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Agilent Technologies
Technologies, Inc.
Inc
Rita Steed
Application Engineer
J
January 22
22, 2010
Page 1
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 2
HPLC Components
• Pump
• Injector/Autosampler
• Column
C l
• Detector
• Data
D t System/Integrator
S t /I t t
Slide 3
Categories of Column Problems
A Pressure
A.
B. Peak shape
3
C Retention
C. 4
5
1
2
6
0 1 2 3 4 5 6 7 8 9
Time (min)
Slide 4
1 Pressure Issues
1.
Page 5
Determining the Cause and
Correcting High Back Pressure
Page 6
Column Cleaning:
Flush
Fl h with
ith stronger
t solvents
l t than
th your mobilebil phase.
h
Make sure detector is taken out of flow path.
Reversed-Phase Solvent Choices
in Order of Increasing Strength
Use at least 10 x Vm of each solvent for analytical columns
1. Mobile phase without buffer salts (water/organic)
2. 100% Organic (MeOH or ACN)
3. Is pressure back in normal range?
4. If not, discard column or consider more drastic conditions:
75% Acetonitrile:25% Isopropanol, then
5. 100% Isopropanol
6
6. 100% Methylene Chloride*
7. 100% Hexane*
* When using either Hexane or Methylene Chloride the column must be flushed
with Isopropanol before returning to your reversed-phase mobile phase.
Page 7
Column Cleaning
Normal Phase Solvent Choices
In Order
O off Increasing Strength
S
Page 8
Preventing Column
Back Pressure Problems
• Filter mobile phase:
o Non-HPLC g grade solvents
o Buffer solutions
• Install an in-line filter between auto-sampler and column
o Use 2 um frit for 3.5 um columns,, use 0.5 um frit for
1.8um columns.
• Filter all samples and standards
• Perform sample clean-up
clean up (i.e. SPE, LLE) on dirty samples.
• Appropriate column flushing –
o Flush buffers from entire system at end of day with
water/organic mobile phase
• Use Mobile Phase Miscible Sample Solvents
Page 9
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 10
Why Filter the Sample?
E t
Extreme P
Performance
f R
Requires
i B
Better
tt S Sample
l “H
“Hygiene”
i ”
Page 11
Mini-UniPrep Syringeless Filters
Page 12
Key Reminders
Page 13
2. Peak Shape Issues in HPLC
• Split peaks
• Peak tailing
• Broad peaks
Page 14
Split Peaks
• Column contamination
• Partially plugged frit
• Column void (gap in packing bed)
• Injection solvent effects
Page 15
Determining the Cause of Split Peaks
3. Injection
j solvent stronger
g than mobile phase
p -
likely split and broad peaks, shape dependent on
injection volume and k value.
Page 16
Split
p Peaks
Column Contamination
C l
Column: St bl B d SB-C8,
StableBond SB C8 4 6 x 150 mm, 5 μm
4.6 M bil Ph
Mobile Phase: 60% 25 mMM Na
N 2HPO4, pHH3
3.0
0 : 40% MeOH
M OH Fl
Flow R
Rate:
t 1.0
1 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
Injection Solvent Effects
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)
• Injecting in a solvent stronger than the mobile phase can cause peak
shape problems, such as peak splitting or broadening.
• Note: earlier peaks (low k) most affected
Page 18
Peak Tailing, Broadening
and
d Loss
L off Efficiency
Effi i (N,
(N plates)
l t )
Page 19
Peak Tailing
Column “Secondary
Secondary Interactions
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/minTemperature: 35°C Sample: 1. Phenylpropanolamine 2. Ephedrine 3.
Amphetamine
p 4. Methamphetamine
p 5. 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 20
Peak Tailing
C l
Column “S
“Secondary
d Interactions”
I t ti ”
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 21
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 22
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 23 January 21, 2010Month ##,
200X
Peak Broadening, Splitting
Column Void
I iti l
Initial
After 30 injections
Page 24
Broad Peaks
Unknown “Phantom” Peaks
Column: Extend-C18, 4.6 x 150 mm, 5 μm Mobile Phase: 40% 10 mM TEA, pH 11 : 60% MeOH Flow Rate: 1.0 mL/min
Temperature: R.T. Detection: UV 254 Sample: 1. Maleate 2. Pseudoephedrine 3. Chlorpheniramine
1
Sample 1: Chlorpheniramine maleate
1 Sample 2 : Chlorpheniramine maleate
Peak 1: maleate and Pseudoephedrine
2
Peak 1: maleate
Peak 2: pseudoephedrine
Peak 3: chlorpheniramine (from 1st
injection)
Plates
1 5922
1.
2. 9879
3. 779
3
“Phantom”
Phantom peak from
first injection
0 5 10
Time (min)
0 5 10 15
Time (min)
Page 25
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 26
Dwell Volume & Extra Column Volume
Page 27
Peak Tailing
Extra-Column Volume
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 28
Peak tailing/fronting
What Happens If the Connections Poorly Made ?
Wrong … too long
Ferrule cannot seat properly
X
If Dimension X is too short, a dead-
volume, or mixing g chamber, will occur
Page 29
Determining the Cause
off Peak
P kT Tailing
ili
Page 30
3 Retention
3. R t ti IIssues
Page 31
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 32
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 33
Separation Conditions That
Cause Changes in Retention*
Retention
Page 34
Determining the Cause of
R t ti Changes
Retention Ch
Same Column
Page 35
Change in Retention/Selectivity
Column to Column
Column-to-Column
Page 36
Column Aging/Equilibration Causes
Retention/Selectivity Changes
Column 1 - After wash with
Column 1 - Initial Column 1 - Next Day 1% H3PO4 /Equilibration
q
1 1
2 2
0 3 5 9 12 15 0 3 5 9 12 15
Time ((min)) Time (min)
• The p
primaryy analyte
y was sensitive to mobile p
phase aging/
g g
conditioning of the column
• The peak shape was a secondary issue (metal chelating
compound) resolved by “de-activating” the active metal
contamination
Group/Presentation Title
Agilent Restricted
Page 37 January 21, 2010Month ##,
200X
Metal Sensitive Compounds Can Chelate
:
Hint: Look for Lone Pair of Electrons on :O:
or N Which Can Form 5 or 6 Membered
Ri with
Ring ith M
Metal
t l
H O
: : : :
H C O C
M +2
OH + M +22 O
H
: : :
N: M +2 C O
M +2
C N OH
: :
OH
8-hydroxyquinoline a-benzoinoxomine
5-membered ring complex 5-membered ring complex
Page 38
Acid Wash Can Improve Peak Shape
HO OH OH HO OH OH
1. 2. OH 1. 2. OH
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 pproblem to either a bad bottle of TEA or p
phosphoric
p acid. Our p
problem has
been solved.”
Page 40
Determining the Cause of
g
Retention Changes
Column-to-Column
Page 41
Minimize Change in
Retention/Selectivity
Lot-to-Lot
Evaluate:
1. All causes of column-to-column change*
2. Method ruggedness (buffers/ionic strength)
3 pH
3. H sensitivity
i i i ((sample/column
l / l iinteractions)
i )
Page 42
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 43
Evaluate Retention Changes
g
Lot-to-Lot
Page 44
Conclusions:
Page 45
Agilent Technical Support
LC or GC Column Support
800-227-9770 (phone: US & Canada)
Select opt. 3, opt. 3, then option 1 for
GC or option 2 for LC.
www.agilent.com/chem
Page 46
The End – Thank You!
Page 47