TRAC 2508 1-3-99
High-performance liquid chromatography
integrated solid-phase extraction in
bioanalysis using restricted access
precolumn packings
Karl-Siegfried Boos*, Carl-Heinz Grimm
Institute of Clinical Chemistry, University Hospital Grosshadern, D-81366 Munich, Germany
Tailor-made precolumn packings for HPLC
integrated extractive clean-up of ( bio)£uids
are characterized by possessing a de¢ned dif-
fusion barrier and a biocompatible outer par-
ticle surface ( restricted access materials,
RAM). Only analytes of low molecular mass
have free access to the binding centers at
the inner pore surface, and thus can be
retained and extracted selectively. These
adsorbents allow the easy, inexpensive
installation and economic operation of fully
automated coupled-column systems for
repetitive on-line solid-phase extraction
(SPE), and the subsequent analytical separa-
tion of untreated ( bio)£uids. z1999 Elsevier
Science B.V. All rights reserved.
Keywords: Restricted access materials; Solid-phase
extraction; PROSPEKT; Sample clean-up
On-line solid-phase extraction (SPE) procedures
are being used increasingly for sample clean-up,
owing to their convenience and versatility. They are
particularly attractive in situations where, for exam-
ple, large numbers of samples and /or sample series
have to be analyzed routinely, or where hazardous or
highly infectious materials have to be processed ( see
Fig. 1).
Solid-phase extraction (SPE) can be coupled
directly to high-performance liquid chromatography
(HPLC), utilizing dedicated sample preparation
units [ 1,2 ]. These automated cartridge exchange
modules process small ^ typically 5^10U3^4 mm
I.D. ^ SPE cartridges under high pressure £ow condi-
tions, and are available under the brand names LiChro-
graph OSP-2 (On-line Sample Preparation unit,
Merck, Darmstadt, Germany ) and PROSPEKT (Pro-
grammable On-line Solid Phase Extraction Tech-
nique, Spark, Holland ). A major disadvantage of this
on-line concept, besides its complex instrumentation,
is the single use of the extraction cartridge. In addition,
when proteinaceous samples are processed, non-
speci¢c adsorption or denaturation of the protein
matrix occurs. This results in undesired loss in the
capacity and selectivity of silica-based cartridge pack-
ing materials.
The SPE technique can be integrated into an HPLC
system by coupled-column switching (see Fig. 2). For
this purpose, a small ^ typically 5^30U3^4.6 mm I.D.
^ precolumn is connected to a conventional analytical
HPLC column via an electrically or pneumatically
driven (six-port) valve. Technical details and various
system con¢gurations have been described [ 3 ] and
some recent applications summarized [ 4 ]. Practical
guidelines, operational procedures, and method-opti-
mization steps for the analysis of biological specimens
are given in [ 5 ].
In the most common case the (pretreated) bio£uid is
injected onto the cartridge or precolumn, which retains
the target molecules. Then potentially interfering sam-
ple constituents are £ushed into the waste. The ana-
lytes retained on the bonded phase of the cartridge or
precolumn are eluted on-line, via the switching valve,
onto the series-connected analytical column. Simulta-
neously with the analytical separation, an exchange of
the cartridge or reconditioning of the precolumn can
take place. It is obvious that when a precolumn is used
for integrated sample pretreatment the chemically
modi¢ed support has to eliminate a complex sample
matrix repeatedly without destructive accumulation
and it has to extract the target compounds with
unchanged selectivity and capacity. In this context,
the most serious problem encountered in the design
and development of suitable precolumn packing mate-
rials lies in the quantitative removal of the protein
matrix during the integrated work-up of a bio£uid.
0165-9936/99/$ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.
PII: S0165-9936(98)00102-2
*Corresponding author.
trends in analytical chemistry, vol. 18, no. 3, 1999
175