Sample loop volume

[EK8]

Hello,

I got a semi-prep HILIC column (150mm x 10mm) that I need to use to fractionate my sample. Max injection volume in this column is ~200uL

I currently have a 250uL syringe and 100uL loop connected to my autosampler.

I also have a 2mL loop laying around.

Would it be a problem if I inject 200uL using this 2mL loop, for fractionation? Or would it be advisable to stick with the 100uL loop and just inject 100uL of my sample?

[Multidimensional]

Several things do not make sense....
"I got a semi-prep HILIC column":: There is no such thing as a "HILIC" column (that is just some marketing/sales misinformation). HILIC is a MODE of chromatography only.

Your column dimensions are 10mm x 150mm, so your column volume is est to be 8.25 mLs. To avoid overload on a typical silica column, 3% of measured column volume equals "max" inj volume. For your column, this would be ~ 247 ul (estimate). Note: The actual max must be determined experimentally using a "Loading study" as this calculation only provides an estimate.

"I currently have a 250uL syringe and 100uL loop connected to my autosampler" ?? You state you have an "Autosampler", but we will assume you mean Autoinjector in this context. Autoinjector's have fixed loops installed which are selected to be paired with a specific injector. If your autoinjector has a low-pressure glass syringe installed (e.g. Most Waters systems), then the loop should be 2 or 3 times smaller than the syringe volume to allow for over-filling the loop (which provides the most accurate volume when injecting 100ul 'loop' volumes). Accuracy is often poorest at the very low end of the injection range and best near the middle to high end. A 250ul syringe would be fine for use with a 100ul loop. *Note: High pressure injectors function differently (e.g. most Agilent systems) and often have Loop volumes that are 2 or 3x LARGER than the High Pressure injector's capacity.

*Installing a 2mL volume loop in a system with a 250 ul syringe (injector) makes no sense at all. You would greatly increase system dwell volume and also dilute any samples injected (greatly impacting reproducibility and introducing diffusion problems). Loop and Injector (a syringe in your example) must be carefully optimized together for best results.

In your example, overfill your 100 ul Loop with sample to obtain the most accurate injection volume (2 or 3x). However, if you are not concerned with accuracy, only sample loading for fraction collection, then you certainly could load the 100ul loop with only 100ul of sample each time to reduce sample waste (i.e. for collecting fractions).

[Multidimensional]

This free article on column loading estimates may be of interest to you.

"HPLC Injection Volume: What Should I Dilute It In and How Much Sample Can I Inject?"; https://hplctips.blogspot.com/2023/07/h ... uld-i.html

[EK8]

This free article on column loading estimates may be of interest to you.

"HPLC Injection Volume: What Should I Dilute It In and How Much Sample Can I Inject?"; https://hplctips.blogspot.com/2023/07/h ... uld-i.html

Thank you for the info.

Will it be OK to pair a 200uL loop with a 250uL syringe if accuracy in injection volume is not a concern? My only concern is avoiding dilution of my sample as the analyte of interest is present in small quantities and will make it difficult to recover after fractionation if diluted.

[Multidimensional]

Could you please share with us the EXACT make and model of your HPLC system with emphasis on WHICH AUTOSAMPLER (AUTOINJECTOR) you are using. There are many different types of autoinjectors and the answer to your question would require that we know EXACTLY which model of A/S (A/I) you are using. Changing these parts without a complete understanding of how they function in the design may lead to damage.

]n most, not all autoinjector designs, a 250 ul syringe would be used with a 250ul loop. If you installed a smaller loop (200 ul in your examplee) and someone used the HPLC to make an injection that was larger than the loop (i.e. 250ul), then they would be flushing part of the sample to waste and not know it (invalidating their method and data). Check the system to see what is recorded as the "max injection volume". Many are set to 100ul by default (and use larger loops to flush the sample out). This is why it so important to know which injector you have. Someone before you could have also incorrectly installed parts into the injector resulting in wrong volumes. You can review the manual on the injector YOU HAVE to find out which needle size, loop volume and syringe size are the default sizes AND which can be used for other situations. BTW: If the HPLC is computer controlled, this information also needs to be programmed into the instrument software parameters before use (so the injector can deliver the correct volume).

If your goal is to inject large volumes for the purpose of collecting fractions, then you really should not be using the autosampler (A/S). Instead, remove it from the flow path and install an inline MANUAL Injection valve. This will delete all of the extra volume found in the A/S and associated tubing. You could then install a 250ul loop (max size) and inject full loop volumes without the diffusion and wash loss. Of course, doing so assumes many things, which may not be true. For example, have you done a loading study to determine how much sample you can load onto your specific HPLC column using your specific method? You will need to concentrate the samples as much as possible before injection and get them into the smallest volume. Injecting the samples at different concentrations will then show you how much you can load onto the column and still maintain separation for analysis.

[EK8]

Could you please share with us the EXACT make and model of your HPLC system with emphasis on WHICH AUTOSAMPLER (AUTOINJECTOR) you are using. There are many different types of autoinjectors and the answer to your question would require that we know EXACTLY which model of A/S (A/I) you are using. Changing these parts without a complete understanding of how they function in the design may lead to damage.

]n most, not all autoinjector designs, a 250 ul syringe would be used with a 250ul loop. If you installed a smaller loop (200 ul in your examplee) and someone used the HPLC to make an injection that was larger than the loop (i.e. 250ul), then they would be flushing part of the sample to waste and not know it (invalidating their method and data). Check the system to see what is recorded as the "max injection volume". Many are set to 100ul by default (and use larger loops to flush the sample out). This is why it so important to know which injector you have. Someone before you could have also incorrectly installed parts into the injector resulting in wrong volumes. You can review the manual on the injector YOU HAVE to find out which needle size, loop volume and syringe size are the default sizes AND which can be used for other situations. BTW: If the HPLC is computer controlled, this information also needs to be programmed into the instrument software parameters before use (so the injector can deliver the correct volume).

If your goal is to inject large volumes for the purpose of collecting fractions, then you really should not be using the autosampler (A/S). Instead, remove it from the flow path and install an inline MANUAL Injection valve. This will delete all of the extra volume found in the A/S and associated tubing. You could then install a 250ul loop (max size) and inject full loop volumes without the diffusion and wash loss. Of course, doing so assumes many things, which may not be true. For example, have you done a loading study to determine how much sample you can load onto your specific HPLC column using your specific method? You will need to concentrate the samples as much as possible before injection and get them into the smallest volume. Injecting the samples at different concentrations will then show you how much you can load onto the column and still maintain separation for analysis.

I have a Waters 2690/2695 HPLC system. The default syringe is 250uL and default loop is 100uL. They also offer a 2500uL syringe and 2000uL loop.

[Hollow]

*some post havebeen made while I was writting. So some info may be repetion or allready answered*

I need to contradict Multidimensional in one point

Even the Waters system have low pressure glas syringes, they often still have a high pressure injection system under the hood, like on the Alliance 2695. So your combination of 250 ul loop and 100 ul may very well make sense, as it is the standard config on the analytical Alliance system. On them, you always want to have a smaller syringe than the loop to prevent your sample end up in the syringe.

So without knowing your system, we can only speculate.

but as Multidim. said, you may need to test how much you could load under your conditions.
One issue is mass overload, if your sample is highly concentrated, but often volume overload effects are seen earlier, that happens when your sample solvent has a higher elution strength. Then you may even inject much less than the estimated 200 ul.

Depending on your sample amount, you may statt injecting low and increase it 10-25% and so on. If you overload, then only oneinjection is "lost"; has to be recovered.

What is your sample dissolved in? If it's water then it probably won't work with your HILIC-(mode) column.


Probably best may be dry-loading "injection", where you adsorb your sample on silica powder (e.g 15 um, bare or C18) which you the fill into a spare column which you then may install prior the semi-prep column. A manual switch valve may help.

[EK8]

*some post havebeen made while I was writting. So some info may be repetion or allready answered*

I need to contradict Multidimensional in one point

Even the Waters system have low pressure glas syringes, they often still have a high pressure injection system under the hood, like on the Alliance 2695. So your combination of 250 ul loop and 100 ul may very well make sense, as it is the standard config on the analytical Alliance system. On them, you always want to have a smaller syringe than the loop to prevent your sample end up in the syringe.

So without knowing your system, we can only speculate.

but as Multidim. said, you may need to test how much you could load under your conditions.
One issue is mass overload, if your sample is highly concentrated, but often volume overload effects are seen earlier, that happens when your sample solvent has a higher elution strength. Then you may even inject much less than the estimated 200 ul.

Depending on your sample amount, you may statt injecting low and increase it 10-25% and so on. If you overload, then only oneinjection is "lost"; has to be recovered.

What is your sample dissolved in? If it's water then it probably won't work with your HILIC-(mode) column.


Probably best may be dry-loading "injection", where you adsorb your sample on silica powder (e.g 15 um, bare or C18) which you the fill into a spare column which you then may install prior the semi-prep column. A manual switch valve may help.

Thank you. I have an old WAter 2690 HPLC system. My sample is dissolved in acetonitrile. i played around a bit with load on cartridges with a hilic sorbent.

[EK8]

I spoke to a waters tech. They said I should have absolutely no problem using a 250uL syringe to inject 200uL into a 2mL loop. At most I can expect a 5% deviation in RSD.

Do you guys agree now with this process?

[Multidimensional]

No. Injection of 200 ul using a 2 mL or 2.5 mL loop means you are only using the lower end of the volume and diluting the sample a great deal (dilution equals diffusion of sample while loading). This is an example of very poor chromatography. If you have a 250 ul syringe, than you could inject 200 ul of sample into a 200 or 250ul loop. Why would you install a 2mL (or 2.5mL) loop ???

Correct Procedure to Follow: Concentrate your sample first. Inject the highest concentration in the smallest volume possible to load the sample at the head of the column. This reduces diffusion and band spreading. Use a loop that it optimized for the sample volume. These are basic fundamentals of chromatography. Please contact and work with someone who has actual HPLC experience on your project.

[EK8]

No. Injection of 200 ul using a 2 mL or 2.5 mL loop means you are only using the lower end of the volume and diluting the sample a great deal (dilution equals diffusion of sample while loading). This is an example of very poor chromatography. If you have a 250 ul syringe, than you could inject 200 ul of sample into a 200 or 250ul loop. Why would you install a 2mL (or 2.5mL) loop ???

Correct Procedure to Follow: Concentrate your sample first. Inject the highest concentration in the smallest volume possible to load the sample at the head of the column. This reduces diffusion and band spreading. Use a loop that it optimized for the sample volume. These are basic fundamentals of chromatography. Please contact and work with someone who has actual HPLC experience on your project.

I asked the Waters technician about the dilution issue and she said solvent or any other liquid will not be added to the loop that will cause dilution of my sample. I am not sure who to trust at this point.

The only issue she mentioned is that if I use a smaller loop (100uL) and inject 200uL, then it will flow back into the syringe.

even with the standard loop (100uL), Waters claims we can inject 0.1 to 100uL.
https://support.waters.com/KB_Inst/Chro ... -L_syringe

[Hollow]

The Alliance has what nowadays is called a flow through needle. Your sample will sit at the end of the needle and replaces the existing solvent in the needle (loop) in a reversed order. Then will reach the columnf first. Not too much of dilution should happen in the tubing. Of course it's not ideal but you're anyway in preparative mode, not analytical, quantitative one so. So you may be ok with somesuboptimal settings.

Btw: I guess you mixed something up in the prvious post. like in the beginning, the combinations are 100ul syringe+250ul needle-loop or 2 ml syringe + 2.5 ml neddle-loop?
I don't know if there are other combinations available and could be configured in system's setting.

Finally you have to try it out.
If the system can do it, your sample solvent is equal or less strong then the eluent and you're confident about the loading on the semi-prep column, then go on.

[Hollow]

I would trust the Waters tech. They (normally) know their systems...

Interesting WKB article.
I've never done larger injections than the syringe volume. I wasn't aware that this is possible and the system will just draw multiple times.
(yeah, just learned something new )

[Multidimensional]

"EK*: wrote: "Waters claims we can inject 0.1 to 100uL".
- That is not a claim. That is a fact. You can set the injector at those values.That is the Specification range only, It has nothing to do what we are discussing here. A volume Input value in the software is different than actual sample volume on the column. Depending on how the HPLC is setup (the tubing used, the loop size, the sample concentration, the column, the mobile phase, the injection volume and sampling rate etc) will be the determining factor in what the peak will look like (and your separation). For your injector, you can set any number into the software between 0.1 and 100 ul. *Most auto-injectors have a "useful: range which gives the most accuracy between 20 and 80ul (Or for manual injectors, by overfilling the loop 3 or more times for best results). Multiple stacked injections can also be used too. But you are not there yet. What we are concerned with is sample dilution and diffusion using loop sizes that are not optimized for the A/I. As noted, we are less concerned about accuracy in your case. More concerned that you seem fixated on the injector instead of using the stock setup and actually determining what the chromatography looks like using different sample concentrations. Start running some samples using your method to get the process started.

[EK8]

"EK*: wrote: "Waters claims we can inject 0.1 to 100uL".
- That is not a claim. That is a fact. You can set the injector at those values.That is the Specification range only, It has nothing to do what we are discussing here. A volume Input value in the software is different than actual sample volume on the column. Depending on how the HPLC is setup (the tubing used, the loop size, the sample concentration, the column, the mobile phase, the injection volume and sampling rate etc) will be the determining factor in what the peak will look like (and your separation). For your injector, you can set any number into the software between 0.1 and 100 ul. *Most auto-injectors have a "useful: range which gives the most accuracy between 20 and 80ul (Or for manual injectors, by overfilling the loop 3 or more times for best results). Multiple stacked injections can also be used too. But you are not there yet. What we are concerned with is sample dilution and diffusion using loop sizes that are not optimized for the A/I. As noted, we are less concerned about accuracy in your case. More concerned that you seem fixated on the injector instead of using the stock setup and actually determining what the chromatography looks like using different sample concentrations. Start running some samples using your method to get the process started.

The problem is I called Waters and the technician said the sample will not be diluted in the loop, even when using a low injection volume in a large loop. How this works, I don't know, but it is contradictory to what you are saying. I need to find out who is being factual.

To add - why should I care if the sample is diluted with mobile phase in the loop, assuming when I collect fractions for bioactivity assessment, I reduce each collected fraction to the original volume of my injected sample?