HPLC -2

Dr. Md Ashraful Alam

Associate Professor
 Retention Time (tR)

The time between injection and the

appearance of the peak maximum.

This time is measured from the time at which the

sample is injected to the point at which the display
shows a maximum peak height for that compound.
 The time taken for a particular
compound to travel through the column to
the detector is known as its retention time.
Different compounds have different
retention times.
Each solute has a characteristic
retention time.
The conditions have to be carefully
controlled if you are using retention times
as a way of identifying compounds.
 For a particular compound, the retention
time will vary depending on:
• the pressure used (because that affects the flow
rate of the solvent)
• the nature of the stationary phase (not only
what material it is made of, but also particle
size)
• the exact composition of the solvent
• the temperature of the column
tR Total retention time of the compound
t′R Corrected retention time of the compound (r.t. -stationary phase)
tM "Dead time" (retention time-mobile phase)
W0,5 Peak width at half height
h Height of a signal
• The baseline is any part of the chromatogram
where only mobile phase is emerging from the
column.
• The peak maximum is the highest point of the
peak.
• The injection point is that point in time/position
when/where the sample is placed on the column.
• The dead point is the position of the peak-
maximum of an unretained solute.
• The corrected retention time (t′R) is the time
elapsed between the dead point and the peak
maximum.
• The dead time (tM) is the time elapsed
between the injection point and the dead
point.
• The retention time (tR) Total retention time of
the compound in the whole chromatographic
system
tR = t′R + tM
• The retention volume (VR) is the volume of
mobile phase passed through the column
between the injection point and the peak
maximum.
Thus, VR = F x tR
where F is the flow rate in ml/min.
• The band width (tw) of the chromatographic
band during elution from the column.
Small band widths usually represent
efficient separations. Also referred to as peak
width.
 HPLC Separation Modes

• In general, three primary characteristics of

chemical compounds can be used to create
HPLC separations.
• Polarity
• Electrical Charge
• Molecular Size
• First, let’s consider polarity and the two primary
separation modes that exploit this characteristic:
normal phase and reversed-phase
chromatography.
 Separations Based on Polarity

• A molecule’s structure, activity, and

physicochemical characteristics are determined
by the arrangement of its constituent atoms
and the bonds between them.
• Within a molecule, a specific arrangement of
certain atoms that is responsible for special
properties and predictable chemical reactions
is called a functional group.
• This structure often determines whether the
molecule is polar or non-polar.
• Organic molecules are sorted into classes
according to the principal functional group(s)
each contains.
• Using a separation mode based on polarity,
the relative chromatographic retention of
different kinds of molecules is largely
determined by the nature and location of
these functional groups.
• Water [a small molecule with a high dipole
moment] is a polar compound.
• Benzene [an aromatic hydrocarbon] is a
non-polar compound.
• Molecules with similar chromatographic
polarity tend to be attracted to each other;
those with dissimilar polarity exhibit much
weaker attraction, if any, and may even
repel one another.
• This becomes the basis for
chromatographic separation modes based
on polarity.
• Another way to think of this is by the familiar
analogy: oil [non-polar] and water [polar] don’t
mix.
• Unlike in magnetism where opposite poles attract
each other, chromatographic separations based on
polarity depend upon the stronger attraction
between likes and the weaker attraction between
opposites.
Remember, “Like attracts like” in polarity-based
chromatography.
• Silica has an active, hydrophilic [water-loving]
surface containing acidic silanol [silicon-
containing analog of alcohol] functional
groups.
• The activity or polarity of the silica surface
may be modified selectively by chemically
bonding to it less polar functional groups
[bonded phase].
 In order of decreasing polarity,
• cyanopropylsilyl-[CN],
• n-octylsilyl-[C8],
• n-octadecylsilyl-[C18, ODS]
moieties on silica.

The latter is a hydrophobic [water-hating],

very non-polar packing.
 To summarize, the best combination of a
mobile phase and particle stationary phase
with appropriately opposite polarities must be
chosen.

Then, as the sample analytes move

through the column, the rule like attracts like
will determine which analytes slow down and
which proceed at a faster speed.
 Normal-Phase HPLC

• In his separations of plant extracts, Tswett was

successful using a polar stationary phase [chalk in
a glass column] with a much less polar [non-
polar] mobile phase. This classical mode of
chromatography became known as normal phase.

Normal-Phase Chromatography
• The stationary phase is polar and retains the
polar yellow dye most strongly.
• The relatively non-polar blue dye is won in the
retention competition by the mobile phase, a
non-polar solvent, and elutes quickly.

A normal-phase chromatographic separation of

our three-dye test mixture.
• Since the blue dye is most like the mobile
phase [both are non-polar], it moves faster.

It is typical for normal-phase

chromatography on silica that the mobile
phase is 100% organic; no water is used.
 Reversed-Phase HPLC
• The term reversed-phase describes the
chromatography mode that is just the opposite
of normal phase, namely the use of a polar
mobile phase and a non-polar [hydrophobic]
stationary phase.

Figure illustrates the black three-dye mixture being

separated using such a protocol.
 Now the most strongly retained compound
is the more non-polar blue dye, as its attraction
to the non-polar stationary phase is greatest.
The polar yellow dye, being weakly
retained, is won in competition by the polar,
aqueous mobile phase, moves the fastest
through the bed, and elutes earliest like attracts
like.
• Today, because it is more reproducible and has
broad applicability, reversed-phase
chromatography is used for approximately
75% of all HPLC methods.
• Most of these protocols use as the mobile
phase an aqueous blend of water with a
miscible, polar organic solvent, such as
acetonitrile or methanol. This typically ensures
the proper interaction of analytes with the
non-polar, hydrophobic particle surface.
• A C18
–bonded silica [sometimes called ODS] is the
most popular type of reversed-phase HPLC
packing.

• Octadecylsilane phases are bonded to silica or

polymeric supports. Both monomeric and
polymeric phases are available.
 Table presents a summary of the phase
characteristics for the two principal HPLC
separation modes based upon polarity.
Remember, for these polarity-based modes,
like attracts like.

Phase Characteristics for Separations Based on

Polarity
 Ultra Performance Liquid Chromatography
(UPLC)

UPLC increases in resolution, speed,

and sensitivity in liquid chromatography.

• Columns with smaller particles [1.7 micron]

• and instrumentation with specialized
capabilities designed to deliver mobile phase
at 15,000 psi [1,000 bar]
 Hydrophilic-Interaction Chromatography
[HILIC]

• HILIC may be viewed as a variant of normal-phase

chromatography.
• In normal-phase chromatography, the mobile phase is 100%
organic. Only traces of water are present in the mobile
phase and in the pores of the polar packing particles.
• HILIC may be run in either isocratic or gradient elution
modes.
• Polar compounds that are initially attracted to the polar
packing material particles can be eluted as the polarity
[strength] of the mobile phase is increased [by adding more
water].
 Hydrophobic-Interaction Chromatography
[HIC]

• HIC is a type of reversed-phase chromatography that is

used to separate large biomolecules, such as proteins.
• It is usually desirable to maintain these molecules intact
in an aqueous solution, avoiding contact with organic
solvents or surfaces that might denature them.
• HIC takes advantage of the hydrophobic interaction of
large molecules with a moderately hydrophobic
stationary phase, e.g., butyl-bonded [C4], rather than
octadecyl-bonded [C18], silica. Gradient separations are
typically run by decreasing salt concentration. In this
way, biomolecules are eluted in order of increasing
hydrophobicity.
 Separations Based on Charge:
Ion-Exchange Chromatography [IEC]

• For separations based on polarity, like is attracted

to like and opposites may be repelled.
• In ion-exchange chromatography and other
separations based upon electrical charge, the rule
is reversed. Likes may repel, while opposites are
attracted to each other.
• Stationary phases for ion-exchange separations
are characterized by the nature and strength of
the acidic or basic functions on their surfaces and
the types of ions that they attract and retain.
• Cation exchange is used to retain and separate
positively charged ions on a negative surface.
Conversely, anion exchange is used to retain and
separate negatively charged ions on a positive
surface. With each type of ion exchange, there are at
least two general approaches for separation and
elution.
• Strong ion exchangers bear functional groups [e.g.,
quaternary amines or sulfonic acids] that are always
ionized. They are typically used to retain and
separate weak ions. These weak ions may be eluted
by displacement with a mobile phase containing ions
that are more strongly attracted to the stationary
phase sites. Alternately, weak ions may be retained
on the column, then neutralized by in situ changing
the pH of the mobile phase, causing them to lose
their attraction and elute.
 Size-Exclusion Chromatography [SEC] –
Gel-Permeation Chromatography [GPC]
• All of these techniques are typically done on
stationary phases that have been synthesized
with a pore-size distribution over a range that
permits the analysts of interest to enter, or to be
excluded from, more or less of the pore volume
of the packing.
• Smaller molecules penetrate more of the pores
on their passage through the bed. Larger
molecules may only penetrate pores above a
certain size so they spend less time in the bed.
• The biggest molecules may be totally excluded
from pores and pass only between the particles,
eluting very quickly in a small volume.