No it isn't because MS adds only a 2nd dimension for identification and quantification. It does not affect the LC separation

No. LC-MS is simply HPLC with an MS as detector instead of the usual UV/Vis or other type of detector. If you are using the same method conditions between the two techniques (HPLC vs LC-MS), then you will not achieve better separation. Try changing method conditions or use a different type of column to achieve purification.

In LCMS the fractions from HPLC are directly going to the source of MS and providing you the mass of each peak. In principle you have same trouble like your analytical HPLC. I would suggest to play around with HPLC methods even think of changing to other column types. Very polar or very non polar compound may need special type of columns. Merck has good collection.

The MS is only another detector and won't help the purification as pointed out above. However, one can collect the mixture only containing the mass-ion of interest and potentially enrich the fractions containing the desired compound. This material can be purified again until the required purity is obtained. Again, as others mentioned, try to improve the chromatography; this will pay dividends in time.

As others have said the MS is just a really expensive yet potentially very specific detector. You should improve your chromatography and may want to try another type of stationary phase. If you insist on using MS you will have to keep your flow rates to 2mL/min or less if you are using ESI. However, you can set up a splitter to divert most of the flow to a fraction collector while the remaining goes into the MS for detection. I have done this with semi-preparative C8 RP columns. You can also automate the entire process. The problem is the injection volume that you use will be determined by the loop in your auto-sampler which likely wont be more than 100uL. Depending on how much you have to purify you could be doing many injections.

What do you mean "isolate"? You want to collect the fraction, or you only want to identify what were in the pre-fraction? If only identification, the LC-MS/MS is my primary choice.

Thank everybody for nice suggestions. I will do as your suggestions.

Dear colleague, it is important to know that the LC-MS technique is a destructive technique, if you mean overcome the co-elution of some molecules inseparable with the common HPLC, then in this case it is useful LC-MS because you will follow the ion parents or its fragments in SIM, but if you intend to purify a small molecule then you will have to use an HPLC with a column SEC (Size exclusion column) with appropriate stationary phase in relation to your molecular size class.

the function of MS is to detect what is seperated by the chromatograph. Separation is carried out in the coloumn . the varables are the mobile phase and ofcourse the coloumn that you use.use proper solvent gradientsand find out what solvent system will give an effective seperation.

Somehow you know you have a mixture of 5 compounds. That is important to eventually solve your problem. The technique that told you that there are 5 could help you decide on the stationary phase to separate your compound from the other 4. The mistake most make is using a C18 separation (a good first choice) that does not work, and then trying to improve it by substituting another flavor or brand of C18. If you have 5 coeluting compounds changing the mobile phase conditions on your existing column is unlikely to achieve the separation you need, a change in column type to get the selectivity you need is probably required. As others have noted, the MS is destructive, but it can help you decipher the identity of your impurities and once you have a separation it can be set up to do mass-directed fraction collection. That is a topic for another day.

Thank Dr. Jeffrey Rohrer for your nice fruitfulll explanation. I appreciated your ideas and try to do this. I already used C18 analytical and preparative column to separate my targeted compound but this is a mixture of at least 5 compounds and two are coeluting. My next plan to rechromatography at same column just change mobile phase conditions like slow flow rate and increasing aqueous solvent percentages. Actualy I want to isolate a growth promoting compound from bacteria. Another problem is that my sample is very small amount (about 5 mg after HPLC separation). Other problem is, when I run my sample in preparative HPLC, I was collected several fractions and subjected to all for bioassay. interestingly all fractions was showed activities i.e means all fractions contained my active compound. Is it possible? I dont understand, why every fraction showed activity?

Mohammad, Unless you were running your prep column at a higher flow rate than recommended I would not bother changing the flow rate as it will have little benefit. Making your gradient more shallow (slower increase of the % organic) could benefit your separation. Without knowing your compound or bioassay it is difficult to answer your question, but it is certainly possible to have multiple fractions with activity. First it depends on how specific your assay is. If the assay had low specificity, multiple compounds could exhibit activity. For example, Gentamicin has at least 5 compounds (close in structure) that will give a positive signal on a bioassay. If those 5 or more are resolved they would appear in multiple fractions and hence give positive assay results. It could also be that the compound you are interested in has poor chromatography (e.g. peak with extreme tailing) and hence it shows up in multiple fractions.

Thank again Dear Dr. Jeffrey Rohrer,I think, you found my problem. Yes, my flow rate is 5 ml/min, and peak are tailing. Two fractions were shown high activities in bioassay and the rest of the fractions were shown lower activities.Is it possible of isomer or some thing? My preparative HPLC conditions as: 94% water (0.1% acetic acid) and 6% acetronitrile (0.1% acetic acid)---120 minutes and from 120.1 minutes to 240 minutes used only MeOH. Total program is 240 minutes. I was collected 10 fractions like time interval 10 minutes, 15 min, 20 min, 25 min, 27 min (peak), 32 min, 40 min, 49 min, 145 min and 240 min. Here 20 to 27 min, this two fractions are most active and was shown 5 peaks. Now I was collected two most active fractions and concentracted and again rechromatography. Am I use same c18 column or change another band of c18?Please give me suggestions.

Its not isolation technique its identification and estimation technique. Preparative HPLC when hyphenated with MS may be called as isolation technique.

LC-MS is not for isolation, it is for detection and identification.

Preparative HPLC is for isolation.

Using a split in the post column, one small portion goes MS, another goes collector.

But you need to adjust the ret time and ratio based on ID of tubing, to make sure the peak come from MS is almost the time in the collect.