The three most effective techniques for your immediate problem are to increase the sample size extracted, decrease the reconstitution volume, or increase the injection volume. As mentioned several times above, narrower bore columns can increaase sensitivity, but often lower injection volumes are needed and many fluorescence cells (especially on older instruments) have relatively large cell volumes compared to the flow rate of narrower columns which negates the advantage. You might also check if you haven't the the excitation and emission wavelenghts have been optimized and aren't historical values from old filter based instruments. Finally, please remember that nanogram is not a concentration unit. Best wishes

Improving sensitivity can be achieved in three fronts.

1. Improving the enrichment technique: liquid-liquid extraction is a great technique which offers various advantages depending with the analyte. There are other extraction techniques which you can use which could help minimize baseline noise and boost your sensitivity. Some of the instrumental techniques such as membrane extraction might not be accessible to you, so the next best option will be using an additional clean-up step with an appropriate technique, such as florisil, alumina, C-18 solid phase extraction.

2. Separation phase: Locatelli et al. (2012) say "the most common choice for improving chromatographic performance is the use of columns packed with low-diameter particles to obtain efficiency improvement, optimal velocity, and mass transfer. The main issue in using low-diameter stationary-phase packing is that pressure increases with the square of the particle diameter."

3. Sensitive detector: well the best option though the most expensive, difficult to access approach is changing the detector to a more sensitive one.

You can increase your signal to noise ratio by using solid-core particle columns (Supelco Ascentis or equivalent). These will simulate a smaller diameter particle (more efficient) without a large increase in backpressure.

Since you are already using LL prep methods, follow that with a column cleanup step and then evaporate away the solvent to a smaller volume. Concentration never goes out of style.

Hi. I belive you are going for 3x S/N = LOQ. What is your mobile phase? any stuff like TEA or TFA are increasing noise due to relatively high absorbance. Anotherone is temperature. I never w orked with fluorescence detectors but I had some expierience with RI -I had to cover all lines to detector with insulation. Last one is the column - ensure that everything is well separated from your matrix and ref to Edmond comment.

Dear sabin,

which particle size column u are using. try to get 1.7 µm column so u may got better separation in peaks as well sharper peaks also. if the peaks are getting good then the detection is also getting better against signal to noise.

as proposed before, you can use core-shell columns

with these columns, the diameter of the particles is lower but because they are non 100% porous, the back pressure is not highly increased what enables to use the columns with classical HPLC systems.

Thanks to these columns, the efficiency is highly increased : from a column with 100% porous particle of 5µ (3.5µ), to semi porous particles of 2.7µm, the efficiency is tripled (doubled).

Since the efficiency is higher, the peak will be thiner. And since the area remains constant, the height of the peak will also be higher, and so the sentitivity will be increased.

Hi Sabin, To optimised the method, you should try with differents pH values, for the best results , the pH value should be +/- 2 pH Unit of the analyte pKa value. to work with "No Ionized" form. in HPLC with fluorescence detection. and select the column of diameter of the particle smaller, if your equipment is HPLC try with 3.5 µm, but if you have a UHPL system you can use columns with 2.1 or 1.7 µm. other way to improve the sensibility, is spend time to clean the sample with Solid phase Extraction. (SPE).

Hi Sabin, I'm not sure what your units are in your question, ng/mL of plasma or ng injected onto the column. The above suggestions about smaller particles/solid core are all good, but be aware that if your detector has a traditional flow cell you will give back most of the benefits due to broadening in the detector at the resulting lower flow rate. Better cleanup will help if S/N is your limiting factor. If your extraction recovery is

You may improve the sensitivity in hplc by using a mass spectrometer as a detector and preferably lc-ms.ms tandem mass spectrometry. Also you can use coloums with small silica partice size with a diameter ranging from 2.7-1.7 micrometer.

As it mentioned several times above, the key is to increase the concentration in the detector. Supposing that the injected volume and concentration are given, the best way to get higher concentration in the detector to decrease the column diameter. Just half of the diameter will result in 4 times higher concentration in the detector (theoretically). Of course the column capacity is reduced the same time. Also reducing the particle size will increase the sensitivity (narrower peak), and if the necessary pressure is too high just elevate the analysis temperature. If you need an estimation on the column head pressure with a certain column and eluent, just send me the information on the selected column and gradient (if there is), and I will give you the expected pressure profile (maybe at several column temperature).

hey, try with small particle size column with higher injection volume. If u have ELSD detector, just check it for better responce.

Least case, add ion pair reagent or pre derivatise the anlyte.

Fluorescence can be a problem, I would suggest the cleanest highest quality solvents or even post column or other derivatization methods to better detect your compounds of interest. Try LCMS as well or a different kind of detector. THis may increase concentration in your detector if thats what you are really after. Good luck

The three most effective techniques for your immediate problem are to increase the sample size extracted, decrease the reconstitution volume, or increase the injection volume. As mentioned several times above, narrower bore columns can increaase sensitivity, but often lower injection volumes are needed and many fluorescence cells (especially on older instruments) have relatively large cell volumes compared to the flow rate of narrower columns which negates the advantage. You might also check if you haven't the the excitation and emission wavelenghts have been optimized and aren't historical values from old filter based instruments. Finally, please remember that nanogram is not a concentration unit. Best wishes

 Good question and very good responses to a very important analysis for measuring drugs in blood (HPLC) concentration and how to maintain reliable, accurate and improve technical sensitivity!

I agree, with the comments about the chromatographic methods, you can change the HPLC columns by UPLC or change the detector if you have possibility.  But another way you can increase the sensitivity is by changing sample prep method, when you work with plasma, SPE (Solid Phase Extraction)  is the most efficient method to decrease the noise and increase de sensitivity. if you need more information about it, don´t hesitate to contact me

Thanks alot. I have also learnt