Normal phase HPLC mobile phases

As opposed to normal-phase HPLC, reversed-phase chromatography employs mainly dispersive forces (hydrophobic or van der Waals interactions). The polarities of mobile and stationary phases are reversed, such that the surface of the stationary phase in RP HPLC is hydrophobic and mobile phase is polar, where mainly water-based solutions are employed. 

Figure 12.18 LC-SFC analysis of mono- and di-laurates of poly (ethylene glycol) ( = 10) in a surfactant sample (a) normal phase HPLC trace (b) chromatogram obtained without prior fractionation (c) chromatogram of fraction 1 (FI) (d) chromatogram of fraction 2 (F2). LC conditions column (20 cm X 0.25 cm i.d.) packed with Shimpak diol mobile phase, w-hexane/methylene chloride/ethanol (75/25/1) flow rate, 4 p.L/min UV detection at 220 nm. SFC conditions fused-silica capillary column (15 m X 0.1 mm i.d.) with OV-17 (0.25 p.m film thickness) Pressure-programmed at a rate of 10 atm/min from 80 atm to 150 atm, and then at arate of 5 atm/min FID detection. Reprinted with permission from Ref. (23).

Normal-phase HPLC An HPLC system in which the mobile phase is less polar that the stationary phase. 

Normal phase HPLC consists of methods that utilize a nonpolar mobile phase in combination with a polar stationary phase. Adsorption HPLC actually fits this description, too, since the adsorbing solid stationary phase particles are very polar. (See discussion of adsorption columns in Section 13.5.3.) Normal... 

Normal-phase HPLC has also found application in the analysis of pigments in marine sediments and water-column particulate matter. Sediments were extracted twice with methanol and twice with dichloromethane. The combined extracts were washed with water, concentrated under vacuum and redissolved in acetone. Nomal-phase separation was performed with gradient elution solvents A and B being hexane-N,N-disopropylethylamine (99.5 0.5, v/v) and hexane-2-propanol (60 40, v/v), respectively. Gradient conditions were 100 per cent A, in 0 min 50 per cent A, in 10 min 0 per cent A in 15 min isocratic, 20 min. Preparative RP-HPLC was carried out in an ODS column (100 X 4.6 mm i.d. particle size 3 jum). Solvent A was methanol-aqueous 0.5 N ammonium acetate (75 25, v/v), solvent B methanol-acetone (20 80, v/v). The gradient was as follows 0 min, 60 per cent A 40 per cent A over 2 min 0 per cent A over 28 min isocratic, 30 min. The same column and mobile phase components were applied for the analytical separation of solutes. The chemical structure and retention time of the major pigments are compiled in Table 2.96. 

A normal-phase HPLC method was employed for the separation of 11 -cis- and all-frany-retinals. Separation was performed in a silica column (150 X 6.0 mm i.d. particle size 3 jum) with an isocratic mobile phase (n-hexane with 15 per cent ethyl acetate and 0.15 per cent ethanol). The flow rate was 1 ml/min and retinols were detected at 360 nm. The method separated well the 11 -cis- and all-/ran.v-retinals as demonstrated in Fig. 2.162. The results emphasize again the decisive role of chromatographic methods in the elucidation of the mechanism of various biochemical processes [334],... 

In high-pressure adsorption chromatography, solutes adsorb with different affinities to binding sites in the solid stationary phase. Separation of solutes in a sample mixture occurs because polar solutes adsorb more strongly than nonpolar solutes. Therefore, the various components in a sample are eluted with different retention times from the column. This form of HPLC is usually called normal phase (polar stationary phase and a nonpolar mobile phase). 

Carotenoid separations can be accomplished by both normal- and reversed-phase HPLC. Normal-phase HPLC (NPLC) utilizes columns with adsorptive phases (i.e., silica) and polar bonded phases (i.e., alkylamine) in combination with nonpolar mobile phases. In this situation, the polar sites of the carotenoid molecules compete with the modifiers present in the solvent for the polar sites on the stationary phase therefore, the least polar compounds... 

Normal-phase HPLC on silica columns are also used extensively in D3 analysis of vitamin products with nonpolar mobile phase containing polar modifiers. Krol et al. (66) separated D3 from pre-D3 and from a mixture of other vitamins using an adsorptive silica support introduced in 1972 (Vydac , supplied at that time by Applied Science Laboratories, Inc. State College, Penn.). The hand-packed column was used in conjunction with a mobile phase of pen-tane tetrahydrofuran (97.5 2.5). Sterule (32) used aluminum oxide as column support with chloroform as the mobile phase. Separation of D3 and its isomers and from vitamin A acetate was achieved. 

Compared to GC columns, HPLC columns are short and thick, ranging from 10 to 25 cm in length and 2 to 4.6 mm in internal diameter. They are filled with an inert material (silica, polymer resin), which is coated with a stationary phase. In normal phase HPLC, the mobile phase is less polar than the stationary phase. In reverse phase HPLC, the opposite is true, and the mobile phase is more polar than the stationary one. Reverse phase HPLC is the technique of choice for environmental applications. Similar to GC columns, analyte-specific HPLC columns are recommended in the published methods. 

Normal phase HPLC has only found limited application in this field because most of the species of interest are polar. It has, however, been used to separate metal chelates, such as dithiocarbamates, that are soluble in the non-polar mobile phases (Steinbrech, 1987). 

Separation of enantiomers of etodolac using two different derivitization agents and three chiral stationary phases has been studied [24]. Etodolac was converted to its anilide derivative with either 1,3-dicyclohexyl-carbodiimide or l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. Etodolac, derivatizing agent, aniline, and dichloromethane were allowed to incubate for 30 minutes, which was followed by addition of 1 M HC1. The organic layer was removed, washed, dried, and then injected into normal phase or reverse phase HPLC. The HPLC system consisted of a 250 x 4.6 mm (5 pm particle size) column packed with chiral stationary phases, and detection was effected by the UV absorbances at 254 and 280 nm. Separation of etodolac enantiomers was achieved on only one of the stationary phases when using 20% 2-propanol in hexane as the mobile phase at a flow rate of 2.0 mL/min. 

Normal-phase HPLC may be used for separation of some aldehyde classes prior to quantitative reversed-phase HPLC. It is possible to separate 4-hydroxyalkenals by normal-phase HPLC as described below using 50% water-saturated dichloromethane as a mobile phase. This is prepared by mixing equal volumes of dry dichloromethane and a solution of dichloromethane stored under a layer of water (100%... 

In recent years several normal-phase HPLC methods have been reported for the quantitative analysis of tocopherols and tocotrienols (Table 11.5). The best of these methods have been able to achieve baseline separation of all four tocopherols and all four tocotrienols, as shown in Figures 11.2 and 11.3. Kamal-Eldin et al. (2000) reported the optimal baseline separation of all eight common tocols using a Diol-bonded phase column and an isocratic mobile phase of hexane/methyl tert-butyl ether (MTBE), 96 4, v/v (Figure 11.2). Similar separations were reported by Moreau et al. (2007) using the same type of column and mobile phase. Schwartz et al. (2008) reported that, with a normal-phase silica column, plastochromanol-8 in rapeseed oil eluted between y-tocopherol and 5-tocopherol. 

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. 

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. 

Warmuth [34] when they adopted NPC for the analysis of supermolecules, such as hemicarcerplexes. Hemicarcerplexes are complexes formed with hemi-carcerand host and guest molecules. As shown in Figure 5-6, hemicarcerands possess a very hydrophobic structure with molecular weight over 2000 and is insoluble in protic solvents. A normal-phase HPLC method was developed using a silica column with dichloromethane and diethylether as the mobile-phase system. The authors demonstrate that the chromatographic retention of hemicarcerplexes is mainly dominated by its size. Furthermore, a linear relationship between the logarithmic retention factor and the size of the hemicarcerplexes was observed for linear guest molecules independent of their polarity. 

In both normal phase and reversed phase HPLC, the eluting power or solvent strength of the mobile phase is mainly determined by its polarity. Although most analysts have a good idea of what the term polarity implies and could rank most common solvents in order of their polarity, a more quantitative description would be very useful in chromatography. 

HPLC analysis of taxanes is achieved almost exclusively in reversed phase mode on various stationary phases. The normal-phase HPLC mode has been applied in very limited cases and resulted in broad peaks and long analysis times (retention times of 45 min for paclitaxel and 38 min for cephalomannine). The namre of the sample is the main criterion for the choice of the stationary phase. Analysis of plant material is performed mostly on phenyl, biphenyl, and pentafluorophenyl materials, but silica-based cyano, Cig, and Cg materials have been used as well. C18 phases are the most common material utilized in pharmacokinetic studies. Mobile phases typically consist of mixtures of methanol, acetonitrile, and water or buffer (mostly ammonium acetate). Detection is performed by UV, mostly in the low region of 225 -230 nm. Taxanes give similar UV spectra with a minimum at 210-215 nm and a maximum at 225-232 nm. Therefore, detection is performed, preferably at 227-228 nm. Dual/multiple UV detection is performed in both low and upper regions, e.g. 227 and 273 nm 230 and 280 nm 227, 254, and 270 nm, etc. (Fig. 2). 

Analysis of 10 crude fish oils from various regions of the world for arsenolipids was performed by normal phase HPLC-ICPMS with various mixtures of organic solvents as mobile phases [172]. All ten fish oils appeared to contain the same 4-6 major arsenolipids, but in varying amounts depending on the origin of the fish. Further chromatography of some of the oils imder both normal phase and reversed-phase conditions indicated the presence of many more minor arsenolipids. Unfortunately, the authors did not provide any data on the structures of the arsenolipids they described. 

Retinoic acid, an endogenous retinoid, is a potent inducer of cellular differentiation. Because cancer is fundamentally a loss of cellular differentiation, circulating levels of retinoic acid could play an important role in chemoprevention. However, physiological concentrations are typically below the limits of HPLC detection. Sensitive techniques, such as negative chemical ionization (NCI) GC/MS have been employed for quantification, but cause isomerization and also fail to resolve the cis and trans isomers of retinoic acid. Normal phase HPLC can resolve the cis and trans isomers of retinoic acid without isomerization, and mobile phase volatility makes it readily compatible with the mass spectrometer. Based on these considerations, a method combining microbore normal phase HPLC separation with NCI-MS detection was developed to quantify endogenous 13-cis and all-trans retinoic acid in human plasma. The limit of detection was 0.5 ng/ml, injecting only 8 pg of retinoic acid onto the column. The concentration of 13-cis retinoic acid in normal, fasted, human plasma (n=13) was 1.6 +/- 0.40 ng/ml. 

In normal-phase HPLC, solute retention is based on the distribution of solute between a polar stationary phase and a nonpolar mobile phase (typically a mixture of hexane and a more polar solvent such as isopropanol). Elution may be promoted by increasing the amount of polar solvent in the mobile phase. In reversed-phase HPLC, retention is based on distribution between a nonpolar stationary phase and a polar mobile phase (typically a mixture of water and acetonitrile or methanol), and elution is promoted by addition of the less polar solvent to the mobile phase. With the exception of extremely polar or ionized compounds, which are not amenable to normal-phase HPLC, and extremely nonpolar compounds such as certain steroids and natural products, which are not amenable to reversed-phase HPLC, both modes of HPLC are potentially applicable to APIs and related substances. However, about 75% of current HPLC analyses are performed using the reversed-phase.This is due not only to safety considerations using nonpolar solvents but also to the differences in sample preparation procedures required for normal-phase versus reversed-phase HPLC. 

For the separation of chiral molecules into their respective enantiomers, several approaches are possible by HPLC. These include precolumn derivatization to form diastereomers, followed by the use of normal-phase or reversed-phase HPLC, or addition of the derivatization reagent to the chromatographic mobile phase to form dynamic diastereomers during the separation process. Alternatively, specialty columns prepared with cyclodextrins or specific chiral moieties as stationary phases may be used. 

Examination of the synthetic route used in production allows for the prediction of potential residual synthetic impurities present in the drug substance. The API structure allows for the postulation of degradation pathways via hydrolytic, oxidative, catalytic, and other mechanisms. Both of these evaluations serve to facilitate the interpretation of (subsequent) identification tests. An examination of the physicochemical properties also allows for the rational establishment of method screening experiments by precluding certain conditions. For example, the use of normal-phase HPLC will be eliminated if the API is a salt or shows limited solubility in nonpolar organic solvents. Similarly, if the API (or suspected related substances) has no significant chromophore above 250 nm, the use of tetrahydrofuran (THE) and other solvents as mobile-phase components is severely limited. For compounds with an ionizable group, variation of pH will have considerable influence on elution behavior and can be exploited to optimize the selectivity of a reversed-phase separation. 

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