Reversed-phase liquid chromatography aqueous samples

Ogan, K., Katz, E., and Slavin, W., Determination of polycyclic aromatic hydrocarbons in aqueous samples by reversed-phase liquid chromatography. Anal. Chem., 51, 1315, 1979. 

Application of Prep-HPLC to PGS Analysis. Reverse phase liquid chromatography has proven to be well suited for cleanup of plant extracts by prep-HPLC (4, 46, 47, 48). When the mobile phase is initially an aqueous buffer at pH 2.8, all but the highly charged (e.g., zeatin ribotide with 5 AMP used as a representative compound for zeatin ribotide) plant hormones are retained at the head of the column (Fig. 1). Since the PGS are retained, samples can be injected onto the column in a dilute form. In-... 

Reversed phase liquid chromatography was performed on a 150 mm x 3.0 mm Waters Symmetry C-8 column at 30 °C and a flow rate of 1.0 mL/min. The solvent phase was acetonitrile tetrahydrofuran 0.1 % aqueous phosphoric acid (50.4 21.6 28v/v/v). Under these operating conditions, most of the UV absorbance occurred as a peak at 3.83 minutes for all samples. Chromatograms of all samples had some small peaks (presumably the more polar compounds) eluting prior to the major peak. Product C presented a small but significant UV absorbing peak that eluted after the peak at 3.83 minutes. 

The focus is on concepts in reversed-phase liquid chromatography (RPLC), though the same concepts are usually applicable to other modes of HPLC. International Union of Pure and Applied Chemistry (IUPAC)10 nomenclature is used. The term sample component is often used interchangeably with analyte and solute in the context of this book. As mentioned in Chapter 1, the most common stationary phase is a hydrophobic C18-bonded phase on a silica support used with a mixed organic and aqueous mobile phase. The terms packing and sorbent often refer to the bonded phase whereas solid support refers to the unbonded silica material. 

A molecularly imprinted column for liquid chromatography can be used not only to separate analytes, but also to selectively extract analytes from complex samples. This technique is called on-line Molecularly Imprinted Solid Phase Extraction (on-line MISPE), and it combines the high extraction efficiency of reverse phase SPE for aqueous samples with the high selectivity of the molecular-imprinted polymers. Examples of successful selective extraction and clean-up are reported in Figs. 9 and 10. 

Parris NA. Non-aqueous reversed-phase liquid chromatography a neglected approach to the analysis of low polarity samples. J Chromatogr A 1978 157 161—70. 

Before considering the special requirements for automated on-line determination of metals from industrial effluents, it is worthwhile examining the features of standard laboratory procedures associated with the off-line determination of copper as a dithiocarbamate complex by liquid chromatography with electrochemical detection. The off-line determination of copper as its diethyldithiocarbamate complex in aqueous samples, zinc plant electrol3d e, and urine have been described [3, 7, 10] using reverse phase liquid chromatography with amperometric detection. A standard instrumental configuration for the conventional laboratory off-line method as used in these studies is depicted in Fig. 7.2. 

The method for chloroacetanilide soil metabolites in water determines concentrations of ethanesulfonic acid (ESA) and oxanilic acid (OXA) metabolites of alachlor, acetochlor, and metolachlor in surface water and groundwater samples by direct aqueous injection LC/MS/MS. After injection, compounds are separated by reversed-phase HPLC and introduced into the mass spectrometer with a TurboIonSpray atmospheric pressure ionization (API) interface. Using direct aqueous injection without prior SPE and/or concentration minimizes losses and greatly simplifies the analytical procedure. Standard addition experiments can be used to check for matrix effects. With multiple-reaction monitoring in the negative electrospray ionization mode, LC/MS/MS provides superior specificity and sensitivity compared with conventional liquid chromatography/mass spectrometry (LC/MS) or liquid chromatography/ultraviolet detection (LC/UV), and the need for a confirmatory method is eliminated. In summary,... 

Despite its potential importance, formic acid has proven difficult to quantify at submicromolar levels in non-saline water samples. Formidable analytical difficulties are associated with its detection in highly saline samples. Ion exclusion, anion exchange, and reversed-phase high performance liquid chromatography techniques based on the direct detection of formic acid in aqueous samples are prone to interferences (especially from inorganic salts) that ultimately limit the sensitivity of these methods. 

As a consequence of the previous considerations Kieber et al. [75] have developed an enzymic method to quantify formic acid in non-saline water samples at sub-micromolar concentrations. The method is based on the oxidation of formate by formate dehydrogenase with corresponding reduction of /3-nicotinamide adenine dinucleotide (j6-NAD+) to reduced -NAD+(/3-NADH) jS-NADH is quantified by reversed-phase high performance liquid chromatography with fluorimetric detection. An important feature of this method is that the enzymic reaction occurs directly in aqueous media, even seawater, and does not require sample pre-treatment other than simple filtration. The reaction proceeds at room temperature at a slightly alkaline pH (7.5-8.5), and is specific for formate with a detection limit of 0.5 im (SIN = 4) for a 200 xl injection. The precision of the method was 4.6% relative standard deviation (n = 6) for a 0.6 xM standard addition of formate to Sargasso seawater. Average re-... 

According to the modified procedure (602), milk is thoroughly mixed in its storage container immediately before transfer of the 1 ml aliquot in the extraction tube. This is necessary because approximately 50% of phenylbutazone in milk is associated with the cream. The sample is extracted with 2.4 ml diethyl ether and 2.4 ml petroleum ether in presence of 1 ml ethanol and 100 1 25% ammonia solution. The organic layer that contains the milk lipids is discarded. Five ml hexane-tetrahydro furan (4 1) is added to the aqueous layer, which is tiien acidified with hydrochloric acid and the layers are mixed. Under the acidic conditions, phenylbutazone partitions quantitatively into tlie organic layer, which is collected, evaporated, and dissolved in the mobile phase to be analyzed by liquid chromatography. Separation is performed on a reversed-phase column using an isocratic 0.02 M phosphate buffer/methanol mobile phase, and determination is by ultraviolet detection at 264 nm (Fig. 29.18.2). The limit of detection and limit of quantification were 3.0 and 5.4 ppb, respectively (Table 29.17). 

Liquid-liquid partitioning is a convenient and often economical method for bioseparations. L. Gu (personal communication, 1999) has shown that an acetonitrile-water system can be used for separation of proteins. This system partitions into two phases under subzero temperatures with the top phase containing more acetonitrile and water. The low temperature and the presence of water in both phases help reduce protein denaturation. An added advantage is that sample solution can be directly applied to reversed-phase high-performance liquid chromatography (HPLC) for further purification. Aqueous liquid-liquid partitioning is likely to remain an attractive choice for the separation of proteins, and exploration of new systems will continue. 

In normal-phase chromatography, the retention is governed by the interaction of the polar parts of the stationary phase and solute. For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample. Therefore, the stationary phase is usually silica and typical mobile phases for normal phase chromatography are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these. In reverse phase the packing is nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as Ci8, Q, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water. In the strictest interpretation, normal and reverse phase are terms which only relate to the polarity of the column and mobile phase with respect to the sample as shown in Table 3-3 and drawn schematically in Figure 3-14. 

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