High-Performance Thin Layer Chromatography: A Technique for Preliminary Separation and Quantification of Lipids From Neutrophils

Abstract

Source: Brogden, G., et al. Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps. J. Vis. Exp. (2017).

This video demonstrates a technique for the preliminary separation and quantification of lipids from human peripheral blood-derived neutrophils. This technique helps in the initial evaluation of target analytes before proceeding with HPLC.

Protocol

1. High-performance thin layer chromatography (HPTLC) to semi-quantify the amount of several lipids, including cholesterol, phospholipids, and sphingomyelin

1. Prepare the following four running solutions and staining solutions.
   1. Solution 1: Mix ethyl acetate (26.6%), 1-propanol (26.6%), chloroform (26.6%), methanol (26.6%), and potassium chloride (9.6%). Prepare KCl by dissolving 0.25 g of KCl in 100 mL of HPLC-quality water.
   2. Solution 2: Mix n-hexane (73%), diethyl ether (23%), and citric acid (2%).
   3. Solution 3: 100% n-hexane.
   4. Prepare the staining solution by mixing distilled water (90 mL) with 7.5 g of copper sulfa....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-propanol	Sigma-Aldrich	33538
10 µL syringe	Hamilton	701 NR 10 µl
Diethyl ether	Sigma-Aldrich	346136
Ethyl acetate	Carl Roth	7336.2
Copper(II)sulphatepentahydrate	Merck	1027805000
Chloroform	Carl Roth	7331.1
CP ATLAS software	Lazarsoftware	2
Methanol	Carl Roth	7342.1
n-Hexane	Carl Roth	7339.1
Phosphoric acid	Sigma-Aldrich	30417
Potassium chloride	Merck	49,361,000
HPTLC silica gel 60	Merck	105553