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.
2.
3.

Ion exchange chromatography

Size exclusion chromatography
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
psis 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 m packed tightly with a
pore size of 70 to 300
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
L to 500 L 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/in
fo_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
(L/min)

Analyte

Typical
Injection

4.6 mm

Standard

500-3000

10-4-10-8

Microbore

20-200

10-6-10-10

30 L
1

0.2

Capillary

10-Jan

10-9-10-13

0.06

0.05

Nanoscale

0.05-0.5

<10-12

0.003

Reference 4

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 L.
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 beams 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 L
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 ECDs are voltammetric and
conductometric
The mobile phase must carry charged
electrolytes eliminating normal phase as an
option
ECDs 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

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
O

OEt
O

1:1 mixture that is hard to separate on HPLC

NaBH4
CH3OH

NaBH4
CH3OH

H
BzC

N
H

OEt
HO

BzC

N
H

OEt
H

OH

O
H

H
BzC

N
H

OEt
HO

H- anti to CH3

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

H
BzC

N
H

OEt
HO

BzC

N
H

OE
t

Et3Si

TESCL

Pyridine

H
BzC

N
H

OEt
HO

BzC

N
H

OEt
Et3Si

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 L injection volume, and a Rheodyne
injection valve.
Reference 9

Standard protein molecular

weight data
Standard Mixture for Size Exclusion Chromatography
Retention
Time (min.)

Protein

Molecular
Weight
(Daltons)

Log MW

Thyroglobulin

670,000

2.83

5.93

-Globulin

158,000

5.2

8.27

Ovalalbumin

44,000

4.64

9.5

Myoglobulin

17,000

4.23

10.7

Vitamin B 12

1,350

3.13

12.38

Reference 9

Protein standard Log MW vs. Retention Time

y = -0.4103x + 8.4451
2

R = 0.9641
7
6
5

Log

MW

3
2
1
0
0

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=100486
93&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