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HPLC, A practical guide for the beginner users

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 ‫ميحرلا نمحرلا هللا مسب‬
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
(HPLC)

A PRACTICAL GUIDE FOR

THE BEGINNER USERS
A. Prof. Sherif M. Taha
Tel: 01004724944
[email protected]
 INTRODUCTION

Chromatography:
Is the separation and detection of certain compounds in a sample (Food, water, blood, urine,
chemicals, …).

based on their different chemical-physical properties

based on different physical interactions between two immiscible phases

Such separation may be accomplished in a liquid-liquid extraction

system or by its transfer through an immobilized stationary phase by a
carrier gas (GC) or a mobile phase (LC).
 INTRODUCTION

Mikhail Semenovich Tswett

https://bitesizebio.com/29947/basics-chromatography-column/
 THE BASIC COMPONENTS OF
HPLC SYSTEM

MOBILE PHASE
At HPLC, The sample is partitioning between
the mobile phase and the stationary phase
(not just being carried by a carrier gas, GC).

COLUMN

Mobile phase
D C B A
Stationary phase, column DETECTOR
Detector
A
C B
D
Sample loading with the mobile phase
(Pump role)
 THE BASIC COMPONENTS OF
HPLC SYSTEM

MOBILE PHASE

COLUMN

DETECTOR

Pilar Campíns-Falcó; Rosa Herráez-Hernández; Pascual Serra-Mora, Liquid Chromatography-Instrumentation

 THE BASIC COMPONENTS OF
HPLC SYSTEM

HPLC separation

Adsorption Partition size-exclusion Ion Exchange Chiral

Polar St phase Non polar St phase,

Normal phase Reversed phase
 THE BASIC COMPONENTS OF
HPLC SYSTEM
B-Strong ads, late eluted
A- Weak ads, early eluted

C
B H x
B x
B A E
C x
A H x
D
A

Adsorption partition size-exclusion Ion Exchange Chiral

A- Analytes are in an adsorption interaction with functional gps on surface of stationary phase
B-Analytes partitions between the mobile phase and the stationary phase depending on its solubility
 [𝑆𝑜𝑙𝑢𝑡𝑒 𝑛𝑜𝑡 𝑖𝑜𝑛𝑖𝑠𝑒𝑑 𝑖𝑛 𝑜𝑐𝑡𝑎𝑛𝑜𝑙]
KOW =
[𝑆𝑜𝑙𝑢𝑡𝑒 𝑛𝑜𝑡 𝑖𝑜𝑛𝑖𝑠𝑒𝑑 𝑖𝑛 𝑤𝑎𝑡𝑒𝑟]

PARTITION
CONSTANT;
(KOW )

http://dx.doi.org/10.1016/B978-0-12-385013-3.00009-4
 C8 RP

STATIONARY
PHASE;
(RP)
C18 and C 8 are the commonly
used ones in HPLC analyses.

https://www.chromacademy.com/lms/sco4/Theory_Of_HPLC_Column_Chemistry.pdf
 Polar Non polar
Stationary phase Stationary phase
Analyte Highly polar Moderately to non
polar
Mobile phase for Low polar solvent High polar solvent
loading
Mobile phase for High polar solvent Low polar solvent STATIONARY
eluting
PHASE;
• Improvement of the obtained selectivity by using a specific (RP // NP)
separation condition (NP or RP) was carried out firstly by
selecting proper mobile phase conditions and then changing the
stationary phase.

• It is also preferred to use previously published conditions

(especially on the used column) and then modify it according to
your own need.
 C18, 250 mm, 4,6 mm, 5 µm, 300 A
Particle ‘s Pores size
• Below 60 A
Bonded phase • 60-150 A
• 150-300 A (proteins)

Length
Increasing L
Inter diameter
Smaller ID Particle size ,
Depending on target
analytes and matrix COLUMN
• Higher peak
• Higher peak
resolution
resolution
• But, higher back
•
•
Smaller PZ introduce
higher surface area,
higher peak resolution
SPECIFICATION
• But, higher back
pressure too • Shorter run time
pressure too
• But, higher back
pressure too

High Vinj and analyst Conc.

May cause fronting of un-retained molecules

Injection volume, Vinj;

Vinj < 0.14 𝐝2 𝐋 x dp

d, L; internal diameter and length of the used column
dp diameter of solid phase particles
 C18, 5 µm, 150 mm × 4.6 mm column

COLUMN
C18, 3 µm, 150 mm × 4.6 mm column SPECIFICATION
HPLC-FLD chromatograms of aflatoxins

C18, 2.7 µm, 100 mm × 4.6 mm

 COLUMN
SPECIFICATION

Pilar Campíns-Falcó; Rosa Herráez-Hernández; Pascual Serra-Mora, Liquid Chromatography-Instrumentation

Sample particle size ≤ pores PZ in sintered frit <Stationary phase PZ
e.g. Acrodisck of 0.2µm< frit of 0.5µm< particles size inside the column 3µm

SAMPLE FILTERING
AND COLUMN
CLOGGING
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Wr4kJeF2xVFsIjmlToXWu8g0mJ6ChKcHczDpHUaJ05Q

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THE ELUTION STRENGTH APPLING RP-HPLC ;

• HEXANE(3.13)>TERT-BUTANOL(0.54),ISOPROPANOL(0.25), ACETONE (0.11),

ETHANOL (-0.16)ACETONITRILE (-0.34), METHANOL(-0.77), WATER (-1.38).

changing between two immiscible first loading of sample MOBILE PHASE, KOW
solvents. It is also used for long components in RP HPLC.
storage of the used columns
Kow Polarity

water and a miscible organic solvent are commonly used mobile phase
mixtures in HPLC since they give a new Kow that rapid the chromatographic
separation run.

Log Kow (A+B)= 1 − 𝑥 𝐿𝑜𝑔 𝐾𝑜𝑤 𝐴 + 𝑋 𝐿𝑜𝑔 𝐾𝑜𝑤 𝐵

A& B; used solvents.
• Acetone has a strong eluting properties in
RP-HPLC but it has a high UV cut-off
value.

• Methylene chloride, chloroform have

medium polarity but in RP HPLC it is
immiscible with water.

• Acetonitrile has a very low UV cut-off,

MOBILE PHASE, KOW

Kow Polarity
 THE BASIC COMPONENTS OF
HPLC SYSTEM
 Gradient elution results in; minimizing the run time, good shape for eluted peaks, and
cleaning the used column for each run.

includes three basic steps:

 Short isocratic start (Solution A, lower elution)

 Gradient program (gradually increase solution B, higher elution).

 Short isocratic for cleaning the used column ( Highest percent of solution B).

 Return to the initial conditions (column conditioning, very critical step).

 Time A (H2O:MeOH 5%, pH 4.6). B (MeOH)

0.01-5.00 10 % B
5.01-7.00 70 % B

7.01-10.00 10 % B MOBILE PHASE,

GRADIENT ELUTION

BENZOIC AND SORBIC ACID

DETERMINATION IN JUICE, MILK
PRODUCTS,…
 BUFFER, HPLC MOBILE PHASE

 In RP- HPLC Solvent A is mainly water as it:

 The highest polar solvent (weakest eluent), suitable for sample loading.
 Buffers can be easily prepared in water at different concentrations.

 The purity of water for HPLC is very important, especially when using MS/ MS.

 Itis preferred to use a percent (About 10 %) of a suitable organic solvent in water

(Solvent A);
 To prevent algae formation.
 Enhance the evaporation in ESI ionization unit (LC-MS/MS).
 BUFFER, HPLC MOBILE PHASE

 Forseparation of acidic or basic compounds, a buffer should be

used in solvent A to keep the ionizable compounds in neutral
form as possible. Where pH of the mobile phase A (using
buffer) should be acidic for basic compounds and the reverse.

 Changing pH by 2 units (lower or higher than its pKa) create a

reverse partitioning condition for this compound.
[𝐴− ]
pH= pKa+ Log ([𝐻𝐴])

At pH= 2,

[𝐴− ]
-2.2= Log ([𝐻𝐴]), [HA]=166 [𝐴− ]
http://dx.doi.org/10.1016/B978-0-12-803684-6.00004-4
 BUFFER, HPLC MOBILE PHASE
Ricinoic acid (castor Oil)
[𝐴− ]
pH= pKa+ Log ([𝐻𝐴])

Hexane

http://dx.doi.org/10.1016/B978-0-12-803684-6.00004-4

MeOH (Acidic mix)

MeOH (basic mix)
 BUFFER, HPLC MOBILE PHASE

 A buffer is
a solution of a weak acid (HA) and its conjugate base (𝐴− ) &
a solution of a weak base (B) and its conjugated acid (𝐵𝐻 + )

 Such buffer system has the capability to resistance changing in pH upon the addition of
small amounts from a strong acid or base.

[𝐴− ]
pH= pKa+ Log ( )
Buffer [𝐻𝐴]
(pH & Concentration)
Cbuf = [𝐴− ] + [𝐻𝐴]
 BUFFER, HPLC MOBILE PHASE
 Buffer capacity, β:
The number of added moles (dn) of a strong acid or a base that change the pH of one-liter
buffer solution.
𝑑𝑛
β=
𝑝𝐻
Since, the addition of n moles from a base (NaOH) leads to the formation of [𝐴− Na] or CNaA

[𝐴− b]
β= 𝑝𝐻 β

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5953930/
 BUFFER, HPLC MOBILE PHASE

[𝐴− ]
pH= pKa+ Log ( )
Fixed [𝐻𝐴]

Cbuf = [𝐴− ] + [𝐻𝐴]

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5953930/
 BUFFER, HPLC MOBILE PHASE
• BUFFER CONCENTRATION IN HPLC UV IS USUALLY MADE WITH A CONCENTRATION OF 10-
200 MM. (LOWER CONCENTRATIONS FOR LC MSMS <50MM, VOLATILE SALTS ARE MORE
FAVORABLE).
• THE SOLUBILITY OF INORGANIC SALTS DEPENDS MAINLY ON THE NATURE OF THE CATION,
AND THEIR SOLUBILITY TREND IN ORGANIC SOLVENTS FOLLOWS (THE SAME AS IN
WATER): NH4 + >𝐾 + >𝑁𝑎+ .
• A HIGHER CONTENT OF ORGANIC PHASE SHOULD BE AVOIDED NOT TO PRECIPITATE THE
BUFFER SALTS.
_____________________________________________________
• FOR PREPARING A BUFFER AT PH 4.5 YOU SHOULD USE:

 A WEAK ACID OF PKA CLOSE TO THE REQUIRED PH (……ACID PKA =….)

 THE CONJUGATE BASE FOR THIS ACID WILL BE ITS (NA, K, AMMONIUM) SALT
 WHAT IS THE TOTAL BUFFER CONCENTRATION AND HOW TO PREPARE ?
BUFFER, HPLC MOBILE PHASE
BUFFER, HPLC MOBILE PHASE
Volatile buffers that can be used for LC MS

http://dx.doi.org/10.1016/B978-0-12-811732-3.00006-6
 HPLC DETECTORS

UV-Vis

Florescence HPLC MS

Others
 SOLVENT OF THE SAMPLE

The solvent of the sample should be with;

- Same polarity as the mobile phase (loading solvent, A)
or weaker (increase solvent compressing, enhance the
retention of solutes).
- pH close to that of the mobile phase (loading solvent, A)

These points are more critical, especially for higher injection volume
and for ionizable solutes solutes

Zorbax Eclipse XDB-

C18, 150 mm, 4.6 mm, 5 µm
with a mobile phase:
35% ACN and 65% H2O (0.2% H3PO4);
flow-rate: 2 mL/min.
A CHROMATOGRAM AND A MASS SPECTRUM
 COLUMN EFFICIENCY
Dead time, Void time

T0 = Time at which mobile phase pass through the chromatographic column

Depending on column length and flow rate

Actual Rt (X)= Obtained Rt (X)+ t0

Jesús Lozano-Sánchez; Isabel Borrás-Linares; Agnes Sass-Kiss; Antonio Segura-Carretero, Chapter 13 - Chromatographic
Technique: High-Performance Liquid Chromatography (HPLC)
 COLUMN EFFICIENCY

Column efficiency:

 High Plate N per unit length (N/L)

or
Height of the theoretical plate H= 𝐿/𝑁≃ 2dp
dp diameter of solid phase particles

 Selectivity (Separation Factor)

A measurement for the separation
of two compounds X and Y

 Resolution
Separation of apexes of two adjacent peaks

Plate Number (N)

2 Selectivity Resolution
N= (𝑅𝑡/ )
N= 16(𝑅𝑡/Wb)2 𝑅𝑡 (𝑥) 2 [𝑅𝑡 𝑥 −𝑅𝑡 𝑦 ]
N= 5.45 (𝑅𝑡/W 0.5)2
=𝑅𝑡 (𝑦) R = (𝑊𝑏 𝑥 +𝑊𝑏 (𝑦)

Wb= 4*x Standard deviation ( )

Δ𝑅𝑡
R=
 Thank You
A. Prof. Sherif M. Taha
Tel: 01004724944
[email protected]

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