BASIC PRINICPALS OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Violeta - - PowerPoint PPT Presentation

BASIC PRINICPALS OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Violeta Ivanova-Petropulos Faculty of Agriculture, University “Goce Delčev” – Štip, R. Macedonia

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What is liquid chromatography?

• Liquid chromatography (LC) is an analytical technique based on the

separation of molecules due to differences in their structure and/or composition.

• Liquid chromatography was defined in the early 1900s by Mikhail S.

Tswett.

• Separation of compounds (leaf pigments) extracted from plants

using a solvent, in a column packed with particles.

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Tswett's Experiment Ether CaCO3 Plant extract

Chromatography

Colors

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Chromatographic methods are applied for:

• SEPARATION OF COMPOUNDS in a mixture
• Identification and determination
• QUALITATIVE ANALYSIS (retention time, UV-Vis spectra, MS

spectra)

• QUANTITAVIE ANALYSIS (peak area or peak height)
• Separation is performed between two phases, mobile and stationary.
• Compounds which are longer retained at the stationary phase will elute

later, compared to those which are distributed into the mobile phase.

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Chromatography Types

Mobile phase Gas Liquid Solid

Stationary phase

Gas Liquid Solid

Gas chromatography Liquid chromatography

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High performance liquid chromatography (HPLC) system

• HPLC is a form of liquid chromatography used to separate compounds that are

dissolved in solution.

• HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a

separation column, detector and data processor.

Pump Sample injection unit (injector) Column Column oven (thermostatic column chamber) Detector Eluent (mobile phase) Drain Data processor Degasser

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HPLC instruments

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HPLC columns

• The column is the “core” of any chromatographic system
• One of the most important components where the separation
• f the sample components is achieved
• Columns are commercially available in different lengths, bore sizes and

packing materials.

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• The most widely used packing materials for HPLC separations are

silica-based.

• The most popular material is octadecyl-silica (ODS-silica), which

contains C18 coating

• materials with C1, C2, C4, C6, C8 and C22 coatings
• The column life can be prolonged with proper maintenance:
• flushing a column with mobile phase of high elution strength

following sample runs is essential.

• When a column is not in use, it should be capped to prevent it

from drying out.

• Particulate samples need to be filtered and when possible a

guard column should be utilized.

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Column types

• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange

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Normal phase

• Stationary phase: High polarity
• Silica or organic moieties with cyano and amino functional groups
• Mobile phase: Low polarity

– Hexan or heptan

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Reverse phase

• Stationary phase: Low polarity

– Octadecyl group-bonded silical gel (ODS)

• Mobile phase: High polarity

– Water, methanol, acetonitrile – Salt or acid is sometimes added.

• Typical stationary phases are nonpolar hydrocarbons (such as C18, C8, etc.) and the

solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water.

Si

• O-Si

C18 (ODS)

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

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Reverse phase

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Normal Phase/Reversed Phase

Type Stationary phase Mobile phase Normal phase High polarity

(hydrophilic)

Low polarity

(hydrophobic)

Reversed phase Low polarity

(hydrophobic)

High polarity

(hydrophilic)

• The polarities of stationary phase and mobile phase have to be different!

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Elution

• Isocratic
• Constant eluent composition, same eluent: for example 50 % methanol
• Gradient

– Varying eluent composition

• HPGE (High Pressure Gradient): High gradient accuracy, complex

system configuration (multiple pumps required)

• LPGE (Low Pressure Gradient): Simple system configuration, degasser

required

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• In isocratic mode

Long analysis time!! Poor separation!!

CH3OH/H2O = 6/4 CH3OH/H2O = 8/2 (Column: ODS type)

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95% 30%

Concentration of methanol in eluent

• In gradient mode

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Detector requirements

• Sensitivity

– The detector must have the appropriate level of sensitivity.

• Selectivity

– The detector must be able to detect the target substance without, if possible, detecting other substances.

• Adaptability to separation conditions
• Operability, etc.

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Types of Detectors

 UV-Vis absorbance detector  Photodiode array-type UV-VIS absorbance detector (DAD)  Fluorescence detector  Refractive index detector  Electrical conductivity detector  Electrochemical detector  Mass spectrometer

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• UV-Vis detector has only one sample-side light-receiving section
• DAD has multiple (1024 for L-2455/2455U) photodiode arrays to
• btain information over a wide range of wavelengths at one time

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UV-Vis spectra of anthocyanin monoglucosides

243.4 276.5 348.0 528.0 357.4 515.9 276.5 345.7 525.6 290.8

AU 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 nm 250.00 300.00 350.00 400.00 450.00 500.00 550.00

Mv-Glc UV max = 528.0 nm Dp-Glc UV max = 525.6 nm Cy-Glc UV max = 520.7 nm Pt-Glc UV max = 525.6 nm Pn-Glc UV max = 515.9 nm

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UV-Vis spectra of vitisin A and vitisin B

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• The most sensitive among the existing modern HPLC

detectors.

• Typically, fluorescence sensitivity is 10 -1000 times higher

than that of the UV detectors

• Fluorescence detectors are very specific and selective

among the others optical detectors.

• Roughly about 15% of all compounds have a natural

fluorescence - derivatization is necessary

Fluorescence detector

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Refractive index detector

• Measures the refractive index of an analyte relative to the

solvent

• They can detect anything with a refractive index different from

the solvent, but they have low sensitivity

• Very sensitive to slight changesd of the mobile phase, not

compatible for gradient elution

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Mass spectrometer

• Mass spectrometry (MS) is an analytical technique that ionizes

chemical species and sorts the ions based on their mass to charge ratio.

• Mass spectrum measures the masses within a sample.
• Mass spectrometry is used in many different fields and is applied to pure

samples as well as complex mixtures.

• Used for:
• characterization of complex structures of compounds
• detection of new compounds in different matrices
• ………

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(a) (b) 50 100 150 Intens. [mAU] 50 100 150 200 (c) (d) 10 20 30 40 10 20 30 40 Time [min] 50 20 40 1 2 3 4 4 5 4 6

Anthocyanin- p-coumaroylglucosides Anthocyanin- acetylglucosides Anthocyanin- monoglucosides

7 8 10 9 11 12 13

UV and visible chromatograms of polyphenols: (a) 280 nm, (b) 320 nm, (c) 360 nm, (d) 520 nm

Intens . (a) (b) (c) 1 2 3 4 5 1’ 2’ 3’ 4’ 5’ 1’’ 2’' 3’’ 4’’ 5’’ 1 2 3 4 0.0 1.0 x108 10 20 30 40 50 Time [min] 1 2 3 x107 x107

Extracted ion chromatograms at different m/z values, which correspond to the M+ signals of the anthocyanins

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Mass spectrum of catechin (m/z 291) obtained under positive mode

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Mass spectrum of procyanidin (m/z 577) obtained under negative mode

Intensity

200 300 400 500 600 700 800 900 1000 1100

m/z

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

425 407 451 289 559 [M-H-H2O]-

[M-H-170]- [M-H-152]- [M-H-126]-

O HO OH OH OH O OH OH OH OH OH OH

[M-H]- = 577 Dimer

152 289

m/z 577 559, 451, 425, 289

• H2O

126

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Quantitative analysis

• Quantitation performed with peak area or height.
• Calibration curve created beforehand using a standard.

– External standard method – Internal standard method – Standard addition method

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External standard method

• The simplest method
• The accuracy of this method is dependent on the reproducibility of the

injection volume.

• Standard solutions of known concentrations of the compound of

interest are prepared with one standard that is similar in concentration to the unknown.

• A fixed amount of sample is injected.
• Peak height or area is then plotted versus the concentration for each
• compound. The plot should be linear and go through the origin.
• The concentration of the unknown is then determined according to the

following formula: Area unknown Area known conc.known Conc.unknown =

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Internal standard method

• The internal standard method tends to yield the most accurate and precise results
• An equal amount of an internal standard, a component that is not present in

the sample, is added to both the sample and standard solutions.

• The internal standard selected should be chemically similar, to have similar

retention time and derivatize similarly to the analyte, to be stable and does not interfere with any of the sample components.

• The internal standard should be added before any preparation of the sample so

that extraction efficiency can be evaluated.

• Quantification is achieved by using ratios of peak height or area of the component

to the internal standard.

Area Internal Std in known Area Internal Std in unknown conc.known Conc.unknown = x Area known Area unknown x

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Fields in Which High Performance Liquid Chromatography Is Used

• Biogenic substances

– Sugars, lipids, nucleic acids, amino acids, proteins, peptides, steroids, amines, etc.

• Medical products

– Drugs, antibiotics, etc.

• Food products

– Vitamins, food additives, sugars, organic acids, amino acids, polyphenols, biogenic amins

• Environmental samples

– Inorganic ions – Hazardous organic substances, etc.

• Organic industrial

products

– Synthetic polymers, additives, surfactants, etc.

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Conclusion

• HPLC offers high sensitive, accurate and fast analysis of various non-volatile

compounds

• It is currently the most widely used method of quantitative analysis in the

pharmaceutical industry, food and beverages analysis laboratories, environmental application etc.

• Connected to MS detectors is one of the most sophisticated techniques for

quantification of very low concentrations of various compounds.

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Thank you for your attention!