanol/water (5 95). 

Figure 9.112 Separation of substrates and products of reaction catalyzed by adenylosuccinate synthetase. Column Prepacked C18 /xBondapak, 10 /im particle size. Mobile phase 65 mM potassium phosphate, 1 mM tetrabutylammonium phosphate, 10% methanol at pH 4.4. Absorbance was measured at 254 nm. (From Rossomando, 1987.)...

Column Dynamax Cl8, 5 x 250 mm, 8 pm particle size Guard Tube Dynamax Cl8 Guard, 8 pm particle size Mobile phase Acetonitrile water 70 3 0 (v/v)... 

The experimental optimization procedures outlined above can be replaced with others based on computer simulations [64,65], which make use of the chromatographic theory and of one or two prior experiments intended to define critical parameters such as the sample, mobile phase, column, temperature, flow-rate and pressure. Simulated chromatograms are obtained for different experimental conditions (column dimensions, particle size, mobile phase composition, flow-rate, temperature, etc.) until the required resolution is achieved. In essence, the procedure is similar to experimental optimization, although the chromatograph functioning is replaced with programming. The information obtained can be checked experimentally or be used for designing new approached to experimental optimization. 

The equation, though complex, shows the importance of particle size, mobile phase flow rate and diffusion coefficients and indicates that the deleterious effects on H and thus the column performance can be minimised by reducing the packing particle size, the stationary phase thickness and the solvent viscosity (thus decreasing Dyi and D ). The latter can be achieved by using elevated column temperatures. 

Column and injection port temperature, sample size, mobile phase flow-rate, column efflciency. 

Ca. 0.5 g sample was extracted into 30 mL methanol and 0.05% tropolone mixture. 200 pL of extract were injected onto a cationic exchange HPLC column (Partisil, cation exchange, 25 cm length, 4.6 mm internal diameter, 10 pm particle size, mobile phase consisting of 80% methanol-water, 0.1 mol ammonium acetate and 0.1% tropolone). The eluent was mixed with 0.3 molL HCl and 0.25 molL NaBILi in 1 mol L NaOH. The organotin hydrides were drained to a gas-liquid separator. Final detection was by ICP-AES. Recoveries were evaluated by spiking the material results were (108 + 3)% for MBT, (79 8)% for DBT and (102 11)% for TBT. Calibration was performed by standard additions. 

The field of liquid chromatography is well established, and reliable methods have been developed for analytical and preparative separations. Column miniaturization improves performance for analytical separations. Numerous stationary phases have been developed to separate analytes based on a wide variety of molecular properties including hydrophobicity, ionic interactions, and molecular size. Mobile-phase modifiers can be used to aid in the niinumzation of unwanted interactions with the solid support. Although the field is well established, current research continues to improve separations for both microscale analytical and larger preparative separations. Recent publications will be highlighted that demonstrate the developments toward integrating HPLC components and separation techniques onto microfabricated devices. 

Figure 19 RPC of a mixture of synthetic peptide polymers. Column SynChropak RP-P Ci8 (250 X 4.6 mm ID, 6.5-pm particle size, 300-A pore size). Mobile phase linear gradient, where eluent A is 0.1% aqueous trifluoroacetic acid (TFA), pH 2.0, and eluent B is 0.1% TFA in acetonitrile gradient rate, 1% acetonitrile/min flow rate, 1 mL/min temperature, 26°C. (A) Elution profile of five peptide polymers (10-50 residues sequences shown in Fig. 2). (B) Plot of predicted minus peptide observed retention time (t - versus the sum of the retention coefficients (H o 6t al. [164] times the logarithm of the number of residues (In N). (C)...

HILIC Silica (unbonded or bonded) Porous or core-shell silica 3—5-pm particle size Mobile phase high organic solvent concentration Stationary phase attracts H20-enriched layer of stagnant aqueous mobile phase allowing retention of polar compounds Amino acids, monoamines, acetylcholine... 

Another example is the purification of a P-lactam antibiotic, where process-scale reversed-phase separations began to be used around 1983 when suitable, high pressure process-scale equipment became available. A reversed-phase microparticulate (55—105 p.m particle size) C g siUca column, with a mobile phase of aqueous methanol having 0.1 Af ammonium phosphate at pH 5.3, was able to fractionate out impurities not readily removed by hquid—hquid extraction (37). Optimization of the separation resulted in recovery of product at 93% purity and 95% yield. This type of separation differs markedly from protein purification in feed concentration ( i 50 200 g/L for cefonicid vs 1 to 10 g/L for protein), molecular weight of impurities (<5000 compared to 10,000—100,000 for proteins), and throughputs ( i l-2 mg/(g stationary phasemin) compared to 0.01—0.1 mg/(gmin) for proteins). 

Conductivity detectors, commonly employed in ion chromatography, can be used to determine ionic materials at levels of parts per million (ppm) or parts per bUHon (ppb) in aqueous mobile phases. The infrared (ir) detector is one that may be used in either nonselective or selective detection. Its most common use has been as a detector in size-exclusion chromatography, although it is not limited to sec. The detector is limited to use in systems in which the mobile phase is transparent to the ir wavelength being monitored. It is possible to obtain complete spectra, much as in some gc-ir experiments, if the flow is not very high or can be stopped momentarily. 

It is clear that the separation ratio is simply the ratio of the distribution coefficients of the two solutes, which only depend on the operating temperature and the nature of the two phases. More importantly, they are independent of the mobile phase flow rate and the phase ratio of the column. This means, for example, that the same separation ratios will be obtained for two solutes chromatographed on either a packed column or a capillary column, providing the temperature is the same and the same phase system is employed. This does, however, assume that there are no exclusion effects from the support or stationary phase. If the support or stationary phase is porous, as, for example, silica gel or silica gel based materials, and a pair of solutes differ in size, then the stationary phase available to one solute may not be available to the other. In which case, unless both stationary phases have exactly the same pore distribution, if separated on another column, the separation ratios may not be the same, even if the same phase system and temperature are employed. This will become more evident when the measurement of dead volume is discussed and the importance of pore distribution is considered. 

The stationary phase constitutes about 12% of the column volume, which is equivalent to only about 17% of the mobile phase content of the column. The values given in Table 2 are probably representative of most reverse phase columns but will differ significantly with extremes of pore size and pore volume. 

The explicit form of those equations that satisfy the preliminary data criteria, must then be tested against a series of data sets that have been obtained from different chromatographic systems. As an example, such systems might involve columns packed with different size particles, employed mobile phases or solutes having different but known physical properties such as diffusivity or capacity ratios (k"). 

The results obtained were probably as accurate and precise as any available and, consequently, were unique at the time of publication and probably unique even today. Data were reported for different columns, different mobile phases, packings of different particle size and for different solutes. Consequently, such data can be used in many ways to evaluate existing equations and also any developed in the future. For this reason, the full data are reproduced in Tables 1 and 2 in Appendix 1. It should be noted that in the curve fitting procedure, the true linear velocity calculated using the retention time of the totally excluded solute was employed. An example of an HETP curve obtained for benzyl acetate using 4.86%v/v ethyl acetate in hexane as the mobile phase and fitted to the Van Deemter equation is shown in Figure 1. 

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