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HPLC Problems
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HPLC
Performance Monitoring
Use Your Test Method
(Known Performance)
Troubleshooting
Troubleshooting
Detector
1. Fucose
2. Galactosamine
3. Glucosamine
4. Galactose
5. Glucose
6. Mannose
300
mV
4
6
Control &
Data
Processing
Problem
0.00
5.00
Minutes
20.00
Waste
CHEMISTRY
COLUMN/GUARD COLUMN
SOLVENT
SAMPLE
Fraction
Collector
a b
cd
Pump
flows 50-5000L/min)
Auto
Sampler
HPLC Column
in Oven
Hardware/
Software
PUMP
INJECTOR
DETECTOR
INTEGRATION
Performance Monitoring
Use Your Test Method
(Known Performance)
Determination Of Both
The Column AND Instruments'
Performance
Performance Monitoring
Column Efficiency:
N = the number of Theoretical Plates
a = is a constant depending on the Method used
tr = retention time of peak
W = the peak width (time units) at a given peak height
Performance Monitoring
Band Spreading
tR
N=a
tr
W
t0
w
METHOD
Peak Width at Half Height
Peak Width at 4.4% Peak Height (5 Sigma)
Tangent
a
5.54
25.0
16.0
Band Spreading
* Column Contribution
2
TOT
COL
2
EC
COLUMN
2 =
EC
2
TUBING
+ 2
CONNECTIONS
+ 2
+ 2
INJECTORS
Performance Monitoring
Extra-Column Band Spreading
(Instruments' Contribution)
1. Injection Volume
2. Injector
3. Connection Tubing
a. from Injector to Column
b. from Column to Detector
c. Endfittings and Frits
4. Detector Volume
Connectors
Waters SpherisorbR and
many other brands
0.090"
Parker
Style
0.130"
Waters
Style
DETECTORS
Good Seal
Performance Monitoring
Effect of Connecting Tubing on System Bandspreading
Tubing Contribution
.009"
.020"
.009"
.040"
.040"
.020"
Performance Monitoring
To perform a measurement:
- disconnect column from system
- connect injector directly to detector
Parameter
Flow Rate
Chart Speed
Setting
1.0 mL/min
20 cm/min
Detector Sensitivity
Time Constant
Performance Monitoring
Using 5 sigma efficiency method, measure the peak width
at 4.4% of peak height
Convert to microliters using the following equation:
Performance Monitoring
Impact of System Band Spread on a Plate Count:
( )( ) ( ) (
2cm
PW
1min
20 cm
1 mL
min.
L
1000
mL
) = 100 (L)
where:
1min/20cm = chart speed
1 mL/min = flow rate
1000 L/mL= volume correction factor
L +/- 30
L
Typical LC System should be 100
L
Microbore System should be no greater than 20
Performance Monitoring
Use Your Test Method
(Known Performance)
0.006
Sample in MeOH
0.005
0.004
Minocycline
AU 0.003
Demeclocycline
Tetracycline
0.002
0.001
0.000
20.0
10.0
30.0
Minutes
0.006
0.005
Minocycline
0.004
Tetracycline
AU 0.003
Demeclocycline
0.002
0.001
0.000
10.0
20.0
30.0
Minutes
Column Use
Silicas hydrolyze at high pH
Instability of bonded phase at low pH
Elevated temperatures decrease
column lifetime
C18 approximately 1000 times more
stable than CN
Silica
Polymer
pH 2 - 8
pH 2 -12
pH
10
Column Collapse
Column Collapse
voided column
Mass Overload
Column/Volume Overload
0.60
500
L
0.40
300
L
100
L
0.20
0.00
5.00
5.00
10.00
15.00
Minutes
10
L
Volume Overload
Performance Monitoring
Use Your Test Method
(Known Performance)
* 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
Solvent Composition
Clearly
Clearly specify
specify HOW the
the Mobile Phase
Phase is to be prepared
Drifting Retention
Solvent Composition
Temperature
pH-Control
Ion Pairing
Equilibration
Stationary Phase Stability
Column Contamination
Hydrophobic Collapse
60/40
Temperature Control
23.5C
pH
26.3C
Neutrals: No Influence
Acids: Reduced Retention with Increasing pH
Bases: Increased Retention with Increasing pH
10% Change in Retention per 0.1 pH Units
pH Control
35
Un-ionized Acid
30
6% Methanol, 6% THF
25
Small Change
in pH = Large
change in k
(potential
reproducibility
problems)
20
15
10
pKa
Imp. 1
AU
0.010
Imp. 3
0.008
Ionized Acid
Phoebe, Tran
10
11
0.004
12
pH
Imp. 4
0.002
19
pH 2.5
Imp. 2
0.006
1.7
25
Un-ionized Base
5.0
6.7
8.4
10.
11.
13.
15.
16.
18.
Imp. 3
0.008
pH 2.7
Imp. 2
0.006
20
Imp. 4
0.002
0.000
15
pKa
-0.002
10
1.7
3.4
5.0
6.7
8.4
10.
11.
13.
15.
16.
Time [min]
Ionized Base
0
0
1
2
10
11
12
pH
23
15 cm
After injection
12 cm
21.
0.004
Phoebe, Tran
20.
Time [min]
0.010
30
3.4
Imp. 1
AU
35
0.000
-0.002
COLUMN REGENERATION
REVERSE PHASE
1. Wash with unbuffered mobile phase
2. Wash with 100% water
3. Wash with methanol (or ACN)
4. Wash with THF or IPA
5. Wash with methylene chloride
6. Wash with N-Heptane
7. Wash with methylene chloride
8. Wash with methanol (or ACN)
9. Wash with water
10. Return to solvent
18.
20.
Length (mm)
2.0
2.0
3.9
3.9
3.9
3.9
3.9
4.6
4.6
5
8
7.8
19
25
30
40
47
50
150
300
50
75
100
150
300
150
250
100
100
300
150
100
300
100
300
300
.47
.94
.6
.9
1.2
1.8
3.6
2.5
4.2
2.0
5.0
4.3
43
49
212
125
520
589
Solvent Viscosities
Solvent
Solvent Viscosities
Viscosity [cP] at 20 C
Acetone
Acetonitrile
Cyclohexanone
Di-isopropylether
Diethyl ether
Dimethyl acetamide
Dimethyl formamide
Dimethyl sulfoxide
Dioxane
Ethanol
Ethyl acetate
Hexafluoroisopropanol
iso-Propanol
Isooctane
Methanol
0.32
0.37
0.98
0.37
0.23
2.1
0.92
2.2
1.54
1.2
0.45
1.0
2.5
0.5
0.6
Solvent
Viscosity [cP] at 20 C
Methyl acetate
0.37
Methylene chloride
0.44
Methylethyl ketone
0.4
n-Heptane
0.42
n-Hexane
N-Methyl pyrrolidone
n-Pentane
0.33
1.67 (25? C)
0.235
n-Propanol
2.3
o-Dichlorobenzene
1.41
Tetrahydrofuran
0.46
Toluene
1.2.4-Trichlorobenzene
0.59
1.89 (25? C)
Water
1.0
m-Xylene
0.62
o-Xylene
0.81
Column Protection
Column Protection
Major cause of column deterioration is contamination.
Use of guard columns may increase column life-time
to > 10,000 analyses
column coupler
30mm guard
column
Column Protection
Column Protection
1: Sulfanilamide
Column:
Mobile Phase:
Sentry Symmetry C8
2: Sulfadiazine
Conditions
Symmetry C 8 3.9 mm X 150
mm with Sentry Guard
Column 3.9 mm X 20 mm
water/methanol/glacial acetic
acid 79:20:1
Adsorbosphere C18
3: Sulfathiazole
Upchurch ODS
Brownlee NewGuard RP-18
Alltech Econosil C18
4: Sulfamerazine
Zorbax Reliance Rx C8
Injection 5020
0%
20%
40%
60%
80%
100%
5: Sulfamethazine
1
Start
% of original efficiency
0
6: Succinylsulfathiazole
10
Minutes
Column Protection
Performance Monitoring
Extension of column lifetime with Guard Column using a mixture of sulfa drugs as the sample
- Integration Software
0.010
** Electronic
Electronic Peak
Peak Generator
Generator
** Poor
Poor Peak
Peak Shape
Shape
AU
** Cell
Cell Problem
Problem
** Lamp
Lamp Failing
Failing
0.005
0.000
51.8 52.0 52.2 52.4 52.6 52.8
Minutes
0.01
- Detector
S
R
AUFS
Offset
Reference
Energy
Sample Energy
Absorbance
Offset
Extraneous Peaks
Unusual Phenomena
Extraneous Peaks
Problems with Baseline
Isocratic LC - Extra Peak - Sharp - Contaminant
Extraneous Peaks
Sample
0.100
Blank
Extraneous Peaks
0.028
0.026
0.090
0.022
0.030
0.020
0.010
0.018
0.016
0.014
0.012
AU
0.040
TBHQ - 4.525
AU
0.050
BHT - 10.633
0.020
0.060
BHA - 6.896
0.070
PG - 2.919
0.024
0.080
0.010
0.008
0.006
0.004
0.002
0.000
0.000
0.00 2.00 4.00 6.00 8.00 10.0012.0014.00 16.0018.0020.0022.0024.0026.0028.00 30.00
Minutes
-0.002
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
Waters
1.0
HPLC Pump
Manual
Injector
Guard
Column
HPLC Column
Detector
Degas Solvents
Vacuum
Ultrasonic bath
Time = 1 minute
BASELINE TROUBLESHOOTING
NOISY BASELINE
Noisy baseline
Cyclic
INSTRUMENTAL
WEAK DETECTOR LAMP
Synchronous noise
Spikes
Replace lamp
CHEMICAL
TRASH ELUTING OFF COLUMN
Flush column with strong solvent
LEAKS
Stop leaks. Replace fittings
No peaks
Asynchronous noise
Drift
ELECTRONIC NOISE
SYNCHRONOUS NOISE
ALMOST ALWAYS CAUSED BY THE PUMP
Air in pump head - Prime pump and degas solvent
Check valve problem - Rebuild or replace
Broken plunger - Replace (blame it on someone else)
Mixing problem - Increase system volume
Electrical noise - Change circuits, remove source
ASYNCHRONOUS NOISE
BUBBLES
Degas mobile phase
GAS CAUGHT IN DETECTOR
Degas mobile phase. Put
backpressure on cell.
LEAKS
Fix leaks, replace fittings
MIXING PROBLEMS
Increase system volume
PLUGGED LINES
Remove plug, flush system
ELECTRICAL PROBLEMS
Remove source, change circuits
BASELINE DRIFT
INSTRUMENTAL
GRADIENT - SOLVENT B ABSORBS MORE
THAN SOLVENT A
Try a new mobile phase, use baseline
subtraction
SOLVENT CHANGING (GAS ABSORPTION,
EVAPORATION
Helium sparge, enclose solvents
SOLVENT LEAKS
Tighten, replace fittings
THERMAL EFFECTS (ESPECIALLY RI,
CONDUCTIVITY, ECD)
Cell temperature regulation
BACKPRESSURE CHANGES
Filter solvents and samples. Sample too
viscuous
SIPHONING (RI, CONDUCTIVITY, ECD)
Increase system volume
MIXING PROBLEMS
CHEMICAL
COMPOUNDS ELUTING OFF COLUMN
Run strong solvent until baseline is stable
SOLVENTS IN GRADIENT ARE NOT PURE
Change the solvent batch or
manufacturer.
Check if the solvents are grandient
grade.
SPIKES
BUBBLES
Degas solvent
POOR ELECTRICAL CONNECTION, LOOSE WIRING
Clean and tighten detector leads, check wiring,
replace spade lugs.
LAMP RELAY TRYING TO FIRE A DEAD LAMP
Replace lamp
ELECTRICAL NOISE
Change circuits, remove source
Common sources include switching valves,
compressors, muffle furnaces, fraction collectors,
power conditioners, lighting, poor power source.
CYCLIC BASELINE
TEMPERATURE FLUCTUATIONS
Thermally insulate. Move away from ventilation.
Increase cell temperature.
MIXING PROBLEMS
Increase system volume
GAS IN MOBILE PHASE
Degas solvents
ELECTRICAL PROBLEMS
Change circuits, remove source
ERRATIC PUMP
Repair pump
PLUG
Remove obstruction, flush system
NO PEAKS
INSTRUMENTAL
Injector not making injections
Pump not pumping
Dead detector
Integrator/recorder not wired
correctly
Gain setting too low
Leaks
WHAT TO DO:
Inject acetone solution to make a peak
CHEMICAL
Column retaining all compounds
Bad or wrong mobile phase
Bad or wrong standard or sample
Wrong guard column
WHAT TO DO:
Remove column and inject acetone
solution to make a peak
CHEMICAL
Some eluting compounds
absorb less than solvent
Use a different or cleaner
solvent
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
5. The detector has a good lamp in it
6. The solvent bottle doesn't have a vacuum on it
7. You're not using acetone for solvent at 195 nm
8. You're not injecting rocks
9. You're not doing a water to hexane gradient
10. Your're not trying to detect sugars at 254 nm
11. You're not mixing MEOH and water without degassing
12. You're not sparging with nitrogen or air
13. You're not running water through a silica column
14. Solvent pH is not 13 on a silica base column
15. You're not running a 1M NaCl to 100% ACN gradient
16. You're not doing gradients with an RI detector
17. You're RI is not under the air conditioner vent
18. No buffer stalagtites on your pump heads
19. HCl vapors are not blowing onto your HPLC
20. You're having a wonderful time!