Transcript 1) The

Principles of HPLC
Part 1: Overview
Lab Methods
Fall, 2013
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.
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.
COLUMN
INJECT HERE
MOBILE
PHASE
DETECTOR
TYPICAL HPLC SYSTEM: The operator can choose the
mobile phase, flow rate, injector volume, column, and detector
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.
IN THE LOAD POSITION, THE
MOBILE PHASE GOES
THROUGH A BYPASS
IN THE RUN POSITION, THE
MOBILE PHASE ALL GOES
THROUGH THE LOOP
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
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.
The packing is made beads (5 microns in size) coated
with C18 groups. This coating is VERY HYDROPHOBIC.
C18
C18
C18
C18
C18
C18
C18
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.
Alpha-tocopherol
3 methyls on ring:
most hydrophobic
Gamma-tocopherol
2 methyls on ring
Delta-tocopherol
1 methyl on ring
Tocol (internal standard, our work)
0 methyls on ring:
least hydrophobic
MeOH flowing through column, packed with C18 beads
Tocol
a-toc
Inject tocol and a-toc at same time
At start
SEE NEXT SLIDE!
After 1 minute
After 4 minutes
After 6 minutes
a-toc stuck most to the beads, and came off later
Tocol did not stick to much, and came off earlier
DAD1 B, Sig=295,2 Ref=600,100 (100705\VITA0001.D)
14
0.
81
4
mAU
Ar
ea
:
Tocol
12
10
Ar
ea
:
51
.1
42
1
8
60
.3
5
6
Ar
ea
:
a-Tocopherol
4
2
0
-2
-4
0
2
4
6
8
10
12
14
16
18
min
START RUN
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.
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).
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
a-Tocopherol
maximum
absorbance
*DAD1, 13.464 (4.1 mAU, - ) of VITEA002.D
mAU
4
3.5
3
2.5
2
1.5
1
0.5
0
260
280
300
320
340
360
380 nm
Alpha-tocopherol spectrum: the best data is achieved
by setting the detector at 295 nm
Structure of vitamin A (all-trans-retinol)
Retinol
maximum
absorbance
*DAD1, 5.517 (10.8 mAU, - ) Ref=4.857 & 6.137 of VITEA002.D
mAU
10
8
6
4
2
0
260
280
300
320
340
360
380 nm
Retinol spectrum: the best data is achieved by setting the
the detector at 325 nm, but other wavelengths can be used
Vitamin C releases electrons very easily as it passes
through the detector, so it can be measured.
+200 mv
oxidizing
voltage
+ 2 electrons
The electrons
generate
a current, which
is measured
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.
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.
You could not easily measure decane (C10H22) by HPLC. Examine the
structure of decane.
H
H
H
H
H
H H
H
H
H
H– C – C – C – C – C – C – C – C – C – C –H
H H
H H
H H H
H 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.
C
CH3
OH2+
CH3
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.