Pickering Laboratories Pinnacle PCX - Design of a HPLC system

1.2
Section 1
IntroductIon
Pinnacle Operators Manual
Pickering Laboratories Inc.
will manifest itself in the baseline of a chromatogram unless it is properly dampened. Below are the basic
requirements for a successful automated post-column method:
1)
Reagent Stability. The minimum reagent stability sufficient for routine work is one day. This means that
the yield and signal-to-noise ratio for a given sample must remain constant for at least 8 hours.
2)
Reaction Speed. The analytical separation is complete when the reagent is mixed with the column effluent.
Therefore it is important that the analyte react as quickly as possible. The longer the reaction time, the larger
the reactor volume required. With larger volumes, the peak shape will become distorted. To minimize band
spreading, it is important to keep the overall time (and therefore volume) as low as possible between the
column and detector. If the reaction is slow (in excess of one minute), an elevated temperature can be used to
decrease the reaction time.
3)
Reproducibility. Because the reaction is occurring “on the fly,” as the combined column and reagent
stream flows toward the detector, the reproducibility is linked to the flow rate precision of the pumps and to the
temperature. Accordingly, even an incomplete reaction will be as repeatable as the retention time for any given
species. Therefore, it is important that the pumps maintain a constant flow rate, and that the reactor maintain a
constant temperature. It is also very important that the column be maintained at constant temperature to ensure
that the analytes are properly separated and identified.
4)
Minimal Detector Response of Reagents. The color or background fluorescence of the reagent (or
its by-products) represents a continuous noise source. Because the reagent is present in excess relative to the
analyte, the analyte’s signal could be obliterated by the reagent’s strong background signal. The baseline noise
is proportional to the background signal.
5)
Solubility. All species must remain in solution, including the combined components of the eluants and the
reagent(s), as well as the newly formed derivative(s). Precipitates can block capillary tubes, burst reactors, and
foul detector flow cells.
6)
Uniformity of flow. The baseline noise is a function of the flow-noise in the eluant and reagent pumps.
Non-uniform flow causes non-uniform mixing leading to modulation of the background signal which appears
as noise. Refractive index noise can be even more objectionable than absorbance noise. Common techniques
for evening the flow of the pumps is the addition of a pulse dampener, or the use of a syringe pump.
Design of a HPLC system
This next section is a simplified view of a HPLC, followed by the ideas behind a post-column system. This
section is intended to help novice HPLC operators.