Methods by Liquid-Chromatography

Column separation of proteins and peptides is used for both preparative and analytical purposes. Generally, in the former case low- or medium-pressure systems are preferred, whereas in the latter, high-performance-liquid-chromatography (HPLC) is the method of choice. These systems are coupled with a spectrophotometric detector normally set at wavelengths 280 and (around) 220 nm for proteins and peptides, respectively. Coupling of HPLC with mass spectrometry allows structural identification of compounds after the separation. 

The molecular characteristics exploited for the separation of proteins and peptides include molecular size (size exclusion chromatography, SEC), net charge (ion-exchange chromatography, IEC), polarity (reverse-phase chromatography, RPC), hydrophobicity (hydrophobic interaction 

SEC of grape and wine proteins has to be performed on samples deprived of phenolics because these compounds tend to bind irreversibly the stationary phase. Phenolic compounds are removed by passing the protein extract through a C18 cartridge (see above). As an alternative to removing the interfering substances, it is also possible to pre-treat the sample with small amounts of the same stationary phase used for SEC. 

Generally, the mobile phase used for SEC should have a pH similar to that of the wine, in order to avoid pH-dependent protein solubility changes. It should also contain some salt, typically NaCl (higher than OHM, Somers and Ziemelis, 1973), in order to prevent possible protein aggregation or adsorption on the stationary phase due to electrical interactions. 

In peptides analysis, SEC is generally used as a pre-fractionation step to remove high MW molecules such as proteins and polysaccharides. A Sephadex G10 column can be used to separate peptides in two fractions with MW lower and higher than 700 Da (Moreno-Arribas et al., 1998). 

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Li H, Kijak PJ, Turnipseed SB, Cui W, Analysis of veterinary drug residues in shrimp a multi-class method by liquid chromatography-quadrupole ion trap mass spectrometry, J. Chromatogr. B 2006 836(l-2) 22-38. 

Table 1.2 Structure of EN/ISO 15061 (2001). EN/ISO 15061 Water quality - Determination of dissolved bromate Method by liquid chromatography of ions...

ISO 15061, Water Quality—Determination of Dissolved Bromate—Method by Liquid Chromatography of Ions. 2001. 

Different parameters can be employed to eharacterize the extracted com germ oil such as physical parameters (e.g. refraetive index, density, color) and chemical parameters, including composition in terms of fatty acids profile by gas ehromatography (AOAC, 1995) and neutral lipids, that can be determined, among other analytical methods, by liquid chromatography (Schaefer et al., 2003). 

ASTM Standard E 755-90, "Standard Test Method for Assay of Dicumyl Peroxide by Liquid Chromatography," in Ref. 253. 

Farre M, Ferrer I, Ginebreda A, Figueras M, Olivella L, Tirapu L, Vilanova M, Barcelo D (2001) Determination of drugs in surface water and wastewater samples by liquid chromatography-mass spectrometry methods and preliminary results including toxicity studies with Vibrio fischeri. J Chromatogr A 938 187-197... 

A further extension of the DFG S19 method was achieved when polar analytes and those unsuitable for GC were determined by LC/MS or more preferably by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Triple-quadrupole MS/MS and ion trap MS" have become more affordable and acceptable in the recent past. These techniques provide multiple analyte methods by employing modes such as time segments, scan events or multiple injections. By improving the selectivity and sensitivity of detection after HPLC separation, the DFG S19 extraction and cleanup scheme can be applied to polar or high molecular weight analytes, and cleanup steps such as Si02 fractionation or even GPC become unnecessary. 

The enforcement methods provided by the applicants give basic information about appropriate cleanup steps and specific determination procedures. Typically, direct use of this developmental work occurred when a GC multi-residue method was found appropriate. Owing to the recent developments in the field of MS/MS with atmospheric pressure ionization, an alternative approach for those compounds that can be analyzed by liquid chromatography (LC) will soon be possible. It is important that some fundamental considerations for such method(s) should be agreed at the outset. Considerations include the most suitable extraction solvents and cleanup steps and some standard HPLC conditions. 

Ethylenethiourea (ETU) is a toxic decomposition product/metabolite of alky-lenebis(dithiocarbamates). This compound could be generated during processing of treated crops at elevated temperature. Different chromatographic methods to determine the residue levels of ETU have been published. After extraction with methanol, clean-up on a Gas-Chrom S/alumina column and derivatization (alkylation) with bro-mobutane, ETU residues can be determined by GC with a flame photometric detector in the sulfur mode. Alternatively, ETU residues can also be determined by an HPLC method with UV detection at 240 nm or by liquid chromatography/mass spectrometry (LC/MS) or liquid chromatography/tandem mass spectrometry (LC/MS/MS) (molecular ion m/z 103). ... 

Jelic A, Petrovic M, Barcelo D (2009) Multi-residue method for trace level determination of pharmaceuticals in solid samples using pressurized liquid extraction followed by liquid chromatography/quadrupole-linear ion trap mass spectrometry. Talanta 80 363-371... 

Related substances. Examine by liquid chromatography, as directed in general method (2.2.29). 

T.H. Kao and B.H. Chen, An improved method for determination of isoflavones in soybean powder by liquid chromatography. Chromatographia 56 (2002) 423 430. 

Di Corcia A, Marchetti M. 1992. Method development for monitoring pesticides in environmental waters liquid-solid extraction followed by liquid chromatography. Environ Sci Technol 26(1 ) 66- 74... 

Toyo oka T. 2002. Resolution of chiral drugs by liquid chromatography based upon diastereomer formation with chiral derivatization reagents. J Biochem Biophys Methods 54 25-56. 

Toyo oka T, Kashiwazaki T, Kato M. On-line screening methods for antioxidants scavenging superoxide anion radical and hydrogen peroxide by liquid chromatography with indirect chemiluminescence detection. Talanta. 2003 60 467-475. 

Analytical Methods. Various analytical methods involve titration with oxidants, eg, hexacyanoferrate (ferricyanide), which oxidize dithionites to sulfite. Iodimetric titration to sulfate in the presence of formaldehyde enables dithionite to be distinguished from sulfite because aldehyde adducts of sulfite are not oxidized by iodine. Reductive bleaching of dyes can be used to determine dithionite, the extent of reduction being determined photometrically. Methods for determining mixtures of dithionite, sulfite, and thiosulfates have been reviewed (365). Analysis of dithionite particularly for thiosulfate, a frequent and undesirable impurity, can be done easily by liquid chromatography (366). 

Tagliacozzi D, Mozzi AF, Casetta B, Bertucci P, Bernardini S, Di Ilio C, Urbani A, Federici G (2003) Quantitative analysis ofbile acids in human plasma by liquid chromatography-electrospray tandem mass spectrometry a simple and rapid one-step method. Clin Chem Lab Med 41 1633-1641... 

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