summary of analytical separation methods 3e bach chemistry part 2
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Course
Analytische scheidingsmethoden (C003986)
Institution
Universiteit Gent (UGent)
Summary of analytical separation methods, part 2 (Gas chromatography). Will be taught in the 3rd bachelor, 1st semester at Ghent University, in chemistry. There may be minor errors, feel free to send a message because I still have my own summary with extra notes on it.
H2 High performance Liquid
Chromatography (HPLC)
2.1 Introduction
moleculen met > 1000 g/ mol, thermisch labiele moleculen niet meer geschikt
voor GC ⇒LC
from LC to HPLC
Column chromatography HPLC
inexpensive (+) expensive (-)
Easy preparative work (+) Automated preparative separation
“new column” for each analysis re-use of columns (>1000 analyses)
Large particles size (20-100 μm) Particle size 1.7-7 um
Low pressure (gravity or 1 or 2 bar High applicable pressures (up to
pressure) 1300 bar)
Off line detection On-line (continuous) detection
Injection difficult to reproduce accurate and precise analysis
Sample can’t be recovered Sample can be recovered
Poor for quantitation Excellent for quantitation
Only normal phase Many separation modes
simple (+) Automation, versatile
2.2 Principles & theoretical aspects
2.2.1 Column efficiency
open capillaries (used in GC) not suitable for LC because of slow difussion of molecules in
liquids ⇒
columns packed with irregular of spherical particles (often silica) (dia: 2-20 μm)
→ minimal plate height: Hmin ≈ 2dp with dp = diameter of particle
→ efficiency in packed columns independent of column diameter
→ max plate number (column efficiency): N = L
H
≈ L
2dp
met
L= column length
→ H = length of one plate, height equivalent of one theoretical plate = HETP packed
column
particle size reduction
H2 High performance Liquid Chromatography (HPLC) 1
, 1. see y-axis ⇒ verification Hmin ≈ 2dp
2. slope in high velocity area becomes larger as function of particle
diameter ⇒
so using 2/3 μm: curves ± flat over broad range ⇒
elution speeds can be increased without loss of efficiency (fast
analysis)
3. larger particles: less theoretical plates (voor een lengte-eenheid
Van Deemter curve van een kolom)
→ difficult to reach very high plate numbers with HPLC (± 25000 in
general columns) because plate number N limited by pressure drop
over the column (ΔP )
ϕηL ϕ0 ηNH
ΔP = u= u [Darcy equation]
2 d2p
dp
→ Lot of E required to mobilize liquid through densely packed bed
ϕ0 = dimensionless constant,
→ higher velocity for smaller particle sizes
η= viscosity of the mobile
phase, dp = particle size, u=
lineair velocity (cm/s)
Consequences
HPLC: if column is blocked (contamination) or packed with to small particles: higher pressures
necessary to move mobile phase at correct velocity ⇒
introduction of Ultra High Performance Liquid
Chromatography (UHPLC) (smaller particles used, typical ∼ 1.8μm+ shorter columns): more
plates introduced in less analysis time (shorter columns + flatter VD curve obtain UHPLC ⇒
chromatograms in about 1/7th to 1/10th of the time necessary to record an HPLC chromatogram) =
speed of analysis main benefit (Hmin is lower: higher efficiency for same length compared to HPLC),
but higher pressure drop [p.125]
lowering η→ ηof liquid decreases with increasing T; so use of higher T in HPLC: use longer
columns (!! for gases it increases with increasing T)
2.2.2 Influence of column diameter on efficiency
H independent of internal diameter for packed columns, but reduction of column diameter → mobile
phase consumpton reduces (quadratically) ⇒
to obtain same lineair velocity in narrower column:
MC 2
flowMC = flowCC ( i.d.
i.d.CC )
(MC: microcolumn dia , CC: conventional column dia)
increase of sensitivity in combination with concentration sensitive detection when reducing column
diameter (HPLC-ESI-MS, HPLC-UV-MS)
i.d.CC 2
sensMC = sensCC ( i.d.
MC
)
2.3 Modes in LC
H2 High performance Liquid Chromatography (HPLC) 2
, subdivision based on type of interaction
2.3.1 Normal phase LC (Adsorption chromatography, NPLC)
→ oldest but poorer reliability, asymmetric peak shape + longer column regeneration times
→ requires use of organic solvents only (less green + more expensive compared to reversed phase LC)
→ Polar stationary phase - organic (apolar-medium polar) mobile phase
separation based on different affinity for surface of adsorbent → hydroxylgroups are the active sites
here affinity of molecules for the polar surface (determined through their polarity: higher polarity:
higher interaction ⇒
longer retention (denk aan TLC op polair eluens bv)
position of groups important too ! (eg meta, para op benzeen ⇒ different order of eluting)
interaction with SiO2 (silicagel) or Al2O3 (aluminiumoxide)
⇒
eluent strength (ϵ) adsorption energy of eluent / unit of
area of specific adsorbent, eluotropic series (arranged
if retention factor too small with specific eluens ⇒
⇒
according to increasing ϵ)
decrease in ϵby 0.05, kx3
water problem
→ water (content) in eluent adsorbed strongly at surface & controls retention of components
→ adsorption chromatography becomes irreproducible due to the variable moisture content in the
atmosphere !
H2 High performance Liquid Chromatography (HPLC) 3
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