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HPLC Problems
http://www.hplc1.com/shodex/english/dd.htm
HPLC Performance Monitoring
Use Your Test Method
(Known Performance)
Troubleshooting * Monitor at least One Peak in one injection
- Plate Count (Peak width relative to RT),
- Peak Asymmetry,
- Retention Time and/or Retention parameter
- Relative Retention Time for Critical Pair of Analytes.
- Peak Response
300
mV
1
2
3
1. Fucose
2. Galactosamine
3. Glucosamine
4. Galactose
5. Glucose
6. Mannose
Detector
Troubleshooting
4
6
0.00
5.00 20.00
Minutes
Data
Processing Waste
CHEMISTRY Hardware/
Software
COLUMN/GUARD COLUMN
SOLVENT PUMP
SAMPLE INJECTOR
DETECTOR
INTEGRATION
Fraction
a b
cd Collector
Pump Auto HPLC Column
flows 50-5000µL/min) in Oven
Sampler
Performance Monitoring
Column Efficiency:
N = the number of Theoretical Plates
a = is a constant depending on the Method used Performance Monitoring
tr = retention time of peak
W = the peak width (time units) at a given peak height
tR Band Spreading
2
tr * Band Spreading Impacts Chromatographic
N=a ( W ) h
Performance -- The Greater The Band Spreading,
t0
The Poorer The Performance (ie; Resolution)
w
σ2 = σ2 + σ2 + σ2 + σ2
σ2 = σ2 + σ2
TOT COL EC
EC TUBING CONNECTIONS INJECTORS DETECTORS
Performance Monitoring
Good Seal
.020"
.009"
.040"
.040" .020"
Performance Monitoring
Using 5 sigma efficiency method, measure the peak width
at 4.4% of peak height Performance Monitoring
Convert to microliters using the following equation:
Impact of System Band Spread on a Plate Count:
( )( ) ( ) (
2cm
PW
1min
20 cm
1 mL
min. mL
µL
1000µ
) = 100 (µL) - System with 70µl Band Spread >> 10,000 plates
where:
1min/20cm = chart speed
1 mL/min = flow rate
µl Band Spread >> ~8,000 plates
- System with 130µ
1000 µL/mL= volume correction factor
On the Same Column!
µL +/- 30µ
Typical LC System should be 100µ µL
Assumption: <40% loss in resolution at k' = 5 and N= 10,000 and <20% loss
µL
Microbore System should be no greater than 20µ in resolution at the preferred value
0.20
0.00
5.00
5.00 10.00 15.00 EFFECT OF INJECTION VOLUME
Minutes ON PEAK DISTORTION
Lift-off Point Moves Earlier
Retention times are shorter
Temperature Control
Non-Column Influences:
pH
26.3°C Neutrals: No Influence
Small Change
20 in pH = Large
change in k pKa
15 (potential
reproducibility AU Imp. 1
problems) + 1 pH unit = 91% ionized 0.010
10
Imp. 3
5 Ionized Acid
0.008
pH 2.5
0.006 Imp. 2
0
0.004
0 1 2 3 4 5 6 7 8 9 10 11 12
±1 pH 0.002 Imp. 4
Phoebe, Tran
19
Reversed-Phase Retention Behavior of Basic
© Waters Corporation 2000
(101300)
0.000
Column Protection
Column Protection
1: Sulfanilamide Conditions
Column: Symmetry™ C 8 3.9 mm X 150
Sentry Nova-Pak C18 mm with Sentry™ Guard
Column 3.9 mm X 20 mm
Mobile Phase: water/methanol/glacial acetic
Sentry Symmetry C8
2: Sulfadiazine acid 79:20:1
Adsorbosphere C18
Zorbax Reliance Rx C8
2 Injection 5020
0% 20% 40% 60% 80% 100% 5: Sulfamethazine
1
% of original efficiency Start
0 2 4 6 8 10
Extension of column lifetime with Guard Column using a mixture of sulfa drugs as the sample * Inject Multiple Runs
- Precision (at least 5 injections)
- Accuracy (Use Control Samples)
Reference
0.01
Offset
R
Unusual Phenomena
Extraneous Peaks
Problems with Baseline
Extraneous Peaks
Extraneous Peaks
Sample Blank
0.100
0.028
0.090 0.026
0.024
0.080
PG - 2.919
0.022
0.070 0.020
0.018
BHT - 10.633
0.060
BHA - 6.896
0.016
0.050 0.014
TBHQ - 4.525
0.012
AU
AU
0.040
0.010
0.030 0.008
0.006
0.020
0.004
0.010 0.002
0.000
0.000
-0.002
0.00 2.00 4.00 6.00 8.00 10.0012.0014.00 16.0018.0020.0022.0024.0026.0028.00 30.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.0020.00 22.00 24.00 26.0028.00 30.00
Minutes Minutes
1.0
SYNCHRONOUS NOISE
ALMOST ALWAYS CAUSED BY THE PUMP ASYNCHRONOUS NOISE
SPIKES NO PEAKS
BUBBLES
INSTRUMENTAL CHEMICAL
Degas solvent
POOR ELECTRICAL CONNECTION, LOOSE WIRING Injector not making injections Column retaining all compounds
Clean and tighten detector leads, check wiring, Pump not pumping Bad or wrong mobile phase
replace spade lugs. Dead detector Bad or wrong standard or sample
LAMP RELAY TRYING TO FIRE A DEAD LAMP Integrator/recorder not wired Wrong guard column
Replace lamp correctly
ELECTRICAL NOISE Gain setting too low WHAT TO DO:
Change circuits, remove source Leaks Remove column and inject acetone
Common sources include switching valves, solution to make a peak
compressors, muffle furnaces, fraction collectors,
WHAT TO DO:
power conditioners, lighting, poor power source.
Inject acetone solution to make a peak
Basic assumptions
1. The HPLC is plugged in and turned on
2. Solvent is in the reservoir
3. The pumps are primed and in good working order
4. The HPLC is plumbed and wired correctly
Things not to do:
5. The detector has a good lamp in it
6. The solvent bottle doesn't have a vacuum on it * Plug the outlet of your RI detector
7. You're not using acetone for solvent at 195 nm * Flush your system with methanol after running buffer
8. You're not injecting rocks
* Inject samples that may precipitate in the eluent
9. You're not doing a water to hexane gradient
10. Your're not trying to detect sugars at 254 nm
* Run long durations with HCl on your stainless steel HPLC
11. You're not mixing MEOH and water without degassing * Filter organic solvents through aqueous filters
12. You're not sparging with nitrogen or air * Spill buffers onto HPLC electronics
13. You're not running water through a silica column * Try to change the column frits while it still has pressure in it
14. Solvent pH is not 13 on a silica base column * Store THF on the shelf, uncapped, for weeks
15. You're not running a 1M NaCl to 100% ACN gradient * Pump cyclohexane above 2000 psi
16. You're not doing gradients with an RI detector
* Tightly seal your mobile phase container
17. You're RI is not under the air conditioner vent
18. No buffer stalagtites on your pump heads
* Cut tubing with a wire cutter
19. HCl vapors are not blowing onto your HPLC
20. You're having a wonderful time!