HPLC in MCAL Solvents Fluorescence Detector Autosampler Manual injector Pumps UV Vis diode array Detector

HPLC Image System control- Data storage HPLC column (packing) Injector Autosampler Sample Detector(s) (Diode Array & Fluorescence) Solvent(s) Waste Pump(s)

Separation in HPLC Seperation depends on : Polarity of molecule RP verses NP, Hilic, Hydrophilic-Interaction Chromatography Hic, Hydrophobic-interaction Chromatography Electrical charge (ion exchange). Anion exchanger attracts” –” charged analyte Cation exchanger attracts “+” charged analyte Molecular size Size exclusion or gel permeation

Columns - Heart of System Solid Support (column packing) - Backbone for bonded phases. Usually 10µ, 5µ, 3µ, 2.5µ siloxane or polymeric particles. Porous or solid core Packing has different degrees of polarity depending on functional groups attached Bonded Phases - Functional groups chemically bound to the solid support.

HPLC - Modes Normal Phase - Polar stationary phase and non-polar solvent. • Reverse Phase (used for caffeine and quinine separation) - Non-polar stationary phase and a polar solvent. - “Like dissolves like”. In Normal Phase the analyte will be partitioned preferentially in the mobile phase and provide little interaction with the stationary phase. This is not desirable since selective retention on the column will be very hard to control. It can be controlled by modifying the stationary phase, a very time consuming and expensive proposition even if feasible. In Reverse Phase the opposite is true. The analyte will be partitioned preferentially in the stationary phase (“Like dissolves like”). and by simply modifying the mobile phase, by adjusting the polarity, ionic strength or pH, selectivity can be virtually fully controlled.

Normal Phase verses Reverse Phase HPLC Packing Polar packing Non-polar packing C4 C8 C18

HPLC Solid Support C18RP CH3 CH2 (CH2)14 CH2 CH2 CH2 Si O Si CH3 O O OH CH3 O O Can be some polar OH groups that are not bonded. Some of these are end capped. The manufacturer ususually tells you the % of endcapping. O Si (CH2)18 Si CH3 Gel Surface Bonded phase

HPLC Solid Support C18RP End Capping CH3 CH2 (CH2)14 CH2 CH2 CH2 Si O Si CH3 O O + Si (CH2)2 Si OH CH3 O O O Si (CH2)18 Si CH3 Gel Surface Bonded phase

HPLC Solid Support C18RP Polymeric CH3 CH2 (CH2)14 CH2 CH2 CH2 Si O Si CH3 O O Si O Si (CH2)18 CH3 O O O Si (CH2)18 Si CH3 Gel Surface Bonded phase

HPLC Solid Support C18RP Polymeric OH CH2 (CH2)14 CH2 CH2 CH2 Si O Si O O O Si O Si (CH2)18 CH3 O O O Si (CH2)18 Si CH3 Gel Surface Bonded phase

Solid Core Particle Solid Core Porous Outer Layer

Core-Shell Particle

Core-Shell Particle

HPLC Columns (MCAL) C18 Microsorb (Varian)5 u 100 A 250 x 4.5 mm C18 Polar Synergi (Phenomenex) 4 u 80 A 250 x 4.5 mm C18 RP Particil column (Varian) 3 u 300 A 100 x 2.0 mm C18 Hilic column (Phenomenex) 5 u 200 A 150 x 4.6 mm XB-C18 RP Kinetex (Phenomenex) 2.6 u 100 A 100 x 4.6 mm C18 Microsorb (Varian) 5 u 100 A 250 x 4.5 C8 Polaris (Varian) 3 u –A 50 x 20 mm

Manual Injector HPLC columns

Solvents for RP H2O most polar with decreasing polarity as solvent B added to mix. Solvent B usually MeOH, acetonitrile, iso-propanol Can be isocratic mixture - 1 or 2 pump system or Gradient – need at least two pumps and mixing apparatus. Caffeine-quinine exp. uses buffer-acetonitrile or buffer- methanol gradient mixing.

The Theoretical Plate Model of Chromatography The plate model supposes that the chromatographic column is contains a large number of separate layers, called theoretical plates (N). Separate equilibrations of the sample between the stationary and mobile phase occur in these "plates". The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next.

Chromatographic Resolution Depends On: 1.Column efficiency (N) peak width Particle diameter Flow rate Sol. Viscosity Temp. Col. Length Col. diameter 2. Solvent efficiency (α) retention time Stationary phase Mobile phase Solvent composition Temperature

Column Efficiency -Theoretical plate numbers (N) Theoretical plate numbers are indirect measure of peak width for a peak at a specific retention time. Columns with high plate numbers are considered to be more efficient, that is, have higher column efficiency, than columns with a lower plate count. A column with a high number of theoretical plates will have a narrower peak at a given retention time than a column with a lower N number. N=5.545(tr/wh)2 Column efficiency is a function of the column dimensions (diameter,length and film thickness), the type of solvent and its flow rate or average linear velocity, and the compound and its retention.

Column Efficiency -Height equivalent to theoretical plates Measure of the resolving power of the column HETP = L/N L = Length of column N = Theoretical plates At each theoretical plate one equilibrium distribution of solute between mobile phase and stationary phase occurs.

Peak Seperation (α) On stationary phases where the alphas (α) are small, more efficient columns (higher N) are beneficial. Column efficiency is a function of: a) the column dimensions (diameter, length and film thickness), b) the type of carrier solvent and its flow rate or average linear velocity, and c) the compound and its retention.

Band broadening theory (Van Deemter equation) column band broadening originates from three main sources: multiple path of an analyte through the column packing; molecular diffusion; effect of mass transfer between phases.

Multiple path (Eddy diffusion) The velocity of mobile phase in the column may vary significantly across the column diameter, depending on the particle shape, porosity, and the whole bed structure.

Measure of the Resolving Power of the Column HETP = A + B / u + C u A = eddy diffusion take different paths (different lengths). B = longitudinal diffusion analyte diffuses from centre to outer edge. C = resistance to mass transfer: analyte strong affinity for stationary and veloscity of mobile phase is high. U = avg veloscity of mobile phase

Resolving Power α α N N

Column efficiency is reported as plates per meter (N/Meter). Smaller average packing particle size = Larger N Broader particle size distribution = Smaller N Better packing procedures = Larger N

Polarity - Elotropic Series Water is at the polar end of mobile-phase-solvent scale, Hexane, an aliphatic hydrocarbon, is at the non-polar end. In between, single solvents, as well as miscible-solvent mixtures Which end of the scale represents the ‘strongest’ mobile phase depends upon the nature of the stationary phase surface where the competition for the analyte molecules occurs.

Stationary Phase Polarity Spectrum Silica has an active, hydrophilic [water-loving] surface containing acidic silanol [silicon-containing analog of alcohol] The polarity of the silica surface may be modified selectively by chemically bonding to it less polar functional group like n-octylsilyl- [C8], and n-octadecylsilyl- [C18, ODS] moieties. The latter is a hydrophobic [water-hating], very non-polar packing. the chromatographer will choose the best combination of a mobile phase and particle stationary phase with appropriately opposite polarities. Then, as the sample analytes move through the column, the rule like attracts like will determine which analytes slow down and which proceed at a faster speed.

HPLC Experiment Seperation and identification of caffeine, quinine and added theophylline (internal standard) in commercial drinks Comparison of different quantitation methods (external standard, internal standard) Uv verses fluorescent detection Diode array detection

HPLC Experiment The separation is done on a C18 or C12 RP column. The solvent is a 20% methanol in water (low ph with TEA added) increased to 25% methanol. The caffeine comes off column before the quinine.

HPLC Exp: Caffeine and Quinine Quinine Rt = 11.6 min Caffeine wavelength 206 Rt = 9.2 min Caffeine wavelength 273 Rt = 9.2 min

Diode Array Detector Caffeine

Detection with HPLC Ultraviolet light source (deuterium lamp 190 to 950nm) Visible light source (quartz iodide 350 nm and beyond) Flurescent light (Xenon lamp) Excitation and emmission wavelength selection

Concentration Of Analytes Concentration is determined by measuring the area of known concentrations of caffeine and quinine, and making a standard curve. Concentration of caffeine is also determined by adding an internal standard (IS) and calculating caffeine based on the response factor of the IS.