PEARLS of LABORATORY MEDICINE Liquid Chromatography

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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

• 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

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

• Types of 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

3 LC Components

4 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

5 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 6 Columns: Dimensions

Common Common Common Particle Common Pore IDs (mm) Lengths (mm) Sizes (um) Sizes (Å) • Available in a variety of (smaller) 2 1.3 20 lengths and internal 0.2 4 1.7 25 diameters (IDs) 0.5 5 1.8 50 1 10 2.5 60 2 15 2.6 65 3 20 3 70 • Variability in particle and 4 30 3.5 80 pore sizes 4.6 35 3.6 90 5 50 4 100 6 60 5 110 10 75 6 120 • Packing is determined (larger) 100 7 125 by the type of separation 150 8 (larger) 200 10 desired 250 (larger) 300 (longer)

7 Columns: Particle Sizes

Particle # of Back Size Theoretical Pressure Plates 35 5µm 12000 1100psi 30 25 20 15

3µm 22000 2900psi 10 # of Plates Plates of #

5 thousands) ( 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 8 Chromatography Pressure Regimes

6000 psi • Low Pressure (400 bar) • Gravity is the “Pump” • Used for sample prep 20,000 psi (1400 bar) • Used for synthesis purification 0 psi 6000 psi (400 bar) • High Pressure – HPLC 20,000 psi • Traditional pressure (1400 bar) 0 psi • Routine (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) 9 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

10 Types of Separation

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

• Ion Exchange • Chiral Separation

11 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

12 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

C18 13 Reverse Phase: Partition and Separation

90% H2O Mobile Phase 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 Elution90% Organic

Mobile Phase

Non Polar Elution

14 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

15 SEC / GPC / GFC

• Size Exclusion Chromatography / Gel Permeation Chromatography / Gel Filtration Chromatography

• Separation of polymers (SEC) or Stationary phase: gel or biopolymers (GPC/GFC) polymer with tightly controlled pore size • Separation based on molecular size (Stokes radius) Separation Mechanism: – based on molecules path through the column • Used for large molecule – small molecules travel separations: protein MW, polymer easier into pores of the stationary phase, “retained” MW, and polymer distributions longer than larger molecules

16 Ion Exchange Chromatography + Analyte – Anions • Stationary phase: resin with and Cations + - covalently bound charged functional - groups + + • Different columns for analyzing Injection + + anions and cations + + + + • Used for separating ionic species • F-, Cl-, Br-, NO -, SO 2- etc. 3 4 + + 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

18 Summary

LC - Basics

LC – Separation Mechanisms

LC – Method Development

19 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

20 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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