CM3007
Chromatography: Theory, Sampling and Detection
Dr. Alan O’Riordan
Nanotechnology Group
Tyndall National Institute.
Email: alan.oriordan@tyndall.ie
Learning Outcomes
•
Define and explain the theory underpinning chromatography.
•
To be able to explain how partition of an analyte between
stationary phase and mobile phase effects separation.
•
To be able to identify and explain the factors influencing
chromatographic separation in terms of resolution and
specificity.
•
Identify the factors influencing different sample injection
techniques and be able to discuss the advantages and
disadvantages of each type.
•
Identify the factors influencing different analyte detection
systems and be able to discuss the advantages and
disadvantages of each type.
Classification of Chromatography Methods
Introduction to Chromatography
Separation of a mixture of components
A and B by column elution
chromatography.
Sample
M obile phase
The detector signal at each stage is
shown.
Column
The effectiveness of a column in
separating two analytes depend in part
on the relative rates at which the two
components are eluted.
These rates are determined by the
magnitude of the equilibrium constants
for the components partioned between
the stationary phase and mobile phase.
Detector
A mobile ⇆ A stationary
Time
Mechanisms of Partition to Stationary Phase
Equipment Overview
Sample injection
Detector
Mobile
phase
Packed column GC & HPLC
Chem station
Capillary column GC only
Partition in Chromatography
• Stationary phase, mobile phase, & analyte form a ternary
system.
• Each analyte is distributed between the two phases (in
equilibrium):
– Partition Coefficient
– CS: concentration of analyte on the stationary phase
– CM: concentration of analyte on the mobile phase
Using the Partition Coefficient: Plate Theory
Column
Column divided into theoretical pieces
(plates). Analytes are partitioned between
SP and MP in each plate. Separation occurs
as analytes move down the column.
Definitions: Prototype Chromatogram
Factors Influencing Retention
• Are those that influence distribution
– Stationary phase: type & properties
– Mobile phase: composition & properties
– Intermolecular forces between
• Analyte & mobile phase
• Analyte & stationary phase
– Temperature
Intermolecular Forces I
• Based on electrostatic forces
– “Like-attracts like” or “oil and water” (similar
electrostatic properties)
• Polar/polar & non-polar/non-polar
– Molecules with dissimilar properties are not
attracted (They are NOT repelled)
• Polar retention forces
– Hydrogen bonding
(permanent dipoles)
– Dipole-Induced dipole
Component molecule
+
-
-
+
Stationary phase
Intermolecular Forces II
Polar forces (cont.):
– Energy of dipole-dipole interaction
: dipole moment, A: analyte,
S: stationary phase
– Factor of 10 variation on permanent dipole
moment
Factor of 104 variation on interaction energies
– As r6 => mainly at the surfaces of stationary
phase
Intermolecular Forces III (London)
London’s Dispersion Forces (Van Der Waals)
– Most universal interaction between molecules
– Only one for non-polar species
– Relatively weak
– Energy of interaction:
: is the polarisability, I: ionisation potential, A:analyte,
S: stationary phase
– As r6 => mainly at the surfaces of stationary phase
How do Van Der Waal forces occur?
Definitions I
•VR, Retention Volume
– Volume required to carry a band of component
molecules
– Primary quantity, but hard to measure!
•tR, Retention Time
• FC, volume flow rate of mobile phase
– Calculated from cross section of column (dC),
average velocity of mobile phase (u), and term to
account for particle volume in columns (εT ; =1 if no
particles)
Definitions II
• u, linear velocity of the mobile phase:
– L: length of the column
– tM: flow time of mobile phase
• Measure with non-retained component
e.g. CH4 in GC
• Correction for dead volume
– Retention time is measured since injection
– Correct for mobile phase in column at injection ( VM)
t´R
Retardation
Recall partition coefficient
RF, retardation:
Substituting:
– 0 < RF < 1
– If RF = 1, analyte is not retained at all
– If RF = 0, analyte is completely retained
Retention (or Capacity) Factor
• k is like K except:
– k is the ratio of total amounts, rather than of concentrations
– Describes the ability of the stationary phase to retain
components (same as K)
– But it is a measure of actual retention properties
Separation Factor
Separation Efficiency – Peak Width
Peak heights of a Gaussian peak and width as a function of standard deviation
Separation Efficiency – Column Efficiency
Peak Band Broadening Processes
Initial
Time, t1
Time, t2
What physical or chemical processes cause broadening of the peaks in chromatography
Peak Band Broadening Processes II
Band Broadening Van Deemter Model
Recall
Van Deemter Model “A” Term
Illustration of Eddie Diffusion
Van Deemter Model “B” Term
Illustration of band broadening due to molecular
diffusion. T3 > T2 > t1
Van Deemter Model “B” Term
D gas ≥ 104 D liquid
Van Deemter Model “C” Term
Van Deemter Model “C” Term II
Optimum Mobile Phase Velocity
How would this plot differ for packed columns versus capillary columns?
Gas Chromatography:
Gas Chromatography – Overview
•
Sample is vaporised and injected onto head
of a chromatography column.
•
Elution is effected by the flow of an inert
gaseous mobile phase.
•
Separation is based upon the partition of the
analyte between a gaseous mobile phase
and a liquid phase immobilised on the
surface of an inert solid (GLC) at a
temperature above boiling point of analyte
(multi-analyte: temperature programming).
•
Mobile phase does not interact with
molecules of the analyte.
•
Eluted analyte detected by a detector and
recorded by PC – Chemstation.
•
GC columns are either packed (with silica
particles coated in stationary) or capillary in
nature.
Sample Injection
•
GC column efficiency requires that the sample be of suitable size (to prevent column over loading) and be
introduced as a plug of vapour.
•
Two common approaches include for introduction of 0.01 – 50 l include: Microsyringe and valve loop.
•
The syringe technique is most common and can be used with both gas and low viscosity liquid samples by
inserting the needle through a rubber septum to the column inlet port.
•
The region into which the needle projects must be heated in order to flash vaporise the sample.
•
However, overheating of the rubber septum must be avoided to prevent out gassing.
•
The most popular inlet for capillary GC is the split/splitless injector.
•
If this injector is operated in split mode, the amount of sample reaching the column is reduced (to prevent
column overloading) and very narrow initial peak widths can be obtained.
•
For maximum sensitivity, the injector can be used in so-called splitless mode, then all of the injected
sample will reach the column.
•
Injection may be manual or automated.
Split – Splitless Injection
•
Septum purge outlet prevents components of previous
injections from entering the column and minimizes the
effect of septum bleed (low flow rate ~3 ml/min).
•
The sample is injected into the liner region where it is
completely vaporised. Mostly glass liners – zero dead
volume
•
The sample volume is then split between the column
and the split outlet. Split injection is employed to dilute
the sample and prevent column overloading. Typically
1:100 split ratios are employed with 99% of sample
being vented to atmosphere.
•
Method development: Some parameters of split/splitless
injection that require optimisation, apart from
instrumental design, are injector temperature, split ratio,
split delay, injection volume, sample solvent and initial
temperature of the column.
Sample Valve Injection
•
Sample valves are convenient for on-line gas stream
analysis.
•
In position (a) the stream to be sampled flows through a
loop of calibrated volume while the carrier gas alone
passes through the column.
•
In position (b) the loop is placed in the carrier gas
stream and the entrapped sample is swept along to the
column.
•
Sample valves are becoming more prevalent for
quantitative work employing both liquids and gases to
introduce a reproducible volume of sample onto a
column.
•
They are typically employed for smaller volumes, e.g.,
to prevent over loading of a column > 0.01 l of a liquid
sample is preferred volume - a precision syringe for this
volume is both expensive and fragile.
•
Valves may also be used in split – splitless mode.
(a) Sample mode
(b) Inject mode
Pyrolysis Gas Chromatography (PGC)
•
A version of reaction chromatography in which a
sample is thermally decomposed to simpler fragments
before entering the column. 1993, 65, 827
IUPAC Compendium of Chemical Terminology
•
Many non-volatile solids can be decomposed thermally
to produce characteristic gaseous products that can be
chromatographed.
•
Samples are placed directly on a small coil of Pt wire
where it can be heated to several hundred degrees in a
few milliseconds while the carrier gas is flowing over it.
•
The pyrolysis products are swept directly onto the
column.
Column Configuration
Packed Columns
Capillary Columns
•
2 to 4 mm I.D. and 1 to 4 meters long.
•
100 mm to 500 mm I.D. and 10 m to 100 m long
•
Packed with a suitable adsorbent.
•
•
Mostly used for gas analysis.
Stationary phase is coated on the internal wall of
the column as a film 0.2 m to 1 m thick
•
•
Sharper peaks – no eddy diffusion.
Peak broadening due to zone (eddy) diffusion
resulting from multitude of pathways a molecule
can pass through column.
•
Up to 500,000 theoretical plates – excellent
separations.
•
Most popular type of column in use.
Characteristics of Ideal GC Detector
•
Good stability and reproducibility.
•
Linear response to analytes that extends over several orders of magnitude.
•
Similarity in response toward all analytes.
•
Temperature range from room temperature to 400 ºC.
•
A short response time that is independent of flow rate.
•
Non-destructive.
•
High reliability and ease of use.
•
No one detector exhibits all of these characteristics
Thermal Conductivity Detector
•
Exploits the changes in the thermal conductivity of a gas
stream brought about by the presence of analyte molecules.
•
The resistance of either a heated platinum wire or a heated
semiconductor thermistor gives a measure of the thermal
conductivity of the gas.
•
Twin detector pairs are typically incorporated into two arms
of a Wheatstone bridge.
•
In the presence of a relatively small concentration of
analyte a large decrease in thermal conductivity of carrier
gas occurs resulting in a temperature rise in detector.
•
Thermal conductivities of He and H2 are ~ 6 – 10 times
higher than most organic compounds. Necessitates the use
of these gases as carrier gas.
•
Linear range of 10 and is suitable for organic and
inorganic samples.
•
Non-destructive and allows collection of sample after
-8
detection but low sensitivity ~ 10 g/s analyte/gas
5
Flame Ionisation Detector
•
Most organic compound pyrolyse in H2-air flame and
produce ions and electrons.
•
A potential of a few hundred volts is applied across the
burner tip and a collector electrode located above the
flame.
•
•
•
•
The resulting current is amplified and proportional to the
number of carbon atoms in the flame.
General detector for GC. However, carbonyl, alcohol,
halogen and amine groups yield few electrons. Also
insensitive to H20 CO2 SO2 NOX.
Collector
electrode
Insulator
Connector nut
Air
H2-air
flame
Grounded
jet
Inside oven wall
7
Large linear response range (~ 10 ) and low noise (once
detector has settled). Needs to be burning 24 hours before
analysis.
Exhibits very high sensitivity ~ 10
-13
g/s of analyte/second
H2
Exit end
of column
Advantages and Disadvantages of GC
• Fast analysis
• Limited to volatile samples
– Typically minutes (even sec.)
• High Resolution
– Record N~1.3 x 10
•
•
•
•
6
Sensitive detectors (easy ppm, often ppb)
Highly accurate quantification (1-5 % RSD)
Automated systems
Non-destructive
– Allows online coupling to Mass Spec.
• Small sample (L)
• Reliable and relatively simple
• Low cost (~€20,000)
– T limited to ~ 380 °C
• Need Pvap ~ 60 Torr at that
temperature
• Not suitable for thermally labile
samples
• Some samples may require extensive
preparation
• Requires spectroscopy (usually MS)
to confirm peak identify
CM3007
Gas Chromatography:
Method Development and Validation
Dr. Alan O’Riordan
Nanotechnology Group
Tyndall National Institute.
Email: alan.oriordan@tyndall.ie
Learning Outcomes
•
Be able to differentiate between different GC column types.
•
Explain how mobile phase flow rate, temperature and type of
column can affect column resolution and sensitivity.
•
Determine/identify suitable stationary phase for analytes.
•
Distinguish between different quantitative approaches to GC.
•
Specify and explain the eight requirements necessary for
method validation.
•
Identify and explain the different approaches to analyte
sampling and injection.
•
Identify two application areas for GC.
Choice of GC Columns
Packed GC Columns
Open (capillary) GC Columns
Column Type Vs Separation
Comparison of Columns
Effect of Column Diameter & Film Thickness
Optimum Mobile Phase Velocity
Effect of Column, Film and Carrier Gas
Effect of Temperature on Retention Time
Two Decisions
How to Choose a Stationary Phase
Most Important: Stationary Phase Polarity
Effects of Stationary Phase Polarity
=>
Can change the order of
elution, so need to verify
elution times using standards
Separating Efficiency – Peak Asymmetry
Resolution
• Objective: accurate measurement of individual peak areas
Can One Have Too Much Resolution
RS = 2.5
Resolution and Selectivity
Quantification in GC
Quantification: Normalizing Peak Areas
Requires identical samples – not very robust
Quantification: Internal Standard
Popular method: may be used as internal QC
Why?
Quantification: External Standard
Normally performed in conjunction with internal standard
Quantification: Standard Addition
•
`
•
•
Different concentrations of standard are added to
sample aliquots.
Samples are analysed
Can be used to verify linearity
Q: why are separate sample
aliquots prepared?
Method Validation
Organisations regulating pharmaceutical industry:
FDA (CFR 21)
Irish Medical Board (IMB)
Official Analytical Chemists (AOAC)
US Environmental Protection Agency (USP)
American Association of Official Analytical Chemists (AOAC)
Resource Conservation and Recovery Act (RCRA )
European Pharmacopoeia
Japanese Pharmacopoeia
US Pharmacopoeia
The International Conference on Harmonization (ICH) of Technical
Requirements for the Registration of Pharmaceuticals for Human Use has
developed a consensus text on the validation of analytical procedures (1995)
ICH Guidelines
Validation requirements include:
1.
2.
3.
4.
5.
6.
7.
8.
Specificity: Ability to measure desired analyte in complex mixture.
Accuracy: agreement between measured and real value.
Linearity: proportionality of measured value to the concentration.
Precision: agreement between a series of measurements.
Range: concentration interval where method is precise accurate and linear.
Detection limit: lowest amount of analytes that can be detected (LOD).
Quantitation limit: lowest amount of analyte that can be measured (LDQ).
Robustness: reproducibility under normal but variable laboratory conditions.
What is the difference between repeatability and reproducibility?
What is GMP?
Specificity
Selectivity in chromatography is obtained by choosing optimal columns and setting
chromatographic conditions, such as mobile phase composition, column temperature and
detector wavelength. UV-vis spectroscopy may be used to identify pure peaks
Accuracy and Recovery
Active Ingred. [ %]
Analyte ratio
Unit
Mean recovery [%]
100
1
100%
98-102
>=10
10-1
10%
98-102
>=1
10-2
1%
97-103
>=0.1
10-3
0.1 %
95-105
0.01
10-4
100 ppm
90-107
0.001
10-5
10 ppm
80-110
0.0001
10-6
1 ppm
80-110
0.00001
10-7
100 ppb
80-110
0.000001
10-8
10 ppb
60-115
The accuracy of an analytical method is the extent to which test results generated by the method and
the true value agree. Accuracy can be assessed by analyzing a sample with known concentrations,
e.g., a certified reference material
Linearity
The linearity of an analytical method is its ability to elicit test results that are directly, or by means of
well-defined mathematical transformations, proportional to the concentration of analytes in samples
within a given range. Linearity is determined by a series of three to six injections of five or more
standards whose concentrations span 80-120 percent of the expected concentration range.
Precision
Analyte %
Analyte ratio
Unit
RSD (%)
100
1
100%
1.3
10
10-1
10%
2.8
1
10-2
1%
2.7
0.1
10-3
0.1 %
3.7
0.01
10-4
100 ppm
5.3
0.001
10-5
10 ppm
7.3
0.0001
10-6
1 ppm
11
0.00001
10-7
100 ppb
15
0.000001
10-8
10 ppb
21
0.0000001
10-9
1 ppb
30
The precision of a method is the extent to which the individual test results of multiple injections of a
series of standards agree. The acceptance criteria for precision in pharmaceutical quality control of
better than 1 % RSD is required.
Range, LDQ & LDQ
Range: The range of an analytical method is the interval between the upper and lower levels
(including these levels) that have been demonstrated to be determined with precision, accuracy and
linearity using the method as written. The range is normally expressed in the same units as the test
results (e.g. percentage, parts per million) obtained by the analytical method.
LDQ: Normally three times the baseline noise .
LDO: The limit of quantitation is the minimum injected amount that gives precise measurements.
This may be set as twenty times the baseline noise or determined using the Eurochem method viz A
number of samples with decreasing amounts of the analyte are injected six times. The calculated
RSD% of the precision is plotted against the analyte amount. The amount that corresponds to the
previously defined required precision is equal to the limit of quantitation.
Head Space Analysis
•
Head space analysis is a technique where the vapours in
the gas above, and in equilibrium with, a solid or liquid
is sampled.
•
The advantage of this approach is that GC can be used
instead of HPLC, thus providing four to five orders of
magnitude greater sensitivity.
•
Procedure involves the extraction of a volume of the
equilibrium gas over the sample (usually about 10 ml)
by a syringe through either a vial containing a bed of an
appropriate absorbent or a cryogenic trap.
•
The vial/trap is the placed in line with a GC column,
heated and the vaporised sample swept onto the column
and the components separated.
•
Used to identify spoiled food, fragrances from botanical
material, the determination of plasticizers in plastics
and for forensic samples involving arson.
Purge and Trap – EPA method 5030C
•
5030C can be used for most volatile organic compounds
that boiling points below 200 ºC and are insoluble or
slightly soluble in water.
•
Multiple sample aliquots are collected in sealed
containers with minimum headspace and stored at 4 ºC
or less in solvent free area.
•
An inert gas is bubbled through aqueous sample and
room or elevated temperature depending on the target
analytes.
•
The vapour is swept through a sorbent column where
the analytes are captured.
•
After purging, the sorbent column is heated (thermal
extraction) and back-flushed with inert gas to desorb the
components onto a GC column.
Solid Phase Microextraction
•
Solid phase microextraction (SPME) is suitable for
sampling environmental contaminants with a wide
range of physical properties in air, water and soil.
•
A fused silica fibre with a polymer coating is
exposed to the sample or the headspace above
the sample.
•
Organic analytes adsorb to the coating on the
fibre. After adsorption equilibrium is attained,
usually in 2 to 30 minutes, the fibre is withdrawn.
•
The fibre is introduced into a GC injector, where
the adsorbed analytes are thermally desorbed and
delivered to the GC column.
•
The amount of analyte adsorbed by the fibre
depends on the thickness of the polymer coating
and on the distribution constant for the analyte.
•
Fibres with a range of different polarities are now
commercially available.
Direct Thermal Extraction
•
Permits the direct thermal extraction of volatile and
semi-volatile organics directly from small sample sizes
(mg) without the need for solvent extraction or other
sample preparation requirements.
•
The sample is maybe trapped on sorbent resisn or
placed inside a preconditioned glass-lined stainless steel
desorption tube.
•
The desorption tube containing the sample is then
connected to a short path thermal desorption system.
•
The desorption tube is ballistically heated and carrier
gas carries the analytes through the injection port and
onto the GC column for analysis.
Comparison of Techniques
Gas Chromatography – Mass Spectrometry
•
Mass Spectrometer systems operate under UHV
conditions. GC systems may use flow rates as high 50
ml/min
•
Flow rate from capillary columns is generally low
enough that the column can be directly interface with
the detector.
•
For packed columns a jet separator is typically
employed to remove most of the carrier gas from the
analyte.
•
Exit gasses flow through a nozzle of an all-gas separator
which increases momentum of heavier analyte
molecules. ~50% travel in more or less a straight path in
to the skimmer.
•
In contrast, the lighter helium molecules are deflected
by the vacuum and pumped away
Mass Spectrometry Operation
http://www.chem.agilent.com/scripts/generic.asp?lpage=5450&indcol=N&prodcol=N
GC Environmental Applications
•
Local government regulate and monitor emissions from
industrial chimney stacks.
•
On-site audits are performed by local authorities to
police and ensure emissions are within licensing limits.
•
Samples of stack emissions are taken by pumping a
know volume of gas through a desorption tube packed
with an appropriate sorbent material.
•
A second tube is placed in series with the first tube to
trap any break through analytes.
•
Thermal desorption-GC-MS analysis is performed on
trapped sample to:
–
–
•
Determine constituents (qualitative)
Determine concentrations (quantitative)
Hand-held and portable GC-MS systems are now
becoming more prevalent for in-the-field analysis.
GC Forensic Applications
•
GC-MS employed in the identification of illicit (illegal)
drugs.
•
GC-MS analysis of biological specimens for the presence of
alcohol, drugs, and/or poisons and their corresponding
metabolites.
•
Pyrolysis GC employed in paint analysis especially useful
in hit-and-run cases, where the paint chips are directly
analysed.
•
Forensic arson analysis deals with the analysis of fire debris
for the presence of accelerants. Identification of
hydrocarbon constituents is performed by comparison to
known standards by GC-MS.
•
GC detection of explosives and residues by direct head
space analysis.
•
Determination of carbon monoxide in postmortem blood
using GC.