Dear Dr.Oyeyemi,

In TLC, there are a number of critical factors that one needs to look at to achieve repeatability. I will list a few of them.

1. If you are using pre-coated prep TLC plates, there will be plate uniformity. I am not sure whether you are making your own plate or you are using -pre-coated plates supplied by say 'Merck'? Even if you use pre-coated prep plates, it will be ideal to pre-elute the plates completely with methanol, dry and then reactivate at ~100 C for an hour every time.

2. Make sure you dry the solvent from the sample 'load'. any residual solvent will change the separating conditions.

3. The sample loads should be the same every time (wt/voume). Overloading may reduce resolution and result in band overlaps.

3.. The most critical is the mobile phase saturation of the Chamber. In TLC the separation occurs in a chamber where the 'plumes' of mobile phase mixture is in a 'gradient' from top to bottom. Again, there is a 'mobile phase gradient' on the plate as well while eluting. The time for chamber saturation with the mobile phase should be strictly followed. Otherwise, you will create different separating conditions every time.

If you critically observe some of these factors, I am sure you will have good repeatability in prep TLC.

Good Luck

I agree with Suresh- it is a comprehensive reply.

Thank you Dr Govindaraghavan, please what do you think should be the minimum time for mobile phase saturation

It depends on

1. the volume of the chamber! For a 10 cm X 10 cm analytical HPTLC plate chamber (refer to CAMAG Chamber dimensions), one uses 5 mL mobile phase! The normal saturation time would be around 15 minutes.

2. it depends on the mobile phase mixture. If you are using polar solvent mixtures (water, formic acid, methanol,chlorofomr, ethyl acetate mixutres) 15 minutes to 20 minutes should be sufficient. If you are using less polar solvent mixtures (for eg.hexane: ethyl acetate), chamber saturation should not be more than 5 minutes. For a highly volatile non-polar solvent mixtures (for eg pet ether hexane mixtures), do not leave it forchamber saturation!

I am not sure which chamber you are using. Since you have indicated prep TLC, I assume the plates are 20 cm X 20 cm. For larger chambers, the volume of mobile phase and the time of chamaber saturation will be longer. Again this will depend on the type of solvent mixture that you use for separation!!

I forgot to mention! The quick and best way to get the chamber saturated is to use 'square' filter papers wet with the mobile phase stuck to the two opposite inner vertical walls of the chamber.

Thank you Dr Govindaraghavan · for the pieces of information

TLC reproducibility can be affected by a number of different factors such as layer thickness, moisture on the TLC plate, vessel saturation, temperature, depth of mobile phase, nature of the TLC plate, sample size, and solvent parameters

These are all excellent responses and reproducibility critically depends on many of these factors and consistent technique. I have had fewer issue with glass backed plates although aluminum or plastic are cheaper and can be cut to smaller sizes that utilize less sample and run faster. The glass plates can be heated for activation and stored over a desiccant in a drying chamber. One issue that critically altered plate activity and plate-to-plate run consistency was laboratory humidity depending on the season and quality of air handling. I found that cutting aluminum backed plates using a paper cutter to the desired size and placing in a small beaker that was then placed in a tightly sealed glass jar over KOH pellets efficiently dried and activated these plates for use. That is how I always stored plates after learning that technique. Furthermore, depending on how well the plates were cut, I often had issues with solvent advancing faster at the edges. Hence, I would use scissors to clip each corner at the bottom that sits in the solvent, and this method removed the problem with bowing of the solvent front.

If using the large plates, sometimes I would load the sample, dry the plate, and then sit the loaded plate carefully on a platform of small beakers in the saturated chamber in order to make sure that the plate is equilibrated prior to placing the plate into the solvent. A large piece of filter paper on each side of the chamber can be each easily wetted by tilting the chamber to either side, Next, I would typically run two plates facing each other (even using a blank if necessary) to keep the solvent/chamber atmosphere at the highest saturation at the plate interface. Usually these exhaustive precautions are not required when running small plates to QC reactions. When running prep plates, however, consistency is an absolute and make sure that you use all possible helping hints to get the best and most reproducible separations. I don't know too many people that use those nowadays though.

To add to the already discussed points:

One of the best ways of getting repeatable separations with correct chamber saturation is to use a horizontal chamber instead of a vertical one. In the horizontal chamber the plate can be inverted with the silica layer facing the base partly filled with the mobile phase. By running the plate this way, one gets the same moble phase plumes through out the entire plate. Refer to the following link that would be quite useful

http://www.camag.com/en/tlc_hptlc/products/chromatogram_development/horizontal_developing_chamber.cfm

For most of my QC based analytical hptlc, I have used horizontal chambers with really excellent results in terms of repeatability

All the best

I must agree with the suggestions given by Dr, Govindraghavan. He has covered most of the points concerning TLC which provide a comprehensive solution to your problem.

In adsorption chromatography processes including TLC, the mixture to be separated moves through the mobile phase and falls out into the stationary phase. So, to be reproducible, different elements of the mix must move by the same amount in the mobile phase and fall out into the stationary at the same point every time. The phases must therefore be as uniform as possible. External conditions (temperature, pressure) should be the same. Quality of adsorbent in terms of particle size (that may differ among the product of different manufacturers), thickness and uniformity of adsorbent layer, activation time and temperature of TLC plate, ambient temperature & humidity, amount of sample applied, quality of mobile phase, equilibrium between liquid and vapour in the tank and other operating conditions like angle at which the plant lies affect TLC reproducibility.

For pingpointing the concrete question, in addition to the above listed good suggestions, I think you might as well checking the qualities of the silica gel plate,the solvents used for preparing the mobile phase to ensuring all being the same as you used in the first time. Further concrete suggestion is likely put forward based on your concrete detail information which you experimented. Confused question can only obtain the confused reply.

Several factors. Infact you could write a book on your questions. However, just to mention a few:

1. Saturation of your solvent chamber

2. Thickness of the silicagel on the plate.

3. Activation of your plate etc.

I agree to Jai Ghosh.

I am wondering if all the good suggestions (almost covered the factors of TLC experiment) could solve the concrete problem questioned by Oyeyemi?

Let's repeat the question:"I did a preparative TLC to isolate some compounds and the bands resolved well with the mobile phase. After checking the activity about a week later, i couldn't reproduce the TLC, either the bands are not moving away from each other or they are clumping close to the spotting line"

Q1: The bands are not moving away from each other. .

A: It might be caused due to the sample solution application were overloaded; or the room temperature was higher than the first time experment, particularly if the Rf value of the bands were originally very close in the first testing.

Q2： The bands "are clumping close to the spotting line".

A: It seems another situation as the above mentioned. The solvent system was the same to the first testing completely? or the activity of the plates used were different each other.

If some detail description plus the TLC images available will very helpful to discuss how to solve it. I don't think it is a hard nut to crack.

I agree the points covered by Dr.Govindaraghavan, in addition I think volatile nature of compounds is also important issue. In this regard, as in your case the bands are lost after a week, try to redissolve in another solvent say pyridine recrystallize/ evaporate the solvent and go for TLC in your solvent system.

I think the major reason might be caused from the problem of his operation procedure, because the two problems completely differ each other according to the description of the questioner who seems to escape from the discussion.

Looking for solution to similar problem I found this discussion. Excellent replies! But I have 1 minor question to Suresh Govindaraghavan ...... like you mentioned sir, the mobile phase saturation time varies based on polarity. For hexane:ethyl acetate mixtures you mentioned that "saturation should not be more than 5 minutes". Did you mean that this 5 min time is sufficient ? or you meant that more time is not good for the optimum TLC separation?

Dear Shekhar,

it depends on the ambient temperature (at which the HPTLC is run).  Most GLP labs have air-conditioners where there is temperature control, say for eg. ~25 C.  In Labs not having controlled temperature, the ambient temperature will vary.  This factor will decisively affect the evaporation rates of low boiling solvents such as n-hexane and ethyl acetate. The 5 minutes saturation was set at ~25 C and repeated for all experiments to have uniformity in saturation levels. 

One more aspect you need to consider:  Even though the HPTLC tank is a closed system, it is not an air tight system.  Hence you have to consider the solvent loss over time.

Considering these two factors, I suggested shorter time durations (5 min) for low boiling mobile phases and longer (~15 minutes) for high boiling mobile phases (that includes water or acetic acid for example).

What ever the time duration you give for saturation, maintain it for all the repeat experiments to get consistent separation.

All the best.

Thank you for this prompt reply Govindaraghavan Sir!