Escolar Documentos
Profissional Documentos
Cultura Documentos
Troubleshooting of
HPLC Columns
Columns and Consumables
Edward Kim
Applications Engineer
January 17, 2008
Page 1
Chip LC
Page 2
Nano LC
Capillary LC
Analytical LC
Prep LC
mA
U
Troubleshooting in HPLC
20
00
15
00
10
5
00
0
0
0
Page 3
1
0
1
5
Time
(min)
2
0
2
5
Page 4
1. Pressure Issues
Column Observations
Potential Problems
Plugged inlet frit
Column contamination
Plugged packing
Page 5
Pressure Measurement
Pressure Problem I
Pressure Too
High
Page 7
Pressure Measurement
Pressure Problem II
Pressure Too Low
Solvent inlet
frits
Page 8
Outlet Valve
Plunger Housing
Pump Housing
Seals
Pistons
Page 9
Purge
Valve
Cartridge
5062-8562
5062-8568
Old style
Page 10
5
2
Purge
Valve
G1312-60009
5. Gold Seal
5001-3707
2. Plastic cap
01018-21207
6. Cap(4pk)
5062-2485
3. Gold Seal
5001-3707
4. Cap(4pk)
5062-2485
Column Cleaning
Flush with stronger solvents than your mobile phase.
Make sure detector is taken out of flow path.
Reversed-Phase Solvent Choices
in Order of Increasing Strength
Page 11
Column Cleaning
Normal Phase Solvent Choices
in Order of Increasing Strength
Page 12
Pre-Column
Injector
Guard
Filter Column
Analytical
Column
To Detector
Page 13
Page 14
Page 15
Split Peaks
Page 16
Split Peaks
Column Contamination
Column: StableBond SB-C8, 4.6 x 150 mm, 5 m
Mobile Phase: 60% 25 mM Na2HPO4, pH 3.0 : 40% MeOH
Temperature: 35C Detection: UV 254 nm
Sample: Filtered OTC Cold Medication: 1. Pseudoephedrine 2. APAP
Injection 1
Injection 1
After Column Wash
with 100% ACN
Injection 30
4
1
1
4
4
3
3
Time (min)
10
15
Time (min)
10
15
Time (min)
10
15
Column washing eliminates the peak splitting, which resulted from a contaminant on the column.
Page 17
Split Peaks
A. Injection Solvent
100% Acetonitrile
B. Injection Solvent
Mobile Phase
2
2
1
10
Time (min)
10
Time (min)
Page 18
Normal
Page 19
Doublet
Peaks
Page 20
Page 21
Peak Tailing
1
3
2
No TEA
USP TF (5%)
1.
2.
3.
4.
5.
USP TF (5%)
4
5
1.29
1.91
1.63
2.35
1.57
0.0
10 mM TEA
2.5
5.0
TIme (min)
0.0
2.5
1.
2.
3.
4.
5.
5.0
Time (min)
1.19
1.18
1.20
1.26
1.14
Peak Tailing
1
1
pH 3.0
USP TF (5%)
4. 1.33
0.0
4
5
4
2.5
5.0
Time (min)
0.0
2.5
pH 7.0
USP TF (5%)
4. 2.35
5.0
Time (min)
Peak Tailing
Column Contamination
Column: StableBond SB-C8, 4.6 x 250 mm, 5m
Temperature: R.T.
Detection: UV 254 nm
QC test forward
direction
Plates
1.
2.
3.
4
7629
12043
13727
13355
TF
Plates
1.
2.
3.
4
2.08
1.64
1.69
1.32
7906
12443
17999
17098
TF
Plates
3
1.
2.
3.
4
1.43
1.21
1.19
1.252
Page 24
2.5
Time (min)
1.06
1.21
1.11
1.17
4
4
0.0
7448
12237
15366
19067
TF
5.0
0.0
2.5
Time (min)
5.0
0.0
2.5
Time (min)
5.0
Analytical
Prep
Peak Width
k
Retention
Amount injected
Page 25
Peak Tailing/Broadening
Sample Load Effects
Columns: 4.6 x 150 mm, 5m
Mobile Phase: 40% 25 mM Na2HPO4 pH 7.0 : 60% ACN
Flow Rate: 1.5 mL/min
Temperature: 40C
Sample: 1. Desipramine 2. Nortriptyline 3. Doxepin 4. Imipramine 5. Amitriptyline 6. Trimipramine
Tailing
Eclipse XDB-C8
USP TF (5%)
1.
2.
3.
4.
5.
6.
1.60
2.00
1.56
2.13
2.15
1.25
1.70
1.90
1.56
1.70
1.86
1.25
Broadening
Competitive C8
Plates
C.
5
Time (min)
B.
Page 26
High Load
x10
A.
10
5
Time (min)
Low Load D.
5
Time (min)
10
1.
2.
3.
4.
5.
6.
850
815
2776
2539
2735
5189
D
5941
7842
6231
8359
10022
10725
5
Time (min)
Group/Presentation Title
Agilent Restricted
October 28, 2008Month ##,
200X
Column Void
Mobile Phase: 50%ACN: 50% Water : 0.2% TEA
(~ pH 11)
Initial
After 30 injections
Page 27
Peak Tailing
Before
Plates
USP TF (5%)
1. 2235
2. 3491
3. 5432
1.72
1.48
1.15
1. 3670
2. 10457
3. 10085
1.45
1.09
1.00
0.0
0.5
1.0
Time (min)
1.5
0.0
0.5
1.0
Time (min)
1.5
2.0
Page 28
Peak Tailing
Extra-Column Volume
Column: StableBond SB-C18, 4.6 x 30 mm, 3.5 m
Mobile Phase: 85% H2O with 0.1% TFA : 15% ACN
Flow Rate: 1.0 mL/min
Temperature: 35C
Sample: 1. Phenylalanine 2. 5-benzyl-3,6-dioxo-2-piperazine acetic acid 3. Asp-phe 4. Aspartame
10 L extra-column
volume
50 L extra-column
volume (tubing)
2
4
4
0.0
Page 29
0.5
1.0
1.5
Time (min)
2.0
0.0
0.5
1.0
1.5
Time (min)
2.0
Page 30
Reproducibility
Typically,
Area and Peak Height problems together point to the autosampler system
Area and Retention Time problems together point to the pump
Page 31
Page 32
Seal
Pump
Column
Rotor Seal
3. Retention Issues
Page 33
Selectivity factor
= k2/k1
Page 34
Column aging
Column contamination
Insufficient column equilibration
Poor column/mobile phase combination
Change in mobile phase
Change in flow rate
Change in column temperature
10
Time (min)
20
Time (min)
30
Page 36
1%
1% tr
Temp
1 C
1 to 2% tr
%Organic
1%
5 to 10% tr
pH
0.01%
0 to 1% tr
Page 37
Page 38
3.
4.
5.
6.
Change in Retention/Selectivity
Column-to-Column
1. Different column histories (aging)
2. Insufficient/inconsistent equilibration
3. Poor column/mobile phase combination
4. Change in mobile phase
5. Change in flow rate
6. Other instrument issues
7. Slight changes in column bed volume (tr only)
Page 39
4
Time (min)
Column 2 - Fresh
mobile phase
Column 2
3 4 5
Time (min)
4
Time (min)
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 to either a bad bottle of
TEA or phosphoric acid. Our problem has been solved.
Page 40
Evaluate:
1.
2.
3.
Page 41
pH 4.5 - Lot 1
1
2-Base
3
3
4-Base
8
10 12
Time (min)
14
16
18
8
10 12
Time (min)
14
16
18
pH 3.0 - Lot 2
pH 4.5 - Lot 2
1
2-Base
1
3
4-Base
4
0
8
10 12
Time (min)
14
16
18
8
10 12
Time (min)
14
Page 42
16
18
Page 43
Seal
Pump
Column
Rotor Seal
Pump Problems
Mobile phase composition problems
Valves AIV, ball valve defective
Flow rate problems
Column Oven Problems
Temperature fluctuations
Other
Column equilibration
Column deterioration
Page 44
Autosampler
Principle of Operation
Standard loop volume300l
Total delay volume
300l + Vinj
Minimal (bypass) delay volume
6.2ul
Metering device
Vial gripper
Sampling
unit
Rheodyne 7750
From pump
To column
O
To waste
Page 45
Page 46
Conclusions
HPLC column problems are evident as:
1. High pressure
2. Undesirable peak shape
3. Changes in retention/selectivity
These problems are not always
associated with the column and may
be caused by instrument and
experimental condition issues.
Page 47
Page 48
Page 49
Separation Fundamentals
Agilent Restricted
December 11, 2007
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Separation Fundamentals
Agilent Restricted
December 11, 2007
www.SeparationsNOW.com/agilentwebinar
Be sure to register after todays event.
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Separation Fundamentals
Agilent Restricted
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Upcoming LC e-Seminars
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Separation Fundamentals
Agilent Restricted
December 11, 2007