Liquid Chromatography Flashcards
Effect of decreasing size of SP particles
Decreasing size of SP particles increases the efficiency of packed columns (but require high pressure)
Describe Modern LC/HPLC
High Performance (Pressure) Liquid Chromatography
Uses high pressure to force liquid solvent through a packed column containing fine particles giving high resolution separations
LC vs GC characteristics
LC = mobile phase is a liquid that interacts with the solutes, the stationary phase is is coated onto small particles in packed column
GC = mobile phase is an inert gas, stationary phase is on column walls
What kind of analytes will stay on a normal phase column longer?
More polar analytes
Reverse phase LC
-SP/MP
Non-polar SP
Polar MP
Normal phase LC
-SP/MP
Polar SP
Non-polar MP
Order of elution for normal phase LC
Most non-polar (first)
Most polar (last)
How to increase retention times of a normal phase LC?
Decrease MP polarity
Why has normal phase been replaced by reverse phase?
Normal phase SP (Si-OH or Al2O3) have poor selectivity
Normal phase has poorer column efficiency (low N, high H) compared to reverse phase
Often observe peak tailing (particularly for more polar compounds due to differences in activity of silanol sites)
Many MP organic solvents are hazardous
List some SP’s and MP’s for reverse phase LC
SP = octyldecyl silane (C18) or octyl silane (C8)
MP = MeOH, ACN, THF
What kind of analytes will stay on a reverse phase column longer?
More non-polar analytes
How can we alter polarity of SP in reverse phase LC?
Increase R (alkyl) chain length, SP will become more non-polar
Carbon load
Refers to the % (by weight) carbon content of the stationary phase bonded to the support material
What does carbon load increase with?
Alkyl group chain length (more non-polar SP)
The amount of R-group bonded to the silanol groups
How does alkyl chain length of SP effect retention times (for non-polar analytes)?
Retention times increase (for non-polar analytes) with increasing alkyl chain length
What does increasing carbon loading do (RPLC)?
Increases k’ and tr
What does increasing polarity of SP do (RPLC)?
Decrease k’ and tr
Endcapping
If residual Si-OH groups are present on a RPLC column then peak tailing will be observed, often worse for more polar analytes such as alcohols and amines
Polar analytes can interact (eg H bond) with SiOH groups
Endcapping deactivates these groups
(residual SiOH groups)
For what analytes is endcapping more likely needed?
Analytes with OH or NH2 groups
How can we increase tr of analytes in RPLC by varying the MP?
To increase retention of the analytes (keep analytes longer on the column), you need to increase the polarity of the mobile phase (remember water is the most polar)
1. increase the % of water
2. decrease % of organic solvent
How does increasing the percent of organic solvent change P’mix and k’ in RPLC?
As analyte polarity decreases, k’ increases
As MP polarity decreases, k’ decreases
Increasing the amount of organic solvent will decrease P’ and tr
Can different solvents cause different elution order in RPLC?
Yes
Solvents have different proton acceptor/donor properties as well as dipole-dipole interactions that can impact selectivity
What is (generally) the greatest effect on solute retention in HPLC?
MP composition
Isocratic elution
MP composition is constant
Gradient elution
MP composition is varied
HPLC Injectors
Manual injector
(low cost, few samples)
Autosampler injection system
(automated injection, more costly, good for large number of samples, unattended sample injection)
HPLC Detectors
Ultraviolet/Visible (UV/Vis) Detectors
- Fixed wavelength
- Variable wavelength
- Photodiode array
Fluorescence Detector
Refractive Index Detector
Conductivity Detector (Ion-exchange chromatography)
Mass Spectrometry (not discussed in the course)
How do photodetectors work?
The absorbance of a sample will be proportional to the number of absorbing molecules in the detector cell
Molar absorptivity due to organic chromophores
What functional groups are chromophores?
Double (pi) bonds, functional groups, and aromatic systems
Ultraviolet/Visible (UV/Vis) Detectors
Most common, inexpensive, rugged
Wide range of solute detection
Good linear range, good sensitivity
Analytes must absorb in UV or visible region
Different types of ultraviolet/visible (UV/Vis) detectors
Fixed Wavelength: measures at one wavelength, usually 254 nm (Mercury Lamp)
Variable Wavelength: measures at one wavelength at a time, but can detect over a wide range of wavelengths (you can adjust what wavelength you want)
Photodiode Array: measures a spectrum of wavelengths simultaneously
Advantages/disadvantages of photodiode array detector
Advantage: greater selectivity than single or variable wavelength detectors
Disadvantage: slightly lower sensitivity
Fluorescence Detector
Greater sensitivity and selectivity over UV-Vis but the analyte must fluoresce
High absorbance, aromatic, fused rings, electron donating groups all make good fluorophores
What causes a molecule to fluoresce?
Several combined aromatic groups, or plane, or cyclic molecules with several π bonds
More rigid structures
Refractive index detector
Universal detector for LC applications
- responds to differences in refractive index of mobile phase and solute(s)
Advantage - response to almost all analytes
- reliable and unaffected by flow rate (pressure changes)
Disadvantage - low sensitivity, cannot use MP gradients (µg levels as compared to ng levels with UV-vis)
- not used with gradient elution
