Basics of Liquid Chromatography for Clinical LC/MS/MS

PEARLS OF LABORATORY MEDICINE

Liquid Chromatography: Separation Mechanisms

Y. Victoria Zhang, PhD, DABCC

University of Rochester Medical Center

DOI:10.15428/CCTC.2015.244665

• LC Mechanisms • Overview • Mechanism: Reverse Phase • Isocratic vs. Gradient Separations

• LC Separation Parameters

• Retention Time, tR • Resolution, Rs • Efficiency, N • Retention Factor (Capacity), κ • Selectivity, a

• AND – learn to “think like a molecule!”

2 Mechanism: Overview Flow Direction

T = 0

T = 2

T = 5

The analytes (RGY) partition between the mobile phase and the stationary phase

3 Reverse Phase Overview

Packing materials are based on silica gel, a network polymer with –OH groups on the surface

Hydrocarbon chains of various lengths are bonded to the silica gel C4 bonded to – different lengths give different Silica particle

retention properties C8

C18 Any one silica gel particle has many, many hydrocarbon chains bonded to it

C8 on silica

4 Mechanism of Reverse Phase Stationary Phase: C18 bounded silica | Mobile Phase: Organic Hydrophobic Compound

Loading Low Organic Elution 1 Elution 2 High Organic Re- Washing Washing equilibration

C18 Flow Flow

Time (min) Time (min)

5 Partitioning 90% H2O Mobile Phase

Polar Retention

Non Polar

Mobile Phase 50% Organic Non Polar Non Polar Partitioning

Non Polar Non Polar Elution90% Organic

Mobile Phase

Non Polar Elution

Isocratic vs. Gradient Separation

Mobile B concentration (%) concentration MobileB (%) concentration MobileB

Time (min) Time (min) Advantages: Advantages: Simple – one bottle Higher resolution Larger range of components eluted Disadvantages: Disadvantages: Peak spreading Programing more complicated Some compounds may not be eluted Column needs recovery time

7 Isocratic vs. Gradient Separation

1 2 3 4 Isocratic 30% ACN

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

1,2 3 4 Isocratic 60% ACN

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

2 3 4 Gradient 1 10% to 80% ACN 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

The gradient is reset to initial conditions to re-equilibrate post-analysis 8 Chromatographic Parameters

• Basic concepts

• Retention time, tR

• Resolution, Rs

• Column variables that impact separation • Efficiency, N • Retention (Capacity), κ • Selectivity, a

9 Retention Time, tR

tR = 0.75 minutes tR = 1.25 minutes

10 Resolution, Rs Complete Resolution 1 8.42 8.95

0.8 tR1 tR2 0.6

0.4 Wb1 Wb2 0.2

0 7.8 8 8.2 8.4 8.6 8.8 9 9.2 9.4 0 0.5 1 1.5 2 Wb1 = Wb2 = 0.33 Incomplete Resolution

W = the width of a peak

Rs ≥ 1.5  two peaks are baseline resolved; the signal returns to baseline before the response for the second 0 0.5 1 1.5 2 analyte starts 2(8.95 − 8.42) 2 ∗ 0.53 푅푠 ≅ = ≅ 1.6 2 ∗ 0.33 0.66 11 Three Major Chromatogram Factors Impacting Resolution, Rs

1 푘 훼 −1 푅 = 푁 × × 푠 4 1+푘 훼 Efficiency Retention Selectivity Efficiency (N) Retention (κ)

Selectivity (α) Good N

Poor N

12 Efficiency, N The ability of a column to produce narrow peaks

Better 2 푡 2 푡 푁 = 16 푅 = 5.54 푅 푊퐵 푊1/2 W1/2

13 Determination of Retention Factor (Capacity), κ How well any one analyte is retained

Injection Valve 풕 − 풕 κ = 푹 ퟎ Closes t % Organic R 풕ퟎ

Lower κ The blue line illustrates t0 the gradient profile in a reverse phase separation showing the difference in retention time for the same tR Higher κ analyte resulting from a different gradient.

Solvent Front or Dwell time 14 Selectivity (Separation) Factor, 훼 The ability to distinguish between species being separated

tR2

tR1

t0 Inject

15 Parameter Summary Parameter N k a (Efficiency) (Capacity) (Selectivity) % Organic Solvent Organic Solvent Choice Column Type Column Length Particle Size Mobile Phase pH * Buffer Concentration * Ion-pair Reagent Concentration * Flow Rate Large effect Small effect Little to no effect Dark arrows indicate parameters commonly used to control N, k or a. Light arrows indicate parameters not commonly used for control * Most applicable to acidic or basic analytes 16 Chromatography Method Development

• Chromatography method development is a balance between efficiency, retention, and selectivity • Goal: obtain an adequate separation within the desired timeframe

Method

Selectivity Efficiency Retention [analyte(s) of (narrow peaks are (shorter separations interest separated good) desirable) from other compounds]

17 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, 912 p.pp. 5. Snyder LR, Kirkland JJ, Glajch JL. Practical hplc method development. 2nd ed. New York: Wiley, 1997:xxvi, 765 p.pp.

18 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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