What are the general steps in developing as isocratic separation for reversed-phase chromatography with one organic solvent and temperature as variable?

Short Answer

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General steps in developing as isocratic separation.

To develop an isocratic separation for reversed-phase chromatography with one organic solvent .

Step by step solution

01

definition of general steps

To develop an isocratic separation for reversed-phase chromatography with one organic solvent.

And temperature as variables we need to change %B and temperature (T) using this steps

02

following this steps

in run Ahigh %B and low T

in rub Bhigh %B and high T

in rub C low %B and high T

in rub D low %B and low T

Using the chromatograms, determine the conditions between runs A, B, C and D.

Hence,

To develop an isocratic separation for reversed-phase chromatography with one organic solvent and temperature as variables we need to change %B and temperature (T) using this steps

in run Ahigh %B and low T

in rub Bhigh %B and high T

in rub Clow %B and high T

in rub Dlow %B and low T

Using the chromatograms, determine the conditions between runs A, B, C and D.

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Chromatography–mass spectrometry. Cocaine metabolism in rats can be studied by injecting the drug and periodically with drawing blood to measure levels of metabolites by HPLC–mass spectrometry. For quantitative analysis, isotopically labelled internal standards are mixed with the blood sample. Blood was analysed by reversed-phase chromatography with an acidic eluent and atmospheric pressure chemical ionization mass spectrometry for detection. The mass spectrum of the collisionally activated dissociation products from the m/z 304 positive ion is shown in the figure on the next page. Selected reaction monitoring (m/z 304 from mass filter Q1 and m/z 182 from Q3 in Figure 22-33) gave a single chromatographic peak at 9.22 min for cocaine. The internal standard H52-cocaine gave a single peak at 9.19 min for m/z 309 (Q1) 182(Q3).

(a) Draw the structure of the ion at m/z 304.

(b) Suggest a structure for the ion at m/z 182.

(c) The intense peaks at m/z 182 and 304 do not have C2isotopic partners at m/z 183 and 305. Explain why.

(d) Rat plasma is exceedingly complex. Why does the chromatogram show just one clean peak?

(e) Given that H52-cocaine has only two major mass spectral peaks at m/z 309 and 182, which atoms are labelled with deuterium?

(f) Explain how you would use H52-cocaine for measuring cocaine in blood.

Spectrum for Problem 25-25.

Left: Mass spectrum of collisionally activated dissociation products from m/z 304 positive ion from atmospheric pressure chemical ionization mass spectrum of cocaine.

Right: Chromatograms obtained by selected reaction monitoring. [Data from G. Singh, V. Arora, P. T. Fenn, B. Mets, and I. A. Blair, “Isotope Dilution Liquid Chromatography Tandem Mass Spectrometry Assay for Trace Analysis of Cocaine and Its Metabolites in Plasma,” Anal. Chem. 1999, 71, 2021.]

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