columns, HPLC, high performance liquid chromatography, guard, derivatizing, separation, stationary phase, stationary, phase, capillary, fast, preparatory, Microbore, small-bore, Capillary Electrophoresism, back-pressure regulator, fraction collector, university of kentucky, college of pharmacy, primary, derivatization, sample, analytical, analysis, chemistry, analytical chemistry, microcolumns
There are various columns that are secondary to the separating column or stationary phase. They are: Guard, Derivatizing, Capillary, Fast, and Preparatory Columns.
Guard Columns are placed anterior to the separating column. This serves as a protective factor that prolongs the life and usefulness of the separation column. They are dependable columns designed to filter or remove: 1) particles that clog the separation column; 2) compounds and ions that could ultimately cause "baseline drift", decreased resolution, decreased sensitivity, and create false peaks; 3) compounds that may cause precipitation upon contact with the stationary or mobile phase; and 4) compounds that might co-elute and cause extraneous peaks and interfere with detection and/or quantification. These columns must be changed on a regular basis in order to optimize their protective function. Size of the packing varies with the type of protection needed.
Derivatizing Columns- Pre- or post-primary column derivatization can be an important aspect of the sample analysis. Reducing or altering the parent compound to a chemically related daughter molecule or fragment elicits potentially tangible data which may complement other results or prior analysis. In few cases, the derivatization step can serve to cause data to become questionable, which is one reason why HPLC was advantageous over gas chromatography, or GC (Brown, 1990). Because GC requires volatile, thermally stabile, or nonpolar analytes, derivatization was usually required for those samples which did not contain these properties. Acetylation, silylation, or concentrated acid hydrolysis are a few derivatization techniques.
Capillary Columns- Advances in HPLC led to smaller
analytical columns. Also known as microcolumns, capillary columns
have a diameter much less than a millimeter and there are three
types: open-tubular, partially packed, and tightly packed. They
allow the user to work with nanoliter sample volumes, decreased
flow rate, and decreased solvent volume usage which may lead to
cost effectiveness. However, most conditions and instrumentation
must be miniaturized, flow rate can be difficult to reproduce,
gradient elution is not as efficient, and care must be taken when
loading minute sample volumes (Brown,
1990).
Microbore and small-bore columns are also used for
analytical and small volumes assays. A typical diameter for a
small-bore column is 1-2 mm. Like capillary columns, instruments
must usually be modified to accommodate these smaller capacity
columns (i.e., decreased flow rate). However, besides the
advantage of smaller sample and mobile phase volume, there is a
noted increase in mass sensitivity without significant loss in
resolution (Simpson,
1987).--Capillary Electrophoresis
Fast Columns- One of the primary reasons for using these columns is to obtain improved sample throughput (amount of compound per unit time). For many columns, increasing the flow or migration rate through the stationary phase will adversely affect the resolution and separation. Therefore, fast columns are designed to decrease time of the chromatographic analysis without forsaking significant deviations in results. These columns have the same internal diameter but much shorter length than most other columns, and they are packed with smaller particles that are typically 3 µm in diameter. Advantages include increased sensitivity, decreased analysis time, decreased mobile phase usage, and increased reproducibility (DiCesare, 1987).
Preparatory Columns- These columns are utilized when
the objective is to prepare bulk (milligrams) of sample for
laboratory preparatory applications. A preparatory column usually
has a large column diameter which is designed to facilitate large
volume injections into the HPLC system.
Accessories important to mention are the back-pressure
regulator and the fraction collector. The
back-pressure regulator is placed immediately posterior to the
HPLC detector. It is designed to apply constant pressure to the
detector outlet which prevents the formation of air bubbles
within the system. This, in turn, improves chromatographic
baseline stability. It is usually devised to operate regardless
of flow rate, mobile phase, or viscosity.The fraction collector
is an automated device that collects uniform increments of the
HPLC output. Vials are placed in the carousel and the user
programs the time interval in which the machine is to collect
each fraction. Each vial contains mobile phase and sample
fractions at the corresponding time of elution. Packings for
columns are diverse since there are many modes of HPLC. They are
available in different sizes, diameters, pore sizes, or they can
have special materials attached (such as an antigen or antibody
for immunoaffinity chromatography). Packings available range from
those needed for specific applications (affinity, immunoaffinity,
chiral, biological, etc.) to those for all-purpose applications.
The packings are attached to the internal column hull by resins
or supports, which include oxides, polymers, carbon,
hydroxyapatite beads, agarose, or silica, the most common type(Brown,
1990).
Brown, P.R. Analytical Chemistry, 1990, Vol. 62, p. 995
Simpson, R.C. and Brown, P.R.; J. Chromatogr., 1987, No. 400, p. 297.
DiCesare, J.L.; Dong, M.W.; Vandermark, F.L.; Am. Lab., 1981, No. 13, p. 52.
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columns, HPLC, high performance liquid chromatography, guard, derivatizing, separation, stationary phase, stationary, phase, capillary, fast, preparatory, Microbore, small-bore, Capillary Electrophoresism, back-pressure regulator, fraction collector, university of kentucky, college of pharmacy, primary, derivatization, sample, analytical, analysis, chemistry, analytical chemistry, microcolumns