I believe that you can isolate the material through a HPLC technique and the identification coudl be by MS or RMN... it will depends on if you already have a primary standard or not. If you have, the MS is maybe much easier.

i think it is better to go for LC-MS or HPLC analysis

1. What is your objective, please?

2. Do you want steroids? Or you want to study the plant chem?

3. You may get more quantity of steroids and others if you go to more polar solvents.

pl clarify your intention, then peers could help better.

Why do you use hexan to extract steroids? I suggest you get the steroids with 80% EtOH, and separated with macroporous resin to obtain the 55% and 75% fractions, then go for pr-HPLC method, the pure compounds should be characterized by 1D,and 2D-NMR methods.

I think you should use other solvent like methanol or ethanol to extract steroids not hexane, hexane is too non-polar

Can you clarify it ? Do you want analyze steroids ? or the other secondary metabolites in the hexan extract?

I've purified steroidal compounds with HPLC (C18). "Steroids" cover a lot of ground and depending on the substituents, you may wish to use a more polar solvent to extract them as mentioned above. The compound I purified had some sugars attached and it was extracted into methanol. If your compound doesn't have conjugated bonds so it is visible in UV, you may need ELSD (Evaporative Light Scattering Detection) to see your compound. If you can follow the activity from fractions, you can use silica gel flash chromatography to clean the steroids from other material.

The steroids can be isolated by solvent extraction and column chromatography. you can read the following article for help.

http://www.anabolicsteroidcalculator.com/resources/articles/chemical_analysis/article6.pdf

The steroids including their glycosides can be extracted by polar solvents like MeOH. The isolation can be carried by eluting the silica gel column with chloroform by increasing polarity of MeOH.

your aim is not clear from the question if you wish to quantify secondary metabolites then you have to follow specific standard procedure, if you are only analysing the steroidal compounds then you may use HPLC for analysis.

I think it will be better separation by Column chromatography. Solvent system may be Hexane:DCM=9:1........DCM 100% then DCM :EtOAc=98:2, 95:5..........30%EA and finally Eluted by MeOH. and run the column very slow 1 drop in 2 second.

First make a fractionation by CC and then, those fractions containing steroids can be separated by HPLC using RP_C-18 columns and MeOH as eluent

Your objective is paramount. From the question it appears you desire other compounds but only steroids were found. it is important you identify your objective and the target compounds. this detrmines your extractant solvent. and then follow standard procedure

Just try to use another solvent like methanol for extraction, of you want the steroides that you get from hexane phase, avoid usin RP-18 becouse all material will stucked into your chromatography column

Hexane is genrally used to remove fats and waxes from plant material , some steroidal compound may also dissolve in hexane.For extraction of steroids first extract ur plant material in EtOH , recover ur EtOH , dry the extract and then dissolve in Chloroform and filter , most of the steroids will dissolve in chloroform.If u want to test either steroids is present in or not. take 0.5ml chloroform extract and add 1ml of Acid anhydride in a test tube and add 1 or 2 drpos of sulphuric acid. a red or brown ring will appear in the tube, which indicates the presence of stroids.

Well the best method to involve extraction of fatty to medium polar compounds is extract your dried plant material with MeOH and partition it with hexane and chloroform. Partitioning with hexane would help to remove waxes, fatty acids and lipids. The rest left over aqueous layer should be partitioned with ChCl3. Check the TLC for a comparative profile and spray with 5% H2SO4- MeOH spray reagent . On heating steroids will give pink color but almost color will change to fade green with some time on cooling.Purify through column by Hexane/ Ethyl acetate gradients.Perform LB test for the respective purified fractions.

Sorry I do not know because my thesis is about Sorghum Halepens.

The method you are going use in the isolation of steroids from n-hexane extract is by culumn chromatography (cc) using gradient elution. Starting from n-hexane through chloroform. The eluates containing similar compounds from the TLC profile will be pooled together into fractions. Fraction containing the steroids will be subjected to further cc if it is not a single compound or preparative tlc using silca gel plates.

I think u should go for column chromatography using using siligal column. U se solvnent system with increasing polarity with carbinol and chloroform. also use a combination of solvents with increasing polarity. for solvent system consult stal's book

Most of the plant hexane extracts contain few common steroids like B-sitosterol, Stigmasterol, Campsterols .....which can be easily tested...After verification for these you can proceed for presence of newer steroids ,if any.

u can also go for GLC

Arnab's response is probably nearest to the best. If you want small quantities of pure fractions, carry out optimised preparartive layer TLC on alufoil. cut 1-2 cm strips on both sides of the plate- spray with steroid spray reagent- Liebermann Burchard. Cut the bands in the central plate matching blue, blue green, green bands/spots in the test strips.

This is probably the simplest and very sure way of getting steroids.

In agreement with Bajubin for the small quantities ( qualitative work). For large amounts use of petroleum ether may also be used initially as De-fatting solvent ( instead of hexane). For clean up of CC fractions, size exclusion chromatography may be used. For example, use of sephadex LH-20 with methanol- dichloromethane mobile phase. LB reagent more specific than the sulfuric acid in methanol spray reagent.

I used DCM-EtOAc mixtures on the hexane fraction (Silica gel simple column). with 90:10 polarity, I got stigma-sterol in the sub-fractions which yielded in a very huge amount.

I am agree with the answer of Bapuji Maringanti with this you can also get a UV spectra of the dilute solution and can compare with the possible steroids present in your drug.

The best option is go for GC MS where u quantify the constituent. and also identify on the basis of mass.

if any query free to ask

After using hexane use solvents of higher polarity mixtures , than compounds with higher polarity will elude out . Also we can use subcolumns for further separation

Excuse me. . But can anyone please tell me which particular kind of silica gel should have to use for CC (for example, mesh size, etc). What solvent system should have to use for CC elution, when I have hexane extarct? And what is it for the TLC mobile phase?

@ Shriniwas

70-230 mesh for gravity columns, 230-400 for Flash columns (uses air pressure or pumps). Flash chromatography instruments often use smaller particle sizes in their columns. Smaller particles generally mean improved resolution provided the column is properly packed and there is sufficient solvent flow through the column.

Before taking the time to isolate them, look into purchasing a few standards or a phytosterols standard kit for cheap and common phytosterols such as beta-sitosterol, and then you can compare retention times using whatever separation tools you have available.

If you do decide to isolate them, you won't likely need any specialty separation materials. Work up a procedure from the separation tools you already have available by consulting the literature rather than buying new separation tools. Steroids are common lipophilic fraction plant constituents - they usually turn out to be common and known.

most of the hexane extracts of plant material contain fatty acids and waxes or steroids

u can go for GC studies of your extract which will tell you fatty acid profile as well steroids

Excuse me. . But can anyone please tell me which particular kind of silica gel should have to use for CC (for example, mesh size, etc). What solvent system should have to use for CC elution, when I have hexane extarct? And what is it for the TLC mobile phase?

You can isolate steroidal compund by extracting the plant plant extract using either pet- ether of n-hexane as solvent