An Introduction to ChromatographyThe Retention Index in Temperature-Programmed Chromatography •...
Transcript of An Introduction to ChromatographyThe Retention Index in Temperature-Programmed Chromatography •...
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Gas Chromatography
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Schematic Diagram of a Gas Chromatograph
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Open Tubular Columns
• The vast majority of analyses use long narrow open tubular columns made of fused silica (SiO2) and coated with polyimide
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Porous-layer open tubular column (porous carbon)
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Chromatogram of vapors from the headspaceof a beer can on a porous carbon column
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Capillary Columns
• Inner diameters are typically 0.10 to 0.53 mm and lengths are 15 to 100 m
• Narrow columns provide higher resolution but require higher operating pressure and have less sample capacity
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Effect of OT column inner diameter on Rs
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Resolution Increases in Proportion to NN
N
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Effect of Thickness of Stationary Phase on Resolution
• Increasing the thickness of the stationary phase increases retention time, sample capacity and resolution of early peaks(k’ ≤ 5)
• Thick films of stationary phase can shield analytes from the silica surface and reduce tailing but can also increase bleed of the stationary phase at elevated temperature
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Effect of Thickness of Stationary Phase on Resolution (cont.)
• A thickness of 0.25 μm is standard, but thicker films are used for volatile analytes
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Effect of Stationary Phase Thickness on OT Column Performance
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The Choice of Liquid Stationary Phase
• Based on the rule “like dissolves like”: nonpolar columns are best for nonpolar solutes and strongly polar columns are best for strongly polar solutes
• To reduce the column bleed, it is usually bonded (covalently) to the silica surface or covalently cross-linked to itself
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Chiral Phases for Separating Optical Isomers
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Enantiomers of an amino acid
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Volatile derivative for gas chromatography
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Structure of β-cyclodextrin, a cyclic sugarmade of seven glucose molecules
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Primary –OH groups lie on one face and the secondary –OH groupslie on the other face. The hydroxyls are capped with alkyl groups
to decrease the polarity of the faces
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Chlorinated pesticide impurity separated on the chiral stationary phase
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Chiral separation on a 25-m x 0.25 mm OT column; 0.25-μm phase:10% methylated β-cyclodextrin chemically bonded to PDMS
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Adsorbents Used for PLOT Columns
• Alumina• Silica gel• Porous polymers• Graphitized carbon blacks• Molecular sieves
– Inorganic (zeolites)– Organic (carbon)
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Structure of the Zeolite Molecular Sieve
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Packed Columns
• Provide greater sample capacity, but give– Broader peaks– Longer retention times– Less resolution
• Typically 3-6 mm in diameter and 1-5 m in length
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Alcohols separated on a 2 mm x 76 cm column with 20%Carbowax 20M on Gas-Chrom R
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Retention Index
• Relative retention times of polar and nonpolar solutes change with the polarity of the stationary phase
• On nonpolar stationary phases, solutes are eluted in order of increasing boiling points (retention is determined by the volatility of the solutes)
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Retention Index (cont.)
• On strongly polar stationary phases, the order of elution is determined by the intermolecular forces between the solutes and the stationary phase (hydrogen bonding, dipole-dipole)
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PDMS – nonpolarstationary phase
Poly(ethylene glycol) – strongly polarstationary phase
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Retention Index (cont.)
• The Kovats retention index, I, for a linear alkane equals 100 times the number of carbon atoms (for octane, I = 800; for nonane I = 900)
• A compound eluted between octane and nonane has a retention index between 800 and 900 computed by the formula
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( ) ⎥⎦
⎤⎢⎣
⎡−
−+=)('log)('log
)('log-(unknown)'log100ntNt
nttnNnIrr
rr
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n – the number of carbon atoms in the smaller alkane (8 in octane)N – the number of carbon atoms in the larger alkanet’r(n) and t’r(N) – the adjusted retention times of the smaller and larger alkane, respectively
The above formula is valid for isothermal conditions only.
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Example
tr(CH4) = 0.5 min; tr(octane) = 14.3 mintr(unknown) = 15.7 min; tr(nonane) = 18.5 minThe retention index for the unknown is
( ) 8368.13log0.18log8.13log-2.15log898100 =⎥⎦
⎤⎢⎣
⎡−
−+=I
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The Retention Index in Temperature-Programmed Chromatography
• Uses total (unadjusted) retention times and nottheir logarithms
• The value of IT will usually differ from the value of I measured under the same conditions
( ) ⎥⎦
⎤⎢⎣
⎡−
−+=)()(
)(-unknown)(100rr
rrT
ntNtnttnNnI
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Useful Practical Properties of I
• By definition a methylene group adds 100 to the retention index
• A functional group (phenyl, hydroxyl) adds an increment (Xn) to the retention index
• Generally the Xn are additive if the groups are separated by a few C atoms in a chain
• Retention indices are independent of flow rate and film thickness
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Useful Practical Properties of I (cont.)
• RI are independent of column dimensions and can be extrapolated even from packed column data to capillary columns
• RI is only slightly dependent on column temperature (generally within 5 units over a 50 °C temperature range)
• RI is a characteristic of the liquid phase type and the solute
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McReynolds Classification of Stationary Phases
• McReynolds selected 10 probe solutes of different functionality, each designated to measure a specific interaction with a liquid phase
• For each probe, a ΔI value is calculated:ΔI = Iliquid phase – Isqualane
• As ΔI increases, the degree of specific interaction associated with that probe increases
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McReynolds Classification of Stationary Phases (cont.)
• The cumulative effect, when summed for each of the ten probes, is a measure of overall “polarity” of the stationary phase
• In tables of McReynolds constants, the first five probes usually appear
• Each probe is assigned a value of zero with squalane as reference liquid phase
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2,6,10,15,19,23-Hexamethyltetracosane
The Structure of Squalane (C30H62)
Hydrogenated squalene from shark liver oil. Completelynonpolar. The only interactions with the solute are throughdispersion (Van der Waals) forces
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Practical Applications for McReynolds Values
• Comparison of phases for similarity• Ranking phases by degree of polarity:
– ΔI values between 0 and 100 – nonpolar phase– ΔI between 100+ and 400 – moderately polar– ΔI over 400 – a highly polar phase
• Prediction of analyte elution order: ΔI for the probe indicates the degree of shift from a boiling point order
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If we have an aromatic/alcohol coelution, switch from a PDEAS column to a LAC-2R-446 column (the shift of 61)
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Temperature Programming
• Used to solve general elution problem (GEP) for mixtures with a wide range of boiling points
• Temperature of a column is raised during the separation to increase solute vapor pressure and decrease retention times of late-eluting components
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Pressure Programming
• Increasing the inlet pressure increases the flow of mobile phase and decreases retention time
• At the end of a run, the pressure can be rapidly reduced to its initial value for the next run (no need for column cooling)
• Programmed pressure is useful for thermally labile analytes
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Carrier Gas
• Helium – most common, compatible with most detectors
• For FID – N2 gives a lower detection limit than He
• H2, He, and N2 give essentially the same Hmin at significantly different flow rates: uoptincreases in the order N2 < He < H2
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Carrier Gas (cont.)
• H2 cannot be used with an MS detector – it breaks down vacuum pump oil
• H2 and He give better resolution because the solutes diffuse more rapidly through them than through N2 (larger diffusion coefficients) – the Cm term is smaller
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Method Development in Gas Chromatography
Order of decisions:1. Goal of analysis2. Sample preparation3. Detector4. Column5. Injection
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Selecting the Column
• Stationary phase – nonpolar most useful• Diameter and length• The thickness of stationary phase• Thick-film, narrow-bore columns provide a
good compromise between resolution and sample capacity
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Choosing the Injection Method
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In a Nutshell
• Split – routine for introducing small sample volume into open tubular column
• Splitless – best for trace levels of high-boiling solutes in low-boiling solvents
• On-column – best for thermally unstable solutes and high-boiling solvents; best for quantitative analysis
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Split Injection
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Choosing the Injection Method (cont.)
• Split injection:– Concentrated sample (or gas analysis)– High resolution– Dirty samples (use packed liner)– Could cause thermal decomposition– Poor quantitative analysis– Less volatile components can be lost during
injection
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Choosing the Injection Method (cont.)
• Splitless injection:– Dilute sample– High resolution– Poor quantitative analysis (less volatile
components can be lost during injection)– Requires solvent trapping or cold trapping– Cannot be use with isothermal chromatography
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Choosing the Injection Method (cont.)
• On-column injection:– Best for quantitative analysis– Thermally sensitive compounds– Low-resolution technique
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MIBK
CH2Cl2
p-xylene
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