Chp 6.1_hplc.pdf

ANNOUCEMENT QUIZ 2 - ONLINE • • • •

Week 14 Date: 18/10/18 – 21/10/18 Time: 2.00 PM – 2.00 AM Chapter: Chap 4 – 5

TEST 2 - WRITTEN • • • •

WEEK 16 Date: 2/11/18 Time: 10.45 AM – 11.45 AM Chapter: Chap 4 - 6

U N I V E R S I T I

K U A L A

CHAPTER 6.1 CHROMATOGRAPHY

L U M P U R

SUBTOPIC 6. 1 High Performance Liquid Chromatography

6.1.1 High Performance Liquid Chromatography Introduction 6.1.2 Separation modes in HPLC

6.1.3 Developing a separation 6.1.4 Sample preparation and data evaluation

HISTORY OF HPLC • • -

Early LC carried out in glass columns diameters: 1-5 cm Lengths: 50-500 cm Size of solid stationary phase Diameters: 150-200 µm

Flow rates still low, separation times long

Improved Technology • Decrease particle size of packing causes increase in column efficiency -Diameters 3-10 µm • This technology required sophisticated instruments. -new method called HPLC

ADVANTAGES OF HPLC • Speed - 30 min or less

• Improved resolution - can adjust a variety of parameters, including types of stationary phase • Greater sensitivity - various detectors • Reusable column - many analyses • Ideal for ionic species and large molecules (substances with low volatility • Easy Sample recovery - fraction collectors

HPLC Introduction • High performance liquid chromatography can be applied to the analysis of any compound with solubility in a liquid that can be used as the mobile phase. • Separate any compound with solubility in a liquid/ solvent that can be used as a mobile phase. • Analytical or preparative (highly purified compound in small or large quantities). • Applications include analysis of sugars, pesticide residues, organic acids, lipids, amino acids, toxins, and vitamins.

Basic Flow diagram of HPLC Eluent

Column

Detection cell

Sample injection valve

F-

ClNO2Br-

SO42-

NO3-

HPO42-

Pump Detector

Pumping Injection

Separation

Detection

Recording 7

HPLC Instrumentation Overview Principle Pattern

An Example Solvent Reservoirs Controller

Solvent Cabinet

Vacuum Degasser Binary Pump

Autosampler

Thermostatted Column Compartment Detector

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Composition of HPLC System • Solvent • Solvent Delivery System (Pump) • Injector • Sample • Column • Detectors • Waste Collector • Recorder/integrator/Data System

Solvent Filters Guard column Injector Precolumn Filter

Analytical Column

Solvent Inlet Filter

Solvent Inlet Filer • Stainless Steel or glass with 10 micron porosity. • Removes particulates from solvent. 10

Precolumn Filter • Used between the injector and guard column. • 2 to 0.5 micron • Removes particulates from sample and autosampler wear debris. • Must be well designed to prevent dispersion.

HPLC Pump • function is to deliver mobile phase through the system » 1 ml/min - needs to be controlled, accurate and precise • 2 main type of pump: Constant volume and Constant pressure • System and connecting lines are made from stainless steel (can handle pressure and is resistant to corrosion by most mobile phases except mineral acids and halide ions • Pumps are sensitive to particles in the mobile phase and air - filter and degas mobile phase  Commercial HPLC pumping system and connecting lines are made of grade AN51316 stainless steel.  withstand the pressure generated and is resistant to corrosion by oxidizing agents, acids, bases and organic solvents.  Mobile phase filter using 0.45 or 0.22 µm porosity porous.  Degassing HPLC eluent using vacuum/ultrasonication/sparging with helium. • to prevent the problems that can be caused by air bubbles in a pump or detector.

Functions of the Solvent Delivery System The solvent delivery system has three basic functions:

1. Provide accurate and constant flow. 2. Provide accurate mobile phase compositions.

3. Provide the force necessary to push the mobile phase through the tightly packed column.

Injector • To place the sample into the flowing mobile phase. • Valve injector • trouble free, good precision and can change loop for different volumes • Place in load position and load sample into an external fixed volume loop at atmospheric pressure • Rotate to inject position - loop becomes part of the eluent flow stream and the sample is carried to the column • Auto samplers • fully automatic injection systems enabling greater productivity and the highest level of precision. • Small vials with a septum placed in a tray • Needle penetrates septum and withdraws the sample

Auto Samplers

Sample Injectors Requirements: Reproducible introduction of the sample volume into the mobile phase flow. Two major designs: •Automatic Injectors or •Manual Injectors

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Manual Injectors Sample Loop

Load - Inject

Front View

Rear View

Inject 15

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Manual Injectors Sample Load From Pump To column

From Pump To column

Solvent in Solvent out

Sample in

Solvent in Solvent out

Sample in

Sample Inject

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Automatic Injectors

Step 1

Step 2

Step 3 18

Column

Columns • Actual tool to separate molecules • External hardware - stainless steel tube with terminators for connection • Precolumns - auxillary columns that precede the analytical columns • Presaturates -contains large particles of bare silica and presaturate the mobile phase with silica (slow down dissolution of silica based packing materials during the use of an aqueous mobile phase) • Guard columns - protect analytical column (same diameter, small total volume, and minimal dead space 20

ANALYTICAL COLUMNS CHARACTERISTICS: – Made of Stainless steel tubing for high pressure – Heavy-wall glass or PEEK tubing for low P (< 600 psi) – Analytical column: straight, Length (5 ~ 25 cm), diameter column (3 ~ 5 mm), diameter particle (35 μm). N (40 k ~ 70 k plates/m)

-Micro column: L (3 ~ 7.5 cm), d (1 ~ 5 mm), dp: 3 ~ 5 μm, N: ~100k plates/m, high speed and minimum solvent consumption – Guard column: remove particulate matter and contamination protect analytical column, similar packing – T control: < 150 °C, 0.1 °C -Column packing: silica, alumina, a polystyrene-di vinyl benzene synthetic or an ion-exchange resin – Pellicular particle: original, Spherical, nonporous beads, proteins and large biomolecules separation (dp: 5 μm) – Porous particle: common used, dp: 3 ~ 10 μm. Narrow size distribution, porous micro particle coated with thin organic films 21

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Column and Solvent Filters Guard column Injector Precolumn Filter

Analytical Column

Solvent Inlet Filter

Solvent Inlet Filer • Stainless Steel or glass with 10 micron porosity. • Removes particulates from solvent.

Precolumn Filter • Used between the injector and guard column. • 2 to 0.5 micron • Removes particulates from sample and autosampler wear debris. • Must be well designed to prevent dispersion. 23

Packing material • Silica based & polymeric • General requirements • Available in well defined particle sizes with narrow distribution - small particles decrease the solute distance between stationary and mobile phases facilitates equilibration and increases efficiency, but more flow resistance and higher pressure • Can withstand pressure • Chemical stability

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Silica based column packing • Porous silica - half of volume consists of pores • Bonded phases - silicon dioxide tetrahedron with silanol groups on surface - (disadvantage is that silica skelton slowly dissolves in aqueous solution especially at pH >8 – remember use of presaturates column) • Pellicular packing - thin coating (polyamine is physically absorbed to surfaces and then cross-linked into a permanent polymer layer) on a microparticulate core (core can be porous or nonporous). 25

Polymeric column packing • synthetic organic resins - have better chemical stability and the potential to change properties by direct chemical modification • Microporous resins • Macroporous resins

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Types of Packing Materials Utilized in HPLC

A) Bonded-phase silica

B) Pellicular packing

C) Microporous polymeric resin;

C) macroporous polymeric resin

Detectors • UV/VIS absorption detectors • Refractive index detector • Fluorescence detectors • Electrochemical detectors • Evaporative light scattering detectors • Viscosity detectors • Radioactive detectors • Transport detectors • Chemiluminescent nitrogen detectors 28

UV cell

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UV-Detectors • Photodiode

• Diode array

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HPLC Detectors Characteristics of Absorbance Detectors: • Eluent Measurement Cell •

•

Minimum Volume • Reduces Extra Column Broadening • Volume : 1 to 10 mL • Path Length (b): 2 to 10 mm Pressure Limited • Maximum Typically 600 psi • Usually Requires Pressure Reducer

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32

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Light path in a micro flow cell of a spectrophotometric detector. Cells that have a 0.5-cm pathlength and contain only 8μL of liquid are common. 34

Refractive Index Refractive index detectors: • Nearly universal but poor detection limit • Passes visible light through 2 compartments, sample & reference. • When the solvent composition are the same the light passed through the compartments the light beam that passes through is recorded as zero. • When a solute is in the sample compartment, refractive index changes will shift the light beam from the detector. • Limit of detection (LOD) 10 ng of solute •The Refractive Index Detection is strongly influenced by: Pressure changes Temperature changes Flow pulse Gradient elution is not possible! 35

SEPARATION MODES

Separation Modes • Based on physiochemical principles - adsorption, partition, ion exchange, size exclusion, and affinity • Two types of separation modes popular in HPLC application: • Normal phase • stationary phase in polar, • mobile phase in non polar

• Reverse phase • stationary phase non polar and • mobile phase is polar (more than 70% of all HPLC)

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Types of Separation Modes

Normal phase • Stationary phase • Chemically attached polar nonionic functional group (alcoholic hydroxy, nitro, nitrile, or amino) • The terminal polar group is linked to the silica through a short hydrocarbon spacer • Mobile phase • Nonpolar solvent (like hexane) with a more polar modifier (like methylene chloride) • Solvent strength and selectivity is controlled which influences solute retention (weak solvents increase retention and strong solvents decrease retention)

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Normal phase Application • Best for compounds high soluble in organic solvent (fat soluble vitamins) or that have low solubility in an aqueous mobile phase • Also can be used for compound classes, isomers, and highly hydrophilic species like carbohydrates

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Reverse phase • Stationary phase • Silica • Chemically bound to silica via surface silanol • R3 group is usually an octadecyl (C18) • Bonded phase (non polar ocatdecylsilyl) can be monomeric or polymeric depending on interaction with silica surface

• Polymeric • Highly cross-linked polystyrene-divinylbenzene

• Mobile phase • Usually water mixed with methanol, acetonitrile, or a hydrofuran • Solute retained due to hydrophobic interactions with the non polar stationary phase

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Reverse phase • Solute eluted in order of increasing hydrophobicity (least hydrophobic first, most hydrophobic last) • Increasing polarity of the mobile phase increases solute retention • Increasing organic solvent content decreases solute retention • Additives: an amine to deactivate silanols, aqueous buffers to suppress ionization of sample components, ion-pair reagents to neutralize charged solutes and make them more lipophilic

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Reverse phase Application • Proteins - including cereal proteins • Water soluble and fat soluble vitamins • Carbohydrates using ion-pairing reagents • Antioxidants, dyes, pigments, and phenolic flavor compounds

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DEVELOPING A SEPARATION

Evaluating the sample and Define Goal of the separation • • • • • •

How many components need to be resolved? What degree of resolution is needed? Is qualitative and quantitative information needed? Molecular weight Polarity Sample Ionic character

Choose a separation mode of sample • • • •

Must select an appropriate column Elution conditions Detection method Trial experimental conditions may be based on - literature search -analyst’s previous experience -recommendations from expert

Resolution Optimization • Manipulation of mobiles phase variables (nature and percentage of organic components) • pH • Ionic strength • Nature and concentration of additive • temperature

SAMPLE PREPARATION & DATA EVALUATION

Sample Preparation and Data Evaluation • General purpose is to remove interfering material prior to chromatography • Considerations - extraction efficiency, analyte stability, and consistency of chemical and enzymatic pretreatment • Peak identification - equivalent retention time does not prove identity • Identification techniques - spike, diode array detector, rationing procedure, collect fraction and further analyze • Quantitative validity - analyte recovery (addition of known quantity before extraction), check sample (has known quantitiy) 49

Limit of Detection (LOD) The limit of detection for a detector can be characterized by its signal to noise ratio (S/N) for an analyte under a given set of conditions. Peak

Noise

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Limit of Detection - Limit of Quantitation Response

Linear range

Slope = sensitivity

MQL MDL Intercept

e.g.,RSD<10%, S/N > 20

e.g., S/N > 3

Amount

• Limit of detection (LOD) is a result of the whole chromatography system, not only the detector performance • Limit of quantification (LOQ) is a defined limit for a method used for a specific purpose. 51

UV-Vis Detectors Principles: The fraction of light transmitted through the detector cell is related to the solute concentration according to Beer’s Law. Detector Flow Cell

c

I0

I

b

Log I0 = A = abc I

Characteristics: Specific, Concentration Sensitive, good stability, gradient capability. Special: UV-Vis Spectral capability (Diode Array Technology ).

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CHROMATOGRAPHIC TERMS

CONCLUSION HPLC is widely used for the analysis of • small molecules and ions (eg: sugars, vitamins, and amino acids) • separation and purification of macromolecules, such as proteins, nucleic acids, and polysaccharides.