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Introduction to High Performance 
Liquid Chromatography and its parts 
1
Chromatography equipment looks 
rather intimidating to anyone who has 
not handled it before, but on a closer look and as 
you get familiar with the equipment you realize 
that behind the network of wires, complex 
plumbing and circuitry is a simple machine with 
only a few major parts. 
2
Different combinations of these parts namely : 
• Pumps 
• Detectors 
• Injectors 
yield an infinite number of configurations based 
on the application. 
3
Just like an understanding of human anatomy 
makes you conscious of the vital role of each 
and every body organ towards your well being 
and vitality. Similarly you need to have a good 
understanding of the parts of your HPLC 
system to generate data of highest reliability. 
4
A conceptual understanding of the function of 
each component will add to your comfort level 
with your HPLC system. You will ensure long 
time usage with high reliance on output data. 
5
What is HPLC? 
6
7
HPLC is a technique for separation, identification 
and quantification of components in a 
mixture. It is especially suitable for 
compounds which are not easily volatalised, 
thermally unstable and have high molecular 
weights. 
8
Mobile Phase 
9
The liquid phase is pumped at a constant rate to 
the column packed with the stationary phase. 
Before entering the column the analysis 
sample is injected into the carrier stream. 
10
On reaching the column the sample components 
are selectively retained on the basis of 
physico-chemical interactions between the 
analyte molecules and the stationary phase. 
11
Mobile Phase 
12
13
Mobile phase serves to transport the sample to the 
system. Essential criteria of mobile phase are 
inertness to the sample components. Pure 
solvents or buffer combinations are commonly 
used. The mobile phase should be free of 
particulate impurities and degassed before use. 
14
Mobile Phase Reservoirs 
15
16
These are inert containers for mobile phase storage 
and transport. Generally transparent glass bottles 
are used so that so as to facilitate visual 
inspection of mobile phase level inside the 
container. Stainless steel particulate filters are 
provided inside for removal of particulate 
impurities in the mobile phase if any. 
17
Pumps 
18
19
Variations in flow rates of the mobile phase 
effect elution time of sample components and 
result in errors. Pumps provide constant flow 
of mobile phase to the column under constant 
pressure. 
20
Injectors 
21
22
Injectors are used to provide constant volume 
injection of sample into the mobile phase 
stream. Inertness and reproducibility of 
injection are necessary to maintain high level 
of accuracy. 
23
Column 
24
25
A column is a stainless steel tube packed with 
stationary phase. It is a vital component and 
should be maintained properly as per supplier 
instructions for getting reproducibility and 
separation efficiency run after run. 
26
Column Oven 
27
28
Variation of temperature during the analytical run 
can result in changes of retention time of the 
separated eluting components. A column oven 
maintains constant column temperature using air 
circulation. This ensures a constant flow rate of 
the mobile phase through the column 
29
Detector 
30
31
A detector gives specific response for the components 
separated by the column and also provides the 
required sensitivity. It has to be independent of any 
changes in mobile phase composition. Majority of the 
applications require UV-VIS detection though detectors 
based on other detection technique are also popular 
these days. 
32
Data Acquisition & Control 
33
Modern HPLC systems are computer based and 
software controls operational parameters such 
as mobile phase composition, temperature, 
flow rate, injection volume and sequence and 
also acquisition and treatment of output. 
34
Specialized Versions 
35
Specialized HPLC Systems might also have solvent 
selection valves, vacuum degasser, auto 
samplers, column switches, pre or post 
column derivatization and fraction collectors. 
These are all covered at length in our online 
certificate program on HPLC. 
Certificate Course on HPLC 
36
What Next? 
• Keep Learning to Keep Succeeding – visit 
www.lab-training.com/hplc to learn more about 
HPLC 
• Get online certificate training on HPLC – 
www.lab-training.com/product/join-our-certificate- 
course-on-hplc/ 
• Subscribe to our newsletter and get free training 
delivered straight to your inbox – www.lab-training. 
com 
• Don’t have a HPLC? Need samples to be tested 
visit www.testing-lab.com and get a proposal! 
37

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Introduction to High Performance Liquid Chromatography (HPLC)

  • 1. Introduction to High Performance Liquid Chromatography and its parts 1
  • 2. Chromatography equipment looks rather intimidating to anyone who has not handled it before, but on a closer look and as you get familiar with the equipment you realize that behind the network of wires, complex plumbing and circuitry is a simple machine with only a few major parts. 2
  • 3. Different combinations of these parts namely : • Pumps • Detectors • Injectors yield an infinite number of configurations based on the application. 3
  • 4. Just like an understanding of human anatomy makes you conscious of the vital role of each and every body organ towards your well being and vitality. Similarly you need to have a good understanding of the parts of your HPLC system to generate data of highest reliability. 4
  • 5. A conceptual understanding of the function of each component will add to your comfort level with your HPLC system. You will ensure long time usage with high reliance on output data. 5
  • 7. 7
  • 8. HPLC is a technique for separation, identification and quantification of components in a mixture. It is especially suitable for compounds which are not easily volatalised, thermally unstable and have high molecular weights. 8
  • 10. The liquid phase is pumped at a constant rate to the column packed with the stationary phase. Before entering the column the analysis sample is injected into the carrier stream. 10
  • 11. On reaching the column the sample components are selectively retained on the basis of physico-chemical interactions between the analyte molecules and the stationary phase. 11
  • 13. 13
  • 14. Mobile phase serves to transport the sample to the system. Essential criteria of mobile phase are inertness to the sample components. Pure solvents or buffer combinations are commonly used. The mobile phase should be free of particulate impurities and degassed before use. 14
  • 16. 16
  • 17. These are inert containers for mobile phase storage and transport. Generally transparent glass bottles are used so that so as to facilitate visual inspection of mobile phase level inside the container. Stainless steel particulate filters are provided inside for removal of particulate impurities in the mobile phase if any. 17
  • 19. 19
  • 20. Variations in flow rates of the mobile phase effect elution time of sample components and result in errors. Pumps provide constant flow of mobile phase to the column under constant pressure. 20
  • 22. 22
  • 23. Injectors are used to provide constant volume injection of sample into the mobile phase stream. Inertness and reproducibility of injection are necessary to maintain high level of accuracy. 23
  • 25. 25
  • 26. A column is a stainless steel tube packed with stationary phase. It is a vital component and should be maintained properly as per supplier instructions for getting reproducibility and separation efficiency run after run. 26
  • 28. 28
  • 29. Variation of temperature during the analytical run can result in changes of retention time of the separated eluting components. A column oven maintains constant column temperature using air circulation. This ensures a constant flow rate of the mobile phase through the column 29
  • 31. 31
  • 32. A detector gives specific response for the components separated by the column and also provides the required sensitivity. It has to be independent of any changes in mobile phase composition. Majority of the applications require UV-VIS detection though detectors based on other detection technique are also popular these days. 32
  • 33. Data Acquisition & Control 33
  • 34. Modern HPLC systems are computer based and software controls operational parameters such as mobile phase composition, temperature, flow rate, injection volume and sequence and also acquisition and treatment of output. 34
  • 36. Specialized HPLC Systems might also have solvent selection valves, vacuum degasser, auto samplers, column switches, pre or post column derivatization and fraction collectors. These are all covered at length in our online certificate program on HPLC. Certificate Course on HPLC 36
  • 37. What Next? • Keep Learning to Keep Succeeding – visit www.lab-training.com/hplc to learn more about HPLC • Get online certificate training on HPLC – www.lab-training.com/product/join-our-certificate- course-on-hplc/ • Subscribe to our newsletter and get free training delivered straight to your inbox – www.lab-training. com • Don’t have a HPLC? Need samples to be tested visit www.testing-lab.com and get a proposal! 37