Instrumentation of HPLC

Columns
 Instrumentation
Columns

• Analytical - determine chemical composition of a sample

• Preparative - purify and collect one or more components of a
sample
HPLC columns (Stationary Phases)
 Two basic types of packings are used in liquid
chromatography :
1. Pellicular, and
2. Microporous particles.
 Most HPLC packings are microparticles of varying size,
shape, and porosity.
 Silica packings are popular - can withstand high pressure,
is abundant, and inexpensive. Functional groups can be
bonded to the silica.
- Disadvantage- unstable at high and low pH
Silica = SiO2 (from sand)

Pure silicon dioxide

HPLC columns (Stationary Phases)
Totally porous Pellicular Particle Totally porous microparticle
particles (20-40 m) (20-40 m) (5-10 m)
Long pores filled with Thin layer of adsorbent or Most common stationary phase
stagnant mobile phase stationary phase particles
These have relatively Solid spherical bead with Fully porous materials that can
low efficiency and so thin outer surface of be either irregular or spherical in
are not used much. stationary phase shape. Particles with small pores
exhibit a high surface area and
have greater retention.
Give higher efficiencies than Spherical materials have better
porous particles of the same stability at high pressures, larger
size but restricted to small sample volume capacity, and
sample loadings (low surface better detection sensitivity.
area).
(glass or polymer beads) Have high efficiency and speed
for trace analysis and large peak
capacity

thin porous layer of silica,

alumina or ion-exchange resin The surface are coated with thin organic
films (chemically bonded)
HPLC columns (Stationary Phases)
 Column packings can be described in terms of their chemical
composition, whether silica, alumina, or carbon.
 The silicas are more acidic and therefore good for the
separation of basic materials
 The aluminas are more basic and therefore good for the
separation of acids.
 Retention and separation on these two adsorbents (silicas or
aluminas) are generally similar, with the more polar sample
components being preferentially held.
 Chemically bonded stationary phase is now the most
common column packing for reverse phase HPLC. These
packings are almost all prepared from rigid silica or silica-base
compositions.
 HPLC columns
Synthesis of Chemically bonded stationary phase
 The most useful bonded-phase particles are siloxanes (Si-O-Si)
formed by reaction of the hydrolyzed surface of the silica particles
with an organochlorosilane such as octadecylsilane (ODS).
surface
Bonded stationary phase
= a liquid stationary phase
that is chemically bonded
to a particulate packing
organochlorosilane C18
material.
surface
HPLC columns
Chemically bonded stationary phase
Silica Gel Derivatized Silica Gel
O O O
| | | O O O
OSiOSiOSiOH | | |
| | | OSiOSiOSiOR Where R = C18H37
O O O | | |
| | | O O O
hydrocarbon chain
OSiOSiOSiOH | | | (octadecylsilyl deriv.
| | | OSiOSiOSiOR silica or “C18”)
O O O | | |
O O O

bulk (SiO2)x surface bulk (SiO2)x surface

relatively polar surface relatively nonpolar surface

“normal phase” “reversed phase”

Columns - Stationary Phases

Microporous particles
HPLC columns
Chemically bonded stationary phase
 The properties of a stationary phase are determined by the
nature of the organosilane’s alkyl group.
 If R is a polar functional group, then the stationary phase will
be polar.
 Examples of polar stationary phases include:
 R = cyano (-C2H4CN),
diol -(C3H6OCH2CHOHCH2OH), or
amino (-C3H6NH2) functional group.
Useful for separations of mixtures consisting of very different components that
might have a broad range of retention times examples involving ethers, esters,
nitro compounds, double-bond isomers, and ring compounds that differ in
double-bond content
To separate polar compounds: such as carbohydrates and peptides
To separate polar organic polar compounds
HPLC columns
Chemically bonded stationary phase
 The most common nonpolar stationary phases use an
organochlorosilane for which the R group is an n-octyl (C-
8) or n-octaldecyl (C-18) hydrocarbon chain.
 Bonded octyl packings (C-8): to separate of compounds
with low to high polarity and samples with wide-ranging
polarities.
 Octadecyl packing (C-18) can be used for applications in
which maximum retention is required.
 The silica substrate is subject to hydrolysis in basis
solutions, the pH of the mobile phase must be less than
7.5.
 Most reverse-phase separations are carried out using a
buffered aqueous solution as a polar mobile phase.
maintain the pH, preserve the column, so have to run the buffer
Buffer for HPLC
uffer pKa pH Range

Trifluoroacetic acid << 2 1.5 -2.5

KH2PO4 / Phosphoric acid 2.12 1.1 - 3.1
Tri-K-Citrate / hydrochloric acid 1 3.06 2.1 - 4.1
Potassium formate / formic acid 3.8 2.8 - 4.8
Tri-K-Citrate / hydrochloric acid 2 4.7 3.7 - 5.7
Potassium acetate / acetic acid 4.8 3.8 -5.8
Tri-K-Citrate / hydrochloric acid 3 5.4 4.4 - 6.4
Ammonium formate 3.8 & 9.2 2.8-4.8 & 8.2-10.2
Bis-tris propane HCl/Bis-tris propane 6.8 5.8 - 7.8
Ammonium acetate 4.8 & 9.2 3.8-5.8 & 8.2-10.2
KH2PO4 / K2HPO4 7.21 6.2 - 8.2
Tris HCl / Tris 8.2 7.3 - 9.3
Bis-tris propane HCl/Bis-tris propane 9.0 8.0 - 10.0
Ammonium hydroxide / Ammonia 9.2 8.2 - 10.2
Borate (H3BO3 / Na2B4O7.10H2O) 9.24 8.2 - 10.2
Glycine.HCl / glycine 9.8 8.8 - 10.8
1-methylpiperidine.HCl / 1-methylpiperidine 10.1 9.1 - 11.1
Diethylamine.HCl / diethylamine 10.5 9.5 - 11.5
Triethylamine.HCl / triethylamine 11.0 10.0 - 12.0
Pyrollidine.HCl/pyrollidine 11.3 10.3 - 12.3
Common buffers for HPLC
Buffer pKa values
Phosphate 2, 7
Acetate 4.75
Citrate 3.08, 4.77, 6.40

Useful buffering between pH 2-8.

HPLC columns
Chemically bonded stationary phase
Octadecyl C-18
50: 50 MeOH /H2O Octyl C-8
50: 50 MeOH /H2O
HPLC columns
Chemically bonded stationary phase

 Chiral-phase HPLC columns are a relatively new and used

for resolving optically active isomers.
 Separation is based on the degree of interactions
between functional groups in the optically active sample
components and an optically active bonded phase

Types of interactions:
• hydrogen bonds
• dipole-dipole
interactions
• - interactions etc.

Chiral-phase HPLC columns

 HPLC columns
Chemically bonded stationary phase
 The most important advantage of bonded-phase packings:
1. Enhanced stability that results from chemically bonding
of the stationary phase to the solid support. Losses of
the stationary phase owing to solubility in the mobile
phase are not encountered.
2. They permit gradient elution, because of their stability.
HPLC columns
Effect of different temperatures

 Elevated temperatures result in either better resolution or

faster analysis. k = (tR – tM) / tM
 the k values are relatively smaller and the peaks become
sharper
HPLC columns
Limits of column performance
(1) Pressure Drop
 A very high pressure can seriously degrade column
performance. Pressures above 5000 psi (340 atm) do not
appear worthwhile for most HPLC separations.

(2) Particle Diameter of Stationary Phase

 The analytical performances improve dramatically when the
particle diameter is reduced.
 Each time the particle diameter is halved, the pressure drop
required is raised by approximately a factor of four.
Use a shorter column
 Commercial columns are available with packing materials
with particle diameters of 3, 5, and 10 µm.
HPLC columns
Limits of column performance
(3) Viscosity
 A solvent with low viscosity is always preferred in HPLC. An
increase in the viscosity of the solvent always decreases
the flow rate of the mobile phase.
 The diffusion coefficients (D) of solutes are also affected by
the viscosity of the mobile phase.
 For example, D for 1-propanol = 0.3 x 10-3 N sec m-2, and
hexane = 3.0 x 10-3 N sec m-2 .
0.31 2.00

Viscosity: hexane < 1-propanol

Flow rate: hexane > 1-propanol
Diffusion coefficient, DM: hexane > 1-propanol
Viscosity

lower
viscosity,
higher flow
rate
HPLC columns
Limits of column performance

(4) Extra-Column Band Broadening

 This spreading includes the dilution factor caused by:
the injector, any column dead (void) spaces, the volume
of connecting tubing before and after the separation
column, and the detector volume.
HPLC columns
Isocratic Elution
 In isocratic elution, a sample is injected into a column and
the mobile phase composition remains unchanged
throughout the whole analysis.
Isocratic elution : A separation that employs a single solvent
of constant composition.
 If a sample containing components with wide range of k’
value is chromatographed by isocratic elution:
 Early bands (low k’ values) are usually badly resolved
 Middle bands (k’ = 2 to 5) well resolved, and
 Late bands (k’ > 5) only poorly detected. k = (tR – tM) / tM
 The analysis time is unnecessarily long.
 This problem can be solved by employing the technique of
gradient elution or solvent programming.
HPLC columns
Gradient elution
 This is performed by changing the composition of the
mobile phase either stepwise or continuously during the
analysis.
 The chromatogram is started with a pure solvent 1 which
may give low k’ values eluting at the start of the
chromatogram.
 The addition of a second solvent 2 to solvent 1 varies the
eluent strength until a concentration of pure solvent 2 is
reached.
 Solvent 2 must be strong enough to elute the strongly
retained solutes with high k’ values in a reasonable time.
Solvent 2: Depends on types of column used: normal- or reversed-
phase. Best k = 2 to 5
HPLC columns
Gradient elution
 Compared to isocratic elution, gradient elution has the
following advantages:
 More nearly equal bandwidths
 Faster overall separation
 Increased sensitivity
 Better resolution

 Gradient elution has the disadvantages:

 it is necessary to regenerate the column to the
initial starting conditions before another separation
can be attempted
 time-consuming
 HPLC
Gradient elution

Decreasing elution strength,

samples less dissolve in mobile
phase. Retention time increases.

acetonitrile
HPLC Instrumentation
Sample Injection System
 This consists of a stainless steel ring with six different
ports, one to the column.
 Samples of a few microliters can be injected at pressures
up to 6000 psi. Rear View

Sample Loop

Load - Inject
Front View

Inject
Manual Injectors
HPLC Instrumentation
Sample Injection System

Sample in
the loop

Sample size: 0.5 L to 2 mL

Sample is
swept into
the column
AUTOMATED INJECTER
HPLC Instrumentation
Common HPLC Detectors
•UV-VIS
•Diode Array
•Multiple Wavelength
•Variable Wavelength
•Mass Spectrometers
•Refractive Index
•Fluorescence
•Electrochemical
detectors
HPLC Instrumentation
Common HPLC Detectors
 UV detectors are divided into 3 types:
(a) fixed wavelenght – measures one wavelength, usually 254
nm. use mercury vapor lamps. Up to 20x more sensitive than
variable wavelength detectors. Compounds containing
carbonyl groups, multiple double bonds, or aromatic rings can
be detected at this wavelength. manual, journal, UV spectrometer
(b) variable wavelength – measures one wavelength at a
time, for a wide range of wavelengths. Use deuterium or
similar lamp that produce a broad spectrum of
wavelengths that are separated by a diffraction grating. Only
selected wavelength of interest passes through the slit.
 Both fixed and variable spectrophotometers select a single
wavelength of light to pass through the sample.
HPLC Instrumentation
Common HPLC Detectors
 UV detectors are divided into 3
types:
(c) Diode array - measures a
spectrum of wavelength
Spectrum of a diode
simultaneously. arrays detectors

 The PDA or DAD passes a wide spectrum of light through the

sample and then the light is separated into individual
wavelengths after passing the sample. The spectrum of light is
directed to an array of photosensitive diodes. Each diode can
measure a different wavelength which allows for the monitoring
of many wavelengths at once.
 It can provide information on the purity of the peak.
HPLC Instrumentation
Absorbs at one wavelength
Common HPLC Detectors and re-emit light at the
other wavelength
 Fluorescence detectors
 The most sensitive HPLC detectors and depending on the
compound, can be from 10 – 1000 times more sensitive than
UV detectors analyzing strong UV absorbing compounds.
 Very specific and selective, which can be used as an advantage
to reduce interference but this also limits the number of
compounds that can be analyzed by florescence detection.
 Typical fluorescing compounds are polynuclear aromatics,
steroids, plant pigments, vitamins, alkaloids, catecholamines,
aflatoxines, and porphyrins.
 Xenon lamp for light source
 Excitation wavelength range: 200-650 nm
 Emission wavelength range: up to 900 nm depending on
photomultiplier installed
HPLC Instrumentation
Common HPLC Detectors
 Electrochemical detectors have found their greatest
applications when polar mobile phases are used.
 Aromatic amines and phenolic compounds are the most
important classes of compounds to which electrochemical
detection has been applied. These classes include a large
number of compounds of biochemical interest.
Usually used to measure compounds that undergo
oxidation or reduction (electrochemical reaction).
Electrons are produced and the electrical current is
measured.

Example: neurotransmitters such as

dopamine, serotonin and
noradrenalin.
HPLC Instrumentation
Common HPLC Detectors A universal detector
 Refractive Index (RI) detectors measure the ability of sample
molecules to bend or refract light. This property for each
molecule or compound is called its refractive index.
 The least sensitive, 1000x less sensitive than UV detectors. It
is also sensitive to changes in ambient temperature, pressure
,and flow rate and is generally not compatible with gradient
elution.
 For samples with little or no UV Absorption
 Alcohols, sugars, saccharides, fatty acids, polymers
 Best results when RI of samples is very different from RI of
mobile phase
 Flow cell is temperature controlled with a double insulated
heating block.
HPLC Instrumentation
Common HPLC Detectors
HPLC Detectors – Mass Spec

Heated Q-array Octapole Electron Multiplier

capillary Detector

Orthogonal source Quadrupole mass analyser

geometry
HPLC Applications
• Biogenic substances • Food products
Sugars, lipids, nucleic Vitamins, food additives,
acids, amino acids, sugars, organic acids,
amino acids, etc.
proteins, peptides,
steroids, amines, etc.
• Environmental samples
polyaromatic hydrocarbons,
herbicides, Inorganic ions,
Hazardous organic substances, etc.

• Organic industrial
• Medical products products
Synthetic polymers,
Drugs, antibiotics, etc.
additives, surfactants,
dyes, phthalates, etc.

34
 Types of Liquid Chromatography

(TLC) Paper Gravity Chrom. Flash Chrom. HPLC 1952 UPLC 2004
Chrom. Tsvett, 1903 1978 ultra-high
performance liquid
chromatography
Chromatograms of simvastatin
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

Q: what type of detectors. All five detectors must know

 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