High Performance Liquid Chromatography HPLC- UV-VIS Detector Animation — Transcript

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1. 0:00high-performance liquid chromatography
2. 0:01is a technique and analytical chemistry
3. 0:04used to separate identify and quantify
4. 0:06each component in a mixture as well as
5. 0:09other forms of chromatography in HPLC
6. 0:12there is a mobile phase which forced by
7. 0:14a pump to pass through the system and
8. 0:16there is a stationary phase called a
9. 0:18column which located in an oven where
10. 0:20the temperature can be controlled
11. 0:24the sample can be automatically injected
12. 0:27into an HPLC system by the use of HPLC
13. 0:29auto sampler
14. 0:33or can be manually introduced into the
15. 0:35injector using a syringe
16. 0:38there is also a detector attached to the
17. 0:40HPLC system which measures the analytes
18. 0:43after its separation in the column
19. 0:47the pump forces the mobile phase through
20. 0:49the column and then the detector under
21. 0:52high pressures
22. 0:57in the HPLC system a vacuum pump and a
23. 1:00dagger sir are connected to the pump and
24. 1:02used to remove dissolved gases from the
25. 1:04solvents the pump drives each solvent to
26. 1:07the mixing chamber where mixing takes
27. 1:09place under higher pressures HPLC
28. 1:13analysis commonly uses isocratic or
29. 1:15gradient elution an isocratic mode the
30. 1:18mobile phase composition remains
31. 1:20constant throughout the procedure
32. 1:24whereas in gradient mode the mobile
33. 1:27phase composition is changed during the
34. 1:29separation process
35. 1:34sample introduction can be accomplished
36. 1:37in various ways the simplest method is
37. 1:40to use an injection valve in the load
38. 1:42position the high-pressure el Yuting
39. 1:43solvent flows to the column directly the
40. 1:46loop is loaded at the atmospheric
41. 1:47pressure from a syringe via the needle
42. 1:49port excess sample exits the loop via
43. 1:52vent port after loading the sample the
44. 1:55valve is switched to the inject position
45. 1:57the flow delivered by the pump flows
46. 1:59through the loop forcing the sample
47. 2:01ahead of it flowing to the column
48. 2:08then the valve returns to the load
49. 2:10position and the mobile phase moves the
50. 2:12sample through the column
51. 2:18the separation is based on differential
52. 2:20partitioning of the sample components
53. 2:22between the mobile and stationary phases
54. 2:24the component which has more affinity to
55. 2:27the mobile phase consequently less
56. 2:29affinity to the stationary phase travels
57. 2:31faster and eluded out first and the
58. 2:34component which has more affinity to the
59. 2:36stationary phase consequently more
60. 2:38interaction travels slower and diluted
61. 2:40later this separation can be carried out
62. 2:43according to which type of HPLC used
63. 2:46there are two main types of HPLC reverse
64. 2:49phase and normal phase reversed phase
65. 2:52has a non-polar stationary phase and
66. 2:54moderately polar mobile phase one common
67. 2:57stationary phase is a silica which has
68. 2:59been modified by attaching a straight
69. 3:01chain alkyl group to its surface such as
70. 3:04the ITAT as'll group c18 or the octal
71. 3:06group c8
72. 3:11in this case the more hydrophobic the
73. 3:14analytes are the more retained they will
74. 3:16be on the stationary phase the more
75. 3:19polar they are the more they will prefer
76. 3:21the mobile phase
77. 3:30then normal phase chromatography the
78. 3:32stationary phase is polar and the mobile
79. 3:35phase is non-polar this method separates
80. 3:39analytes based on their affinity for a
81. 3:40polar stationary surface such as silica
82. 3:46in this case the more polar the analytes
83. 3:49are the more retained they will be on
84. 3:51the stationary phase the more
85. 3:53hydrophobic they are the more they will
86. 3:55prefer the mobile phase
87. 4:04as compounds ellyiot from the column
88. 4:06they interact with the detector
89. 4:08different types of detectors can be used
90. 4:10such as the UV this detector which
91. 4:12showing an absorption spectrum in the
92. 4:14ultraviolet or visible region for UV
93. 4:17detection a deuterium discharge lamp as
94. 4:19a light source is used and four
95. 4:21components detection and visible region
96. 4:23a tungsten lamp is used then UV this
97. 4:27detector we can also find entrance slit
98. 4:30lens prism or diffraction grating exit
99. 4:34slit flow cell and detector for
100. 4:38absorption measurements
101. 4:41light from the lamp is shown onto the
102. 4:43prism and dispersed according to
103. 4:45wavelength when the measurement is
104. 4:47performed with a specific wavelength the
105. 4:49angle of the prism is adjusted so that
106. 4:51the light of this wave can shine on the
107. 4:53flow cell as the compound zealot from
108. 4:56the column they enter the flow cell
109. 4:58where the bonding and non-bonding
110. 5:00electrons of these compounds can absorb
111. 5:03energy in the form of ultraviolet or
112. 5:04visible light
113. 5:08the manner in which the final data is
114. 5:10displayed is based on the computer in
115. 5:12software
116. 5:14the number of Peaks present can indicate
117. 5:17how many components are in the mixture
118. 5:21usually the x axis of the HPLC
119. 5:24chromatogram shows the amount of time
120. 5:27taken for the analytes to pass through
121. 5:28the column and reach the detector
122. 5:30typically the y-axis or the area of the
123. 5:33peak is a reflection of the amount of a
124. 5:35specific analyte that's present