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HPLC originally referred to the fact that high pressure was needed to generate the flow required for liquid chromatography in packed columns. Because sample compounds have differing characteristics, several different types of detectors are available. For example, if the compound can absorb ultraviolet light, then UV Absorbance Detector is used. If the compounds can fluorescence, then a Fluorescence Detector is used. If the compounds do not have either of these characteristics, then a more universal type of detector is used, such as Evaporative Light Scattering Detector (ELSD). In general, three primary characteristic of chemical compounds can be used to create HPLC separation:

Polarity Electrical Charge Molecular Size (see GPC)

Separation based on Polarity

Water [a small molecule with a high dipole moment] is a polar compound. Benzene [an aromatic hydrocarbon] is a non-polar compound. Molecules with similar chromatographic polarity tend to be attraction, if any, and may even repel one another. This becomes the basis for chromatographic separation modes based on polarity. Compounds in the sample that are similar in polarity to the stationary phase [column packing material] will be delayed because they are more strongly attracted to the particles. Compounds whose polarity is similar to that of the mobile phase will be preferentially attracted to it and move faster.

1. Normal Phase HPLC:
   For a normal-phase chromatographic separation the stationary phase is polar silica and the polar sample compounds will be attracted to the Polar Stationary Phase and slow down. The Non-Polar compounds in the sample will be attracted to the Non-Polar Mobile Phase and move faster to create separation. The mobile phase will be 100% organic solvent. No water is present.
2. Reversed-Phase HPLC:
   If we “reverse” the conditions used in Normal Phase chromatography, now the Stationary Phase is Non-Polar (hydrophobic), and the Mobile Phase is Polar. This is called Reversed-Phase chromatography. The sample compounds, which are Non-Polar, will be attracted to the Non-Polar Stationary Phase and slow down, while the Polar compounds in the sample will be attracted to the Polar Mobile Phase and move faster. Today, approximately 75% of all HPLC methods utilize Reversed-Phase chromatographic conditions because they tend to provide reproducible results. The most popular type of reversed-phase columns is a “C18” stationary phase.
3. Hydrophilic-Interaction [HILIC]:
   HILIC utilize a blend of water (aqueous) with a miscible organic solvent to insure the proper interaction of the sample compounds with the non-polar, hydrophobic particle surface. In normal-phase chromatography, the mobile phase is 100% organic. Only traces of water are present in the mobile phase and in the pores of the polar packing particles. Polar compound bind strongly to the polar stationary phase and may not elute.
   Adding some water [<20%] to the organic mobile phase [typically acetonitrille] make it possible to separate and elute polar compounds that are strongly retained in the normal-phase mode [or weakly retained in the reversed-phase mode]. Water competes effectively with polar analytes for the stationary phase. HILIC may be run in either isocratic or gradient elution modes. Polar compounds that are initially attracted to the polar packing material particles can be eluted as the polarity [strength] of the mobile phase is increased [by adding more water]. Analytes are eluted in order of increasing hydrophilicity [chromatographic polarity relative to water]. Buffers or salts may be added to the mobile phase to keep ionizable analytes in a single form.
4. Hydrophobic-Interaction Chromatography [HIC]:
   HIC is a type of reversed-phase chromatography that is used to separate large biomolecules, such as proteins. It is usually desirable to maintain these molecules intact in an aqueous solution, avoiding contact with organic solvents or surface that might denature them. HIC takes advantage of the hydrophobic interaction of large molecules with modearately hydrophobic stationary phase, e.g., butyl-bonded [C4], rather than octadecyl-bonded [C18], silica. Initially, higher salt concentrations in water will encourage the proteins to be retained [salted out] on the packing. Gradient separations are typically run by decreasing salt concentration. In this way, biomolecules are eluted in order of increasing hydrophobicity.

Separation Based on Charge

1. Ion-Exchange Chromatography [IEC] :
   For separation based on polarity, like is attracted to like and opposites may be repelled. In ion-exchange chromatography and other separations based upon electrical charge, the rule is reversed. Kikes may repel, while opposites are attracted to each other. Stationary phases for ion-exchange separations are characterized by the nature and strength of the acidic and basic functions and the types of ions that they attract and retain. Cation exchange is used to retain and separate positively charged ions on a negative surface. Conversely, anion exchange is used to retain and separate negatively charged ions on a positive surface. With each type of ion exchange, there are at least two general approaches for separation and elution. Strong ion exchange bear functional groups [e.g., quartenary amines or sulfonic acids] that are always ionized. They are typically used to retain and separate weak ions. These weak ions may be eluted by displacement with a mobile phase containing ions that are more strongly attracted to the stationary phase sites. Alternately, weak ions may be retained on the column, then neutralized by in situ changing the pH of the mobile phase, causing them to lose their attraction and elute. When weak ion exchangers are neutralized, they may retain and separate species by hydrophobic [reversed-phase] or hydrophilic [normal-phase] interactions; in these cases, elution strength is determined by the polarity of the mobile phase. Thus, weak ion exchangers may be used for mixed-mode separation [separations based on both polarity and charge]. For example, to retain a strongly basic analyte [always positively charged], use a weak-cation-exchange stationary phase particle at pH>7; this assure a negatively charged particle surface. To release or elute the strong base, lower the pH of the mobile phase below 3; this removes the surface charge and shuts off the ion-exchange retention mechanism. Note that a pKa is the pH value at which 50% of the functional groups is ionized and 50% is neutral. To assure an essentially neutral, or fully charged, analyte or particle surface, the pH must be adjusted to a value at least 2 units beyond the pKa, as appropriate.
   
   Do not use a strong cation exchanger to retain a strong base; both remain charged and strongly attracted to each other, making the base nearly impossible to elute. Very strong acids and bases are dangerous to work with, and they may be corrosive to materials of construction used in HPLC fluidics.

Equipment
Pump: Waters Alliance HPLC System (Waters 2695 Separation Module) Stationary phase: See Manual Columns: See Manual Detection:       Waters 2998 Photodiode Array Detector w/ICS       Waters 2424 ELS Detector Solvent: Organic solvents, water, water/acetonitrile mixtures. Flow Rate: 1 mL/min Injection: 10 µL. Autosampler: five carousels with total capacity of 120 vials

Please contact Krystyna Brzezinska (kbrzez@mrl.ucsb.edu) to schedule training. Before training starts please read HPLC MANUAL or HPLC Tips.

Manufacturer:

Waters Corporation 43 Maple Street Milvord, MA 01757, USA Telephone: 800-252-4752 Internet - http://www.waters.com

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