Troubleshooting HPLC Systems

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Troubleshooting HPLC Systems
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Sample Injection
Figure 1. Influence of sample solvent on peak
shape. The sample is dissolved in buffer with
(a) 0, (b) 30, (c) 50, and (d) 70
acetonitrile.
(Mobile Phase 92 buffer pH 3.5, 8 acetonitrile)
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Column Testing
The best way to evaluate a columns performance
is to use the same test that was used by the
manufacturer before it was shipped. By
comparing the efficiency, retention and peak
shape of the peaks in the sample and very
importantly the pressure under these
experimental conditions, you will be able to tell
if your column has changed over time.
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Column Regeneration
Cleaning Reversed-Phase Columns Use 10 column
volumes of each 100 methanol 100
acetonitrile 75 acetonitrile/25
isopropanol 100 methylene chloride 100
hexane 100 isopropanol
Aqueous based solvents such as acetic acid,
trifluoroacetic acid, urea, guanidine, sodium
phosphate, or dimethylformamide can aid in the
removal of proteinaceous material from reversed
phase columns.
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Gradient Testing
Mobile Phase A Methanol Mobile Phase B 1000
mL methanol with 2 mL acetone Flow 2
mL/min Detector UV _at_ 280nm
GRADIENT 0 min 97.5 A and 2.5
B 10 min 2.5 A and 97.5 B 12 min
2.5 A and 97.5 B 12.1 min 97.5 A and
2.5 B 20 min 97.5 A and 2.5 B
The acetone in Mobile phase B is detectable by UV
at 280 nm. Therefore, as the amount of Mobile
Phase B is increased, the detector response
increases.
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Gradient Testing
Repeat 4-5 Times and note standard deviations
of times and detector response.
Evaluation of the Gradient Test.
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Gradient Testing
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Gradient Testing
Not too much dwell time
Not too much curvature
Should be linear
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Dwell Time
Dwell Time is the time it takes the mobile phase
to movethrough the dwell volume. The dwell
volume is the volume of the mobile phase between
where the solvents are mixed and the column.
This includes the mixing chamber, the injector,
and tubing. Depending on the instrument, dwell
volumes can be as low as 0.5 mL or as high as 8.0
mL. Differences in dwell volume can cause
considerable differences in chromatography and
may be one reason that it is difficult to
reproduce a separation reported in
the literature. In documenting a method, the
dwell volume of the instrument should be
reported.
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Extra-Column Volume
Extra-column volume is volume of the system other
than the column that contributes to band
broadening outside the column, (in addition to
the dwell volume, the detector flow cell, post
column reactors, and tubing are the main sources).
The relative amount of allowable extra column
broadening is proportional to the column volume.
The relative volumes of 2 columns can be
calculated based on the formula for the volume
of a cylinder. Length x pR2. Example (Column 1,
diameter 4.6 mm, extra column volume 100 mL,
Column 2, diameter 2 mm, What is the
equivalent extra-column volume?) Since the
radius(R) is the only thing changing in this
example, relative volume can be calculated as
the ratio of the two radii ( 2.32/12 5.3) which
means that the 2 mm column has a volume 5.3 times
less than a 4.6 mm column therefore, the
extra-column volume should be 5.3 times less
(100 mL/5.3 18 mL).
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Problem 1. No Peaks/Very Small Peaks
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Problem 2. No Flow
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Problem 3. No Pressure or Pressure Lower than
Usual
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Problem 4. Pressure Higher than Usual
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Problem 5. Variable Retention Times
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Problem 6. Loss of Resolution
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Problem 7. Split Peaks
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Problem 8. Peaks Tail on Initial and Later
Injections
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Problem 9. Tailing Peaks
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Problem 10. Fronting Peaks
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Problem 11. Rounded Peaks
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Problem 12. Baseline Drift
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Problem 13. Baseline Noise (Regular)
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Problem 14. Baseline Noise (Irregular)
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Problem 15. Broad Peaks
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Problem 16. Change in Peak Height (one
or more peaks)
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Problem 17. Change in Selectivity
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Problem 18. Negative Peaks
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Problem 19. Ghost Peaks
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