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HPLC System Performance Factors
If the desired separation quality and
reproducibility can be achieved, then quality and reproducibility of
the post-column derivatization will follow. However, this statement
presupposes that the HPLC system used will meet all of the basic
requirements for holding and injecting a sample, forming the eluant
gradient or step-change, pumping eluant through the column against
high pressure, and detecting the signal from the post-column
reaction with low background noise and high sensitivity. For most
HPLC brands these assumptions are valid.
HPLC Pumps
Ion-exchange chromatography of amino acids
followed by post-column derivatization places higher performance
demands upon the HPLC system than do standard reversed-phase
applications. Thus, for methods requiring an elution gradient,
eluant pumps with low-pressure mixing are preferred. These are the
exemplified by a ternary gradient pump with a proportioning valve
which delivers a pre-programmed percentage of each eluant per pump
stroke, in order to form the desired elution gradient.
Since all Pickering gradient methods require
three eluants-- two buffers and a high-pH column regenerant-- a
ternary HPLC pump is required. If a piston-wash accessory is
available for that system, we strongly recommend its use, since it
will prolong the life of the piston seal. The additional expense of
a more inert pump and flow-path than stainless steel, e.g. titanium,
while desirable, is not needed for these applications. The advances
in HPLC technology over the last ten years have resulted in
commercially-available HPLC gradient pumps which are capable of
stable, highly-reproducible operation against high pressures.
Caution: Some HPLC pumps require special
seals, check-valves or other components to be compatible with high
pH or high-concentration salt buffers. Contact the pump manufacturer
or Pickering Laboratories for guidance.
For special step-gradient applications such as
PKU and all of the Beckman System 6300 methods, pumps delivering
eluants via high-pressure mixing have an advantage over the HPLC
ternary gradient systems. Since each pump delivers 100% of one
eluant at a time, the "mixing" takes place after the pump outlet,
before the column. Discrete step changes and return to equilibrium
conditions can be sharp and rapidly effective, because the liquid in
each pump head is not a mixture, and consequently, the volume does
not need to be swept out beforehand. For the 5-10 minute high-volume
clinical screening analyses, especially, it is highly desirable to
be able to recycle the column as rapidly as possible for the next
sample injection.
Depending upon the design and volume of the
flow-path in the HPLC pump head, it may be necessary to "dead-head"
the pressure transducer and/or capacitor. It is important to ensure
that there is no "dead volume" where unswept buffers may reside and
adversely affect the reproducibility of the early part of the
chromatogram.
Before ordering a post-column amino acid
analysis system, be sure to let us know what brand of HPLC equipment
you intend to use. Our Customer Service department can advise you of
the required modifications, if any.
Sample Injector
Whether using a manual injector or autosampler,
the injection valve in most brands (usually Rheodyne) contains a
rotor seal which will not tolerate the high pH column regenerant
used in the amino acid methods. Some, like Hewlett-Packard's, also
have an injection needle seat of the same material. Lithium,
Potassium or Sodium Hydroxide will dissolve the standard seal, and
the dissolved polymer will be distributed eventually throughout the
column, post-column system, and the detector. Besides causing
chronic baseline perturbations, such contamination may result in the
near-term replacement of the column, guard, and reactor. This
unfortunate outcome can be prevented by substituting the original
rotor seal (usually Vespel®) with one made of either Tefzel® or
PEEK.
While all-titanium construction is not
necessary, a titanium sample needle will extend the service interval
significantly.
Buffer Reservoirs
Dip-tubes, gas dispersers and filters in the
eluant reservoirs are subject to corrosion by the sodium and lithium
buffers. All such metal parts in contact with these liquids must be
replaced by inert equivalents.
Detector Flowcell
In post-column amino acid analysis, the reagent
and eluant stream meet at the mixing Tee, are heated, and then
directed to the detector. If OPA is the post-column reagent of
choice, most major brands of fluorescence detectors will work
without modification-- some more sensitively than others. The
post-column instrument applies a nominal 7 bar (100 psi) to the
flowcell outlet in order to prevent bubble formation, but this
pressure can be adjusted downwards if the flowcell will not tolerate
7 bar.
For the visible-range detector required for the
Ninhydrin reagent, the same pressure specification applies. In
addition, the geometry of the flowcell should reject refractive
index (RI) effects. Expect a very noisy baseline if the detector
flowcell is not RI insensitive. Fortunately, most modern brands of
detector flowcells are designed to reduce or eliminate the effects
of RI.
For detection at 570 and 440 nm a detector with
a tungsten lamp is recommended, but not strictly necessary.
Background subtraction at 690 nm is a useful feature for improving
the signal/noise ratio.
Some detector flow-cells have a very fine inlet
capillary or a heat-exchanger. If they are too restrictive, the
excess back-pressure on the post-column reactor will cause the
safety relief valve to open. Such tubing can be safely replaced by
0.25 mm (0.010 inch) i.d. tubing. Consult Pickering or the
manufacturer of the detector for specific information.
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