Home » Process-descriptions » HILIC Chromatography

HILIC Chromatography

What topics are discussed on this page

  1. Why do we need chromatography for polar compounds
  2. Historical evolution of HILIC
  3. What is HILIC
  4. HILIC separation mechanism
  5. What type of phases can be used for HILIC
  6. Suppliers of HILIC Phases
  7. Dual process possibilities with PolyLC Columns
  8. Operating Recommendations for HILIC Separations with PolyHYDROXYETHYL Aspartamide
  9. Operating Recommendations for SEC Separations with PolySULFOETHYL Aspartamide
  10. Aqueous normal-phase chromatography (ANP) with Silica Hydride Phases
  11. Features of Silica Hydride phases
  12. Application examples
  13. Zwitter Ionic HILIC Phases
  14. How to work effectively with HILIC and ANP

Why do we need chromatography for polar compounds

HILIC is used for separation of polar (hydrophilic) and apolar (hydrophobic) compounds
Polar compounds play a significant role as:

Pharmaceuticals
Components in biological processes
Metabolites
Clues in forensic identification
Biomarkers of disease
Indicators in food safety and nutrition

Analysis of polar compounds often involves complex matrices
These include:

plasma
urine
saliva
tissue
food products
other biological materials
crude extracts
peptide digests,

And they usually require some sample preparation such as:

selective precipitation
liquid-liquid extraction
solid phase extraction

In these matrices we find a wide range of highly polar molecules including

Peptides, Peptide Mapping
Amyloid Peptide
Lipopeptides
Synthetic and Natural Peptides
Glycopeptides
Phosphopeptides
Carbohydrates, Complex Carbohydrates
Histones
Polar lipids
Phospholipids
Oligonucleotides and their antisense analogs
Nucleic acids and
Many proteins. membrane proteins,
Organic acids
Dyes

Some call it Aqueous Normal Phase (ANP) Chromatography

Historical evolution of HILIC

First HILIC experiments were done in 1975 separating sugars on an amino column.
The term HILIC was invented by Dr. Andrew Alpert of PolyLC, Colubia, MD, in his original paper (J. Chrom. 499, pp 177 – 196, 1990). Alpert defines Hydrophilic Interaction Chromatography as variant of normal-phase chromatography that can be performed using polar stationary phases with partially aqueous eluents. Solutes elute in order of increasing hydrophilicity (polarity), this is opposite of reversed-phase LC. The main interaction of HILIC may be “electrostatic interaction”.HILIC began to expand rapidly after several commercial manufacturers introduced columns like silica, amino and cyano that retained polar compounds in high organic content mobile phases.

What is HILIC?

Hydrophilic Interaction Chromatography sometimes refered to as Aqueous Normal Phase Chromatography (ANP) is a variant of normal-phase chromatography is performed using polar stationary phases with aqueous buffers and organic modifiers as they are used in RP Chromatography.

HILIC comprises of

  • A column with a hydrophilic stationary phase
  • An eluent with water, buffer and a high concentration of water-miscible organic solvent

Solutes elute in order of increasing hydrophilicity (polarity), that is the opposite of reversed-phases. A compound that elutes in the void volume on a RPLC column typically has high retention in HILIC, and vice versa. HILIC is also used for separating some biomolecules by their differences in polarity. Compounds such as, acids, bases, ions, sugars, and other charged and neutral hydrophilic compounds that are troublesome to separate in RPLC, are much easier to separate in HILIC due to the different separation selectivity. 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. HILIC can be used with many detection techniques, but when combined with electro spray ionisation mass spectrometry (ESI-MS) for example, HILIC will also enable higher sensitivities. A low proportion of water in the eluent generates a stagnant aqueous layer on the surface of the stationary phase. This enables solutes to partition between the eluent and aqueous layers. In addition, weak electrostatic interactions between solute and stationary phase contribute to overall selectivity

HILIC Mechanism

HILIC MechanismSchematic picture of a adsorbed diffuse water layer at the surface of a phase in a highly organic environment. Analytica Chimica Acta 692 (2011) 1 – 25

What type of phases can be used for HILIC

Any polar chromatographic surface may be used for Water / Solvent (ACN) mixture separations. Even non-polar bonded silicas have been used with extremely high organic solvent composition, when the silica used for the chromatographic media was particularly polar.

The following commercial columns are being sold for HILIC application:

Un-bonded silica, Diol and Cyanopropyl bonded phases
Amide bonded phases
Amino or anionic bonded phases
Cationic bonded phases
Zwitter-ionic bonded phases (+ and – charge)
Silica hydride based phases
Polymer material with polar functional groups

Suppliers of HILIC Phases

Phase
Manufacturer
Functional groups
Particle Size (um)
Pore Size A
BioBasic AX
Thermo Scientific
Polyethyleneimine
5
300
Cosmosil HILIC
Nacalai Tesque
Triazole
5
120
Epic HILIC-HC
ES Industries
Polyhydroxylated polymer
3,5,10
120
HALO HILIC
Advanced MaterialsTechnology
?
2,7
90A on solid core
Hypersil GOLD HILIC
Thermo Scientific
Proprietary
1.9, 3.5
175
Inertsil HILIC
GL Sciences
Propyl alcohol
5
100
NUCLEODUR HILIC
Macherey-Nagel
Zwitterionic ammonium sulphonic acid
1.8, 3.5
110
Obelisc N
SIELC
Proprietary
5, 10
100
PolyGLYCOPLEX
PolyLC5, 12PolyHYDROXYETHYL A
Hydroxyethylaspartamide
3, 5, 12
60, 100, 200, 500, 1000
PolySULFOETHYL A
Sulfoethylaspartamide
3, 5, 12
300, 1000
A TSKgel Amide-80
Tosoh Bioscience
Carbamoyl
3,5,10
80, 100
Ultisil HILIC Amide
Welch MaterialUltisil Amphion
tertiary amine and sulfonic acid
3,5,10
120
ZIC-HILIC
Merck SeQuant
Sulfobetaine
3.5, 5,10
100, 200
ZIC-pHILIC
Sulfobetaine
5
200

Silica based columns are not ideal for HILIC application because of the intrinsic problems with the Silanol groups. Better are phases with coated or chemically modified Silanol groups. Below we like to discuss successful peptide separation with products from PolyLC, – the inventors of HILIC. Silicahydride based columns from Microsolv are ideal HILIC products with exponential growth in the field of metabolomics. Dual process possibilities with PolyLC Columns 

The inventor of HILIC .(Dr. Andrew Alpert ) recommends two column choices to perform HILIC separations, and can be extended to perform additional separation mechanism for added versatility.

The PolyHYDROXYETHYL Aspartamide column (HEA) will retain solutes solely through hydrophilic interactions when using mobile phase concentrations in the range of 40-85% acetonitrile. Under non-HILIC conditions (mobile phase concentrations less than 40% acetonitrile) the column will perform small-molecule size exclusion separation. (SEC)

The second column is the PolySULFOETHYL A spartamide SCX column which performs either hydrophilic interaction separations superimposed upon electrostatic effects under HILIC conditions as above, or a cation exchange mixed-mode separation where resolution is enhanced for peptides with the same net positive charge under non-HILIC conditions.

Furthermore some organic and some inorganic molecules[2] can also be separated successfully with HILIC.

Its utility has increased due to the simplified sample preparation for biological samples, when analysing for metabolites, since the metabolic process generally results in the addition of polar groups to enhance elimination from the cellular tissue. For the detection of polar compounds with the use of electrospray-ionization mass spectrometry as a chromatographic detector, HILIC can offer a tenfold increase in sensitivity over reversed-phase chromatography[2] because the organic solvent is much more volatile
Mobile phase to be used

A typical mobile phase for HILIC chromatography includes acetonitrile (“MeCN”, also designated as “ACN”) with a small amount of water. However, any aprotic solvent miscible with water (e.g. THF or dioxane) can be used. Alcohols can also be used, however, their concentration must be higher to achieve the same degree of retention for an analyte relative to an aprotic solvent – water combination.

Additives

Ionic additives, such as ammonium acetate and ammonium formate, are usually used to control the mobile phase pH and ion strength. In HILIC they can also contribute to the polarity of the analyte, resulting in differential changes in retention. For extremely polar analytes (e.g. aminoglycoside antibiotics (gentamicin or Adenosine triphosphate), higher concentrations of buffer (ca. 100mM) are required to assure that the analyte will be in a single ionic form. Otherwise asymmetric peak shape, chromatographic tailing, and/or poor recovery from the stationary phase will be observed. For the separation of neutral polar analytes (e.g. carbohydrates), no buffer is necessary.
Use of other salts such as 100-300mM sodium perchlorate, which are soluble in high-organic solvent mixtures (ca. 70% acetonitrile), can be used to increase the mobile phase polarity to effect elution. These salts are not volatile, so this technique is less useful with a mass spectrometer as detector. Usually a gradient (to increasing amounts of water) is enough to promote elution
All ions partition into the stationary phase to some degree, so an occasional “wash” with water is required to ensure a reproducible stationary phase.

As shown in the Mechanism above the mobile phase forms a water-rich layer on the surface of the polar stationary phase vs. the water-deficient mobile phase, creating a liquid/liquid extraction system. The analyte is distributed between these two layers. However, HILIC is more than just simple partitioning and includes hydrogen donor interactions between neutral polar species as well as weak electrostatic mechanisms under the high organic solvent conditions used for retention. This distinguishes HILIC as a mechanism distinct from ion exchange chromatography. The more polar compounds will have a stronger interaction with the stationary aqueous layer than the less polar compounds. Thus, a separation based on a compound’s polarity and degree of solvation takes place.

Operating Recommendations for HILIC Separations with PolyHYDROXYETHYL Aspartamide

Initial Use
When using either column to perform HILIC separations, flush the new column with 25 ml water, and then condition with at least 60 ml of a buffer solution with a salt concentration of 0.2 – 0.4 M and a pH in the range of 3 – 6 (exact figures are not important here). Flush again with another 20 ml water, then equilibrate with 30 ml of the mobile phase before injecting samples. (These volumes relate to 4.6 mm ID columns. For 9.4 mm ID columns, the volumes above should be multiplied by a factor of four).

To prepare the HEA column for size exclusion chromatography or the SCX column for ion exchange chromatography, see the appropriate section below).

Routine Use

Filter samples and mobile phases before use. Flush and store HILIC columns in water when not in use. Operation at room temperature is recommended, since elevated temperature shortens column lifetime.

General Mode of Operation

Salt is not required with solutes that are not electrolytes. In the case of electrolytes, use at least 5-10 mM buffer in the mobile phase. Gradient elution can be accomplished by a decreasing organic gradient (starting from 80-85% acetonitrile for peptides or 95% for phospholipids) or an increasing salt gradient (which typically gives flatter baselines). Solubility of salts can be a problem with mostly organic mobile phases, but sodium perchlorate works well and is transparent at low wavelengths . Buffer salts with reasonable solubility in 80% acetonitrile include triethylamine phosphate (TEAP) and sodium methylphosphonate (from methylphosphonic acid).

Isocratic retention is typically several times greater with TEA salts than with the corresponding sodium or potassium salt. With 80% acetonitrile, concentrations of 75 mM (pH 5.0) or 100 mM (pH 2.8) TEAP are attainable.

Gradient elution in HILIC generally requires one-fifth to one-tenth the concentration of salt required in ion-exchange chromatography.
A stock solution of TEAP is prepared by making a concentrated aqueous solution of phosphoric acid, adding TEA until the desired pH is attained, then diluting to give a known final concentration (e.g., 2 M in phosphate). Similar methods are used with phosphonate buffers. Prepare stock solutions fresh monthly and store in the refrigerator. For preparation of the mobile phase, add the appropriate amount of stock solution and water to a volumetric flask. Next, add the acetonitrile to a level several ml short of the mark. Mix, then put the flask in a sonication bath for 5 minutes, this degasses and warms the solution. Finally, add acetonitrile to the mark and mix.

Organic solvents such as isopropanol can be used as alternatives to acetonitrile, but higher concentrations are usually required to attain the same degree of retention, and the resulting mobile phases are appreciably more viscous.
HILIC of AminoacidsHydrophilic Interaction Chromatography (HILIC) of Amino Acids
COLUMN: PolySULFOETHYL Aspartamide, 200 x 4.6-mm
Mobile Phase: 5 mM TEAP, pH 2.8, in 80% ACN
Order of elution: Least to most polar
HILIC of Peptide and Proteins
A good mobile phase to try is 10 mM TEA, pH 2.8, containing 80% acetonitrile. Run gradients as described above. If retention in inadequate, try 85% acetonitrile.
The following factors affect retention of peptides in HILIC:
1. Retention is proportional to the hydrophilicity of a peptide: Basic groups are the most hydrophilic, followed by phosphorylated residues. Thereafter, retention follows the opposite trend seen with reversed-phase HPLC: Asn promotes retention the most, followed by Ser-, Gly-, etc., with Phe- and Leu- promoting retention the least.
2. Juxtaposition of an acidic and basic residue: An acidic and a basic residue, or an acidic residue as the N-terminus, largely eliminates the normal retention effects of a basic residue.
3. Change in polarity with a change in pH: At pH 2.8, only basic and phosphorylated groups will be charged. At pH 5.0, both acidic and basic residues will be charged. This factor can be used to manipulate selectivity.
4. Retention proportional to the number of basic residues: In general, at pH 2.8 peptides will elute in order of increasing number of basic residues, as do cation-exchange separations. However, unlike cation-exchange, a particularly hydrophilic peptide can be retained more strongly than a hydrophobic peptide with more basic residues. Thus, the selectivity of the two methods is complementary.
HILIC of Sugars and Oligosaccharides:
No salt is necessary unless the carbohydrate is charged. The mobile phase should contain 80-85% acetonitrile (with much lower levels used with amino- sugars). Anomeric forms of reducing sugars are resolved.
HILIC of Oligonucleotides
Try a salt gradient in 75% acetonitrile. C and G are retained much more than A and T, and may necessitate lower levels of acetonitrile.
HILIC of Phospholipids:
Try a mobile phase of 15 mM ammonium formate pH 6.5 and 95% acetonitrile decreasing to 50%. Selectivity depends upon the pH and ionic strength.HILIC of Drugs, Small Molecules and Miscellaneous Metabolites
Retention will be the opposite of that with reverse-phase HPLC. Initially, try mobile phases with 80% acetonitrile. Some experimentation with the salt level and pH will be necessary in each case.
Volatile Mobile Phases and Sequencing
The presence of 5-10 mM nonvolatile buffer salt does not interfere with many sequencing techniques for peptides. If a completely volatile mobile phase is needed, as for mass spectroscopy, then ammonium formate can be used as the buffer salt, with a descending acetonitrile gradient. Unfortunately, formate absorbs and gives baseline artifacts in gradient elution at low wavelengths.
No such problems are encountered at 254 or 280 nm.
NOTE: If the mobile phase contains over 80% organic solvent, then the sample should contain at least 70%. Otherwise, pure solutes may elute in multiple peaks.
Operating Recommendations for SEC Separations with PolySULFOETHYL Aspartamide
Background on SEC Use
The PolyHYDROXYETHYL Aspartamide column was created specifically to perform HILIC and retain solutes solely through hydrophilic interactions. However, when used with a mobile phase which does not contain enough organic solvent to induce hydrophilic interaction, then it functions in the size exclusion chromatography mode. Swelling the coating with a suitable mobile phase causes the effective pore diameter to become the spacing between polymer chains of the coating (~15Å), allowing solutes as small as water to be separated by size.
The HEA column is available in three pore sizes, 200Å, 300Å and 1000Å. A column with 200Å pores has a fractionation range of 20-10,000 MW allowing resolution of the smallest of bioorganic molecules. The 300Å pore size accommodates a range of 20-80,000, and the 1000Å pore material can fractionate over a size range of 1,000 – 2,000,000, (over 5 orders of magnitude). The HEA columns may also be used with volatile mobile phases.
Start-up
Flush new columns (4.6 mm ID) with 25 ml water, then condition with at least 60 ml of a buffer solution with salt concentration of 0.2 M and a pH in the range of 3-6 (exact figures are not important here). Flush with another 20 ml water, then equilibrate for six hours (flow rate 0.5 ml/min) using one of the mobile phases recommended below before injecting samples. (For 9.4 mm ID columns, the above volumes should be multiplied by a factor of 4, and the flow rate for equilibration is 2 ml/min.) It is not necessary to repeat this conditioning step thereafter unless the column is flushed with organic solvent for long-term storage or used under HILIC conditions.
HEA Columns will exhibit two different fractionation ranges, depending on the mobile phase used. For a mobile phase of 0.2 M (Na)2SO4 + 5 mM K-PO4, pH 3.0, containing 25% acetonitrile, the fractionation range will be approximately Mol. Wt. 400-10,000 for columns with 200Å pores. For HEA columns with 300Å pores, the fractionation range is approximately MW 1000-200,000.
For a mobile phase of 50 mM formic acid, the fractionation range will be approximately MW 20 – 1000 for columns with 200Å pores. For 300Å columns, MW 20 – 80,000. The same column can be used for both fractionation ranges, simply by switching between these two mobile phases. The formic acid mobile phase is volatile, but precludes detection below 240 nm. The use of volatile mobile phases which are transparent at 215 nm (e.g. hexafluoro-isopropanol, HFIP) is experimental and hazardous, although it has the same effect on the fractionation range as formic acid.
Sample Composition
The sample solvent should not differ greatly from the mobile phase in ionic strength or organic solvent content, in order to prevent a significant difference in viscosity of the two. With high viscosity, solute molecules might not diffuse from the mobile phase to the stationary phase before the sample passes through the column. The loading capacity of a 4.6 mm I.D. column is roughly 0.4 – 0.8 mg peptide with no significant loss of resolution, but this number depends on the composition of the sample.
Operating Recommendations for SCX Separations
The PolySULFOETHYL Aspartamide SCX column in the ion exchange mode is useful for n-terminal variant analysis, neuropeptides, growth factors, CNBr peptide fragments, and synthetic peptides as a complement to RPC.
Initial Use
Flush the methanol storage solvent from the column with at least 40 ml water before elution with salt solution to prevent salt precipitation. Then elute the column with a strong buffer for at least one hour prior to its initial use. A convenient solution to use is 0.2 M monosodium phosphate + 0.3 M sodium acetate
Routine Use
Filter samples and mobile phases before use. Flush and store the column in water when not in use. Operation at room temperature is recommended, since elevated temperature shortens column life-time. Use of 0.1% TFA or high concentrations of formic acid in the mobile phase is not recommended. The conditioning process is reversed by exposing the column to pure organic solvents. Accordingly, to minimize the time to start the column for a 1-2 day storage, the column should be flushed with at least 40 ml of deionized water (not methanol), and the ends should be plugged. For extended storage it is recommended that a 100% methanol storage be used to prevent bacterial growth and contamination. Exercise care when using organic solvents to prevent precipitation of salts.
General Operation in the SCX Mode
By varying the pH, ionic strength or organic solvent concentration in the mobile phase, selectivity can be significantly enhanced. Mobile phase modifiers help to improve peptide solubility or to mediate the interaction between peptide and stationary phase. For more strongly hydrophobic peptides, a non-ionic surfactant (at a concentration below its CMC) and/or acetonitrile or n-propanol as mobile phase modifiers, can substantially improve resolution and recovery over conventional reverse phase methods. You may obtain additional selectivity by simply changing the slope of the KCl or (NH4)2SO4 gradient.
Using this column at pH 3 is better for retention of neutral to slightly acidic peptides. Use of a higher pH may be considered for basic hydrophobic peptides.
Adding MeCN or propanol to the A&B solvents changes the separation mechanism and results in a separation based not only on positive charge, but also on hydrophobicity. Experimentation with organic content is encouraged.
One set of operating conditions for these applications for an analytical column would be:
Buffer A: 5 mM K-PO4 + 25% MeCN;
Buffer B: 5 mM K-PO4 + 25% MeCN + 300-500 mM KCl;
Linear gradient, 30 min at 1 ml/min.Peptides are retained by the positive charge of at least the n-terminal amine and eluted by a combination of total charge, charge distribution and hydrophobicity. If your peptide does not stick to the column, be sure it is in a small amount of buffer, or decrease the concentration of organic in the A&B solvents to 10 or 5%. (Organic solvent concentration is empirically determined).
Since total binding capacity is on the order of 100 mg/gm of packing (for nonresolved materials) there will be a considerable Donan effect present. To prevent exclusion from the column put your sample in 5-15 mM of salt or buffer. Additionally, the gradient at the outlet of the column will be much more concave than that observed on the chart paper. Consequently, if you have had no prior experience using this column, we recommend following a standard methods development protocol to be sure that your protein is eluting properly (request publication P4). We recommend an upper load limit of 1 milligram for an analytical column, although up to 40 mg of a soluble peptide has been separated on one.
When using a guard column as a methods development column, we recommend a load limit of one-tenth of a milligram with gradient times shortened to 8-10 min at the same flow rate since the void volume is only 0.3 ml.
References
1. Alpert, A.J., “Hydrophilic Interaction Chromatography (HILIC): A New Method for Separation of Peptides, Nucleic Acids, and Other Polar Solutes,” J. Chromatogr. 499, 177-196 (1990). doi:10.1016/S0021-9673(00)96972-3.
2. Zhu, B., Mant, C., Hodges, R., “Mixed-Mode Hydrophilic and Ionic Interaction Chromatography Rivals Reversed-Phase Chromatography for the Separation of Peptides,” J. Chromatog., 594, 75-86 (1992).
3. Boutin et al., HILIC of phosphorylated peptides and tyrosine kinase reaction mixtures, submitted to Anal. Biochem.
4. Fong et al., HILIC and SCX of Hydroxyproline-rich peptides from Douglas Fir cell wall proteins, submitted to Plant Physiol.
5. Kieliszewski et al., HILIC and SCX of Hydroxyproline-rich peptides from cell wall proteins of Zea maize (corn), submitted to Plant Physiol.
6. Przysiecki et al., HILIC, SEC and SCX of recombinant antistasin with a preproleader sequence, submitted to Arch. Biochem. Biophys.
7. Przysiecki et al., HILIC, SEC and SCX of recombinant antistasin with a preproleader sequence, submitted to Arch. Biochem. Biophys.
8. Przysiecki et al., Characterization of Recombinant Antistasin Secreted by Saccharomyces cerevisiae, “Protein Expression & Purification”, 3,.185-195 (1992).
a b Eric S. Grumbach et al. (October 2004). “Hydrophilic Interaction Chromatography Using Silica Columns for the Retention of Polar Analytes and Enhanced ESI-MS Sensitivity”. LCGC Magazine. http://www.lcgcmag.com/lcgc/issue/issueDetail.jsp?id=4734. Retrieved on 2008-07-14.Silica Hydride Phases for Aqueous Normal-Phase chromatography (ANP)
What’s the difference?
The “Silicahydride surfaces” are different from silica surfaces. Silica materials used for chromatography have a surface composed primarily of silanols (-Si-OH). In a “hydride surface” the terminal groups are primarily -Si-H. The hydride surface can also be functionalized with carboxylic acids[1] and long-chain alkyl groups[2]. Mobile phases for ANPC are organic solvent (such as methanol or acetonitrile) with a small amount of water; hence, the mobile phase is both “aqueous” (water is present) and “normal” (less polar than the stationary phase). Thus, polar solutes (such as acids and amines) are most strongly retained, with retention decreasing as the amount of water in the mobile phase increases.
Typically the amount of the nonpolar component in the mobile phase must be 60% or greater with the exact point of increased retention depending on the solute and the organic component of the mobile phase. A true ANP stationary phase will be able to function in both the reversed phase and normal phase modes with only the amount of water in the eluent varying. Thus a continuum of solvents can be used from 100% aqueous to pure organic. ANP retention has been demonstrated for a variety of polar compounds on the hydride based stationary phases (J.J. Pesek, M.T. Matyska, J. Sep. Sci., in press[citation needed]).
Features of Silica Hydride phases
An interesting feature of Hydride phases is that both polar and nonpolar compounds can be retained at high concentration over a broad range of mobile phase composition (organic/aqueous). The retention mechanism of polar compounds has not been elucidated yet for the silica hydride phases. This property distinguishes it from a pure HILIC (hydrophilic interaction chromatography) column where separation by polar differences is obtained, or a pure RP stationary phase on which separation by nonpolar differences in solutes is obtained with very limited secondary mechanisms operating.
With hydride-based phases most polar compounds such as bases can be analysed in neutral to acidic conditions. it is usually not necessary to use a high pH mobile phase for analysis. The aqueous component of the mobile phase usually contains from 0.1 to 0.5% formic or acetic acid, which is compatible with detector techniques that include mass spectral analysis.
Silica hydride offer great separation flexibility
Most silica hydride particularly those with hydrophobic ligands (C18, C8, Cholesterol) also retain non-polar compounds. This dual retention mechanism is excellent for retaining mixtures of polar and apolar compoundsCompound with high hydrophobicity such as Glyburide
Highly polar Compounds such as Metformin
Ideal for mixtures of highly polar and apolar compounds
Easy to use
Step 1. Pre-Conditioning the column with a typical reverse phase run e.g.
Eluent A : DI Water + up to 0.5% Acid (Formic, Methyl Phosphonic Acid or Acetic Acid if you dont use LCMS, For LC/MS applications use up to 0.1 % TFA )
Eluent B: Acetonitrile + up to 0.5% Acid (Formic, Methyl Phosphonic Acid or Acetic Acid if you dont use LCMS, For LC/MS applications use up to 0.1 % TFA )
Run 6 column volume of the mobile phase at 95% Eluent A
Step 2. Reverse Phase Mode run
Set up your instrument to run a shallow gradient from 95 % Eluent A to 40 % Eluent A over 20 minutes for 75 mm column or 40 minutes for a 150 mm column
Step 3. Solvent Run
Run column with 100 % Eluent B for 2 minutes. for 75 mm or 4 minutes a 150 mm column
Step 4 Aqueous Normal Phase Mode run
Set up your instrument to run a shallow gradient from 90 % Eluent B (Acetonitrile) to 40 % Eluent B over 20 minutes for 75 mm column or 40 minutes for a 150 mm column
Step 5. Evaluation
Evaluate both gradient runs for retention time, peak shape and elution order. Some compounds may not retain either in Reverse Phase or ANP mode. Revert to a isocratic mode
What to do if above method does not work satisfactorily
1. Some Organic Acids do not retain at low pH in ANP mobile phase. Change to high pH ANP mobile phase for retention. A rule of thumb is that for acids you would want to be from 1 – 2 pH units above the pKa. Test the following Buffers: Ammonium Acetate, Ammonia, Sodium Acetate , Ammonium Formate or Sodium Formate.
2. Solvents used in ANP have different resolving power for acids and bases. Acetone, Acetonitrile, THF, Ethanol, Acetonitrile/Methanol, Methanol and then Water will have a decending strength of retention in ANP.
3. All Cogent Type C column have the ability to to perform in ANP mode. Depending on your sample mixture and matrix, different columns will produce different peak shapes, retention power and selectivity. Ranging from Silica C, Bidentate C8, UDC-Cholesterol and Bidentate C18, the amount of reverse phase (hydrophobicity) will increase in this order.
4. Equilibration between gradient runs. Although the Cogent Type C columns all equilibrate quickly, the mobile phase solvent you are using will determine how much equilibration time you may need between runs. Hydride surface has a strong affinity to Methanol and thus it may take longer to equilibrate if Methanol is used in a gradient and not at the beginning. To equilibrate use at the starting point
Column Care : See under section Column Care
ANP is highly efficient to retain polar compoundsThe powerful anticancer drug, methotrexate (4-amino-N10-methylpteroyl glutamic acid) acts as an antimetabolite and is used for the treatment of many neoplastic diseases including acute leukemia, osteosarcoma, non-Hodgkins lymphoma, and breast cancer.
Inverse Gradient Program
A: DI Water + 0.5% Formic Acid
B: AcetonitrileANP for Fast Equilibration and RepeatabilityREPEATABILITY AND FAST EQUILIBRATION ARE COMMON FEATURES OF AQUEOUS NORMAL PHASE ANALYSES ON SILICA HYDRIDE TEN CONSECUTIVE ANALYSES OF GLUCOSAMINE ON LOW CARBON SILICA HYDRIDE PHASE WITH A ONE COLUMN VOLUME EQUILIBRATION PERIOD
Application examples
ANP in Drug Quality Control
Determination of impurities in tablet formulation containing metforminColumn: low carbon silica hydride, 4 μm ,100 Å Dimensions: 2.1 x 150 mm
Solvents:
A: 50% 2-propanol/50% DI water/0.1% acetic acid
B: acetonitrile/0.1% acetic acid
Gradient: Time (min) % B 0.0 100 2.0 100 5.0 20 9.0 20 10.0 100
Peaks:
1.Cyanoguanidine 85.0509 m/z (M +H)+
2.Melamine 127.0727 m/z (M +H)+
3.Metformin 130.1087 m/z (M +H)+A: Single gradient run
B: 4 overlaid injectionsANP for Food AnalysisHistamine, determination in red and white wine, analysis done without derivatization
Method conditions
Column: low carbon silica hydride column, 4 μm, 100 Å
Dimensions: 2.1 x 150 mm
Solvents
A: 50%DI water+50% 2-propanol / 0.1% formic acid
B: acetonitrile / 0.1% formic acid
Gradient:
Time (min) % B
0.0 80
5.0 10
7.0 10
8.0 80
Peak: 1 – histamine 112.0869 m/z,
Figure A – histamine standard
Figure B- white wine
Figure C – red wine. Some red wines are low in histamine
Detection: ESI – POS – TOF mass spectrometer.ANP for Toxin in Biological Matrix
Toxic Pseudo-nitzschia australis diatoms (algae), neurotoxin domoic acid ,L- tryptophan. L-leucine and L-histidineANP for Environmental AnalysisAPPLICATIONS OF ANP – ENVIRONMENTAL
Column:
low carbon silica hydride
Dimensions: 2.1 x150 mm
Solvents:
A: DI water + 20 mM ammonium acetate pH adjusted to pH 3.3 with formic acid
B: Acetonitrile
Mobile phase: 70% A
NORMAL PHASE WITH 70% WATER
Peaks
1. Chlormequat, 122.0737 m/z (M)+
2. Mepiquat, 114.1277 m/z (M)+
CQ and MQ are charged in solution and with ESI the mass spectra show an abundant molecular ion (M)+. Zwitter Ionic HILIC Phases
Phase
Manufacturer
Functional groups
Particle Size (um)
Pore Size A
BioBasic AX
Thermo Scientific
Polyethyleneimine
5
300
Cosmosil HILIC
Nacalai Tesque
Triazole
5
120
Epic HILIC-HC
ES Industries
Polyhydroxylated polymer
3,5,10
120
HALO HILIC
Advanced MaterialsTechnology
?
2,7
90A on solid core
Hypersil GOLD HILIC
Thermo Scientific
Proprietary
1.9, 3.5
175
Inertsil HILIC
GL Sciences
Propyl alcohol
5
100
NUCLEODUR HILIC
Macherey-Nagel
Zwitterionic ammonium sulphonic acid
1.8, 3.5
110
Obelisc N
SIELC
Proprietary
5, 10
100
PolyGLYCOPLEX
PolyLC5, 12PolyHYDROXYETHYL A
Hydroxyethylaspartamide
3, 5, 12
60, 100, 200, 500, 1000
PolySULFOETHYL A
Sulfoethylaspartamide
3, 5, 12
300, 1000
A TSKgel Amide-80
Tosoh Bioscience
Carbamoyl
3,5,10
80, 100
Ultisil HILIC Amide
Welch MaterialUltisil Amphion
tertiary amine and sulfonic acid
3,5,10
120
ZIC-HILIC
Merck SeQuant
Sulfobetaine
3.5, 5,10
100, 200
ZIC-pHILIC
Sulfobetaine
5
200