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12/1/2005 Chem 253 - Chapter 25 1

High Performance Liquid
Chromatography (HPLC)
Harris Chapter 25
12/1/2005 Chem 253 - Chapter 25 2
HPLC
 Separation of nonvolatile or thermally unstable
compounds. If the analyte/sample can be
found to be sufficiently soluble in a solvent
system, then that system can usually to used
as the m.p. in an HPLC separation.
 Common method used for analysis of
 Biological compounds
 Pharmaceuticals
 Low- or Non-volatile environmental cpds. e.g. PCB,
DDT
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LC Origins.
 Michael Tswett (1906) separation of plant pigments by organic
solvent mobile phase & chalk stationary phase.
 Martin and Synge (1941) liquid-liquid partition chromatography,
1952 Nobel Prize in chemistry.
 Other variants – Paper chromatography
Thin-layer chromatography (TLC)
Preparative column chromatography
Medium pressure chromatography
Ion-exchange chromatography*
Size-exclusion chromatography*
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HPLC components:
 Also an integrator usually records the detector
response.
 We will discuss each component, but let’s first
discuss the band broadening aspects of LC.
This discussion tells us why high pressures
are required for analytical separations.
Liquid
Mobile => Pump => Injection => Separation => Detector
Phase Valve Column
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Band Broadening in LC
 Back to the van Deemter Equation,
H = A + B/u + Cu
 Which of the three components is the largest
contribution to H? Consider the following:
 B/u effects – Diffusion is usually 100x less in liquids
than in the gas phase.
 Cu effects – By process of elimination we will assume
that mass transport effects are the largest contribution
to H in LC.
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Cu – Mass Transfer (MT) Effects.
 This is the effect of the kinetics of mass transfer to/from the
mobile phase to/from the stationary phase.
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Review of MT effects
MT in the m.p.
m
p
m D
f k d
C
( ) 2
α
Where dp is the di
meter of the p
cking p
rticle in LC,
Sm
ller dp incre
ses the surf
ce
re
/volume r
tio
nd thus
incre
ses M.T. in the m.p.
P
cking p
rticle
(silic
)
St
tion
ry
Mobile ph
se
Ph
se Flow
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pter 25 9
Sm
ller dp incre
ses the surf
ce
re
/volume
r
tio
nd thus incre
ses M.T. in the m.p.
 Volume = 4/3 π r3 Surface Area = 4 π r2
 Surface area/volume = 1/3×r
 The effect is dramatic in figures 25-2 & 25-3
 The cost of small acking articles is that the ressure required to
force liquid through the column follows as:
ΔP α 1/dp
3
 The typic
l p
rticle sizes in HPLC is 3-10 μm. In order to

chieve flow r
tes of 0.5 to 5 mL/min, for
10-30 cm column,
pressures of 70 to 400
tm (1000 to 6000 psi)
re required.
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pter 25 10
Figure 25-3
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pter 25 11
Figure 25-2
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pter 25 12
HPLC pumps
 Requirements for HPLC
• pressures to 6000 psi
• pulse free, prevents remixing of solutes
• control flow r
te from 0.1 to 10 mL/min
 Types of HPLC pumps
 Reciproc
ting pumps most commerci
l systems
re
b
sed on this design.
 Syringe pumps
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pter 25 13
Reciproc
ting pumps
 Dis
dv
nt
ges – pulses from single piston. See
du
l piston design in figure 25-14 of your text.
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pter 25 14
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pter 25 15
Syringe Pumps
 Pulse-free
output,
limited
mobile
ph
se
c
p
city.
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pter 25 16
Pulse D
mpers.
 Di
phr
gm:
 Coil – 3 to
20 meters in
length:
Gel
St
inless
Steel
J
cket
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pter 25 17
http://www.chromtech.com/2001c
t
log/Sep
r
tePgs/303.pdf
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pter 25 18
Injection (S
mpling) V
lves
Introduces s
mple to the column.
Mobile => Pump => Injection => Column
Ph
se V
lve
V
lve consists of
rotor
nd st
tor (st
tion
ry b
ck-pl
ne). See
schem
tics below
nd figure 25-15 of your text.
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pter 25 19
Fl
sh Anim
tion of Injection V
lve
 http://www.restek.com/info_sixport.
sp
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pter 25 20
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pter 25 21
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pter 25 22
www.vici-jour.se/ 10
ccessories_06.html
 HPLC Syringes – unbeveled tips
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pter 25 23
Precolumn filters
- 2 types porous st
inless frit 0.5 to 2 μm or
little piece of
s
crifici
l column.
Injection => Precolumn => Column => Detector
V
lve
Prevents the cont
min
tion of the expensive
n
lytic
l columns
with fine p
rticles th
t c
n eventu
lly clog the mobile ph
se
flow.
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pter 25 24
An
lytic
l Columns
 Common configur
tion to the right.
 Gener
lly st
inless steel
nd teflon
components.
 The st
tion
ry ph
se p
ckings
re
microporous silic
2-10 μm in di
meter.
 Unmodified silic
is very pol
r.
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pter 25 25
SiO2
OH OH OH OH OH
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pter 25 26
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pter 25 27
Fig 25-5 silic
p
rticles
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pter 25 28
 Where R c
n v
ry, typic
lly…. C18, C8, -CH2-C6H5
 -{CH2}3-NH2, -{CH2}5-CN
SiO2
OH OH OH OH O
Cl
Me Me
R
Me Me
R
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pter 25 29
Fig 25-8 protection from hydrolysis
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pter 25 30
“Reversed” & “Norm
l” Ph
se
Sep
r
tions.
 Norm
l Ph
se – Pol
r s.p. & Nonpol
r m.p.
 E
rly HPLC work w
s conducted on unmodified
silic
(highly pol
r) this required the use of nonpol
r
mobile ph
ses in order to get
dequ
te
sep
r
tions.
 Reversed Ph
se – Nonpol
r s.p. & Pol
r m.p.
 L
ter HPLC rese
rch led to silic
C18 modified
surf
ces which required the use of pol
r mobile
ph
ses.
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pter 25 31
P
rtition vs. Adsorption
Chrom
togr
phy
 Adsorption Chrom
togr
phy – B
sed on the unmodified silic
surf
ce,
which is very pol
r. Solute
n
lyte species is
dsorbed to this surf
ce.
 P
rtition Chrom
togr
phy – B
sed on modified silic
surf
ces. The C-18
bonded ph
ses dissolve r
ther th
n
dsorb the
n
lyte solute species, thus
p
rtitioning of the solute between two essenti
lly liquid ph
ses.
SiO2
C-18
function
l
groups
Am
As
Am
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pter 25 32
When should we use P
rtition (nonpol
r s.p.) vs. Adsorption
(pol
r s.p.) ph
ses in chorm
togr
phic sep
r
tions?
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pter 25 33
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pter 25 34
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pter 25 35
HPLC solvents.
 Oper
tor experience pl
ys
l
rge role in the design

nd selection of
n HPLC solvent system.
 Gener
lly we w
nt
signific
nt difference between
the pol
rities of the s.p.
nd the m.p., the re
son
being is th
t sep
r
tion is b
sed on solubility
differences between the m.p.
nd s.p. (p
rtitioning)
K = Cs/Cm
 Almost
ll reversed ph
se sep
r
tions (pol
r m.p. &
nonpol
r s.p.) c
n be c
rried out with combin
tion of

cetonitrile (CH3CN),
nd/or meth
nol,
nd w
ter

s
m.p.
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pter 25 36
W
ter is the most pol
r of
ll possible
solvents
UV cutoff is
import
nt to keep in
mind when we get to
detectors
Incre
sing
Pol
rity
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pter 25 37
Pr
ctic
l notes
Sep
r
tion of most org
nic compounds c
n be
h
ndled by C-18 st
tion
ry ph
ses.
Most mobile compositions c
n be h
ndled by either
CH3CN/H2O or CH3OH/H2O
Solvents must be miscible e.g. w
ter/eth
nol. An
immiscible solvent system such
s w
ter/toluene
would cre
te
mess in the column!
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pter 25 38
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pter 25 39
Mobile Ph
se Compositions
 Isocr
tic Elutions – Const
nt solvent composition, mobile ph
se
pol
rity st
ys const
nt throughout elution process. This is
equiv
lent to isotherm
l sep
r
tions in GC.
 Gr
dient Elutions – Mobile ph
se composition (
nd thus pol
rity)
v
ries throughout elution. This is equiv
lent to temper
ture
progr
mming in GC.
 Consider the series of isocr
tic elutions on the next p
ge.
 We c
n see th
t
n efficient sep
r
tion is never
chieved.
A = H2O B = CH3CN
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pter 25 40
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pter 25 41
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pter 25 42
The pump system for gr
dient elution is more expensive th
n for
isocr
tic systems. The metering v
lves require electronic control:
 Solute elution times under gr
dient progr
ms
re not
s
reproducible
s isocr
tic elutions.
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pter 25 43
Column He
ters in HPLC
 He
ting the column in HPLC will improves m
ss tr
nsport, decre
ses the Cu term i
n the v
n
Deemter equ
tion.
 Consider the following ex
mple:
 Notice th
t the tr for e
ch solute ch
nges with temper
ture, this is bec
use of
the solubility
ch
nges we should expect with T.
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pter 25 44
Detectors in HPLC
 Ide
l Ch
r
cteristics
 Univers
l
 Sm
ll volume, prevents remixing & b
nd
bro
dening
 F
st response to flowing system
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pter 25 45
Refr
ctive Index (RI) detector
 Ne
rly univers
l but poor detection limit
 P
sses visible light through 2 comp
rtments, s
mple &
reference.
 When the solvent composition
re the s
me the light p
ssed
through the comp
rtments the light be
m th
t p
sses through is
recorded
s zero.
 When
solute is in the s
mple comp
rtment, refr
ctive index
ch
nges will shift the light be
m from the detector.
 Limit of detection (LOD) 10 ng of solute
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pter 25 46
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pter 25 47
UV-vis
bsorb
nce detector
 B
sed on electronic tr
nsitions within molecules. (Ch
pter 19)
 - Most common type of detector for LC
 - Fixed w
velength, Hg l
mp 254 nm (π => π*)
 - Tunable wavelength, selectable for secific wavelengths,
monochromators or filters. Still limited to single wavelegths.
 - 1 g LOD
 Solvent limitations with UV-vis abs. Detectors
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Diode array detector
 See lecture notes
on the diode array
sectrometer,
Chater 21.
 Allows for the
recording of the
entire sectrum of
each solute as it
assed through the
diode array
detector
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Tyical Diode Array Signal Outut
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Fluorescence Detectors
 Review - based on emission of excited
state molecules.
 Detector 900 from excitation axis.
 LOD 10 fg
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From Chater 18 - Emission Instrumentation
 Note that the signal is measured at 900 relative to the light source
axis. Why?
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IR Detectors
 FT-IR allows for sectrum records of
flowing systems analgous to the diode
array system.
 Water/alcohols can be major
interferences to solute detection
 LOD 100 ng
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Evaorative Light Scattering Detector
 Resonds to any analyte that is significantly
less volatile than the mobile hase.
 Eluate is mixed with N2(g) and forms a fine
mist.
 Solvent (m..) evaorates leaving fine
articles of analyte. The articles themselves
are detected by light scattering.
 Resonse is roortional to analyte mass.
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Electrochemical Detectors
 Based on amerometric resonse of
analyte to electrode usually held at
constant otential.
 If the analyte is electroactive, can be
highly sensitive since resonse is based
on a surface henomenon rather than a
solution bulk roerty (e.g. UV-vis
absorbance)
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LC-MS
 LOD 1 g
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Selected ion-monitoring focuses the mass sec onto one
articular m/e ratio. S/N enhancement occurs when scanning mode is off.
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Summary of LC Detectors
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