hplc column troubleshooting - agilent€¦ · 4 soc 6 c 12-oc j.c. 268(1983) 1 j.c. 111(1975) 149...
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HPLC Column Troubleshooting
Page 1
Gulf Coast Conference 2014
John Palmer
Page 2
Pump
Injector/Autosampler
Column
Detector
Data System/Integrator
HPLC System Components
Problems Can Be Related to All Components in the System
There Can Be Many Causes Start Asking Questions That Lead to The Cause
•1st Did System Suitability or the Sample Fail?
•2nd Review Method for Compliance - Is The Procedure Being Followed Properly?
- Are Instrument Settings Correct?
•3rd Ask More Questions! - When Did the System Last Function Properly?
- Has Anything Been Changed?
•4th Review ALL parameters! - The Obvious Is Not Always the Cause
- Was There More Than One Change?
Page 3
Page 4
Categories of Column and System Problems
A. Pressure
B. Peak shape
C. Retention
Page 5
Column Observations Potential Problems
High pressure - Plugged frit
- Column contamination
- Plugged packing
Low Pressure - Leak
- Flow Incorrect
Pressure Issues
Determining the Cause and Correcting High Back Pressure
Page 6
• Check pressure with/without column
• Remove Guard
• Remove Column
• If Column pressure is high:
• Back flush column – Clear “dirty” frit surface • • Wash column – high molecular weight/adsorbed compounds – precipitate from sample or buffer
• Replace Column
Change frit – Clear plugged frit PREVENT THIS!
Peak Splitting Caused By Disrupted Sample Path
Page 7
Split or Double Peaks
Normal Double
Peaks
Tip: Similar Effect Can be Caused by Partially Plugged Frit
•Flow Path Disrupted by Void
•Sample Allowed to Follow Different Paths
Through Column
•Poorly Packed Bed Settles in Use
•High pH Dissolves Silica
Split Peaks from Injection Solvent Effects
September 28, 2014Month
##, 200X
Group/Presentation Title
Agilent Restricted
Page 8
Column: StableBond SB-C8, 4.6 x 150 mm, 5 mm Mobile Phase: 82% H2O : 18% ACN
Injection Volume: 30 mL Sample: 1. Caffeine 2. Salicylamide
A. Injection Solvent
100% Acetonitrile
B. Injection Solvent
Mobile Phase
Tip: Injecting in a solvent stronger than the mobile phase can cause peak shape
problems such as peak splitting or broadening
Trick: Keep Organic Concentration in Sample Solvent < Mobile Phase
0 10Time (min)
1
2
0 10Time (min)
1
2
Split Peaks from Column Contamination
Page 9
0 5 10 15
1
3
4
2
Time (min)0 5 10 15
1
3
4
2
Time (min)0 5 10 15
1
3
4
2
Time (min)
Column: StableBond SB-C8, 4.6 x 150 mm, 5 mm Mobile Phase: 60% 25 mM Na2HPO4, pH 3.0 : 40% MeOH Flow Rate: 1.0 mL/min
Temperature: 35°C Detection: UV 254 nm Sample: Filtered OTC Cold Medication: 1. Pseudoephedrine 2. APAP 3. Unknown 4. Chlorpheniramine
Injection 1 Injection 30 Injection 1
After Column Wash
with 100% ACN
Tip: Column washing eliminates the peak splitting, which resulted from a contaminant on the column
How could this be prevented? (Guard Column, SPE clean up of samples, Periodic column wash)
Peak Tailing - Column Contamination
Page 10
0.0 2.5 5.0
2
41
3
Time (min)
2
4
1
3
0.0 2.5 5.0
Time (min)
2
41
3
0.0 2.5 5.0
Time (min)
Column: StableBond SB-C8, 4.6 x 250 mm, 5mm Mobile Phase: 20% H2O : 80% MeOH Flow Rate: 1.0 mL/min
Temperature: R.T. Detection: UV 254 nm Sample: 1. Uracil 2. Phenol 3. 4-Chloronitrobenzene 4. Toluene
Plates TF
1. 7629 2.08
2. 12043 1.64
3. 13727 1.69
4 13355 1.32
Plates TF
1. 7906 1.43
2. 12443 1.21
3. 17999 1.19
4 17098 1.25
Plates TF
1. 7448 1.06
2. 12237 1.21
3. 15366 1.11
4 19067 1.17
QC test forward
direction QC test reverse direction
QC test after cleaning
100% IPA, 35°C
Tip: Quick Test to Determine if Column is Dirty or Damaged
Trick: Reverse Column and Run Sample –If Improved, Possible Cleaning Will Help
- No improvement - Column Damaged and Needs to be Replaced
Column Cleaning
Page 11
Use at least 25 mL of each solvent for long analytical columns
Flush with stronger solvents than
your mobile phase.
Reversed-Phase Solvent Choices in Order of Increasing Strength
• Mobile phase without buffer salts
• 100% Methanol
• 100% Acetonitrile
• 75% Acetonitrile:25%
Isopropanol
• 100% Isopropanol
• 100% Methylene Chloride*
• 100% Hexane*
Must Reverse
to
Re-Equilibrate
This Is Time Consuming
Often Performed Offline
Changing a Frit May Not Be a Good Idea May not be possible with new generation columns
May damage high performance columns
Page 12
Column
Inlet Frit
Compression
Ferrule
Column Body
Female End Fitting
Male End Fitting
Wear gloves
Do not allow bed to dry
Do not touch the column -
body heat will extrude packing
Do not overtighten
Tip: Prevention is a Much Better Idea!
The Trick: Prevention Techniques - A Better Choice!
Page 13
• Filter samples
• Filter buffered mobile phases
• Use column protection
- In-line filters
- Guard columns
• Sample clean-up (i.e. SPE)
• Appropriate column flushing
Easy
Not As Easy
Inexpensive Filters Prevent Column Frit Plugging
Page 14
Regenerated Cellulose (RC) Recommended •Universal hydrophilic membrane, compatible
with most solvents - aqueous and organic
•High purity, extremely low extractables and
binding
•More Uniform Surface
•Different than Other Cellulose Filters!!
In-line Filters Easy to Use and replace
Frits Available in 0.2,0.5 and 2.0µ Porosity
Much Less expensive than a Column
Easier and Faster to Replace than a Column Frit
Mini-UniPrep Filtering Vials
•Pre-assembled filtration devices for removing particulate matter from samples.
•Single disposable unit can replace the combination of syringe filters, syringes, auto-sampler vials, transfer containers, septa and caps.
•Quick, environmentally conservative way to filter samples prior to HPLC analysis.
Page 15 Manufactured by Whatman, a division of GE Healthcare
Separation Conditions and the Effect on Retention*
16
Flow Rate +/- 1% +/- 1% Tr
Temp +/- 1 deg C +/- 1 to 2% Tr
%Organic +/- 1% +/- 5 to 10% Tr
pH +/- 0.01% +/- 0 to 1% Tr
*excerpted from “Troubleshooting HPLC Systems”, J. W. Dolan and L. R. Snyder, p 442.
Mobile Phase: Aqueous Component Experimental Variables That Impact Resolution
Column
Mobile Phase
• Aqueous Component - Importance of Buffers
- Considerations for Buffer Selection
- Buffer pH
- Buffer Concentration
• Organic Component
Sample
Instrument
Gradient Separations
September 28, 2014
CI0126C
17
Your opportunity to improve
robustness and ruggedness
Mobile Phase pH and pH Buffers Why Are These So Important in HPLC?
•pH Effects Ionization
- Silica Surface of Column
- Sample Components of Interest
• Buffers
- Resist Changes in pH and Maintain Retention
- Improve Peak Shape for Ionizable Compounds
• Effects Column Life
- Low pH strips Bonded Phase
- High pH Dissolves Silica
Page 18
Why Worry About pH? pH, pKa and Weak Bases
Page 19
At pH 9 – the sample exists as protonated and unprotonated diphenhydramine
in a ratio of 1:1. Peak shape can be poor.
At pH 10 – 91% of the sample exists as unprotonated diphenhydramine.
At pH 8 – 91% of the sample exists as protonated diphenhydramine.
Ka = [R3N][H+]
[R3NH+]
Ka = 1 x 10-9
pKa = 9
R3NH+ R3N + H+
CHOCH CH N2 2
CH3
CH3
+
HCH
3
CH3
CHOCH CH N2 2 + H+
Why Worry About pH? pH, pKa and Weak Acids
Page 20
At pH 4.2 – the sample exists as benzoic acid and the benzoate ion in a ratio
of 1:1. Peak shape can be poor
At pH 5.2 – 91% of the sample exists as the benzoate ion. RP retention decreases.
At pH 3.2 – 91% of the sample exists as benzoic acid. RP retention increases.
RCOOH RCOO- + H+
Ka = 6.4 x 10-5
pKa = 4.2
Ka = [RCOO-][H+]
[RCOOH]
+ H+
COOH COO _
pH vs. Selectivity for Acids and Bases
Page 21
5
SCD
+
+
2 +
1
1.5
1.0
0.5
0.0
-0.5
log
k«
3 4 5 6 7 8 pH
A B C
2
4
5
3
3 5 7 9
ELUENT pH
40
30
20
10
10
Ret
entio
n
+++
3
17,12 - OC
UDC
SOC4
6
C12-OC
J.C. 268(1983) 1
J.C. 111(1975) 149
Column: Nucleosil-C18
Mobile Phase: 45% ACN/55% phosphate buffer
Sample: Bile Acids
Column: mBondapak-C18
Mobile Phase: 60% 25 mM phosphate buffer
40% Methanol
1. Salicylic acid
2. Phenobarbital
3. Phenacetin
4. Nicotine
5. Methamphetamine
• Retention and selectivity can change dramatically when pH is changed.
Why Use Buffered Mobile Phases?
September 28, 2014
CI0126C
22
A = pH 7.0
water A = pH 7.0, 25 mM phosphate
buffer
Column: ZORBAX Rapid Resolution Eclipse XDB-C8, 4.6 x 75 mm, 3.5 µm Mobile Phase: 44% A : 56% methanol
Flow Rate: 1.0 mL/min Temperature: 25°C Detection: UV 250 nm
Sample: 1. ketoprofen 2. ethyl paraben 3. hydrocortisone 4. fenoprofen 5. propyl paraben 6. propranolol 7. ibuprofen
• Buffered mobile phases enhance retention, resolution, and peak shape.
0 1 2 3 4 5Time (min)
1
2
3
4
6
7
5
0 1 2 3 4
1, 4, 6, 7
2
3
5
Time (min)
Considerations For Buffer Selection
Buffer Type
• Inorganic vs. organic buffers—choice can affect resolution, column lifetime and MS compatibility
Buffer pH
• Select buffer based on desired pH and optimum buffer pH range.
• Measure pH of buffer solution before mixing with organic modifier.
• Compare resolution at desired pH ± 0.1–0.2 pH units. Buffer Concentration and Ionic Strength
• Start at 20 – 25 mM.
• Prepare buffer according to accepted procedures.
• Avoid overshoot and readjustment when setting pH.
• Compare resolution at desired buffer concentration ± 5–10 mM.
September 28, 2014
CI0126C
23
Changes in Buffer Concentration Can Affect Retention, Peak Width and Peak Shape
September 28, 2014
CI0126C
24
USP Tf
(5%)
1. 1.62
2. 1.65
3. 1.63
4. 1.77
5. 1.83
6. 1.12
0.0 0.02.5 2.55.0 5.0
6
6
5
54
4 3
3
2
2
1
1
7.5
Time (min) Time (min)
10 mM Phosphate 25 mM Phosphate
Column: ZORBAX Eclipse XDB-C8, 4.6 x 150 mm, 5 µm
Mobile Phase: 40% phosphate buffer (pH 7.0) : 60% ACN Flow Rate: 1.5 mL/min Temperature:
40°C
Sample: Tricyclic Antidepressants, 1. Desipramine 2. Nortriptyline 3. Doxepin 4. Imipramine 5. Amitriptyline 6.
Trimipramine
USP Tf
(5%)
1. 1.41
2. 1.50
3. 1.33
4. 1.39
5. 1.36
6. 1.00
Effect of pH on Peak Shape at or Near the Sample pKa
Page 25
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10
Time (min) Time (min)
Column: ZORBAX SB-C8 4.6 x 150 mm, 5 mm Mobile Phase: 40% 5 mM KH2PO4: 60% ACN
Flow Rate: 1.0 mL/min. Temperature: RT
pH 4.4 pH 3.0
Ibuprofen
pKa = 4.4
CH3CHCOOH
CH2CH(CH3)2
• Inconsistent and tailing peaks may occur when operating close to an analyte
pKa and should be avoided.
Lot-to-Lot Selectivity Change Related to pH Choice
September 28, 2014Month
##, 200X
Group/Presentation Title
Agilent Restricted
Page 26
0 2 4 6 8 10 12 14 16 18
Time (min)
2-Base
3
4-Base
1
0 2 4 6 8 10 12 14 16 18
Time (min)
2
3
4
1
• pH 4.5 shows selectivity change from lot-to-lot for basic compounds
• pH 3.0 shows no selectivity change from lot-to-lot
• Indication of poorly controlled ionization
pH 4.5 - Lot 1 pH 3.0 - Lot 1
pH 4.5 - Lot 2 pH 3.0 - Lot 2
0 2 4 6 8 10 12 14 16 18
Time (min)
2-Base
3
4-Base
1
0 2 4 6 8 10 12 14 16 18
Time (min)
2
3
4
1
Test for pH Robustness
September 28, 2014
CI0126C
27
1 2
3
4
6 7
5
0 1 2 3 4 5 6
Time (min)
0 1 2 3 4 5 6
1 2
3
4
6
7
5
Time (min)
pH 7.00
pH 7.25
Column: ZORBAX Rapid Resolution Eclipse XDB-C8, 4.6 x 75 mm, 3.5 µm
Mobile Phase: 44% 25 mM phosphate, pH 7.00 : 56% methanol Flow Rate: 1.0 mL/min Temperature: 25°C
Detection: UV 250 nm
Sample: 1. ketoprofen 2. ethyl paraben 3. Hydrocortisone 4. fenoprofen 5. propyl paraben 6. Propranolol 7.
ibuprofen
• The resolution of ionizable compounds can change
markedly with pH changes—even as small as 0.05–
0.25 pH units.
Don’t Forget - Match Column to pH of Mobile Phase for Maximum Column Lifetime low pH and high temperature (pH 0.8, 90°C)
Page 28 000023P1.PPT
Purge Solvent:
50% methanol/water with
1.0% TFA
Solute: Toluene
Kirkland, J.J. and J.W. Henderson, Journal of Chromatographic Science, 32 (1994) 473-480.
Don’t Forget - Match Column to pH of Mobile Phase for Maximum Column Lifetime High pH and Room Temperature (pH 11 RT)
Page 29
Tip: Use Columns Designed for chosen pH
Mobile Phase: 50%ACN: 50% Water : 0.2% TEA
(~ pH 11)
After 30 injections
Initial
Mobile Phase: Organic Component
Column
Mobile Phase • Aqueous Component
• Organic Component
- % Organic Modifier
Sample
Instrument
Gradient Separations
September 28, 2014
CI0126C
30
Even a Small Change in % Organic Modifier Can Change Resolution
September 28, 2014
CI0126C
31
12
34
5
6
0 1 2 3 4
Time (min)
0 1 2 3 4
12
34
5
6
Time (min)
1 2
34
5
6
0 1 2 3Time (min)
4
• Verify that resolution doesn’t change
significantly around desired
conditions (for example, %B ± 1–
2%).
59% MeOH
57.5% MeOH
56% MeOH
Column: ZORBAX Rapid Resolution Eclipse
XDB-C8
4.6 x 75 mm, 3.5 µm
Mobile Phase: A: 25 mM phosphate, pH
7.00 (10 mM TEA)
B: methanol (10 mM TEA)
Flow Rate: 1.0 mL/min
Temperature: 25°C controlled
Injection: 5 mL
Detection: 275 nm
Sample: 1. ketoprofen 2. ethyl paraben 3.
hydrocortisone
4. fenoprofen 5. propyl paraben 6.
propranolol
Rs (5,6) =
2.5
Rs (5,6) =
1.7
Rs (5,6) =
1.2
The Sample Experimental Variables That Impact Resolution
Column
Mobile Phase
Sample
• Injection volume
• Sample solvent
strength
Instrument
Gradient Separations
September 28, 2014
CI0126C
32
Injection Volume and Sample Solvent Strength
Injection Volume
• Lack of ruggedness typically seen
– when Vinj is increased to improve signal-to-noise (S/N) ratio, or,
– when column size is decreased.
• Use minimum Vinj for required repeatability and limit of detection.
• Compare resolution, peak shape and repeatability at 0.2X, 1X and 2–5X Vinj.
Sample Solvent Strength
• Match % organic modifier in mobile phase (or weaker).
• If stronger sample solvent needed (solubility, stability), keep Vinj to
minimum.
• Compare resolution, peak shape and width at desired solvent strength ±50% relative.
September 28, 2014
CI0126C
33
Test For Injection Volume Robustness
September 28, 2014
CI0126C
34
0 1 2 3 4 5
0 1 2 3 4 5
1 2
3
4 5
T im e (m in)
6 7
1 2
3
45
6
7
1
2
3
45
6
7
0 1 2 3 4 5
1 µL
5 µL
Column: ZORBAX Rapid Resolution Eclipse XDB-C8 4.6 x 75 mm, 3.5 µm
Mobile Phase: 44% 25 mM phosphate,
pH 7.00
56% methanol
Flow Rate: 1.0 mL/min
Temperature: 25°C
Detection: UV 250 nm
Sample: 1. ketoprofen 2. ethyl paraben 3. hydrocortisone 4. fenoprofen 5. propyl paraben 6. propranolol 7. ibuprofen
Rs(6,7) =
2.5
Rs(6,7) =
2.1
Rs(6,7) =
1.6
10 µL
• Varying injection volume can sometimes reveal lack of robustness for resolution and peak shape.
Peak Tailing, Broadening and Loss of Efficiency
Page 35
May be caused by:
• Column “secondary
interactions”
• Column contamination
• Column aging
• Column loading
• Extra-column effects
Peak Shape: Tailing Peaks
Page 36
Normal Tailing
Normal Tailing
Symmetry > 1.2
All Peaks Tail:
Extra-Column Effects.
Build up of Contamination on Column
Inlet.
Bad Column.
Causes
Some Peaks Tail:
Secondary - Retention Effects.
Residual Silanol Interactions.
Small Peak Eluting on Tail of Larger Peak.
Peak Tailing
Identifying Column “Secondary Interactions”
Page 37
Tip: Mobile phase modifier (TEA) competes with Sample for surface ion exchange
sites at mid-range pH values
Column: Alkyl-C8, 4.6 x 150 mm, 5mm Mobile Phase: 85% 25 mM Na2HPO4 pH 7.0 : 15% ACN Flow Rate: 1.0 mL/min
Temperature: 35°C Sample: 1. Phenylpropanolamine 2. Ephedrine 3. Amphetamine 4. Methamphetamine 5. Phenteramine
No TEA USP TF (5%)
1. 1.29
2. 1.91
3. 1.63
4. 2.35
5. 1.57
10 mM TEA USP TF (5%)
1. 1.19
2. 1.18
3. 1.20
4. 1.26
5. 1.14
TIme (min) Time (min)
0.0 2.5 5.0
54
32
1
5
4
3
2
1
0.0 2.5 5.0
Peak Tailing
Low pH Minimizes “Secondary Interactions” for Amines
Page 38
Column: Alkyl-C8, 4.6 x 150 mm, 5mm Mobile Phase: 85% 25 mM Na2HPO4 : 15% ACN Flow Rate: 1.0 mL/min
Temperature: 35°C Sample: 1. Phenylpropanolamine 2. Ephedrine 3. Amphetamine 4. Methamphetamine 5. Phenteramine
pH 3.0
USP TF (5%)
4. 1.33
pH 7.0
USP TF (5%)
4. 2.35
Tip: Reducing mobile phase pH reduces interactions with silanols and peak tailing.
Time (min)
0.0 2.5 5.0
54
32
1
5
4
32
1
Time (min)
0.0 2.5 5.0
Peak Tailing
High pH Eliminates “Secondary Interactions” for Amines
Peak Shape and Retention of this sample of basic compounds improves at high pH where column has high IEX activity. Why?
Page 39
0 5
1
2,3
4
5
Time (min)
7
6
0 5 10
1
2
3
4
Time (min)
6
5
7
Column: ZORBAX Extend-C18, 4.6 x 150 mm, 5 m m Mobile Phase: See Below Flow Rate: 1.0 mL/min Temperature: RT
Detection: UV 254 nm
Sample: 1. Maleate 2. Scopolamine 3. Pseudoephedrine 4. Doxylamine 5. Chlorpheniramine 6. Triprolidine 7. Diphenhydramine
pH 7
30% 20 mM Na2HPO4
70% MeOH
pH 11
30% 20 mM TEA
70% MeOH
tR = 8.5 tR = 11.4
40
Peak Shape: Broad Peaks
All Peaks Broadened:
• Loss of Column Efficiency.
• Column Void.
• Large Injection Volume.
Some Peaks Broadened:
• Late Elution from Previous Sample
(Ghost Peak).
- High Molecular Weight.
- Sample - Protein or Polymer.
Page 40
Unknown “Phantom” Peaks
Page 41
Time (min)
3
1
2
0 5 10 15
Column: Extend-C18, 4.6 x 150 mm, 5 mm 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
Plates
1. 5922
2. 9879
3. 779
Tip: The extremely low plates for moderately retained peaks are an indication of a
very late eluting peak from a preceding run.
Time (min)
1
0 5 10
Sample 1: Chlorpheniramine maleate
Peak 1: maleate
Sample 2 : Chlorpheniramine
maleate
and Pseudoephedrine
Peak 1: maleate
Peak 2: pseudoephedrine
Peak 3: chlorpheniramine (from 1st
injection)
“Phantom” peak from
first injection
Changes in Retention Can Be Chemical or Physical
Page 42
May be caused by:
• Column aging
• Column contamination
• Insufficient equilibration
• Poor column/mobile phase combination
• Change in mobile phase
• Change in flow rate
• Different Gradient Delay Volumes
Column Aging/Equilibration Causes Retention/Selectivity Changes
September 28, 2014Month
##, 200X
Group/Presentation Title
Agilent Restricted
Page 43
Column 1 - After Cleaning
with 1% H3PO4
/Equilibration
• The primary analyte was sensitive to mobile phase aging/
conditioning of the column
• The peak shape was a secondary issue (metal chelating
compound) resolved by “de-activating” the active metal
contamination
Column 1 - Next Day Column 1 - Initial
0 3 5 9 12 15Time (min)
2
1
0 3 5 9 12 15Time (min)
2
1
Column Temperature Adequate Temperature Control is Essential
Laboratory temperatures can vary by ±5°C or more. Column temperature changes affect resolution and repeatability. Useful tool for changing selectivity, retention and efficiency when developing separations. Important parameter to control during method development and validation. Compare resolution, peak width and peak shape at desired temperature ±5°C.
September 28, 2014
CI0126C
44
Small Temperature Changes Can Cause Dramatic Changes in Resolution
September 28, 2014
CI0126C
45
12
34 6
5
0 0.5 1 1.5 2 2.5 3 3.5
Time (min)
0 0.5 1 1.5 2 2.5 3 3.5
12
34 6
5
Time (min)
0 0.5 1 1.5 2 2.5 3 3.5
1 2
34 6
5
Time (min)
Column: ZORBAX Rapid Resolution Eclipse XDB-C8 4.6 x 75 mm, 3.5 µm
Mobile Phase: Isocratic, 28%B : 72%A
A: 5/95 methanol/pH 7.00 buffer 25 mM, 10 mM TEA
B: 80/20 methanol/pH 7.00 buffer 25 mM, 10 mM TEA
Flow Rate: 1.0 mL/min.
Temperature: See Figure
Injection: 5 mL
Detection: 275 nm
Sample: 1. ketoprofen 2. ethyl paraben 3. hydrocortisone 4. fenoprofen 5. propyl paraben 6. propranolol
20°C
25°C
30°C
• Column temperature
control will produce the
most consistent
results.
CASE STUDIES
Page 46
Example: Change in Retention/Selectivity
September 28, 2014Month
##, 200X
Group/Presentation Title
Agilent Restricted
Page 47
Unintended Mobile Phase Variation
Tip: The Source of the Problem is Often Not the Obvious Change
“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.”
Column 1 Column 2 - Fresh
mobile phase Column 2
0 4 6Time (min)
0 2 3 4 5 6 7
Time (min)
0 4 6Time (min)
Case History: Problem – Selectivity Does Not Appear the Same from Column-to-Column
Details:
• 3 Columns with the Same Bonded Phase were used
• They were the same dimensions, just different particle sizes (and therefore
different lots of material)
• They were tested on the same day on the same instrument and with the
same mobile phase
• Isocratic elution; binary (two channel) pump generated mobile phase
Problem:
• The selectivity was different on each of the columns
48
49
Selectivity between particle sizes of Eclipse Plus C18, 4.6 x 50 mm, using same test solutes
John W. Henderson JrGroup/Presentation
Title
Agilent Restricted Month ##, 200X
min 0 1 2 3 4 5
mAU
-5
0
5
10
15
20
25
DAD1 B, Sig=280,16 Ref=360,20 (F:\EPC18SELECTIVITY\ANALINE000015.D)
0.2
93
0.3
60
0.7
12
0.9
61
1.2
29
1.6
35
3.9
07
min 0 1 2 3 4 5
mAU
-5
0
5
10
15
20
25
DAD1 B, Sig=280,16 Ref=360,20 (F:\EPC18SELECTIVITY\3P5MICRON200001.D)
0.0
17
0.3
00
0.3
69
0.7
25
0.9
96
1.2
90
1.7
22
4.1
98
min 0 1 2 3 4 5
mAU
-5
0
5
10
15
20
25
DAD1 B, Sig=280,16 Ref=360,20 (F:\EPC18SELECTIVITY\ANALINE000019.D)
0.2
99
0.7
84
1.0
71
1.3
89
1.9
00
4.8
97
2 3
5 1 4
6
5 um, UXE01033, NEP0652003
3.5 um, UXF01309, B08111
1.8 um, UXG03882, B08021
m.p.: A: water, B: acetonitrile (60:40 A:B)
Flow : 1.5 mL/min
Temp: 25 C
Detection: UV 280nm,16, ref=360,20
Flow cell: 6mm, 5uL
Data rate: 0.2s
Inj. Vol 2 uL
N=3800
N=7100
N=9900
tr k alpha
uracil 0.3 0.00
aniline 0.78 1.60 #DIV/0!
o-toluidine 1.07 2.57 1.60
4-chloroaniline 1.39 3.63 1.42
2-naphthylamine 1.9 5.33 1.47
3,3'-dichlorobenzidine 4.9 15.33 2.88
tr k alpha
uracil 0.3 0.00
aniline 0.73 1.43 #DIV/0!
toluidine 1 2.33 1.63
chloroaniline 1.29 3.30 1.41
naphthylamine 1.72 4.73 1.43
dichlorobenzidine 4.2 13.00 2.75
tr k alpha
uracil 0.29 0.00
aniline 0.71 1.45 #DIV/0!
toluidine 0.96 2.31 1.60
chloroaniline 1.23 3.24 1.40
naphthylamine 1.63 4.62 1.43
dichlorobenzidine 3.91 12.48 2.70
What Questions Should We Ask and What Should We Try to diagnose problem?
Compare more columns – see if they are different
Try another LC
Premix the mobile phase
50
51
One channel premixed mobile phase shows similar α whereas previous two channels doesn’t show similar α!
John W. Henderson JrGroup/Presentation
Title
Agilent Restricted Month ##, 200X
0 1 2 3 4
mAU
-5
0
5
10
15
20
DAD1 B, Sig=280,16 Ref=360,20 (E:\ECLIPSE SELECTIVIY INVESTIGATION\ONECHANNEL00002.D)
0.2
49
0
.28
8
0.3
52
0.6
96
0.9
46
1.2
14
1.6
15
3.8
79
DAD1 B, Sig=280,16 Ref=360,20 (E:\ECLIPSE SELECTIVIY INVESTIGATION\ONECHANNEL00003.D)
0.2
88
0.3
53
0.6
95
0.9
44
1.2
13
1.6
12
3.8
73
5 um, UXE01033, NEP0652003
min 0 1 2 3 4 5 6
mAU
-5
0
5
10
15
20
25
30
DAD1 B, Sig=280,16 Ref=360,20 (E:\ECLIPSE SELECTIVIY INVESTIGATION\ONECHANNEL00005.D)
0.2
90
0.3
51
0.4
17
0.7
14
0.8
48
0.9
71
1.2
41
1.6
68
4.0
73
DAD1 B, Sig=280,16 Ref=360,20 (E:\ECLIPSE SELECTIVIY INVESTIGATION\ONECHANNEL00006.D)
0.2
87
0.3
48
0.7
13
0.9
70
1.2
41
1.6
66
4.0
72
1.8 um, UXG03882, B08021
one channel tr k alpha
uracil 0.29 0.00
aniline 0.69 1.38 #DIV/0!
o-toluidine 0.94 2.24 1.63
4-chloroaniline 1.21 3.17 1.42
2-naphthylamine 1.61 4.55 1.43
3,3'-dichlorobenzidine 3.87 12.34 2.71
one channel tr k alpha
uracil 0.29 0.00
aniline 0.71 1.45 #DIV/0!
o-toluidine 0.97 2.34 1.62
4-chloroaniline 1.24 3.28 1.40
2-naphthylamine 1.67 4.76 1.45
3,3'-dichlorobenzidine 4.07 13.03 2.74
m.p.: A: water, acetonitrile (60:40 v/v)
Flow : 1.5 mL/min
Temp: 25 C
Detection: UV 280nm,16, ref=360,20
Flow cell: 6mm, 5uL
Data rate: 0.2s
Inj. Vol 2 uL
46 bar
246 bar
52
Repeat experiment of two channel isocratic mobile phase confirms different selectivity probably due to wrong composition of mobile phase being delivered
John W. Henderson JrGroup/Presentation
Title
Agilent Restricted Month ##, 200X
min 0 1 2 3 4 5 6
mAU
-5
0
5
10
15
20
DAD1 B, Sig=280,16 Ref=360,20 (E:\ECLIPSE SELECTIVIY INVESTIGATION\TWOCHANNEL00005.D) DAD1 B, Sig=280,16 Ref=360,20 (E:\ECLIPSE SELECTIVIY INVESTIGATION\TWOCHANNEL00006.D)
min 0 1 2 3 4 5 6
mAU
-5
0
5
10
15
20
25
DAD1 B, Sig=280,16 Ref=360,20 (E:\ECLIPSE SELECTIVIY INVESTIGATION\TWOCHANNEL00002.D)
0.3
00
0.3
68
0
.38
7
0.7
81
1.0
75
1.4
01
1.9
18
4.9
91
DAD1 B, Sig=280,16 Ref=360,20 (E:\ECLIPSE SELECTIVIY INVESTIGATION\TWOCHANNEL00003.D)
0.3
00
0.3
67
0
.38
7
0.7
82
1.0
74
1.4
00
1.9
16
4.9
85
5 um, UXE01033, NEP0652003
1.8 um, UXG03882, B08021
45 bar
238bar
m.p.: A: water, B: acetonitrile (60:40 A:B)
Flow : 1.5 mL/min
Temp: 25 C
Detection: UV 280nm,16, ref=360,20
Flow cell: 6mm, 5uL
Data rate: 0.2s
Inj. Vol 2 uL
two channel (2nd exp) tr k alpha
uracil 0.29 0.00
aniline 0.72 1.48 #DIV/0!
o-toluidine 0.98 2.38 1.60
4-chloroaniline 1.25 3.31 1.39
2-naphthylamine 1.67 4.76 1.44
3,3'-dichlorobenzidine 4.03 12.90 2.71
two channel (2nd exp) tr k alpha
uracil 0.3 0.00
aniline 0.78 1.60 #DIV/0!
o-toluidine 1.07 2.57 1.60
4-chloroaniline 1.4 3.67 1.43
2-naphthylamine 1.92 5.40 1.47
3,3'-dichlorobenzidine 4.98 15.60 2.89
Typical Case History: Lack of Efficiency on Poroshell 300
53
Agilent Publication Customer Results
Looked Bad – What Did We Do?
1. Customer needed to understand that the column used – 2.1
x 75mm, Poroshell 300 column---is a low volume column, and
therefore, low column volume guidelines should be followed
2. What about the instrument and sample ?
3. Turns out that instrument was quaternary pump 1100, not set
up appropriately for low volume use (difficult to optimize
completely)
4. Customer provided with details on how to achieve a better
low volume set-up, based on 1100-RRHT Kit documents
5. Customer now achieving acceptable results
54
Conclusions
Page 55
• High pressure (prevention better than the cure)
• Undesirable peak shape
• Changes in retention/selectivity
Often these problems are not associated with the column and
may be caused by instrument and chemistry issues.
•pH of mobile Phase
•Instrument Connections
•Detector Settings
•Metal Contamination
Start With the Correct Questions
•Find the Answers
•The Answers will Lead to Solutions
HPLC column problems are evident as
APPENDIX
Page 56
Important Buffer Systems
September 28, 2014
CI0126C
57
Buffer Selection
Buffer Selection
Buffer pKa pH Range UV Cutoff (A > 0.5)
Trifluoroacetic acid <<2 (0.5) 1.5-2.5 210 nm (0.1%)
KH2PO4/phosphoric acid 2.12 1.1-3.1 <200 nm (0.1%)
tri-K-Citrate/hydrochloric acid 1 3.06 2.1-4.1 230 nm (10 mM)
Potassium formate/formic acid 3.8 2.8-4.8 210 nm (10 mM)
tri-K-Citrate /hydrochloric acid 2 4.7 3.7-5.7 230 nm (10 mM)
Potassium acetate/acetic acid 4.8 3.8-5.8 210 nm (10 mM)
tri-K-Citrate /hydrochloric acid 3 5.4 4.4-6.4 230 nm (10 mM)
Ammonium formate 3.8
9.2
2.8-4.8
8.2-10.2 (50 mM)
Bis-tris propane·HCl/Bis-tris propane 6.8 5.8-7.8 215 nm (10 mM)
Ammonium acetate 4.8
9.2
3.8-5.8
8.2-10.2 (50 mM)
KH2PO4/K2HPO4 7.21 6.2-8.2 <200 nm (0.1%)
Tris·HCl/Tris 8.3 7.3-9.3 205 nm (10 mM)
Bis-tris propane·HCl/Bis-tris propane 9.0 8.0-10.0 225 nm (10 mM)
Ammonium hydroxide/ammonia 9.2 8.2-10.2 200 nm (10 mM)
Borate (H3BO3/Na2B4O7·10 H2O) 9.24 8.2-10.2
Glycine·HCl/glycine 9.8 8.8-10.8
1-methylpiperidine·HCl/1-methylpiperidine 10.1 9.1-11.1 215 nm (10 mM)
Diethylamine·HCl/diethylamine 10.5 9.5-11.5
Triethylamine·HCl/triethylamine 11.0 10.0-12.0 <200 nm (10 mM)
Pyrollidine·HCl/pyrollidine 11.3 10.3-12.3
Adapted from Practical HPLC Method Development, 2nd Edition, Snyder, L.R., Kirkland, J.J. and
Glajch, J.L., page 299.
Separation Ruggedness Buffer Preparation
1. Dissolve salt in organic-free water in 1- or 2-L beaker. Use appropriate volume to leave room for pH
adjustment solution. Equilibrate solution to room temperature for maximum accuracy.
2. Calibrate pH meter. Use 2-level calibration and bracket desired pH. Use appropriate audit solution to
monitor statistical control (for example, potassium hydrogen tartrate, saturated solution, pH = 3.56).
3. Adjust salt solution to desired pH. Minimize amount of time electrode spends in buffer solution
(contamination). Avoid overshoot and readjustment (ionic strength differences can arise).
4. Transfer pH-adjusted buffer solution quantitatively to volumetric flask, dilute to volume, and mix.
5. Filter through 0.45 µm filter. Discard first 50 – 100 mL filtrate. Rinse solvent reservoir with small
volume of filtrate and discard. Fill reservoir with remaining filtrate or prepare premix with organic
modifier.
– Agilent Solvent Filtration Kit, 250-mL reservoir, 1000-mL flask, p/n 3150-0577
– Nylon filter membranes, 47 mm, 0.45 µm pore size, p/n 9301-0895 (not for proteins!)
September 28, 2014
CI0126C
58
Using Buffers Successfully Initial Column and System Equilibration
In an appropriate vessel, test highest % organic/buffer ratio to verify
that buffer will not precipitate. With stirring, add organic to buffer
first, not vice versa.
Equilibrate column with, in order:
• 100% organic modifier (if brand new)
• mobile phase minus buffer
• buffered mobile phase containing highest % organic modifier (gradient high end)
• buffered mobile phase containing lowest % organic modifier (gradient low end).
Inject standard or sample several times until RTs stable, or for
gradient methods, precede former with 1 or 2 blank gradients.
September 28, 2014
CI0126C
59
Using Buffers Successfully Shutdown State and Instrument Flushing
Next day use—using same buffers
• Pump mobile phase very slowly (for example, 0.01 – 0.1mL/min). When flushing column or for longer term column storage • Flush with 20/80 organic/water, then 80/20 organic/water or 100%
organic.
Instrument flushing
• Replace column with capillary tubing. Leave disconnected from detector.
• Flush pumps with water, then connect capillary tubing to detector. • Inject water 2-3 times at maximum injection volume setting. • Flush all pumps with 100% organic for long term storage.
September 28, 2014
CI0126C
60
Importance of pH and Buffers A Practical Example
•Why the Sample Dictates Use
•What Happens When Buffer Used Effectively
•What Happens When Buffer Ignored or Used Improperly
Page 61
Importance of pH and Buffers - A Practical Example Optimized Isocratic Conditions for Cardiac Drugs
Page 62
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time (min)
5
4
32
1
0 1 2 3 4 5 6Time (min)
5
4
32
1Column: StableBond SB-C18, 4.6 x 150 mm, 5 mm Mobile Phase: 45% 25 mM NaH2PO4, pH 3.0 55% MeOH Flow Rate: 2.0 mL/min.
Temperature: 35°C
Detection: UV 254 nm
Sample: Cardiac Drugs 1. Diltiazem 2. Dipyridamole 3. Nifedipine 4. Lidoflazine
5. Flunarizine
I Don’t Have Time to Make Buffers or Adjust pH …
Page 63
0 5 10 15 20 25
Time (min)
Column: StableBond SB-C18 4.6 x 150 mm, 5 mm
Mobile Phase: A: 20% H2O B: 80% MeOH
Flow Rate: 1.0 mL/min.
Temperature: 35°C
UV Detection: 254 nm
Sample: Cardiac Drugs
• Buffers are critical to good retention and peak shape in many separations.
Even at very high % MeOH Most Components
Strongly Retained with Poor peak Shape Due to
IEX at Surface
What If You Work Outside the Buffer Range?
Page 64
0 5 10 15 20 25
5
1
2
34
Time (min)
Columns: StableBond SB-C18
4.6 x 150 mm, 5 mm
Mobile Phase:A: 30% 25 mM NaH2PO4, pH 4.8 unbuffered B: 70% MeOH Flow Rate: 1.0 mL/min.
Temperature: 35°C
UV Detection: 254 nm
Sample: Cardiac Drugs 1. Diltiazem 2. Dipyridamole 3. Nifedipine 4. Lidoflazine 5. Flunarizine
Unsuitable Peak Shape
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