BASIC PRINICPALS OF HIGH

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

Plant extract Colors

CaCO3

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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

Gas

Stationary
phase Liquid
Gas Liquid
chromatography chromatography
Solid

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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.

Detector

Column

Pump Column oven

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

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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
of 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.

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2

Si -O-Si
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

C18 (ODS)

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 Reverse phase

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 Normal Phase/Reversed Phase

Type Stationary phase Mobile phase

Normal High polarity Low polarity

phase (hydrophilic) (hydrophobic)

Reversed Low polarity High polarity

phase (hydrophobic) (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
CH3OH/H2O = 6/4
Long analysis time!!

Poor CH3OH/H2O = 8/2

separation!!

(Column: ODS type)

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Page  17
Concentration of methanol in eluent

30%
95%
In gradient mode
 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
obtain information over a wide range of wavelengths at one time

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 UV-Vis spectra of anthocyanin monoglucosides

0.20 528.0
Mv-Glc UV max = 528.0 nm
0.18 Dp-Glc UV max = 525.6 nm
Cy-Glc UV max = 520.7 nm
0.16
Pt-Glc UV max = 525.6 nm
0.14 Pn-Glc UV max = 515.9 nm

0.12 276.5
AU

0.10

0.08 243.4

0.06

0.04 525.6
276.5 348.0 515.9
0.02 357.4
290.8 345.7
0.00
250.00 300.00 350.00 400.00 450.00 500.00 550.00
nm

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 UV-Vis spectra of vitisin A and vitisin B

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Fluorescence detector

 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

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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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 UV and visible chromatograms of Extracted ion chromatograms at different m/z
polyphenols: (a) 280 nm, (b) 320 nm, (c) 360 values, which correspond to the M+ signals of
nm, (d) 520 nm the anthocyanins
Intens
Intens. . 8
x10
[mAU] 4 (a) 5 (a)

150

100
1 2 3 1.0
5
50

0 3 4
4
200 (b) 1
2
150 0.0
6 x107 (b)
100
5’
50 4

0 3
4 11 (c)

9 2
40
7 10 13 3’ 4’
1
8 1’ 2’
20
12
0
0 x107 (c)
5’’
0
Anthocyanin- (d)
40 monoglucosides 3

30 Anthocyanin-
acetylglucosides 2
Anthocyanin- 4’’
20 p-coumaroylglucosides
3’’
10 1 1’’
2’'
0
0 10 20 30 40 50 Time [min]
0
0 10 20 30 40 50 Time [min]
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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
m/z 577 559, 451, 425, 289

425
100 [M-H]- = 577
95 [M-H-152]-
90 Dimer
85
80
152 OH
75 OH
70
65 HO O
60
Intensity

-H2O
55 OH
50 126 OH
OH
45
[M-H-170]- OH
40 407 289 OH O
35
30
25 OH
20
451 [M-H-126]-
289 OH
15
-
10
559 [M-H-H2O]
5
0
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m/z
 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
Conc.unknown = conc.known
Area known

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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 unknown

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

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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.
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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.
 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!

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