Mobile phase in NP HPLC

Mobile phases in NP HPLC are based on nonpolar solvents (such as hexane, heptane, etc.) with the small addition of polar modiher (i.e., methanol, ethanol). Variation of the polar modifier concentration in the mobile phase allows for the control of the analyte retention in the column. Typical polar additives are alcohols (methanol, ethanol, or isopropanol) added to the mobile phase in relatively small amounts. Since polar forces are the dominant type of interactions employed and these forces are relatively strong, even only 1 v/v% variation of the polar modiher in the mobile phase usually results in a signihcant shift in the analyte retention. 

Mobile phases in NP-HPLC are mostly apolar solvents (or solvent mixtures) such as -hexane, n-heptane, dichloromethane, dichloroethane, diethyl ether, methyl acetate, ethyl acetate, acetone, isopropanol, ethanol, or methanol. In NP-HLPC more polar solvents represent higher solvent strength and these elute compounds faster from the column. The typical order of solvent strength is hydrocarbons < ethers < esters < alcohols < acids < amines (going from weak to strong). 

Selection of using normal-phase HPLC as the chromatographic method of choice is usually related to the sample solubility in specific mobile phases. Since NP uses mainly nonpolar solvents, it is the method of choice for highly hydrophobic compounds (which may show very stronger interaction in re versed-phase HPLC), which are insoluble in polar or aqueous solvents. Figure 1-5 demonstrates the application of normal-phase HPLC for the separation of a mixture of different lipids. 

The most common requirements of an HPLC pumping system are delivery of a constant flow of mobile phase in both isocratic and gradient modes in the range from a few mL.min to 10(xL.min , with an inlet pressure generally np to 5000 psi ( 35 MPa) thongh considerably higher valnes (up to 15 000 psi) are required for columns packed with ultra-small particles (Section 3.5.7) moreover, pressure pulses from piston-driven pnmps must be no larger than 1% of the total flow rate for normal and reverse phase separations. 

Toluene is frequently used as a mobile phase in gel permeation chromatography (GPC). In GPC, trace levels of benzene, xylenes, linear and cyclic alkenes, thiophene, and carbon disulfide are of little consequence unless they can react with analytes of interest. However, in normal-phase (NP) HPLC, the polar impurities can be of critical importance since they modify the polar surface and will significantly alter the resulting chromatography. Therefore, a high-purity toluene should be acquired for HPLC use. 

In NP-HPLC the stationary phase is more polar than the mobile phase and the interaction between analyte and column has predominantly polar character (hydrogen bonding, tt-tt or dipole-dipole interactions, etc.). The most commonly used NP stationary phase is silica gel ([Si02]j [HjOJ, ). Alter colunm preparation the surface of silica gel consists mainly of hydroxyl groups bound to silica atoms as shown in Fig. 7. 

The biggest problem in using NP-HPLC is its dramatic sensitivity to water. Even water traces (in the mobile phase or from the sample) may bind to the column, deteriorate its performance, and cause irreproducibility. In addition, particular care must be taken to ensure accurate pH, as in NP-HPLC, retention is very sensitive to the charge state of the analyte. Owing to these practical problems NP-HPLC is relatively rarely used. Its main application fields are separation of polyaromatic hydrocarbons, sterols, vitamins, chlorophylls, ceramides, and other Upid extracts. 

Online LC-ESI-TOF-MS experiments are carried out in a very similar fashion to the off-line NPS-HPLC separations described above, with a few notable exceptions. Firstly, 0.3% (v/v) formic acid is added to each mobile phase to counteract the ionization suppression induced by TFA. Because of the formic acid UV detection must be carried out at 280 nm (as opposed to 214 nm). To aid in normalization between runs 1 jag of Bovine insulin (MW = 5734 Da) is added to each chromatofocusing fraction prior to injection onto the column. Finally, the flow is split postcolumn directing 200 JlL/min into the ion source and the remaining 300 JlL/min through the UV detector and fraction collection. 

The PO mode is a specific elution condition in HPLC enantiomer separation, which has received remarkable popularity especially for macrocyclic antibiotics CSPs and cyclodextrin-based CSPs. It is also applicable and often preferred over RP and NP modes for the separation of chiral acids on the cinchonan carbamate-type CSPs. The beneficial characteristics of the PO mode may arise from (i) the offset of nonspecific hydrophobic interactions, (ii) the faster elution speed, (iii) sometimes enhanced enan-tioselectivities, (iv) favorable peak shapes due to improved diffusive mass transfer in the intraparticulate pores, and last but not least, (v) less stress to the column, which may extend the column lifetime. Hence, it is rational to start separation attempts with such elution conditions. Typical eluents are composed of methanol, acetonitrile (ACN), or methanol-acetonitrile mixtures and to account for the ion-exchange retention mechanism the addition of a competitor acid that acts also as counterion (e.g., 0.5-2% glacial acetic acid or 0.1% formic acid) is required. A good choice for initial tests turned out to be a mobile phase being composed of methanol-glacial acetic acid-ammonium acetate (98 2 0.5 v/v/w). 

FIG U RE 1.13 Gradient separation of polypeptides on silica rod column and particle-packed columns. Mobile phase velocity 4mm/s, gradient 5%-60% ACN in the presence of TFA, gradient time 5min, columns (a) silica rod column, (b) Capcellpak SG (5 pm), (c) LiChrospher WP 300 RP-18e (5 pm), (d) nonporous NPS-ODS-1 HPLC column (1.5pm) (e) polymer-based TSKgel Octadecyl-NPR (2.5pm). (Reprinted from Minakuchi, H. et al., J. Chromatogr. A, 828, 83, 1998. Copyright 1998, with permission from Elsevier.)... 

The combination of normal (silica) and reversed (C18) phase HPLC in a comprehensive 2D LC system was used for the first time for the analysis of alcohol ethoxylates [64] the NP separation was run using aqueous solvents, so the mobile phases used in the two dimensions were miscible, resulting in the easy injection of the entire first-dimension effluent onto the second-dimension column. 

The most common method is RP-HPLC. Microbore-HPLC [613] and narrow bore columns [614] are applied with good results in increasing sensitivity. A recent comparison between NP and RP microbore columns confirms the better suitability of RP for this purpose [615], The mobile phase is usually composed of acetonitrile or methanol. 

Ideally, one wants an orthogonal (nonoverlapping) separation technique for the alternate method. In this respect, CE is a good first choice. Other choices include TLC, NP-HPLC, and RP-HPLC using different columns, different pH conditions, and different mobile phases. Peak purity evaluations using photodiode array (PDA) UV analysis (to evaluate UV-homogeneity) as well as LC-MS analyses are recommended. The ICH supports this approach in suggesting, Peak purity tests may be useful to show... 

Normal phase (NP) separations are comparatively rarely used in environmental analysis. Again, the reasons lie in the range of analytes amenable to this mode of separation, and in the limited compatibility of typical normal phase HPLC (NP-HPLC) mobile phases with mass spectrometric detection (this also applies to IC). Not only for this reason has interest recently grown in hydrophilic-lipophilic interaction chromatography (HILIC), which represents a viable alternative to the separation of very polar compounds with mobile phases that have a much better compatibility with MS detection, for example, acetonitrile/water with a low water content, typically below 10%, 32 Nonetheless, NP chromato-graphy retains its important role in sample preparation, particularly for the cleanup of complex environmental samples. In the off-line approach, fractions are collected and the relevant one is injected into the reversed phase HPLC (RP-HPLC) system, often after solvent exchange. 

In parallel with recent developments in GC, multidimensional HPLC (LC x LC) is now also finding application in environmental analysis.33 The combination of two sufficiently different separation dimensions (e.g., NP-HPLC x RP-HPLC or IC x RP-HPLC), however, remains difficult because of the solvent compatibility issues discussed above. Here, too, HILIC may bring about a significant improvement, since its mobile phase requirements are much closer to RP-HPLC than those of other liquid chromatographic techniques.34 In contrast to GC x GC, LC x LC cannot be implemented with a (thermal) modulator that collects the analytes after the first separation dimension and reinjects them into the second column it is most practically realized with a double-loop interface that alternately collects and transfers the analytes from the first to the second dimension (Figure 13.7). Even though the second dimension chromatogram is also very fast, detection is not normally a problem since the peak widths in the second dimension are usually still of the order of 1-2 s. 

Normal-phase HPLC explores the differences in the strength of the polar interactions of the analytes in the mixture with the stationary phase. The stronger the analyte-stationary phase interaction, the longer the analyte retention. As with any liquid chromatography technique, NP HPLC separation is a competitive process. Analyte molecules compete with the mobile-phase molecules for the adsorption sites on the surface of the stationary phase. The stronger the mobile-phase interactions with the stationary phase, the lower the difference between the stationary-phase interactions and the analyte interactions, and thus the lower the analyte retention. 

Early PG analysis using HPLC techniques was carried out as adsorption chromatography on normal-phase (NP) columns packed with silica or alumina. The nonpolar mobile phase comprizing of organic solvents (hexane, toluene, ethyl acetate, and HOAc) allows separation of PGs which are unstable in aqueous media (e.g., PGH2 on cyano- or phenyl-bonded phases). Usually, the injection medium must be fairly polar to dissolve the PGs. This is achieved by the addition of... 

Over 500 HPLC packings have been described in the Hterature. Nevertheless, as the result of years of development, only a limited number of types of stationary phases remain on the market. Most of the conventional HPLC separations today are performed using monodisperse silica gel 3 or 5 Xm microbeads, especially those grafted with C4, C8, or C18 alkyl chains, as well as with cyano-propyl or amino-propyl groups. The last two bonded silicas and bare silica are used in normal phase (NP) HPLC, where the mobile phase (usually hexane with small amounts of isopropyl alcohol) is less polar than the stationary phase. Even more popular is the reversed phase (RP) mode, which uses polar eluents (mosdy water or methanol with such additives as acetonitrile, methanol, or tetrahydrofuran (THE)) in combination with nonpolar alkyl-bonded stationary phases. 

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