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Gas Chromatography Cheat Sheet (DRAFT) by

Theory of gas chromatography

This is a draft cheat sheet. It is a work in progress and is not finished yet.

Basic Theory

Gas Chroma­tog­raphy
SP Liquid
 
MP Inert gas
No role in separation
Only directs analyte down column (carrier gas)
 
D
m
>>> D
s

C
m
U ~ 0
 
Flow rate
Dictate by choice of SP (thick­ness, proper­ties)
Modest plate height ~1mm ( L = N)

Theory Equations

Column Type

Packed
Packed full of particles
 
Put SP on particles
 
MP pushes through packed bed
 
Tubing
Glass, stainless steel, etc.
Inert = not part of separation
Wall Coated Open Tubular (WCOT)
Inside wall of quartz­/glass tube
Chemically roughen
Surface area
Coated with SP
Fused Silica Open Tubular (FSOT)
SP coating on wall of long thin tube
Smooth wall
 
Diameter ~ 75-200 um

Column Diagram

GC Systems

Sample
Introduce into injector port
Vaporize sample
Vaporized analyte swept into column
Mobile phase
High pressure cylinders
Use a gas flow regulator
Regulate the pressure
Detector
Detect components of the mixture being eluted off the chroma­tog­raphy column
Some may require a reference flow
Oven
Separation occurs
Controlled temper­ature
Fan Circulates air and controls temper­ature

GC System Diagram

Split Flow Injector

Sample dissolved in volatile solvent
Collect sample into syringe and inject through rubber septum
Seals injector for analyte to go into column
Protects from outside atmosphere
Bad peak shapes = hole in septum
Use a heat block to "­fla­sh" sample into vapour
~50-100C hotter than oven
Need to vaporize sample
GC systems design to operate with 3 main columns
Each column has a different flow rate
Adjust based on column used
FSOT/WCOT
Can't handle large sample mass
Small diameter
Limited SP
Limited volume capacity
Control by valve system
Split flow outlet
Avoid overlo­ading the column
Packed Set at 0 (closed)
FSOT/WCOT Split flow ratio Depends on [analyte] in injection volume

Split Flow

Requir­ements of SP

Solvent
Must dissolve analyte
 
Bad SP
Unretained
Affects R'~0
No separation
Volatility
A substance with high volatility is more likely to exist as a vapour
 
A substance with low volatility is more likely to be a liquid or solid
 
Prefer a low volatile solvent Don't want SP to vaporize in oven
Thermal Stability
SP in column
 
Don't want thermal breakdown products
Inert/­Rea­ctive
Don't want analyte to react with SP
 
Only want to interact

Stationary Phase

Siloxane Polymer
Low volatility
 
Thermally stable bond
 
Contains a silicone backbone
Close to inert
Can be deriva­tized
 
Add pendant functional groups
Tune select­ivi­ty/­sol­ubi­lit­y/r­ete­ntion
Adjust polarity
Non-Polar
Poly(d­ime­thy­l)s­iloxane (PDMS)
Good quality
 
Fluroc­arbons
Polar
Can replace dimeth­yl/­methyl groups
CN,CO,OH
Phenyl Groups (Benzene Ring)
Non-polar
 
π e- deloca­lized
 
When approach by polar molecules
e- reorga­nized Induced dipole intera­ctions
Can behave polar with polar molecules (vice versa)
Chiral Moiety
Chiral­-chiral intera­ctions on SP
 
Rise to select­ivity of 1 enantiomer over another

Siloxane Polymer

Minimize Loss of SP

Bonded Phase
Process of the SP polymer is attached to
Silica support particle
Wall of a capillary
 
A liquid­-liquid chroma­tog­raphy method in which a stationary phase is covalently bound to a carrier particle
Cross-­Linked Phase
Polymer attached to wall
 
Polymer cross-­linked with each other
Critical for separation
 
Produce more rigidty, hardness and Melting point
Formation of covalent bonds
Issue
Most SP are non-polar and silica support surface are polar
Not much intert­action
 
Uses phases to prevent issue of contact
 
Use silane reaction to bond/c­ros­s-link
Silane Reaction
Use to anchor­/bond silicones to silica surfaces
In packing materials (parti­cles)
FS capill­aries
 
Use to deactivate silanols
 
Same chemistry for polyme­riz­ation and cross-­linking
 
Silanol
Very polar
Expose on surface of silica
Disagree with SP polarity
Compet­ition for polar analyte
 
Deacti­vation chemistry
Use dichloro dimethyl silane
Use ethano­l/MeOH
Create less polar surface
Inertness of Column
Residual silanols
React strongly to polar compounds
Produce tailing peaks
Undesi­rable intera­ctions in column

Deacti­vation of Silanol

Silane Reaction Mechanism

  

Contro­lling Retention

Retention
Controls resolving power (R')
R' depends on K'
K' depends on separation conditions
 
Want all peaks to fall in "­ideal range" of retention
1-10
 
MP is inert
Only function to control retention
Equili­brium constant = thermo­dynamic property
 
Temper­ature
Alter overall retention
 
Type of SP
Alter select­ivity
Impact of Different Temper­ature
Isothermal separation
A thermo­dynamic process, in which the temper­ature of the system remains constan
 
Temper­ature = Thermal energy available
Less thermal energy
Analyte spends more time in SP
More time in column
Clearer separation
 
Temper­ature = Resolution = Overall time
Can become excessive
Needs to adjust separation as it proceeds
 
Temper­ature Favors SP
 
Temper­ature Favors MP
Different Ramp Rates
Altered t
r
and resolution indepe­ndently
 
Adjust temper­ature during course of separation
 
Resolution imporves under better retention conditions for the analyte
 
Change in gradient steep Improves separation
Shorten separation time
Increase resolution
As a function of temper­ature
Round-Up of T Progra­mming
Powerful tool for contro­lling K'
 
Directly affects distri­bution constant
 
Temper­ature = K'
 
Ramped (gradient) temper­ature is used to adjust K'
 
Make GC less intuitive
R= {{fa-arrow down}} K' (general)
R= K' (T progra­mming)
K'=f(T)
 
Separation limited by ΔT/Δt (ramp rate)
 
Column lifetime is shorter at higher temper­ature
Other Factors
K'=K(V
s
/V
m
)
SP thickness
Total mass of SP
 
FSOT columns
Calculate phase ratio (V
s
/V
m
)

Graphs with Different Temper­ature

Ramp Rate Graphs

GC Detectors

Requir­ements
Sensit­ivity
10-8-10-15 g analyte/s
Packed All sample used Decrease efficiency = broader peaks
FSOT Split flow injector (5-10% sample used) Increase efficiency = narrow peaks
 
Stability
Noise on baseline Smooth {[fa-a­rro­w-r­ight}} Detect the smallest peaks Minimal DL
Drift No baseline (goes up and down)
 
LDR
5-8 orders of magnitude
 
Can accept MP over a wide temper­ature range
T Progra­mming {[fa-a­rro­w-r­ight}} Improves separation
Immune to T change
Compensate T change Require reference gas flow
 
Fast response and indepe­ndent of T
 
Simple to use, maintain, repair
 
Select­ive­/Un­iversal
Detect analyte of interest (S)
Detect all species (U)
 
Non-de­str­uctive

Flame Ionization Detector (FID)

Analyte elute from column
Mix with H2 gas
Combusted
Reduced carbons
Produce ions that alter conduc­tivity of flame and alter current
Signal propor­tional to # of reduced carbons
Mass sensitive
Oxidized and e- capturing species
No-little signal
Cannot be oxidized further
Non-co­mbu­stible gasses
No signal
Already oxidized
High sensit­ivity
10^-13­& g/s
use FSOT/WCOT
Large LDR
7 orders of magnitude
Destru­ctive
No reference flow

FID Diagram

Electron Capturing Detector (ECD)

An ionization detector
response is based upon the ability of molecules with certain functional groups to capture electrons generated by the radioa­ctive source
Radioa­ctive source 63Ni
Emits beta-p­art­icles
When disint­egr­ation occurs
Large energy release
Beta particle emission
Impacts any filler gas and/or MP present in detector and ionize it
Use a N2 make-up gas
Get ionized by high energy
Ionized N2 gas Pass an electric current through detector cell
In absence of analyte with e- capturing groups
A constant current establ­ished through the detector
When analyte with e- capturing groups enters cell
Quench some ionization
Reduce conduc­tivity of gas = reduce current in cell
Selective detector
analytes with a high electron affinity
Sensitive for species that can disrupt ionization of N2 gas
Pesticides halides, peroxides, nitro groups

ECD Diagram

Thermal Conduc­tivity Detect­or(­TCD)

Properties
Signal propor­tional to change in heat capacity
Difference between MP and MP+analyte are relatively small
 
Universal detector
Detect solvent as well
Unders­irable Solvent order of magnitude is more concen­trated than analyte
Result in large solvent peaks and small analyte peaks
If analyte is not well retained Interfered by solvent
 
Modest sensit­ivity ~ 10-9 to -10 g/ml
Less sensitive than FID
 
Modest LDR
Very short linearity
 
Non-de­str­uctive
 
Require a reference flow
Basic Theory
Based on ability of the gas exciting the column to absorb heat
 
Contains thin filament
electr­ically heated
As heat capacity of gas changes (MP vs MP+ana­lyte), so does the T of the filament
 
Resistance of thin filament
T changes the resistance
Resistance changes the current of the circuit
Current is VERY sensitive to T
Reference Flow (Type 1)
To compensate for the T of MP coming from the oven
T is changing with T programmed elution
left section of diagram
 
Equation
V
out1
= V
applied
* (R
ref
/(R
column
+R
ref
))
 
If R
column
= R
ref

two resistors are “balanced”
The signal from the column is coming from the MP
V
out1
=(1/2)V
app
 
If R
column
≠ R
ref

Analyte’s heat capacity changes T
heating or cooling of filament
V
out1
increases as analyte elutes
As R
column
gets closer to 0, V
out1
gets closer to V
app
Reference Flow (Type 2)
Opposite concept as reference flow type 1
right section of diagram
 
Equation
V
out2
= V
applied
* (R
column
/(R
column
+R
ref
))
 
If R
column
= R
ref

two resistors are “balanced”
The signal from the column is coming from the MP
V
out1
=(1/2)V
app
 
If R
column
≠ R
ref

Analyte’s heat capacity changes T
Heating or cooling of filament
V
out2
decreases as analyte elutes
As R
column
gets closer to 0, V
out2
gets closer to 0
Reference Flow (Type 3)
Type 1 and Type 2 TCD operating together
With separate power supplies
 
If R
column
= R
ref

two resistors are “balanced”
The signal from the column is coming from the MP
V
out1
=(1/2)V
app
 
If Rcolumn ≠ Rref
As analyte elutes
V
out1
increases
V
out2
decreases
V
out1
and V
out2
have same magnitude, opposite signs
Taking the difference of the two will double the V measured
Double the signal for the same effort
Reference Flow (Type 4)
Same as Type 3, but with a single power supply
 
Wheatstone bridge
Name of this circuit
Common approach for detecting VERY small change in resistance
Advantage: Doubles the signal magnitude

TCD Diagram

GC-MS

Properties
Versatile
Provide identi­fic­ation power
 
Have to run known standards (spiked)
 
Electron beam ionization
M+ and fragments
 
Excellent DL
Depending on instrument and analyte
~ 2-20 picog injected
 
Concen­tration DL in sample
Depends on sample work-up
 
Long LDR
Dependent on instrument
4-6 orders of magnitude
 
Selective
Less interf­erences
Filters out MP signal
 
Destru­ctive
 
Expensive
Basic Theory
Quadruple MS
Contains 2 positive and 2 negative poles
 
Movement of M+
M+ travels in a sinusoidal path
If M+ is too light or too heavy, it is kicked out of quadrupole
b/c they are not really able to respond to polarity change
How to fix this
Quickly change the frequency and voltage of the poles
Can quickly scan through all m/z ratio to obtain mass spectrum
 
Spectrum generated
Total ion current (TIC)
Easiest way
Sum of all ion signals that passes through
Acts as a universal detector: does not filter out MP signal
Tells you how many species are present
Extracted mass spectra
Take a slice of TIC peak and see its fragments
 
Isotopes
Parent ion
Most prominent and heaviest
Isotopes
Daughter peak from most prominent peaks
can provide more info depending on its ratio with parent peak
Isotop­ically labelled analytes
Replacing parts of molecule with deuterium
Produces a known mass higher than the original mass
Compare spectrum with orignal
 
Positive identi­fic­ation
Compare experi­mental spectrum with the “real” analyte spectrum
3 steps
Correct mass of molecule?
Correct set of fragments?
Correct fragment intens­ities?
 
Quanti­tation
Usually multiple ions monito­red­/me­asured
Validate ratio of peaks at the correct m/z ratio
 
Column bleed
SP is boiling and bleeding out
Leads to a rise in baseline
Not good
How to fix it
Running at low T
Purchase column made specif­ically for MS ($$$)

GC-MS Diagram

Key Factors and Applic­ations

When will GC be useful
1. Analyte
Needs to be volatile
Not proteins Unstable at high temper­ature
Silation reaction Produce volatile products (Risk of contam­ina­tion, loss, produce new products)
Needs to be stable Stable enough to transit the column
 
2. High enough concen­tration to detect
Packed columns: great sample capacity but low resolving power and resolution
FSOT: lower capacity (split flow) but high resolving power and resolution
Detectors: Has a good sensit­ivity
 
3. Does sample require high R' separation
Depends on the type of detector
Universal = high R
Selective = low R
 
4. Generally faster than LC
Applic­ations
Anti-d­opping and forensics
 
BAC (Crime­/fo­rensics labs)
 
Pharma­ceu­ticals
Process control
Quality control
Research and develo­pment
 
Food and Beverages
Wine/alcohol
Pesticides
 
Enviro­nmental
Pesticides
PAH and industrial solvents
Oil/hy­dro­carbon spills
 
R&D
Organic synthesis
Catalysis (monitor products)
 
Industrial
Feedstock
Off gassing