Page 56 - Illustrated Pocket Dictionary of Chromatography

DERIVATIZING REAGENT 51

degassing Before use in HPLC, solvents undergo degassing to
remove dissolved gases. Dissolved gases can lead to the following prob-
lems: Bubbles get trapped throughout the HPLC system, most notably
(1) in the check valves (lead to inconsistent flow) and (2) in the detec-
tor, causing erratic baseline pulsing or unwanted signals (e.g., O 2
absorbs at l< 210nm and reacts in an electrochemical detector).
Degassing can be accomplished through one of many techniques:
sparging (with He or Ar), sonication, refluxing and applying a vacuum.
Whereas refluxing is the most effective technique, it is also incom-
patible with mixed solvents and is impractical (from a time and
safety point of view). Sonication with vacuum seems to be the most
effective, coupled with a helium blanket blown across the top of the
reservoir.
denaturation The loss of the three-dimensional structure of a
protein. This process is often irreversible. In chromatographic separa-
tions, denaturation can be caused by such things as high or low pH, ionic
strength, organic solvent level, and the type of stationary phase. This is
a difficult problem because many organic solvents cause denaturation
when present at the 15–25%v/v level and much higher organic levels are
needed to cause elution. The use of a butyl bonded phase seems to be
most compatible with preservation of the protein structure.

densitometer Frequently used for quantitative work in thin-layer
chromatography. The source light (often UV) is transmitted through
the back of the plate and up through the adsorbed packing. The
plate is scanned by moving it past the source from the point of sample
application to the solvent front. The change in absorbance is read as
a function of the position on the plate.
density, d The density is the weight per unit volume for a given
substance or sample. Typical units are grams/milliliter.

derivatization The process of chemically modifying either (1) a
packing material surface to change its polarity or (2) an analyte
through the addition of a specific functional group and increase
detection specificity or sensitivity.
derivatizing reagent Used to react with a compound or material
in order to change the chemical and/or physical properties of that
compound or material. Examples include the derivatization of silica
to create bonded phase supports or of compounds to incorporate
chromophores or fluorophores or render them volatile.