Full Text Article - Millipore

The R&D Notebook
From tap to ultrapure water: tracking organic contaminants
Daniel Darbouret (1) , Thomas Stone (2)
(1) Research & Development, Laboratory Water Division, Millipore S.A., St Quentin en Yvelines, France.
(2) Central Research & Development, Analytical Lab, Millipore Corp, USA.
RD005
Key Words: Ultrapure Water, Organic Contamination, Reversed Phase Chromatography, U.V. Detection, Baseline.
Abstract: When performing HPLC analysis, the quality and purity of the solvents used are of
primary significance since these reagents are present throughout the experiment. Detection,
identification and quantification of the studied compounds will depend on the detection limits
that are achievable and thus on the baselines obtained while performing the analyses.
In this paper, we will be concentrating on ultrapure water as a reagent. The way water of
differing quality affects analyses is shown. The organic contaminants are monitored throughout
the combination of processes required to produce this ultrapure water. The configuration of this
water purification chain allows delivery of ultrapure water free of organic contaminants that
interfere with sensitive reversed phase chromatography. Careful water purification system
maintenance is also key when performing trace organic analysis.
Introduction
The quality of different types of HPLC analyses
depends largely on the detection system in use and
on the quality of the solvent used 1 . Detection methods
vary from the measurement of physical properties to
photo-emission/absorption, to electroactivity. Each
technique has its own requirements in terms of mobile
phase characteristics, and in particular, the quality of
the ultrapure water used will affect detection limits 2 .
The reversed phase mode in combination with UV
detection is one of the most commonly used
separation methods for liquid chromatography 3,4,5 .
Various organic solvents, such as methanol,
acetonitrile, tetrahydrofuran, and water are usually
used as mobile phase. Because the change in
mobile phase conditions modifies the adsorption
characteristics on the chromatography column,
optimization of the mobile phase is important to
achieve accurate chromatographic separation 6 .
Most organic molecules absorb in the UV region of
their spectrum. Many HPLC-grade solvents are
available on the market. Their grade name indicates
A publication of the Laboratory Water Division of Millipore
that these solvents are specifically prepared to
emphasize low backgrounds when used with a UV
detector. At high wavelengths, > 240 nm, solvent
interference is less critical than in the wider UV region
(190 - 230 nm) where the slightest impurity will be
absorbed.
When performing a gradient from 100% water to
100% organic solvent, impurities present in the water
will cause additional peaks to appear in the
chromatogram, mainly at the beginning of the
gradient, while ghost peaks in the high organic
percentages are often due to impaired organic
solvent purity.
In order to produce water with the lowest possible
organic contamination for mobile phases, a
combination of purification technologies should be
used to produce ultrapure water from tap water. In
this study, organic contamination during the different
water purification steps is tracked.

2
Additionally, technical hints are given as to how to
ensure reproducible high quality ultrapure water,
including after each consumable change.
One of the most widely trusted parameters used to
track ultrapure water quality is resistivity monitoring.
Previous studies: traditional HPLC profiles
The quality of purified water is often questioned,
when poor baselines are obtained (especially at
214 nm, a wavelength which most organic
contaminants absorb).
To illustrate this, a blank gradient (no sample
injected) was run by pumping for 4 minutes at initial
conditions (100% water), then ramping up to final
conditions (100% acetonitrile) over a period of
20 minutes. Next, suspecting water as the contributor
of the contaminant peaks, the same procedure was
run except that initial conditions were kept for
36 minutes, before running the gradient. Since the
size of the peaks increased, one can be certain they
came from the water and not from the acetonitrile
(Figure 1). If any of the peaks of interest were eluted
at the same time as these contaminating peaks, there
would be quantitation errors. There is also a
possibility that the contaminating peaks could be
confused with desired compounds.
1.0
A.U.
0
10 20
Figure 1: Blank analysis profiles at 214 nm.
Time (minutes)
36 minute delay
4 minute delay
Typical chromatograms obtained with different
purified water types are given below.
The first one is a gradient analysis of 50 ml of lab
water that is not ultrapure, concentrated on the head
of a C18 column. A gradient was then run, up to
100% acetonitrile, and the output was monitored at
two different wavelengths at a moderately sensitive
setting. Clearly, as shown in Figure 2 this water
source is not acceptable for HPLC, be it the isocratic
or the gradient method. Peaks due to the water occur
throughout the chromatogram. Two peaks come from
the acetonitrile.
1.0
A.U.
0
contaminants
from water
10 20
Figure 2: Poor baseline profiles at 214 and 254 nm.
contaminants
from acetonitrile
Time
(minutes)
The R&D Notebook: From tap to ultrapure water: tracking organic contaminants
This value, however, does not measure the possible
organic contaminants that may be present in water.
Significant organic contamination will still give an
18.2MΩ.cm resistivity reading 7 .
The second one is a gradient analysis of 50 ml of
triple distilled water, concentrated on the head of a
C18 column. A gradient was then run, up to 100%
acetonitrile, and the output monitored at two different
wavelengths at a moderately sensitive setting.
Clearly, this water source is not acceptable for HPLC.
The early peaks are due to the water, the last two
peaks come from the acetonitrile (see Figure 3).
1.0
A.U.
0
For reverse osmosis water, gradient analysis of 50 ml
concentrated on the head of a C18 column is shown
in Figure 4. A gradient was run, up to 100%
acetonitrile, and the output monitored at two different
wavelengths at a moderately sensitive setting .
Clearly, this water source is not acceptable for HPLC,
neither isocratic nor gradient. The early peaks are
due to the water, two of the last peaks come from the
acetonitrile. Moreover, with this type of purified
water, the residual background is very high.
A.U.
0
contaminants
from water
Figure 3: Triple distilled water.
10 20
contaminants
from water
10 20
contaminants
from acetonitrile
contaminants
from acetonitrile
Time
(minutes)
1.0 214 nm
Figure 4: RO water.
254 nm
Time
(minutes)
In order to get the ultrapure water quality required to
perform organic chromatography analysis, a water
purification chain combining various technologies is
therefore necessary.
In order to highlight the efficiency in removing
organics from tap water, organic studies are
performed on the water delivered after the main
purification steps.

Experimental
Water Purification System
A combination of technologies is necessary to produce
ultrapure water. Methods for tracing classes of
organic matter at different points in a water
purification system were first described in 1987 8 .
Combination techniques were also used to obtain
meaningful information about contamination in the DI
water process 9 . The configuration used during this
qualitative study is made up of two new water
purification systems. The first one combines reverse
Chromatographic conditions
Equipment
RO reject
RO permeate
EDI Product Reservoir
The following equipment was used throughout the
experiment:
HPLC pumps delivering a gradient eluent at
2.0 ml/min
Computer for data acquisition and system control
Injector for 100 µL injection with 250 µL loop, 200 µL
syringe, and appropriate vials.
Detectors: Dual detectors operated at 214 nm and
254 nm (Waters Corp, Milford, Mass, USA).
Column Type: Waters µBondapak ® C-18 column,
3.9 x 300 mm (#27324).
Precolumn Type (optional): C18 (Waters µBondapak
C-18 Guard-Pak TM (#T33552))
Elution solvents:
Water (fresh Milli-Q ® system water (Millipore))
Acetonitrile (HPLC grade: J.T. Baker JT9017 or
equivalent).
The bottles were rinsed in 10% nitric acid, washed
extensively with fresh Milli-Q system water before
sampling.
Figure 5: The water purification chain studied.
osmosis and electrodeionization techniques. This
purified water is stored in a 60 l reservoir. This high
purity water tank is specifically designed for the
optimization of high purity water storage 10 . A second
system combining activated carbon, ion exchange
resins and UV treatment is fed by this pretreated
water.
Sampling ports are ensured throughout the process
(see arrows in Figure 5)
HPLC gradient
Ultrapure
water
The following elution gradient was used throughout
the experiment. 50 ml of each water sample were
injected (except for tap water and RO reject water
where 1 ml was used) in order to concentrate trace
impurities on the column.
Time [minutes] Flow [ml/min.] Water % Acetonitrile %
0.0 2.0 100 0
2.0 2.0 100 0
12.0 2.0 0 100
15.0 2.0 0 100
19.0 2.0 100 0
25.0 2.0 100 0
The acquisition time was fixed at 14 minutes with a
4 point/second acquisition rate and a resolution
over 20 bit.
Chromatograms were compared at different
maintenance conditions and during the different
water purification stages.
The R&D Notebook: From tap to ultrapure water: tracking organic contaminants 3

4
Results
Rinsing of new consumables
Rinsing of a new RO cartridge (first water purification
system)
When installing a new reverse osmosis cartridge in a
water purification system, or when changing
purification media of a polishing system, it is
mandatory to allow all preservative solutions and
possible leachables from materials to be rinsed out.
This is why most pieces of modern equipment have an
automatic rinsing and flushing time after each
maintenance operation. To study the influence of this
RO membrane on water quality, RO water from reject
and permeate was collected at different times during
the regular RO rinse cycle. The water produced during
this cleaning cycle is automatically discarded by the
system itself and does not go through further
purification stages. Additional chromatographic
profiles were obtained after 2 days and 13 days of
system use. Chromatograms are those indicated in
Figure 6.
UV absorbance (A.U.)
UV absorbance (A.U.)
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
%CH 3CN
0.00
0 2 4 6 8 10 12 14 16
0.10
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
RO reject
RO permeate
Time (minutes)
100 %
0.00
0 2 4 6 8 10 12 14 16
Time (minutes)
Figure 6: RO reject and RO permeate water profiles.
30 min
1 h 30
The amount of organics released during the first
30 minutes of the RO rinse is quite significant and
justifies the automatic rinsing function of a new RO
cartridge. The RO reject profiles also give a good
indication of the best efficiency time. The amount of
organic preservative solution coming off the membrane
after a 2 hour rinse does not represent an amount
which could impair further purification processes.
The R&D Notebook: From tap to ultrapure water: tracking organic contaminants
2 h
3 h 30
4 h
30 min
2 h
2 h 30
4 h
2 days
13 days
Rinsing of new purification media (final polishing
system)
The organic quality of the ultrapure water was
checked over time during the installation procedure of
a new polishing cartridge. When installing a new
purification cartridge, it is mandatory to produce
water for 5 minutes to the drain. Then, it is
recommended to leave the system in standby mode
for 6 hours. A 5 minute recirculation per hour during
this period allows good hydration and final rinsing of
the new purification media. After these
6 hours, water is again produced to the drain for
5 minutes. This installation procedure allows any
remaining trace contamination and air present in the
purification cartridge to be removed and ensures
reproducible excellent baselines at 254 nm as well
as 214 nm. Chromatograms shown in figure 7
highlight this high quality water suitable for high
sensitivity HPLC organic analyses. The four first
chromatograms show the efficiency of the installation
procedure of a new polishing cartridge. The last
chromatogram is an example of the baseline that can
be achieved with ultrapure water suitable for organic
trace analysis at 254 or 214 nm UV detection.
UV absorbance
UV absorbance
UV absorbance
0.050
0.040
0.030
0.020
0.010
0.050
0.040
0.030
0.020
0.010
Milli-Q system water, 1 min air purge
214NM
254NM
0.000
0.00 4.00 8.00 12.00 16.00
Gradient Time (minutes)
0.050
0.040
0.030
0.020
0.010
Milli-Q system water, 4 min air purge
214NM
254NM
0.000
0.00 4.00 8.00 12.00 16.00
Gradient Time (minutes)
Milli-Q system water, 1 min after 6 hours
214NM
254NM
0.000
0.00 4.00 8.00 12.00 16.00
Gradient Time (minutes)
Figure 7: Ultrapure water baselines at 254 nm and 214 nm.

UV absorbance
UV absorbance
From tap to ultrapure water
Using the water purification chain described
previously, the various sampling points enable the
organic decrease through the different water
purification processes to be traced. The change in
organic nature during water treatment does not allow
direct contaminant identification.
However, chromatogram profiles obtained, and
especially at 214 nm, the more sensitive wavelength,
highlight organic contamination removal through the
purification stages (see Figure 8). This shows the
importance of having a combination of technologies
when producing ultrapure water from potable water.
Conclusion
0.050
0.040
0.030
0.020
0.010
Milli-Q system water, 6 min after 6 hours
0.000
0.00 4.00 8.00 12.00 16.00
Gradient Time (minutes)
0.050
0.040
0.030
0.020
0.010
214NM
254NM
Milli-Q system water, day 20
214NM
254NM
0.000
0.00 4.00 8.00 12.00 16.00
Gradient Time (minutes)
Figure 7: Ultrapure water baselines at 254 nm and 214 nm.
The presence of impurities in the mobile phase, and
especially in the purified water used for HPLC studies
raises several issues:
- These contaminants create peaks during gradient
runs. This leads to problems with peak
identification and peak quantitation.
- The impurities contaminate and “kill” columns.
- When solutions of standards are prepared with
impurities contained in ultrapure water, inaccurate
results are obtained for sample identification and
quantitation.
- Moreover, when preparative chromatography is
performed, impurities in mobile phase and/or
samples prepared, can contribute to contaminated
fractions.
A significant number of studies have shown the
importance of water quality during HPLC analysis.
Storage of high purity water is inadequate and
inappropriate to obtain high quality baselines 11 .
A.U. (254 nm)
A.U. at 214 nm
0.035
0.030
0.025
0.020
0.015
0.010
0.005
0.000
0.00 5.00 10.00 15.00 20.00
0.035
0.030
0.025
0.020
0.015
0.010
0.005
Time (minutes)
Figure 8: From tap to ultrapure water, chromatographic profiles at
254 nm (top) and 214 nm (bottom) (1 ml injected (Tap and RO)
or 50 ml accumulated (EDI, reservoir and Milli-Q) on the
C18 column).
More and more sensitive detection methods are
being coupled to HPLC in order to improve detection
limits 12,13,14 .
The mobile phase in RP-HPLC generally contains a
“buffer” component, an organic modifier and, often,
an ion pairing agent added to the mobile phase to
influence selectivity. All solvents, buffering salts, ion
pairing agents, as well as the water used to prepare
the mobile phases, must be of high chemical purity.
A water purification chain combining multiple
technologies such as electrodeionization
pretreatment, UV photooxidation and high quality
purification media, and ensuring easy maintenance
with automatic rinsing and flushing capabilities,
delivers ultrapure water suitable for high sensitivity
organic analyses. Additionally, the use of an
ultrapure water system with on line TOC (Total
Organic Carbon) analysis is the best way to monitor
these organic contaminants at the point-of-use 15 .
Tap
RO
EDI
Reservoir
Milli-Q
0.000
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00
Time (minutes)
Tap
RO
EDI
Reservoir
Milli-Q
The R&D Notebook: From tap to ultrapure water: tracking organic contaminants
5

6
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The R&D Notebook: From tap to ultrapure water: tracking organic contaminants
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