This presentation introduces general information for HPLC analysis with more focusing on proper selection for the used column and mobile phase.

1. Dr. Sherif M. Taha
A practical guide for
HPLC beginner users
High performance liquid
chromatography

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

3. Introduction
Mikhail Semenovich Tswett
https://bitesizebio.com/29947/basics-chromatography-column/

4. 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
 Column; through which the sample passed
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
A
C
D
B
B
AD C
http://www.mtsrecreations.com/wp-
content/uploads/2018/09/kids-waterslide-fun-3128-
kna-1.jpg

5. Basic Components
Pilar Campíns-Falcó; Rosa Herráez-Hernández; Pascual Serra-Mora, Liquid Chromatography-Instrumentation

6. Common SeparationsHPLC
separation
Adsorption
Polar St phase
Normal phase
Partition
Non polar St phase,
Reversed phase
size-exclusion Ion Exchange Chiral
B
B
A
C
A
D
xH
x
x
x
H
I II III IV V
C
E
B
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
A

7. partition constant
partition constant: Kow, Dow =
[𝑆𝑜𝑙𝑢𝑡𝑒 𝑛𝑜𝑡 𝑖𝑜𝑛𝑖𝑠𝑒𝑑 𝑖𝑛 𝑜𝑐𝑡𝑎𝑛𝑜𝑙]
[𝑆𝑜𝑙𝑢𝑡𝑒 𝑛𝑜𝑡 𝑖𝑜𝑛𝑖𝑠𝑒𝑑 𝑖𝑛 𝑤𝑎𝑡𝑒𝑟]
Dow =
[𝑆𝑜𝑙𝑢𝑡𝑒 𝑖𝑜𝑛𝑖𝑠𝑒𝑑 𝑖𝑛 𝑜𝑐𝑡𝑎𝑛𝑜𝑙]
[𝑆𝑜𝑙𝑢𝑡𝑒 𝑖𝑜𝑛𝑖𝑠𝑒𝑑 𝑖𝑛 𝑤𝑎𝑡𝑒𝑟]
http://dx.doi.org/10.1016/B978-0-12-385013-3.00009-4

8. Stationary phase, RP
Reverse phase column, especially C18 and C 8, is the commonly
used one in HPLC analyses.
Reaction with analytes
(peak tailing)
C8 RP
https://www.chromacademy.com/lms/sco4/Theory_Of_HPLC_Column_Chemistry.pdf
• Reduce reactions with analytes
• Increase pH stability

9. NP and RP
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.
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

10. 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 analytes and matrixInter diameter
Smaller ID
• Higher peak resolution
• But, higher back pressure too
Length
Increasing L
• Higher peak resolution
• But, higher back pressure too
http://dx.doi.org/10.1016/B978-0-12-385013-3.00009-4
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 , Conc.
Fronting of un-retained molecules

11. 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 for aflatoxin analysis
Journal of Chromatography B, 889– 890 (2012) 138– 143

12. Pilar Campíns-Falcó; Rosa Herráez-Hernández; Pascual Serra-Mora, Liquid Chromatography-Instrumentation
Column specification

13. Sample particle size
Sample filtering and column clogging
https://blog.restek.com/?p=34681
https://www.sigmaaldrich.com/content/dam/sigmaal
drich/product8/135/p000543.tif/_jcr_content/renditio
ns/p000543-medium.jpg
https://encrypted-
tbn0.gstatic.com/images?q=tbn:ANd9GcQVL_oKnG-
Wr4kJeF2xVFsIjmlToXWu8g0mJ6ChKcHczDpHUaJ05Q
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

14.  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.
 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, Kow
Kow Polarity

15. 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

16.  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

17. Benzoic and sorbic acid determination in juice, milk products,…
Gradient Mb phase
Benzoic (4 ppm)
Sorbic acid (2 ppm)
Vinj: 2µl
Flow: 0.50 ml
A (H2O:MeOH 5%, pH 4.6).
B (MeOH)
Time
10 % B0.01-5.00
% B705.01-7.00
10 % B7.01-10.00

18.  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, Mb phase

19.  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.
Solvent A, Buffer
pH= pKa+ Log (
[𝐴−]
[𝐻𝐴]
)
• At pH= 2,
-2.2= Log (
[𝐴−]
[𝐻𝐴]
)
[HA]=166 [𝐴−]
http://dx.doi.org/10.1016/B978-0-12-803684-6.00004-4

20. Effect of pH
pH= pKa+ Log (
[𝐴−]
[𝐻𝐴]
)
Hexane
MeOH (basic mix)
MeOH (Acid mix)
Ricinoic acid (castor Oil)

21.  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.
Solvent A, Buffer
pH= pKa+ Log (
[𝐴−]
[𝐻𝐴]
)
Cbuf = [𝐴−
] + [𝐻𝐴]
Buffer
pH &
Concentration

22.  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
Buffer capacity, β
β
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5953930/

23. Buffer capacity, β
Fixed
http://dx.doi.org/10.1016/B978-0-12-803684-6.00004-4

24.  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

25. Solvent A, Buffer

26. 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

27. Sample ’s solvent
http://dx.doi.org/10.1016/B978-0-12-385013-3.00009-4
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.
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

28. Detectors
HPLC
UV-
Vis
MS
Others
Florescen
ce
https://www.slideshare.net/sherif_taha/mass-spectrometry-for-pesticides-
residue-analysis-l3

29. 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
N
O
NH
Cl
Cl
342

30. 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)

31. 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
Wb
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

32. Dr. Sherif Mohamed Taha