Home
|| HPLC Topics
|| LC-MS
TOPICS || HPLC
Course || Educational
|| Links
Courses&
Events
|| Personal
|| FTIR ||
Contact Me
1. Solvent Delivery System
a. Preparation of the Solvents – Hygiene
Before any analysis by HPLC the solvents for the mobile phase are
prepared. In every HPLC it is possible to identify the solvent
system by
the reservoirs and the Teflon tubing leading from them into the
gradient
proportioning valve in the pump (a low pressure mixing type),
which
should be marked clearly as A,B, C, D (according to the number of
reservoirs
available). One tubing only comes out of the pump, in which the
final
composition flows constantly.
• It is important that on every reservoir will be
clearly labeled!
• In every bottle’s end of the tubing there is a
very fine filter that prevents from colloids such as algae and dust to
enter
the column. The filter should be protected from microorganism’s
growth and take care of it from time to time (at least once a month
when
aqueous buffers are used) by sonication in 10%-20% HNO3 in water.
Selecting water for HPLC
by Millipore
On-line
recipes for Buffers - PFG
• All types of solvents should be filtered before any
use (or verify that they are sold already filtered), to prevent
clogging of the
column. Filtration needs to be performed with a special solvent
filtration system that should exist in every analytical lab that uses
HPLC. There are 3 categories of membranes for filtration,
compatible with
water and compatible for organic solvents and compatible with
both. Pore
diameter in these membranes are 0.2 – 0.45 um
Solvent
Preparation by Millipore Sample
preparation -
filtration products: PALL
• When a solvent is replaced in a reservoir, A, B, C or D it is important not to switch it with a non-compatible solvent that does not mix with it properly. This issue is especially crucial when switching back and forth between reversed phase and normal phase. See: Switching between Normal phase and Reversed phase.
• In many instrument there might be an additional in-line filter between the pump and the injector tha tcleans the solvents from un-washable impuritites in the mobile phase. In special cases (full aqueous mobile phases) it is also recommended to add a small silica-gel column that will saturate the mobile phase before its arrival to the reversed phase column with silica, by that will reduce the hydrolysis of its packing and prolong its life.
2. Programming of
the
mobile phase composition – Mixing between different solvents
• After the preparation of the solutions and solvents
a
protocol is decided, i.e., the composition of the mobile phase during
the
run. For this purpose it there is a need to mix various solvents.
• It is imperative
to verify whether the solvents mix easily with each other before using
them. Mixing
solvents that are not compatible with each other during a separation
program is
strictly forbidden because the
pump, the column and the detector cell
can be
damaged! For example, chloroform is entirely wrong for reversed
phase
chromatography when water is used in the mobile phase as a bulk
solvent.
Hexane does not mix well with methanol and acetonitrile.
Isopropanol mixes with water, acetonitril
and methanol (reversed phase) on one hand, and on the other it also
mixes with
hexane and/or chloroform (normal phase). Therefore, isopropanol
is used as a mediating solvent
in the switch between reversed phase and
normal
phase modes of chromatography.
• It is important to note that not every solvent is
appropriate for HPLC use, for example pentane is too volatile and
bubbles are
release from it during high pressures changes. Isopropanol
or ethanol are very viscous, and cause high pressures in the column
when they
are used at high percentage. A MIXTURE OF 50:50 METHANOL WATER is very viscous and creates high
pressures! Make sure
to use a temperature over 40 when using it.
• Another important thing to remember that acetonitril and methanol can be used as
mediators
between
most aqueous non-buffer type
of mobile phases, therefore it is
recommended to
leave the system in a mixture of water-methanol-or-acetonitril
or pure methanol-or-acetonitril in the end
of
work.
3. Elimination of
gasses
in the solvents – Degassing
• Degassing of solvents is removing all air in them,
and it is imperative in most HPLC methods. There are several ways
to degass solvents,
the most common
one currently is by using in-line vacuum degasser. The other
method is by
sparging helium in the reservoir. The
vacuum
degasser contains vacuum chambers, one for every reservoir, where the
solvents
enter, effuse all the gasses from them instantly, and proceed
degassed.
When using an in-line vacuum degasser there is a need to verify that
the
chambers are washed thoroughly during the purge stage of the sustem. In
some
instruments the volume of such chambers can get up to 12 ml.
• When Helium is bubbled through the solvents it is
important to make sure that the Helium cylinder is always opened to
maintain
positive pressure in the tubings leading
to the
instrument. The Helium is opened/shut
by a
special valve at the HPLC system itself. It is important to have
pressure
regulator to every one of the solvents due to the differences in their
viscosity and solubility of gasses in them. Helium is an inert
gas and
its solubility is so small that it removes the dissolved air and
saturates the
solvents, and then bubbles are not created during mixing of the
solvents and
the change in mobile phase composition during the run (gradient).
Helium degassing should be used only
in system under fume hood, because Acetonitril and Methanol are toxic!
Description of vacuum degasser
Photo:
Purging the System
• When the solvents are ready for work the system is
purged with the degassed solvents to wash the tubing all the way to the
column’s connection. This is done to drive out previous solvents
and/or air from the system. The void volume of the system should
be
accounted for, including the tubings from
the
reservoirs, which could be as high as 5 ml. This wash can be done
at very high flow-rates, up to 10
ml/min for 2-3 minutes for
every reservoir. In every HPLC system there is a special valve
for this
purpose, which diverts the solvents out in between the pump and the
injector.
• It is important to purge all the solvents in the
system, not just those used immediately, because sometimes the user can
make a
mistake and select the wrong solvent and introduce air to the system.
• When switching solvents it is imperative to notice
that they can readily dissolve in each other even in the purge stage,
because
they all pass through the pump, which is the meeting point to all
solvents. When Normal Phase is used it is especially important to
switch
between solvents according to their ability to mix readily.
Introduction
1-3: Solvent delivery system
4. The column
5 - Detector
6- Injector
7-Gradient
and isocratic
8-Data
Processing
Copyright (C) since 1997: Dr. Shulamit Levin