HPLC

1. Analytical Chemistry: Identification and
Quantitation of Compounds in Complex Mixtures
 The General Analytical Strategy
 Spectroscopic Methods
 Mass Spectrometry
 Sample Preparation Methods
 Quantitation
 Application to the Analysis of Flavonoids:
Mass Spectrometry

2. Figure 1. Common flavonoid structures.
O
O
OH
HO
OH
H
O
O
OH
O
sugar
OH
OH
O
OH
hhhOH
OH
Chalcone
Flavanone
Chalcone
Flavone O
O
OH
hhhHO
OH
OH
O
O
OH
hhhHO
OH
Flavonol
Flavone
O
O
OH
HO
OH
Isoflavone
Flavone O-glycoside
Example:
Common Flavonoid
Structures
Analytical Chemistry: Identification and Quantitation of Compounds
in Complex Mixtures

3. The complexity of the problem:
Many flavonoids are glycosylated
 Sugar linkage: O-glycosylflavonoids>>C-glycosylflavonoids
 Positions of glycosylation: 3-OH>7-OH>>3’, 4’, or 5-positions
 Level of glycosylation
 Number of different sugars involved
Common sugars found in flavonoid glycosides
glucose galactose
rhamnose arabinose xylose
OH
OH
OH
HO
O
OH
OH
OH
OH
HO
O
HO
OH
OH
OH
HO
O OH O
OH
OH
HO
OH
OH
OH
HO
O
O
1
2
3
4
5
7
8
2'
3'
4'
5'
6'
6
A C
B
flavonoid aglycone

4. THE ANALYTICAL STRATEGY
1) Evaluate the problem:
--pure component or mixture
--solid, liquid or gas
--organic, inorganic, elemental
--sample size and number of samples
--quantitative or qualitative analysis
--type of matrix
--requirements for accuracy and precision

5. Analytes of interest….
N
CH3
C
O
O
OCH2CH3
C
O
Hg H2SO4
C O
(a protein)

6.  2) Select the appropriate method.
 Select a sample preparation method:
extraction? dilution? acid/base conditions? filtration?
 If it is a mixture, select:
 GC (Gas Chromatography)
 HPLC (High Performance Liquid Chromatography)
 CZE (Capillary Zone Electrophoresis)
 For analysis, select:
 Classical "wet chemistry" methods (titrimetry, gravimetry)
 Electrochemical methods UV/Vis Molecular Absorption Spectroscopy
 Infrared Absorption Spectroscopy Molecular Fluorescence Spectroscopy
 Raman Scattering Spectroscopy Microscopy
 Nuclear Magnetic Resonance X-Ray Spectroscopy
 Electron Spectroscopy Atomic Spectroscopy
 Mass Spectrometry

7. Analytical Strategy continued…
3) Characterize the method with reference compounds
and standards.
Run controls. Establish accuracy and precision for
quantitative applications.
4) Construct a calibration curve and/or standard
addition method or use internal standard for
quantitative analysis.
5) Evaluate samples of interest. Repeat as necessary.

8. Spectroscopic
Method
Wavelength Range Energy Range of
Process
Analytical process Analytical
Information
UV-Vis
Absorption
180-750 nm 1.5 – 6.0 eV transitions of valence
electrons between
molecular orbitals
Quantitation
Infrared
Absorption
2.5 – 15 um
(2500 – 15,000 nm)
0.08 – 0.5 eV changes in
amplitudes of
vibrations of
molecules
Structural fingerprint
Mass
Spectrometry
NA Ionization = 7 – 10 eV,
Fragmentation = 2 – 6
eV above ionization
ionization and
dissociation
Structural fingerprint
and quantitation
NMR 0.6 – 10 m
(6 x 108
- 10 x 109
nm)
0.1 – 2 eV excitation of nuclear
rotational motion
Structural fingerprint
Overview of Spectroscopic Methods

9. Ground
state
Excited
state
CO
bond
length
120 pm 132 pm
CH
bond
length
110 pm 109 pm
H-C-H
angle
116.5o 119o
The absorption of energy….
C
O
H H
C
O
H H
hv

10. Spectrophotometer
Radiation Radiation Wavelength Wavelength Detector
source selector selector
Sample

11. Molecular UV-Vis Absorption
H
H
C O Formaldehyde:
..
.
.
.
.
C-H bonds are sigma
C=O bonds are sigma plus pi
O electrons are non-bonding




n
antibonding
antibonding
nonbonding
bonding
bonding
Energy
**
*
*
*
*
Absorption of UV or
visible light causes
“electronic transitions”
in which electrons are
excited to antibonding
orbitals.
e

12. Po P
.
.
.
.
. .
.
.
.
.
. .
.
.
.
.
.
.
.
.
T = P/Po A = log Po/P
where T = transmittance and
A = absorbance
Po and P represent the power of the
radiation before and after passing the sample
Absorption in Spectroscopy
Electromagnetic
Radiation
Sample

13. UV-Vis Spectra of Four Flavonoids
Rutin Quercetin glycoside
Ploridzin Anthocyanidin
O
HHO
OH O
ORutinose
OH
OH
3'
OH
O
HHO OH
O--D-Glucose
O
HHO
OH
OHH
OH
OH
+
O
HHO
O
OGlu
OH
OHH
OH
Adapted from FEBS Letters, 401 (1997) 78-82, Paganga and Rice-Evans.

14. Beer’s Law:
A = a b c = Absorbance
where a = absorptivity in L/(g-cm)
b = pathlength of radiation through sample in cm
c = concentration of sample in g/L

15.  Absorptivity: the probability that an analyte will
absorb a particular wavelength of energy
(also known as “extinction coefficient”)
--range from 0 – 100,000
--units of L/(g-cm) or L/(mol-cm)
--depends on presence of chromophores in the analytes

16. Absorbance  concentration
Absorbance
Wavelength in nm
Increasing concentration
of analyte  higher absorbance
A = a b c

17. UV-Vis Spectrometer

18. IR Absorption
C
H
O
H
Formaldehyde
Vibrational modes include stretching and
bending (twisting, rocking, scissoring,
wagging)
Stretching: change in distance
between atoms along interatomic axis
Bending: change in angle between two
bonds

19. Functional Group Frequency Range cm-1
Alkyl 2850 - 2960
Alkenyl C-H 3010-3095
Alkenyl C=C 1620 – 1680
Alkynyl C-H 3300
Alkynyl C=C 2100 – 2260
Aromatic Ar-H 3030
Aromatic substituents 690 – 840
Alcohols, Phenols 3200 – 3650
Carboxylic Acids 2500 – 3000
Carbonyls 1630 – 1780
Amines 3300- 3500
Nitriles 2220 - 2260
Characteristic IR Absorption Frequencies

20. Infrared Absorption Spectrum of Naringin
O
O
OH
O
O
CH2 OH
OH
OH
O
OH
CH3
O
OH OH
OH
4'
2
3
4
3'
C=O stretch
OH
aromatic
C-O
stretch
Adapted from Sadtler Index, 1973.

21. FTIR Spectrometer

22. Energy of nuclear
spin whose dipole
is aligned against
the magnetic field
Energy of nuclear
spin whose dipole
is aligned with
the magnetic field
 E
Relative energy
of nuclear spins
with no magnetic
field
C
O
H
H
B (applied magnetic
field)
Nuclear Magnetic Resonance

23. Type of Proton Chemical Shift (ppm)
Alkyl 0.8 – 1.7
On carbon adjacent to ether oxygen 3.3 – 3.9
On carbon adjacent to hydroxyl 3.3 – 4.0
On carbon adjacent to ketone 2.1-2.6
On carbon adjacent to aldehyde 9.5 – 9.6
Aromatic 6.0 – 9.5
Hydroxyl 0.6 – 9.0
Carboxylic acid 10 – 13
Amine 1.0 – 5.0
Phenolic 4.5 – 7.7
Typical NMR Proton Chemical Shifts

24. Proton NMR Spectrum of Morin
O
HHO
O
OH
OH
OHH
HO
3'
5'
6'
8
6
(Hydroxyl protons not shown, from 9 - 13 ppm)
H6’
H3’
H5’
H8 or H6
H8 or H6
Adapted from Biochem. Pharm., 59 (1995) 537-543, Wu et al.

25. NMR Spectrometer

26. Basic Components
Inlet System
sample introduction
Ion Source
ionizes sample
Ion Analyzer
sort ions by m/z
Ion Detector
“counts” ions
Data System
signal processor
Mass Spectrum
Vacuum chamber
Instrument Control Computer
controls timing and voltages
ion
optics
ion
optics
Overview of the Mass Spectrometer

27.  Electron Ionization: “EI”
Common Ionization Method for GC-MS
M = analyte
e = electron
F = fragment

28. Electron Ionization Mass Spectrum of Chalcone:
Molecular Weight 208 amu
O
131
77
89
105
152 165
179
193
207 208
Adapted from Rapid Commun. Mass Spectrom., 12 (1998) 139-143, Ardanaz et al.

29. e
O O
.+
O+ m/z 131
m/z 208
m/z 208
O
+.
m/z 105
O
+
m/z 77
CO
Fragmentation of Chalcone Ion

30. EI-Quadrupole MS

31. Electrospray ionization for Larger, Involatile Molecules
octapole ion
guides
heated
capillary
sheath gas
ESI
capillary
detector
skimmer exit
end-cap
entrance end-
cap
ring electrode
sample
capillary

32. M T
M = analyte molecule
T = target gas molecule
F = fragments of analyte
F1
F2
Collisional Activation Dissociation to Fragment Ions

33. Collisional Activated Dissociation
of Protonated Nomilin
O
O
O
O
CH3
CH3
H3C
H3C CH3
O
O
O
O
H3C
m/z 200 300 400 500
411
393
455
(M + H)+
515
231
455 -- loss of CH3COOH
411 -- loss of CH3COOH and CO2

34. Sample Preparation Methods:
Filtration
Centrifugation
pH Adjustment/Buffering
Extraction
Acid/Base Hydrolysis
Chromatography

35. Contaminants
Compounds of
Interest
1. Add Sample/Wash
Contaminants
2. Elute Compounds
of Interest
Solid-Phase Extraction

36. Gas chromatography for separation of volatile
analytes in mixtures
Retention time
Intensity
Capillary column

37. Pump
Column
Detector
Injector
Valve
Regulated
He Supply
Solvent
Reservoirs
HPLC Apparatus: for involatile analytes in mixtures

38. Si
CH3
CH3
Si
CH3
Si
H3C
Si
C18 Stationary Phase

39. Absorbance
Concentration
o
o
o
o
o
o
o
Calibration Curve
Quantitation

40. Typical Calibration Curves for Flavonoids
Adapted from: FEBS Letters, 401 (1997) 78-82, Paganga and Rice-Evans.

41. HO
OH
O
O
OH
OH
OH
HO
OH
O
O
OH
OH
quercetin (302)
kaempferol (286)
Flavonoids in Kale: An LC-MS/MS Study

42. Kale extraction
 Liquid extraction of flavonoids from kale
extract 1 aqueous phase collected
filter
N2 (l)
Kale leaves kale powder
1:1 acetone vortex centrifuge
supernatant
powder
1:1 30:70 acetone: water
2:1
chloroform
separation
Adapted from: Zhang, Satterfield, Brodbelt, Britz, Clevidence, Novotny Anal. Chem., 75 (2003) 6401-6407.

43.  Acid hydrolysis for cleaving flavonoid glycosides to
their aglycone forms
1 ml kale extract 1 mix 100°C 2h
+ kale extract 2
5 ml 2N HCl in 50:50 water:methanol
 Solid phase extraction for cleanup and concentration
condition kale extract 2 wash elute
C18 cartridge kale extract 3 (final)
Kale extraction (cont’d)
LC-MS/MS analysis

44. Flavonoid separation by HPLC
 Guard column: Waters Symmetry C18, 2.110 mm, 3.5 m
 Analytical column: Waters Symmetry C18, 2.150 mm, 3.5 m
 Mobile phase: Solvent A-water, solvent B-acetonitrile. 0-13 min: 30-100 B;
13-15 min: 100-30 B; 15-25 min: 30 B

45. O
O
O
H
OH
OH
OH
OH
C15H10O7 Mw=302
Identification of flavonoids by ESI-MS/MS:
CAD spectrum of quercetin
100 140 180 220 260 300 340
m/z
0
20
40
60
80
100
Relative
Abundance
107: C7H6O2 - CO - CO2
151: - C7H6O2 – CO
179: - C7H6O2
193 229 239
257: - CO2
273: - CO
301 (M - H+)

46. Detection limit by HPLC-ESI-MS: quercetin-~10 pg; kaempferol-~3 pg
Linear concentration range: 0.03-90 g/ml
Calibration curves of flavonoids by LCMS
0 20 40 60 80 100
0
2
4
6
8
10
Quercetin
y = 0.0556x + 0.199
R
2
= 0.9785
Kaempferol
y = 0.0635x + 0.0876
R
2
= 0.9972
area
ratio
to
int.
std.
flavonoid conc. (g/ml)
Kaempferol
y = 0.0635x + 0.0876
R2 = 0.9972
Quercetin
y = 0.0556x + 0.199
R2 = 0.9785

47. Quercetin in kale: 77 ppm
Kaempferol in kale: 235 ppm
Recovery: ~65%
AU
0.000
0.010
0.020
0.030
0.040
Minutes
4.00 8.00 12.00 16.00 20.00 24.00
quercetin
kaempferol
int. std.
0 4 8 12 16 20 24
Time (min)
0
20
40
60
80
100
Relative
Abundance
quercetin kaempferol
int. std.
Analysis of kale samples by LCMS
A. HPLC-UV chromatogram
B. TIC-MS chromatogram
Adapted from: Zhang, Satterfield, Brodbelt, Britz, Clevidence, Novotny Anal. Chem., 75 (2003) 6401-6407.

48. Flavonoids in Grapefruit:
Monitoring metabolites by LC-MS/MS
OH
O O
OH
OH
CH2
OH
O
OH
CH3
OH OH
O
O
O
OH
O
O
OH
O
O
OH
CH3
OH OH
O
O
OH
CH2OH
OH
OH
OH
O
HO O
OH
OH
COOH
OH
O
O
OH
O
HO
naringin (580)
narirutin (580)
Identification of metabolites
Pharmacokinetics
Bioavailability

50. Relative
Abundance
0
100
RT: 5.6
m/z: 447
RT: 9.7
m/z: 253
RT: 10.7
m/z: 351
Urine at t = 7.5 h
RT: 6.4
m/z 447
Analysis of urine by LCMS after consumption
of grapefruit juice
Time (minutes)
Zhang and Brodbelt, The Analyst., 129 (2004) 1227-1233.

51. Relative
Abundance
100
0
100
0
100
0
100
0
100
0
100
0
527
447 (-SO3)
527
255 (-NE)
351 (-GlcUA)
447 (-SO3)
351
271 (-SO3)
351
271 (-SO3)
351
271 (-SO3)
447
429 (-H2O)
175 (-NE)
271 (-GlcUA)
B) m/z 447 with RT = 6.4 min
C) m/z 351 with RT = 4.4 min
D) m/z 351 with RT = 9.1 min
E) m/z 351 with RT = 11.1 min
F) m/z 527 with RT = 8.2 min
G) m/z 527 with RT = 10.8 min
Fragmentation patterns of components
from previous LCMS chromatogram
A glucuronide
A sulfate
Another sulfate
Another sulfate
A glucuronide-
sulfate
Another sulfate

52. 0
1
2
3
4
0 5 10 15 20 25
Time (h)
Peak
Area
Ratio
naringenin glucuronide with RT = 6.2 min
naringenin glucuronide with RT = 6.6 min
Time plot of major metabolites in urine after consumption
of grapefruit juice (analysis by LC-MS/MS)