High performance liquid

chromatography

A practical guide for

HPLC beginner users

Dr. Sherif M. Taha

 Introduction
HPLC
 Chromatography; Is the separation and
detection of certain compounds in a sample
(Food, water, blood, urine, chemicals, …)
depending on their different chemical-physical
properties and subsequently different physical
interactions between two immiscible phases.
 These compounds may be separated in a
liquid-liquid extraction

 The separation and detection of GC

these compounds carried out by
transferred it through an immobilized
stationary phase by a carrier gas
(GC) or a mobile phase (LC).
 Introduction

https://bitesizebio.com/29947/basics-chromatography-column/

Mikhail Semenovich Tswett

 Basic Components
The basic components of HPLC system
 Mobile phase; sample components are not
only passed through the stationary phase by
the mobile phase as in GC, but also be in
partitioning between the mobile phase and
the stationary phase.
 Pump http://www.mtsrecreations.com/wp-
content/uploads/2018/09/kids-waterslide-fun-
 Column; through which the sample passed 3128-kna-1.jpg

and its components were separated

according to their different partitioning
interactions with the surface of the column
’s particles and the used mobile phase.
 Detector

D C B A
A
C B
D
 Basic Components

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

 HPLC Common Separations
separation

Adsorption Partition size-exclusion Ion Exchange Chiral

Polar St phase Non polar St phase,

Normal phase Reversed phase

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

I II III IV V
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
 partition constant
[𝑆𝑜𝑙𝑢𝑡𝑒 𝑛𝑜𝑡 𝑖𝑜𝑛𝑖𝑠𝑒𝑑 𝑖𝑛 𝑜𝑐𝑡𝑎𝑛𝑜𝑙]
partition constant: Kow, Dow =
[𝑆𝑜𝑙𝑢𝑡𝑒 𝑛𝑜𝑡 𝑖𝑜𝑛𝑖𝑠𝑒𝑑 𝑖𝑛 𝑤𝑎𝑡𝑒𝑟]
[𝑆𝑜𝑙𝑢𝑡𝑒 𝑖𝑜𝑛𝑖𝑠𝑒𝑑 𝑖𝑛 𝑜𝑐𝑡𝑎𝑛𝑜𝑙]
Dow = [𝑆𝑜𝑙𝑢𝑡𝑒 𝑖𝑜𝑛𝑖𝑠𝑒𝑑 𝑖𝑛 𝑤𝑎𝑡𝑒𝑟]

http://dx.doi.org/10.1016/B978-0-12-385013-3.00009-4
 Stationary phase, RP
Reverse phase column, especially C18 and C 8, is the commonly
used one in HPLC analyses.

C8 RP

Reaction with analytes

(peak tailing)

• Reduce reactions with analytes

• Increase pH stability
https://www.chromacademy.com/lms/sco4/Theory_Of_HPLC_Column_Chemistry.pdf
 NP and RP

Polar Non polar

Stationary phase 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

o Improvement the obtained selectivity by using a specific

separation condition (NP or RP) should be carried out firstly with
selecting proper mobile phase conditions then changing the
stationary phase.

o It is also preferred to use previously published conditions

(especially the used column) then modify it according to your own
need.
 Column specification

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

High Vinj , Conc.

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

http://dx.doi.org/10.1016/B978-0-12-385013-3.00009-4
 Column specification
HPLC-FLD chromatograms for aflatoxin analysis

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

Journal of Chromatography B, 889– 890 (2012) 138– 143

 Column specification

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

 Sample particle size

Sample filtering and column clogging

https://encrypted-
tbn0.gstatic.com/images?q=tbn:ANd9GcQVL_oKnG-
Wr4kJeF2xVFsIjmlToXWu8g0mJ6ChKcHczDpHUaJ05Q

https://www.sigmaaldrich.com/content/dam/si
gmaaldrich/product8/135/p000543.tif/_jcr_cont https://blog.restek.com/?p=34681
ent/renditions/p000543-medium.jpg

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
 For RP HPLC, The elution strength for 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). Therefore water used for first
loading of sample components in RP HPLC.
Kow Polarity

 Iso-propanol is used when changing between two immiscible solvents. It is

also used for long storage of the used columns.

 The used solvent should be compatible to the used detector (with no UV

absorbance for HPLC UV,….

 A mixture of solvent is commonly used in HPLC (usually water with a

miscible organic solvent) since it give a new Kow that rapid the
chromatographic separation run.

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

 Mobile phase
• 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,

http://dx.doi.org/10.1016/B978-0-12-803684-6.00013-5
 Gradient Mb phase
 Gradients mobile phase may also be obtained with three or four
solutions for some HPLC instruments, but, with no high additional
advantages.
 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).

 Gradient Mb phase

Benzoic and sorbic acid determination in juice, milk products,…

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

Benzoic (4 ppm)
Sorbic acid (2 ppm)
Vinj: 2µl
Flow: 0.50 ml
 Solvent A, Mb 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 MSMS)
 Solvent A, Buffer
 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
 Effect of pH
Ricinoic acid (castor Oil) [𝐴− ]
pH= pKa+ Log ( )
[𝐻𝐴]

Hexane

MeOH (Acid mix)

MeOH (basic mix)
 Solvent A, Buffer
A buffer is a solution of a weak acid (HA) and its conjugate
base (𝐴− ) or 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 ( )
[𝐻𝐴]
Buffer
pH &
Concentration
Cbuf = [𝐴− ] + [𝐻𝐴]
 Buffer capacity, β

 Buffer capacity, β: 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 https://www.kisspng.com/png-
bouncer-clip-art-bodyguard-security-
guard-royalty-5953930/

[𝐴− b]
β= 𝑝𝐻
 Buffer capacity, β

Fixed

http://dx.doi.org/10.1016/B978-0-12-803684-6.00004-4
 Solvent A, Buffer
 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 ?
Solvent A, Buffer
 Solvent A, Buffer
Volatile buffers that can be used for LC MS

http://dx.doi.org/10.1016/B978-0-12-811732-3.00006-6
 Sample ’s solvent
Sample ’s solvent should be with;
- Polarity is the same as the mobile phase (loading solvent, A) or weaker
(increase solvent compressing, enhance the retention of solutes).
- A pH close to that of the mobile phase (loading solvent, A)
These points are more critical, especially for higher injection volume and for ionized 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.

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

UV-Vis

Floresce https://www.slideshare.net/sherif_taha/mass-spectrometry-for-
nce HPLC MS pesticides-residue-analysis-l3

Others
 HPLC Chromatogram
 A blot of peak intensity versus retention time is a chromatogram
 A blot of peak intensity versus a mass/charge is a mass spectrum

lC N

NH
342

lC
 Column efficiency
Actual Rt (X)= Obtained Rt (X)+ t0
Dead time, Void time

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

Depending on column length and flow rate

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

Plate Number (N) Selectivity Resolution

N= (𝑅𝑡/ )2 𝑅𝑡 (𝑥)
=𝑅𝑡 (𝑦) 2 [𝑅𝑡 𝑥 −𝑅𝑡 𝑦 ]
R = (𝑊𝑏
N= 16(𝑅𝑡/Wb)2 𝑥 +𝑊𝑏 (𝑦)

N= 5.45 (𝑅𝑡/W 0.5)2 Δ𝑅𝑡

R=
Wb= 4*x Standard deviation ( ) Wb
Dr. Sherif Mohamed Taha
[email protected]