Questions and Answers
What does HPLC stand for?
High-Performance Liquid Chromatography
Which of the following is NOT a type of chromatography?
What determines the retention time (Rt) in HPLC?
Stationary phase, mobile phase and its composition, column parameters, flow rate, column temperature.
What type of analysis can be performed with HPLC?
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Which type of HPLC column is made of silica?
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Name one application of HPLC in the pharmaceutical industry.
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The refractive index detector is the most sensitive type of HPLC detector.
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The ______ phase is used to separate compounds based on their affinity in HPLC.
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Study Notes
HPLC Overview
- High-Performance Liquid Chromatography (HPLC) separates, identifies, and quantifies components in mixtures.
- A liquid sample is passed through a column containing a stationary phase, where separation relies on interactions between sample components and the stationary phase.
- Chromatography was invented by M. Tsvet, based on the partition coefficient principle.
Basic Concepts
- Retention Time (Rt): Identifies compounds and is influenced by stationary phase, mobile phase, column parameters, and flow rate.
- Chromatogram: The area under the curve indicates the concentration and quantity of compounds.
Types of Chromatography
- By Mode: Normal Phase, Reverse Phase
- By Elution Technique: Isocratic separation, Gradient elution
- By Analysis Type: Qualitative, Quantitative
- By Scale: Analytical HPLC, Preparative HPLC
Separation Modes
- Includes Normal Phase, Reverse Phase, Ion Exchange, Gel Permeation, and Gel Filtration.
HPLC Components
- Mobile Phase: Carrier liquid for the sample.
- Key equipment: Degassing unit, Solvent delivery pump, Injector, Sample vials, Column, Detector, Drain/Fraction Collector.
HPLC Detectors
- UV-Visible Absorbance Detector: High sensitivity.
- Photodiode Array Detector: Scans wavelengths from 200 to 800 nm.
- Fluorescence Detector: Highly sensitive to fluorescent compounds.
- Refractive Index Detector: Universal but less sensitive.
- Evaporative Light Scattering Detector: Specific application.
- Mass Spectrometer: Advanced detection methods.
Applications of HPLC
- Pharmaceuticals: Assay of active pharmaceutical ingredients (API), content uniformity, and related substance analysis.
- Biopharmaceuticals: Peptide mapping, charge variant, glycan analysis, and size exclusion chromatography.
- Food Industry: Analysis of pesticides, vitamins, and amino acids.
- Chemical Industry: Qualitative and quantitative analysis of synthetic compounds.
- Environmental Analysis: Water and soil assessments.
Column Types
- Polar Columns: Made of silica for normal phase HPLC.
- Non-Polar Columns: Made of C18 for reverse phase HPLC.
Steps in HPLC Procedure
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Mobile Phase Preparation:
- Selecting components based on polarity, solvent strength, and compatibility.
- Accurate measurement and thorough mixing of high-purity solvents.
- Adding buffers and adjusting pH to optimize separation.
Key Considerations for Mobile Phase
- Match the polarity of mobile phase to analytes and the stationary phase.
- Implement additives when necessary for improved performance.
- Ensure components are fully dissolved and pH is adjusted for optimal analyte behavior.
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Description
Explore the fundamentals of High-Performance Liquid Chromatography (HPLC) through this quiz. Learn about retention time, chromatograms, separation modes, and different types of chromatography. This quiz covers key concepts that are essential for understanding HPLC techniques and their applications.
