In addition to the orthogonal selectivity, kromasil hilic d offers a robust column that is 100% ms compatible. Highperformance liquid chromatography hplc has been widely used for years as an analytical method and is a key tool for the separation and analysis of pharmaceutical drugs, for drug monitoring. A simple, generally applicable hilic method development. Provide some tips and cautions as they related to method development in hilic mode aim. Hydrophilic interaction liquid chromatography hilic is a popular technique for the separation. Ace stepbystep hilic method development protocol hplc. Hydrophilic interaction liquid chromatography hilica. Another important analyte parameter is the pk a log k a value. Main interactions and influences of the chromatographic. Hplc column for hydrophilic interaction cosmosil hilic. Thermo scientific hypersil gold hilic, 5m, 150 x 2. Jan 11, 2020 high performance liquid chromatography hplc. Evaluation and optimization of zichilicrp as an alternative. These modes have been previously discussed in detail.
Hilic n phases and poor peak shape on the hilic a at low percentage organic. Hilic method development thermo fisher scientific uk. Describe the hilic system and prevailing theories on retention mechanisms. For historical reasons, it has been reported that hilic is a variant of normal phase liquid chromatography, but the separation mechanism used in hilic is more complicated than that in nplc. Hilic hplc and uhplc columns information thermo fisher. Hilic is typically used for the retention and separation of small, polar analytes. Hydrophilic interaction chromatography or hydrophilic interaction liquid chromatography, hilic is a variant of normal phase liquid chromatography that partly overlaps with other chromatographic applications such as ion chromatography and reversed phase liquid chromatography. Method development tools used in hydrophilic interaction. Hilic method development thermo fisher scientific us. Sequant hilic hydrophilic interaction liquid chromatography hplc columns constitute a range of highperformance tools for separating polar hydrophilic compounds. For many decades, it has played a key role in academic. Retention mechanism in zwitterionic hydrophilic interaction liquid chromatography zichilic studied. Chromabond hilic sequant hilic thf 97 % hilic polypropylene columns.
Selection guide of mobile phase comosil hilic column generates retention and separation by hydrophilic interaction mainly hydrogen bond and anionexchange. Zorbax rapid resolution ht high throughput columns up to 93% decrease in analysis time great resolution extended ph range, ph 1 to 9 a better way to faster separations. We show that zic hilic separation is dramatically dependent on buffer ph in the range from 3 to 8, due to deprotonation of acidic amino acids. The amide hilic bonded phases provide strong hydrogen bonding interaction between the stationary phase and the analytes, resulting in stronger retention compared to other hilic phases. Hilic for food, environmental, and other applications hydrophilic. Pdf retention mechanism in zwitterionic hydrophilic interaction. Hplc tn1039 increasing lcmsms sensitivity with luna hilic introduction the analysis of polar compounds in support of clinical and preclinical pharmacokinetic studies requires an analytical methodology capable of achieving ultralow detection and quantification limits. These columns are well suited for separating a variety of hydrophilic molecules, including carbohydrates and peptides. Principle, types, instrumentation and applications by editorial team on january 11, 2020 in biochemistry chromatography is a technique to separate mixtures of substances into their components on the basis of their molecular structure and molecular composition.
To ensure good chromatographic performance, it is recommended that sample solvent conditions be as close as possible to the initial mobile phase conditions, or at least to have an organic content greater than 50%. Nijo karasuma, nakagyoku, kyoto 6040855 japan tel fax website email. Agilent zorbax hilic plus hplc columns are nonbonded silica columns made with zorbax eclipse plus silica highpurity, type b silica, specially treated for good peak shape and optimized for hydrophilic interaction chromatography hilic separations. Our newest zorbax columns, rapid resolution ht, are. The high sensitivity afforded by coupling hplc with tandem mass. Gradient hplc for practitioners wiley online books. Injection solvent sample solvent too strong for the mobile phase. This column is the best choice for hilic applications, since it is most.
Hilic provides excellent results in the separation of both small and large. At the end of each chapter there is a list of references andor further reading which will help the reader to develop their expertise in the technique. Zic hilic exhibits a mixedmode effect consisting of electrostatic and polar interactions. Refer to following recommendations to select an appropriate mobile phase condition. Aug 31, 2011 hydrophilic interaction liquid chromatography hilic is an alternative highperformance liquid chromatography hplc mode for separating polar compounds. It is a popular analytical technique used for the separation, identification and quantification of each constituent of mixture. Hilic hplc columns hydrophilic interaction liquid chromatography hilic is a subset of normal phase chromatography where some percentage of the mobile phase contains water, and the organic solvents used are watermiscible. The active component of the column, the sorbent or the stationary phase, is typically a granular. Hilic hplc uhplc columns draw and retain a waterenriched layer onto the surface of the silica which facilitates the interaction of polar compounds with the stationary phase for increased retention. Significant improvements in peak shape were obtained at acetonitrile concentrations greater than 60%.
Background, mechanisms, method development 1 frank michel, david s. As a general recommendation for hilic standard methods, select a 100 or 150mm long column, packed with 3 5m particles and with a 4. The hilic a stationary phase is based upon the ultrapure ace silica platform. Highly consistent silica surface providing excellent batch to batch reproducibility. Hilic is a variant of normal phase chromatography 8 and typical stationary phases.
The ace hilic a column is an acidic character phase with an ionisable negative surface charge depending upon mobile phase ph. In addition to providing an introduction to hplc for pharmaceutical analysis it is intended that this book will be a useful resource. Hilic is generally recommended for compounds with negative log p values. Retention mechanism in zwitterionic hydrophilic interaction liquid chromatography zic hilic studied. Hplc is an advanced technique of column liquid chromatography. If manual injection performed, the loop should be rinsed with a volume at least 10. As one of the worlds most recognized hplc brands, luna is a high quality, industry standard that is incredibly dependable, batch to batch and column to column. Hydrophilic interaction chromatography it is a separation mode utilizing the interaction difference of hydrophilic properties between the compounds. With a highly versatile family of selectivities, luna stationary phases will help drive your method development and leave you at ease with existing methods. Retention, thermodynamics, selectivity, zone spreading, kinetics, and resolution torgny fornstedt, patrik forssen, and douglas westerlund liquid chromatography is a very important separation method used in practically all chemistry. An improved hilic hplcmsms method for the determination of. Sensitive detection of oligosaccharides after hplc analysis represents an.
Highly efficient analysis of underivatized carbohydrates. What basic difference is required for me to decide if i should use hilc or rp hplc. My experiment aim is nucleotide separation from serum. Hydrophilic interaction liquid chromatography hilic is gaining a great. All agilent zorbax columns are uniquely optimized for specific applications. The phase is low bleed and the solvents used when running your hplc in hilic mode are optimal for ms. Summary this chapter talks about an introduction of hydrophilic interaction liquid chromatography hilic in food and environmental analysis.
For mandelic acid, the peak shape was less affected by the diluent composition on the hilic b and hilic n phases, whilst on the hilic a, peak shape was poor at higher. Pdf the retention behavior of six highly polar compounds on. Hilic coupled with electrospray ionization esimass spectrometry hilicesims using the 200tpaam column was employed for the analysis of underivatized carbohydrates to achieve fast and efficient separations of mixtures containing mono, di, and. Available in c18, xbc18, c8, pfp, and hilic phases 2. The elution order in the hilic mode is in reverse order of reversed phase mode. High pressure liquid chromatography 20 chem 4 introduction chromatography can be described as a mass transfer process involving adsorption using a nonpolar stationary phase and a mobile polar phase titrating through the column. Hilic has been developing and evolving as a separation mode for retaining neutral and polar analytes poorly. High performance liquid chromatography which is also known as high pressure liquid chromatography. Discuss various hilic stationary phases and the interactions they exhibit 3. A robust hplc uhplc hilic column with acidic character.
Hilic as a first dimension for the analysis of complex peptide mixtures. If hilic is coupled to ms or cad detection, use a smaller column, e. Pharmaceutical formulation by hplc with diode array. Sequant hilic hplc columns are ideal for separating polar hydrophilic compounds, offering higher selectivity, favorable retention and robust performance.
K a gives us an indication of the acidity of a molecule. All columns carry densely bonded, truly zwitterionic functional groups with a charge balance of 1. Depending on the pk a values of the analytes, ph manipulation can greatly affect analyte ionization and thus dramatically change retention and selectivity. The diol derivatized kromasil hilic d provides excellent reproducibility when compared to hilic columns based on standard bare silica. The lc handbook guide to lc columns and method development the lc handbook guide to lc columns and method development agilent crosslab combines the innovative laboratory services, software, and consumables competencies of agilent technologies and provides a direct connection to a global team of scientific and.