1. Principles of HPLC Part 1: Overview Lab Methods Fall, 2013

2. These tutorials will describe several key features of HPLC: -principles of how HPLC works -sample processing (very important) -use of the column to obtain a separation -how the molecules are detected, when they come off the column The first tutorial provides a basic overview.

3. The first slide shows a system diagram of an HPLC. NOTICE WHERE YOU CAN MAKE CHOICES: -Mobile phase -Injection volume -Column -Detector type These are the major decisions that are made when setting up an HPLC analysis.

4. INJECT HERE MOBILE PHASE COLUMN DETECTOR TYPICAL HPLC SYSTEM: The operator can choose the mobile phase, flow rate, injector volume, column, and detector

5. HPLC PUMPS ARE VERY HIGH TECH DEVICES They produce very stable flow rates at very high pressures, that exceed 1,000 psi (atmospheric pressure = only 14 psi) All the fittings in the system have to be very tight, because leaks can easily occur at these high pressures from the pump.

6. IN THE LOAD POSITION, THE MOBILE PHASE GOES THROUGH A BYPASS IN THE RUN POSITION, THE MOBILE PHASE ALL GOES THROUGH THE LOOP

7. Several key principles are followed in making choices for an HPLC measurement: 1)The column and mobile phase have to be able to bring about a separation 2)The molecules need a feature to allow detection, such as an UV-absorbing group, an oxidizable, or the ability to produce charged fragments 3)Sample prep is usually required to allow putting the molecules onto the HPLC column

8. The molecules need a feature that allows separation Some tocopherols (alpha-tocopherol) are more hydrophobic than other tocopherols (tocol, gamma-tocopherol). This difference in their hydrophobic character can be used for separation. The next slides illustrate the principle of some molecules sticking more to the packing than others.

9. The packing is made beads (5 microns in size) coated with C18 groups. This coating is VERY HYDROPHOBIC. C 18 The next two slides show that a-toc sticks to the beads much more, and therefore moves more slowly with the methanol mobile phase flowing through the column.

10. Alpha-tocopherol 3 methyls on ring: most hydrophobic Gamma-tocopherol 2 methyls on ring Tocol (internal standard, our work) 0 methyls on ring: least hydrophobic Delta-tocopherol 1 methyl on ring

11. MeOH flowing through column, packed with C18 beads Tocol a-toc Inject tocol and a-toc at same time SEE NEXT SLIDE!At start

12. After 1 minute After 4 minutes After 6 minutes

13. a-toc stuck most to the beads, and came off later Tocol did not stick to much, and came off earlier

14. a-Tocopherol Tocol On the C18 column that we use, the a-tocopherol sticks better to the column than the tocol, and elutes later. Data from previous measurement in our lab. START RUN

15. The most common type of HPLC is C18, using particles coated with long-chain hydrocarbons as the stationary phase packed into the column. For our class, we chose this packing for vitamin E and vitamin A analysis. And we chose a mobile phase that was mostly acetonitrile (90% total content).

16. If your molecule has a property that allows detection (absorption spectrum, fluorescence, electron release, fragmentation), then - You can CHOOSE your detector. For our class, we chose: -for vitamin E and A, a UV detector -for vitamin C, electrochemical detector -For vitamin D, a mass spec detector

17. a-Tocopherol maximum absorbance Alpha-tocopherol spectrum: the best data is achieved by setting the detector at 295 nm

18. Structure of vitamin A (all-trans-retinol)

19. Retinol maximum absorbance Retinol spectrum: the best data is achieved by setting the the detector at 325 nm, but other wavelengths can be used

20. Vitamin C releases electrons very easily as it passes through the detector, so it can be measured. + 2 electrons +200 mv oxidizing voltage The electrons generate a current, which is measured

21. The electrochemical detection works well for all molecules that are readily oxidized. For vitamin C, we use E=200 mV, a low potential, but in fact vitamin C gives up electrons easily (it’s a good reducing agent, also!). Phenolic compounds in plants are easily oxidized, and this detector is popular for measurement of plant phenolics by HPLC.

22. MANY OTHER COMPOUNDS PASS THROUGH THE COLUMN AND THEN THROUGH THE DETECTOR, SUCH AS AMINO ACIDS AND SUGARS. HOWEVER – they are not oxidized, do not release electrons, and don’t create a signal.

23. You could not easily measure decane (C 10 H 22 ) by HPLC. Examine the structure of decane. C – C – C – C – C – C HH HHHH HH HHHH C – C – C – C HH HH HH HH – – – H H This molecule has no UV or visible groups. It cannot be readily oxidized to release electrons. It has no features that allow simple measurement by HPLC. In fact, MANY of the molecules in biology are difficult to measure because they are not readily detected. But for some molecules, HPLC works really well. Vitamin C, retinol, and tocopherol are examples that are easily measured. We will discuss the mass spectrometric detector, which enables almost every molecules to be detected.

24. C CH 3 OH 2 + For vitamin D, we will actually measure 25-OH-vitamin D, which is formed in the liver from dietary vitamin D. We can measure it by mass spec because it takes on a positive charge during sample analysis.