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Figure 3
A small amount of methanol finely tunes the separation of narcotic analgesics and acetaminophen
on an Ultra C18 column, as indicated by less peak tailing.
• 7 •
800-356-1688
These changes led to a slight increase in tailing for
all compounds on both Ultra C8 and Pinnacle II™
C8 columns, but this was acceptable, especially
because the run time for the analysis was reduced
by a factor of 3 and resolution was improved by
59% to 79%. The system passed the system suitabil-
ity requirements in the USP monograph.
In the next experiment, we re-introduced the ion
pair reagent hexane sulfonic acid into the system
under the control of the pH 2.5 phosphate buffer
system. The run time doubled, relative to the origi-
nal procedure, demonstrating that TEA did affect
the concentration of the ion-pairing agent.
Reducing the concentration of ion pairing agent, or
using a shorter chain length ion-pairing agent,
might have been a better alternative to adding TEA.
The system still passed the system suitability
requirements listed by the USP, but the chro-
matogram was much noisier—and equilibration
problems seen in the USP 25 analysis returned.
After reviewing the monographs for admixtures con-
taining structurally related narcotics and acetamino-
phen, we created a single separation for morphine
sulfate, acetaminophen, codeine phosphate, oxy-
codone HCl, and hydrocodone bitartrate. The goal
was to create an adequate separation while keeping
the method as simple as possible. We chose an Ultra
C18 column and set detection to 235nm. All compo-
nents, including a small unknown peak, were sepa-
rated to baseline (Figure 2).
Next, we increased the amount of buffer to 90% (a
5% increase). This simple increase doubled the
analysis time. Resolution doubled between most
components, with the greatest change between acet-
aminophen and codeine. The unknown peak disap-
peared and probably co-eluted with morphine.
We adjusted the mobile phase ratio to 85:15,
buffer:organic solvent, using a 90:10 mixture of
acetonitrile and methanol as the organic solvent.
Resolution improved, relative to the original mobile
phase composition, analysis again was under 10
minutes, and the unknown peak returned (Figure
3). For this analysis, these conditions provided the
most desirable results.
In summary, the goal of any method should be to
achieve the most stable and robust separation.
Sometimes methods are more complicated than they
need to be, and this can make analysis unnecessari-
ly difficult. Even troubleshooting such methods adds
to production costs. When preparing to follow a
method always attempt to determine the reason a
reagent would be included in a mobile phase. Any
change or modification should have an established
scientific purpose. By creating more universal meth-
ods for analyses of structurally related compounds,
it should be possible to reduce costs for supplies,
increase laboratory analysis efficiency, and reduce
personnel training time.
For chromatograms illustrating the changes in sep-
aration that occur with each change in the mobile
phase, please request Applications Note #59453. If
you encounter problems when analyzing your sam-
ples according to an established method, our expe-
rienced Technical Service chemists will be glad to
help. Contact them at 800-356-1688, ext. 4 or 814-
353-1300, ext. 4, or contact your Restek represen-
tative.
Peak List: Conc. (µg/mL) Ret. Time (min.) Tailing Resolution
U. unknown unknown 3.1 NA NA
1. morphine sulfate 204 3.3 1.0 1.8
2. acetaminophen 92 5.0 1.1 14.1
3. codeine phosphate 216 5.5 1.4 2.6
4. oxycodone HCl 206 7.5 1.4 8.5
5. hydrocodone bitartrate 218 8.9 1.4 5.0
Sample:
Inj.: 4.0µL
Sample: raw material mix
Solvent: mobile phase
LC_0218
Column: Ultra C18
Catalog #: 9174575
Dimensions: 250 x 4.6mm
Particle size: 5µm
Pore size: 100Å
Conditions:
Mobile phase: A: pH 2.8 10mm
potassium phosphate
B: acetonitrile:
methanol (90:10 v/v)
(85A:15B, v/v)
Flow: 1.0mL/min.
Temp.: 27°C
Det.: UV @ 235nm
Ordering Information | Ultra C18 5µm Columns
1.0mm ID 2.1mm ID 3.2mm ID 4.0mm ID 4.6mm ID
Length cat.# cat.# cat.# cat.# cat.#
30mm 9174531 9174532 9174533 — 9174535
50mm 9174551 9174552 9174553 — 9174555
100mm 9174511 9174512 9174513 9174514 9174515
150mm 9174561 9174562 9174563 9174564 9174565
200mm 9174521 9174522 9174523 — 9174525
250mm 9174571 9174572 9174573 — 9174575
Trident™ Direct HPLC Guard Column System
Choose from three levels of protection!
Trident™ Direct
high-pressure filter
✔ Protection against
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Trident™ Direct 1cm guard
cartridge holder with filter
✔ Protection against
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✔ Moderate protection against
irreversibly adsorbed compounds.
Trident™ Direct 2cm guard
cartridge holder with filter
✔ Protection against
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✔ Maximum protection against
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for moreinfo For information about Trident ™ guard columns,
request the Trident™ Fast Facts (lit. cat.# 59314 and
59896).
compare
values to
Figure 2