Chemistry Reference
In-Depth Information
FIgure 3.12
Quadrupole MS schematic. (Reprinted from HPLC method development for
pharmaceuticals, Volume 8 of
Separation Science and Technology
, S. Ahuja, Editor, Chapter 6,
Contemporary liquid chromatographic systems for method development, p. 167, 2007.)
However, similar to ELSD and CAD, using a mass detector places constraints
on mobile phase selection. Proper selection of the mobile phase and any additives
is critical to detection viability (Table 3.3). First and foremost, the mobile phase
must be suitable for the ionization, and must be selected depending on the ioniza-
tion mode (electrospray (ESI) or atmospheric pressure chemical ionization (APCI),
positive or negative mode) and the analyte (e.g., p
K
a). The molecular weight of the
mobile phase components should also be considered. It is not always possible to ana-
lyze compounds whose molecular weight is lower than the one of the mobile phase
or any additives. For routine operation, it is easier to use volatile buffers. Acids such
as HCl, H
2
SO
4
, or methane sulfonic acid might damage the instrument and should
not be used; volatile organic acids (e.g., TFA, formic, acetic) should be used instead.
Some ions (e.g., Na, NH
4
, acetate) from the mobile phase can form adducts. In
the case of phosphate, multiple adducts are observed, which can produce compli-
cated mass spectra. The formation of an adduct is usually not a reason for avoiding
a mobile phase as adducts can sometimes be used to advantage. Ion pairing reagents
can impact the spray formation, the droplet evaporation, and compete in terms of ion
formation, and are generally avoided. Buffer concentration is generally kept as low
as possible (millimolar range). If the buffer concentration is too high, ion suppression
occurs, thus affecting sensitivity.
Common eluents for LC/MS include methanol/water; acetonitrile/water (metha-
nol usually gives a better sensitivity than acetonitrile); pH modifiers (formic, acetic
acids, TFA, NH
4
, TEA, DEA); and buffers (carbonates, ammonium formate, ammo-
nium acetate, ammonium carbonates, and ammonium phosphate (all nonvolatile)).
The column, while of course providing the separation without using a high con-
centration of buffers, or ion pairing reagents, must be stable so that the column will
not “bleed” or shed interfering compounds. Special low- or no-bleed MS versions of
columns are available from most suppliers.
3.4.7 c
olumn
m
odule
Running a column at room temperature, or even “controlled” room temperature, is
a thing of the past. Modern requirements for accuracy and precision require that
columns be thermostated. Because temperature can also be used as a selectivity tool,
