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HPLC Instrument:
reservoir of mobile phase, a pump, an injector, a separation column, a detector.
Compounds are separated by injecting a sample onto the column. Components pass through column due to differences in partition behaviour between mobile and stationary phases.
High Performance Liquid Chromatography (HPLC) is one of the most widely used techniques for identification, quantification and purification of mixtures of organic compounds.
In HPLC, as in all chromatographic methods, components of a mixture are partitioned between an adsorbent (the stationary phase) and a solvent (the mobile phase).
The stationary phase is made up of very small particles contained in a steel column. Due to the small particle size (3-5 um), pressure is required to force the mobile phase through the stationary phase. There are a wide variety of stationary phases available for HPLC. In this lab we will use a normal phase (Silica gel), although reverse phase (silica gel in which a 18 carbon hydrocarbon is covalently bound to the surface of the silica) columns are currently one of the most commonly used HPLC stationary phases.
Uses of HPLC
This technique is used for chemistry and biochemistry research analyzing complex mixtures, purifying chemical compounds, developing processes for synthesizing chemical compounds, isolating natural products, or predicting physical properties. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product stability and monitor degradation. In addition, it is used for analyzing air and water pollutants, for monitoring materials that may jeopardize occupational safety or health, and for monitoring pesticide levels in the environment. Federal and state regulatory agencies use HPLC to survey food and drug products, for identifying confiscated narcotics or to check for adherence to label claims.
Advantages of HPLC
Rapid and precise quantitative analysis
Typical analysis time of 5-20 min, precision <0.5-1% RSD
Automated analysis
Using autosampler and data system for unattended analysis and report generation
Mobile phase
HPLC mobile phases are usually a mixture of one or more solvents with these characteristics
Desirable Physical properties
High purity, low cost, UV transparency, non-corrosive, low viscosity, low toxicity, non-flammable, sample solubility
Strength
Strength is related to polarity of solvent; Water is a strong solvent in normal phase but a weak one in reversed-phase Solvent strengths under normal phase are characterized by Hildebrands scale (Eo)
Selectivity
Depends on dipole moment, induced dipole, H-bonding, and dispersive characteristics of the solvents
Mobile phase
Viscosity
Low viscosity solvents decreases the pressure to achieve a given flow rate Low pressure extends the lifetime of pumps and columns
Boiling point
Low b.p. facilitate solvent removal from collected fractions Some pumps have difficulty in pumping liquids with b.p. <40
Mobile phase
Flammability
Many solvents used in LC are flammable\ LC equipment should be operated in well ventilated area away from sources of ignition
Toxicity
Preferable to use low toxicity solvents for both safety and disposal reasons
Cost
Operation of LC 1 mL/min for 8 hr/day for 5 day/week uses 125 liters of solvent/year Prudent to consider cost of operation when selecting solvent
Toluene
Methylene chloride Tetrahydro furan Acetonitrile 2-propanol Methanol Water
0.29
0.42 0.57 0.65 0.82 0.95 Large
78
40 66 82 82 65 100
0.59
0.44 0.55 0.30 2.30 0.54 1.00
285
233 212 190 205 205 <190
1.49
1.42 1.41 1.34 1.38 1.33 1.33
Acidifiers
To suppress ionization of acidic analytes under RPC (phosphoric acid, acetic acid)
Ionic strength
To control elution of ionic analyte under IEC (i.e., NaCl)
Ion-pair reagents
For separation of ionic compounds under RPC (hexane sulfonate)
Amine modifiers
To reduce tailing of basic analytes under RPC (triethylamine)
http://www.chemistry.nmsu.edu/Instrumentation/Waters_HPLC_MS_TitlePg.html
Eluent
Column
Sample injection valve
Detection cell
F-
ClNO2Br-
NO3-
SO42HPO42-
Pump Detector
Pumping Injection
Separation
Detection
Recording
HPLC columns
The column is one of the most important components of the HPLC chromatograph because the separation of the sample components is achieved when those components pass through the column. The High performance liquid chromatography apparatus is made out of stainless steel tubes with a diameter of 3 to 5mm and a length ranging from 10 to 30cm.
Normally, columns are filled with silica gel because its particle shape, surface properties, and pore structure help to get a good separation. Silica is wetted by nearly every potential mobile phase, is inert to most compounds and has a high surface activity which can be modified easily with water and other agents. Silica can be used to separate a wide variety of chemical compounds, and its chromatographic behavior is generally predictable and reproducible.
Instrument Parameters
Temperature Flow Signal Sample Sensitivity Detector
Normal phase
In this column type, the retention is governed by the interaction of the polar parts of the stationary phase and solute. For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample
Reverse phase
In this column the packing material is relatively nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water.
Size exclusion
In size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size. Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores. The large molecules elute before the smaller molecules.
Ion exchange
In this column type the sample components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase. Separations are made between a polar mobile liquid, usually water containing salts or small amounts of alcohols, and a stationary phase containing either acidic or basic fixed sites.
tR
http://www.labhut.com/education/flash/introduction07.php
HPLC - Resolution
Resolution (RS) of a column provides a quantitative measure of its ability to separate two analytes
Rs = DZ /1/2(WA+WB)
Rs =
HPLC - Resolution
Capacity Factor (k): Also called retention factor. Is a measure for the position of a sample peak in the chromatogram. k = (tR1-to)/to
specific for a given compound and constant under constant conditions A function of column and mobile phase chemistry Primarily applicable under isocratic conditions In general, a change in the k of one peak will move all peaks in the same direction.
Selectivity Factor (a): Also called separation or selectivity coefficient is defined as a = k2/k1 = (tR2-to) / (tR1-to)
A function of column and mobile phase chemistry Primarily applicable under isocratic conditions Changes in selectivity will affect different compounds in different ways.
Skoog and Leary: Principals of Instrumental Analysis, 4th ed. Suanders, 1992
HPLC - Resolution
Theoretical Plates (N): The number of theoretical plates characterizes the quality or efficiency of a column.
N = 5.54 [(tR) / w1/2]2
(N = 16 (tR/W)2)
Skoog and Leary: Principals of Instrumental Analysis, 4th ed. Suanders, 1992
Skoog and Leary: Principals of Instrumental Analysis, 4th ed. Suanders, 1992
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (TLC vs Normal Phase and Reverse Phase HPLC)
a a 0 b
b Time