All approaches have pro and cons:

if you extract fresh plant material you have the highest likelihood that what you extract is actually present in the plant at that moment of the plant growth cycles. However, keep in mind that the moisture in your plant tissue will influence the characteristics of your extraction solvent. i.e. if you work with a water miscible solvent you will not be extracting with the solvent itself but with a water/solvent mix of ill-defined composition. Especially if you want to examine the evolution in time of certain actives during the plant's life cycle you will do this at different moments in time and the amount of moisture in your plant sample can be quiet different and thus also your extraction solvent as it will have different amounts of water in it. Thus fresh leaves high in moisture will be extracted differently compared to fully matured old leaves. Since the extraction parameters are different this might bias your compositional data.

The levels of moisture might also cause problems when you are extracting with non-polar solvents such as hexane.

To avoid this kind of issues you are better of to first dry the plant material, but as already mentioned above this can lead to a change in composition as the drying process can impart oxidative degradation, decomposition of certain components, even enzymatically induced changes can occur.

Also keep in mind especially if you are extracting fresh material and your HPLC-protocol requires the use of non-polar solvents to ensure that your extract is completely dry as small amounts of moisture in some HPLC-setups might lead to pressure buildup in the column.

Yes definitely. You can prepare the extract with 70% methanol of fresh sample and analyse on HPLC or any other analysis.

Gud luck

:)

Depending upon your requirment of analysis you can prepare the extract. In case you are taking up the analysis where the composition of compound changes during drying of tissue, you need to prepare the extract from the fresh tissue. On the other hand if the drying doesnot bring in much chaanges in the composition of target compounds you can go for the analysis from dried tissue. In any case ther is no harm in the analysis fromfresh tissue.

Sure, this is possible. A nice technique is to freeze the plant tissue in liquid nitrogen, grind it (which is much more easier when frozen!), place it in the desired solvent, and eliminate the solid residues using a filter orf a SPE.

look for example here: Glauser et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 50, pp. 34506–34513

Take the plant and do the analysis. Fresh is always gud. I like the suggestions of Robin and Anil kumar. It depends on the composition and your target compound.

Gud Luck

As previously suggested, the extraction of plant tissue depends on your compound of interest. Eg. If you aim for gingerols, extract fresh ginger rhizomes. However, if you are looking forward for shaogols, use dried ginger rhizomes.

As Philipee has suggested, collect your plant tissue at desired growth stage, freeze dry, thaw and extract it with solvent.

I agree all above. Fresh is good and you may avoid some heat impact on certain metabolites. Liquid N2 is also good approach. It is better to compare two techniques both dried and fresh sample used...

Depending on your purpose and target compounds, you can analyse fresh, dried and frozen plant tissue. The yield and selectivity of the extraction process, and consequentely of the analysis results, may often vary considerably. Good luck!

In case you freeze your tissue, wich is much easier to work with as it was said, make sure you never get your sample warm afterwards; in this case the yield of the analysis could be vary between samples and thus the accuracy of the method goes down.

All approaches have pro and cons:

if you extract fresh plant material you have the highest likelihood that what you extract is actually present in the plant at that moment of the plant growth cycles. However, keep in mind that the moisture in your plant tissue will influence the characteristics of your extraction solvent. i.e. if you work with a water miscible solvent you will not be extracting with the solvent itself but with a water/solvent mix of ill-defined composition. Especially if you want to examine the evolution in time of certain actives during the plant's life cycle you will do this at different moments in time and the amount of moisture in your plant sample can be quiet different and thus also your extraction solvent as it will have different amounts of water in it. Thus fresh leaves high in moisture will be extracted differently compared to fully matured old leaves. Since the extraction parameters are different this might bias your compositional data.

The levels of moisture might also cause problems when you are extracting with non-polar solvents such as hexane.

To avoid this kind of issues you are better of to first dry the plant material, but as already mentioned above this can lead to a change in composition as the drying process can impart oxidative degradation, decomposition of certain components, even enzymatically induced changes can occur.

Also keep in mind especially if you are extracting fresh material and your HPLC-protocol requires the use of non-polar solvents to ensure that your extract is completely dry as small amounts of moisture in some HPLC-setups might lead to pressure buildup in the column.

Depending on the targeted extractive you may plan the extraction. Yes you can do extraction in fresh tissue also without drying, but I agree with Patrick's mentioned details though.

I agree with previous comments, the way is largely dependent on the target compoinds stability. For example, for low molecular weight antioxdants I strongly recommended to use only fresh tissue extraction bacause compounds will degrade during drying. Please, alos remebre that ratio of plant materail/solvent should be more than 1/10. In this case you can be usre about good preservation of the compound of interest.

Good luck!

Depend strongly on the targeted components. Anyway, all the extracts to be compared (according the different growth stages) must be prepared either from dried or fresh samples.

Absolutely! You even may luckily find new compound which may not be found from dry one, because the plant tissue will experience various chemical change such as oxidation, condensation, enzyme decomposition etc. during drying process.

I agree completely with Patrick as he has explained it in detail. The moisture content influences extraction of different class of compounds differently and so one needs to consider both options. It is better to make an analytical run for extract both from fresh as well as dry form and then decide.

I agree with Luis, target components will help you determine extraction process. And Patrick said it well for comparative run of both fresh and dried plant.

Of course it can be done in some instances but it depends on the analyte of interest since some compounds are very labile. Others have suggested the use of liquid nitrogen which is an option. I would need to know the compound or class of compounds prior to recommending a sample preparation protocol.

Please also consider that you need to use different extraction procedure for analysis of the different compounds with HPLC. Moreover, if you want to determine concentration of the compound of interest, you need to consider tissue amount you used for extraction, usually as fresh weight. Good luck!

Thanks a lot to everybody for valuable suggestions..this discussion has really became helpful to me..:)

Regards :)

Dear raksha, be sure..although it depends to your desirable compound, but in most cases the amount of that compound is much higher in fresh tissue..... I tried this...

Good luck....

And make sure the extraction method is reproducible; include analytical triplicates to test and ensure that.

What ever process you choose (fresh extract or freeze dried one) the preparation of extract inject-able to HPLC column is bound to eliminate some phyto-chemicals .....

it will be good

1.If at each stage of preparing extract for injecting to HPLC column --monitor the loses / reduction of phyto-compounds by TLC plates .

2.If your HPLC detection is confined to only by UV detection .....HPTLC with a scanner will be a very easy and reliable tool for fresh plant extracts assys.

At least one phytopharmaceutical company (Bioforce (www.bioforce.ch)) produces extracts from fresh plants only, which are treated within 24 hours after harvesting. They use an ethanol/water mixture as sole extraction solvent, but adapt the composition of the mixture to the water content of the plant, so that the final composition of the extractant is always the same. I suggest that you similarly determine the water content of each sample and aim for a fixed final extractant composition.

Yes you can use fresh plant tissue for performing HPLC assay, care should be taken in trems of sampling depneding upon the type of tissue you are going to use for the assay. If you are going to use it later than the tissue should be snap freezed in liquid nitrogen and stored in -80 till further use.

yes you can, even frozen tissue can be used. Frozen tissue could give problems during the handling of the sample though.

Depends on what analyte you want to quantify. Our experience dictates that dry samples suffer drastic changes, and the stability of your metabolite might be compromished. The best thing to do, if you do not have prior experience, is work with almost intact plant material: fresh material or snap frozen material, but kept at -70ºC for shor time. Forzen material trend to lose water, and subsequently metabolites can be altered (i.e. more difficult to disolve or suffer oxidation). And it is very important to perform recovery experiments, adding known concentration of standard compounds to your samples (spiked samples), so you can determine if your protocol is reliable. Also, as commented by other contributors, you must run a number of replicates to ensure reproducibility and accuracy. Once this experiments are performed, you can start working with treatments or growing conditions.

It would be more reliable to perform parallel experiments to compare the analytical results extracts from dry and fresh tissue.

Jianyong Wu has a point. A comparative study will help you to plan the studies in future also.

You can use fresh plant material and as suggested by the others you can also extract the dry (dried under shade avoiding direct sun light) material and compare.

Dr. Alok is right. do a comparative study and let us know. Constituents of plant can be different even during day timings. Environment do have a contribution which is different for different plants. good luck

If you want to do quantitative analysis, the % of water (or at least %moisture) of each sample at each time point should be taken into account as well.

I can suggest to work on lyophylized or freez-dried material. Freeze-drying, also known as lyophilisation, lyophilization, or cryodesiccation, is a dehydration process typically used to preserve a perishable material.

Following on from the answer by Patrick Ricquier, extracting from dry plant material is really your best option as fresh plant material has a lot of moisture in, which is problematic if extracting with organic solvents. Also, it makes it more difficult in the end in the determination of your extraction efficiency when the extraction solvent is dried off with the moisture in the original plant material.

To help avoid degradation of the molecules in your plant material during the drying process you can dry the material at lower temperatures (30 degrees C) for 2-3 days.

Flash freeze in liquid nitrogen and then freeze-dry (lyophilise). It will maintain the integrity and cellular components, including enzymes, and will allow for a better comparison between sample dates. If you analyse on a fresh mass basis, you results will be affected by the moisture content of the tissue which fluctuates during the day and season - resulting in apparent differences in your compound of interest that are actually due to changes in moisture content.

When you are interested in only polar compounds then extraction of Ultra Turax-treated material is possible. The homogenate should first be extracted with hexane (to remove chlorophyll and other nonpolar compounds and then will have after centrifugation an extract directly usable for HPLC analysis.

Dear Raksha

I hope you must have receive right suggestion. Yet I would like to add some more information. You should use the fresh plant material for the HPLC analysis rather than shade/air drying process. As suggested above you can directly freeze your plant tissue in liquid nitrogen and then extract with the appropriate solvent (as desired: which can extract your component of interest from the plant tissue). then directly you can move for filtration and injecting in HPLC.

If your component of interest is thermolabile then definately you should follow this protocol. In other case where thermal sensitivity is not an issue, same process will save much of time (than drying it using several means) .

These are the suggestions. You should consider these issues while selecting the right protocol.

Good luck.

Is HPLC the only option for your detecting an unaltered compound in fresh plant you have in mind? Look at alternative, highly selective techniques which may provide you with an answer - without damaging plant tissue structure - at least in the case of looking at plant leaves. With other plant parts - it will be still a challenge to choose the best option from those options suggested above.

Some contributors suggests the use of hexane or other non-polar solvent to remove chlorophyll should a polar analyte need extraction. Normally, Chl won't be extracted in aqueous extraction buffers. Only thylakoid vesiclcles could appear, which can be removed by filtration. If you use any non-polar solvent you might affect the solubility of some cellular components or metabolites. In any case, every step taken must be validated with appropriate internal standards. This is crucial to develop an accurate analytical procedure.

I think people who suggested that you take your fresh samples put them immediately in liquid nitrogen and lyophilize them before extraction gave you the best option.

We are routinely extracting and HPLCing from fresh samples. If you use high enough extractant to plant material ratio (we usually do 3 times 1:10) the difference in moisture should not matter that much. Nevertheless make sure you know what the water content of the sample is so you can calculate the concentration of your compound. An internal standard is also an important thing so you know how effective your extraction is.

The Dry or fresh material is not the matter mostly we use fresh material for HPLC just you need to know what is your mobile phase and column RP is general usage, and your calcualtion will be united per DW or FW as you applied

Dear Raksha,

The best way is extracting metabolites, pigments and other biochemicals from FRESH PLANT MATERIAL frozen at liquid nitrogen immediately after sampling.

Follow standard protocols, like extraction medium, columns,gradient (if nay), flow rate, detection system etc. for estimations.

Best wishes.

For quantification and reproducibility purpose it is important to start with dry-powder extract, you need to know the exact weight of your extract in the absent of water. Lyophilization might help you in that regards

If you need to measure secondary metabolites, PHWE (Pressure Hot Water Extraction) is a useful approach to consider for plant samples (see attached). For trace phytohormones' extraction from plant tissues, it is best to deactivate enzymes that may break down the various phytohormones' especially the nucleotides (see next message). Good luck!

Here are the few options to extract phytohormones from plants. Adding a tritiated recovery marker is a useful technique when dealing with trace chemical compound analyses from plant tissues.

If you want to analyse/process the fresh plant material, then extract by juicer or hydraulic press, take out the juice/extract in separating funnel, extract it with solvents like, hexane, chloroform and ethyl acetate, three times with each solvent, concentrate these extracts and carry out HPLC analysis using appropriate column and solvent system. You will definitely get good results. By this way you may get many such compounds which are sensitive to air and light.

I think lyophilization is the best way. Extracting from fresh sample is not efficient as it is difficult to reduce the particle size of the sample. Compounds might degrade while reducing the particle size my milling as various degrading enzymes might still be active particularly in aqueous medium. On the other hand lyophilzation drys the sample in subzero temperatures. Therefore there wont be degradation (thermal and enzymatic) of the compounds. After drying you can reduce the particle size by milling easily. Particle size is a very important parameter for efficient extraction. The advantage of lyophilization over liquid nitrogen is scale up. The lyophilized sample can be stored at -20 degree in vacuum pouches for a longer time. Lyophilized samples are equivalent to fresh samples with loads of advantages over others. If you don't have a freeze drier, I think liquid nitrogen is the option. Hope it helps.