Simultaneous analysis of small organic acids and humic acids using high performance size exclusion chromatography

2012 ◽  
Vol 35 (24) ◽  
pp. 3455-3460 ◽  
Author(s):  
Xiaopeng Qin ◽  
Fei Liu ◽  
Guangcai Wang ◽  
Liping Weng
Keyword(s):  
Organic Acids ◽  
Size Exclusion Chromatography ◽  
Humic Acids ◽  
High Performance ◽  
Simultaneous Analysis ◽  
Size Exclusion ◽  
Exclusion Chromatography

scholarly journals High Performance Size Exclusion Chromatography-Inductively Coupled Plasma-Mass Spectrometry to Study the Copper and Cadmium Complexation with Humic Acids

Molecules ◽  
2019 ◽  
Vol 24 (17) ◽  
pp. 3201 ◽  
Author(s):  
Marta Radaelli ◽  
Elisa Scalabrin ◽  
Giuseppa Toscano ◽  
Gabriele Capodaglio
Keyword(s):  
Mass Spectrometry ◽  
Size Exclusion Chromatography ◽  
Humic Acids ◽  
High Performance ◽  
Size Exclusion ◽  
Inductively Coupled ◽  
Complexing Capacity ◽  
Icp Ms ◽  
Exclusion Chromatography ◽  
Detection Window

Dissolved organic matter (DOM) plays an important role in the environment by influencing the transport and distribution of organic and inorganic components through different processes: the retention, mobilization, and bio-availability of potentially toxic elements (PTEs). The aim of the present study is to examine the dimensional characterization of humic acids (HA) extracted from soil matrix, as well as to analyze the metal distribution among different ligand classes. The molecular size distribution of the HA extract from soil showed three dimensional classes: 52 KDa, 4.5 KDa, and 900 Da. HPSEC-ICP-MS measurements demonstrated that the dimensional classes, relative to first two fractions, bind the largest part of metals. The complexing capacity of HA was evaluated to assess the pollutants mobility in the environmental system. In particular, cadmium (Cd) and copper (Cu) complexation was investigated due to the great concern regarding their bio-availability and toxicity in natural waters. The complexing capacity of HA solution (20 mg/L) was measured by titration using a high-performance size exclusion chromatography (HP-SEC) coupled to an inductively coupled mass spectrometry (ICP-MS). Results obtained by this technique are compared with those obtained by anodic stripping voltammetry (ASV) to investigate the effects of kinetic lability of complexes on measurements carried by HPSEC-ICP-MS. In this study, results of ligand concentrations and stability constants obtained via the two techniques are assessed considering the detection window associated to the applied analytical methodology. Results obtained using the two analytical techniques showed that Cd is complexed by two classes of ligands. However, the ligand concentration values obtained using the two techniques are different, because the detection window associated to the two methodologies; the complexing capacity, which was obtained as sum of the two classes of ligands, were 33 nmol/L and 9 nmol/L for ASV and HPSEC-ICP-MS, respectively. The copper complexing capacities determined by the two methodologies are comparable: 166 and 139 nmol/L for ASV and HPSEC-ICP-MS, respectively. However, the results of Cu titration differ for the two techniques, highlighting only one class of ligands when ASV was used, and two classes when HPSEC-ICP-MS was employed. Differences on results obtained by the two techniques are explained considering the kinetic lability of complexes; the results show that, differently from previous studies, also Cu complexes can be kinetically labile, if one technique with high reaction time is used, as well some cadmium complexes are sufficient stable to be determined by HPSEC-ICP-MS.

scholarly journals Periplasmic synthesis and purification of the human prolactin antagonist Δ1-11-G129R-hPRL

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miriam F. Suzuki ◽  
Larissa A. Almeida ◽  
Stephanie A. Pomin ◽  
Felipe D. Silva ◽  
Renan P. Freire ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
High Performance ◽  
Signal Sequence ◽  
Osmotic Shock ◽  
Size Exclusion ◽  
Specific Expression ◽  
E Coli ◽  
Human Prolactin ◽  
Exclusion Chromatography

AbstractThe human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b( +) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 ± 0.015 μg/mL/A600 at a final optical density of 3.43 ± 0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF–MS. The final product presented > 95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.

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