Analysis of plant analytes using capillary electrophoresis and high performance liquid chromatography

Plants contain an enormous array of organic and inorganic components, the analysis for which may involve a wide range of methods. The focus of this study was to develop high performance liquid chromatography and capillary electrophoresis methods for the analysis of three classes of analytes: osmoregulants, minerals and amino acids. Firstly, this study explored the potential of capillary electrophoresis for the analysis of three very common osmoregulants (proline, glycine betaine and mannitol). A diverse array of methods has been reported for determining each of these analytes, however, the literature on osmoregulants and their analysis is quite disjointed and traverses both biological and chemistry fields. Therefore, a comprehensive review of this literature has been completed (Chapter 2). Considerably fewer methods are available for the simultaneous determination of these osmoregulants, compared to individual analysis. In chapter 3, a method is described for the simultaneous analysis of proline and betaine by capillary electrophoresis at low pH and specifically various cationic probes for the indirect detection of proline and betaine were explored. Sulfanilamide was identified as a suitable probe and was employed to quantify proline and betaine in spinach and beetroot. However, this method could not detect mannitol as it is not charged at low pH. In Chapter 4, a high performance liquid chromatography method for the simultaneous determination of all three osmoregulants is described. For separation, a NH2 column with formic acid and acetonitrile as the mobile phase were used. The high performance liquid chromatography evaporative light scattering detection method was applied to determine osmoregulants in Stylosanthes guianensis, Atriplex cinerea and Rhagodia baccata plant extracts. A complementary method, using a C18 column with heptafluorobutyric acid added to acetonitrile was used for verification of the analytes. Secondly, the potential for using capillary electrophoresis was investigated to simplify and shorten the complex sample preparation procedure. Chapter 5 describes a capillary electrophoresis method that allows direct injection from plant tissues. The experiments highlighted that uncontrolled hydrodynamic injection of sample on piercing of food sample resulted in non-reproducibility. The addition of hydroxypropylmethlycellulose to the background reduced the uncontrolled hydrodynamic injection up to 95% for all of the analytes. The sample was injected electrokinetically and an imidazole buffer consisting of hydroxypropylmethlycellulose was used for separation. The issue of reducing the reliance on prior separation is also relevant to minerals, thus the developed capillary zone electrophoresis-UV method was applied for the direct injection of inorganic cations from apple, mushroom, zucchini, green bean and strawberries. The applicability of the method across fruit varieties was determined by analysing four apple varieties including red delicious, fuji, pink lady and royal gala. Thirdly, the potential of the direct injection method was explored for the analysis of amino acids in zucchini. As amino acids are present at low concentrations and lack a chromophore, a more sensitive detector, capacitively coupled contactless conductivity, and pre-concentration of amino acids using isotachophoresis (leading electrolyte = HCl, terminating electrolyte = hydroxyproline) was performed. The separation of amino acids was carried using acetic acid. For minimising uncontrolled hydrodynamic injection poly(ethylene oxide) was used. Using this method sensitive detection of amino acids was possible (Chapter 6). In short, the developed methods allow for quick, inexpensive, sensitive and efficient analysis of plant components

Preoperative Diabetes Mellitus in individuals Undergoing Surgery

Objective: Level of glycaemic management is critical in a surgical patient. The current research will manage in the surgical patients the diabetes mellitus.Study Design: Cross Sectional Study place and duration: The current research was carried out from January 2018 to March 2018 at Outpatient Department of Medicine at Lahore General Hospital, Lahore, Pakistan.Materials and Methods: There were 200 participants of the study. There were 100 men and 100 women. From all participants the informed consent was obtained, a Proforma questionnaire with previous history, investigation and clinical examination was completed for carrying out the research. The study included all patients who are newly diagnosed with diabetes and those Diabetes Mellitus patient having surgical disease. The study excluded diabetic patients not with surgical disease and patients without diabetes but have surgical disease.Results: The research was carried out on 200 participants. The age was ranging between 20-60 years of age and the mean age was 34.63 ± 6.3. All the participants were newly diagnosed patients with diabetes or diabetic patients. HbA1C ranged was from 5.60-10 with mean 8.1 ± 0.45. The range for Random Blood Sugar was 139-510 mg/dl with mean 281.1 ± 77. Fasting Blood Sugar was ranging from 121-231 mg/dl with mean 129.12 ± 24. All the participants were on insulin therapy after the examination and discontinuation of oral hypoglycaemic drug in order to avoid surgery complication.Conclusion: Pre and Post surgery the main problem is the uncontrolled Diabetes Mellitus, the complications can be lessened with the close communication between surgeon, physician and anaesthetic staff. The controlling of glycaemic before operation and blood glucose after operation are important for lessening the complication. In order to perform a better surgery, electrolyte status and acid base balance must be monitored.Key Words: anaesthesia, Diabetes Mellitus Surgery, Glycosylated haemoglobin, Insulin, Oral hypoglycaemic agents, Preoperative care

Preoperative Diabetes Mellitus in individuals Undergoing Surgery

Objective: Level of glycaemic management is critical in a surgical patient. The current research will manage in the surgical patients the diabetes mellitus.Study Design: Cross Sectional Study place and duration: The current research was carried out from January 2018 to March 2018 at Outpatient Department of Medicine at Lahore General Hospital, Lahore, Pakistan.Materials and Methods: There were 200 participants of the study. There were 100 men and 100 women. From all participants the informed consent was obtained, a Proforma questionnaire with previous history, investigation and clinical examination was completed for carrying out the research. The study included all patients who are newly diagnosed with diabetes and those Diabetes Mellitus patient having surgical disease. The study excluded diabetic patients not with surgical disease and patients without diabetes but have surgical disease.Results: The research was carried out on 200 participants. The age was ranging between 20-60 years of age and the mean age was 34.63 ± 6.3. All the participants were newly diagnosed patients with diabetes or diabetic patients. HbA1C ranged was from 5.60-10 with mean 8.1 ± 0.45. The range for Random Blood Sugar was 139-510 mg/dl with mean 281.1 ± 77. Fasting Blood Sugar was ranging from 121-231 mg/dl with mean 129.12 ± 24. All the participants were on insulin therapy after the examination and discontinuation of oral hypoglycaemic drug in order to avoid surgery complication.Conclusion: Pre and Post surgery the main problem is the uncontrolled Diabetes Mellitus, the complications can be lessened with the close communication between surgeon, physician and anaesthetic staff. The controlling of glycaemic before operation and blood glucose after operation are important for lessening the complication. In order to perform a better surgery, electrolyte status and acid base balance must be monitored.Key Words: anaesthesia, Diabetes Mellitus Surgery, Glycosylated haemoglobin, Insulin, Oral hypoglycaemic agents, Preoperative care

Comparative efficacy of locally isolated fungal strains for Pb(II) removal and recovery from water

Abstract The present investigation aimed to study and compare the efficiency of non-viable fungal isolates to remove divalent lead (Pb(II)) from aqueous streams. The selected fungal isolates showed identity with Aspergillus caespitosus, Aureobasidium sp. RBSS-303 and Aspergillus flavus HF5 as confirmed using gene sequencing of ITS regions of the ribosomal DNA (rDNA). The obtained equilibrium data for Pb(II) biosorption of A. caespitosus fitted better to Langmuir isotherm with maximum sorption capacity of 351.0 mg/g and A. sp. RBSS-303 and A. flavus HF5 showed good fit to Freundlich isotherm with maximum sorption capacity of 271.5 and 346.3 mg/g respectively. The values of thermodynamic factors ascertained the nature of adsorption process is endothermic with A. caespitosus and A. flavus HF5 but exothermic with A. sp. RBSS-303. The experimental data for Pb(II) biosorption fits very well to pseudo second order kinetic model. With HCl the maximum 85.5, 75.3, 73.7% recovery of Pb(II) was obtained from A. caespitosus, A. sp. RBSS-303 and A. flavus HF5, respectively. The observed percentage loss in sorption capacity of Pb(II) was 3.9% by A. flavus HF5, 12.2% by A. caespitosus and 26.6% by A. sp. RBSS-303 after five cyclic studies of sorption and desorption. Results from the study confirmed the efficiency order of A. caespitosus > A. flavus HF5 > A. sp. RBSS-303 to remove and recover Pb(II) from aqueous solution. Finally, the fungal biosorbents can be used as soil conditioning agent after compositing into valuables fungal protein

A review of extraction and analysis: Methods for studying osmoregulants in plants

Compatible osmolytes are substances produced by plants exposed to stressful environmental conditions. These protect plants during stress by performing several functions including scavenging of free radicals and maintenance of osmotic balance. The three most commonly examined include proline, mannitol and glycine betaine. Extraction and analysis of osmolytes are essential steps for a number of applications including estimation of the potential of new breeds, genetically engineered plants, and plants exposed to extreme environmental conditions. A wide range of extracting solvents have been used for the three key osmoregulants, and rarely is the selection of the extracting solvent experimentally determined. Furthermore, in many studies involving two or more osmolytes each is extracted individually. Similarly, there are a variety of methods reported for the quantification of these compounds. As with extraction, a separate method is often applied in quantifying each osmoregulant. Therefore, the aim of this review is to provide a detailed overview of the available methods for extraction and quantification of compatible osmolytes for the study of plant stress. Furthermore, the methods available for simultaneous extraction and quantification of key osmoregulants have been described

Evaluation of potential cationic probes for the detection of proline and betaine

Osmoregulants are the substances that help plants to tolerate environmental extremes such as salinity and drought. Proline and betaine are two of the most commonly studied osmoregulants. An indirect UV CE method has been developed for simultaneous determination of these osmoregulants. A variety of reported probes and compounds were examined as potential probes for the indirect detection of proline and betaine. Mobility and UV-absorption properties highlighted sulfanilamide as a potential probe for indirect analysis of proline and betaine. Using 5 mM sulfanilamide at pH 2.2 with UV detection at 254 nm, proline and betaine were separated in less than 15 min. The LODs for proline and betaine were 11.6 and 28.3 μM, respectively. The developed method was successfully applied to quantification of these two osmoregulants in spinach and beetroot samples

Mechanistic study of a diazo dye degradation by Soybean Peroxidase

BACKGROUND: Enzyme based remediation of wastewater is emerging as a novel, efficient and environmentally-friendlier approach. However, studies showing detailed mechanisms of enzyme mediated degradation of organic pollutants are not widely published. RESULTS: The present report describes a detailed study on the use of Soybean Peroxidase to efficiently degrade Trypan Blue, a diazo dye. In addition to examining various parameters that can affect the dye degradation ability of the enzyme, such as enzyme and H(2)O(2) concentration, reaction pH and temperature, we carried out a detailed mechanistic study of Trypan Blue degradation. HPLC-DAD and LC-MS/MS studies were carried out to confirm dye degradation and analyze the intermediate metabolites and develop a detailed mechanistic dye degradation pathway. CONCLUSION: We report that Soybean peroxidase causes Trypan Blue degradation via symmetrical azo bond cleavage and subsequent radical-initiated ring opening of the metabolites. Interestingly, our results also show that no high molecular weight polymers were produced during the peroxidase-H(2)O(2) mediated degradation of the phenolic Trypan Blue

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2014–2016)

One of the most cited limitations of capillary (and microchip) electrophoresis is the poor sensitivity. This review continues to update this series of biennial reviews, first published in Electrophoresis in 2007, on developments in the field of on‐line/in‐line concentration methods in capillaries and microchips, covering the period July 2014–June 2016. It includes developments in the field of stacking, covering all methods from field amplified sample stacking and large volume sample stacking, through to isotachophoresis, dynamic pH junction, and sweeping. Attention is also given to on‐line or in‐line extraction methods that have been used for electrophoresis