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Verfügbare Sprachen
Verfügbare Sprachen
®
NUCLEODUR
CN / CN-RP
®
NUCLEOSIL
CN / CN-RP
Note: All HPLC columns from MACHEREY-NAGEL are supplied with a certificate, which contains specifications
®
and test results of the column. NUCLEODUR
CN and CN-RP columns are quality products based on the high
purity and very pressure stable silica NUCLEODUR
®
NUCLEOSIL
. They are specifically developed for HPLC analysis. If carefully and properly used excellent chro-
matographic results and long column lifetime can be achieved. HPLC columns are designed for qualitative
and quantitative analysis of mixtures of substances and single components. They must exclusively be used
in accordance with universally accepted laboratory regulations and HPLC working methods. Before running
the column the entire analytical system (column and equipment) has to be carefully checked by the opera-
tor. Chromatographic conditions (mobile phase, flow, temperature etc.) must be adapted to the analytical task.
MACHEREY-NAGEL does not give any warranty and is not liable for the success of a separation or application.
If you have any questions after reading this leaflet, please call our service / technical support.
Table of contents
· Safety indication
· Sample
· Description of the column
· Eluent
· Application note
· Flow rate and pressure
· Installation
· Temperature
· Guard columns
· Detection
Safety indication
Follow the general safety instructions for handling of HPLC solvents used as mobile phases (e.g., acetonitrile,
methanol) and take precautions against any kind of injuries or damage to health (e.g., skin and eye protection in
case of broken capillaries). Disposal of used HPLC columns must follow international, national and local environ-
mental protection regulations. The use of HPLC columns is only permitted to staff members, who are qualified in
their field. Keep HPLC columns away from children. MACHEREY-NAGEL disclaims and excludes all warranties
of any kind or nature whatsoever and MN shall not be liable for any damages (whether direct, indirect, foresee-
able, incidental, compensatory, consequential or special), whether based upon warranty, contract, tort or strict
liability, if damages and / or losses occur caused by improper use, maintenance, neglect or improper treatment
(especially opening of the column and exposure of the column bed).
Description of the column
As stationary phases the columns contain spherical silica modified with cyanopropyl by a special procedure. Due
to an exhaustive endcapping of the NUCLEODUR
results is featured.
The normal phase columns NUCLEODUR
®
CN and NUCLEOSIL
They can be applied for the separation of compounds (e.g., steroids) in normal phase chromatography (NP) with
nonpolar mobile phases. As so-called multi-mode columns, they can also be used for reversed phase (RP) ap-
plications in aqueous-organic mobile phases. But then an intermediate flushing is necessary (see eluent).
Eluent in the reversed phase columns NUCLEODUR
Due to a distinct selectivity for polar organic compounds (e.g., organic acids) as well as for molecules contain-
ing π electron systems (e.g., analytes with double bonds, tricyclic antidepressants), this phase clearly shows a
different retention behavior compared to C
or C
18
but is not recommended (see eluent).
Application note
Separation of cold medicine ingredients on different RP phases
Columns:
®
A) EC 250/4 NUCLEODUR
100–5 C
B) EC 250/4 NUCLEODUR
®
100–5 CN-RP
Eluent:
acetonitrile – 100 mmol/L sodium citrate,
pH 2.5 (15:85, v/v)
Flow rate:
1 mL/min
Temp.:
25 °C
Detection:
UV, 270 nm
Inj. volume: 10 μL
Peaks:
1. Maleic acid
2. Norephedrine
3. Ephedrine
4. Acetaminophen
5. Chlorpheniramine
6. Brompheniramine
MN Appl. No. 119340
Installation
The column should be installed in the flow direction indicated on the column label. It is connected with 1/16"
capillaries and fittings, typical for HPLC instruments.
Guard columns
For protection and an extension of column lifetime the column should always be used with a guard column.
The filter elements and the adsorbent in the guard column retain contaminants from the sample or the elu-
ent. Connection of the guard column with the separation column is made by a suitable guard column holder
(see www.mn‑net.com or the MN chromatography catalog). Cartridge replacement is required when increased
column pressure and / or loss of performance is observed.
Sample
Sample solutions should be passed through a syringe filter (e.g., CHROMAFIL
729220) before entering the column. If injected sample solutions are still turbid even after filtration, the lifetime
of the column may be significantly reduced. The sample volume should be as small as possible to achieve an
optimal resolution.
Eluent
NP columns: Eluent in the column is n-heptane. As mobile phases in normal phase mode (NP) n-heptane, hex-
ane, dichloromethane or 2-propanol are used. Eluents should be filtered through a 0.2–0.45 μm membrane filter
and degassed. For changing to the reversed phase mode (RP), columns must be rinsed with 10 column volumes
tetrahydrofuran (THF).
RP columns: They are supplied with the eluent acetonitrile – water (depending on the type 80:20, 70:30 or 60:40,
v/v; see column certificate for details). Typical RP eluents are e.g., acetonitrile or methanol with pure water or
phosphate buffer. They should be filtered and degassed. pH stability of NUCLEODUR
for NUCLEOSIL
®
CN-RP between 2 and 8. Strongly acidic or basic conditions, especially for column tempera-
tures higher than 40 °C, can result in dissolution of the column bed or the organic modification. The amount of
buffer salts should be kept as low as possible. Note the solubility limit of the buffer in the eluent. An increase of the
organic portion can result in precipitation of buffer salts and plugging of the column. Before start of operation with
an eluent containing a buffer the column should be first preconditioned with a minimum of 10 column volumes
acetonitrile – water (25:75, v/v). Always after finishing measurements with buffer containing eluents, the column
should be regenerated (see column regeneration). A changing to NP mode is not recommended. If necessary, it
should only be made with an intermediate flushing step with THF.
Flow rate and pressure
Flow rate (recommended for analytical columns with 2–4.6 mm ID: 0.2–2.0 mL/min) influences the time required,
the resolution and the column lifetime. It is limited by the back pressure, which should not exceed the maximum
of 600 bar (NUCLEODUR
®
) / 400 bar (NUCLEOSIL
maximum at about 40 % methanol. For this reason a reduced flow rate is recommended, when changing the
eluent composition. We recommend controlling back pressure regularly. If a high pressure results from the use of
the column at nominal flow rates, this usually indicates that some contaminants have become deposited on the
packing material, which must be removed (see troubleshooting).
Temperature
Column temperatures up to 60 °C are possible; for a long lifetime 30–40 °C is recommended. However, they
should be at least 30 °C below the boiling temperature of the eluent, in order to ensure proper detection. Variation
of the temperature influences retention times and especially the peak shape. Optimum temperatures for success-
ful separations should be determined empirically.
Detection
Spectrophotometers, refractometers and electrochemical detectors can be used with the columns.
®
NUCLEODUR
CN and CN-RP are also suitable for LC/MS detection. If a higher sensitivity is required, post-
column derivatizations with an appropriate detector for the reaction product can be used.
Equilibration
Prior to measurement of samples the column must be rinsed with the eluent at the same flow rate and tempera-
ture as the method to be applied. Column equilibration is finished, when the baseline of the detector no longer
shows a drift (generally after 10 column volumes).
Column storage
The original eluent (see eluent) is recommended for storage. For long-term storage mobile phases containing
inorganic salts are not recommended (see regeneration). Methanol is also not recommended for a longer stor-
age, because of a possible impurity with metal ions (e.g., iron(III)). For column storage be sure the end fittings are
tightly sealed using column end plugs, because storage without these seals can result in drying of the packing
material. Under these circumstances rinse the column with approx. 10 column volumes of the eluent of storage
at a flow rate of max. 0.2 mL/min.
Deutschland und International:
MACHEREY-NAGEL GmbH & Co. KG
Neumann-Neander-Str. 6–8 · 52355 Düren · Deutschland
Tel.: +49 24 21 969-0 · Fax: +49 24 21 969-199
info@mn-net.com · www.mn-net.com
®
; NUCLEOSIL
®
CN and CN-RP are based on the robust silica
· Equilibration
· Column storage
· Troubleshooting
· Column regeneration
· Abstract
®
cyano phase, an exceptionally high reproducibility of analysis
®
CN are supplied with the eluent n-heptane.
®
®
CN-RP and NUCLEOSIL
CN-RP is acetonitrile – water.
modified RP phases. A changing to NP conditions is possible,
8
ec
18
4
4
3
2
5
3
5
2
1
A
B
1
6
0
4
8
®
Xtra PET, 0.45 μm, 25 mm, REF
®
CN-RP is between 1 and 8,
®
). In mixtures of methanol and water viscosity reaches a
Schweiz:
MACHEREY-NAGEL AG
Hirsackerstr. 7 · 4702 Oensingen · Schweiz
Tel.: 062 388 55 00 · Fax: 062 388 55 05
sales-ch@mn-net.com
Troubleshooting
The following outline describes the symptoms of performance loss and their cause. All columns are subject to the
strict regulation and control of our quality assurance system. Columns based on silica are robust and hold their
separation efficiency for long periods by correct maintenance and treatment. According to experience, column
failures are mostly a result of injection of contaminants to the sorbent bed. The usage of a guard column, as well
as an appropriate sample pretreatment will help to minimize these risks.
Use the outline below to help determine the cause of a possible performance loss:
Symptom / Error / Cause
Baseline drift
· insufficient period for equillibration with the eluent
· contaminated eluent
· temperature
Broad peaks
· mixing and / or diffusion before / behind the column
· too large sample volume
Peak interference; too fast elution
too fast elution and / or insufficient separation by:
· improper column temperature or flow rate
· elution power of eluent is too high
Increasing back pressure; degradation of the
separation performance
contamination of sorbent by:
· particulate accumulation on frit or sorbent bed from
sample, eluent or system
· precipitation of buffer salts
Insufficient separation; degradation of the
separation with regular column pressure
contamination with:
· fats, oils, lipids from sample (coating of sorbent
surface) and other organic substances from im-
properly prepared eluent or matrices
Double peaks (dead volume)
· faulty fittings (capillaries, ferrules, nuts)
· dissolution of silica by too high pH value of eluent
Column regeneration
In some cases the perfomance of the column can be restored by removing contaminants from the sorbent bed
or by regeneration of the phase. It is important, however, to locate the source of contamination before using the
column for the analysis of samples again.
1. Prepare fresh eluent: Sometimes the performance loss is caused by eluent contamination. Therefore, prepare
fresh eluent and flush all liquid lines before using the column again. The eluent should be filtered through a
0.2–0.45 μm membrane and degassed prior to use.
2. Cleaning of sorbent: To remove contamination rinse the column with a minimum of 10 column volumes (see
table below) at the original flow rate and temperature as follows:
NP columns:
· 100 % tetrahydrofuran to remove non or medium polar organic compounds
· if necessary, 100 % tetrahydrofuran with inverse flow direction at 1/5 of original flow rate
· convert column to storage condition with n-heptane at original flow rate
RP columns:
· acetonitrile – water or methanol – water (10:90, v/v) for removal of the buffer
· 100 % methanol to remove polar organic compounds
6
· 100 % acetonitrile to remove medium polar organic compounds (possibly T= 40 °C)
· 100 % tetrahydrofuran to remove non polar organic compounds
· if necessary, 100 % tetrahydrofuran with inverse flow direction at 1/5 of original flow rate
· column is converted to storage condition with acetonitrile – water (80:20, 70:30 or 60:40, v/v) at
12 min
original flow rate
An adequate indicator for a clean column is a constant baseline. At constant temperature you should observe
less than 2–3 mAU drift during a running time of 5 minutes with an isocratic run.
After the usage of buffer, directly after finishing a measurement and always before storage RP columns rinse
with a minimum of 10 column volumes at the original flow rate and temperature as follows:
· acetonitrile – water or methanol – water (10:90, v/v) for removal of the buffer
· increase the organic part in steps of 20 % to the conditions of a new measurement run
· or gradually increase the part of acetonitrile in steps of 20 % to the storage conditions
3. Column replacement: The above procedures will restore performance only in certain cases. Some organic
contaminants are particularly refractory and may not respond to treatment. Also dead volume, due to column
compression can generally not be repaired. Under these circumstances, column replacement is necessary. It
is highly advisable to locate the cause of the problem before installing a new column.
Length [mm]
100
150
250
Abstract
To extend column lifetime, please keep in mind the following:
1. As NP eluents nonpolar organic solvents (e.g., n-heptane, dichloromethane, 2-propanol) and as RP eluents
organic-aqueous eluent systems (e.g., acetonitrile – water or buffer) are recommendable. For a change from
NP to RP mode the column must be always rinsed with THF between the steps. An inverse change is not
recommended. Eluents should be filtered through a 0.2–0.45 μm membrane and degassed.
2. Filter samples through a 0.2–0.45 μm CHROMAFIL
3. Use a guard column for contaminated samples.
4. The recommended flow rate for analytical columns (ID 2-4.6 mm) is 0.2–2.0 mL/min.
5. Adjust flow rate to keep column pressure below 600 (for NUCLEODUR) / 400 (for NUCLEOSIL) bar.
6. Store the NP column in n-heptane and the RP column in acetonitrile – water (70:30, v/v).
7. Use analytical grade reagents and HPLC grade solvents for all work. Discard any solutions that show evi-
dence of bacterial growth.
Please check the full range of MACHEREY-NAGEL chromatography products!
HPLC
GC
TLC
SPE and Flash
Syringe filters
Vials and caps
... for applicative support please visit our website
with more than 4000 chromatography applications: www.mn‑net.com/apps
Frankreich:
MACHEREY-NAGEL SARL à associé unique
1, rue Gutenberg · 67722 Hoerdt · Frankreich
Tel.: 03 88 68 22 68 · Fax: 03 88 51 76 88
sales-fr@mn-net.com
Prevention / Remedy
longer or better equilibration
use freshly prepared solvents and reagents
column temperature control
keep length and ID of capillaries at a minimum
smaller injection volume
optimize concerned parameter
optimize eluent system
prepare fresh eluent; prefilter samples and eluent,
use in-line filter / rinse LC system, clean the sorbent
check solubility of buffer salts before / remove them by
rinsing (see column regeneration)
remove organic substances by sample preparation /
clean the sorbent (see column regeneration)
use "PEEK Fingertight Fittings", REF 718770 or
REF 718778 / replace fittings
consider pH range of column / replace column
Column volume [mL]
Inner diameter [mm]:
2
3
4
4.6
0.30
0.70
1.25
1.65
0.45
1.05
1.90
2.50
0.80
1.75
3.15
4.15
®
Xtra PET syringe filter before injection.
USA:
MACHEREY-NAGEL Inc.
2850 Emrick Boulevard · Bethlehem, PA 18020 · USA
Tel.: 484 821 0984 · Fax: 484 821 1272
sales-us@mn-net.com
www.mn-net.com
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