depending on which are the major components you are interested in.

for terpenoids toluene : ethyl acetate (4:1) or benzene : ethyl acetate (5:1)

for alkaloids CHCl3/MeOH/AcOH (18:1:1, v/v/v), and Dragendorff as a revealing reagent

for coumarins n-BuOH/AcOH/H2O (4:1:5, v/v/v), and acetate of lead (5%) and alcoholic KOH (5%) as a revealing reagent

for flavonoids n-BuOH/AcOH/H2O (4:1:5, v/v/v), and AlCl3 (0,5 g/100 mL of EtOH) as a revealing reagent while for tannins use FeCl3 (10% in MeOH/H2O, 1:1, v/v)

for anthocyanins HCl/ formic acid/water, (19.0/39.6/41.4 v/v/v )

hope it helps

Solvent system depends upon types of compounds that you want to separate. Which types of compounds that you want to separate.???????

Looking for flavonoids and terpenes !!

For flavonoids Chloroform: methanol 8:2 is useful. For terpenes you can use ethyl acetate: chloroform.

In case of tailing add a little formic acid or acetic acid (2-3 drops). Try different variations to get the best mixture for your case. Add toluene if separation in case of resolution problems.

Some times Ethyl acetate: ethanol: formic acid : water (66:30:4:37) and Chloroform: Glacial acetic acid: Methanol : water (16:8:3:2) has proved very useful. Just hit and trial for your case. Hopefully you will get a positive result.

Gud Luck

Regards

Same as above

Dr. Alok, the combination that you mentioned here in the last para is it for flavonoids? would this combination go with DCM extract? should try with NP-TLC/RP-TLC?

@Moyeenul Huq... For flavonoids CGMW (16:8:3:2) would prove useful.

With DCM extract , first try with a normal phase TLC. DCM extract is more likely to contain steroids, terpenoids, sometimes alkaloids and flavonoids also.

So you can try. Will take some additional pains, but possibility of a result can't be ruled out. As a general rule, in TLC never hesitate to try for a new possibility, miracles happen sometimes :)

Gud Luck

regards

@ Dr Alok, Yes I agree with you. Lats 1 week I am struggling with my DCM extract. very difficult to get good separation. I guess this fraction contains more polar compounds. I tried with many combinations. also tried with RP TLC. But now I got some good result with Tol: ACN (8:3), HEX: EA (6.5:3.5). But still trying to use new combinations.

@ Moyeenul Huq

I believe you are very close to your goal now. Try Toluene:Ethyl acetate (8:2). I assure you of a very good result this time because it was my own solvent system during my PhD work. It also seems that your extract contains steroidal compounds. Are you having standards of beta-sitosterol and stigmasterol. If yes, run a co-TLC of your extracts with them in Tol:EtOAc and see the result. I beleive you will be a happy man this time, haha :)

@ Dr Alok, Thank you very much for your inspiration. But unfortunately I don't have standard of sitosterol/stigmasterol. But I have Kaempferol, Quercetin, gallic acid, myrcetin, apigenin.

@Moyeenul Haq

What was the result of co-TLC with quercetin and kaempferol ? Are the standards available with you not present in your extracts?

@Moyeenul Haq ... Send me your email id, I will send you pics of co-TLC with betasitosterol and stigmasterol in the same solvent system as above. You can compare the Rf then. At least you will have an idea about the presence or absence of the compound.

Here is my e-mail: [email protected]

@ Dr Alok,

I am just running TLC as per your suggestion Tol:EA (8:2) and CGMW. i will let you know soon.

@ Dr Alok,

Yes, Tol: EA was good and quite similar with my To:ACN. there is long tailing from the base line to almost middle of the TLC. after that there are some spots. I am using TLC with 10 cm length. But for CGMW the compounds moves together as a big single spot!!! I may try in RP for this combination.

Thanks u so much for wonderful answers, I am sure they will be helpful :)

To determine the best solvent or mixture of solvents or solvent system to run a TLC plate with an extract, vary the polarity of the solvent in several trial runs: a process of trial and error. Carefully observe and record the results of the chromatography in each solvent system. You will find that as you increase the polarity of the solvent system, all the components of the mixture move faster or vis versa.

depending on which are the major components you are interested in.

for terpenoids toluene : ethyl acetate (4:1) or benzene : ethyl acetate (5:1)

for alkaloids CHCl3/MeOH/AcOH (18:1:1, v/v/v), and Dragendorff as a revealing reagent

for coumarins n-BuOH/AcOH/H2O (4:1:5, v/v/v), and acetate of lead (5%) and alcoholic KOH (5%) as a revealing reagent

for flavonoids n-BuOH/AcOH/H2O (4:1:5, v/v/v), and AlCl3 (0,5 g/100 mL of EtOH) as a revealing reagent while for tannins use FeCl3 (10% in MeOH/H2O, 1:1, v/v)

for anthocyanins HCl/ formic acid/water, (19.0/39.6/41.4 v/v/v )

hope it helps

It depends on what compounds you are interested in.The best thing would be to try one polar solvent and one nonpolar solvent.Then remove the solvents from the crude extracts.Take a sample of each and after taking it up in a small amount of solvent, do a TLC with a medium polar solvent or a mixture of two solvents to check on range of separable compounds from each of the two extracts.

you had better to use more than one solvent system. so you can use hexane:chloroform (4:1) to see non polar compound. chloroform:methanol (4:1) to see polar compounds.

Good luck Anuj

Like they mention above- it depends on what compounds you are interested in. Even on assay directed fractionation, one often gets a sense of what compounds thesd to show up as active; for example I tended to find alkaloids in central nervous system assays and would extract them, then clean them with reverse phase.

If you aren't sure of the compound, one thing to try for prep TLC is to run the plate in a non-polar solvent system, visualize the edge of the plate (I'm assuming you are striping the plate with sample), scrape off what has eluted, then try again in a stronger solvent.

Another thing I would try is packing a small silica column and running a range of gradients (step gradients if using an open glass column). I call this "wide polarity range chromatography, and it can be done with high quality silica as well (not silica G either). With silica one can start with hexane, gradient to 100% ethyl acetate, ethyl acetate to 100% ethanol or 2-propanol, alcohol to 50% water (at this point, your compound has come off if it is going to come off the silica). You can then TLC the active fractions and purify them further as you know the approximate purity of the compounds. Here's an app note I wrote on Wide Polarity Range Chromatography: http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN77_Wide_Polarity_Range_Chromatography.pdf

I wrote this for diol and C18, but I later learned how to do silica HILIC so the method can be extended there as well.

It depends on what compounds you are interested in.The best thing would be to try one polar solvent and one nonpolar solvent.Then remove the solvents from the crude extracts.Take a sample of each and after taking it up in a small amount of solvent, do a TLC with a medium polar solvent or a mixture of two solvents to check on range of separable compounds from each of the two extracts.

If you aren't sure of the compound, one thing to try for prep TLC is to run the plate in a non-polar solvent system, visualize the edge of the plate (I'm assuming you are striping the plate with sample), scrape off what has eluted, then try again in a stronger solvent.

i always used home made TLC plate which has a thick layer of silica and its easy to collect spot from plate and dissolve in solvent and now use that compound for second separation procedure

I agree with most of the answers given by others. If you are looking for a specific group of compounds your choice of solvent becomes more specific otherwise you have to try solvents from most polar (water or MeOH) to highly non polar solvents like hexane. You have to fractionate your extract using column chromatography for easy separation of group of compounds. As you know compound identification from crude plant extract is a very laborious job or work that calls for different separation techniques (HPLC, MS, NMR etc.).

Best wishes!!

I recommend the book entitled `` Thin Layer Chromatography in Phytochemistry ``. by Monika Waksmundzka . (2008)

Dear all,

I agree with all the answers. In my experience worked with Verbenaceae especially Stachytarpheta sp I used CHCl3 : MeOH: EtOAc 9 : 3 : 5 to run crude extract on TLC.

I hope it is used. thanks...

Let me refer you to the article entiled: Two-dimensional TLC in the Analysis of Secondary Metabolites by Ciesla, et al in the Journal of Chromatography A 1216(2009) 1035-1052.

Dear Mr. Ranjan

Recent phylogenetic studies have shown that numerous genera traditionally classified in Verbenaceae belong instead in Lamiaceae. The new narrowly circumscribed Verbenaceae family includes some 35 genera and 1,200 species.

References:

Cantino, P.D., Harley, R.M. & Wagstaff, S.J. 1992. Genera of Labiatae: status and classification. Pp. 511-522. In Harley, R.M. & Reynolds, T. (eds) Advances in Labiate Science. Richmond, Royal Botanic Gardens, Kew.

"Angiosperm Phylogeny Website - Lamiales". Missouri Botanical Garden.

Heywood, V.H., Brummitt, R.K., Culham, A. & Seberg, O. 2007: Flowering Plant Families of the World. Royal Botanic Gardens, Kew.

Can you reveal the name of the plant on which you want to work or are working and the nature of the extract (ethanolic, methanolic, aqueous, pet ether, chloroform etc. etc). So instead of suggesting general solvent systems for different chemical categories, all the contributors will feel it easier to post some directly relevant information for you. Might be possible that somebody is working on similar lines and you can get a good guidance. Would be very favorable for you in that case. :)

Regards

A typical plant extract contains several components such as alkaloids, terpenes, flavonoids, steroids, saponins etc. These components have different solubility in different solvents. It may therefore not be easy to have one solvent system that will be effective for TLC analysis of all these components. I think the choice of solvent system will depend on the type of component you have in mind. There are several write ups that give excellent information on the best solvent systems for each class of secondary metabolite. The book by Harbone is a classical one (PHYTOCHEMICAL METHODS: A guide to modern techniques of plant analysis, 1998, by J.B. Harbone. 3edition. Chapman and Hall). The attached article on TLC by Joseph Sherma may also be of help. I wish you the best of luck

OK, i am going to do that right away. Check your email box in the next few minutes.

Mudasir, Pls check your mail box. I have just sent the article to your box. Cheers!

You can refer the following books which are considered milestones in Chromatography, this will definitely end up ur search regarding the querry you have now:

1. Stahl E (1967). Text Book of Chromatography, 2nd Ed., Springer-Verlag, Berlin.

2. Wagner H, Baldt S. (1996). Plants Drug Analysis, Springer Verlag, Berlin Heidelberg,

3. WHO (1989). The Validation of Analytical Procedures used in the Examination of Pharmaceutical Materials, WHO/Pharm. 89541, rev 2 Genf,.

4. Harborne JB (1973). Phytochemical Screening Methods In: A Guide to Modern Techniques of Plant Analysis. 1st Edition, Chapmann and Hall, London.1973 Regards and gud luck

Dear Mudasir, I am glad that you appreciate my little effort. I wish you the best.

Based on my experience, first in my mind > what crude extract is it ? MeOH-, Acetone-, or others. Then, I run TLC for identified non-polar, semi-polar and polar constituents by using hex:ethyl acetate (9:1; 5;5; 1;9) as system solvent, checked the existence of chlorophyll (by UV) and tannin (TLC using CHCl3:MeOH (9:1 & 1:9).

Verbenaceae is rich of essential oils, diterpenoids, triterpenoids and flavonoids. My suggestions for TLC of crude extracts are : diterpenoid (hexane:ethyl acetate), triterpenoid (hexane/toluene:ethyl acetate) and flavonoid (hexane/chloroform:ethyl acetate/methanol). But for general, system solvent of hexane:ethyl acetate actually can cover almost all constituents.

Depends upon what constituents you want to see. In General If it is Petroleum ether extract run the tlc in petroleum ether and increasing polarity with chloroform 10,20, 40, 50 and Chloroform etc if methanol ext run in chloroform and methanol mixture. Increas the polerity of methnol 1 t0 20 % in chloroform.

Dear

Anuj

Medicinal Plants have many secondary metabolites. In a simple way solvent system for TLC is a trial and error procedure. You may go for Non-Polar to Polar combination of solvent system which gives you a perfect RF ( .2 to .3 between two spots) and maximum number of spots You may go for this If there are no particular class of compound. If there are particular class of compound then above suggestion given by Frederick Lia is right way.

Useful information indeed. Thanks all

I would like to thank every one for valuable answers, I just wanna say that, I got better resolution with DCM and Benzene on 95:5 and more enhanced with 99:1 with few drops of toluene and acetic acid. I am still trying to improve with several combinations. Thank you sooooooooo much every one !!

Dear Anuj,

You had asked one of the most important question, I guess which is always in mind of researcher when running TLC. And you received wide variety of response to your query. To be honest, it is a game of polarity, and need permutations and combinations. The solvent systems mentioned above are good as general, but you have to optimize it according to your extracts.

You can take help of the following solvent miscibility table and make your own solvent system.

http://www.erowid.org/archive/rhodium/pdf/solvent.miscibility.pdf

To cite someone else work is very easy Anuj, but to do work which receive citations certainly need some novel efforts.

Sincere Regards,

Rajeev K Singla

http://rajeevksingla.in

Thanks to you all who responded to the question, we are informed that there many options to finding the correct method.

On the other hand, sometimes it is a surprise to researchers to be able to find the best method and get reproducible results.Keep on trying!

Best wishes!

Solvent system of TLC depends mainly on the polarity & chemical nature of constituents in Plant extract & should be adjusted if you use mix of solvent by increasing or decreasing ratio of polar or non polar solvent until reach to best ratio for isolation

Petrolium ether& ethyl acetate in different proportions,eg. 9:1, 8.5:1.5 like that

its also depands on your targeted compound , and K.Meselhy is right Solvent system of TLC depends mainly on the polarity & chemical nature of constituents in Plant extract & should be adjusted if you use mix of solvent by increasing or decreasing ratio of polar or non polar solvent until reach to best ratio for isolation.

and after that you have to analyse eachfraction again for conformation wheter its a single or mixture!!

@ Mudasir MIr

Anisaldehyde sulphiric acid reagent is very good TLC spraying reagent covering almost all the categories of chemical compounds including sterols, triperpenoids, phenolics, flavonoids, saponins, tannins etc.

You can also use methanolic Sulphuric acid for most of the phytoconstituents.

Vanillin Sulphuric acid reagent is also very useful for the above mentioned categories on TLC.

Dragendorff reagent can be used for spraying in case you are expecting alkaloids.

You can also use conc. sulphuric acid in case all of them are not working. It will char anything organic present on the plate and you can easily detect the compound and its Rf.

References

You can refer the following books which are considered milestones in Chromatography, this will definitely end up ur search regarding the querry you have now:

1. Stahl E (1967). Text Book of Chromatography, 2nd Ed., Springer-Verlag, Berlin.

2. Wagner H, Baldt S. (1996). Plants Drug Analysis, Springer Verlag, Berlin Heidelberg,

3. WHO (1989). The Validation of Analytical Procedures used in the Examination of Pharmaceutical Materials, WHO/Pharm. 89541, rev 2 Genf,.

4. Harborne JB (1973). Phytochemical Screening Methods In: A Guide to Modern Techniques of Plant Analysis. 1st Edition, Chapmann and Hall, London.1973

Regards and gud luck

I would suggest to post questions in a separate. not in the trailing..your question was very inportant.

Mudasir, I support the suggestion by Nithya that you should post your question separately so that it can receive wider and adequate attention.

However, the most comprehensive write up I have come across so far on the subject of spray reagents is the one by Hellmut Jork, Werner Funk, Walter Fisch er, Hans Wimmer. Thin-Layer Chromatography Reagents and Detection Methods Volume la

Physical and Chemical Detection Methods:Fundamentals, Reagents I

Translated by Frank and Jennifer A. Hampson. If you give your email address, I will send a copy to your email box.

Let me refer you to the Journal of Chromatography A, 1216 (2009) 1035–1052.Two-dimensional thin-layer chromatography in the analysis of secondary plant metabolites

Łukasz Cie´sla, MonikaWaksmundzka-Hajnos∗

I'd like to add a bit to Frederick Lia's answer. For cinnamic acids; benzene/ acetic acid 9:1.

Dear Mudasir Mir, pls check your email box for the article. Best wishes.

Dear Mudasir Mir, I am delighted that you appreciate my little contribution. I wish you the best.

Thanks you Dr. K. Pathy for the elaborated procedure you provided.

Best regards,

Thank you very much Dr. Pathy (I type fast and sometimes make mistakes in the Grammar! Sorry for that).

I agrre with Fredrick Lia. Every compound have a special character so trial and error always be needed. Base on F. Lia sugestion, you can choose which one is suitable with your compound and expand it especially in the ratio of that mixture solvent. I hope you can find the best one.

i appreciates all the scientist for nice sharing, But we all know fact about Chromatography's principal that Every compound have a special character so separation (Here we can say affinity/ interaction, molecular mass...) so trial and error always be needed , but yes based on reference you reduced trial, so all the best for experiment.

i would like to share one thing "My professor often says me that Chromatography is an art" and that's true.

I am in agreement with Vipul because first it depends on the solvent of extraction. SUPPOSE IT IS A WATER TOTAL EXTRACT

Not only solvent type but also you need, in many case, specific reagent(s) and wavelength of the light (UV254 or 366) to indicate the band(s) on TLC plate.

Let me know which extract you have , like petroleum ether, chloroform, acetone etc.

I agree with what Vipul Davariya has said. Though some previous reference available some time by trial and error we may get better results. Plant extracts have broad range of components so all extracts and group of compounds can not be evaluated by predetermined mobile phase.... There are many factors affecting results of chromatography like... Geographical variations, Seasonal variations, Environmental condition during chromatography etc....

Best way to select good solvent system to run crude plant extract on TLC is optimization of system as per our requirement.

Dear Anuj selection of solvents for TLC is also depends upon the solvent of extract

We tried BEA (benzene/ethanol/ammonia) : 9:1:0.1 and we got good seperation of crude plant extract...

I think based on the previous suggestions some few months ago the questioner must have found a workable TLC system for him. Already a lot of solvent systems have been discussed and suggested. Its entirely my personal view, of course. I believe there should be a time span for a question to remain alive. Do you agree?

I agree with the suggestion of Alok & Mudasir!

regards,

Buhara has picked up on an important point. 366nm UV illumination is used to detect phenolics (those which fluoresce), while 254nm light is used to detect nearly everycompund. Many TLC plates are sold with a 254nm excitation fluorophore in the stationary phase, so that after the run is complete, the analytes appear as dark spots under UV.

Another thing, A number of cinnamic acids have cis- and trans- isomers, so it may be a good idea to run them under 366nm illumination to prevent their resolving into double bands.

I agree the clearly and correctly given suggestions by Frederick Lia in january 2013. But the development of good profile is also dependent on extraction method and other chromatographic conditions mainly the temperature too. These factors results in significant variation.

Agree with Dr Alok...

ALOK, you are right. There is no point over flogging the issue. It should now be laid to rest, since justice has already been done to the issue raised.

Agree with Mr. Alok..

Go to:

http://carlasabandar.files.wordpress.com/2012/10/35577728-plant-drug-analysis-a-thin-layer-chromatography-atlas-2001.pdf

TLC in precoated Silicagel GF 254 plate AND RUN IN SOLVENT SYSTEM Containing Methylene Chloride:Ethyl Acetate:(40:60) This is a universal system to find out Plant constituents namely Glycosides,Alkaloids,Steroids etc.Chromotogram sprayed with 1%Phospho molybdic acid in ethanol and chromotogram heated at 60 degree c.Appearence of blue and blue grey spots indicates the presence of Plant constituents.

Many thanx to Dr. Urzua, I guess this is best answer providing ebook related to almost all compound being isolated from plants and their solvent systems for TLC applications.

Dear, first of all it is necessary to clarify which kind of crude extract you have. An ethanolic extract? n-butanolic? methanolic? chloroformic?

I used to start analysing my crude ethanolic extract with hexane-acetyl acetate (8:2), and then revealing it with a general reactive such as phosphomolybdenum or ceric sulphate or another one. This mixture of solvents usually shows a good initial chromatographic profile for the composition of your crude extract. Then, additional attempts will be probably necessary to find a suitable eluent by means of changing the polarity of your first one.

Its depend upon the constituents present on them... You should properly gone through literature for the particular plant....

Any one high polar solvent + one low polar solvent mixture (eg. hexane: ethyl acetate) (1:9; 2:8;3:7;4:6; 5:5,6:4....9:1) try to different ratio

You will apply your reaction mixture in solution to the plate then "run" , the plate by allowing a solvent to move up the plate by capillary action. Depending on the

polarity of the components of the mixture, different compounds will travel different distances up the plate.

A spot of the mixture to be separated is placed on the baseline near the bottom of the TLC plate

TLC plate show bands clearly speared.

However, many organic compounds are colorless, and the spots on the TLC plate must be visualized differently. Some compounds fluoresce and are visualized under an ultraviolet (UV) lamp.

Another method is to place the developed plate in a jar containing a few crystals of iodine. Iodine vapor stains the organic compounds brown.

hi,i would recomend reading THIN LAYER CHROMATOGRAPHY IN PHYTOCHEMISTRY BOOK ,Edited by Monika Waksmundzka,Joseph Sherma and Teresa Kowalska.

on the basis of nature of extract select the solvent for TLC.

For polar compounds

CHCl3:MeOH:H2O( 66:35:10)

For less Ploae

Acetone:Hexane (8:2)

Hildebert Wagner and Sabine Bladt. “Plant Drug Analysis: A Thin Layer Chromatography Atlas”; Second Edition

This TLC atlas includes about 230 medicinal plants of worldwide interest. The photographs of the TLC fingerprints and the descriptions of the characteristic compounds of each plant extract are a quick and reliable source for the identification and purity check of plant material and phytopreparations.

Most of the TLC systems are standard systems and have been optimized when necessary.

http://carlasabandar.files.wordpress.com/2012/10/35577728-plant-drug-analysis-a-thin-layer-chromatography-atlas-2001.pdf

We have spent substantial time in developing TLC solvent systems that are useful in separating as many as possible compounds by TLC. Our solution is to use three different solvent systems one that would separate the more polar compounds ethylacetate:methanol:water [10:1.35:1], one to separate the intermediate polarity compounds chloroform:ethylacetate:formic acid [5:4:1] and one to separate the non-polar compounds benzene:ethanol:ammonia [9:1:0.1]. The pH can have a major effect on the separation of compounds with phenolic or amino-imino groups. We therefore selected an acidic, a neutral and a basic solvent system. Because benzene is a dangerous solvent TLC should be carried out in a fume cupboard.

It is important that TLC plates should not be overloaded (to prevent streaking) or too little (to ensure that minor compounds are visible) of the extract placed on the TLC plate. Usually a quantity of 100 microgram placed in a thin line with a width of about one cm gives good results. We usually dissolve our extract in acetone (evaporates quickly) because it is nearly impossible to place a thin line on a TLC plate with water or methanol as solvent.

This technique has been used to distinguish between extracts of more than 50 western herbal medicines. In this paper we also compared different spray reagents used. The complete reference in the open acess journal is Eloff JN, Ntloedibe DT and van Brummelen R (2011) A simplified but effective method for the quality control of medicinal plants by planar chromatography. Afr J Tradit Complement Altern Med. 8(S):1-12

The method was also used in the preparation of the African Herbal Pharmacopoeia (Brendler T, Eloff JN, Gurib Fakim A, Phillips D (Eds) (2010) African Herbal Pharmacopoeia, AAMPS publishing, Mauritius ISBN 9789990389098) (See www.aamps.org)

Another advantage of the solvent systems used is that the solvents are relatively volatile so that it is easy to remove all traces of the solvents before bioautography to identify active antimicrobial compounds.

The three solvent systems are fine. However, the proportions can be adjusted as needed. It is diffcult to stick to the ratios as is suggested by Jacobus Eloff.

I would like to request to try a 2D TLC so that the compounds can be separated in 2 different solvents for separation purposes.As mentioned earlier, natural products varied greatly, so the chances are that it is difficult to predict accurately for any run.

We have had limited success in using two dimentional TLC. In contrast to paper chromatography where complex mixtures can be separated well, the higher load that is required to see minor compounds after the second run means that you need to place a large quantity on the TLC plate. This easily leads to overloading and streaking.

To use a precoated TLC plate for each extract makes it very expensive. We can analyze 20 extracts on a 20 x 10 cm plate. Separating compounds placed in a spot is much more inefficient than separating a sample placed as a line. That is why we developed three solvent systems that would separate compounds with different polarities in three parallel analyses. The three solvent systems we developed and single extractant and single spray reagent was in response to the large number of extractants, solvent systems and spray reagents used by Wagner and Bladt “Plant Drug Analysis: A Thin Layer Chromatography Atlas”. This was developed for the quality control of African medicinal plants.

I could suggest optimization of TLC method by statistical method Design of Experiment. Within the State Ease program entitled Design Expert you can find algorithms for optimization of mixtures, which is really easily performed and does not require a skillful mathematician. Free downloads are usually available for trial, you can check it at:http://www.statease.com/software/dx9-trial.html

Good luck and best wishes everyone!

:)