HPLC TROUBLESHOOTING

Avoid Problems with Right Choice of Solvent

Abdullah Dawamuz Zikri

Field Application Specialist for LC and IA, PT MCLS

Webinar PPLPI BKAL - 9 December 2021

The life science business of Merck operates

as MilliporeSigma in the U.S. and Canada.
 OUR
Legacy 1951
ALDRICH
1975
SIGMA-ALDRICH

1935 2015
SIGMA
MERCK
CHEMICAL
COMPANY

1954
2010
MILLIPORE

1668
MERCK
MILLIPORE
ANGEL PHARMACY founded
in Darmstadt, Germany by Friedrich Jacob Merck
2
What is “Troubleshooting”?

„Trouble“ is when e.g. the System Suitability Test cannot be met

• Steps for Troubleshooting are:

• identify the problem
• finding the cause of the problem
• optimizing the system to avoid the problem repeating itself

3 Title of Presentation | DD.MM.YYYY

What Can Cause Trouble?

4 Title of Presentation | DD.MM.YYYY

RP - Mobile Phase
Mobile Phase
Polar, water-miscible, organic solvents
mixed with buffer or water.
Buffers and Acids
Amonium salts of acetate and formate,acetic
acid, formic acid are recommended with most
detectors., including MS.
Inorganic salts like phosphate are superior at
low UV wavelength analysis (highest
transparency).
About 5-20 mM buffer (up to ~250 mM)
Attention: if using high acetonitile content in
the mobile phase inorganic salts have low
solubility.
5 Chromatographic Troubleshooting May 2013
Mixing
Mixing of an organic solvent with aqueous buffers
often result in a temperature change of the solution.
Allow the mobile phase to return to ambient
temperature before use, especially if your
chromatographic system lacks thermostatic control
Filtering
Filter mobile phases before use, preferably through a
0.45 µm filter with or 0.2µm if using UHPLC. Prolongs
column lifetime and protects the pump from damage
Degassing
Mobile phases should be degassed daily, or use an
online degassing device, and purge a stream of inert
gas, i.e. helium, through the eluent (prevents pump
related interferences lowers detector noise)
Why using a special HPLC grade?

 Solvent impurities can result in ghost peaks especially using gradients

→ HPLC peak resolution and quantitative results are effected by solvents with lower quality
Compounds producing artefacts: Additives, Peroxides, Phtalates

NOTE: Use HPLC quality only! (Do not use old or unfiltered buffers!)

Solvents for Headspace GC and LC-MS

Solvent and reagent purity

Solvent/additive quality
 purest solvent and reagent (acids, bases, buffers) quality necessary
(e.g. LiChrosolv® UHPLC-MS, LiChrosolv® hypergrade for LC-MS, EMSURE®, Suprapur® and
Ultrapur solvents and reagents)
Reagent impurities and reagent contamination during handling might
 increase background noise and hence decrease sensitivity (higher LOD)
 increase complexity
 cause “ghost peaks”
 baseline drifts
 cause signal suppression (MS)
 cause adduct formation (MS)

Troubleshooting for improved LC performance

Solvent and reagent purity
Quality - Organic solvents

Organic solvents for HPLC, e.g. LiChrosolv® acetonitrile and methanol, are available in four
qualities:
 isocratic grade for liquid chromatography (for LC-UV isocratic runs)
 gradient grade for liquid chromatography (for LC-UV gradient runs)
 hypergrade for LC-MS (for LC-MS gradient runs)
 LiChrosolv® UHPLC-MS (for UHPLC-MS gradient runs)
4.2E4 399.5

isocratic grade
4.2E4 4 gradient grade direct injection of two
443.5
broad peak range and broad peak range but different ACN qualities
high signal intensity low signal intensity
(„grades“) into the MS
MS intensity via syringe pump (flow
3
MS intensity

355.4

487.5
injection analysis, flow
2
2 12 mL/h)

531.5
413.4
lower signal intensity
→ better quality
1 443.5
1

305.3259.2 487.5
135.1
135.1
575.5 531.5
2172.161.2

217.1

0
0
0 200 400 600 800 1000 1200 1400 1600 1800 0 200 400 600 800 1000 1200 1400 1600 1800

m/z m/z

Troubleshooting for improved LC performance

Solvent Quality Assurance – Basic Absorbtion

Basic absoption is one of the parameter giving information

about the total purity of the solvents

High concentration of impurities influence the baseline and increase

the basic absoption unit. What means at the end...

▪ Loss of detector sensitivity – Less concentrated compounds will not be

detected - The results are not reliable automation of analysis
▪ Drift of baseline in the used gradient elution - the baseline is not stable –
no baseline separation – The results are not reliable

Solvents for Headspace GC and LC-MS

HPLC UV detector

Gradient Methanol - Water gradient UV 215 nm

The wavelength of operation

can influence the organic
solvent type used.

Methanol is satisfactory for

gradient work at 254 nm, but
0 min 40 min
at 215 nm it creates an Acetonitrile - Water gradient UV 200 nm
unacceptable baseline drift.

Acetonitrile, which has much

lower adsorbance at low
wavelength is suitable down to
200 nm.

0 min 40 min

Solvents for Headspace GC and LC-MS

Risks using non-suitable Acetonitrile @210

Baseline drift due to trace

Risk due to enrichment process of
impurities
impurities

No reliable HPLC result

Loss in separation performance Solvent purity problems
Reduced column lifetime

Solvents for Headspace GC and LC-MS

LC-MS Method
Is it necessary to have a better grade of solvent and better column
purity for LC-MS?

Problem: With standard solvents and

HPLC columns, trace impurities cause
unwanted background signals in LC-MS
which reduce sensitivity and cause
complex spectra and low reproducibility,
compared to HPLC with standard UV
detectors.

LC-MS requires
improved
procedures,
compared to HPLC
with UV detectors

Solvents for Headspace GC and LC-MS

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Main problem: low ionization efficiency

HPLC separation of
methyl, ethyl and propyl
paraben

Spectrum Methyl
Ionization intensity is
paraben low, due to ion
suppression →
base peak intensity
< 4 x 102
Background mass signals
appear in the spectra,
making interpretation
more difficult
Solvents for Headspace GC and LC-MS
13
 Preparation of Mobile phase

Four different ways to prepare a

mobile phase of
40% Water
and
60% Methanol
with different chromatographic results

All approaches can be used

but you NEED to DOCUMENT the

PROCEDURE so the RESULTS
can be REPRODUCED

14 Chromatographic Troubleshooting May 2013

Prepare fresh mobile phases daily

Day 1 Day 2

Troubleshooting for improved LC performance

Solvent storage

Solvent storage (water and organic) in

 surface treated amber glass
 borosilicate glass

Bottle caps/adapters
 professional equipment only, directly mounted to original bottle
(see image)
 avoid decanting
 no homemade solvent tubing solutions
→ solvent purity maintained, low background noise

Troubleshooting for improved LC performance

Mobile phase and additive purity

Isocratic separations
 isocratic grade solvent quality
Gradient elution
 gradient grade solvent quality
 no contaminant accumulation on column
(“ghost peaks”, see figure)
Contaminations in solvents
 decrease in sensitivity
 Might accumulate on stationary phase
Column Chromolith FastGradient RP-18 endcapped 50-2
 elute as distinct peaks („ghost peaks“) in Mobile phase A: Acetonitrile + 0.1% formic acid, B:
water/acetonitrile 95/5 (v/v) + 0.1% formic acid
gradient runs Gradient 0 min 100% B, 3 min 5% B, 5 min 5% B
Flow rate 0.4 mL/min
Detection Pos. ESI-MS, TICs
Sample Plasticizers (in eluent B)

Troubleshooting for improved LC performance

Typical HPLC Problems

Retention Recovery
• Changing retention times*
• Poor sample recovery
• Decreasing retention times*
• Increasing retention times* Leaks
• Leak at column or fittings
Equilibration
• Leak at detector
• Slow column equilibration time
• Leak at injection valve
• Varying retention times
• Leak at pump
Peaks Selectivity
• Broad peaks*
• Differences in selectivity*
• Ghost peaks*
• Negative peaks* Baseline
• Peak doubling* • Disturbance at void time*
• Peak fronting* • Drifting baseline*
• Peak tailing* • Noise*
• Spikes Pressure
• No peaks • Decreasing pressure
• Peaks with shoulders, split • Fluctuating pressure
peaks* • High back pressure*

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Retention Time

01 • Cause :
Change in mobile phase composition. (Small changes
can lead to large changes in retention times.)

• Solution :
Check make-up of mobile phase. If mobile phase is
machine mixed using proportioning values, hand mix
and supply from one reservoir.

02 • Cause :
Sample solvent incompatible with mobile phase.

• Solution :
Adjust solvent. Whenever possible, inject samples in
mobile phase.

19 Title of Presentation | DD.MM.YYYY

Baseline

Baseline Drift

• Cause :
Nonhomogeneous mobile phase. (Drift usually to higher
absorbance, rather than cyclic pattern from temperature
fluctuation.)

• Solution :
Use HPLC grade solvents, high purity salts, and additives.
Degas mobile phase before use, sparge with helium during
use.

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Baseline

Baseline Noise

Cause and Solution

1. Air in mobile phase, detector cell, or pump. → Degas

mobile phase. Flush system to remove air from detector
cell or pump.

2. Incomplete mobile phase mixing → Mix mobile phase by

hand or use less viscous solvent.

3. Mobile phase contaminated, deteriorated, or prepared from

low quality materials. → use HPLC grade solvent

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Further Information?
Visit our websites

www.SigmaAldrich.com www.MerckMillipore.com

22 Safety in Laboratory
Further Information?
More information

23 Safety in Laboratory
Further Information?
Contact us

Abdullah Dawamuz Zikri

Field Application Specialist for LC & IA
Email : abdullah-dawamuz.zikri@merckgroup.com
Phone : 0811 9093 707

Utama Putra
Channel Sales Team for LC & IA
Email : Utama.Putra@merckgroup.com
Phone : 0811 1807 216

Fitriah
Sales Manager Channel Sales for LC & IA
Email : fitriah.fitriah@merckgroup.com
Phone : 0811 1807 019