Claudio Corradini

High-performance immobilized-metal affinity chromatography of proteins of iminodiacetic acid silica-based bonded phases

High-performance separations of proteins, based on immobilized-metal affinity chromatography (HPIMAC), are described. The stationary phase consisted of iminodiacetic acid (IDA) chelate groups, bonded to small particle, wide pore silica gel by means of a polyether hydrophilic leash. After loading the column with metal, retention of proteins was achieved by protein-metal complexation at high concentrations of sodium sulfate. Elution was accomplished by addition of competitive complexing ligands, such as ammonia at constant pH, and/or by a decreasing pH gradient of a specially designed buffer system to maintain buffer capacity constant throughout the gradient. Selective separations, based on differences in the number of histidine residues present on the surface of the proteins, are described. The application of HPIMAC in the separation and purification of structurally similar proteins is presented. The potential application of IDA columns in three chromatographic modes (HPIMA, hydrophobic interaction, and cation exchange) is also described.

Electrophoresis of proteins in uncoated capillaries with amines and amino sugars as electrolyte additives

Abstract The effect of triethylamine and triethanolamine in the running electrolyte on the electroosmotic flow and the migration of four standard basic proteins in bare fused-silica capillaries was examined. At pH 2.5 the direction of the electroosmotic flow was anodal with both additives and at constant ionic strength its magnitude increased with increasing additive concentration. The observations are in qualitative agreement with a theoretical model which is based on the Gouy—Chapman—Stern theory and incorporates a Langmuir-type relationship and also the Von Smoluchowski expression for the electroosmotic mobility, with the approximation to identify the zeta potential with the potential at the Stern plane. From the independence of the electrophoretic mobilities of the additive concentration and type the absence of interactions between the four basic proteins and the two alkylamines is inferred. The utility of glucosamine and galactosamine as additives for the capillary electrophoresis of basic proteins is also demonstrated and their effectiveness is compared with that of the alkylamines.

Effects of alkylamines on electroosmotic flow and protein migration behaviour in capillary electrophoresis

This paper reports the use of four closely related alkylamines as running electrolyte additives in capillary electrophoresis that permit the control of electroosmotic flow and protein migration behaviour in uncoated capillaries. At pH 2.5 the direction of the electroosmotic flow was anodic with all additives and at constant ionic strength its magnitude increased with increasing alkylamine concentration. The observations are in qualitative agreement with a previous reported theoretical model that correlates the electroosmotic mobility with the charge density in the Stern region of the electric double layer, arising from the adsorption of the additive, and the charge density at the capillary wall due to dissociation of silanols.

Separation of alditols of interest in food products by high-performance anion-exchange chromatography with pulsed amperometric detection

High-performance anion-exchange chromatography (HPAEC)-pulsed amperometric detection (PAD) employing a CarboPac MA 1 column was investigated with respect to mobile phase composition, linear response characteristics, repeatability, reproducibility and sensitivity with different alditols used as sugar substitutes in food and confectionery products. The energy-reduced bulk sweeteners isomalt and maltitol were well resolved in less than 25 min by isocratic elution with 600 mM sodium hydroxide solution. HPAEC-PAD was also successfully applied to the determination of alditols in sugar-free products and a low-calorie sweetener containing sorbitol, mannitol and fructose at different levels.

Maillard reaction in milk-based foods : Nutritional consequences

Chemical reactions occurring during industrial treatments or storage foods can lead to the formation of epsilon-deoxyketosyl compounds, the Amadori products. Food protein value can be adversely affected by these reactions, and in particular lysine, an essential amino acid having on its side chain a free amino group, can be converted to nonbioavailable N-substituted lysine or blocked lysine. by acid hydrolysis of epsilon-deoxyketosyl compounds, furosine is formed. In this paper furosine prepared from milk-based commercial products has been evaluated by use of a recently developed HPLC method using a microbore column and phosphate buffer as the mobile phase at controlled temperature. Furosine levels have been used, together with protein, total amino acids, and lysine content, as an estimate of protein quality of a few different products such as cooked-cream dessert, yogurt mousse, white chocolate, milk chocolate, milk chocolate with a soft nougat and caramel center, milk chocolate with a whipped white center, chocolate spread, part-skim milk tablets, milk-based dietetic meals, and baby foods. The protein content of the analyzed products ranged from 34.3 gxkg(-1) (milk nougat) to 188.4 g x kg(-1) (milk tablets). The Maillard reaction caused a loss in available lysine that varied from 2.5% (cooked cream) to 36.2% (condensed milk). The contribution to the lysine average daily requirement is heavily affected by this reaction and varied from 13% (milk tablets and soft nougat) to 61% (dietetic meal). Variable results were also obtained for the other essential amino acids.

High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection of Nutritionally Significant Carbohydrates

Abstract The separation of nutritionally significant saccharides in fruit juices was investigated by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD}. The effect of sodium hydroxide concentration in the mobile phase on the retention and selectivity was investigated on three different pellicular anion-exchange columns (all from Dionex). The sugar alcohol sorbitol and the sugars glucose, fructose, and sucrose were well resolved in less then 10 minutes on the Carbopac PA 100 column by using simple isocratic elation with 150 mM sodium hydroxide solution, without column regeneration between runs. Under these conditions the relative standard deviations of the retention times were better than 0.98%. HPAEC-PAD was successfully applied to the determination of the major sugars in a fruit concentrate and in several varieties of commercial fruit juices by an internal standard method without any sample pretreatment.

Separation and determination of 5-hydroxymethyl-2- furaldehyde and 2-furaldehyde in fruit juices by miceliar electrokinetic capillary chromatography with direct sample injection

The separation of 5-hydroxymethyl-2-furaldehyde (5-HMF) and 2-furaldehyde (2-FA), which are recognized indices of deteriorative changes in some commercially processed foods, was investigated by micellar electrokinetic capillary chromatography (MECC), employing sodium dodecyl sulphate as the anionic surfactant. The effects of micellar concentration, temperature and the addition of methanol or acetonitrile on the migration times and selectivity were investigated. MECC was successfully applied to the determination of 5-HMF and 2-FA in grapefruit juice by an internal standard method without any sample pretreatment.

Identification and dosage of 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde in beverages by reversed phase chromatography with a microbore column

Abstract This paper reports the results of a study performed to develop a rapid and straightforward chromatographic method for the identification and dosage of 5-hydroxymethyl-2-furaldehyde (HMF) and 2-furaldehyde (FA), which are recognized indices of deteriorative changes in commercially processed food. The method employs a Supelco microbore reversed phase column (300 × 1.0 mm I.D.), eluted isocratically with a 94:6 (v/v) water/acetonitrile mixture at flow rate of 60 μL/min. Sample is detected at 280 nm in a micro flow cell of 300 nl. Peak purity and identification is assessed by comparing the UV spectra monitored at two points through the chromatographic peak in continuous flow mode in the range from 200 to 400 nm. This method is successfully applied to the identification and quantitative determination of HMF and FA in alcoholic and non-alcoholic beverages by an internal standard method without any sample pretreatment.

DETERMINATION OF FUROSINE IN HYDROLYZATE OF PROCESSED MILK BY HPLC USING A NARROW BORE COLUMN AND DIODE-ARRAY DETECTOR

A simple, rapid, sensitive, reproducible, and accurate HPLC procedure employing a narrow bore reversed phase column and a diode-array detector is proposed for the determination of ϵ-N-2-furoylmethyl-L-lysine (furosine) in acid hydrolyzed of milk and powdered milk samples. After optimization of the variables involved, the method was characterized and validated in terms of selectivity, repeatability, sensitivity, and accuracy and applied to determine furosine content in hydrolyzed milk samples.

Analysis of ∊-N-2-Furoylmethyl-L-lysine (Furosine®) in Concentrated Milk by Reversed Phase Chromatography with A Microbore Column

Abstract This paper reports the results of a study aimed to develop a rapid, accurate and sensitive HPLC method for routine analysis of ∊-N-2-furoylmethyl-L-lysine (furosine) in acid hydrolyzed of processed food products. The method uses a microbore reversed phase HPLC column packed with a polymer coated silica-based octadecyl sorbent (5 μm, 250 × 1 mm I.D) eluted with a mobile phase consisting of 20 mM phosphate buffer, at pH 2.5. The identification of furosine was performed by UV detection at 280 nm in a micro flow cell of 300 nL. The limit of detection was 0.3 ng based on a signal to noise ratio of 3 with a sample volume of 0.5 μL. The separation was achieved by isocratic elution within 15 min. This method was successfully applied to the identification and quantitative determination of furosine in a sample of concentrated milk hydrolyzed with hydrochloric acid. Dosage was performed by the method of external standard.