PEARLS OF LABORATORY MEDICINE

Liquid Chromatography:
LC Basics and Separation Types

Y. Victoria Zhang, PhD, DABCC

University of Rochester Medical Center

DOI:10.15428/CCTC.2015.240044

© Clinical Chemistry
Outline
• Definitions

• LC Components
• Columns; solvents/mobile phase; pumps; autosampler;
detector

• Columns
• Key parameters; stationary phase; dimensions; particle
sizes; pressure regimes

• Types of Separations
• Normal phase; reverse phase; HILIC; size exclusion;
ion exchange; chiral chromatography

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What is Chromatography?
• Chromatography:
• Derived from the Greek words for “color writing”
• Mikhail Tsvet

• Types of Chromatography Supercritical fluid chromatography

• Based on mobile phase (GC, LC, SFC)

• Based on separation type (IC, GPC/SEC)

• Liquid:
• LC is a separation based on a liquid mobile phase
• Other separations use gases or supercritical fluids
as the mobile phase

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LC Components

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Columns: Key Parameters

Length, mm
Inner Diameter (ID), mm
Particle Size, um
Pore Size
Packing
• Packing Material
50 x 2.1mm 2.5µm C18 100Å
2.5µm dia.
• Dimensions 2.1mm ID

• Particle Size 50mm (Length)

Frits

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 Columns: Stationary Phase / Packing Material
Column: where the separation happens Normal Phase
Separation: based on interaction between
- Analyte
- Mobile Phase
- Stationary Phase
Silica Gel
Reverse Phase

C8 on Silica Alumina
C4 on Silica

Cyanopropyl Phenyl-hexyl
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Columns: Dimensions

Common Common Common Particle Common Pore

• Available in a variety of IDs (mm)

(smaller)
Lengths (mm)
2
Sizes (um)
1.3
Sizes (Å)
20
lengths and internal 0.2 4 1.7 25
diameters (IDs) 0.5
1
5
10
1.8
2.5
50
60
2 15 2.6 65
3 20 3 70
• Variability in particle and 4 30 3.5 80
pore sizes 4.6
5
35
50
3.6
4
90
100
6 60 5 110
10 75 6 120
• Packing is determined (larger) 100 7 125
by the type of separation 150
200
8
10
(larger)

desired 250 (larger)

300
(longer)

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Columns: Particle Sizes
Particle # of Back
Size Theoretical Pressure
Plates
35
5µm 12000 1100psi 30

( thousands)
25

# of Plates
20
15
3µm 22000 2900psi 10
5
0
5 0
1.7µm 32500 8600psi Particle Size (µm)

2 X Length

1.7µm 65000 17200psi

Compromise is to go to shorter, narrower columns with smaller particle sizes

Figure courtesy of Mike Wright with modifications
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 Chromatography Pressure Regimes
6000 psi
• Low Pressure (400 bar)

• Gravity is the “Pump”

• Used for sample prep 20,000 psi

• Used for synthesis purification 0 psi (1400 bar)

6000 psi
(400 bar)

• High Pressure – HPLC

• Traditional pressure 20,000 psi
(1400 bar)
• Routine 0 psi
(0 bar)

• Ultra High Pressure – UHPLC or UPLC 6000 psi

• Newest Technology (400
bar)
• Better Performance
• Plumbing Concerns 20,000 psi
0 psi (1400 bar)
• Two pressure units – bar and psi (0 bar)

To convert from bar to psi, multiply by 14.50 (or 15 for quick calculation)
To convert from psi to bar, divide by 14.50 (or 15 for quick calculation)
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Progress in Column Technology
2.1 x 50 mm
Changes in column technologies enhance both Peak 1
sensitivity and separation, with new 1.17 min
opportunities and challenges
Peak 2
1.25 min

Peak 1 Tomorrow
3 x 100 mm 3.62 min

Peak 2
3.77 min

4.6 x Magnified Today

250 mm by 5x
Peak 2
Peak 1 15.6 min
13.2 min

Yesterday

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Types of Separation
• Normal Phase
• Reverse Phase
• Hydrophilic Interaction Liquid Chromatography (HILIC)
• Size Exclusion Chromatography (SEC)
- Gel Permeation Chromatography (GPC)

• Ion Exchange
• Chiral Separation

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Normal Phase
• Polar stationary phase: silica or alumina
- (many exposed hydroxyl groups)

• Non-polar mobile phase

Silica
• Largely supplemented by other techniques

• Good for polar analytes

• Reproducibility can be difficult Alumina

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Reverse Phase
• Most common
• Non-polar stationary phase
• Aqueous or moderately polar mobile phase
• MANY different stationary phases available
• C4, C8, C18
• Cyano, Phenyl, Fluorophenyl, PFP
• Amide, Amino
• Excellent for “normal” organic compounds
C4 bonded to Silica particle

C8

C18
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Reverse Phase: Partition and Separation
Mobile Phase
90% H2O
Polar Retention More mechanisms
in Part 2 of this
series
Non Polar

Mobile Phase
50% Organic
Non Polar
Non Polar Partition

Non Polar Non Polar

Elution
90% Organic

Mobile Phase

Non Polar
Elution

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HILIC (Hydrophilic Interaction Liquid Chromatography)

• Modern adaptation of normal phase chromatography

• Well defined polar stationary phase

NP IC
• Acetonitrile + water mobile phase HILIC

• (or other aqueous-miscible solvent)

RP

• Works well for very polar compounds

• Acids, Bases, Zwiterions
• Glycosylates, Metabolites

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SEC / GPC / GFC
• Size Exclusion Chromatography /
Gel Permeation Chromatography /
Gel Filtration Chromatography
• Separation of polymers (SEC) or
biopolymers (GPC/GFC)
• Separation based on molecular
size (Stokes radius)
• Used for large molecule
separations: protein MW, polymer
MW, and polymer distributions
Stationary phase: gel or
polymer with tightly
controlled pore size
Separation Mechanism:
– based on molecules path
through the column
– small molecules travel
easier into pores of the
stationary phase, “retained”
longer than larger molecules
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Ion Exchange Chromatography
+
• Stationary phase: resin with
Analyte – Anions
and Cations +
- -
covalently bound charged functional
groups + +
• Different columns for analyzing Injection + +
anions and cations + +
+ +
• Used for separating ionic species
• F-, Cl-, Br-, NO3-, SO42- etc. +- +
o Anions in physiological fluids
Retention + -+
• Ammonia, Methylamine, etc. + +
o Cations in physiological fluids + ++
o Transition metal ions in plasma
and blood
+
• Proteins + +
• Carbohydrates + +
Elution
• Oligosaccharides + +
+- +
- 17
Chiral Chromatography

• Separation of enantiomers
• Chiral stationary phase
• Cellulose
• -cyclodextrin
• Imidizole antifungals
• NSAIDS

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Summary

LC - Basics

LC –
Separation
Mechanisms

LC – Method
Development

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References
1. Carr PW, Stoll DR, Wang X. Perspectives on recent
advances in the speed of high-performance liquid
chromatography. Analytical Chemistry 2011;83:1890-900.
2. Chester TL. Recent developments in high-performance
liquid chromatography stationary phases. Analytical
Chemistry 2013;85:579-89.
3. Dong MW. Modern HPLC for practicing scientists.
Hoboken, N.J.: Wiley-Interscience, 2006:xvi, 286 p.pp.
4. Snyder LR, Kirkland JJ, Dolan JW. Introduction to modern
liquid chromatography. 3rd ed. Hoboken, N.J.: Wiley,
2010:xli, p. 912
5. Snyder LR, Kirkland JJ, Glajch JL. Practical HPLC method
development. 2nd ed. New York: Wiley, 1997:xxvi, p. 765

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Disclosures/Potential Conflicts of Interest

Upon Pearl submission, the presenter completed the Clinical Chemistry

disclosure form. Disclosures and/or potential conflicts of interest:
▪ Employment or Leadership: None declared
▪ Consultant or Advisory Role: None declared
▪ Stock Ownership: None declared
▪ Honoraria: None declared
▪ Research Funding: None declared
▪ Expert Testimony: None declared
▪ Patents: None declared

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