Applications of uv with hplc

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. National Centre of Excellence in Analytical Chemistry University of Sindh Presentation Topic: Presented by: Assigned By: APPLICATIONS OF UV WITH HPLC DR.AYAZ ALI MEMON DADU MAL(DK) Zubair Ali Shah 2

3. CONTENTS Spectroscopy UV-Visible Region Hyphenated techniques Chromatography HPLC HPLC Instrumentation UV Detector Advantages and Limitations Application of UV with HPLC References Acknowledgement 3

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

5. Why we need Spectroscopic Technique ? Detection of functional groups. Detection of impurities. Qualitative analysis Quantitative analysis. Characterization of species Structure elucidation. Determination of conjugation etc. 5

6. Introduction Of UV-Visible Spectroscopy 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. Chromatography 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 techniques 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. Instrumentation HPLC Mobile Phase Degasser Pressure Pump Sample Injector Guard Column Analytical Column Detector Data Analyzer Waste Container 11

12. Detectors of HPLC 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. Components of UV Detector S lit P hotodiode D euterium L amp D iffraction G rating F low C ell 14

15. What is Flow Cell 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. Principle of HPLC with UV-Visible 16

17. Function Of UV Detector 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. Advantages of UV Detector 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. limitations of UV Detector 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. APPLICATIONS OF UV WITH HPLC 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. REFRENCES 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. AKNOWLEDGMENT 22

23. THANK YOU 23 COMMENTS? Queries? SUGESTIONS?