The document discusses the applications of ultraviolet (UV) spectroscopy with High-Performance Liquid Chromatography (HPLC), explaining key concepts such as separation techniques, instrumentation, and detector types. It highlights the advantages of UV detection, including sensitivity and wide applicability in various industries such as pharmaceuticals and food. The content also acknowledges contributors and references relevant literature on the topic.

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2. Presentation Topic:
Presented by:
Assigned By:
APPLICATIONS OF UV WITH HPLC
DR.AYAZ ALI
MEMON
DADU MAL(DK)
Zubair Ali Shah

3.  Spectroscopy
UV-Visible Region
Hyphenated techniques
Chromatography
 HPLC
HPLC Instrumentation
UV Detector
Advantages and Limitations
 Application of UV with HPLC
References
Acknowledgement
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4. The study of interaction of
electromagnetic radiations with
matter is called “Spectroscopy.
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5.  Detection of functional groups.
 Detection of impurities.
 Qualitative analysis
 Quantitative analysis.
 Characterization of species
 Structure elucidation.
 Determination of conjugation etc.
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6. This is the earliest method of molecular
spectroscopy.
A phenomenon of interaction of molecule with
ultraviolet and visible light(EMR).
Absorption of photons results in electronic
transition of a molecule and electrons are promoted
from ground state to the excited state.
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7.  Russian botanist Mikhail Tswett in 1903
 Chromatography is a physical method of
separation that distributes components to separate
between two phases, one stationary (stationary
phase), the other (the mobile phase) moving in a
definite direction.
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CHROMATOGRAPHY
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9.  HPLC is a technique in analytical chemistry used to separate,
identify, and quantify each component in a mixture.
BASIC PRINCIPLE
 When a mixture of components is introduced into a HPLC column,
they travel according their relative affinities towards the
stationary phase.
 The components which has more affinity towards the adsorbent
travel slowest the component which has less affinity towards the
stationary phase travels faster.
 Since two components have not the same affinity towards the
stationary phase, the components are separated.
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HPLC

10.  Hyphenated technique is a combination or coupling of two
different analytical techniques with the help of proper
interface.
 Hirschfield (1980) introduced the term “hyphenation” to
refer to the on-line combination of separation technique and
one or more spectroscopic detection techniques .
 The hyphenated technique is developed from the coupling of
a separation technique and an online spectroscopic detection
technology.
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11.  Mobile Phase
 Degasser
 Pressure Pump
 Sample Injector
 Guard Column
 Analytical Column
 Detector
 Data Analyzer
 Waste Container
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12. UV/VIS Detector
Photo diode array Detector
Fluorescence Detector
Refractive index Detector
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13.  UV detection is the most usual and widespread detection.
 The instrument is simple and is available from numerous
manufacturers
 Many analytes suitable for HPLC analysis and also absorb
in the UV region.
 The wavelength range can be easily expanded to the region
of visible light (Vis) for the detection of colored Analytes.
 UV/Vis detection is more sharp than many other detection
systems.
UV DETECTOR
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14. SlitPhotodiode
Deuterium
Lamp
Diffraction Grating
Flow Cell
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15. The heart of an efficient HPLC detector
is the flow cell.
Light passes through the flow cell for
detection.
Flow cell has capacity 10-25 µL with a
narrow depth(1.07 mm) and large
surface area for excitation-emission
collection
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17. The detector measures the concentration of sample bands as they
leave the column and pass through the detector flow cell.
When no band is passing through the detector, a constant signal is
recorded -- called the Baseline of the chromatogram or detector.
When a sample band reaches the detector, the detector responds to
the difference in the mobile phase properties caused by the
presence of the sample compound, giving rise to a change in
detector signal, seen as a Peak.
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18.  They have much better sensitivity.
 Reproducibility.
 Wide linear dynamic range.
 Short response time.
 Minimum volume detected.
 Similarity in response towards all analytes.
 Non-destructive method.
 They are not temperature sensitive.
 They can be used with gradient elution.
 They are sensitive to large number of organic compounds.
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19. Samples that are thermally labile may decompose or react
in heated nebulizer of the source and should be avoided.
Samples that have charges in solution reduce the
sensitivity such as protein, peptide or amino acids should
be avoided.
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20. Determination of conjugation.
Analysis of colored and colorless compounds.
Used in food Industries.
Purification of Biological samples.
Drug Discovery.
Pharmaceutical Industries.
Oil Refineries.
Petrochemical Industries etc.
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21.  ORGANIC SPECTROSCOPY AND
CHROMATOGRAPHY,
Third edition, By M.Younis
 ELEMENTRY ORGANIC SPECTROSCOPY, By
YR SHARMA
 Wikipedia
 Quora
 Slideshare 21

22.  We are thankful to Almighty ALLAH for
his divine support.
 We are thankful to Sir Dr: AYAZ ALI
MEMON for giving such a good platform
to enhance our skills and also thanks for his
kind support.
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23. THANK YOU
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