HPLC_A practical guide for the beginner users.pdf

HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
(HPLC)
A PRACTICAL GUIDE FOR
THE BEGINNER USERS
A. Prof. Sherif M. Taha
Tel: 01004724944
sherif2taha@gmail.com
‫ميحرلا نمحرلا هللا مسب‬

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
COLUMN
DETECTOR
Sample loading with the mobile phase
(Pump role)
A
C
D
B
B
A
D C
Mobile phase
Stationary phase, column
Detector
At HPLC, The sample is partitioning between
the mobile phase and the stationary phase
(not just being carried by a carrier gas, GC).

HPLC SYSTEM
Pilar Campíns-Falcó; Rosa Herráez-Hernández; Pascual Serra-Mora, Liquid Chromatography-Instrumentation
MOBILE PHASE
COLUMN
DETECTOR

HPLC SYSTEM
HPLC separation
Adsorption
Polar St phase
Normal phase
Partition
Non polar St phase,
Reversed phase
size-exclusion Ion Exchange Chiral

HPLC SYSTEM
B
B
A
C
A
D
x
H
x
x
x
H
Adsorption partition size-exclusion Ion Exchange Chiral
C
E
B
A
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
B-Strong ads, late eluted
A- Weak ads, early eluted

PARTITION
CONSTANT;
(KOW )
KOW =
[𝑆𝑜𝑙𝑢𝑡𝑒 𝑛𝑜𝑡 𝑖𝑜𝑛𝑖𝑠𝑒𝑑 𝑖𝑛 𝑜𝑐𝑡𝑎𝑛𝑜𝑙]
[𝑆𝑜𝑙𝑢𝑡𝑒 𝑛𝑜𝑡 𝑖𝑜𝑛𝑖𝑠𝑒𝑑 𝑖𝑛 𝑤𝑎𝑡𝑒𝑟]
http://dx.doi.org/10.1016/B978-0-12-385013-3.00009-4

STATIONARY
PHASE;
(RP)
C8 RP
https://www.chromacademy.com/lms/sco4/Theory_Of_HPLC_Column_Chemistry.pdf
C18 and C 8 are the commonly
used ones in HPLC analyses.

Polar
Stationary phase
Non polar
Stationary phase
Analyte Highly polar Moderately to non
polar
Mobile phase for
loading
Low polar solvent High polar solvent
Mobile phase for
eluting
High polar solvent Low polar solvent STATIONARY
PHASE;
(RP // NP)
• Improvement of the obtained selectivity by using a specific
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.

COLUMN
SPECIFICATION
C18, 250 mm, 4,6 mm, 5 µm, 300 A
Bonded phase
Particle size ,
Smaller PZ introduce
• higher surface area,
• higher peak resolution
• Shorter run time
• But, higher back
pressure too
Particle ‘s Pores size
• Below 60 A
• 60-150 A
• 150-300 A (proteins)
Depending on target
analytes and matrix
Inter diameter
Smaller ID
• Higher peak
resolution
pressure too
Length
Increasing L
• Higher peak
resolution
pressure too
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
High Vinj and analyst Conc.
May cause fronting of un-retained molecules

COLUMN
SPECIFICATION
C18, 5 µm, 150 mm × 4.6 mm column
C18, 3 µm, 150 mm × 4.6 mm column
C18, 2.7 µm, 100 mm × 4.6 mm
HPLC-FLD chromatograms of aflatoxins

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

SAMPLE FILTERING
AND COLUMN
CLOGGING
https://www.sigmaaldrich.com/content/dam/sigmaaldrich/pro
duct8/135/p000543.tif/_jcr_content/renditions/p000543-
medium.jpg
https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcQVL_oKnG-
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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

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

MOBILE PHASE, KOW
Kow Polarity
• 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,

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).

MOBILE PHASE,
GRADIENT ELUTION
A (H2O:MeOH 5%, pH 4.6). B (MeOH)
Time
10 % B
0.01-5.00
% B
70
5.01-7.00
10 % B
7.01-10.00
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.
 It is 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).

 For separation 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.
pH= pKa+ Log (
[𝐴−]
[𝐻𝐴]
)
At pH= 2,
-2.2= Log (
[𝐴−]
[𝐻𝐴]
), [HA]=166 [𝐴−
]

pH= pKa+ Log (
[𝐴−]
[𝐻𝐴]
)
Hexane
MeOH (basic mix)
MeOH (Acidic mix)
Ricinoic acid (castor Oil)

 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.
pH= pKa+ Log (
[𝐴−]
[𝐻𝐴]
)
Cbuf = [𝐴−] + [𝐻𝐴]
Buffer
(pH & Concentration)

 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.
β=
𝑑 𝑛
𝑝𝐻
Since, the addition of n moles from a base (NaOH) leads to the formation of [𝐴−
Na] or CNaA
β=[𝐴−
b]
𝑝𝐻
https://www.kisspng.com/png-bouncer-clip-
art-bodyguard-security-guard-royalty-
5953930/
β

https://www.kisspng.com/png-bouncer-clip-
art-bodyguard-security-guard-royalty-
5953930/
β
Fixed
pH= pKa+ Log (
[𝐴−]
[𝐻𝐴]
)
Cbuf = [𝐴−
] + [𝐻𝐴]

• 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 ?

Volatile buffers that can be used for LC MS

HPLC DETECTORS
HPLC
UV-Vis
MS
Others
Florescence

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

COLUMN EFFICIENCY
Plate Number (N)
N= (𝑅𝑡/ )2
N= 16(𝑅𝑡/Wb)2
N= 5.45 (𝑅𝑡/W 0.5)2
Wb= 4*x Standard deviation ( )
Selectivity
=
𝑅𝑡 (𝑥)
𝑅𝑡 (𝑦)
Resolution
R =
2 [𝑅𝑡 𝑥 −𝑅𝑡 𝑦 ]
(𝑊𝑏 𝑥 +𝑊𝑏 (𝑦)
R=
Δ𝑅𝑡
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

A. Prof. Sherif M. Taha
Tel: 01004724944
sherif2taha@gmail.com
Thank You

HPLC_A practical guide for the beginner users.pdf

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