High Performance Liquid Chromatography

Picture of HPLC instrument

Introduction

• Form of liquid chromatography • Separate compounds dissolved in

solution.

HPLC Instrument:• reservoir of mobile phase, • a pump, • an injector, • a separation column, • a detector.

Compounds are separated by injectinga sample onto the column.

Components pass through column due to differences in partition behaviourbetween mobile and stationary phases.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

• 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

• High sensitivity detection– Detection limits of ng to pg

• Quantitative sample recovery– Preparative technique from mg to kg quantities

• Amenable to diverse samples– Can handle >60% of all existing compounds vs. 15% for GC– Can analyze samples with little or minimal preparation

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

Hildebrand’s 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

Common Solvents for HPLC Mobile Phase

Solvent Solvent strength

(Eo)

Bp( )℃

Viscosity (cP)

UV cut off (nm)

Refractive index

N-Hexane 0.01 69 0.31 190 1.37

Toluene 0.29 78 0.59 285 1.49

Methylene chloride

0.42 40 0.44 233 1.42

Tetrahydrofuran

0.57 66 0.55 212 1.41

Acetonitrile 0.65 82 0.30 190 1.34

2-propanol 0.82 82 2.30 205 1.38

Methanol 0.95 65 0.54 205 1.33

Water Large 100 1.00 <190 1.33

Common Mobile phase Modifiers

• Buffers– To stabilize pHs of mobile phase under RPC or IEC (phosphate, acetate,

citrate)• 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

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Basic Flow diagram of LCEluent

Pump

Column

Detector

Pumping Separation Detection Recording

F-

NO3- SO4

2-

Cl-

NO2-

Br-

HPO42-

Sample injection valve

Detection cell

Injection

HPLC Chromatograph injectors

• The function of the injector is to place the sample into the high-pressure flow in as narrow volume as possible so that the sample enters the column as a homogeneous, low-volume plug. To minimize spreading of the injected volume during transport to the column, the shortest possible length of tubing should be used from the injector to the column.

• When an injection is started, an air actuator rotates the valve: solvent goes directly to the column; and the injector needle is connected to the syringe. The air pressure lifts the needle and the vial is moved into position beneath the needle. Then, the needle is lowered to the vial.

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.

Picture of an HPLC column

WHAT AFFECTS SYSTEM

Column Parameters

• Column Material• Deactivation• Stationary Phase• Coating Material

Instrument Parameters

• Temperature• Flow• Signal• Sample Sensitivity• Detector

WHAT AFFECTS SYSTEM

Sample Parameters

• Concentration• Matrix• Solvent Effect• Sample Effect

Several column types(can be classified as )

• Normal phase

• Reverse phase

• Size exclusion

• Ion exchange

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.

http://www.labhut.com/education/flash/introduction07.php

TLC vs High Performance Liquid Chromatography (HPLC)HPLC Optimization

tRtRtR

Schematic Presentation of a Chromatogram

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, 4 th 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, 4 th ed. Suanders, 1992

Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992

x

a

b

c

a b c

c b a

0

0

Time

Time

Normal Phase (SiO2)

Reverse Phase (C18)

Normal Phase (SiO2) TLC

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(TLC vs Normal Phase and Reverse Phase HPLC)