High Performance Liquid Chromatography Separation of Epigenetic Cytosine Variants
Abstract
:1. Introduction
2. Materials and Methods
3. Results and Discussion
3.1. Resolution of Cytosine Analogs Using C18 Column
3.2. Resolution of Cytosine Analogs Using Phenyl Hexyl Column
3.2.1. Performing HPLC Runs at a pH of 4.0
3.2.2. Performing HPLC Analysis at a pH of 7.0
3.2.3. Adjusting Methanol Concentration to Improve Resolution
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Figure | Column | Mobile Phases | Elution (%, v/v) | Flow Rate mL/min |
---|---|---|---|---|
2 | Agilent C18 (50 × 3 mm, 1.8 μm) | A: CH3OH B: 50 mM NH4H2PO4 (pH 4.0) C: H2O | 3 min 5% A, 15% B 6 min 20% A, 15% B 6.05 min 30% A, 15% B 9 min 30% A, 15% B 9.05 min 5% A, 15% B 13 min 5% A, 15% B | 1.4 |
3 | Luna Phenyl Hexyl (150 × 4.6 mm, 5 μm) | A: CH3OH B: 50 mM NH4H2PO4 (pH 4.0) C: H2O | 4 min 5% A, 15% B 6 min 20% A, 15% B 6.05 min 30% A, 15% B 9 min 30% A, 15% B 9.05 min 5% A, 15% B | 1.4 |
3 (insert) | Luna Phenyl Hexyl (150 × 4.6 mm, 5 μm) | A: CH3OH B: 50 mM NH4H2PO4 (pH 4.0) C: H2O | 8 min 5% A, 15% B 11 min 20% A, 15% B 11.05 min 30% A, 15% B 16 min 30% A, 15% B 16.05 min 5% A, 15% B | 1.4 |
4a | Luna Phenyl Hexyl (150 × 4.6 mm, 5 μm) | A: CH3OH B: 50 mM NH4H2PO4 (pH 7.0) C: H2O | 8 min 5% A, 15% B 11 min 20% A, 15% B 11.05 min 30% A, 15% B 16 min 30% A, 15% B 16.05 min 5% A, 15% B | 1.4 |
4b | Luna Phenyl Hexyl (150 × 4.6 mm, 5 μm) | A: CH3OH B: 50 mM NH4H2PO4 (pH 7.0) C: H2O | 8 min 3 % A, 15% B 11 min 20% A, 15% B 11.05 min 30% A, 15% B 16 min 30% A, 15% B 16.05 min 3% A, 15% B | 1.0 |
4c | Luna Phenyl Hexyl (150 × 4.6 mm, 5 μm) | A: CH3OH B: 50 mM NH4H2PO4 (pH 7.0) C: H2O | 8 min 1% A, 15% B 11 min 20% A, 15% B 11.05 min 30% A, 15% B 16 min 30% A, 15% B 16.05 min 1% A, 15% B | 1.4 |
Column | Luna Phenyl Hexyl (150 × 4.6 mm, 5 μm) | ||
---|---|---|---|
Mobile phases | A: Methanol | ||
B: 50 mM ammonium phosphate (pH of 7.0) | |||
C: Deionized water | |||
Gradient elution | Time (min) | %A | %B |
0 | 1 | 15 | |
6 | 1 | 15 | |
14 | 30 | 15 | |
16 | 30 | 15 | |
16.05 | 100 | 0 | |
18 | 100 | 0 | |
18.05 | 1 | 15 | |
21 | 1 | 15 | |
Injection volume | 50 μL | ||
Actual injection | 40 μL | ||
Flow rate | 1.4 mL·min−1 | ||
Detection | Diode-Array Dectector 1 A, Signal = 280 nm Reference = 360 nm |
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Roberts, C.E.; Raner, G.M.; Isaacs, G.D. High Performance Liquid Chromatography Separation of Epigenetic Cytosine Variants. Methods Protoc. 2018, 1, 10. https://doi.org/10.3390/mps1020010
Roberts CE, Raner GM, Isaacs GD. High Performance Liquid Chromatography Separation of Epigenetic Cytosine Variants. Methods and Protocols. 2018; 1(2):10. https://doi.org/10.3390/mps1020010
Chicago/Turabian StyleRoberts, Caroline E., Gregory M. Raner, and Gary D. Isaacs. 2018. "High Performance Liquid Chromatography Separation of Epigenetic Cytosine Variants" Methods and Protocols 1, no. 2: 10. https://doi.org/10.3390/mps1020010
APA StyleRoberts, C. E., Raner, G. M., & Isaacs, G. D. (2018). High Performance Liquid Chromatography Separation of Epigenetic Cytosine Variants. Methods and Protocols, 1(2), 10. https://doi.org/10.3390/mps1020010