Thermo Scientific Acclaim HILIC-10 User manual

Thermo Scientific
Acclaim HILIC-10 Columns
Product Manual
P/N: 065339-02 January 2013
Part of Thermo Fisher Scientific

Product Manual
for
Acclaim HILIC-10 Analytical Column
Acclaim HILIC-10, 3 µm, Analytical, 4.6 x 150 mm (P/N 074257)
Acclaim HILIC-10, 3 µm, Analytical, 3.0 x 150 mm (P/N 074258)
Acclaim HILIC-10, 3 µm, Analytical, 2.1 x 150 mm (P/N 074259)
Acclaim HILIC-10, 5 µm, Analytical, 4.6 x 150 mm (P/N 079693)
Acclaim HILIC-10, 5 µm, Analytical, 4.6 x 150 mm (P/N 079694)
Acclaim HILIC-10, 5 µm, Analytical, 2.1 x 150 mm (P/N 070695)
Acclaim RSLC HILIC-10, 1.9 µm, Analytical, 2.1 x 150 mm (P/N 074260)
Acclaim RSLC HILIC-10, 1.9 µm, Analytical, 2.1 x 250 mm (P/N 075709)
Acclaim HILIC-10 Guard Cartridge
Acclaim HILIC-10, 3 µm, Guard 3.0 x10 mm, 2ea (P/N 074261)
Acclaim HILIC-10, 3 µm, Guard 4.6 x10 mm, 2ea (P/N 074262)
Acclaim HILIC-10, 3 µm, Guard 2.1 x10 mm, 2ea (P/N 074263)
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© 2013 Thermo Fisher Scientific Inc. All rights reserved.
All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.
Thermo Fisher Scientific Inc. provides this document to its customers with a product purchase to use in the product
operation. This document is copyright protected and any reproduction of the whole or any part of this document is
strictly prohibited, except with the written authorization of Thermo Fisher Scientific Inc.
The contents of this document are subject to change without notice. All technical information in this document is for
reference purposes only. System configurations and specifications in this document supersede all previous
information received by the purchaser.
Thermo Fisher Scientific Inc. makes no representations that this document is complete, accurate or error free and
assumes no responsibility and will not be liable for any errors, omissions, damage or loss that might result from any
use of this document, even if the information in the document is followed properly.
This document is not part of any sales contract between Thermo Fisher Scientific Inc. and a purchaser. This
document shall in no way govern or modify any Terms and Conditions of Sale, which Terms and Conditions of Sale
shall govern all conflicting information between the two documents.
Revision History:
Revision 02, January, 2013, Rebranded for Thermo Scientific. Added 5 µm and 1.9 µm column formats.
For Research Use Only. Not for use in diagnostic procedures.
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Safety and Special Notices
Make sure you follow the precautionary statements presented in this guide. The safety and
other special notices appear in boxes.
Safety and special notices include the following:
SAFETY
!
Indicates a potentially hazardous situation which, if not avoided, could result in death or
serious injury.
W
ARNING
!
Indicates a potentially hazardous situation which, if not avoided, could result in damage to
equipment.
CAUTION
!
Indicates a potentially hazardous situation which, if not avoided, may result in minor or
moderate injury. Also used to identify a situation or practice that may seriously damage the
instrument, but will not cause injury.
NOTE
!
Indicates information of general interest.
IMPORTANT Highlights information necessary to prevent damage to software, loss of data, or invalid test
results; or might contain information that is critical for optimal performance of the system.
Tip Highlights helpful information that can make a task easier.

Contents
Contents
1. Introduction.........................................................................................................7
1.1 The main features of the Acclaim HILIC-10 columns include:.....................................................................7
1.2 Physical data ..................................................................................................................................................8
1.3 Specifications and Recommended Operational Parameters...........................................................................8
2. Step-By-Step User Guide....................................................................................9
Step 1 – Visually inspect the column ..........................................................................................................................9
Step 2 – Mobile phase preparation ..............................................................................................................................9
Step 3 – Set up the LC system...................................................................................................................................10
Step 4 – Condition the column ..................................................................................................................................10
Step 5 – Reproduce the chromatogram in the Quality Assurance Report .................................................................10
Step 6 – Real sample analysis ...................................................................................................................................10
3. Considerations in Method Development.........................................................11
3.1 Selection of Organic Solvents......................................................................................................................11
3.2 Buffer Types ................................................................................................................................................11
3.3 Mobile Phase pH..........................................................................................................................................11
3.4 Isocratic or Gradient Method .......................................................................................................................11
3.5 Injection Volume .........................................................................................................................................11
4. Column Care .....................................................................................................12
4.1 Column storage ............................................................................................................................................12
4.2 Operating pH range: pH 2.0 to 8.0...............................................................................................................12
4.3 Operating temperature limit: 70 ºC..............................................................................................................12
4.4 Pressure limit: ..............................................................................................................................................12
4.5 Flow rate ......................................................................................................................................................12
4.6 Column washing procedure .........................................................................................................................12
5. Frequently Asked Questions............................................................................14
5.1 What is the difference between the Acclaim HILIC-10 column and other HILIC columns? ......................14
5.2 Why do I need an Acclaim HILIC-10 column? ...........................................................................................14
5.3 When do I need an Acclaim HILIC-10 column? .........................................................................................14
5.4 What factors should I consider for method development using this column?..............................................14
5.5 What mobile phases should I use with this column?....................................................................................14
5.6 What should I do before starting to use the Acclaim HILIC-10 column?....................................................15
5.7 Can I use this column in normal-phase mode? ............................................................................................15
5.8 How to store the column? ............................................................................................................................15
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Contents
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6. Applications.......................................................................................................16
6.1 Hydrophilic Pharmaceuticals .......................................................................................................................16
6.2 Aspirin and Degradation Product.................................................................................................................16
6.3 Melamine and Cyanuric Acid ......................................................................................................................17
6.4 Solvent Effect of Melamine .........................................................................................................................17
6.5 Carbohydrates ..............................................................................................................................................18
6.6 Sugar Alcohols.............................................................................................................................................18
6.7 Acrylamide and Acrylic Acid ......................................................................................................................19
6.8 Methacrylic Acid and Acrylic Acid .............................................................................................................19
6.9 Acrylic Acid and Oligomers ........................................................................................................................20
6.10 Good’s Buffer Salts......................................................................................................................................20
6.11 Sodium Chloride..........................................................................................................................................21
6.12 Tris•HCl.......................................................................................................................................................21
6.13 α-Tocopherol and α-Tocopherol Acetate .....................................................................................................22
6.14 Vitamins K1 and K2 ....................................................................................................................................22
6.15 Retinol Palmitate and Retinol Acetate .........................................................................................................23

1– Introduction
1. Introduction
The Thermo Scientific™ Acclaim™ HILIC-10 is a general-purpose silica based HILIC
(Hydrophilic Interaction Liquid Chromatography) column for separating highly hydrophilic
analytes. It is based on high-purity, spherical, porous silica gel that is covalently modified with a
proprietary hydrophilic layer. HILIC is a variation of normal-phase chromatography with the
advantages of being compatible with water. It is sometimes called “reverse reversed-phase” or
“aqueous normal phase” chromatography. The HILIC mode of separation is used to retain very
polar compounds. Stationary phase is polar and the mobile phase is highly organic (>80%) with
a small amount of polar solvent (e.g, H2O).
HILIC is a complementary technique to reversed-phase liquid chromatography (RPLC), and has
several benefits over RPLC. It retains polar analytes that cannot be retained on a reversed-phase
column. For electrospray LC/MS applications with very polar compounds, HILIC (using an
organic-rich mobile phase) provides a ten to twenty-fold sensitivity improvement. Moreover,
by eliminating the need for evaporation and reconstitution of a sample dissolved in a non-
aqueous solvent, the sample analysis throughput can be greatly increased.
HILIC is useful for separating polar solutes and has been applied to a wide variety of
application including carbohydrates, peptides, proteins, pharmaceuticals, as well as other highly
polar molecules such as urea, biurea, choline and butyrobetaine, tromethamine, ascorbic acid
and related compounds, folic acid and its metabolites, nicotine and its metabolite, saponins,
aminoglycoside antibiotics, glucosinolates, ionic liquids, organophosphonate nerve agent
metabolites, etc.
1.1 The main features of the Acclaim HILIC-10 columns include:
1. Ability of retaining highly polar molecules that would be un-retained by reversed-
phase chromatography
2. Unique selectivity, complementary to reversed-phase columns or other HILC columns
3. Excellent chemical stability
4. Rugged column packing
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1– Introduction
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1.2 Physical data
Bonding Chemistry: Proprietary covalently bonded hydrophilic layer
Silica Substrate: Spherical, high-purity
Particle Type Column Dimensions P/N
Analytical
3 µm particle size
120 Å pore size
300 m2/g surface area
4.6 x 150 mm 074257
3.0 x 150 mm 074258
2.1 x 150 mm 074259
5 µm particle size
120 Å pore size
300 m2/g surface area
4.6 x 250 mm 079693
4.6 x 150 mm 079694
2.1 x 150 mm 079695
1.9 µm particle size
175 Å pore size
200 m2/g surface area
2.1 x 150 mm 074260
2.1 x 250 mm 075709
Guard
(requires holder
P/N 069580)
3 µm particle size
120 Å pore size
300 m2/g surface area
3.0 x 10 mm 074261
4.6 x 10 mm 074262
2.1 x 10 mm 074263
1.3 Specifications and Recommended Operational Parameters
Shipping solution: 90/10 acetonitrile/100 mM ammonium acetate
Storage solution: 90/10 v/v acetonitrile/10-50 mM ammonium acetate or 100%
acetonitrile
pH Range: pH 2 – 8
Temperature Range: < 70° C
Particle
Size Column Dimension Maximum Recommended Pressure Typical Flow Rate
3 µm
4.6 x 150 mm 8500 psi 0.8 – 1.5 mL/min
3.0 x 150 mm 8500 psi 0.4 – 0.8 mL/min
2.1 x 150 mm 8500 psi 0.2 - 0.4 mL/min
5 µm
4.6 x 250 mm 6000 psi 0.8 – 1.5 mL/min
4.6 x 150 mm 6000 psi 0.8 – 1.5 mL/min
2.1 x 150 mm 6000 psi 0.2 – 0.4 mL/min
1.9 µm 2.1 x 150 mm 10,000 psi 0.2 – 0.4 mL/min
2.1 x 250 mm 15,000 psi 0.2 – 0.4 mL/min

2– Step-By-Step User Guide
2. Step-By-Step User Guide
Thermo Fisher Scientific recommends that you perform an efficiency test on your Acclaim
HILIC-10 column before use. The purpose of column performance validation is to ensure no
damage has occurred during shipping. Steps 1 – 5 below outline the necessary steps to perform
this validation test. Test the column using the conditions described on the Quality Assurance
(QA) Report enclosed in the column box. Repeat the test periodically to track the column
performance over time. Slight variations may be obtained on two different HPLC systems due to
system electronic, plumbing, operating environment, reagent quality, column conditioning, and
operator technique.
Step 1 – Visually inspect the column
Report any visual damage to Thermo Fisher Scientific Corporation.
Step 2 – Mobile phase preparation
Obtaining reliable, consistent and accurate results requires mobile phases that are free of ionic
and spectrophotometric impurities. Chemicals, solvents and de-ionized water used to prepare
mobile phase should be of the highest purity available. Maintaining low trace impurities and low
particle levels in mobile phases helps to protect your columns and system components. Thermo
Fisher Scientific cannot guarantee proper column performance when the quality of the
chemicals, solvents and water used to prepare the mobile phase has been compromised.
Typically, the mobile phase system consists of an organic solvent (e.g. usually acetonitrile)
portion and an aqueous buffer (e.g. ammonium acetate, ammonium formate, phosphate buffer,
etc) portion. Both pre-mixed and proportioning valve generated mobile phases give satisfactory
results. The use of proportioning valve provides better flexibility in method optimization, while
the pre-mixed mobile phase provides more reproducible results.
Solvents
The solvents used must be free from ionic and UV-absorbing impurities. Use of ultrahigh purity
solvents, HPLC grade, will usually ensure that your chromatography is not affected by
impurities in the solvent.
De-ionized Water
The de-ionized water used to prepare the mobile phase should be Type 1 Reagent Grade water
or HPLC Grade water. The de-ionized water should be free of ionized impurities, organics,
microorganisms and particulate matter larger than 0.2 µm. Many commercial water purifiers are
designed for HPLC applications and are suitable for these applications.
NOTE
!
Degas the aqueous component of the mobile phase and then add the solvent component.
Avoid excessive purging or degassing of mobile phases containing solvents, if possible,
since the volatile solvent can be ‘boiled’ off from the solution.
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2– Step-By-Step User Guide
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Mobile Phase for Column Performance Test (QA test):
Mobile phase can be generated either by pre-mixing or by using a proportioning valve, both
give satisfactory results. The use of proportioning valve provides better flexibility in method
optimization, while the pre-mixed mobile phase provides less baseline noise.
100 mM ammonium acetate, pH5.0 Weigh 7.75 ± 0.03 g ammonium acetate, 2.00 ±
0.01 g acetic acid and 998.0 ± 0.5 g of D.I. water to
an eluent bottle. Mix well by sonication.
Mobile Phase A
(for both Column Performance Test and
Lot Qualification Test)
Acetonitrile/10 mM (total) ammonium
acetate, pH5, v/v 90/10
Weigh 100.0 ± 0.2 g of 100 mM ammonium acetate
buffer and 704.0 ± 0.5 g acetonitrile. Mix well by
sonication.
Step 3 – Set up the LC system
Use a standard LC system equipped with a LC pump, a column oven, a UV detector (or an ELS
detector depending on the application), and an injector (or an autosampler). The system should
be thoroughly primed before use.
Step 4 – Condition the column
Each new column is shipped in the mobile phase used for column performance test (containing
90% acetonitrile). Before use, the column should be washed thoroughly with the mobile phase
(~20 to 50 column volumes depending on the aqueous content in the mobile phase) before any
injection is made.
When switching to a different mobile phase, make sure that the new mobile phase is compatible
with the existing mobile phase in the column to avoid column clogging due to precipitation. A
good practice is to purge the column with 50% acetonitrile in D.I. water (v/v) for approximately
10 column volumes before switching to a new mobile phase
Step 5 – Reproduce the chromatogram in the Quality Assurance Report
Perform the column QA test using the conditions described in the Quality Assurance Report
(QAR), and compare the result with the reported values. The column should be fully
equilibrated before any injection. At least three injections should be made until reproducible
results are obtained.
NOTE
!
Due to various reasons, such as difference of LC systems, mobile phases, oven temperature
control, etc, you may observe somewhat different separation from that in the report.
Step 6 – Real sample analysis
Once you are satisfied with the column performance report result, the column is ready for your
application.