Lecture 5 - Gas Chromatography And HPLC Flashcards
GC column / oven
Capillary (usually)
Flow of gas through system is dependent on size of column
Separation based on the affinity of compounds for the
stationary…. and mobile phase (In GC its mainly the stationary phase)
Isothermal - keeping it at one temperature
Working outside to the inside:
There is a polyimide protective coating (copper coloured)
Then fused silica
Then the stationary phase (thin film of the high bp liquid)
Capillary column properties
• Length: doubling the length gives small improvements in separating power, but increases run time
• Inner diameter: a narrower i.d. provides a much higher resolution but can hold less sample
• Film thickness: a thicker film retains compounds for longer, so is good for analysis of more volatile compounds
• Stationary Phase chemistry: the make-up of the inner coating defines how compounds will separate
What does each part of this mean (talking about capillary column)
SH-Rtx^tm - 5 MS 30m x 0.25 mm x 0.25 um
1 2 3 4 5 6
1- brand
2- stationary phase type
3- stationary phase modifications (eg lower bleed, suited for acids etc)
4- length
5- inner diameter
6- film thickness
Stationary phase
Analyte interactions: same ideas as CC and TLC
-dispersion
-dipole-dipole
-hydrogen bond
- pi-pi
-chiral (to separate enantiomers
Non polar: dimethylsiloxane: -[-Si(CH3)2(O)-]-n
Polar diphenylsiloxane (PEG): -[-Si(Ph)2(O)-]-n
Non to mid polar - mix of both
GC seperation in column
Physical separation process
Partitioning of analyses between mobile gas phase and stationary phase
Mobile phase sweeps vaporised sample over stationary phase, analytes seperated by their differences in affinity to the stationary phase (ie must interact with the stationary phase to be retained)
Greater interaction = more time on column
Analytes seperation occurs
Heating column oven helps to elute analytes from column
Equilibrium constant K in terms of seperation
If K is large = increased retention
If K is small = less retention
GC oven
• Isothermal analysis: constant temp
- Analytes have similar volatility, separated by polarity
- however late eluters = broad peaks
- Constant baseline
• Gradient analysis: temperature ramped
- Analytes with different volatilities
- Sharper peaks
- Higher background (inc column bleed) -Column disintegrating as its heated up and then when swept into mobile phase it will be detected by the detector
• Typically in range of 30 - 300 °C
How to pick what temp to use for isothermal
Balance between good resolution (sharp and symmetrical) of early peaks and how unsymmetrical and broad later peaks are (caused by lower temps)
As temp increases shorter retention time peaks resolution decrease and high retention peaks resolution increases
Retention time is prop to temp
GC types of detectors
Ones with - are most important
• Flame Ionisation Detector (FID) - Most common and cheap
• Electron Capture Detector (ECD)
• Thermal Conductivity Detector (TCD) - For gas analysis
• Flame Photometric Detector (FPD)
• Nitrogen Phosphorus Detector (NPD)
• Photo Ionisation Detector (PID)
• Atomic Emission Detector (AED)
• Electrolyte Conductivity Detector (ELCD)
• Infra-Red Detector (IRD)
• Isotope Ratio Monitoring (IRM)
• Mass Selective Detector (MSD) - Can identify the molecules
Flame ionisation detectors (FID) - graph on slide 20
• Universal
• Non-selective
• Column effluent mixed with hydrogen and air then ignited
• Organic compounds burn producing ions and electrons which conduct electricity through flame
• Electrical potential is applied at burner tip and collector electrode is at tip of flame
• Current resulting from pyrolysis of organic compounds is measured
• Response proportional to number of Carbon-Hydrogen bonds
• Large dynamic range
Measure potential across the flame
Gas or liquid chromatography - graph on slide 22
Volatile and hydrophobic tend to be GC
Hydrophilic and non-volatile tend to be LC
High performance liquid chromatography (HPLC)
• Analytical and Preparative tool
• Physical method of separation
• Components distributed between stationary phase and a mobile liquid phase moving in definite direction
• An on-line detector monitors the response of each eluting component and generates a trace called the chromatogram
• Used for
- Qualitative analysis
- Quantitative analysis
- Purification
Scalable (analytical ug), semi-preparative (mg), preparative (g))
HPLC advantages
• Wide applicability - Amenable to diverse samples including labile organics, biomolecules, polymers, and ions (~80% of all existing compounds)
• High-resolution - Resolves hundreds of components in complex samples
• High sensitivity detection - pg - ng detection limits
• Rapid and precise analysis - 1 - 60 min analysis, Precision < 1% RSD
• Automated analysis - Using autosampler and data system for unattended analysis and report generation
• Quantitative sample recovery - Preparative technique from ug to kg quantities
HPLC instrument
Starts in resevoirs
Then travels to LC pump (solvent manager, mixes ratio of solvents - usually 2 but can be from 4-6)
Travels to auto sampler / manual injector - injects known volume of sample
Then to guard column - protects chromatography column
Into the column surrounded by a column oven to control column temp - column is the stationary phase which seperates and retains components
Then leaves oven onto the detector - produce a signal when component passes through it and onto the data system
Mobile phase
• Desirable physical properties
- High purity, low cost, UV transparency, non-corrosive, low viscosity (for low back pressure), low toxicity, non-flammable, sample solubility (to mobile phase)
• Isocratic - Solvent composition constant
• Gradient - Solvent composition changed so all species are eluted from column in reasonable time
HPLC columns
Can have up to 6 in there - switch in and out
Column hardware
- dimension: length (L) and inner diameter (i.d.)
Packing characteristics
- type: silica or polymer
- particle size eg 5 um
- pore size eg 80 A
- bonded group (determines the chromatographic mode (C18/C8/C4)
Compression things at the ends of column
Types of HPLC
Determined by Column Chemistry
• Normal Phase
• Reversed Phase - Mobile phase is more polar than stationary phase
• Ion Exchange - Rely on ionic interaction to seperate
• Ion Pair - Reversed Phase
• Size exclusion - Separated according to size - big pore sizes where small polymers get trapped in - longer rf times
• Chiral - Seperate enantiomers
• Affinity - Biospecific interactions
• Hydrophobic Interaction - Seperate proteins
Column types eg glass PEEK, stainless steal are used for different purposes
Effect of column length and particle size
The shorter the lenth the sharper the peaks and the shorter the retention time. Resolution gets better (more theoretical plates) but much more solvent needed, more expensive as column gets longer, time gets longer.
The smaller the particle the better the seperation of peaks / resolution - slide 33
Van Deemter plot - slide 34 to determine what size particle to use
Smaller particles are efficient over a wider range of flow rates
Normal Phase (NP)
• Polar stationary phase
(e.g. silica, amino/cyano)
• Non-polar mobile phase
(e.g. hexane, dichloromethane, acetonitrile)
• Non-polar compounds
elute first followed by more polar compounds
Reverse phase
Non-polar stationary phase
(e.g. C18, C8, C4)
• Polar mobile phase
(e.g. water, methanol, acetonitrile, THF)
• Polar compounds elute first followed non-polar compounds
