Liquid Chromatography
HPLC/UPLC
Kevin Lankford
Chem 6200
Topics in Analytical
Liquid Chromatography
There are many ways to classify
Liquid Chromatography (LC).
Normally it is described by the
nature of the stationary phase
and separation process.
1. Ion exchange chromatography
2. Size exclusion chromatography
3. Adsorption chromatography
Reference 1
Ion exchange
 Stationary bed has particles with a
charged surface opposite of sample
ions.
 Exclusively used for ionizable
samples
 Buffers are used as the mobile
phase using pH and ionic strength to
control the elution time.
Reference 1
Size exclusion
chromatography
 These columns are filled with a particular
pore size serving to filter the sample
 Large molecules wash through the
column faster than the smaller ones
giving the larger molecules a lower
retention time.
Reference 1
Adsorption Chromatography
 These columns are packed with a
stationary phase such as silica that
serves as an adsorbent to the specific
compound.
 This separation is based on adsorption
and desorption steps using different
solvent ratios to interact with the sample.
Reference 1
Different Phases
 Normal Phase – This is where the
stationary bed is strongly polar (silica gel)
and the mobile phase is largely non-polar
such as hexane or THF.
 Reverse Phase – The stationary phase
is non-polar and the mobile phase are
polar liquids such as methanol,
acetonitrile, or water. The more nonpolar substances have longer retention.
Reference 1
Elution Types
 Isocratic – where the eluent is at a fixed
concentration.
 Gradient – where the eluent
concentration and strength are changing.
Reference 1
Types of Liquid
Chromatography
(TLC) Paper Gravity Chrom. Flash Chrom.
Chrom.
Tsvett, 1903
1978
HPLC 1952
UPLC 2004
HPLC Characteristics
 Columns have small internal diameters
(2-10 mm) usually made with a reusable
material like stainless steel
 High inlet pressures of several thousand
psi’s and controlled flow of mobile phase
 Precise sample introduction and small
sample requirements
 Special continual flow detectors that use
small flow rates and low detection limits
 Some are equipped with automated
sampling devices
 Rapid analysis with high resolution
Reference 3
Stationary Phase in HPLC
 Particle size 3 to 10 µm packed tightly with a
pore size of 70 to 300 Å
 Surface area of 50 to 250 m2/g
 Bond phase density – number of adsorption
sites per surface unit (1 to 5 per 1 nm).
 Typical surface coatings:
Normal phase (-Si-OH, -NH2)
Reverse phase (C8, C18, Phenyl)
Anion exchange (-NH4+)
Cation exchange (-COO-)
Reference 3
Mobile Phase in HPLC
 Purity of the solvents
 Detector compatibility
 Solubility of the sample
 Low viscosity
 Chemical inertness
 Reasonable price
Reference 3
Path of Mobile Phase
Mobile Phase
degassing
Mobile Phase
reservoir
Mobile Phase
mixing
HPLC Column
Rotary Sample
Loop injector
HPLC Pump
HPLC Detector
Mobile phase degassing and
storage
 It is recommended that
you degas your solvents
for several minutes before
use (Helium); Special
containers can prevent
exchange with the ambient
air (shown in this figure).
 This Waters 1525 HPLC is
set up to do solvent
gradients; alternatively,
you could premix the
solvent and use one
reservoir for isocratic runs.
Reference 3
Mobile Phase mixing
 Solvent proportioning valves allow for
gradient elution by being programmed to
mix the solvents with respect to time
HPLC Pump
 Reciprocating
piston pumps are
commonly used
which have
pistons that pull
the mobile phase
in and push it out
into the head of
the column
Reference 4
Rotary Sample Loop Injector
 Injector needles are
used ranging from 10
µL to 500 µL to inject a
sample onto the
sample loop
 Upon a 60° rotation the
pump introduces the
sample onto the
column in a reverse
direction that it was
loaded.
Reference 5
 http://www.restek.com/inf
o_sixport.asp
HPLC Columns
 HPLC Columns come in
various sizes and many
factors involving your
analyte or the function of the
column should be
considered when selecting
the appropriate one. Some
common dimensions: 10,
15, and 25 cm in length; 3,
5, or 10 mm diameters; 4 to
4.6 internal diameters
Reference 3
Column Cost and Sensitivity
 Costs generally range from $200 to $1000 per
column.
Column
Flow Range
Typical
Flow Rates
(mL/min)
Analyte
4.6 mm
Standard
500-3000
10 -10
1
Microbore
20-200
10 -10
0.2
Capillary
10-Jan
10 -10
0.05
Nanoscale
0.05-0.5
<10
Reference 4
-4
-8
-6
-10
-9
-13
-12
Typical
Injection
30 mL
1
0.06
0.003
HPLC Detectors
 Most HPLC instruments are equipped
with optical detectors.
 Light passes through a transparent low
volume “flow cell” where the variation in
light by UV Absorption, fluorescent
emission, or change in refractive index
are monitored and integrated to display
Retention Time and Peak Area.
 Typical flow rates are 1 mL/min. and a
flow cell volume of 5-50 µL.
Reference 3
Common HPLC Detectors
 Refractive Index (RI) - universal
 Evaporative Light Scattering Detector
(ELSD) – universal
 UV/VIS light – selective
 Fluorescence – selective
 Electrochemical (ECD) selective
 Mass Spec (MS) - universal
Reference 3
Refractive Index detector
 Analytes change the refractive index of the
light in a proportional amount to the
concentration.
 Heat can change the RI of the mobile phase so
thermo control important
 RI changes cause a shift in a beam’s focal
location which is detected on a photo-sensor.
 RI is ideal for analyzing complex sugars and
carbohydrates which have no chromophores,
fluorescence or electrochemical activities
Reference 3
ELSD
 Light scatters in response to the
dimension of the analyte particles.
 Light does not scatter in the mobile
phase and must be nebulized and
evaporated
 This universal detector is more sensitive
that RI and shows a response to
compound lacking UV absorption or
fluorescence.
 Downfall is the sample is destroyed.
Reference 3
UV/VIS Detectors
 Scan a range of UV light to detect molecules
with chromophores. Commonly 254 nm.
 Usually having a range of 190 nm to 600 nm
 Low flow cell volume 1 – 10 µL
 Single wavelength filter photometers -uses a
source lamp to emit a single wavelength (Hg, 254
nm)
 Dispersive monochromator detectors -selects a
narrow wavelength band
 Diode array detector -light from flow cell disperses
and is directed towards different diodes
Reference 3
Fluorescent Detectors
 Higher signal to noise ratio than UV/VIS
 Greater sensitivity than UV/VIS
 Many compounds do not fluoresce and
are derivatized with chemicals such as
Dansyl chloride. This works well with
primary and secondary amines, amino
acids and phenolic compounds.
Reference 3
Electrochemical Detectors
 Selective detection commonly used with
reverse phase and isocratic elution with buffers
and salts as the mobile phase
 The two types of ECD’s are voltammetric and
conductometric
 The mobile phase must carry charged
electrolytes eliminating normal phase as an
option
 ECD’s respond to analytes that are oxidizable
or reducible at an electrode surface.
Reference 3
Mass Spectrometer
 Problem interfacing the mobile phase with a MS
detector
 The first interface system was a moving conveyer belt
that passed through vacuum systems leaving the
analyte on a solid adsorbent material
 Thermospray – mobile phase is directed to a capillary
column that is heated and points at a skimmer cone.
(Too much build up on orifice)
 Electrospray (ESI) – analytes are charged upon exiting
the capillary tube and cross sprayed with nitrogen. The
charge particles cause a “Coulomb explosion” making
smaller droplets of analyte to enter the skimmer cone.
 Atmospheric Pressure Chemical Ionization (APCI) –
Analyte is heated by a ceramic tip on the column, cross
flow of nitrogen decreases the droplet size, and a
“corona discharge” charges the particles to enter the
detector. Reference 3
Detector Summary
Detector
Type
RI
ELSD
UV/VIS
Fluorescent
ECD
MS
LOD (ng/
injection)
100
1
1
0.010.1
0.01
0.01
Selectivity
No
No
Moderate
Very
High
High
High
Gradient
Elution
No
Yes
Yes
Yes
No
Yes
Reference 3
Automated Waste Collection
Typical Program Screen
Waters software: Breeze
Why HPLC?
 HPLC works with compounds of higher
molecular weights and polarity.
 Many biological samples are charged such
as DNA and proteins.
 HPLC can be used in a prepatory manner
with larger sample sizes and sample
recovery to continue synthesis
 Good at separating stereoisomers;
techniques that employ heat (GC) can
cause racemization during analysis.
Reference 3
Contrasting HPLC and UPLC
 UPLC gives faster results with better
resolution
 UPLC uses less of valuable solvents like
acetonitrile which lowers cost
 The reduction of solvent use is more
environmentally friendly
 Increased productivity can increase you
revenue in an industrial setting
Reference 6
Chromatograms of simvastatin
Reference 6
Why is UPLC more efficient
 Peak capacity (P) is the number of peaks
that can be resolved in a specific amount
of time.
 P is proportional to the inverse of the
square root of the Number of theoretical
plates (N): N = L/H
 Lower plate heights generate a smaller
number of plates
 Plate heights are correlated through the
Van Deemter equation
Reference 8
Van Deemter Eqn.
 H = A +B/u +C*u
 A is related to the mobile phase
movement through paths in the stationary
phase.
 B describes longitudinal diffusion
 C relates the analyte to mass transfer
between the pores of the stationary
phase
 Halasz eqn.:
Reference 8
Haslaz Eqn.
 Eqn.:
 u relates to the
velocity of the mobile
phase
 dp depends on the
particle size
 This formula
implicates that
decreasing particle
size decreases the
plate height which
increases resolution.
Reference 8
Synthetic Application
Semi-Prep
+
BzC
OEt
SMe2
N
H
O
O
DBU
Syn
Anti
O
H
O
H
+
BzC
N
H
BzC
N
H
OEt
OEt
O
O
1:1 mixture that is hard to separate on HPLC
NaBH4
CH3OH
NaBH4
CH3OH
O
O
H
BzC
H
N
H
OEt
HO
BzC
N
H
OEt
H
H
OH
+
+
O
O
H
H
BzC
N
H
OEt
HO
H- anti to CH3
H
BzC
N
H
OEt
H
OH
H- syn to CH3, not likely formed
Mixture shows two signals on HPLC, but the problem is poor recovery
Therefore we are derivatizing the compound into a less polar Silane
Derivatization
O
O
H
H
BzC
N
H
OEt
HO
BzC
N
H
OE
t
Et3Si
H
TESCL
+
H
+
Pyridine
O
O
H
H
BzC
N
H
OEt
HO
H
BzC
N
H
OEt
Et3Si
H
Typical Chiral Separation
 Biological activity depends on the stereochemistry of a
particular enantiomer
 A common column is a cytodextrin packing with various
glucopyranose units; this column creates hydrophobic
cavities with hydrophilic surfaces.
 The analyte is trapped in the cavity and can be
separated from the polar solvents
Reference 8
Quantitative Analysis Application
 HPLC can be use in conjunction with size exclusion to
determine the molecular weight of proteins
 In this application molecules with larger weights have
lower retention times.
 By plotting standard retention times in excel you can
extrapolate the molecular weight (MW) of your protein
 Lactate Dehydrogenase MW analysis in an experiment
was determined using a 280 nm wavelength with a
TSKGel Super SW 3000 size exclusion 4.6mm × 30 cm
column, 50 mM phosphate buffer pH 7.2 containing 0.3
M NaCl as a mobile phase, a flow rate of 0.6 mL/min.,
20 µL injection volume, and a Rheodyne injection
valve.
Reference 9
Standard protein molecular
weight data
Standard Mixture for Size Exclusion Chromatography
Protein
Molecular
Weight
(Daltons)
Log MW
Thyroglobulin
670,000
2.83
5.93
γ-Globulin
Ovalalbumin
Myoglobulin
158,000
44,000
17,000
5.2
4.64
4.23
8.27
9.5
10.7
Vitamin B 12
1,350
3.13
12.38
Reference 9
Retention
Time (min.)
Protein standard Log MW vs. Retention Time
y = -0.4103x + 8.4451
2
R = 0.9641
7
6
5
Log
4
MW
3
2
1
0
0
5
10
Retention Time (min)
15
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
http://mtsu32.mtsu.edu:11233/471toc.html (accessed 06/19/09).
http://en.wikipedia.org/wiki/Chromatography (accessed
06/25/09).
Robinson, J. W.; Skelly Frame, E.M.; Frame II, G.M.
Undergraduate Instrumental Analysis, 6th ed.; Marcel Dekker
Inc.: NY, 2005; pp 797-835.
http://www.chem.queensu.ca/courses/08/CHEM321/LectureNot
es/Chapter%2025%20part%20one.doc (accessed 06/23/09).
http://www.restek.com/info_sixport.asp (accessed 06/20/09).
http://www.waters.com/waters/promotionDetail.htm?id=1004869
3&ev=10007792&locale=en_US (accessed 06/20/09).
Dionex, Technical Note 75. “Easy Method Transfer from HPLC
to RSLC with the Dionex Method Speed-Up Calculator”
Levin, S.; Abu-Lafi, S. “The Role of Enantioselective
Liquid Chromatography Separations Using Chiral
Stationary Phases in Pharmaceutical Analysis”, in
Advances in Chromatography. Grushka, E.; Brown, P. R.,
Ed.; Marcel Dekker Inc.: NY, 1993; Vol. 33; pp 233-236.
Kline, P. “Analysis of Lactate Dehydrogenase: Determination of
Molecular Weight and Purity”, Middle Tennessee State
University, Murfreesboro, TN, 2009.