Waters ACQUITY UPLC I-Class, Системное руководство, Страница: 28 / 68

2-4 Optimizing Performance
with 2.5 mL for a typical 4.6 × 150 mm HPLC column. The smaller column
and particle size require a system with low dispersion that reduces dilution
and band broadening, thus maintaining the symmetrical peak shape, height,
and sensitivity produced by the high efficiency column.
An ACQUITY UPLC I-Class system typically exhibits a bandspread between 4
and 7 µL, or between 6 and 9 µL, depending on system configuration. An
Alliance HPLC system can exhibit a bandspread between 35 µL and 50 µL.
Because of the dispersion differences, a band on an Alliance system
experiences a significant increase in dilution, compared with an ACQUITY
UPLC I-Class system. As a result, UPLC peak concentrations are higher than
HPLC concentrations. Because solubility effects are more apparent in low
dispersion, high pressure systems, it is important to adjust column load
appropriately.
Carryover
You observe carryover in chromatographic systems when a previously injected
analyte appears as a peak in the chromatogram of subsequent samples.
Carryover tends to occur when a small amount of analyte remains in the
system after a sample is injected. You can measure carryover by observing
analyte peaks that appear when you run a blank sample immediately after an
analytical sample.(See ACQUITY UPLC I-Class System Specifications for
details on carryover performance).
A common cause of carryover is inadequate washing of the system. Choosing
an appropriate wash solvent can minimize carryover for a particular analysis.
The wash solvent must be strong enough to dissolve any remaining sample
and the wash duration must be long enough to remove the residue from the
system.
Method conditions also affect carryover. Too short a hold-time at the final
conditions of a gradient, especially if the gradient is steep, can fail to remove
all analytes from the system. It is important to completely flush the system
and reequilibrate the column before proceeding to a subsequent analysis. Use
caution when choosing the load-ahead and loop-offline options. Initiating
these options before the highly organic part of the gradient reaches the needle
can leave sample residue in the system, and whatever time savings you gain
can be lost in terms of inadequate system cleaning.
The hydrophobicity and solubility of samples are additional factors to consider
when trying to minimize carryover.