Difference: TLCprocedure (1 vs. 3)

Revision 313 Jan 2011 - Main.NicolasCoudray

 
META TOPICPARENT name="2dxOverView"
Changed:
<
<

TLC of lipids and detergents – Martins procedure

>
>

TLC of lipids and detergents – Martin's procedure

 

Materials

  • TLC Silica gel 60 W F254 These instructions are relevant for these plates only. They can be ordered from VWR (#EMD-16485-1) or directly from the manufacturer EMD Chemicals Inc. (formerly MERCK). Other plates have been tested but performed differently.
  • Disposable calibrated glass micropipettes (1-5μl), e.g. VWR # 53432-604
  • Glass cutter
  • Glass vials with Teflon caps. Needed especially for stock solutions for standard curves. #HP-5182-0716 from VWR is amber with write-on spots for labeling and #HP-5182-0720 are suitable caps for these vials.
  • TLC-spray applicator
  • Anilinonaphthalenesulfonic acid (ANSA) for visualization of lipid spots

Mobile phase

  • For general use with most detergents and lipids: 65% chloroform, 30%MetOH, 5% ammonia solution (25%)
  • For polyoxyethylene-derived detergents (such as C12E8): 100% MetOH? was found to work best (might be others, but need to test)

Set-up

  • Cut the TLC plate into a suitable size. For the width of the plate, calculate a distance of at least 8mm between each sample spot to be run.
  • Prepare your samples by mixing one part sample with 3 parts of a MetOH?::Chloroform (2::1) mixture. If you need to prepare reference samples for quantification, do it now as well – these can be stored in the Teflon-capped glass vials for use next time you need to quantify this particular lipid/detergent.
  • Using a graphite pencil and a ruler, mark the spots for where the samples will applied in a straight line approximately 1.5cm above the base of the plate.
  • Use the glass micropipettes to transfer 2-3μl of sample onto each spot on the TLC plate. Capillary action will draw the sample into the micropipette when the bottom end of the pipette is inserted into the sample. If it is required to apply more than 3μl sample onto one spot, perform the application procedure several times, allowing the solvent to evaporate from the TLC plate between successive applications.
  • When all samples have been applied, allow the plate to dry for a short while. During this time, pour the mobile phase into the TLC tank. Thereafter place the plate into the tank, spots down, and make sure that the spots are ABOVE the surface of the mobile phase at the bottom of the tank.
  • Put the lid onto the tank and monitor the progress of the mobile phase up the plate.
  • When the front of the mobile phase has reached around 1cm from the top of the TLC plate, the run is terminated by lifting out the TLC plate from the tank and allowing it to dry in the fume hood. The mobile phase is collected and can be reused several times.

Visualization of spots

  • To develop the spots on the plate after the run, the nature of the substance(s) to be visualized is very important:
    • Detergents are visualized by spraying the plate with pure water and developing the plate under UV light. The detergent spots appear as dark spots on a lighter background caused by the intrinsic fluorescence of the plate.
    • If lipids are studied, the plate is sprayed with 0.1% ANSA in water before imaging the plate under UV-light. In this case, lipids appear as brightly fluorescent spots on a slightly fluorescent background.
    • When both lipids and detergent are analyzed in the same TLC run, start by spraying water onto the plate to visualize the detergent content in the samples. Allow thereafter the plate to dry and spray ANSA onto the plate to visualize the lipid content.
  • For quantification, use ImageJ? and their gel analysis tools or other suitable software.

Revision 228 Dec 2010 - Main.NicolasCoudray

 
META TOPICPARENT name="2dxOverView"
Changed:
<
<		TLC of lipids and detergents – Martins procedure
>
>

TLC of lipids and detergents – Martins procedure

 

Materials

  • TLC Silica gel 60 W F254 These instructions are relevant for these plates only. They can be ordered from VWR (#EMD-16485-1) or directly from the manufacturer EMD Chemicals Inc. (formerly MERCK). Other plates have been tested but performed differently.
  • Disposable calibrated glass micropipettes (1-5μl), e.g. VWR # 53432-604
  • Glass cutter
  • Glass vials with Teflon caps. Needed especially for stock solutions for standard curves. #HP-5182-0716 from VWR is amber with write-on spots for labeling and #HP-5182-0720 are suitable caps for these vials.
  • TLC-spray applicator
  • Anilinonaphthalenesulfonic acid (ANSA) for visualization of lipid spots

Mobile phase

  • For general use with most detergents and lipids: 65% chloroform, 30%MetOH, 5% ammonia solution (25%)
  • For polyoxyethylene-derived detergents (such as C12E8): 100% MetOH? was found to work best (might be others, but need to test)

Set-up

  • Cut the TLC plate into a suitable size. For the width of the plate, calculate a distance of at least 8mm between each sample spot to be run.
  • Prepare your samples by mixing one part sample with 3 parts of a MetOH?::Chloroform (2::1) mixture. If you need to prepare reference samples for quantification, do it now as well – these can be stored in the Teflon-capped glass vials for use next time you need to quantify this particular lipid/detergent.
  • Using a graphite pencil and a ruler, mark the spots for where the samples will applied in a straight line approximately 1.5cm above the base of the plate.
  • Use the glass micropipettes to transfer 2-3μl of sample onto each spot on the TLC plate. Capillary action will draw the sample into the micropipette when the bottom end of the pipette is inserted into the sample. If it is required to apply more than 3μl sample onto one spot, perform the application procedure several times, allowing the solvent to evaporate from the TLC plate between successive applications.
  • When all samples have been applied, allow the plate to dry for a short while. During this time, pour the mobile phase into the TLC tank. Thereafter place the plate into the tank, spots down, and make sure that the spots are ABOVE the surface of the mobile phase at the bottom of the tank.
  • Put the lid onto the tank and monitor the progress of the mobile phase up the plate.
  • When the front of the mobile phase has reached around 1cm from the top of the TLC plate, the run is terminated by lifting out the TLC plate from the tank and allowing it to dry in the fume hood. The mobile phase is collected and can be reused several times.

Visualization of spots

  • To develop the spots on the plate after the run, the nature of the substance(s) to be visualized is very important:
    • Detergents are visualized by spraying the plate with pure water and developing the plate under UV light. The detergent spots appear as dark spots on a lighter background caused by the intrinsic fluorescence of the plate.
    • If lipids are studied, the plate is sprayed with 0.1% ANSA in water before imaging the plate under UV-light. In this case, lipids appear as brightly fluorescent spots on a slightly fluorescent background.
    • When both lipids and detergent are analyzed in the same TLC run, start by spraying water onto the plate to visualize the detergent content in the samples. Allow thereafter the plate to dry and spray ANSA onto the plate to visualize the lipid content.
  • For quantification, use ImageJ? and their gel analysis tools or other suitable software.

Revision 128 Dec 2010 - Main.NicolasCoudray

 
META TOPICPARENT name="2dxOverView"
TLC of lipids and detergents – Martins procedure

Materials

  • TLC Silica gel 60 W F254 These instructions are relevant for these plates only. They can be ordered from VWR (#EMD-16485-1) or directly from the manufacturer EMD Chemicals Inc. (formerly MERCK). Other plates have been tested but performed differently.
  • Disposable calibrated glass micropipettes (1-5μl), e.g. VWR # 53432-604
  • Glass cutter
  • Glass vials with Teflon caps. Needed especially for stock solutions for standard curves. #HP-5182-0716 from VWR is amber with write-on spots for labeling and #HP-5182-0720 are suitable caps for these vials.
  • TLC-spray applicator
  • Anilinonaphthalenesulfonic acid (ANSA) for visualization of lipid spots

Mobile phase

  • For general use with most detergents and lipids: 65% chloroform, 30%MetOH, 5% ammonia solution (25%)
  • For polyoxyethylene-derived detergents (such as C12E8): 100% MetOH? was found to work best (might be others, but need to test)

Set-up

  • Cut the TLC plate into a suitable size. For the width of the plate, calculate a distance of at least 8mm between each sample spot to be run.
  • Prepare your samples by mixing one part sample with 3 parts of a MetOH?::Chloroform (2::1) mixture. If you need to prepare reference samples for quantification, do it now as well – these can be stored in the Teflon-capped glass vials for use next time you need to quantify this particular lipid/detergent.
  • Using a graphite pencil and a ruler, mark the spots for where the samples will applied in a straight line approximately 1.5cm above the base of the plate.
  • Use the glass micropipettes to transfer 2-3μl of sample onto each spot on the TLC plate. Capillary action will draw the sample into the micropipette when the bottom end of the pipette is inserted into the sample. If it is required to apply more than 3μl sample onto one spot, perform the application procedure several times, allowing the solvent to evaporate from the TLC plate between successive applications.
  • When all samples have been applied, allow the plate to dry for a short while. During this time, pour the mobile phase into the TLC tank. Thereafter place the plate into the tank, spots down, and make sure that the spots are ABOVE the surface of the mobile phase at the bottom of the tank.
  • Put the lid onto the tank and monitor the progress of the mobile phase up the plate.
  • When the front of the mobile phase has reached around 1cm from the top of the TLC plate, the run is terminated by lifting out the TLC plate from the tank and allowing it to dry in the fume hood. The mobile phase is collected and can be reused several times.

Visualization of spots

  • To develop the spots on the plate after the run, the nature of the substance(s) to be visualized is very important:
    • Detergents are visualized by spraying the plate with pure water and developing the plate under UV light. The detergent spots appear as dark spots on a lighter background caused by the intrinsic fluorescence of the plate.
    • If lipids are studied, the plate is sprayed with 0.1% ANSA in water before imaging the plate under UV-light. In this case, lipids appear as brightly fluorescent spots on a slightly fluorescent background.
    • When both lipids and detergent are analyzed in the same TLC run, start by spraying water onto the plate to visualize the detergent content in the samples. Allow thereafter the plate to dry and spray ANSA onto the plate to visualize the lipid content.
  • For quantification, use ImageJ? and their gel analysis tools or other suitable software.
View topic  |  History: r3 < r2 < r1  |  More topic actions...
 
Copyright © by the contributing authors. All material on this collaboration platform is the property of the contributing authors.
Ideas, requests, problems regarding this intranet, Send feedback