Transcript Important Criteria for HPLC Detectors An ideal

Important Criteria for HPLC Detectors
An ideal HPLC detector might be considered to be one with the following
characteristics:
(1) High Sensitivity, (2) Negligible Baseline Noise,(3) Large Linear Dynamic
Range, (4) Non-destructive of the Sample, (5) Stable over Longer Period of Time,
(6) Convenient and Reliable to Operate, (7) Inexpensive to Purchase and Operate
(8) Capable of Providing Information on the Identity of the Solute
Characteristics same as discussed
earlier for GC detectors
(9) Response Independent of Mobile Phase Composition
--For example, if UV-detector is used in HPLC changing the M.P composition
from 80/20 acetonitrile/water to 60/40 acetonitrile water should not change the
background UV absorbance
(10) Low Dead-Volume –Dead volume in the detector adds to extra-column
dimensions and cause dispersion so it must be kept to minimum. This
includes cell volume of the detector itself and length/bore of any tubing
associated with it.
Although the HPLC detectors do not meet all 10 criteria, but most of them are
still used in many HPLC applications
Major Types of HPLC Detectors
Fixed l
Variable l
UV-Vis
Solute Property
Electrochemical
Photodiode Array
Amperometry
Pulse Amperometry
Fluorescence
Voltammetry
Coulometry
Deflectance Type
Refractive Index
Bulk Property
Reflectance Type
Suppressed
Conductivity
Non-suppressed
UV-Vis Absorbance Detector
Most commonly employed detector in HPLC “awa. Workhorse Detector for HPLC”
Principle of Operation Operates on exactly same principle as UV-Vis spectrophotometer
a) Light from the lamp passes through a UV transmitting flow cell (through which
M.P flows and is connected to the column) and falls on a diode measures the light
intensity I.
b) Usually, light from the lamp is also directed to the reference diode for
measurement of light intensity I0
c)The detector electronics than convert the signal from the two diodes into
absorbance A, which is transmitted to the data system and is measured
A = log I0/I (ratio of the intensity of absorption b/w ref and sample diode)
d)Analyte concentration (C) in the flow cell is related to the absorbance of the
analyte (A), molar absorptivity (e), and flow cell length (Lfc) by Beer’s Law:
A = e Lfc
What are the two general criteria in selecting sensitive conditions that can be use
to maximize signal of sample components of interest in UV detection in HPLC?
a) Selection of Suitable Wavelength (l)
--l max is ideal to work but a careful knowledge of UV-Spectra is necessary
--UV spectra should be measured if standards are available
--Diode-Array detector provides spectra of eluted peak
b) Select of l where sample have minimum absorption interferences from
matrix/solvent
---Usually wavelength >240 nm is best
The figure to the right shows a UV-spectra of Azobenzene(Az, concentration = 3.73 x 10-3g/10mL and
phenanthrene (P, 3.23 x10-3g/10mL) both recorded in isooctane on a standard UV/Vis spectrophotometer. What
wavelength would you select on your HPLC detector?
251 nm
(i) Assume that Az is the contaminant in
the sample and you are only interested in
sensitive detection of P, what l would
you choose for detecting P without
detecting Az? and Why?
It cannot be done, but at 251 nm the ratio
261 nm
of A and e of P to Az would be greatest.
342 nm
(ii) Assume that you have purified your
sample and now it contains only P and you
want to determine the concentration of P
carefully in your sample. Which l would
you choose for quantitation of P?
For sensitive detection, 251 nm is ideal for P. For quantitation we must choose a l,
where A or e changes with l is not rapid (choose plateau) 261 nm is better.
(iii) Assume that P is the contaminant in the sample and you are only interested in
sensitive detection of Az, what l would you choose for detecting Az and Why?
The shoulder at 342 nm
(iv) Assume you are interested in detecting both Az and P after the HPLC separation
Which l would you choose? And Why?
~ 300 nm (270-320 nm) for precise quantitation as Beer’s Law is followed.
Fixed Wavelength Detectors
--is the most common and inexpensive detector. The use of suitable l is
determined by the nature of the light source used as shown in Table below:
--The above source (Hg, Cd, Zn and Mg) mention in table shows sharp emission
lines at l indicated in the above table. These l’s can be used for sample that
absorbs strongly at these wavelengths
--Deuterium lamp can be used over a range of wavelength (covers a continuum of
wavelengths), hence covering most of the UV spectral region
Variable Wavelength Detectors (provide detection of eluted peak at any selected l.
--Less sensitive than fixed wavelength but the detection wavelength can be varied
--Deterium source is mostly used because it provides continum source. This can
be combined with a suitable monochromator in dual beam mode.
Photodiode Array Detectors (PDA) or DAD
--Even much more rapid scanning of the absorption spectra of the eluted peak is
possible using a photodiode array detector
--The optical arrangement of the photodiode array detection is shown below:
--Optical arrangement is referred to as
“reverse optics”. This is because the
dispersion device (holographic gratings) is
placed after the flow cell (opposite to UV-Vis)
Working of DAD
a) Light from a continum source (e.g., D2
lamp) passes through a lens system which
focuses polychromatic light onto the
flow cell (containing the sample)
b) The transmitted light then falls on a
holographic gratings where it is dispersed
into a photodiode array (PDA).
c)PDA is a several hundreds of photodiodes
arranged in a linear fashion. A typical
photodiode array has 512 diodes to cover a
range of wavelength (190-800 nm), each photodiode has a bandwidth of 2 nm.
d) A range of wavelengths of light falls on a photodiode array and each diode picks
up a different wavelength of light. ( http://www.youtube.com/watch?v=zbTM36_7jIg)
Name one advantage and one disadvantage of DAD over single l detector in HPLC?
Advantage: DAD provides absorption spectra of each peak and can be used for
peak purity analysis
Disadvantage: DAD is less sensitive and more expensive than single l detector
Applications of DAD
a) A 3-D spectra of each peak eluting from the column can be obtained
(b) Peak Purity Analysis
--A chromatogram is shown with 5 peaks and spectra is taken for peak “a” and
“b” at three points
(i) half-way up the rising side (i.e., the leading edge of the peak)
(ii) top of the peak
(iii) Halfway down the trailing side (i.e trailing edge of the peak)
Which peak “a” or “b” is pure?
Peak “a” is impure and peak “b” is pure. This is because in peak “b” the
absorption spectra at each points of a peak matches the l and does not occurs
for peak “a”
ELECTROCHEMICAL DETECTION IN HPLC
www.youtube.com/watch?v=JP2dGDUFkvg
--Electrochemical detection (ECD) is a range of detection techniques, which
involves the application of electric field (via a suitable electrode) to a sample
solution, followed by measurement of resultant current.
--ECD includes the technique of Voltammetry, Amperometry and Coulometry.
--Common characteristics in ECD: A chemical reaction (e.g., Faradaic oxidation
or reduction occurs) during ECD. To be capable of ECD solutes much be easily
oxidize or reduced. One example is:
+
+ eH
OH
O +
--Basic Instrumentation for ECD
Detector is assembled using four
basic components
(1) Potential power supply
--Used for application of voltage
(2) Appropriate circuitory (amplifier
for measurement of current).
(3) A suitable flow-through sample cell. The cell consists of three electrodes
(a)Working electrode (WE): potential is applied. WE is glassy carbon or a precious
metal (Au, Ag, Pt), which is located in a suitable flow cell through which M.P flows
(b) Auxillary electrode: which measures the flowing current
(c) Reference electrode: in contact with electrolyte and sample solution
(4) Current-Voltage convertor: Convert signal generated by oxidative or reductive
current back to voltage to be read by a recorder
1. Voltammetry detection. If the applied voltage is varied over the course of
measurement and we measure “the current resulting from retention (Oxid/Red) of
analyte” species than the ECD is called Voltammetry
2. Amperometry detection. If a fixed voltage is applied over the course of
measurement and we measure the current resulting from reaction of analyte
species than the ECD is called Amperometry.
--Surface area of the working electrode is quite small (0.5 cm2 or less)
--With small surface area faradaic reaction of analyte is incomplete---only a fraction
of the analyte reacts -------- <10% of analyte reacts in a flow cell
3. Coulometric detection. If a fixed voltage is applied over the course of
measurement and we measure the current resulting from reaction of analyte species
than the ECD Is called Coulometry.
So what is the difference between Amperometry and Coulometry?
--Coulometry employs working electrode of large surface area (> 0.5 cm2), this
result in a complete and quantitative reaction of the analyte at the electrode
surface. Thus, amperometry and coulometry can be differentiated on the basis
of Faradaic reaction at the working electrode
Current-Potential Curve for two Electroactive Solutes
The Figure to the right show the currentpotential curve for ascorbic acid and an
organic disulfides.
--The potential is applied and the current
produced is measured.
-Oxidation and Reduction of electroactive
species results in different directions of the
current flow based on this we can generate
oxidation or reduction current.
Oxidation current (Anodic Current)
When compound is oxidized the oxidation
current flows out of the electrode giving
oxidation (anodic)current which is negative. For e.g., ascorbic acid is readily
oxidized giving an anodic current at +0.23 V due to oxidation of enediol
system.
Reduction current (Cathodic Current) When compound is reduced, reduction
electrons flows into the electode giving (cathodic) current or cathodic peak, which
is positive. For e.g., organic disulfides are reduced giving a cathodic peak at
-1.0 V due to reduction.
To use reduction as a method of ECD in HPLC is more difficult than using
oxidation as a method of ECD, why?
--Because M.P present in HPLC may have dissolve oxygen (O2)
--O2 if present in the M.P is very easily reduced and create background current,
which is much larger than the current produced by reduction of analytes.
--Traces of O2 has to be removed carefully if ECD in reductive mode is possible.
Application of ECD
“ECD is useful for organic molecule containing functional group capable of being
oxidized or reduced.”
Some typical functional
groups sensed by
electrochemical detectors are
shown to the right
How can one tell if the electroactive
species (species that can be
electrochemically oxidized or reduced)
has the potential to detected in ECD?
If the applied voltage > Ehalf of the
electroactive species then ECD
of that species is possible
Disadvantages of ECD
1) Technique is not very suitable for electroreducible compounds
This is because of high background current which is generated by dissolve
O2 in the M.P.
Therefore, both M.P and the sample needs to be highly degassed before use
2) Metal ion impurities interfere for electroreductible compound in ECD
What kind of molecules show fluorescence?
*Molecule that are aromatic, contain multiple double bonds, i.e., double bond with
high degree of resonance structure
Anthracene
Benzo[a]pyrene
* Molecules with rigid structures (e.g., fffluorene > ffbiphenyl)
C
H2
Biphenyl
ff = 0.2
Fluorene
ff = 1.0
*Molecules with delocalized p-electrons (e.g., benzo[a]pyrene)
*Molecule with donating group present on aromatic ring
FLUORESCENCE DETECTION IN HPLC
Background
--From our background in spectroscopy we
know that molecules have different energy
states. The two important states are:
Ground State
Excited State
What happened when light energy is absorbed
by a molecule?
--Energy of a molecule increases and the molecule is promoted to excited state.
This is called Absorption or Excitation.
What happens when energy is released by a molecule?
--Energy of molecule decreases and the molecule returns to ground state from
Excited state. This is called “Emission”
Hence, there are two steps in measurement of fluorescence
Fluorescence molecule absorbs radiation at one l and emit radiation at a longer l.
Fundamental Equation for Fluorescence Detection
--Because fluorescence is an optical technique, it is also subjected to Beer’s
Law. For dilute solutions where ecl <0.01,
Intensity of incident
the fluorescence intensity (If) can be written as:
radiation
If = 2.303 ff I0 ecl
Quantum Efficiency
-A linear relationship exist between If and concentration (c)
of the solute provided that ecl is small
--What does the above equation tell us?
--If can be increased (i.e., S/N can be improved) by working at higher ff, and
high excitation power
Q. Why lasers instead of lamp provide high sensitivity of detection in HPLC?
--Lasers produce high excitation power (I0)
--Laser light is monochromatic (little loss of incident light)
Schematic of a Fluorescence Detector
--To avoid problems of differentiating
between excitation and emission
fluorescence detector operates in
“right angle configuration”
Working
1. Radiation from a xenon or
deuterium lamp passess through
an excitation filter, (which provides
essentially monochromatic light)
of desired wavelength to excite the
sample.
2. This excitation l of light then
passes through the column effluent
in the flow cell.
3. When the sample molecule passes
through the column effluent they are excited
and emit light (fluorescence) at a
longer l.
4. A second (emission) filter is positioned at 900 to the first filter to collect the
emitted light. In this way only the light emitted from the sample fluorescence will
pass on PMT for quantitation of the emission signal
Advantages of Fluorescence Detection in HPLC
1. Inherent advantage is higher sensitivity
~2-3 orders of magnitude greater than UVdetection. For example, polycyclic aromatic
hydrocarbons (PAHs) are important air pollutant
(needs to be detected at low concentration).
Chromatogram on the right
compares the UV and fluorescence
detection.
2. Derivitization with fluorescent
reagent o-phthaldehyde will enhance
the detectability
O
R
CH
+
H2N
COOH
H
CH
O
SR'
N
R'-SH
H
C COOH
R
Disadvantage of Fluorescence Detection in HPLC
*Careful choice of M.P. pH and M.P composition quench If
For e.g., aniline is cationic at acidic pH and do not fluoresce, but in pH
range of 7-12 it exist as a neutral species and fluoresce
..
NH2
+ NH
3
fluorescent
non-fluorescent
*Dissolve oxygen and impurities in M.P also quench fluorescence resulting in
self absorption
2. Non-linear calibration curve results at higher
Concentrations of the analyte
self
absorption
REFRACTIVE INDEX DETECTION IN HPLC
-Closest to the ideal of a universal detector (show respond to most solutes)
--Magnitude and the direction of the response depends on the difference in RI
between M.P and the solute(s)
Therefore, sensitivity reaches a maximum when DRI is greatest
Is RI is a bulk or solute property detector?
It is a bulk property detector
•Output of RI detection may show a positive or a negative peak in the same run
for several compounds. If the alkanes in the above table are analytes separated
by HPLC using tetrahydrofuran as the M.P. Which alkane in RI detector will show
positive, negative or no peak.
Nonane = No peak
Pentane = negative
Decane = positive
Types of RI detector
--Two major types are available
(a) Deflection type (most popular)
(b) Reflection type (measure changes in % reflected light at glass-liquid interface
*Reflection type is less popular than deflection type RI detectors
Deflection type RI detector. The reason its called deflection type is because
deflection is created in a
rectangular sample cell
by separating the
compartment into two
parts with a diagonal
glass divider.
P1
Operating principles
P2
--Light from the source is
focussed onto the sample
cell, which consist of
sample and the reference chamber.
b) After deflection from the mirror, light is diverted through an optical zero
adjustment (beam splitter) into the detector, which actually consist of two photocells, P1 and P2.
c) When a solute elutes off the column the RI of the sample compartment changes
d) This causes a change in the amount of deflected light, which in turn
changes the relative the relative amount falling on P1 and P2. Therefore,
differences in relative output P1 and P2 is measured.
*Key: Beam splitter movement, which is proportional to the difference in RI that
cause the splitter to change its angle. The difference is amplified by the amplifier
and the change is measured by the recorder.
What is the most important factor, which influence the performance of RI
detector?
Temperature has a profound effect on RI detection signal. A small change in
temperature (e.g., 0.001 0C) cause a change in 10-6 RI units.
--Most commerical detectors have heat sink and temperature control facilities,
but this may lead to undesired dead volume. Hence, N is affected.
--Other important advantages of RI detector is Universal (respond to all
types of compounds). Therefore, nochromophoric analytes (carbohydrates,
alcohols and polymers) can be detected using RI detection.
Disadvantages of RI detector
a) Not suitable for gradient elution.
Changes in solvent composition
change RI. Therefore, baseline shifts
and S/N is affected.
b) Require careful control of the
column and detector temperature.
c) Moderate sensitivity 10-9--10-10g.
“Not useful for trace analysis”