One question, if you are using phosphate buffer at pH 7-7.2, why are you also employing TEA?? Phophate buffers in this range alone - remove the TEA. The main cause of tailing for basic drugs is the interac tion of the acidic silanols and any residual metal ions in the silica with the drugs. What you describe as a 2nd hump I don't believe is tailing.

Thouroughly clean your column with a 4 solvent wash overnight. The sequence is to run 20 column volumes each of 1.water, then 2. methanol, 3. THF, 4. chloroform or dichloromethane and finally 5. methanol for storage. Keep flow rates about half what you normally run at and allow a slight gradient when moving between water/methanol due to the rise in backpressure.

Having cleaned the column equilibrate with phosphate/organic and reinject your sample. You should always use a guard column - it is cheaper to replace than an analytical column!

can you tell me your compound pka value?,did you checked the spectra of those peak?

If you use more organic solvent there will be a peak splitting due to secondary ionic equilibrium.

Not very much information, Bhupendra, but unfortunately this seems to be common here at RG.... Which type of column? Which pH? Which pka of analytes? Which sample matrices do you analyze? How many samples did you already analyzed with this column?

Peak tailing can be caused by a lot of things. Aging or contamination of the HPLC-column: How old is the column? Did you analyze a problematic matrix recently? If matrix contamination, you may backwash it according to the manufactures advice. If you work with silica columns at high pH the silica may dissolve causing void etc.

Ah, do you use pre-columns? I highly recommend it. Sometimes a new pre-column solves the problem.

Use freely washed column and digest the solvent mix. properly

Many problems in an LC system show up as changes in the chromatogram. Some of these can

be solved by changes in the equipment; however, others require modification of the assay procedure.

Selecting the proper column type and mobile phase are keys to “good chromatography.”

Possible Cause Solution

1. Blocked frit

a. Reverse flush column (if allowed)

b. Replace inlet frit

c. Replace column

2. Column void . Fill void

3. Interfering peak

a. Use longer column

b. Change mobile-phase and/or column/

selectivity

4. Wrong mobile-phase pH 4.

a. Adjust pH

b. For basic compounds, a lower pH

usually provides more symmetric peaks

5. Sample reacting with active sites

a. Add ion pair reagent or volatile basic

modifier

b. Change column

just a few tips included above another very useful technique run methanol over a long period to maintain the column Thanks

Thanks everybody. I think some of the suggestions given y you going to help me. I also doubt column contamination, otherwise how the same method gives tailing peaks just another day. The column is not old just 100 injections may be. I am not using any guard column. I will try that also. I could not understand the meaning of "digest the solvent mix". I will give more specific details regarding my separation. The sample pKa is 3.4, the column type is Dionex acclaim C18(250x4.6,5u) and the sample matrix is sterile dry powder for injection. Kindly suggest the solution

why you used / choose TEA? any particular need? what is your method for? i mean it is for assay / related substances? what is the column, type, dimension, phase, make? what is the tailing factor you got?, how many peaks observed in your sample?, all the peaks are tailing or only your analyte? what is the pH range of your column? how old your column or how many injection over?

you answer for my answer or use the questions for your troubleshooting.

i hope this helps!

regards

Selvan

https://www.researchgate.net/topic/Analytical_Chemistry/post/how_can_we_develope_HPLC_method_for_unknow_sample

change any other column of same phase to check the peak shape, if the peak shape is good, column is the problem

if really the column is problem, it has gone bad because of bad pH or overload due to bad wavelength

flush well the column with ACN & use

This might be due to over loading or something has retained in the column. You should regenerate the column properly.

It happens at times after quite some time one might experience some Tailing. Especially if your prosedure involves pH control. Homogenise your solutions/solvents using ultrasonic bath, check the pH and rewash your column. All the best.

I suggest once more do a routine maintenance of the column after running a days sample run methanol solvent for sometime before shutting down HPLC and it may improve the column performance.

Thanks everyone for your comments kindly suggest me the ways to regenerate the column

Senthamizh : The method is for the assay. The reason behind choosing TEA is that the drug is stable in the pH range of 7-7.2 and also without using any modifier the drug gives tailing peak. So to reduce the tailing as well as to adjust the ph to 7.0 I chose TEA as I am using phosphate buffer. The ph range for my column is 2-8 and the column is only 100 injections old. There are two analytes in my sample and both the peaks are tailing. I am not getting a typical type of tailing but it is something like a small hump towards the end of the peak. Anyways, it is not allowing me to quantitate properly. Kindly give your suggestions

murugesan: The pka value of the drug is 3.4. The organic content in the mobile phase is just 20%. By telling check the spectra do you mean 3D by PDA or UV scanning?

A "hump" sounds seriously like void volume somewhere! Check all connections and if this won't work try a new column. If the problem is solved with a new column I highly (!!!) recommend to use a precolumn. It saves you time, money, and nerves, Bhupendra! Good luck!

PS: While working with TEA I also recommend injecting the analytes in (start) eluent. This may also have a significant positive effect for your peak shape.

One question, if you are using phosphate buffer at pH 7-7.2, why are you also employing TEA?? Phophate buffers in this range alone - remove the TEA. The main cause of tailing for basic drugs is the interac tion of the acidic silanols and any residual metal ions in the silica with the drugs. What you describe as a 2nd hump I don't believe is tailing.

Thouroughly clean your column with a 4 solvent wash overnight. The sequence is to run 20 column volumes each of 1.water, then 2. methanol, 3. THF, 4. chloroform or dichloromethane and finally 5. methanol for storage. Keep flow rates about half what you normally run at and allow a slight gradient when moving between water/methanol due to the rise in backpressure.

Having cleaned the column equilibrate with phosphate/organic and reinject your sample. You should always use a guard column - it is cheaper to replace than an analytical column!

Use of guard column (precolumn) is recommended; especially for semi-preparative chromatography work. I guess, you are carrying out semi-preparative chromatography, right? And not analytical? Though, even for analytical chromatography, guard column is good, the amount of sample injection would be less in case of analytical as compared to semi-preparative. Therefore, in some cases, analytical chromatography work can be performed without guard column also. Are you ensuring that you are not overloading the column? Hope you are working with the optimum concentration level of the analytes.

If you are using guard column, if you are working with optimum concentration levels of the analyte (i.e. without overloading), if you have washed the column thoroughly and then still observe the hump(s), then the hump(s) can be due to the matrix (i.e. the solvent/carrier which contains the analyte) or it can be due to some reaction (i.e. some chemical reaction between the analytes and column packing material or between one analyte and the other) or it can be due to conformational inter-conversion of the analyte(s). If this is due to conformational inter-converstion, then the disappearance of hump(s) at some elevated temperature can occur. When the hump(s) get disappeared, it may be possible to see the peaks of the analytes becoming more sharper (i.e. of lesser peak width). Let me know, if these suggestions work out or not.

if the method is for assay, it is very simple, you are going to worry about only one compound, is the compound is ionisable? if not ,no need of restricted pH at all.

i can say clearly that the mobile phase is not right one, use min 30 % of organic to increase RP column life

use buffer or modifier if the analytes are ionisable because ionisable compounds will show as two peaks or split peak as it is existing in two form (HA and A-); so if you buffered the mobile phase into basic or acidic the analyte will go to single form. maximum use acidic pH (2-4) as the mostly column will hydrolyse more in basic condition

HA------------>H+ + A-

0.1% TFA, H3PO3, H2SO4, 1-2 % Acetic acid are the simple modifiers to restrict the pH into acidic side to improve the peak shape) and well known phasphate buffer (10 mmol – 100 mmol phasphate buffer), optimum is 30-50 mmol

change your buffer as simple phasphate buffer of 2-3 pH or 7.1-7.3 pH, never worry about your compound pKa for pH selection,

pH is just to suppress the ionisation of analyte in to single side,

use gradient to optimize the peak shape,

use buffer in A, Methanol in B channel, optimize the peak shape with start of 70 A ; 30 B

If again you get the same really your column gone bad, try simple molecule caffeine with simple mobile phase water:ACN, check the peak shape & understand the column

Regards

Selvan

Vartharajan:I increased the column oven temperature upto 45degree but the hump is not separating from the analyte peak. The concentration of analyte is 400microgram/ml. Does this concentration cause overloading of a C18, 250x4.6,5um column? Yes I am not using guard column.

Senthamizh: I will check the column performance using caffeine and update you.

Leo: After checking the performance of the column I will wash the column as suggested by you.

I guess, your sample is having some other analyte (i.e. contaminant) because you are observing the hump, even after increasing the temperature up to 45 degree Celsius. Is the sample that you are injecting onto the column, alright? Yes, you need to check with a standard (such as caeffine) or even "a cephalosporin standard" or its analogue can be used to assess the performance of the column.

Hi Bhupendra,

you should have several compounds in your test injection - uracil for a void marker, caffeine and sevral others to check separation of different eluates. Check this before and after column cleaning as well. One step I left out of the wash cycle is you may need an intermediate polarity solvent between the chloroform /CH2Cl2 and methanol (MeOH) that is soluble to both phases in case the MeOH and chlorinated solvent are immisible. Alternatively you can substitute hexane for the chlorinated solvent, and again I would recomend say THF to avoid immiscibility problems with MeOH.

Whatever system you choose, if it works I reccomend a weekend wash cycle for routine use if this is part of your regular QC procedure. I would apply this to most columns with bonded phases on a weekly/fortnightly basis to ensure conformity.

Thnx everyone...i will update you later

If you have a longer C18 column, or one with more theoretical plates, try that and see if you can separate the hump from the analyte peak. If it does, then it is likely a contaminant and not a peak trailing issue. Another option is to try the same LC separation using a mass spec as the detector. A Q1 scan of the LC region of interest will tell you if it is one analyte or two analytes with similar RT.

Use different column. Their acidity if different use as much acidic column as you can. Make sure that column is encapped as well. My advice Thermo Gold C18. I had similar problem in the past. Don't use TEA if you don't need it.

Regards

My problem is solved....the conclusion is column contamination. As suggested by you I washed the column for two days using ACN, methanol, water, IPA and dichloromethane. There is a small hump but it is well separated from the analyte peak

Great! Now use a guard-column filled with the same material as your analytical column and most likely you won't waste time again because of this. ;)