an introduction to chromatographythe retention index in temperature-programmed chromatography •...

Gas Chromatography

Schematic Diagram of a Gas Chromatograph

Open Tubular Columns

• The vast majority of analyses use long narrow open tubular columns made of fused silica (SiO2) and coated with polyimide

Porous-layer open tubular column (porous carbon)

Chromatogram of vapors from the headspaceof a beer can on a porous carbon column

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

Effect of OT column inner diameter on Rs

Resolution Increases in Proportion to NN

N

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

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

Effect of Stationary Phase Thickness on OT Column Performance

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

Chiral Phases for Separating Optical Isomers

Enantiomers of an amino acid

Volatile derivative for gas chromatography

Structure of β-cyclodextrin, a cyclic sugarmade of seven glucose molecules

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

Chlorinated pesticide impurity separated on the chiral stationary phase

Chiral separation on a 25-m x 0.25 mm OT column; 0.25-μm phase:10% methylated β-cyclodextrin chemically bonded to PDMS

Adsorbents Used for PLOT Columns

• Alumina• Silica gel• Porous polymers• Graphitized carbon blacks• Molecular sieves

– Inorganic (zeolites)– Organic (carbon)

Structure of the Zeolite Molecular Sieve

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

Alcohols separated on a 2 mm x 76 cm column with 20%Carbowax 20M on Gas-Chrom R

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)

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)

PDMS – nonpolarstationary phase

Poly(ethylene glycol) – strongly polarstationary phase

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

( ) ⎥⎦

⎤⎢⎣

⎡−

−+=)('log)('log

)('log-(unknown)'log100ntNt

nttnNnIrr

rr

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.

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

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

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

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

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

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

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

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

If we have an aromatic/alcohol coelution, switch from a PDEAS column to a LAC-2R-446 column (the shift of 61)

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

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

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

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

Method Development in Gas Chromatography

Order of decisions:1. Goal of analysis2. Sample preparation3. Detector4. Column5. Injection

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

Choosing the Injection Method

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

Split Injection

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

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

Choosing the Injection Method (cont.)

• On-column injection:– Best for quantitative analysis– Thermally sensitive compounds– Low-resolution technique

MIBK

CH2Cl2

p-xylene