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Troubleshooting of
HPLC Columns
Columns and Consumables
Edward Kim
Applications
pp Engineer
g
January 17, 2008
Page 1
Goals for this presentation:
Prep LC
Analytical
Nano LC Capillary
Chip LC LC
LC
Page 2
Troubleshooting in HPLC
20
00
15
00
U
mA
10
5
00
0
0
0 1 1 2 2
0 5 0 5
Time 0 5
(min)
Page 3
Major Areas of Column Problems -
Dramatic Changes in 3 Key Areas:
Page 4
1. Pressure Issues
Page 5
Determining the Cause and
Correcting High Back Pressure
• Check pressure with/without column - many
pressure problems
p p are due to blockages
g
elsewhere in the system.
If Column pressure remains high:
• Rinse column (remove detector from flow path!)
– Eliminate column contamination and
plugged packing
– high
g molecular weight/adsorbed
g compounds
p
– precipitate from sample or buffer
• Back flush column – may clear plugged column
inlet frit
• Change column inlet frit (… or discard column)
Eliminate pressure issues – add a disposable 0.5 or 2 um
in line filter to system.
in-line s stem
Page 6
Pressure Measurement
Pressure Problem I
Pressure Too
High
g
Page 7
Pressure Measurement
Pressure Problem II
• Column defective
(stationary phase)
Page 8
1100 and 1200 Pumps
Exploded
p View
Holding Screw
Purge
Outlet Valve Valve
P
Pump H
Housing
i
Plunger Housing Seals
Page 9
Pump Check Valves
New style
G1312-60010
Cartridge
5062- 7
8562
Page 10
Column Cleaning
Flush with stronger solvents than your mobile phase
phase.
Make sure detector is taken out of flow path.
Reversed-Phase Solvent Choices
in Order of Increasing Strength
Page 11
Column Cleaning
Page 12
Preventing Back Pressure Problems:
In-Line
In Line Devices
Guard
Injector
Pre-Column Filter Column
Mobile
M bil Ph
Phase
From Pump
Analytical
Column
To Detector
Page 13
Preventing Column Back Pressure Problems
1 Filt
1. Filter mobile
bil phase:
h
- filter non-HPLC grade solvents
- filter buffer solutions
- Install an in
in-line
line filter between auto
auto-sampler
sampler and column
(removes pump seal debris, ALS rotor debris, and sample
particulates). Use 2 um frit for 3.5 um columns, use 0.5 um frit for
1 8 um columns
1.8 columns.
2. Filter all samples and standards
3. Perform sample clean-up (i.e. SPE, LLE) on dirty samples.
4. Appropriate column flushing – flush buffers from entire system at
end of day with water/organic mobile phase.
Page 14
2 Peak Shape Issues in HPLC
2.
• Split
p peaks
p
• Peak tailing
• Broad peaks
• Poor efficiency
y ((low N))
• Inconsistent response
Page 15
Split Peaks
• Column contamination
• Partially plugged frit
• Column void (gap in packing bed)
• Injection solvent effects
Page 16
Split
p Peaks
Column Contamination
C l
Column: St
StableBond
bl B d SB
SB-C8,
C8 4 6 x 150 mm, 5 μm
4.6 M bil Ph
Mobile Phase: 60% 25 mMMN Na2HPO4, pHH3
3.0
0 : 40% M
MeOH
OH Fl
Flow R
Rate:
t 1 1.0
0 mL/min
L/ i
Temperature: 35°C Detection: UV 254 nm Sample: Filtered OTC Cold Medication: 1. Pseudoephedrine 2. APAP 3. Unknown 4. Chlorpheniramine
Injection
j 1 Injection
j 30 Injection 1
After Column Wash
with 100% ACN
2 2 2
4
1
1
4
1
3
4
3
3
0 5 10 15 0 5 10 15 0 5 10 15
Time (min) Time (min) Time (min)
• Column washing
g eliminates the p
peak splitting,
p g which resulted from a contaminant on the column.
Page 17
Split Peaks
I j ti Solvent
Injection S l t Effects
Eff t
Column: StableBond SB-C8, 4.6 x 150 mm, 5 μm Mobile Phase: 82% H2O : 18% ACN
Injection Volume: 30 μL Sample: 1. Caffeine 2. Salicylamide
1
0 10 0 10
Time (min) Time (min)
Page 18
Peak Shape Problems - Doublets
N
Normal
l Doublet
D bl t
Peaks
Page 19
D t
Determining
i i the
th Cause
C off Split
S lit Peaks
P k
1 Complex
1. C l sample
l matrix
t i or many samples
l analyzed
l d
- likely column contamination or partially plugged
column frit.
Page 20
Peak Tailing, Broadening
and
d Loss
L off Efficiency
Effi i (N,
(N plates)
l t )
Page 21
Peak Tailing
Column “Secondary Interactions”
Column: Alkyl-C8, 4.6 x 150 mm, 5μm Mobile Phase: 85% 25 mM Na2HPO4 pH 7.0 : 15% ACN Flow Rate: 1.0 mL/min
Temperature: 35°C
35 C Sample: 11. Phenylpropanolamine 22. Ephedrine 3
3. Amphetamine 4 4. Methamphetamine 55. Phenteramine
1 1
3
2
No TEA
2 3 10 mM
M TEA
USP TF (5%) 4 USP TF (5%)
5
1. 1.29
4
5 1. 1.19
2. 1.91 2. 1.18
3. 1.63 3
3. 1
1.20
20
4. 2.35 4. 1.26
5. 1.57 5. 1.14
Page 22
Peak Tailing
Column “Secondary
Secondary Interactions
Interactions”
Column: Alkyl-C8, 4.6 x 150 mm, 5μm Mobile Phase: 85% 25 mM Na2HPO4 : 15% ACN Flow Rate: 1.0 mL/min
Temperature: 35°C
35 C Sample: 1 1. Phenylpropanolamine 22. Ephedrine 3
3. Amphetamine 4 4. Methamphetamine 5 5. Phenteramine
1
3
2
pH 3.0
30 pH 7.0
70
2
USP TF (5%) 4
3
USP TF (5%)
5
4. 1.33 4
5
4. 2.35
Page 23
Peak Tailing
C l
Column C
Contamination
t i ti
Column: StableBond SB-C8, 4.6 x 250 mm, 5μm Mobile Phase: 20% H2O : 80% MeOH Flow Rate: 1.0 mL/min
Temperature:
p R.T. Detection: UV 254 nm Sample:
p 1. Uracil 2. Phenol 3. 4-Chloronitrobenzene 4. Toluene
3
1. 7629 2.08 1. 7906 1.43 1. 7448 1.06
2. 12043 1.64 2. 12443 1.21 2. 12237 1.21
3. 13727 1.69 2 3. 17999 1.19 3. 15366 1.11 2
4 13355 1.32 4 17098 1.252 4 19067 1.17
4
1
4
1 1 4
Page 24
Analytical vs. Preparative Scale HPLC. Non-linear
Adsorption Isotherms, or Overload Conditions:
Analytical Prep
Peak W
Width
k’
Retention
Amount injected
Page 25
Peak Tailing/Broadening
S
Sample
l Load
L d Effects
Eff t
Columns: 4.6 x 150 mm, 5μm Mobile Phase: 40% 25 mM Na2HPO4 pH 7.0 : 60% ACN Flow Rate: 1.5 mL/min
Temperature: 40°C Sample: 1. Desipramine 2. Nortriptyline 3. Doxepin 4. Imipramine 5. Amitriptyline 6. Trimipramine
A B C D
1. 1.60 1.70 1. 850 5941
2. 2.00 1.90 2. 815 7842
0 5 10 0 5 10 3. 2776 6231
3. 1.56 1.56 Time (min) Time (min)
4. 2.13 1.70 4. 2539 8359
5. 2.15 1.86 B. 5. 2735 10022
6. 1.25 1.25 Low Load D. 6. 5189 10725
0 5 0 5
Time (min) Time (min)
Group/Presentation Title
Agilent Restricted
Page 26 January 17, 2008Month ##,
200X
Peak Broadening, Splitting
Column Void
I iti l
Initial
After 30 injections
Page 27
Peak Tailing
Injector Seal Failure
Column: Bonus-RP, 4.6 x 75 mm, 3.5 μm Mobile Phase: 30% H2O : 70% MeOH Flow Rate: 1.0 mL/min
Temperature: R.T. Detection: UV 254 nm Sample: 1. Uracil 2. Phenol 3. N,N-Dimethylaniline
00
0.0 00.55 11.00 11.55 00
0.0 00.55 11.00 11.55 22.00
Time (min) Time (min)
Page 28
Peak Tailing
E t C l
Extra-Column V
Volume
l
Column: StableBond SB-C18, 4.6 x 30 mm, 3.5 μm Mobile Phase: 85% H2O with 0.1% TFA : 15% ACN Flow Rate: 1.0 mL/min
Temperature: 35°C Sample: 1. Phenylalanine 2. 5-benzyl-3,6-dioxo-2-piperazine acetic acid 3. Asp-phe 4. Aspartame
1
1
10 μL extra-column 50 μL extra-column
volume volume (tubing)
3
3 2
2
4
4
0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0
Time (min) Time (min)
Page 29
Determining the Cause of Peak Tailing
Page 30
Reproducibility Peak retention time precision:
⇒ with oven: < 0.3%
⇒ without oven: < 0.7%
Peak area precision: <1.5%
Typically,
• Area and Peak Height problems together point to the autosampler system
Page 31
Problems with Reproducibility – Peak Areas
Seal
Peak Areas not
Reproducible Pump
Page 32
3 Retention
3. R t ti IIssues
Page 33
Retention time tR, Retention factor k’, and
Selectivity factor α
Selectivity factor α
α = k2/k1
Page 34
Changes
g in Retention (k)
( )-
Same Column, Over Time
Ma be caused
May ca sed b
by:
1. Column aging
2. Column contamination
3. Insufficient column equilibration
4
4. Poor col
column/mobile
mn/mobile phase combination
5. Change in mobile phase
6
6. Change in flow rate
7. Change in column temperature
8. Other instrument issues
Page 35
Mobile Phase Change
Causes Change in Retention
60% MeOH: 40% 0.1%TFA Fresh TFA Added to
Mobile Phase
0 10 0 20 30
Time (min) Time (min)
Page 36
Separation Conditions That
Cause Changes in Retention*
Flow Rate ± 1% ± 1% tr
Temp ± 1° C ± 1 to 2% tr
%Organic ± 1% ± 5 to 10% tr
pH ± 0.01% ± 0 to 1% tr
*excerpted
p from “Troubleshooting
g HPLC Systems”,
y J. W. Dolan and L. R. Snyder,
y p 442.
Page 37
Determining the Cause of Retention
Changes
Same Column
Page 38
Change in Retention/Selectivity
C l
Column-to-Column
t C l
Page 39
Example Change in Retention/Selectivity
Column-to-Column
Mobile Phase Variation
Column 1 Column 2 Column 2 - Fresh
mobile phase
0 4 6 0 2 3 4 5 6 7 0 4 6
Time (min) Time (min) Time ((min)
Ti i )
“I have experimented with our mobile phase, opening new bottles of all
mobile phase components. When I use all fresh ingredients, the problem
ceases to exist,, and I have narrowed the problem
p to either a bad bottle of
TEA or phosphoric acid. Our problem has been solved.”
Page 40
Minimize Change in Retention/Selectivity
Lot-to-Lot
Evaluate:
Page 41
Lot-to-Lot Selectivity Change - pH
pH 4.5 - Lot 1 pH 3.0 - Lot 1
1
2-Base 2 1
3
4-Base 4
0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18
Time (min) Time (min)
2-Base
1
3
3
4-Base
4
0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18
Time ((min)) Time ((min))
Page 42
Problems with Reproducibility – Peak Areas
Seal
Peak Areas not
Reproducible Pump
Page 43
Problems with Reproducibiliy – Retention Time
• Pump Problems
–Mobile phase composition problems
–Valves AIV, ball valve defective
–Flow rate problems
Column Oven Problems
•Column
–Temperature fluctuations
•Other
–Column equilibration
–Column deterioration
Page 44
Autosampler
Principle of Operation
Standard loop volume300µl
Total delay volume 300µl + Vinj
Minimal (bypass) delay volume 6.2ul
Sampling
unit
Rheodyne 7750
From pump
To column
z 4-port rotor seal
Page 45
Evaluate Retention Changes
L tt L t
Lot-to-Lot
Page 46
Conclusions
Page 47
The End – Thank You!
Page 48
Agilent LC Columns and Agilent J&W GC
Columns Scientific Technical Support
302-993-5304 (phone)
For LC columns
Select option 4, then option 2
For GC Columns
* Select option 4, then option 1.
www.agilent.com/chem
Separation Fundamentals
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Page 49 December 11, 2007
Looking for More Information on Agilent’s LC Systems
and Software?
C ll 800.227.9770,
Call 800 227 9770 OOption
ti 5 or visit
i it
www.agilent.com/chem/education
g
to register today!
y
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