Thin Layer Chromatography-Based Separation of Mycolic Acid Variants: A Method to Separate Mycobacterial Cell Wall Lipids Based on Polarity

Overview

This video demonstrates the thin-layer chromatography-based separation of mycolic acids. This technique separates the different mycolic acids—long-chain lipids present in the cell wall of mycobacteria—based on their polarity.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Extraction of the total non-covalent-linked lipids from mycobacteria (Figure 1)

1. Scratch 0.2 g of mycobacteria from a solid media and add to a glass tube with a polytetrafluoroethlene (PTFE) liner screw caps. Add a solution consisting of 5 mL of chloroform and 10 mL of methanol (chloroform:methanol, 1:2).
   NOTE: When organic solvents are used, only glass recipient should be used. No plastic containers are allowed. Moreover, PTFE liner screw caps for bottles are needed.
   CAUTION: Chloroform is a potentially toxic and extremely hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
   CAUTION: Methanol is a potentially toxic and extremely hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
2. Leave the tube in constant stirring overnight to extract non-covalent-linked lipids from the mycobacterial cell surface.
   NOTE: If an orbital shaking platform is not available, constant stirring can be replaced by periodic manual stirring as frequently as possible.
3. Cover a glass funnel with a filter paper, filter the organic solvents, and collect them in a glass tube.
4. Use a nitrogen gas flux to evaporate the liquid phase in the tube. Fill the tube with nitrogen gas, cover and store it at 4 °C.
   NOTE: Connect a glass Pasteur pipette to the stream of nitrogen gas to specifically evaporate the desired tube. Additionally, maintain the tube inside a dry block heater for tubes at 37 °C. When the solvent evaporates, fill the tube with nitrogen gas before closing it.
5. Add 15 mL of a solution of chloroform:methanol (2:1) to the cellular debris. Leave the tube in constant stirring overnight to extract non-covalent-linked lipids from the mycobacterial cell surface.
   NOTE: If an orbital shaking platform is not available, constant stirring can be replaced by periodic manual stirring as frequently as possible.
6. Let the mixture rest for 1 h. With a Pasteur pipette, recover the organic solvents. Cover a glass funnel with a filter paper and filter the organic solvents and collect them in the same glass tube previously used in step 1.3. Use a nitrogen gas flux to evaporate the liquid phase in the tube. Fill the tube with nitrogen gas, close it and store it again at 4 °C.

2. Mycolic acid extraction by acid methanolysis (Figure 2A)

1. Add 2-5 mL of esterifying solution into a hermetic glass tube with a PTFE liner screw cap. Add 0.2 g of mycobacteria biomass into the glass tube.
   NOTE: Esterifying solution is formed by mixing 30 mL of methanol, 15 mL of toluene, and 1 mL of sulfuric acid. Mycobacteria cells can be taken from solid cultures or, even from delipidated cells after performing extraction of total non-covalent-linked lipids from mycobacteria (remaining cells after filtering in step 1.6).
   CAUTION: Toluene is a flammable and extremely hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
   CAUTION: Sulfuric acid is a corrosive and hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
2. Mix the content by vortexing. Let the mixture stand inside a dry bath at 80 °C overnight.
3. Allow the tube to cool until it reaches the room temperature and then add 2 mL of n-hexane to the tube. Mix the contents by vortexing for 30 s and allow the tube to settle until two clear phases appear.
   CAUTION: n-hexane is a potential flammable, irritant, environmentally damaging, and extremely hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
4. Recover the upper phase corresponding to the n-hexane phase. Transfer it to a new tube.
5. Repeat the step 2.3. Recover the upper phase again and transfer it to the same tube used in step 2.4.
6. Evaporate the contents of the tube using a nitrogen gas flux. Fill the tube with nitrogen gas, close it, and store it at 4 °C.

3. Mycolic acid extraction by saponification and methylation (Figure 2B)

1. Scratch 0.2 g of mycobacteria from a solid media and add to a glass tube with a PTFE screw cap.
2. Add 2 mL of methanol-benzene solution (80:20) containing 5% potassium hydroxide. Mix the contents by vortexing. Heat the mixture for 3 h at 100 °C.
   CAUTION: Benzene is a flammable, carcinogenic, and hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
3. Allow the tube to cool to room temperature. Add 20% sulfuric acid to acidify the samples to achieve pH = 1.
4. Add 3 mL of diethyl ether. Gently mix the contents by vortexing.
5. Let the two phases form by settling. Recover the diethyl ether phase and transfer to a new tube. Repeat the wash step for a total of three times.
6. Wash the diethyl ether extract with 2 mL of distilled water and transfer the upper part corresponding to the diethyl ether to a new tube. Repeat the wash step for a total of three times.
7. Add 2 g of anhydrous sodium sulfate over the diethyl ether extract to dry it.
8. Filter the suspension. Evaporate the content using a nitrogen gas flux.
9. To perform the methylation step, dissolve 3 g of N-nitroso-N-methyl urea in a precooled solution formed by 45 mL of diethyl ether and 9 mL of 40% KOH in distilled water.
   CAUTION: N-nitroso-N-methylurea is a toxic, irritant, carcinogenic, and hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
10. Transfer the supernatant (diazomethane) to a new flask cooled in ice containing potassium hydroxide pellets (approximately 30 g).
    NOTE: If the supernatant is not immediately used, it can be stored at -20 °C for a maximum of 1 h.
    CAUTION: Potassium hydroxide pellets are an irritant and corrosive substance. This material must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
    CAUTION: Diazomethane is highly toxic and potentially explosive. It must be used in a laminar flow hood with safety glass wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
11. Add 2 mL of the ether solution containing diazomethane, obtained in step 3.10, into the dried diethyl ether extract that contains mycolic acids, obtained in step 3.8. Incubate for 15 min at room temperature.
12. Evaporate the suspension at 40 °C. Fill the tube with nitrogen gas, close it, and store the methylated lipids at 4 °C.
    NOTE: Evaporate the diazomethane from the ether solution under the laminar flow hood, until the ether loses the yellow color.

4. Thin layer chromatography (TLC) analysis

1. Saturate the glass TLC chamber. To do this, cover one of the walls of the TLC chamber with a piece of filter paper and allow it to be in contact with the mobile phase composed by the mixture of solvents. Place the remaining volume of the solvent onto the bottom of the TLC chamber.
   NOTE: The bottom of the TLC chamber must be covered by at least 1 cm of the mobile phase. In the present experiments, different mobile phases were used to develop the TLCs. They consisted of 85 mL of n-hexane plus 15 mL of diethyl ether; 100 mL of dichloromethane; 90 mL of chloroform, 10 mL of methanol, and 1 mL of water; 30 mL of chloroform, plus 8 mL of methanol, and 1 mL of water; 60 mL of chloroform, plus 35 mL of methanol, and 8 mL of water; 95 mL chloroform plus 5 mL of methanol; and 90 mL of petroleum ether (60-80 °C) plus 10 mL of diethyl ether.
   NOTE: In the two-dimensional TLC, use n-hexane:acetone (95:5) in the first direction three times, and use a single development with toluene:acetone (97:3) in the second direction to analyze mycolic acid composition. To analyze PIMs, use chloroform:methanol:water (60:30:6) in the first direction once, and use chloroform:acetic acid:methanol:water (40:25:3:6) in the second direction. To analyze PDIM and AG, use petroleum ether (60-80 °C):ethyl acetate (98:2) in the first direction three times, and use a single development with petroleum ether (60-80 °C):acetone (98:2) in the second direction.
   CAUTION: Diethyl ether is a potentially toxic and hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
   CAUTION: Dichloromethane is a potentially toxic and hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
   CAUTION: Petroleum ether is a potential flammable, environmentally damaging and extremely hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
   CAUTION: Acetic acid is a potential flammable and corrosive substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
   CAUTION: Ethyl acetate is a flammable and hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
   CAUTION: Acetone is a flammable and hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
2. Close the TLC chamber to saturate it for at least 20 min. Meanwhile, dissolve the lipids present in the glass tube in 0.2-1 mL of chloroform.
   NOTE: The volume used to dissolve the lipids can be modified depending on the desired or expected concentration of the sample.
3. Apply 10 µL of each suspension using a capillary glass tube directly on the TLC plate and let the sample dry for 5 min at room temperature.
   NOTE: Samples must be applied at the bottom part of the plate leaving 1 cm on each side. Samples must be separated one from another for at least 0.5 cm. Once the sample is applied on the plate, tubes can be evaporated again with nitrogen gas and stored at 4 °C for further use.
4. Insert the plate into the saturated TLC chamber containing the mobile phase. Allow the mobile phase to run through the TLC.
   NOTE: Any movement applied to the TLC chamber affects the running solvent on the plate and affects lipid mobility. In the case of performing two-dimensional TLC, two TLC chambers are required to contain both elution systems.
5. Remove the plate from the TLC chamber when the solvent reaches 1 cm distance from the upper end of the plate. Leave the plate under laminar flux until the silica is totally dried.
   NOTE: In the case of analyzing the mycolic acid composition, using n-hexane and diethyl-ether (85:15), repeat steps 4.4 and 4.5 two times more, until running the mobile phase three times over the TLC plate.
6. Reveal the plate with the required stain; heat the plate if required.
   NOTE: In the present experiment, 15-20 mL of the following solutions were used to spray the TLC plates: 10% Molybdatophosphoric acid hydrate in ethanol until the plate is bright yellow, followed by heating the plate at 120 °C; 5% in ethanol of 20% α-naphthol in sulfuric acid followed by heating the plate at 120 °C; Molybdenum Blue reagent (1.3% molybdenum oxide in 4.2 M sulfuric acid) until phosphate bands appeared or 1% anthrone in sulfuric acid.
   CAUTION: Molybdatophosphoric acid hydrate is a flammable and corrosive substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
   CAUTION: Ethanol is a potential flammable and hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
   CAUTION: 1-Naphthol is a flammable, corrosive, and extremely hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).
   CAUTION: Molybdenum Blue Spray Reagent is a corrosive, toxic, and extremely hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).

Representative Results

Figure 1: Scheme of the procedure of extracting lipid content of mycobacteria grown on solid media. Main steps to decipher lipids present on mycobacteria cells.

Figure 2: Scheme of the procedure for extracting mycolic acid content of mycobacteria grown on solid media. Main steps to decipher mycolic acids present on mycobacteria cells using either (A) acid methanolysis or (B) saponification.

Materials

Name	Company	Catalog Number	Comments
Acetic Acid	Merck	100063	CAUTION. Anhydrous for analysis EMSURE® ACS,ISO,Reag. Ph Eur
Acetone	Carlo Erba	400971N	CAUTION. ACETONE RPE-ACS-ISO FOR ANALYS ml 1000
Anthrone	Merck	8014610010	Anthrone for synthesis.
Benzene	Carlo Erba	426113	CAUTION. Benzene RPE - For analysis - ACS 2.5 l
Capillary glass tube	Merck	BR708709	BRAND® disposable BLAUBRAND® micropipettes, intraMark
Chloroform	Carlo Erba	412653	CAUTION. Chloroform RS - For HPLC - Isocratic grade - Stabilized with ethanol 2.5 L
Dry block heater	J.P. Selecta	7471200
Dicloromethane	Carlo Erba	412622	CAUTION. Dichloromethane RS - For HPLC - Isocratic grade - Stabilized with amylene 2.5 L
Diethyl ether	Carlo Erba	412672	CAUTION. Diethyl ether RS - For HPLC - Isocratic grade - Not stabilized 2.5 L
Ethyl Acetate	Panreac	1313181211	CAUTION. Ethyl acetate (Reag. USP, Ph. Eur.) for analysis, ACS, ISO
Ethyl Alcohol Absolute	Carlo Erba	4146072	CAUTION. Ethanol absolute anhydrous RPE - For analysis - ACS - Reag. Ph.Eur. - Reag. USP 1 L
Glass funnel	VidraFOC	DURA.2133148 1217/1
Glass tube	VidraFOC	VFOC.45066A-16125	Glass tube with PTFE recovered cap
Methanol	Carlo Erba	412722	CAUTION. Methanol RS - For HPLC - GOLD - Ultragradient grade 2.5 L
Molybdatophosphoric acid hydrate	Merck	51429-74-4	CAUTION
Molybdenum Blue Spray Reagent, 1.3%	Sigma	M1942-100ML	CAUTION
n-hexane	Carlo Erba	446903	CAUTION. n-Hexane 99% RS - ATRASOL - For traces analysis 2.5 L
n-nitroso-n-methylurea	Sigma	N4766	CAUTION
Orbital shaking platform	DDBiolab	995018	NB-205L benchtop shaking incubator
Petroleum ether (60-80ºC)	Carlo Erba	427003	CAUTION. Petroleum ether 60 - 80°C RPE - For analysis 2.5 L
Sprayer	VidraFOC	712/1
Sodium sulphate anhydrous	Merck	238597
Sulfuric acid 95-97%	Merck	1007311000	CAUTION. Sulfuric acid 95-97%
TLC chamber	Merck	Z204226-1EA	Rectangular TLC developing tanks, complete L × H × W 22 cm × 22 cm × 10 cm
TLC plate	Merck	1057210001	TLC SilicaGel 60- 20x20 cm x 25 u
TLC Plate Heater	CAMAG	223306	CAMAG TLC Plat Heater III
Toluene	Carlo Erba	488551	CAUTION. Toluene RPE - For analysis - ISO - ACS - Reag.Ph.Eur. - Reag.USP 1 L
Vortex	Fisher Scientific	10132562	IKA Agitador IKA vórtex 3
1-naphthol	Sigma-Aldrich	102269427	CAUTION