Reversed-phase HPLC hydrocarbons

Figure 8. Reversed-phase HPLC separation of SRM 1647, priority pollutant polynuclear aromatic hydrocarbons (In acetonitrile), using UV detection.

If simple sample pretreatment procedures are insufficient to simplify the complex matrix often observed in process mixtures, multidimensional chromatography may be required. Manual fraction collection from one separation mode and re-injection into a second mode are impractical, so automatic collection and reinjection techniques are preferred. For example, a programmed temperature vaporizer has been used to transfer fractions of sterols such as cholesterol and stigmasterol from a reversed phase HPLC system to a gas chromatographic system.11 Interfacing gel permeation HPLC and supercritical fluid chromatography is useful for nonvolatile or thermally unstable analytes and was demonstrated to be extremely useful for separation of compounds such as pentaerythritol tetrastearate and a C36 hydrocarbon standard.12... 

Fetzer, J.C. and Biggs, W.R., The use of large polycychc aromatic hydrocarbons to study retention in non-aqueous reversed-phase HPLC, Chromatographia, 27, 118, 1989. 

Additionally, the combination of trace enrichment and microbore columns can effectively increase the maximum sample volume injectable without seriously degrading efficiency. Slais et al. (29) evaluated this combination for the determination of polynuclear hydrocarbons and chlorinated phenols in water. By using reversed-phase HPLC and am-perometric detection, Slais et al. (29) reported lower limits of detection from 20 to 280 ng/L of water (parts per trillion) when 1-mL sample enrichments were carried out directly on the analytical microbore column. 

Jinno, K., and K. Kawasaki, Correlation Between the Retention Data of Polycyclic Aromatic Hydrocarbons and Several Descriptors in Reversed-Phase HPLC. Chromato-graphia, 1983 17, 445-449. 

As regards the lipids, two types of adsorbents are available, one of which is a form of silica gel and is utilized in normal-phase HPLC, and the other of which can be a silica gel bonded to a hydrophobic chain and is employed in reverse-phase HPLC. In normal-phase HPLC the phospholipids appear to be separated based on the molecular classes present (PE, PC, Sph, etc.), whereas in reverse-phase HPLC the separation is closely related to the lipophilic character of the acyl (fatty acyl, hydrocarbon chain) of the particular phospholipids. High-quality adsorbents suitable for HPLC are easily available from commercial companies. 

QSRR differentiate in a quantitative (statistical) manner the stationary phase materials of different chemical natures. However, when the stationary phases are compared which belong to the same chemical class, like hydrocarbon-bound silicas for reversed-phase HPLC, the results obtained are not that unambiguous. 

The phenyl group offers a difference in selectivity to ODS for many compounds in reversed phase HPLC, and has been used in the analysis of polycyclic aromatic hydrocarbons. For most compounds, retention on a phenyl column is similar to that obtained on a C8 packing material. 

Jinno, K. Kawasaki, K. Correlations between the retention data of polycyclic aromatic hydrocarbons and several descriptors in reversed-phase HPLC. Chromatographia 1983, 77(8), 445-449. 

With normal-phase HPLC, oil samples were analyzed as is by simple dilution in n-hexane. A Du Pont Zorbax amino-bonded phase column, 25 cm x 0.46 cm ID, was used, with n-hexane and dichloromethane as solvents. For reversed-phase HPLC, Vydac 201TP5 columns were used (25 cm x 0.46 cm ID for analytical scale and 25 cm x 1 cm ID for preparative scale). Samples for reversed-phase HPLC were fractionated in order to remove the saturated hydrocarbons which can interfere with the separation mechanism. The samples dissolved in n-hexane were passed Baker silica solid-phase extraction cartridges. The PAH fraction was then collected by eluting with a 1 1 mixture of dichloromethane and methanol. Acetonitrile and dichloromethane were used in the HPLC gradient. 

Stationary phases have also been developed, the most common being octadecyl or octyl hydrocarbon groups. Use of these columns is referred to as reverse-phase HPLC. 

Polynuclear aromatic hydrocarbons at trace concentrations may be analyzed by HPLC, GC and GC/MS by US EPA Methods 610, 8100, 8270 and 8320 (US EPA 1997). GC techniques involve the use of a flame ionization detector and a fused silica capillary column or a packed column such as 3% OV-17 on Chromosorb W-AW or equivalent. Certain PAH compounds may coelute on a GC column. This problem does not occur in the reversed phase HPLC analysis using UV and fluorescence detectors. A column such as HC-ODS Sil-X or equivalent having a 5-/Lim particle size is suitable for separating PAH mixtures. GC/MS is a confirmative method to identify individual compounds although some of the compounds produce the same characteristic masses. The compounds should be identified from fheir characferisfics mass ions and refenfion times. Various analytical methods and the characteristic mass ions of some... 

The use of small spherical particles for NPC results in plate numbers in excess of 10,000 which compares favourably with the efficiencies of stationary phases currently in use for reversed phase HPLC. A list of the common functional groups used in NPC are shown in Table 6.1. Most of the popular supports used in NPC are silica based since alumina undergoes undesirable side reactions (e.g. irreversible reaction with carboxylic acid). The introduction of bonded phase supports has led to the availability of many other polar stationary phases, some of which can also be used in the reversed phase mode (Lochmuller et al., 1979). A bonded diamino phase was found to be optimal for aromatic hydrocarbons (Chmielowiec and George, 1980). 

Ultrasphere Cis column acetonitrile in water (v/v) Baweja R, et al. (1987) Application of reversed-phase HPLC for the separation of deuterium and hydrogen analogues of aromatic hydrocarbons. Analytica Chimica Acta 192 345-348. 

The nonsteric interactions in ipc depend on the chemical structure of the analyte, and also on nature of stationary and mobile phases. In normal- or reversed-phase hplc, neutral solutes are separated on the basis of their polarity. In the former case, polar stationary phases are employed (eg, bare sihca with polar silanol groups) and less polar mobile phases based on nonpolar hydrocarbons are used for elution of the analytes. Solvent selectivity is controlled by adding a small amoimt of a more polar solvent, such as 2-propanol or acetonitrile or other additives with large dipole moments (methylene chloride and 1,2-dichloroethane), proton donors (chloroform, ethyl acetate, and water), or proton acceptors (alcohols, ethers, and amines). Correspondingly, the more polar the solute, the greater is its retention on the column, yet increasing the polarity of the mobile phase results in decreased solute retention. 

The first hydrocarbon described from the B race was a C34 compound called botryococcene (31 one of the most widespread members of the botryococcene family as demonstrated by subsequent studies. Due to its large predominance in the total hydrocarbon fraction isolated from some strains, ca 90%, no additional purification was needed for identification of the C34 compound. This simple case, however, occurs rarely and isolation of pure or at least enriched fractions of the other botryococcenes was required. This purification was obtained by reversed-phase HPLC using analytical columns and by repeated injections of botryococcene mixtures various eluents such as acetone-acetonitrile 2 3 (52), acetonitrile (55) and acetone-methanol 3 2 were used 34). This technique was however ineffective for separation of hydrocarbons exhibiting only regio- and (or) stereo-isomerism of double bonds. 

Silica can be used as is for normal phase HPLC in which the mobile phase is nonpolar in nature, such as hex-ane/chloroform and the column-packing surface is polar. For reversed-phase HPLC in which the mobile phase is polar, such as water or methanol, and the column-packing surface is nonpolar, the silica must be chemically altered. Silanization reactions are carried out to covalently attach long chain hydrocarbon groups to the silica surface. Further details of the importance of silica supports are provided in Section III, Separation Techniques. ... 

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