DIODE ARRAY AND FLUORESCENCE DETECTOR

1. HPLC
Diode Array and Fluorescence Detector
Tejaswini k. Mane
G.N. Khalsa college
M.Sc part 1
Analytical Chemistry

2. Chromatography
 Chromatography is a separation technique which is used to separate a
mixture of compounds into its individual components.
 It separate a mixture of compounds into its individual components
based on certain physical and chemical properties.
 The compounds bind at specific regions of stationary phase based on
certain physical and chemical properties.

3. HighPerformanceLiquidChromatography
 HPLC is a form of liquid chromatography used to separate compounds that
are dissolved in solution.
 HPLC instruments consist of a reservoir of mobile phase, a pump, an injector,
a separation column and a detector.
 Compounds are separated by injecting a sample mixture into the column.
 The different component in the mixture pass through the column at
differentiates due to differences in their partition behavior between the mobile
phase and the stationary phase.

4. DETECTOR
• The detector can detect the individual molecules that come
elute from the column.
• A detector serves to measure the amount of those molecules so
that the chemist can quantitatively analyze the sample
components.
• The detector provides an output to a recorder or computer that
results in the liquid chromatogram.

5. DetectorCharacteristics
• High sensitivity and reproducible, predictable response
• Respond to all solutes, or have predictable specificity
• Response unaffected by changes in temperature and mobile
phase flow
• Respond independent of the mobile phase
• Reliable and convenient to use
• Nondestructive of the solute
• Provide qualitative and quantitative information on the
detected peak
• Fast response

6. CommonHPLC Detectors
 UV-VIS
•Diode Array •Multiple Wavelength •Variable
Wavelength
 Fluorescence
 Mass Spectrometers
 Refractive Index
 Light Scattering
 Electrochemical
 Radioactivity
 Conductivity

7. DiodeArrayDetector
 UV/UV-VIS detectors are most frequently used to measure components
showing an absorption spectrum in the ultraviolet or visible region.
 A DAD detects the absorption in UV to VIS region. While a UV-VIS detector
has only one sample-side light-receiving section, a DAD has multiple
photodiode arrays to obtain information over a wide range of wavelengths at
one time, which is a merit of the DAD.

8.  spectra are measured at intervals of 1 second or less
during separation by HPLC with continuous eluate
delivery.
 If the measurement is performed at a fixed wavelength,
components are identified from only their retention time.
 In such a case, the DAD can be used to identify
components by a comparison of the spectrum.
 Light from the lamp is shown onto the diffraction grating,
and dispersed according to wavelength.

9.  PDA detection offers the following advantages:
 Determination of the correct wavelengths in one run.
 After all peaks have been detected, the maximum absorbance wavelength for each
peak can be determined.
 A PDA detector can collect spectra of each peak and calculate the absorbance
maximum.
 PDA detector can analyze peak purity by comparing spectra within a peak.
 Scan spectrum very quickly: entire spectrum in <1 second
 Provides single beam.
 Useful for kinetic studies.
 Useful for qualitative and quantitative determination of the components exiting
from a liquid chromatographic column.
 Disadvantages: large noise because the amount of light is small; the DAD is also
susceptible to various changes, such as lamp fluctuations, because the reference
light cannot be received.

10. FluorescenceDetector

11. Two general types of instruments exist:
 filter fluorometers that use filters to isolate the incident light and
fluorescent light.
 spectrofluorometers that use a diffraction grating monochromators to
isolate the incident light and fluorescent light.
Both types use the following scheme:
 The light from an excitation source passes through a filter or
monochromator, and strikes the sample.
 A proportion of the incident light is absorbed by the sample, and some of
the molecules in the sample fluoresce.
 The fluorescent light is emitted in all directions.
 Some of this fluorescent light passes through a second filter or
monochromator and reaches a detector, which is usually placed at 90° to
the incident light beam to minimize the risk of transmitted or reflected
incident light reaching the detector.

12.  This is normally used as an advantage in the measurement of specific
fluorescent species in samples.
 When compounds having specific functional groups are excited by shorter
wavelength energy and emit higher wavelength radiation which called
fluorescence. Usually, the emission is measured at right angles to the
excitation.
 Fluorescence intensity depends on both the excitation and emission
wavelength, allowing selectively detect some components while
suppressing the emission of others.

13. Advantages
• Sensitivity:
The sensitivity of fluorescence detection is approximately 1,000 times greater
than absorption spectrophotometric methods. This leads to greater limits of
detection, while potentially using less sample material. This is important especially
when working with precious or limited-quantity materials.
• Specificity:
Only molecules that fluoresce are detected by this method, resulting in greater
specificity compared with UV/Vis absorption.
• Wide concentration range:
Fluorimetry generally can detect more than three to six log orders of
concentration without sample dilution or modification of the sample.
• Accurate results:
The sensitivity and specificity of fluorescence measurement leads to potentially
more precise and accurate readings.

14. Disadvantages
• Economic value: The price of the instrument, as well as the assay reagents, can be a
challenge to cost-conscious scientists. The light source and filters are key components
of the instrument, providing sensitivity and selective detection, but these options
also can affect purchase price. Price per assay or even per data point should be
considered when evaluating the methodology.
• Impurities: Although fluorimeters are not affected by turbidity, light scatter may
artificially increase the measured readings. On the other hand, if interfering
substance or impurities absorb light, fluorescent readouts may be reduced.
• Bubbles: Bubbles in the sample preparation can result in erratic or fluctuating
readings, as measurement depends on light cleanly exciting the sample’s molecules.
• pH: Fluorescence can be affected by the pH of the sample solution, leading to
inaccurate measurements. Using the same conditions for standards, blanks and
samples helps eliminate this effect.

15. References :
• Instrumental Analysis , Skoog, Holler and Croouch Pg.
no. 901 – 906.
• Principles of Instrumental Analysis by Skoog, Holler,
Nieman 5th edition Pg. no 147 – 164, 650 – 652,
Harcourt college Publications, 1998.
• Handbook of Instrumental Technology, Pg. no 491 – 493.
• Vogel’s text book of quantitavive chemoical analysis, 6th
edition, pearson education LTD. Pg. no 318 – 325.

16. THANK YOU…!