Sensitivity of Peaks

[mobunvar]

How can I increase the sensivity of the peaks? currently I am working on 4 drugs which can go up to 50ng/ml only, but i want to go up to 500pg/ml. I tried variations in pH and Organic solvent but there is no USE. So please help/suggest me, what can be other posibilities to increase sensivity ? I am working on Agilent UHPLC-UV. and pH is 2.3 with 0.05% TEA and Buffer is Heptanesulfonic sodium salt (0.05mM)

[mobunvar]

please some one reply me...

[HPLCaddict]

Changing the percentage organic in the solvent won't give you larger peaks, unless not everything was going into solution before. Changing pH might change peak areas considerably, if the absorption of your analyte is pH-dependent, but in this case you should change the mobile phase pH not the solvent.

I would focus on the chromatography. Things you might want to consider:
-Increase injection volume. Easist thing to do, but your chromatography (peak shapes!) might deteriorate
-Change detection wavelength. Check what's the absorption maximum of your analyte.
-Decrease flow rate. Will give you larger peaks in a UV-detector. Chromatography will change too, of course, and might have to be adjusted. Best thing, move to a column with a smaller inner diameter and scale the flow rate accordingly (what column dimensions are you using now?).
- For improving signal/noise ratio, you might want to consider instead of increasing the signal to decrease the noise. What does your baseline look like?
- Different detection method possible (fluorescence etc.)?

[nschwartz]

I would also make sure that your method of detection and wavelength is appropriate. If you don't have any aromatic rings or at least conjugated double bonds, then UV is not the best detector to use. If you can scan across all wavelengths and find the one for maximum absorbance that would be best.

[tom jupille]

Without details (such as your detector), it's hard to be specific but:
With a decent chromophore (extinction coefficient around 10^4), and a decent column, UV detection will take you down to about 1 ng (mass-on-column). You are *not* going to be able to inject 2 mL on a UHPLC column so you have only two choices:
- a more sensitive dector (MS, ECD, or fluorescence - with the viability of the last two depending on the structure of your analytes)
- preconcentration (SPE, liquid-liquid exctraction, or evaporation/reconstitution - assuming you have reasonably large volumes of sample to begin with).

The most certain would be LC-MS/MS.

[mobunvar]

Thank You for the replies

@HPLC addict

-I already increased injection volume from 20 microlit to 40 microlit. Yes there was considerable change. But the problem was my sample has 5 different compounds in one shot. I can't increase more than 40 microlit. That's the maximum with the system.

- Agilent UHPLC can detect at various wavelenghts in single injection. so after screening i selected 210nm to be best for all the four compounds.

- right now i am using 0.5ml/min and i tried 0.3ml/min too but with 0.3 ml/min retention time of drugs is extending more than 12 minutes. so 12 minutes with UHPLC is not proper. so i chose 0.5ml/min. and Column dimensions are 150mm X 4.5mm (ID), 5micro met particle size

-noise in the base line is very less...its less than 0.2mAU, so its pretty ok. but any more suggestions to decrease further

- unluckily, few compounds can't be detected by FLD. So i want to simultaneous detection so i can't use.

Apart from all this,

-change of conc of buffer and type of buffer will effect any change in sensitivity ? right now i am using 0.05mM Heptane sulfonic sodium salt with pH 2.8

- any problem in flow cell can effect sensitivity ?
- any problem in injection can effect sensitivity ?

[tom jupille]

You asked for a 100-fold improvement in detectability.

-change of conc of buffer and type of buffer will effect any change in sensitivity ? right now i am using 0.05mM Heptane sulfonic sodium salt with pH 2.8

No

- any problem in flow cell can effect sensitivity ?

You would need to make the flow cell 100 times longer, so again, no.

- any problem in injection can effect sensitivity ?

You would need to inject 100 times the volume so for a third time, no.

I'm not trying to be sarcastic here. Getting detection limits on the order of 50 pg (mass-on-column) is not feasible with UV detection. If fluorescence or electrochemical detection are not feasible, your remaining options are sample precentration (least expensive) or LC-MS/MS (easiest, believe it or not).

How much sample volume (total) do you have available?

[mobunvar]

Tom, Thank You for reply....

LC-MS/MS, we don't have that provision to use.
I didn't understand ur question...how much sample volume....

I inject 40 micro lit, in plasma extraction i use 400 micro lit of plasma.

that means there is no way to increase the sensitivity in UHPLC-UV?

[bisnettrj2]

Can you concentrate your plasma extract? You said 400 microliters of plasma - to what final volume? Perhaps you can look into concentrating the final extract to a smaller volume. Then you can use the extraction multiplier to relate the instrument response to the amount of drug in solution. However, I don't think you'll be able to work with 4-uL of final extract (100-fold concentration), but maybe a 10-fold concentration would be possible?

You said you have an Agilent UHPLC, so I'm going to assume a 1290 with a Diode Array detector and the well plate sampler. If that's incorrect, then please give your actual hardware (Agilent model numbers per module).

Make sure you are using the MaxLight (60-mm) Flow Cell cartridge in your 1290 diode array.

You have a UHPLC, but are not using a column that will allow you to take full advantage of that system. I can't imagine you're seeing much backpressure with that column (150x4.5mm 5-micron) and that flow rate (0.5mL/min) - maybe 100 bar? Unless you're running a very special phase (like some sort of polymer-based stationary phase), ask Agilent to recommend one of their ZORBAX RRHD 1200-bar columns as a replacement. I would get a 2.1x50mm 1.8-micron column to start, at 0.2mL/min. In fact, one probably came with your system (if it is a 1290) as a checkout column. If you have it, it was probably used once and put back in the box. Find it, use it. But don''t ever expect to use it again as a checkout column - dedicate the column you're using to ion-pairing.

Finally, you can increase the injection volume on the 1290 sampler to 120uL, but I think you need the OpenLab CDS software to use that functionality.

http://www.chem.agilent.com/Library/tec ... 9533EN.pdf

I can't guarantee this will get you 100x more sensitivity, especially with the mobile phase you're using, but it might help. I would start with the possibility of concentrating the extract (or extracting more plasma!), then the flow cell cartridge (if you are using the 1290 Infinity Diode Array Detector), then the column, then the injection volume upgrade if it would work with your software. However, you will most likely not be able to inject more than 10-20uL if you switch to a 2.1x50mm column, and that would be pushing it.

[mobunvar]

Thank You for the reply....But i am using Agilent 1200 series with binary pump (G1312B), G1315C DAD. I am using Purospher RP-18 Column. I am not sure about flow cell and I need to check about it. Right now pressure is only 82bar.

right now in the auto sampler also i can increase injection volume to 100 uL (but i am unable to do it, its not accepting it)

The column which u mentioned, is it really increases the sensitivity ?
U told the mobile phase which I use is not helpful, can u suggest me any other mobile phase. I already test Pot Di Hyrogen Phosphate but it didn't work. Any new buffers ??

So today I shifted my method from Isocratic to Gradient. Surprisingly, sensitivity of the 1st drugs raised predominantly. But I am unable to separate the peaks. they are clumped together and base line become more worst.

[tom jupille]

i want to go up to 500pg/ml

Here's what you said in the original post. Now the question is "500 pg/mL in *what*? The original sample? The prepared sample as injected into the column?

As far as the chromatography is concerned, the relevant quantity is mass-on-column: what quantity of analyte actually gets injected, irrespective of what it is dissolved in. UV detection will get you down to the low nanogram range (maybe 100pg in a very favorable case -- and if you have to detect at 210 nm, you do *not* have a favorable case!). That's it. No buffer in the world will change that. And that level assumes that you have optimized things like column efficiency.

If you have large samples (say, 5-10 mL) then you can extract the analyte(s) and concentrate them into a much smaller volume for injection, but there is a reason why the majority of bioanalytical (drugs and metabolites in biological fluids) work is done by LC-MS/MS: that's the only way to get good quantitation at the picogram level from small samples.

[Alexandre]

"Cosmetic" improvements of LC/UV will not give you 100 fold increase. Your method of 50 ppb can not be converted to 0.5 ppb (500 ppt) easily.
You need drastically change your protocol.
So – you may need to start with 4 mL or so of blood. Can you?
Do concentration, SPE etc. Evaporate to 100 uL.
Decrease ID of your column, try 1 mm id, increase inj. vol.
Use the best UV detector on the market.

Can you do derivatization LC/FLD? Or GC?

[HPLCaddict]

- right now i am using 0.5ml/min and i tried 0.3ml/min too but with 0.3 ml/min retention time of drugs is extending more than 12 minutes. so 12 minutes with UHPLC is not proper. so i chose 0.5ml/min. and Column dimensions are 150mm X 4.5mm (ID), 5micro met particle size

Not primarily related to your problem, but you are NOT doing UHPLC!

[mobunvar]

you are NOT doing UHPLC!

i didn't understand, i am working on UHPLC

[Peter Skelton]

UHPLC requires the use of sub-3um particle columns, you are using a 5um particle column so you are not doing UHPLC