Sepax Technologies, Inc.
Delaware Technology Park
5 Innovation Way, Suite 100 Newark DE 19711 USA
Phone: (302) 366-1101; Fax: (302) 366-1151
Toll Free:(877) SEPAX-US; www.sepax-tech.com
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Polar-Imidazole
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Sepax HILIC Column Manual
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Column Information
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Polar-Silica
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To solve the challenges of more and more highly polar
pharmaceuticals and small biological molecules, Sepax developed
a series of chemistries of weak acidic, neutral, and basic
stationary phases for separating basic, neutral and acidic
compounds of high polarity. Utilizing highest purity and enhanced
mechanical stability silica and pure bonding reagents, Sepax
HILIC bonded phases have been innovatively and specially
designed to ensure maximum surface coverage, resulting in high
stability of the stationary phases. The chemistry of monolayer
formation is completely controlled that results in very reliable lotto-lot and column-to-column reproducibility. The uniform,
spherical Sepax HILIC particles have a nominal surface area of
2
300 m /g with a controlled pore size of 120 Å.
With the chemical structures shown in Figure 1, Polar-100 and
Polar-Diol are neutral, polar phases, while Polar-Diol is more
polar than Polar-100. Polar-Silica is a weak acidic phase. PolarPyridine and Polar-Imidazole are basic phases, while Imidazole
phase is more basic than Pyridine phase.
Polar-100
SiO2
OCH2CH2OH
Polar-Diol
SiO2
OCHCH2OH
OH
Polar-Silica
SiO2
SiOH
Polar-Pyridine
SiO2
N
Polar-Imidazole SiO2
N
N
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Polar-Diol
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1 2
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Polar-Pyridine
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0
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Min
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Figure 2. A typical QC chromatogram for Sepax HILIC column.
Column: Sepax HILIC, 4.6mm I.D.×150mm, 5µm
Mobile Phase: CH3CN: 10mM NH4Ac =90:10 (v/v)
Flow rate: 1.0mL/min
Detection: UV 254nm
Injection Volume: 1µL
Temperature: Ambient
Sample: 1.Uracil, 2. Adenosine, 3. Uridine, 4.Cytidine, 5.Guanosine
Characteristics
• HILIC phases with unique chemistries of acidic, neutral, and
basic surfaces
• Ultra-pure silica particles with controlled pore sizes
• Available in particle size of 1.8, 2.2, 3, 5, and 10 μm
• High chemical stability for low leaching
• Available columns with ID in the range of 75 μm to 30 mm and
length from 1 to 30 cm
• Available packings from grams to multi-Kilogram
• pH stability: 1.5 - 8.0
• Suitable for separations of polar pharmaceuticals, peptides,
amino acids, and other compounds
Figure 1. Chemical structures of HILIC phases.
Safety Precaution
Column Performance
Sepax HILIC columns are packed with a proprietary slurry
technique to achieve uniform and stable packing bed density for
maximum column efficiency, can be used as a tool to change the
selectivity or to improve peak resolution for polar and hydrophilic
compounds such as carbohydrates, metabolites, acids and bases,
organic and inorganic ions, metal complexes, amino acids,
peptides and protein digests. A typical test chromatogram for
quality control is shown in Figure 2 for a 4.6x150mm Sepax HILIC
column.
Sepax HILIC columns are normally operated under high
pressure. Loose connections will cause leaking of organic solvents
and injected samples, all of which should be considered as the
hazards. In the case of leaking, proper gloves should be worn for
handling the leaked columns. When open the columns, proper
protections should be used to avoid inhalation of the small silica
particles.
Column Installation and Operation
When column is shipped or not in use, it is always capped at
both ends. When install the column to the system, first remove the
end caps. Make the flow direction as marked on the column.
Unless a user has special purpose to reverse the flow direction, for
example, removal of the inlet pluggage, follow the flow direction
as labeled. Column connections are an integral part of the
chromatographic process. If ferrules are over tightened, not set
properly, or are not specific for the fitting, leakage can occur. Set
the ferrules for column installation to the HPLC system as follows:
(a) Place the male nut and ferrule, in order, onto a 1/16" o.d.
piece of tubing. Be certain that the wider end of the ferrule is
against the nut.
(b) Press tubing firmly into the column end fitting. Slide the nut
and ferrule forward, engage the threads, and fingertighten the nut.
(c) While continuing to press the tube firmly into the endfitting,
use a 1/4" wrench to tighten the nut 90 degrees past
fingertightness.
(d) Repeat this coupling procedure for the other end of the column.
Column Care
PH
Sepax HILIC column can be operated in the pH range 1.5
to 8, while strongly alkaline solutions and washing with sodium
hydroxide should be avoided.. The optimum performance and
operation for longest lifetime are at pH 2.0 – 7.5.
Pressure Even though Sepax HILIC can operate at pressure up
to 5,000 psi, the normal operation is usually under 3,000 psi.
Continuous use at high pressure may eventually damage the
column as well as the pump. Since the pressure is generated by
the flow rate. The maximum flow rate is limited by the
backpressure. It is expected that the backpressure might gradually
increase with its service.
Temperature The maximum operating temperature is 60oC.
Continuous use of the column at higher temperature (>70oC) can
damage the column, especially under high pH (>8.0).
New Sepax HILIC columns are delivered filled with 90 %
(v/v) acetonitrile in ammonium acetate buffer (10mM, pH 6.8).
During stocking and shipping, the silica packing could be dried
out. It is recommended that 10-20 column volumes of acetonitrile
be purged to activate the column. Flush the column with your
mobile phase with gradual increasing the flow rate from 0.1
mL/min to your operation condition, until the baseline is stable.
Solvent replacement should be performed at a flow rate one-half
of the normal operating flow rate. Note: if a column has been run
with a formate-containing mobile phase(e.g., ammonium formate,
formic acid, etc.) and is flushed to remove the buffer, slightly
longer equilibration times may be required after the column is reinstalled and run again with a formate-containing mobile phase.
Note that a drastic change of solvent composition or frequent
solvent replacements may shorten the lifetime of the column.
Storage When not in use for extended time, store the column in
95:5 acetonitrile:water prior to storage. Completely seal column to
avoid evaporation and drying out of the bed.
Cleaning and Regeneration A sudden increase in backpressure
suggests that the column inlet frit might be plugged. In this case it
is recommend that the column be flushed with reverse flow in an
appropriate solvent. If the backpressure increases or a shift in
selectivity is observed, flush with 50:50 acetonitrile:water to
remove the polar contaminant(s). If this flush procedure does not
solve the problem, purge the column with 5:95 acetonitrile:water.
Samples and Mobile Phases
To avoid clogging the column, all samples and solvents
including buffers should be filtered through 0.45 m or 0.2 m
filters before use. Sample solvents should consist of 60-100%
organic solvent, or initial eluent composition. Water should be
minimized. Weak HILIC solvents such as acetonitrile are
favoured. It is recommended to have about 5% water in the auto
sampler wash solution. The relative solvent strength for HILIC is:
Acetone<Acetonitrile<Isopropanol<Ethanol<Methanol<Water
Suitable buffer systems for HILIC separations are formate and
acetate, due to their excellent solubility even in very high
concentrations of organic solvent. Avoid phosphate, and other low
solubility buffers, to prevent precipitation on the column bed. A
buffer concentration in the range 5-20 mM is recommended for
most analytes, with an upper limit of 200-300 mM, depending on
the solubility in the eluent. TFA and other ion pair reagents
should be avoided, as they can interfere with the HILIC separation
mechanism, and suppress MS signals.
A typical HILIC application uses acetonitrile at a concentration
between 50-95% in an aqueous buffer such as ammonium formate,
ammonium acetate or their acids, which have high solubility in
organic solvents. If not familiar with the column, the
recommended initial starting and conditioning procedure is to run
a gradient from 90 % (v/v) acetonitrile/ 10 % (v/v) buffer solution
(e.g., 10 mM ammonium acetate), and ending with 40% (v/v)
acetonitrile. Initially, use a relatively low flow rate to ensure a
suitable linear flow to obtain maximum separation efficiency.
Always degas the mobile phase. A simple way for degassing is to
sonicate it for 5 minutes under water pumped vacuum.
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