vogel's textbook of quantita
TRANSCRIPT
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OLUMN ND
THlrH YER
LIQUID HROM TOGR PHY
packing techniques are used in which the particles
ar e
suspended in a suitable
solvent
an d
the suspension or slurry) driven into the
column under
pressure.
Th e essential features for successful slurry packing of columns have been
summarised.P Many analysts will, however, prefer to purchase the commercially
available
HPLC
columns, for which
the appropriate manufacturer s
catalogues
should be consulted.
Finally, the useful life of an analytical column is increased by introducing a
guard column. This is a
short column
which is placed between
th e
injector
an d
the HPLC column to
protect the
latter from
damage
or loss of efficiency caused
by particulate
matter
or strongly adsorbed substances in samples or solvents.
ma y also be used to saturate th e eluting solvent with soluble stationary phase
[see Section 8.2 2)J. Guard columns may be packed with microparticulate
stationary phases or with porous-layer beads; the latter are cheaper and easier
to pack than the microparticulates, bu t have lower capacities and therefore
require changing more frequently.
Detectors. Th e function
of
the
detector
in HPLC is to monitor the mobile phase
as it emerges from th e column. Th e detection process in liquid
chromatography
has presented more problems
than
in gas chromatography; there is, for example
no equivalent to th e universal flame ionisation detector of gas chromatography
for use in liquid
chromatography.
Suitable detectors ca n be broadly divided
into
th e
following two classes:
a
Bulk property
detectors
which measure the difference in some physical
property of the solute in the mobile phase compared to th e mobile phase
alone, e.g. refractive index and conductivity* detectors. T he y a re generally
universal in application bu t tend to have poor sensitivity an d limited range.
Such detectors ar e usually affected by even small changes in the mobile-phase
composition which precludes
th e use
of
techniques such as gradient elution.
b Solute
property detectors,
e.g. spectrophotometric, fluorescence an d electro
chemical detectors. These
respond
to a particular physical
or
chemical
property of
the solute, being ideally independent of the mobile phase. In
practice, however, complete independence of the mobile phase is rarely
achieved, bu t the signal discrimination is usually sufficient to permit
operation
with solvent changes, e.g.
gradient
elution.
They
generally provide
high sensitivity
about
1 in 10
9
being attainable with UV and fluorescence
detectors) an d a wide linear response range but, as a consequence of their
more selective natures, more than one detector may be required to meet
the demands of an analytical problem. Some commercially available
detectors have a number of different detection modes built into a single
unit, e.g. the Perkin-Elmer
3D
system which combines UV absorption,
fluorescence
and
conductimetric detection.
Some of th e important characteristics required of a detector
ar e
the following.
a Sensitivity,
which is often expressed as the noise equivalent concentration,
i.e. the solute concentration,
Cn,
which produces a signal equal to th e
detector noise level.
Th e
lower the value of C
for a particular solute, th e
more
sensitive is the detector for
that
solute.
The conductance detector is a universal detector for ionic species and is widely used in ion
chromatography see Section 7.4).
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EQUIPMENT FOR
HPLC
8
b A linear response The linear range of a detector is the concentration range
over which its response is directly proportional to the concentration of
solute. Quantitative analysis is more difficult outside the linear range of
concentration.
(c)
Type ofresponse
i.e.whether the detector is universal
or
selective. A universal
detector will sense all the constituents of the sample, whereas a selective
one will only respond to certain components. Although the response of the
detector will
not
be independent of the operating conditions, e.g. column
temperature
or
flow rate, it is advantageous if the response does
not
change
too much when there are small changes of these conditions.
A summary of these characteristics for different types of detectors is given
in Table 8.2.
Table 8.2 Typical detector characteristics in
HPLC
Type
Amperometric
Conductimetric
Fluorescence
UV
/visible absorption
Refractive index
Response
Selective
Selective
Selective
Selective
Universal
10
10
10
7
z
10
8
10
6
Linear range
10
4 - 1 0
5
10
3 -10 4
10
3 - 1 0
4
10
4 - 1 0
5
10
3 - 1 0
4
The range
over
which the response is essentially linear is expressed
as the factor by which the lowest concentration
en
must be
multiplied to obtain the highest
concentration.
A detailed description of the various detectors available for use in
HPLC
is
beyond the scope of the present text
and
the reader is recommended to consult
the monograph by Scott.55 A brief account of the principal types of detectors
is given below.
Refractive index detectors
These bulk property detectors are based on the
change of refractive index of the eluant from the column with respect to pure
mobile phase. Although they are widely used, the refractive index detectors
suffer from several disadvantages - lack
of
high sensitivity, lack of suitability
for gradient elution, and the need for strict temperature control 0.001 C
to operate at their highest sensitivity. A pulseless pump, or a reciprocating pump
equipped with a pulse dampener, must also be employed. The effect of these
limitations may to some extent be overcome by the use of differential systems
in which the column eluant is compared with a reference flow of pure mobile
phase. The two chief types of RI detector are as follows.
The deflection refractometer (Fig. 8.4), which measures the deflection of a
beam of monochromatic light by a double prism in which the reference
and
sample cells are separated by a diagonal glass divide. When both cells contain
solvent of the same composition, no deflection
of
the light beam occurs; i
however, the composition of the column mobile phase is changed because
of the presence of a solute, then the altered refractive index causes the beam
to be deflected. The magnitude of this deflection is dependent on the
concentration of the solute in the mobile phase.
2. The Fresnel refractometer which measures the change in the fractions of
reflected and transmitted light at a glass-liquid interface as the refractive
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COLUMN
ND
THIN t YER
LIQUID CHROM TOGR PHY
Mirror
i .
Light beam
~
Reflected beam
Reference
solvent
Fig. 8.4 Refractive index detector.
index
of
the liquid changes. In this detector both the column mobile phase
and a reference flow of solvent are passed through small cells on the back
surface of a prism.
When
the two liquids are identical there is no difference
between the two beams reaching the photocell,
but
when the mobile phase
containing solute passes through the cell there is a change in the
amount
of
light transmitted to the photocell,
and
a signal is produced. The smaller cell
volume about 3 ,uL in this detector makes it more suitable for high-efficiency
columns but, for sensitive operation, the cell windows must be kept
scrupulously clean.
ltraviolet detectors
The
UV absorption detector is the most widely used in
HPLC being based
on
the principle of absorption of UV visible light as the
effluent from the column is passed
through
a small flow cell held in the radiation
beam. is characterised by high sensitivity
detection
limit of
about
1 x
10-
9
g mL for highly absorbing compounds and, since it is a solute
property detector, it is relatively insensitive to changes of temperature and flow
rate. The detector is generally suitable for gradient elution work since many of
the solvents used in
HPLC
do not
absorb
to
any
significant extent at the
wavelengths used for monitoring the columneffluent. The presence of air bubbles
in the mobile phase can greatly impair the detector signal, causing spikes on
the chromatogram; this effect can be minimised by degassing the mobile phase
prior to use, e.g. by ultrasonic vibration. Both single
and
double beam
Fig. 8.5 instruments are commercially available. Although the original
detectors were single- or dual-wavelength instruments 254
and/or
280 nm ,
some manufacturers now supply variable-wavelength detectors covering the
range
2 ~ 8
nm so
that more
selective detection is possible.
No account
ofUV
detectors would be complete without mention of the diode
array multichannel detector, in which polychromatic light is passed
through
the flow cell. The emerging radiation is diffracted by a grating and then falls
on to an array of photodiodes, each photodiode receiving a different narrow
wavelength band. A microprocessor scans the
array
of diodes many times a
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\
Reference
photocell
Compound
UV filter
QUIPM NT FOR HPL
8 3
Hg lamp Quartz
Movable
source
lens \
calibrated
~
t
+
filter
Sample
~ h o t o e l l
+
Dual-
channel
cell
Fig. 8.5 Block diagram of a double-beam UV detector.
second
and
the spectrum so obtained may be displayed on the screen of a VDU
or s to red in the i ns tr um en t for subsequent print-out. An i mp or ta nt feature of
the multichannel detector is
that
it can be programmed to give changes in
detection wavelength
at
specified points in the chromatogram; this facility can
be used to clean
up
a chromatogram, e.g. by discriminating against interfering
peaks due to compounds in the sample which are not of interest to the analyst.
Fluorescence detectors These devices enable fluorescent compounds solutes)
present in the mobile phase to be detected by passing the column effluent through
a cell irradiated with ultraviolet light
and
measuring any resultant fluorescent
radiation. Although only a small proportion of inorganic and organic compounds
are naturally fluorescent, many biologically active compounds e.g. drugs)
and environmental contaminants e.g. polycyclic aromatic hydrocarbons) are
fluorescent and this, together with the high sensitivity of these detectors, explains
their widespread use. Because both the excitation wavelength
and
the detected
wavelength can be varied, the detector can be made selective. The application
of fluorescence detectors has been extended by means of pre- and post-column
derivatisation of non-fluorescent or weakly fluorescing
compounds
see
Section 8.4).
lectrochemical detectors The
term electrochemical detector in HPL
normally refers to amperometric or coulometric detectors, which measure the
current associated with the ox id ation or reduction of solutes. In practice it is
difficult to use electrochemical reduction as a means of detection in HPL
because of the serious interference large background current) caused by
reduction of oxygen in the mobile phase. Complete removal of oxygen is difficult
so
that
electrochemical detection is usually based on oxidation of the solute.
Examples of co mp ou nd s which can be conveniently detected in this way are
phenols, aromatic amines, heterocyclic nitrogen compounds, ketones, and
aldehydes. Since not all compounds undergo electrochemical oxidation, such
detectors are selective
and
selectivity may be further increased by adjusting the
potential applied to the detector to discriminate between different electroactive
species. may be noted here that an anode becomes a stronger oxidising agent
as its electrode potential becomes more positive. Of course, electrochemical
detection requires the use of conducting mo bile phases, e.g. containing inorganic
salts or mixtures of water with water-miscible organic solvents, but such
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COLUMN AND
THIN LAYER L nUID CHROMATOGRAPHY
conditions are often difficult to apply to techniques other than reverse phase
nd
ion exchange chromatography.
The amperometric detector is currently the most widely used electrochemical
detector, having the advantages of high sensitivity nd very small internal cell
volume. Three electrodes are used:
1. the working electrode, commonly made of glassy carbon, is the electrode at
which the electroactive solute species is monitored;
2. the reference electrode, usually a silver-silver chloride electrode, gives a stable,
reproducible voltage to which the potential of the working electrode is
referred;
nd
. the auxiliary electrode is the current-carrying electrode nd usually made of
stainless steel.
Despite their higher sensitivity nd relative cheapness compared with ultraviolet
detectors, amperometric detectors have a more limited range of applications,
being often used for trace analyses where the ultraviolet detector does not have
sufficient sensitivity.
8
ERIV TlS TlON
In liquid chromatography, in contrast to gas chromatography [see Section 9.2 2 J,
derivatives are almost invariably prepared to enhance the response ofa particular
detector to the substance of analytical interest. or example, with compounds
lacking an ultraviolet chromophore in the 254 nm region but having a reactive
functional group, derivatisation provides a means of introducing into the
molecule a chromophore suitable for its detection. Derivative preparation can
be carried
out
either prior to the separation pre-column derivatisation or
afterwards post-column derivatisation . The most commonly used techniques
are pre-column off-line
nd
post-column on-line derivatisation.
Pre-column off-line derivatisation requires no modification to the instrument
and, compared with the post-column techniques, imposes fewer limitations on
the reaction conditions. Disadvantages are that the presence of excess reagent
nd by-products may interfere with the separation, whilst the group introduced
into the molecules may change the chromatographic properties of the sample.
Post-column on-line derivatisation is carried
out
in a special reactor situated
between the column
nd
detector. A feature of this technique is that the
derivatisation reaction need not go to completion provided it can be made
reproducible. The reaction, however, needs to be fairly rapid at moderate
temperatures nd there should be no detector response to any excess reagent
present. Clearly n advantage of post-column derivatisation is that ideally the
separation
nd
detection processes can be optimised separately. A problem
which may arise, however, is that the most suitable eluant for the chromatographic
separation rarely provides an ideal reaction medium for derivatisation; this is
particularly true for electrochemical detectors which operate correctly only
within a limited range of pH, ionic strength
nd
aqueous solvent composition.
Reagents which form a derivative that strongly absorbs UV/visible radiation
are called chromatags;
n
example is the reagent ninhydrin, commonly used
to obtain derivatives of amino acids which show absorption at bout 570 nm.
Derivatisation for fluorescence detectors is based on the reaction of non
fluorescent reagent molecules ftuorotags with solutes to form fluorescent
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