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Chromatographic Separations

Paper type: Essay
Pages: 3 (673 words)
Categories: Biology, Experiment, Science
Downloads: 17
Views: 325

Hemolymph samples were obtained from chilled, surface-sterilized two-day-old 5th instar larvae of Mythimna separata that had been deprived of food for 4 hours [22]. After puncturing the propterothoracic membrane at the base of the coxa of the metathoracic leg, hemolymph was drawn from the hemocoel and collected in a 10-fold volume TNM-FH medium containing 1.0 mM phenylthiourea. Then, the anti-cancer QDP and the pesticide EMB were added to the medium until the final concentrations were 0, 2.5, 5, 10 and 20 ?g/mL to QDP, and 0, 2.

5, 5 and 10 ?g/mL to EMB, respectively. Followed by the incubation at 28 oC for 2 hours, the mediums were centrifuged (500 g, 4 °C, 3 min) and the supernatants were removed. The hemocyte pellets were washed twice with PBS (10 mM, pH6.8), re-suspended in 0.6 mL of 10% trifluoroacetic acid solution and sonicated with an ultrasound for three times of ultrasound, 20 seconds each time. The lysates were placed at 4 oC for 2 hours and then centrifuged (8,000 g, 4 °C, 10 min), the supernatant was taken and served as samples for organic acid detection.

Chromatographic conditions

Chromatographic separations were performed on an ICS-6000 instrument (Thermo Fisher Scientific, Waltham, MA, USA) composed of an EGC-KOH eluent generator system, a six-port valve, a column oven and DS-6 conductivity detector. The chromatographic column was IonPac®AS18 (250 ? 4.0 mm) having an anion-exchange layer functionalized with very hydrophilic quaternary ammonium groups, that was safeguarded with IonPac® AG18 (4 ? 50 mm) guard column. The mobile phase was potassium hydroxide gradient where the concentration was 8 mM from zero to 30 min, 26 mM from 30.1 to 42 min and 8 mM from 42.1 to 45 min. Mobile phase was delivered by an Eluent Generator which automatically produces the concentration of potassium hydroxide from deionized water. The flow rate of the mobile phase was set at 1.0 mL/min. The injection volume was 25 ?L. The samples were diluted 10 times with Milli-Q water. After being filtrated by using 0.22 micron filter, the samples were put into injection bottles and injected into the IC instrument.

 Method validation

During method optimization, all chromatographic parameters were found to prove specificity, precision, linearity and accuracy for citrate, ?-ketoglutarate, fumarate and oxalate anions. For specificity, the developed ion-exchange chromatographic method was established in the presence of 4 anions and 4 detected organic ions, namely chloride, carbonate ion (CO32-), tartrate, citrate, isocitrate, ?-ketoglutarate, fumarate and oxalate. For precision, the method was performed on 6 different preparations of the test sample. The percentage relative standard deviation of the content of all 4 anions in the 6 preparations was calculated. The instrument precision was also performed on 6 repeated injections of one standard solution; thereafter the percentage of relative standard deviation (RSD) was calculated.

For linearity, the method was checked at 5 concentration levels: from 0.1 to 5 mg/L of citrate, from 0.1 to 5 mg/L of ?-ketoglutarate, from 0.02 to 1 mg/L of fumarate and from 0.1 to 2 mg/L of oxalate. The calibration curve was drawn by plotting the peak areas of all 4 organic anions against the corresponding concentrations. The correlation coefficients of the regression lines of the calibration curves were calculated, and the limit of detection (LOD) and the limit of quantitation (LOQ) for all 4 acids were also calculated.

For accuracy, the amount of added analytes was 50%, 100%, and 150% of the analytes concentration in the samples. Accuracy was calculated as the percentage of recovery by the assay of the known added amount of analyte in the sample. Theoretical concentrations of the anions in their different prepared samples were injected in triplicate at each level. The % recoveries and % RSD of all 4 acids were calculated.

Statistical analysis

Data analysis was performed using the Thermo Scientific™ Chromeleon™ 7.2 Chromatography Data System software. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) post hoc test analysis be tween the treatment groups to the control. Statistical probability (p) at 0.01 was considered significant.

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Chromatographic Separations. (2019, Dec 12). Retrieved from https://studymoose.com/chromatographic-separations-essay

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