©2014 Waters Corporation 1€¦ · ©2014 Waters Corporation 10 Column Volume in 4.6 x 50 mL =0.8 mL (800 µL)* 0.625 mg injected in 5 µL % of column volume: 0.625 2 Minutes 4 6

©2014 Waters Corporation 1


Troubleshooting Common Chromatography Problems

Richard DawField Technical Support Specialist


OutlineOutline

Diagnostic Flow Chart -Peak Problems-Retention Time Shift-Pressure Problems-Resolution Loss-Sensitivity-Ghost Peaks

Benchmarking using QCRM

Practice


Peak ProblemPeak Problem

Tailing Peak

Fronting Peak

What do the peaks look like?

Broad PeakSplitting Peak


Peak ProblemPeak Problem

Are All Peaks Affected?

Yes – Physical No - Chemical


Fronting PeakFronting Peak

Could be Caused by: Chemical•Mass Overload•Equilibration

Fronting Peak

Could be Caused by: Physical•Connections•Column Void•Clogged Frit•Volume Overload


ConnectionsConnections

Bad Connections can cause voids in the flow

path. Voids are bad because they will cause variable concentrations

across a band.


VoidingVoiding

Voided ColumnNormal Column

All Peaks Distorted


Clogged FritClogged Frit

As you can see from these two frits, bands will become distorted passing through the clogged frit.

Unblocked

Blocked

Unblocked

Remember Good Mobile Phase and Sample Preparation!


Column Volume in 4.6 x 50 mL =0.8 mL (800 µL)*

0.625 mg injected in 5 µL

% of column volume: 0.625

2 4Minutes 6

Ab

sorb

ance

0.625 mg injected in 500 µL

% of column volume: 62.5

2 4Minutes 6

Ab

sorb

ance

Volume Overload

*Guideline for Injection Volume: < 5% of column volume

• Wider peaks first observed at low retention

• Peak position shifts to higher retention in proportion to the injection volume


Encountered when mass injected onto column exceeds a certain limit—note earlier lift-off point

• Analytical load of 6µg yields efficient peak shape

• Preparative load of 25mg generates mass overload peak shape

Note that the back of the peaks of the analytical and prep loads are at the same retention (———-)

6.25 µg injected

2 Minutes 4 6

Ab

sorb

ance

Ab

sorb

ance

25 mg injected

2 4Minutes 6

Mass Overload-Concentration


Equilibration/WettingEquilibration/Wetting

In order for chromatography to take place, you need to have the right mobile phase within the pore. The pore pictured has elution strength solvent within it.

This pore contains a low elution strength mobile phase. Analytes can now be loaded into the pores and will retain.


Tailing PeakTailing Peak

Tailing Peak

Could be Caused by: Physical•Connections•Column Void•Clogged Frit•Volume Overload

Could be Caused by: Chemical•Mass Overload•Equilibration•Secondary Interactions•Ligand Loss


Minutes0 5 10 15 20 25

Secondary InteractionsSecondary Interactions

Acids and Neutrals have good peak shape --Basic Analyte “Tails” - Chemistry Problem - Cation Exchange


Secondary Interactions

Time (min) 50

Conventional C18

Modern C18

Neutral

Base

Neutral

Base

0

pH 7

Differences in Silanol Activity of Column Brands (Cation Exchange)

-End Capping? Tri-functionally Bonded? Bridged Carbon Chains?

High Silanol Activity

Low Silanol Activity


Hydrophobic Interaction with Bonded Phase

Hydrophobic Interaction andIon exchange Interaction

with Charged SitesO-SiO-SiO-O-SiO-SiO-O-SiO -O-SiO-SiO-SiO-O--Si

O-Si

O-SiO-SiOHO-SiO-SiOHO-SiOHO-SiO-SiO-SiOH

O-SiO-Si

(CH3)2HN+(CH3)2

HN+Mobile

Phase pH < 3

Si - OH

-Mobile Phase pH 6-8Si – O

BaseVery Early Elution—IonizedNo Cation Exchange

Base

Tailing and excessive retention in HPLC

Same “Conventional” C8 Column used in both experiments

Cation Exchange

Secondary Interactions


IonIon--Pairing ReagentsPairing Reagents

Additives that pair with charges on a molecule to dampen the negative effects of those charges on reversed phase retention are

called Ion-Pairing Reagents.

Ion Pairing Agent: Pairs With:TriEthyl Amine (TEA) Negative Charges

TriFluoroacetic Acid (TFA) Positive ChargesHexylamine Negative Charges

Octane Sulfonic Acid Positive Charges

These additives require long equilibration times to ensure consistent concentrations throughout the pores of a chromatographic material.


Ligand Loss: Hydrolysis of a Bonded Phase Material

Note: Sometimes called “Column Bleed”

+ HCl

+

+

Low pH (hydrolysis of ligand)

SiC

CC

CC

CC

CH3

CH3

H3C

Cl

OHSi

O

O

O

SiC

CC

CC

CC

CH3

CH3

H3C

OSi

O

O

O

SiC

CC

CC

CC

CH3

CH3

H3C

HOOH

SiO

O

O

Note: as hydrolysis of ligand continues, you will obtain less retentivity and potentially poor peak shape for bases.Cannot “regenerate”

BONDING

Mobile phase


Modern Stationary PhasesModern Stationary Phases--

Anal. Chem. 2003, 75, 6781-6788

U.S. Patent No. 6,686,035 B2 Bridged Ethanes within a silica matrix


Broad PeakBroad Peak

Could be Caused by: Chemical•Mass Overload•Secondary interactions•Equilibration•Sample Solvent•Temperature

Broad Peak

Could be Caused by: Physical•Connections•Column Void•Clogged Frit•Volume Overload•System Volume•Data Acquisition


System VolumeSystem Volume

Initial Band Band over Time


Data Acquisition RateData Acquisition Rate

10pt/1sec3pt/1sec

If the data rate is increased, more fine detail can be seen.


Sample SolventSample Solvent

Column: SymmetryShield™ RP18, 3.5 µm, 3.9 x 150 mm

Guard Column: Sentry™ Guard Column SymmetryShield RP18, 5µmTemperature: 30°CMobile Phase: 0.1% TFA:Methanol

(60:40)Detection: UV at 210 nmFlow Rate: 1 mL/minInj. Volume: 30 µL

Sample Identification1.EDDP2. Diphenhydramine (IS)3. Methadone

5 1510 Min.

Sample in Water

0.04AU

1

23

Sample in 80% MeOH, 2% HAc

15

12

30.02AU

Need to focus sample on head of the column!!

This is hindered by using a poor diluent choice


Splitting PeakSplitting Peak

Could be Caused by: Physical•Connections•Column Void•Clogged Frit

Splitting Peak

Could be Caused by: Chemical•Impurity/Coelution


Retention Time Shift Retention Time Shift

Loss of Retention

Gain of Retention

Which way are they shifting?

Peaks Move Both Ways


Retention LossRetention Loss

Could be Caused by:•De-Wetting•Ligand Loss•pH effects•Equilibration•Temperature•Mobile Phase Composition•Mobile Phase Delivery

Loss of Retention


De-wetting/ “Hydrophobic Collapse”

Change in pressure ejects aqueous mobile phase from the pore


1,500 psi

1,500 psi

Mobile phase: Aqueous 0.1% Acetic Acid

Note: Column is not broken—just stopped working—Rewet to restore performance

Minutes0 2 4 6 8 10

Initial(column was wetted first with organic)

Amoxicillin

Vo: No retentivity for analyte

Total Loss of Retention

De-wetting/ “Hydrophobic Collapse”

After Flow Stoppage Re-Start in Mobile Phase (Pores de-wet 100%)

x


pH effects pH effects -- Retention mapRetention map

pH Range for Silica ParticlespH

0

5

10

15

20

25

30

35

40

0 2 4 6 8 10 12

Rete

ntio

n Fa

ctor

(k)

Acid

Base

Neutral

Maximum acidic compound retention range

Maximum basic compound retention range

pH Range for Hybrid Particles


pH EffectspH Effects

You can see that by changing the pH you can have peaks change retention times. This is due to a change in their

charged states, and happens to acids and bases.

Base Acid

Neutral

Acid Base Acid Base

NeutralNeutral

pH = 2 pH = 6 pH = 10


TemperatureTemperature

The Rate of Mass Transfer in and out of the pores of the stationary phase increases with increasing temperature.

25oC

90oC


Mobile Phase CompositionMobile Phase Composition

If these are mixed such that the bottom liquid is added first, which way will give a 60/40 mixture?

400mL MeOH

600mL Water

or

40% MeOH

60% Water

600mL Water

400mL MeOH


Mobile Phase CompositionMobile Phase Composition

If the liquids are measured out separately and then mixed the percentages will be more accurate. In practice

it is important that regardless how this is done, it is always done the same to avoid errors.

60:40 MeOH/water = 960mL

400mL MeOH

600mL Water

+

40% MeOH

60% Water


Mobile Phase DeliveryMobile Phase Delivery

Pump

If the pump is not delivering the appropriate amount of solvent over time, you will not get reproducible retention times. This can happen if the pump is improperly serviced, or not serviced at all.


Retention GainRetention Gain

Gain of Retention Could be Caused by:

•De-Wetting•Ligand Loss•Equilibration•pH effects•Temperature•Mobile Phase Composition•Mobile Phase Delivery


Both WaysBoth Ways

Could be Caused by:•Temperature•Mobile Phase Delivery•Mobile Phase Composition•Mixing

Peaks Move Both Ways


Gradient Formation/MixingGradient Formation/Mixing

In order for a gradient to be effective, small changes in the gradient need to be consistently presented to the head of the column.

How well are you mixing different solvents together?

VS


Pressure ProblemsPressure Problems

Pressure Problem

What happens?

Low Pressure

High Pressure

Fluctuating Pressure


Pressure Problem Pressure Problem -- HighHigh

High Pressure

•Clogged Frit, column, check valve, tubing…etc…

•Pump Problem•Viscosity•Flow Rate•Temperature


Pressure Problem Pressure Problem -- FluctuatingFluctuating

Fluctuating Pressure

•Pump Problem•Check Valve Issue


Pressure Problem Pressure Problem -- LowLow

Low Pressure

•Leak

•Flow Rate•Temperature


Resolution LossResolution Loss

Could be Caused by: Chemical •Temperature•Mobile Phase Composition•pH effects

Could be Caused by: Mechanical •Changed system volume•Mobile Phase Delivery•Equilibration


SensitivitySensitivity

All Peaks decreased?

Yes – Chromatography System

No – Sample/Sample Prep


Sensitivity Sensitivity –– Chromatography System Chromatography System

Possible Causes:•Old Lamp•Dirty Flow Cell•Wrong Wavelength•Dirty Cone•MS Tuning•Autosampler


Sensitivity Sensitivity –– Sample/Sample PrepSample/Sample Prep

Causes:•Matrix Effects•Adsorption to Vial/Filter•Sample Prep•Solubility


Ghost PeakGhost Peak

Unknown Peak

Blank Injection

Unknown Peak

Blank Injection



Unknown Peak

Blank Injection

0 µL Injection

Unknown Peak

Blank Injection

0 µL Injection



Unknown Peak

Blank Injection

0 µL Injection Contamination of Sample during

prep?



Unknown Peak

0 µL Injection

Blank Injection



Unknown Peak

0 µL Injection

Blank Injection

Contamination of Chromatography System/Detector


OutlineOutline



Practice


System Standards AvailableSystem Standards Available

System Performance

Kits andStandards

ApplicationSpecific

Standards

QualityControl

Reference Materials


Quality Control Reference Material Quality Control Reference Material OverviewOverview

Requirements for a QCRM Material– Reproducible Lot to Lot– Accurate– Appropriate to analysis type (LCMS, LC-UV)

The quality of the reference material is critical to the evaluation of analytical data and instrument performance

QCRM’s can be used to benchmark a system when it is known to be in good working order

The most powerful way to use QCRM’s is by analysing them routinely on the analytical system and control charting the data. The current and historical data will then allow for identification of areas of excess variation which may warrant concern and action


New Catalog for 2015/2016New Catalog for 2015/2016


New Catalog for 2015/2016New Catalog for 2015/2016


OutlineOutline



Practice


Retention Time ShiftRetention Time Shift

Day 2Day 1 Conditions:

BEH C18, 186002350

• Col Dimension : 2.1x50mm• Temperature : 35C• Mobile Phase A : 100mM Na2HPO4• Mobile Phase B : Methanol• Flow Rate : 0.45mL/min.• Detection : PDA



Day 2Day 1

Mobile Phase Delivery – Check Pumps!

Conditions:

BEH C18, 186002350

• Col Dimension : 2.1x50mm• Temperature : 35C• Mobile Phase A : 100mM Na2HPO4• Mobile Phase B : Methanol• Flow Rate : 0.45mL/min.• Detection : PDA

-Temperature-pH effects


Split PeaksSplit Peaks

Original Peak Bad Peak



Original Peak Bad Peak Chromatography

Physical?


A new column was tried and the poor peak shape remained.


Original Peak Bad Peak Chromatography

Physical?


A new column was tried and the poor peak shape remained.


ChromatographyOriginal Peak Bad Peak

Physical?



At Start1 Day Conditions:

SymmetryShield RP18; 186000174

• Col Dimension : 2.1x150mm• Temperature : 35C• Mobile Phase A : 100mM H3PO4• Mobile Phase B : Methanol• Flow Rate : 0.45mL/min.• Detection : Mass Spec ESI+



At Start1 Day

Ligand Hydrolysis-Dewetting

-Mobile Phase composition, mobile phase delivery, pH

Conditions:

SymmetryShield RP18; 186000174

• Col Dimension : 2.1x150mm• Temperature : 35C• Mobile Phase A : 100mM H3PO4• Mobile Phase B : Methanol• Flow Rate : 0.45mL/min.• Detection : Mass Spec ESI+



Does not meet Spec



Does not meet Spec

Not All Peaks TailStrong Base



Does not meet Spec


Conditions: •Symmetry C18 3.5um 2.1x50mm•Temperature 25C•Flow Rate : 0.3mL/min.•Mobile Phase A : Water•Mobile Phase B : ACN



Does not meet Spec


Conditions: •Symmetry C18 3.5um 2.1x50mm•Temperature 25C•Flow Rate : 0.3mL/min.•Mobile Phase A : Water•Mobile Phase B : ACN

-Add some buffer!



Why is this peak fronting?




Why are all the peaks fronting?



Conditions:•Column : Atlantis T3 1x50mm 3µm•Injection : 20µL @ 5mg/mL Std.•Flow Rate : 0.1mL/min.•Mobile Phase A : 20mM Phosphate pH 3•Mobile Phase B : ACN





Conditions:•Column : Atlantis T3 1x50mm 3µm•Injection : 20µL @ 5mg/mL Std.•Flow Rate : 0.1mL/min.•Mobile Phase A : 20mM Phosphate pH 3•Mobile Phase B : ACN

Overload!

Too much Injection volume!




QUESTIONS???

Zach KoontzField Chemistry [email protected]

Richard Daw and Steven ParkerField Technical Support [email protected][email protected]


Documents

©2014 Waters Corporation 1€¦ · ©2014 Waters Corporation 10 Column Volume in 4.6 x 50 mL =0.8 mL (800 µL)* 0.625 mg injected in 5 µL % of column volume: 0.625 2 Minutes 4 6