IA 2 - HPLC at Hochschule Bonn-Rhein-Sieg

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Exemplary flashcards for IA 2 - HPLC at the Hochschule Bonn-Rhein-Sieg on StudySmarter:

What is liquid chromatography?

Exemplary flashcards for IA 2 - HPLC at the Hochschule Bonn-Rhein-Sieg on StudySmarter:

What are the differences between classical liquid chromatography and HPLC?

Exemplary flashcards for IA 2 - HPLC at the Hochschule Bonn-Rhein-Sieg on StudySmarter:

What is the principle of HPLC setup?

Exemplary flashcards for IA 2 - HPLC at the Hochschule Bonn-Rhein-Sieg on StudySmarter:

What are the building blocks of HPLC and what is their function?

Exemplary flashcards for IA 2 - HPLC at the Hochschule Bonn-Rhein-Sieg on StudySmarter:

What are important applications of HPLC?

Exemplary flashcards for IA 2 - HPLC at the Hochschule Bonn-Rhein-Sieg on StudySmarter:

Which requirements must a HPLC sample meet?

Exemplary flashcards for IA 2 - HPLC at the Hochschule Bonn-Rhein-Sieg on StudySmarter:

Why is the separation time of an analyte based on the time spent in the mobile and in the stationary phase ?

Exemplary flashcards for IA 2 - HPLC at the Hochschule Bonn-Rhein-Sieg on StudySmarter:

Peak height (h)

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Peak area (F)

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How can one determine if a single peak corresponds to more than one analyte?

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Retention factor k

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How can the retention factor be expressed in terms of retention times or retention volumes?

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Exemplary flashcards for IA 2 - HPLC at the Hochschule Bonn-Rhein-Sieg on StudySmarter:

IA 2 - HPLC

What is liquid chromatography?
- a solvent based analytical method to separate analytes - the solvent (mobile phase) containing the analyze is pumped through a chromatography column - the column is filled by a solid material (stationary phase) with a high surface (several 100 m2/g)

IA 2 - HPLC

What are the differences between classical liquid chromatography and HPLC?
Classical liquid chromatography (since 1906): - inner diameter of the column in cm range, column length: ca. 1 m - hydrostatic flow or low pressure pump - long analysis times (several hours) - soft packing material, column material: glass - Analyte amount: mg to g range HPLC (since 1970): - inner diameter of column in mm range, column length: up to 25 cm - high pressure pump (ca. 10-200 bar) - short analysis times (usually below 30 minutes) - rugged packing material, column material: steel - Analyte amount: pg to microgram range

IA 2 - HPLC

What is the principle of HPLC setup?
We have: - solvent reservoir - pump system - injection valve - column - detector

IA 2 - HPLC

What are the building blocks of HPLC and what is their function?
- Solvent reservoir and pump: provides constant solvent flow rate (ca. 0.5-2ml/min) - Injection valve: defined sample introduction (5-500microgram) - Separation column and oven: optimal resolution at reasonable pressure for extended life times - Detector: High sensitivity, large linear range of detector - Control unit: Adjustment of separation and instrument parameters

IA 2 - HPLC

What are important applications of HPLC?
- standard technique for purity and manufacturing control - Analysis of drug-like molecules in drug development - Analysis of toxins in the environmental sciences - Separation and isolation of biopolymers such as proteins and DNA - Drug analysis in the forensic sciences - Analysis in food chemistry

IA 2 - HPLC

Which requirements must a HPLC sample meet?
- sufficient solubility of mobile phase - particle free - sufficient stability - detectable using one of the standard detector types

IA 2 - HPLC

Why is the separation time of an analyte based on the time spent in the mobile and in the stationary phase ?
- All analytes spend the same amount of time in the mobile phase --> Tm or T0: dead time/mobile time - Analytes can only be separated if the spend different times in the stationary phase tR = t0 + tS tR: retention time (time spent from injection to detection) tS: time spent in the stationary phase tM/t0: time spent in the mobile phase

IA 2 - HPLC

Peak height (h)
= Distance between baseline and peak maximum

IA 2 - HPLC

Peak area (F)
= area between peak curve and baseline

IA 2 - HPLC

How can one determine if a single peak corresponds to more than one analyte?
- Change HPLC conditions (i.e. composition of mobile phase) - use various wavelengths of UV detector - use of different detector (i.e. fluorescence detector) - collect the mobile phase volume corresponding to the peak, evaporate the solvent, then analyze by spectroscopic methods

IA 2 - HPLC

Retention factor k
k = (tR-t0)/t0 - k is standardized to the dead time and thus independent on column dimensions - k allows to compare results based on different column dimensions (length, ID) and flow rate --> for an unretained analyte, the retention factor is equal to zero. For irreversibly bound analytes, the retention factor approaches infinity

IA 2 - HPLC

How can the retention factor be expressed in terms of retention times or retention volumes?
k = (tR-t0)/t0 and VR = tR * f --> k = (VR-V0)/V0 VR: retention volume of analyte (ml) V0: dead volume of column (ml) f = flow rate (ml/min)

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