HPLC is Analytical technique that is used for separating the mixture of substances,so there is a number of promising application of HPLC-UV here uv detector is used which record the absorbance

1. 1

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
3

4. The study of interaction of
electromagnetic radiations with
matter is called “Spectroscopy.
4

5.  Detection of functional groups.
 Detection of impurities.
 Qualitative analysis
 Quantitative analysis.
 Characterization of species
 Structure elucidation.
 Determination of conjugation etc.
5

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.
6

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.
7

8. 8
CHROMATOGRAPHY
8

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.
9
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.
10

11.  Mobile Phase
 Degasser
 Pressure Pump
 Sample Injector
 Guard Column
 Analytical Column
 Detector
 Data Analyzer
 Waste Container
11

12. UV/VIS Detector
Photo diode array Detector
Fluorescence Detector
Refractive index Detector
12

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
13

14. SlitPhotodiode
Deuterium
Lamp
Diffraction Grating
Flow Cell
14

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
15

16. 16

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.
17

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.
18

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.
19

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.
20

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.
22

23. THANK YOU
23
COMMENTS? Queries?
SUGESTIONS?