Giorgio Raspi

Separation and determination of denatured αs1-, αs2-, β- and κ-caseins by hydrophobic interaction chromatography in cows’, ewes’ and goats’ milk, milk mixtures and cheeses

Abstract Caseins α s1 -, α s2 -, β- and κ- from raw cows’, ewes’ and goats’ milk were separated and determined by hydrophobic interaction chromatography (HIC) by using a Propyl column (Eichrom) in the presence of 8.0 M urea in the mobile phase. The method is based on fast and easy solubilization of real raw samples by 4.0 M guanidine thiocyanate followed by the HIC analysis, without any preliminary precipitation or separation of the casein fraction. Elution conditions have been optimized by analyzing commercial single bovine standard caseins and their mixture. In the optimized chromatographic conditions the four casein fractions were separated in less than 45 min. A linear relationship between the concentration of casein and peak area (UV absorbance detector at 280 nm) has been obtained over the concentration range of 0.5 to 40 μ M . The detection limit for α-, β- and κ-caseins ranged between 0.35 and 0.70 μ M . The precision of the method was evaluated, the coefficient of variation for α-, β- and κ-casein determination ranging between 3.0 and 6.0%. The method has been validated by the analysis of reference skim milk powder (BCR-063R) certificated for total nitrogen content. The method was applied to commercial casein mixture and to the qualitative and quantitative analysis of casein fractions in unprocessed, raw cows’, goats’ and ewes’ milk (10 samples analyzed for each species), in one sample of unprocessed buffalos’ milk and in commercial cheeses (mozzarella, robiola, ricotta and stracchino). Binary mixtures of milk (cow/goat and cow/ewe) were also analyzed and the ratio between casein peak areas (α s1 /κ, α s2 /β, β/κ and α s2 /α s1 ) of the HIC chromatograms was proposed and discussed in order to evaluate a possible application of this method to detect milk adulteration.

Mercury speciation by liquid chromatography coupled with on-line chemical vapour generation and atomic fluorescence spectrometric detection (LC-CVGAFS)

Reverse phase chromatography (RPC) coupled on-line with UV-vis diode array detector (DAD) and cold vapour generation atomic fluorescence spectrometry (CVGAFS) is proposed for the speciation and determination of inorganic and organic mercury (methylmercury, ethylmercury and phenylmercury) in the form of cysteine, penicillamine and glutathione complexes. The mercury-thiol complexes are separated on a C(18) Reverse Phase column and oxidized on-line with bromine, generated in situ by KBr/KBrO(3) in HCl medium, in order to fast convert organic mercury species to inorganic mercury in less than 2.5s, at room temperature, in a 30cm knitted coil. Hg(II) is selectively detected by AFS in a Ar/H(2) miniaturized flame after sodium borohydride reduction to Hg(0). Under optimized conditions, on-line bromine treatment gives recoveries of thiol-complexed methylmercury, ethylmercury and phenylmercury with respect to inorganic mercury ranging between 79 and 85%, 80 and 85%, 63 and 76%, respectively, depending on the complexing thiol employed. Optimized elution conditions were provided in the three complexing agents. The detection limits (LODc) for inorganic mercury, methylmercury, ethylmercury and phenylmercury, in the optimized conditions complexed with thiols were about 16, 18, 18 and 20pg (as mercury), respectively, a relative standard deviation (R.S.D) ranging between 1.5 and 2.0%, and a linear dynamic range between 0.1 and 100ng injected. LC-DAD-CVGAFS method has been validated by analysing two certificate reference material, DORM-2 and NIES CRM 13, obtaining 98+/-6 and 97+/-5% of methylmercury recovered, respectively.

Selective determination of thiolic proteins by hydrophobic interaction chromatography coupled with on-line cold vapour atomic fluorescence spectrometry

A new analytical method is proposed for the determination and characterization of thiolic proteins, based on hydrophobic interaction chromatography (HIC) coupled on-line with cold vapour atomic fluorescence spectrometry (CVAFS). Thiolic groups are derivatized pre-column by p-hydroxymercurybenzoate (PHMB) and the derivatized proteins are separated on a TSKgel Ether-5PW column. Post-column on-line reaction of derivatized proteins with bromine, generated in situ by KBr/KBrO3 in HCl medium, allowed the fast conversion of protein-bound PHMB to inorganic mercury, Hg(II), which is selectively detected by AFS after sodium borohydride reduction to Hg0. Under optimized conditions, on-line bromine treatment gives a 85 ± 2% recovery of both free and protein-complexed PHMB in less than 2.5xa0s and at room temperature. Glyceraldehyde-3-phosphate dehydrogenase, aldolase, pyruvate kinase, trioso phosphate isomerase and phospho-glucose isomerase have been examined. Sensitivity and limit of detection of proteins depends on the number of –SH groups reacting with PHMB and are in the range of 10−8–10−9xa0molxa0dm−3 with calibration curves spanning over four decades of concentration.

Hydrophobic interaction chromatography coupled with atomic fluorescence spectrometric detection: Effect of the denaturation on the determination of thiolic proteins

Hydrophobic interaction chromatography coupled online with chemical vapour atomic fluorescence spectrometry (HIC-CVGAFS) has been optimized for the analysis of thiolic proteins in denaturing conditions. Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0moldm(-3) urea and p-hydroxymercurybenzoate (PHMB) and the derivatized denatured proteins are separated on a silica HIC Eichrom Propyl column in the presence of 8.0M urea in the mobile phase. Post-column online reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO(3) in HCl medium, allowed the fast conversion of the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury also in presence of urea. Hg(2+), present in solution as Hg(2+)-urea complex, is selectively detected by AFS in a Ar/H(2) miniaturized flame after sodium borohydride reduction to Hg. Under optimized conditions, online bromine treatment gives a 100+/-2% recovery of both free and protein-complexed PHMB. Denatured glyceraldehyde-3-phosphate dehydrogenase, aldolase, lactate dehydrogenase, trioso phosphate isomerase and beta-lactoglobulin have been examined. As the sensitivity and limit of detection of proteins in the HIC-CVGAFS apparatus depends on number of SH groups reacting with PHMB, the denaturation process, which increases the number of PHMB-reactive thiolic groups in proteins, improves the analytical performances of the described system in protein analysis. The detection limit for the denatured proteins examined was found in the range of 10(-10)-10(-12)moldm(-3), depending on the considered protein, with linear calibration curves spanning over four decades of concentration.