Mobile phase column

Considering that the separation system is fully characterized, i.e., adsorbent and mobile phases, column dimensions, SMB configuration and feed concentration, the optimization of the TMB operating conditions consists in setting the liquid flow rates in each section and also the solid flow rate. The resulting optimization problem with five variables will be certainly tedious and difficult to implement. Fortunately, the... 

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. 

TECHNIQUE MOBILE PHASE COLUMN SPECIES/PLANT MATERIAL SAMPLE IRIDOID REF... 

Mobile phase" Column n/ad separation Temperature (°C) Wavelength (nm) Ref. 

An isocratic HPLC method for screening plasma samples for sixteen different non-steroidal anti-inflammatory drugs (including etodolac) has been developed [29]. The extraction efficiency from plasma was 98%. Plasma samples (100-500 pL) were spiked with internal standard (benzoyl-4-phenyl)-2-butyric acid and 1 M HC1 and were extracted with diethyl ether. The organic phase was separated, evaporated, the dry residue reconstituted in mobile phase (acetonitrile-0.3% acetic acid-tetrahydrofuran, in a 36 63.1 0,9 v/v ratio), and injected on a reverse-phase ODS 300 x 3.9 mm i.d. column heated to 40°C. A flow rate of 1 mL/min was used, and UV detection at 254 nm was used for quantitation. The retention time of etodolac was 30.0 minutes. The assay was found to be linear over the range of 0.2 to 100 pg/mL, with a limit of detection of 0.1 pg/mL. The coefficients of variation for precision and reproducibility were 2.9% and 6.0%, respectively. Less than 1% variability for intra-day, and less than 5% for inter-day, in retention times was obtained. The effect of various factors, such as, different organic solvents for extraction, pH of mobile phase, proportion of acetonitrile and THF in mobile phase, column temperature, and different detection wavelengths on the extraction and separation of analytes was studied. 

FIGURE 5-52. Examples of normal phase separations, (a) Corn-oil tocopherols. Sample 10 /xL of corn oil in 100 /xL of mobile phase. Column Nova Pak Silica (4 jam), 3.9 mm ID x 150 mm. Mobile phase 0.3% isopropyl alcohol in isooctane. Flow rate 1.0 mL/min. Detection fluorescence 290 nm excitation and 335 nm emission. (b) Separation of vitamin E from vitamin A. Mobile phase 0.5% isopropyl alcohol in isooctane. Other conditions are the same as those in a with the exception that retinol was detected with 365 nm excitation and 510 nm emission, (c) Structures of compounds. 

FIGURE 7-30. Effects of undegassed mobile phase. Column /uBondapak C,8, 3.9 mm ID x 30 cm. Mobile phase 50 50 MeOH water. Flow rate 2.0 mL/min. Detector UV at 254 nm, 0.01 AUFS. 

Stationary phases for modern, reversed-phase liquid chromatography typically consist of an organic phase chemically bound to silica or other materials. Particles are usually 3, 5, or 10 p,m in diameter, but sizes may range up to 50 p,m for preparative columns. Small particles thinly coated with organic phase allow fast mass transfer and, hence, rapid transfer of compounds between the stationary and mobile phases. Column polarity depends on the polarity of the bound functional groups, which range from relatively nonpolar octadecyl silane to very polar nitrile groups. 

Figure 18 CEC separation of explosives with addition of SDS to mobile phase. Column 34 cm x 75 mm i.d., 21 cm packed with 1.5-pm nonporous ODS II particles. Mobile phase 20% methanol, 80% 10 mM MES, 5 mM SDS. Running potential 12 kV (480 V/cm in packed portion). Injection 2 s at 2 kV of 12.5 mg/L (each component) sample. (Reprinted from Ref. 89, with permission.)...

Column (CSP) Mobile Phase Column Length Column I.D. ... 

Fig. 7. Dependence of the capacity factor of the protonated peptides on the concentration of D-camphor-10 sulphonate (A) and -hexyl sulfonate (B) in the mobile phase. Column /i.-Bondapak Cig flow rate 2 ml/min temperature 20°C, mobile phase 507c methanol-50% water-50 mM NaHiP04 with HjPOi added to pH 3.0, containing various concentrations of the ion-pairing reagents. The protonated peptides were as follows 1, Arg-Phe 2, Arg-Phe-Ala 3, Met-Arg-Phe 4, Met-Arg-Phe-Ala 5, Leu-Trp 6, Leu-Trp-Met-Arg 7, Leu-Trp-Met 8, Leu-Trp-Met-Arg-Phe. Reproduced from Hearn and Grego (34).

Analyte Mobile Phase Column Temperature Reference... 

Analyte(s) Samples Internal standard Column Mobile phase Column temperature (°C) Detection (nm) Solvent Limit of detection (LOD), limit of quantitation (LOQ), and recovery (Rec) Reference... 

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. During the development phase of the analytical procedure, susceptible parameters should be identified, for example, stability of analytical solutions, extraction time, pH and composition of mobile phase, column lots and suppliers, temperature, flow rate, etc. A factorial design is encouraged. 

Preferably, the sample is dissolved in a solvent mixture weaker than the mobile phase and introduced (concentrated) on the column in a narrow zone. Although a higher concentration of the sample contributes to a narrower zone after injection, it is not recommended to use a solution of more than twice the viscosity of the mobile phase. Column hardware should provide an even distribution of the injected solution throughout the cross section of the column. 

Description of the test method (e.g., mobile phase, column, flow rate, injection size, and so on). 

Fig. 1 Use of polymer-coated silica stationary phase with a neat carbon dioxide mobile phase. Column Deltabond SFC Methyl (150 X 2.0 mm) mobile phase CO2 pressure gradient 75 bar initial for 2 min, then 50 bar/min ramp to 180 bar, then 15 bar/min ramp to 300 bar flow 0.5 mL/min temperature 100°C injection 5 jjlL detection flame ionization detector sample solvent methylene chloride. Peak key 1 BHT 2 dimethyl azelate 3 triethyl citrate 4 tributyl phosphate 5 methyl palmitate 6 methyl stearate 7 diethylhexyl phthalate 8 Tinuvin 327 9 Spectra-Sorb UV531 10 tri(2- ethylhexyl) trimellitate 11 dilauryl thiodipropionate ...

Reproducibility, as defined by ICH, represents the precision obtained between laboratories with the objective of verifying if the method will provide the same results in different laboratories. The reproducibility of an analytical method is determined by analyzing aliquots from homogeneous lots in different laboratories with different analysts, and by using operational and environmental conditions that may differ from, but are still within the specified, parameters of the method (interlaboratory tests). Various parameters affect reproducibility. These include differences in room environment (temperature and humidity), operators with different experience, equipment with different characteristics (e.g., delay volume of an HPLC system), variations in material and instrument conditions (e.g., in HPLC), mobile phases composition, pH, flow rate of mobile phase, columns from different suppliers or different batches, solvents, reagents, and other material with different quality. 

Retention times tend to vary over time because of a number of causes, e.g., differences between batches of mobile phase, column performance and different columns, and ambient temperatures of laboratories. The RSD of retention time, especially important because it is used for peak identification, is influenced by 1) Pump flow 2) composition precision 3) mobile phase composition of solvent delivery systems and 4) column temperature. Imprecise retention time indicates problems within the HPLC system such as piston seals, check valves, etc. 

Figure21.1 Analytical separation ofthe reaction mixture. Conditions sample, 20(j-l solution in mobile phase column, 25cm x 3.2 mm i.d. stationary phase.

Perhaps the biggest limitation to RP-LC is the difficulty with adequate retention for very polar analytes. Three undesirable issues are associated with poor retention (1) poor chromatographic separation, (2) greater ionization suppression, and (3) reduced sensitivity due to poor analyte desolvation in highly aqueous mobile phases. Column manufacturers have used several formats to improve retention of polar solutes that include extended alkyl phases, polar-endcapped alkyl phases, polar-embedded alkyl phases, nonendcapped short alkyl phases, and wide pore-diameter phases [94]. Interested readers are referred to a recent two-part review on this important subject [94,95]. 

This gives the overall relationship between solute retention, distribution of a solute across stationary and mobile phases, column length, and mobile phase flow rate. 

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. 

Figure 7.7. System constants of the solvation parameter model for retention on a porous polymer stationary phase with a binary mixture of carbon dioxide and 1,1,1,2-tetrafluoroethane as the mobile phase. Column 25 cm X 4.6 mm I.D. Jordi-Gel RP-C18 with a 5 pm average particle diameter. The total fluid flow rate was 1.0 ml/min, backpressure 200 bar and ternperamre I25°C.

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