Download 2015/1/6 1 Chapter 28 Liquid Chromatography High

2015/1/6
Chapter 28 Liquid Chromatography
High-performance Liquid Chromatography
(HPLC)
Types classified by separation mechanism or by stationary
phase:
1) Partition chromatography: Normal phase or Reversed phase.
2) Adsorption or liquid-solid chromatography: replaced by normal phase.
3) Ion-exchange or ion chromatography: for ionic compounds.
4) Size-exclusion chromatography: for high MW compounds (polymers).
5) Affinity chromatography: for biomolecules.
6) Chiral chromatography: for separating of enantiomers.
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28A Scope of HPLC
FIGURE 28-1 Selection of
LC modes. Methods can be
chosen based on solubility
and molecular mass. In most
cases for nonionic small
molecules (M < 2000),
reversed-phase methods are
suitable. Techniques toward
the bottom of the diagram
are best suited for species of
high molecular mass (M >
2000).
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28B Column Efficiency in LC
Effects of stationary phase particle size (packing).
van Deemter equation: H = A + (B/u) + CSu + CMu
TABLE 26-3 Processes That Contribute to Band Broadening
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FIGURE 28-2 Effect of particle size of packing and flow rate on plate height H in LC.
Column dimensions: 30 cm × 2.4 mm. Solute: N, N’-diethyl-p-aminoazobenzene.
Mobile phase: mixture of hexane, methylene chloride, isopropyl alcohol.
van Deemter equation: H = A + B/u +CMu + CSu
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28C LC Instrumentation:
FIGURE 28-3 Block diagram showing components of a typical
apparatus for HPLC.
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From MeOH/H2O = 40/60
FIGURE 28-4 Improvement in separation effectiveness by gradient elution. Column: 1 m × 2.1
mm inside-diameter, precision-bore stainless steel; packing: 1% Permaphase® ODS(C18).
Sample: 5 μL of chlorinated benzenes in isopropanal. Detector: UV photometer (254 nm).
Conditions: temperature, 60℃, pressure, 1200 psi.
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MeOH/H2O = 50/50
FIGURE 28-4 (continued)
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28C-2 Pumping Systems (5 requirements)
• Reciprocating Pumps
• Displacement pumps (limited capacity ~250 mL)
FIGURE 28-5 A reciprocating pump for HPLC.
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28C-3 Sample-Injection Systems:
* Sampling loop
* Auto injector
FIGURE 28-6 A sampling loop (1-100 L) for
LC. With the valve handle as shown on the
left, the loop is filled from the syringe, and
the mobile phase flows from pump to column.
When the valve is placed in the position on
the right, the loop is inserted between the
pump and the column so that the mobile
phase sweeps the sample onto the column.
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28C-4 Columns for HPLC
1) Analytical columns （4.6mm x
10 or 15 cm; microcolumn: 3-7.5 cm)
2) Guard columns
3) Preparative columns（製備型）
microcolumn
28C-5 Column Packings
1) Pellicular particle (薄殼型顆粒）
2) Porous particle (3-10 m)
Silica, alumina, or cross-linked
polystyrene
FIGURE 28-7 High-speed isocratic separation. Column dimensions: 4 cm long, 0.4
cm inside diameter; packing: 3-μm spherisorb; mobile phase: 4.1% ethyl acetate
in n-hexane. Compounds: (1) p-xylene, (2) anisole, (3) benzyl acetate, (4) dioctyl
phthalate, (5) dipentyl phthalate, (6) dibutyl phthalate, (7) dipropyl phthalate, (8)
diethyl phthalate.
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28C-6 Detectors
Bulk-property detector: refractive index, dielectric constant, density,…
Solute-property detector: absorbance, fluorescence, ….
TABLE 28-1 Performance of HPLC Detectors
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UV Absorption Detectors with Filters:
(Chapter 7C: wavelength selectors, page 175)
1) Mercury lamp sources with filters (interference filter or absorption filter).
2) Deuterium or tungsten filament sources with interference filters.
3) Scanning spectrophotometer with grating optics.
4) Photodiode-array detector.
Quartz cell
Io
I
c
b
Absorbance (吸收度):
A = log(Io/I) = - log(I/Io) = -logT
Beer's Law: A = bc
T: transmittance
: molar absoyptivity
b: path length
c: molar concentration
Flow-through cell: 1-10 L, 2-10 mm
FIGURE 28-8 A UV-visible absorption cell for HPLC.
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n’ = 2d/cos
n’ = 2d
’n
 = 2dn/n
FIGURE 7-13 Transmission characteristics
of typical Interference filters.
FIGURE 7-12 (a) Schematic cross section of an interference filter. Note that the drawing
is not to scale and that the three central bands are much narrower than shown. (b)
Schematic to show the conditions for constructive interference.
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FIGURE 7-18 Two types of monochromators (單色器): (a) Czerney-Turner grating
monochromator and (b) Bunsen prism monochromator. (In both instances, λ1 >λ2.)
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FIGURE 13-25
A multichannel diode-array spectrophotometer, the Agilent Technologies 8453.
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Ch13 An Introduction to Ultraviolet-Visible Molecular Absorption Spectrometry
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FIGURE 28-9 Absorption spectra of the eluent from a mixture of three steroids taken
at 5-second intervals.
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Fluorescence Detectors:
high sensitivity, one order higher than absorption detectors.
1) Mercury excitation source + filters to isolate emitted radiation
2) Xenon source + grating monochromator to isolate emitted radiation
3) Laser-induced fluorescence
* Analysis of pharmaceuticals, natural products, clinical samples, and
petroleum products.
* Non-fluorescing samples can be treated with reagents to form as
fluorescent derivatives.
Ex.: Dansyl chloride reacts with 1o and 2o amines, amino acids, and phenols to
produce stable fluorescent compounds.
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FGIURE 15-10 A typical fluorometer.
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FIGURE 7-29 A phototube and op amp
readout.
FIGURE 7-31 Photomultiplier tube.
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FIGURE 15-11 A spectrofluorometer.
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Refractive-index Detectors (RI) :
1)
2)
3)
4)
general detector (respond to nearly all solutes).
highly temperature dependent.
not as sensitive as other detectors.
not compatible with gradient elution.
FIGURE 28-10
Schematic diagram of a differential refractive-index (RI) detector.
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FIGURE 28-13 Block diagram of an LC/MS system. The effluent from the LC column is
introduced to an atmospheric pressure ionization source, such as an electrospray or a
chemical ionization source. The ions produced are sorted by the mass analyzer and
detected by the ion detector.
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28D Partition Chromatography
Liquid-liquid columns
Liquid-bonded-phase columns
Rigid silica: 1.5~10 m (3- and 5-m最常用)
Silanol: 8 mmol/m2, 100-300 m2/g
Determine polarity of stationary
phase.
Silanization: limited to
4 mmol/m2, capped by
further reaction with
chlorotrimethylsilane.
Normal-phase: polar stationary phase (R: contains polar CN, diol, amino, and
dimethylamino); relatively non-polar mobile phase (ethyl ether, chloroform, and
n-hexane.
Reversed-phase: no-npolar stationary phase [n-octyl (C8), n-octyldecyl (C18)];
relatively polar mobile phase (water + CH3OH, acetonitrile, or tetrahydrofuran).
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A
M
S
FIGURE 28-14 Relationship between polarity and elution times for normal-phase
and reversed-phase chromatography.
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Longer chain length:
* more retentive
* larger capacity
FIGURE 28-15 Effect of chain length on performance of reversed-phase siloxane
columns packed with 5-μm particles. Mobile phase: 50:50 methanol-water. Flow
rate: 1.0 ml/min.
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28D-2 Method Development in Partition Chromatography
Column Selection: the polarity of the stationary phase is matched roughly with
that of the analytes; a mobile phase of considerably different polarity is then used
for elution.
In summary, polarities for solute, mobile phase, and stationary phase must be
carefully blended if good partition chromatographic separations are to be realized
in a reasonable time.
What’s polarity?
Hildebrand:
Ecoh = Hvap – RT (Cohesive energy)
CED = Ecoh/V (Cohesive Energy Density)
 = (CED)1/2 (Solubility Parameter
No specific chemical interactions:
Hsoln = 12 (1 - 2)2 ≧ 0
Gmix = Hmix - T Smix
溶劑之溶解參數()
n-Hexane
n-Octane
Cyclohexane
Benzene
Toluene
Acetone
Methylethylketone (MEK)
Methyl acetate
Ethyl acetate
Butyl acetate
Tetrahydrofuran (THF)
Methanol
Ethanol
n-Butanol
Water
(cal/ml)1/2
7.3
7.6
8.2
9.2
8.9
9.8
9.3
9.6
9.1
8.5
9.2
14.5
12.7
11.4
23.4
Rs = (√N/4)[(-1)/[kB/(1+kB)]
Mobile-phase selection:
1) Vary k between 2-10 by altering
mobile-phase polarity.
2) Vary : altering mobile-phase
composition or using different
column packing.
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Polarity index P’ is based on solubility measurements in three
solvents: dioxane (a low dipole proton acceptor), nitromethane (a
high dipole proton acceptor), and ethyl alcohol (a high dipole
proton donor).
dioxane
nitromethane
ethyl alcohol
Mixture: PAB’ = APA’ + BPB’
Typically, a two-unit change in P’ results (very roughly) in a ten-fold
change in k.
Normal phase: k2/k1 = 10(P1’ – P2’)/2
Reversed phase: k2/k1 = 10(P2’ – P1’)/2
TABLE 28-2 Properties of Common Chromatographic Mobile Phases
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FIGURE 28-16 Systematic approach to the separation of six steroids. The use of water to
adjust k is shown in (a) and (b). The effects of varying α at constant k are shown in (b), (c),
(d), and (e). column: 0.4*150 mm packed with 5 μm C8 bonded, reversed-phase particles.
Temperature: 50℃. Flow rate: 3.0 cm3/min. Detector: UV 254 nm. THF= tetrahydrofuran.
CH3CN = acetonitrile. Compounds: (1) prednisone, (2) cortisone, (3) hydrocortisone, (4)
dexamethasone, (5) corticosterone, (6) cortoexolone.
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Solvent selectivity triangle
OEt2
Reversed-phase
Normal-phase
CH2Cl2
CHCl3
Use hexane to adjust k.
FIGURE 28-17 Solvent selectivity triangle for optimization of reversed-phase
separations. Ten mixtures of the three organic solvents (methanol, acetonitrile,
and tetrahydrofuran) are shown with the relative proportions indicated in
parentheses (MeOH, MeCN, and THF). Water is used to maintain solvent strength
and keep the k value within an appropriate range.
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FIGURE 28-16 (continued)
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TABLE 28-3 Typical Applications of Partition Chromatography
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FIGURE 28-18(a) Typical applications of bonded-phase chromatography.
(a) soft-drink additives. Column: 4.6 × 250 mm packed with polar (nitrile)
bonded-phase packing. Isocratic elution with 6% AcOH to 94% H2O.
Flow rate 1.0 mL/min.
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FIGURE 28-18(b)
Organophosphate insecticides. Column
4.5 × 250 mm packed with 5 μm C8
bonded-phase particles. Gradient
elution: 67% CH3OH to 33% H2O, to
80% CH3OH to 20% H2O.
Flow rate: 2 mL/min. Both used 254nm-UV detectors.
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Derivative formation
FIGURE 28-19 Chromatogram of orthophthalaldehyde derivatives of 30
amino acids of physiological importance. Column: 5 μm C18, reversedphase. Solvent A: 0.05 M Na2PHO4, pH 7.4, 96:2:2 CH3OH, THF, H2O.
Fluorescence detector: excitation 334 nm; emission 425 nm.
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Post-column reaction with
colorimetric reagent.
Pre-column derivative formation
Purple color
http://en.wikipedia.org/wiki/Ninhydrin
Excitation: 334 nm
Emission: 425 nm
http://www.biotek.com/resources/articles/total-protein-opa.html
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Ion-pair chromatography
(paired-ion chromatography)
Ion-pairing agent: large organic
counterion, such as RSO3-M+ or
R4N+X-
(1)
(2)
FIGURE 28-20 Chromatograms illustrating separations of mixtures of ionic and
nonionic compounds by ion-pair chromatography. Compounds: (1) niacinamide, (2)
pyridoxine, (3) riboflavin, (4) thiamine. At pH 3.5, niacinamide is strongly ionized, and
riboflavin is nonionic. Pyridoxine and thiamine are weakly ionized. Column: μBondapak, C18, 4 mm*30 cm. Mobile phase: (a) MeOH and H2O with C7-alkyl
sulfonate; (b) MeOH and H2O with C5-alkyl sulfonate; (c) MeOH and H2O with 1:1
mixture of C5- and C7-alkyl sulfonates.
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Chiral Chromatography
1) chiral stationary phase
2) chiral resolving agent
A
D
B
C
D
E
E
F
F
R
S
Three-point interaction
FIGURE 28-21 Chromatogram of a racemic mixture of N-(1-naphthyl)leucine
ester on a dinitrobenzene-leucine chiral stationary phase (CSP). The R and S
enantiomers are seen to be well separated. Column: 4.6 × 50 mm; mobile
phase, 20% 2-propanol in hexane; flow rate 1.2 mL/min; UV detector at 254 nm.
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28F Ion Chromatography
FIGURE 28-22 Structure of a cross-linked polystyrene ion-exchange resin. Similar
resins are used in which the – SO3–H+ group is replaced by –COO-H+, –NH3+OH-,
and –N(CH3)3+OH- groups.
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Na+ + HCO3-
Na+(aq) + HCO3-(aq)+ Resin-H+(s)
Resin-Na+(s) + H2CO3(s)
Cation-exchange resin: -SO3H
HCl
Resin-Na+(s) + H+ + Cl-
Resin-H+ + Na+Cl-
FIGURE 28-23 A micromembrane suppressor. Eluent flows through a narrow
channel that contains a plastic screen that reduces the void volume and appears to
increase mass-transfer rates. The eluent is separated from the suppressor solution
by 50-μm exchange membranes. Regenerant flow is in the direction opposite to
eluent flow.
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FIGURE 28-24 Typical applications of ion
chromatography. (a) Separation of
anions on an anion-exchange column.
Eluent: 0.0028 M NaHCO3 to 0.0023 M
Na2CO3. Sample size: 50 μL. (b)
Separation of alkaline earth ions on a
cation-exchange column. Eluent: 0.025
M phenylene-diamine dihydrochloride to
0.0025 M HCl. Sample size: 100μL.
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FIGURE 28-25 Separation of amino acids on an ion-exchange column. Packing
cation exchange with particle size of 8 μm. Pressure: 2700 psi.
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FIGURE 28-26
An ion-exclusion chromatogram for a mixture of six weak acids.
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28G Size-Exclusion Chromatography (SEC)
Column Packings: 5-10 m of polymer beads or silica-based particles.
Hydrophobic packing: gel permeation chromatography (GPC).
Hydrophilic packing: gel filtration chromatography.
Total volume: Vt = Vg + Vi + Vo
elution volume: Ve = Vo + KVi → distribution constant K = (Ve-Vo)/Vi = cS/cM (K= 0~1)
TABLE 28-4 Properties of Typical Packings for Size-Exclusion Chromatography
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K=0
FIGURE 28-27 (a) Calibration
curve for a size-exclusion
column. (b) Chromatogram
showing peak A containing
all compounds with molecular
masses greater than the
exclusion limit, peaks B and C
consisting of compounds within
the selective permeation region,
and peak D containing all
compounds smaller than the
permeation limit.
K=1
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Applications of SEC:
1) Separation of high MW natural-prroduct from low MW species.
2) Separation of homologs and oligomers.
3) Rapid determination of MW and MWD of polymers or natural products.
FIGURE 28-28 Applications of size-exclusion chromatography.
(a) Separation of fatty acids. Column: polystyrene based, 7.5 × 600 nm, with
exclusion limit of 1 × 103. Mobile phase: tetrahydrofuran. Flow rate: 1.2 mL/min.
Detector: refractive index. (b) Analysis of a commercial epoxy resin (n = number of
monomeric units in the polymer). Column: porous silica 6.2 × 250 mm. Mobile
phase: tetrahydrofuran. Flow rate: 1.3 L/min. Detector: UV absorption.
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Molecular Weight of Polymers
常用平均分子量定義:
• Number-average molecular weight (colligative properties)
Mn= W/N = ∑(NiMi)/ ∑Ni = ∑NiMi
• Weight-average molecular weight (light scattering)
Mw = ∑(NiMi2)/ ∑(NiMi) = ∑(wiMi)/ ∑wi = ∑wiMi
• Z-average molecular weight
Mz = ∑(NiMi3)/ ∑(NiMi2) = ∑(wiMi2)/ ∑(wiMi)
• Viscosity-average molecular weight
Mv = [∑(NiMi1+a)/ ∑(NiMi) ]1/a

Polydispersity Index: PDI = Mw/Mn
Mn < Mv < Mw < Mz < Mz+1
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Molecular Weight and MWD by Gel Permeation Chromatography
(GPC)
Broken gel particle
TEM of gel particle
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Flow system of GPC
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