COLUMN WARNING
Essential to any scientist's experimentation is reproducibility of results
in order to further support or reject data or hypotheses. For a chromatographer,
a separation that has been accomplished or optimized must be able to be
repeated in subsequent HPLC analyses. Any anomaly that occurs can usually
be attributed to either 1) the age of the column, 2) the chromatographer
or 3) the manufacturer. Certain measures can limit the effects of age and
the user. For example, proper column storage with the appropriate liquid
interface, such as methanol for certain reverse-phase columns, with each
end capped tightly assures no drying of the resin and increases "life"
of the column. Use of a guard column can prevent the user from potentially
contaminating or introducing unwanted particles into the column.Identifying
the manufacturer as the chief culprit is many times unwarranted among peers
or supervisers. However, there are certain instances where the irregularities
can be aimed at the manufacturers. In these cases, column-to- column or
batch-to-batch variations in the commercially available products must be
addressed.Recently, a disparity in results originated from separation of
proteins using reverse-phase HPLC (Welinder,
1995). Aprotinin, a 58-amino acid polypeptide, was the protein separated
using a C4 column (Column 1) at pH 5 with sodium phosphate/ammonium sulfate
buffer and an acetonitrile gradient. A new column (Column 2) from the same
batch was acquired from the manufacturer and the resultant separation was
characterized by different retention time, "tailing", and some loss of
efficiency:
(Welinder,
1995)The manufacturer admitted to changing the column packing procedure
(using distilled water instead of tap water) upon inquiry.A similar study
was performed using a C4 column produced by another manufacturer. In this
case, biosynthetic human growth hormone (bhGH), a 191-amino acid protein,
and TS-bhGH, a derivative of bhGH containing a tri-sulfide bridge (instead
of a di-sulfide bridge), were separated at neutral pH with high resolution
and efficiency (Welinder,
1995). However, when another column (Column 2) was purchased and tested,
the separation of hGH and bhGH was totally absent:
(Welinder,
1995)The company had no explanation for the occurance. However, it
was determined that the original company had been taken over by another
company since the first column was acquired, and the parent company could
not trace the difference in batches.A scientist, more than any other user,
should not assume a simplified situation where consistent results will
be attained from the same model unless proven. In the case of chromatography,
stationary phases should constantly be tested, calibrated, or analyzed.
By no means should unwanted results be immediately attributed to faults
of the manufacturer. On the other hand, the manufacturer should not be
immune to reproach regarding divergent columns. In either case, the onus
is placed on the user. Welinder,
B.S.; Kornfelt, T.; Sorensen, H.H. Analytical Chemistry, 1995, Vol.
67, pp. 39-43.Return
to columnsReturn
to Theory of OperationReturn
to Users Guide PageExit to the University
of Kentucky College of Pharmacy
(Welinder,
1995)The company had no explanation for the occurance. However, it
was determined that the original company had been taken over by another
company since the first column was acquired, and the parent company could
not trace the difference in batches.A scientist, more than any other user,
should not assume a simplified situation where consistent results will
be attained from the same model unless proven. In the case of chromatography,
stationary phases should constantly be tested, calibrated, or analyzed.
By no means should unwanted results be immediately attributed to faults
of the manufacturer. On the other hand, the manufacturer should not be
immune to reproach regarding divergent columns. In either case, the onus
is placed on the user. Welinder,
B.S.; Kornfelt, T.; Sorensen, H.H. Analytical Chemistry, 1995, Vol.
67, pp. 39-43.Return
to columnsReturn
to Theory of OperationReturn
to Users Guide PageExit to the University
of Kentucky College of Pharmacy