C3110 Midterm 2 Flashcards
What are the 6 main components of an LC?
Autosampler, solvent delivery system, sample injection valve, high-pressure chromatography column, detector, computer to control system and display
What compounds are chosen for LC over GC?
Compounds that are not sufficiently volatile for GC
Why do smaller particles give better resolution? (2 reasons)
- More uniform flow reduces A term
- Less distance the solute must diffuse reduces C.
What is a consequence of smaller particle size?
Requires a higher pressure
What are the benefits of smaller particles? (3)
Higher plate number, shorter tun time, and lower detection limits
What contributes to extra column broadening in HPLC? (4)
Connecting tubing, extra column variance, column frits, and sample filtration
Describe the HPLC column
Filled with stationary phase; highly pure, spherical, microporous particles of silica
What is a guard column
When samples are filtered or centrifuged, the guard column protexts the main chromatographic phase
Most common bonded phase (sp)
C18 (ODS)
Describe reversed-phase chromatography
Most common for HPLC.
Sp is nonpolar and solvent is polar.
Analyte is nonpolar.
Less polar solvent is a stronger mobile phase.
Polarity Index
ranks solvents based on their ability to displace polar solutes; higher index=more polar solvent
Describe normal-phase chromatography
Polar sp and a nonpolar solvent
Analyte is polar
More polar solvent has a higher eluent strength
Eluent strength
measure of the solvent absorption energy on base silica. Pentane is defined as zero.
What is the acronym HILIC stand for?
Hydrophilic interaction chromatography
Describe HILIC
Separates compounds too polar for RPLC
Strongly polar sp (CH3CN with buffer)
Polar sp is coated with a layer of water, and the polar analytes partition
Why do analytical chemists dislike silica? (4)
- Variance
- Surface Complexity
- Not uniform
- Highly pH dependent
What are the problems with HPLC solvents?
Pure HPLC grade solvents are expensive.
What is a sparging system in HPLC
Removes dissolved gases by sweeping them out of solution by fine bubbles of an inert gas.
Why is sparging required?
Air bubbles create difficulties for pumps, columns, and detectors.
Dissolved oxygen can absorb UV.
How (3 steps)/when are HPLC columns cleaned?
When: before storage or after a series of runs.
How:
1. Replace buffer with water and wash with 5-10 mobile phase volumes (Vm)
2. Wash with 10-20 Vm of a strong eluent
3. Store the column with the solvent to inhibit bacterial growth.
What is the general elution problem?
One set of conditions is not suitable to separate a complex mixture of analytes in a reasonable amount of time.
What is gradient elution and why is it used?
Gradient elutions uses a continuous change of solvent composition to increase the mobile phase strength.
Used as a solution for the general elution problem.
What common detectors are used for HPLC?
UV, refractive index, evaporative light scattering, charged aerosol, electrochemical, fluorescence, nitrogen chemiluminescence, conductivity, mass spectrometry
Explain how spectrophotometric detectors work.
UV detectors – common because many solutes absorb UV light.
Photodiode array records spectrum of each solute as it elutes.
This is matched with library spectra to identify the analyte.