Download The Importance of Water Quality in HPLC and LC-MS

The Importance of Water Quality
in HPLC and LC-MS
Dr. Estelle Riché
Senior Scientist
EMD Millipore
Guyancourt, FRANCE
The role of water in chromatography
Preparation of
samples and
standards
Preparation of
mobile phases
Chromatography
Washing, rinsing of
sample/reagent
containers
Blanks
Data
This water needs to be free of any contamination
that could compromise experimental results
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Water contaminants
Water is H2O and …
Ions
Organics
Particles
Bacteria
Gases
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Water for HPLC
 HPLC grade
 LC-MS grade
 ULC/MS grade
Purification
steps
Tap water
 Water purification system
Purified water for HPLC
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What ’s in your HPLC water?
Ions
Organics
Particles
Bacteria
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Examples
 “… HPLC solvents are glass distilled,
submicron filtered…”
 “… LC/MS Grade Water is specially purified
by a proprietary method and tested to ensure
lot-to-lot consistency with a low UV
absorbance to provide you with the most
sensitive detection across all wavelengths”
 “… LC/MS Water is 0.2-micron filtered,
packaged in solvent-rinsed amber glass
bottles and sealed under a nitrogen
atmosphere with Teflon TFE-lined
fluorocarbon caps for ultimate protection”
 “… LC-MS solvents… are designed to have
low contents (max. 100 ppb) of alkaline
impurities, such as calcium, magnesium,
potassium and sodium, which can form
clusters with the analyte
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HPLC
Grade
LC-MS
Grade
ULC-MS
Grade
Agenda
7
1
Water purification technologies
2
Monitoring water quality
3
Impact of water quality on HPLC
4
PPCPs in water
5
Tips for maintaining water quality
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Water Purification Technologies
8
Distillation
Benefits
 Removes a wide array of
contaminants (organics, ions, bacteria)
 Low/moderate capital cost
Limitations
 Some contaminant not fully removed
(organic carryover)
 High maintenance
 High operating cost
 Low product flow
 Water storage needed
 No control over quality of water
produced
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Basic principle of water purification
Tap water
Pretreatment
Polishing
Pure water
(Type II or III)
Ultrapure water
(Type I)
 Reverse osmosis
 Ion exchange resins
 Electrodeionization
 UV photo-oxidation
 Germicidal UV
 Activated carbon
The quality and reliability of the ultrapure water depend on
the quality of the pretreatment
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Reverse Osmosis
Benefits
 About 95% of contaminants removed: ions,
organics, bacteria, particulates & colloids,
pyrogens, viruses
 Low operating cost
 Minimum maintenance
Limitations
 RO membrane sensitivity to plugging,
fouling, piercing, scaling
 Initial rinsing of membrane required
 Some water rejected
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Ion Exchange Resins
-
+
+
Negatively charged
water contaminant
+
+
+
+
+
 Efficient ion removal
+
+ +
 Easy to use
Anion exchange resin
-
-
-
+
Benefits
Positively charged
water contaminant
 Low capital cost
Limitations
-
 No removal of other contaminants
-
 Potential contamination with bacteria
Cation exchange resin
 Multiple regenerations can result in water
contamination by organic and particulates
 Operating cost potentially high
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Electrodeionization (EDI)
Benefits
 Very efficient ion removal
 No particulate or organic contamination
 High water recovery
 No chemical regeneration
 Low maintenance
Limitations
 Good feed water quality required to
prevent plugging and fouling of ionexchange (RO feed water is ideal)
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Activated Carbon
Benefits
 Natural AC: removal of residual chlorine
 Synthetic AC: efficient removal of organics
(used in “Polishing”)
Limitations
 Weak effect on other contaminants
 Natural AC: may release ions and particles
 Bacteria may develop
 Efficiency depends on flow rate
Natural AC
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Synthetic AC
UV Photooxidation (185 / 254 nm)
 UV radiation causes the
formation of reactive
hydroxyl radicals (OH*)
Benefits
 Reduces organic contamination
 Limited energy use
 Easy to operate
 Radicals react with
organic compounds,
turning them into charged
species (can be removed
by ion-exchange resins)
Limitations
 Polishing technique only: may be
overwhelmed if organic concentration in
feed water is too high
 Limited effect on other contaminants
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Germicidal UV light
Benefits
 Inactivates bacteria
 Easy to operate
Limitations
 Does not reduce organic
contamination
Effect of 254 nm wavelength on DNA
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Screen Filtration
Benefits
 Remove all particles and microorganisms
greater than their pore size.
Limitations
 May clog when the surface is covered by
contaminants. Should be used as a last
purification step.
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Overview of Technologies & Contaminant Removal
Distill.
RO
DI
AC
UV
µFiltr.
Ions
Organics
Particles
Bacteria
Gases
100% removed
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100% present
Monitoring laboratory water quality
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Monitoring: Conductivity & Resistivity Measurements
 Electrical conductivity is a measure of a material's ability to conduct an electric current
 Water does not conduct electricity very well
 Water conductivity reflects the extent of ionic contamination of the water
H3O+(aq) +
2 H2O(l)
Conductivity
Conductivity
(Siemens/cm)
OH-(aq)
Kw = 10-14 = [H3O+] [OH-]
  F  ci zi i
Faraday
constant
Concentration of each
ionic species (eq/ml)
Mobility
(cm2 V-1 s-1)
Valence
Minimum theoretical conductivity is 0.055 µS/cm at 25 °C
Resistivity
R
1

Maximum theoretical resistivity is 18.2 M.cm at 25 °C
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Monitoring: TOC (total oxidizable carbon) Measurement
• Measure of all organic substances that can be oxidized
• Gives no indication on the concentration of specific molecules
• Expressed in parts per billion (ppb)
Sample
OXIDATION
• Combustion
• High temp. persulfate oxid.
• UV Oxidation
• UV / Persulfate Oxidation
• UV / TiO2
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DETECTION
• Direct measurement of
conductivity (non-selective)
• Selective conductometric
(permeation through a mb)
• Non Dispersive Infra Red detector
The Potential Impact of Water Quality
on HPLC
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Effects of water contaminants on RP HPLC
Contaminants
Effects
Organics
 Noisy or drifting baselines
 Ghost peaks
 Extensive contamination could result to
shifting retention times and distorted peak
shapes
Ions
 Some ions absorb in the UV range (e.g.,
nitrites and nitrates)
 Metal ions can form adduct peaks (if MS
detection)
Particles


Bacteria
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Damage HPLC pump and injector
Increase system back-pressure
 Behave as particles (back-pressure)
 By-products include organics and ions
Effect of organic contamination on HPLC baselines
60 mL of water were
pre-concentrated before
elution with an
acetonitrile/water gradient
20 ppb
13 ppb
 Increasing TOC levels
result in more
extraneous peaks
9 ppb
5 ppb
2 ppb
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The misunderstood laboratory
solvent : reagent water for HPLC.
Mabic S., Regnault C., Krol J.
LCGC North America 2005, 23
(1), 74-82.
Evaluation of TOC levels of HPLC-grade waters
TOC levels measured off-line
Water Source
TOC (ppb)
Bottled Water A
100
Bottled Water B
87.0
Bottled Water C
777
Bottled Water D
16.5
Bottled Water E
32.4
Bottled Water F
25.5
Fresh ultrapure water
7.0*
* 5 ppb online
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Gradient elution of ULC/MS water and fresh
ultrapure water
UHPLC chromatogram (210 nm) of
ULC/MS grade water
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UHPLC chromatogram (210 nm) of
fresh Milli-Q water
Background mass spectra of ULC/MS grade water
and fresh ultrapure water
ULC/MS grade
6
6 10
6
5 10
6
4 10
6
114
142
MS2 ES+
6.67E6
CPS
182
3 10
6
6 10
6
5 10
6
4 10
6
3 10
6
CPS
7 10
7 10
6
Milli-Q water
MS2 ES+
1.21E6
155
6
2 10
6
2 10
1 10
6
1 10
6
99
0
0
100
200
300
400
500
600
m/z
ES+ mass spectrum of 98% Biosolve ULC/MS water, 2% Acetonitrile
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100
200
300
400
500
600
m/z
ES+ mass spectrum of 98% Mill-Q Integral water, 2% Acetonitrile
Long term impact of water quality
Goal of the experiment:
to illustrate the benefits of using ultrapure water in a routine HPLC separation
Repeated injection of
drug mixture
Acetonitrile
HPLC
Pump
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Detector
Water
Analytical
Column
1.
HPLC-grade bottled water
Waters SymmetryShield™
RP18, 4.6 x 150 mm, 3.5 µm
2.
Ultrapure water from a Milli-Q
system (TOC  5 ppb)
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Separation of a drug mixture – 254 nm detection
0.005
1
0.004
0.003
4
2
7
A
254
0.002
inj50
inj290
inj530
inj770
inj1010
inj1310
5
3
 Appearance of
extraneous peak
0.001
0
1 – acetaminophen
2 – acetazolamide
3 – phenobarbital
4 – carbamazepine
5 – phenytoin
6 – secobarbital
7 - nabumetone
-0.001
-0.002
-0.003
4
8
12
16
20
t , min
R
Mobile phase A: HPLC-grade bottled water
Mobile phase B: HPLC-grade acetonitrile
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 Considerable
baseline drift
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Separation of a drug mixture – 254 nm detection
0.005
1
inj50
inj290
inj530
inj770
inj1010
inj1310
0.004
0.003
2
4
A
254
0.002
0.001
3
7
5
1 – acetaminophen
2 – acetazolamide
3 – phenobarbital
4 – carbamazepine
5 – phenytoin
6 – secobarbital
7 - nabumetone
0
-0.001
-0.002
-0.003
4
8
12
16
20
Time, min
Mobile phase A: Ultrapure water
Mobile phase B: HPLC-grade acetonitrile
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An “emerging” issue: Pharmaceuticals and Personal
Care Products (PPCPs) in water
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http://nicolasrapp.com/pharmawater_update/
Levels of PPCPs in drinking water
Ten most frequently detected PPCPs in drinking water samples analyzed by
Underwriters Laboratories (UL)*
http://www.waterworld.com/index/display/article-display/articles/waterworld/drinking-water/water-quality/2011/01/managing-micro-pollutants.html
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Concentrations of PPCPs in HPLC grade water
Compound
Concentration (ng/L)
Lincomycin
0.37
Trimethoprim
0.35
Carbamazepine
0.21
Caffeine
11.24
Data courtesy of Dr Chuan Wang and Dr Yinfa Ma. Department of Chemistry,
Missouri University of Science and Technology, Rolla, MO
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Concentrations of PPCPs in “LC/MS Water”*
Purification Steps
Potable water
Pre-filtration
Softening
Compound
Conc. (ng/L)
Activated carbon
UV sterilization
Lincomycin
0.44
Reverse osmosis
Trimethoprim
0.37
Carbamazepine
0.17
Caffeine
10.08
Storage tank
recirculation
Deionization
(mixed bed
deionizers)
Ultraviolet TOC
reduction
Final filtration
(0.1 µm)
LC/MS Water
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Ionic contamination –
Metal concentrations in different types of water
Concentration, µg/L
Element
ULC/MS Grade#
LC-MS Grade#
Ultrapure*
Na
< 100
< 10
< 0.003
K
< 50
<100
< 0.001
Al
< 20
< 50
< 0.0015
Ca
< 100
< 50
< 0.005
Fe
< 30
< 10
0.001
Mg
< 20
< 10
< 0.002
#
Specification given with the bottle
* Freshly delivered; 18.2 M.cm resistivity; measured by ICP-MS
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Effect of ion contamination on Mass Spectra
Direct infusion of a peptide onto ESI+ MS (Bradykinin fragment 1-7)
 Na adduct peaks
 Additional peaks
• 96% Milli-Q water
• 4% Acetonitrile
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• 96% Milli-Q water + NaCl
• 4% Acetonitrile
Tips for maintaining the quality of ultrapure water
to avoid HPLC contamination issues
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Maintaining the quality of ultrapure water for HPLC
Do not store ultrapure water (or aqueous solvents)
 Ultrapure water is an excellent solvent:
– it will absorb contaminants from the lab atmosphere and from containers
 Once exposed to the atmosphere, it may become contaminated with
airborne bacteria and bacterial nutrients.
– Bacteria start to grow and multiply
– Solvent becomes contaminated
• living and dead bacteria
• bacterial by-products such as cell wall fragments and protein/peptide
fragments
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Contamination of LC-MS grade water after opening
Newly opened
After a few days
Rapid Commun. Mass Spectrom. 2010; 24: 1502–1506
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If using a water purification system –
effect of water storage
 Milli-Q water was stored in a plastic
carboy, then HPLC analysis was
performed (214 nm)
 Water samples : 60 mL trace
enrichment by accumulation on a C18
column at 1 mL/min
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9 days
6 days
2 days
0 days
If using a water purification system –
Maintaining the quality of ultrapure water for HPLC
 Use freshly purified water – avoid storage
– If water is stored, prefer glass containers, and get fresh water every day
 Discard the first 1 or 2 L before collecting water for HPLC
 Do not attach a plastic tubing (e.g., Tygon) at the delivery point
 A water purification system has to be properly and regularly maintained
– Cartridges, UV lamps, and the point-of-use purifiers have to be replaced as
prescribed.
– Do not ignore warning lights!
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Maintaining the quality of ultrapure water for HPLC
 Use dedicated glassware*
– Wash glassware with clean solvents only.
Do not use detergent.
– Do not dry glassware in racks, or let the
internal surface touch anything other than
the solvent.
– Avoid using label tapes on solvent bottles
because they are a very good source of
phthalates.
*Adapted from Dr Robert Classon, Shimadzu Corporation
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Maintaining the quality of ultrapure water for HPLC
 Use the highest purity solvent
additive/modifier (e.g. TFA, formic acid,
acetic acid, etc)
 Filter water or aqueous eluent to minimize
particulates and bacterial contamination
… or not?
– if using small particle columns
– If using evaporative detector
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UHPLC back pressure
ULC/MS grade water (0.1 µm filtered) : ΔP = 37 psi
Ultrapure water (0.22 µm filtered) : ΔP = 27 psi
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And also…
 Make sure the sample preparation step does not
introduce contaminants !
Chromatograms of a drug mixture filtered using
(A) nylon membrane and (B) polytetrafluoroethylene, PTFE
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Conclusions
 Water is a Reagent !
 Not all high purity waters for HPLC or LC-MS are equivalent.
 Freshly purified ultrapure water is preferable.
Water purification systems efficiently combine different
technologies to produce high purity water for HPLC.
 Follow simple practices to maintain the quality of the high purity
water in your laboratory.
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Thank you !
[email protected]
www.millipore.com
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