There is normal phase HPLC and reverse phase HPLC. Which one you use depends on your samples. I'll assume that you mean reverse-phase HPLC, using C18.

If you have partially purified your compound, you should have an idea of the polarity of the compound. For example, if the compound was extracted in hexane, and purified on silica using a hexane/toluene gradient, the compound is fairly non-polar and you would use a solvent system on C18 that uses a high percentage of organic solvent, with no modifiers. In this example, you may even need to use non-aqueous reverse phase.

At the other polarity extreme, you may have purified the compound from an ion exchange column. This compound would elute from C18 using a low concentration of organic solvent and would probably need the acid modifiers mentioned earlier in the thread for better peak shape.

Ethyl acetate extracts are medium non-polar, methanol extracts are medium polar, and aqueous extracts generally contain very polar compounds. Ethyl acetate extracts generally have compounds that elute in highly organic solvent, methanol extracts generally use more polar solvents on C18.

Also, look up the plant or organism you are extracting from in the literature. There's a good chance that you may have the same, or similar compounds and so have a purification method already.

The biology you are working with also guides your purification. for example, I noticed the central nervous system assays to which i was submitting extracts tended to screen for alkaloids- my HPLC systems were fairly polar solvent systems with an acid modifier.

For flash columns, one can use "wide polarity range" chromatography, both normal phase and reverse phase: http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Wide%20Polarity%20Range%20Flash%20Purifications.pdf

These are Mili Que water (HPLC grade), methanol, acetone, benzene, acetonitrile, chloroform and Petroleum ether.

I agree with Dr. Upadhyay. U can use a gradient system and monitor the elution

I think this depends on the whole conditions not just the nature of compounds.

The column used, type of extracts , etc.

Searching for published literature in your domain will help you greatly.

Kindly suggest me a reference. 

http://www.vedamsbooks.com/no44878/phytochemical-techniques-n-raaman

http://www.crcnetbase.com/isbn/9781420092615

http://www.phytojournal.com/vol2Issue1/Issue_may_2013/40.pdf

Sometimes also tetrahydrofuran - yet as it is said above - the whole experimental conditions are significant, especially type of metabolite and type of column. Often also the acidifing modifiers are used 

Good luck !

There is normal phase HPLC and reverse phase HPLC. Which one you use depends on your samples. I'll assume that you mean reverse-phase HPLC, using C18.

If you have partially purified your compound, you should have an idea of the polarity of the compound. For example, if the compound was extracted in hexane, and purified on silica using a hexane/toluene gradient, the compound is fairly non-polar and you would use a solvent system on C18 that uses a high percentage of organic solvent, with no modifiers. In this example, you may even need to use non-aqueous reverse phase.

At the other polarity extreme, you may have purified the compound from an ion exchange column. This compound would elute from C18 using a low concentration of organic solvent and would probably need the acid modifiers mentioned earlier in the thread for better peak shape.

Ethyl acetate extracts are medium non-polar, methanol extracts are medium polar, and aqueous extracts generally contain very polar compounds. Ethyl acetate extracts generally have compounds that elute in highly organic solvent, methanol extracts generally use more polar solvents on C18.

Also, look up the plant or organism you are extracting from in the literature. There's a good chance that you may have the same, or similar compounds and so have a purification method already.

The biology you are working with also guides your purification. for example, I noticed the central nervous system assays to which i was submitting extracts tended to screen for alkaloids- my HPLC systems were fairly polar solvent systems with an acid modifier.

For flash columns, one can use "wide polarity range" chromatography, both normal phase and reverse phase: http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Wide%20Polarity%20Range%20Flash%20Purifications.pdf

I would say use ZIC-HILIC with ACN and Water; depending on what you are interested in you can acidify both the solvents. 

Is your LC connected to MS ?

If you are connected to MS don't use salt containing solvents. 

In fact, it will depend greatly on what kind of phytochemicals you are looking for?? This depends on the whole conditions not just the nature of the compounds. The column used, type of extracts, etc. Mostly, These are HPLC grade water (Mili Que), methanol, acetone, benzene, acetonitrile, chloroform and Petroleum ether.

HPLC is an analytical method to separate the components of a given mixture sample, with the identification of the separated components made possible with a calibration curve of an appropriate standard that is either introduced singly or mixed with the sample (spiking). The basis of selecting solvent (mobile phase) would depend on the very nature of the mixture component to be isolated, that separation if effected sharply when the different components of the sample have differential solubility in the solvent to use and so with differential adsorption on the material packed (stationary phase) inside  the column. When the component to be isolated is more of a nonpolar, a nonpolar solvent is prefered but when it is more of a polar, a polar  solvent is selected.

Dear

There is a wide range of Solvents for HPLC. It depends upon your sample which and what kind of solvent should be used. So first of all you have to check the misciblity of solvents with your samples.

one of the list of solvents is as below;

Acetone ,  Methylene Chloride 

Acetone, Low Water ,  Methyl Ethyl Ketone ,

Acetonitrile , Pentane ,

Acetonitrile ,2-Propanol ,

Chloroform, Hydrocarbon Stabilized , Pyridine, 

Chloroform , Tetrahydrofuran,

Cyclohexane, Tetrahydrofuran (Stabilized) ,

o-Dichlorobenzene , Tetrahydrofuran, 

Ether, Anhydrous , Toluene ,

Ethyl Acetate , 1,2,4-Trichlorobenzene ,

n-Heptane , 2,2,4-Trimethylpentane ,

Hexanes (95% n-hexane) , Water ,

Isobutyl Alcohol,

Methanol , 

Methyl tert-Butyl Ether.

Regards

Hello!

First you need have on mind what type of metabolics do you want to get. Your metabolics show high polarity? Low polarity? If show high, are soluble in what proportion of water? Take some milligrams of extract and test with different solvents in TLC.

In HPLC we work with two type of mode: Normal and Reverse. The difference between the modes is the polarity of silica. The nomal the filling of column is just silenol groups. The reverse, there is a group alkyl linkage in the silicon that can be C8 and C18. But what is the solvent that I can use?

In normal phase use just apolar solvent how hexane, dicloromethane, Acetato de Etila . For example some class of diterpenes how clerodane and caurane are separate with this solvents.

In reverse just polar solvent. Non polar solvent destroys the phase of the column. In the reverse phase, the most common solvents are water, methanol, acetonitrile, acetone and tetrahidrofuran in this order of power. If you want become the interations more powerful you can put a little of acid our base (in general 0,1 a 1 % of a acid our base freak) that depend of your methabolics.

You need have on mind that the separation in reverse phase the proportion of water and organic solvent are the ‘’Cat Leap”. Some friends my, sometimes, arrive to say that the separations is just in water, in the illusion that little changes in proportion of water do a ''miracle'' in the separation.

Greetings and Regards;

I work on fecal Sterols and look for a suitable method for extracting fecal Sterols from the water for analysis with HPLC.

Thank you very much for your attention.

Sterol alcohols?

These are generally non-polar. I'd try C18 solid phase extraction, wash with acetonitrile and maybe dichloromethane. The literature has lots of HPLC techniques for sterols. My preference for detection would be ELSD (evaporative light scattering detection) or APCI MS (atmospheric pressure chemical ionization mass spectroscopy). APCI MS will probably see [M+H-H2O]+ . These often show weak UV absorbance.

Greetings and Regards;

Thank you for your answer, but for the extraction of the Soxhlet method, or another method, I want to examine the stool from sting water from the aqueduct, in which there are copesterin and a few stool sterols.