Agilent needle wash port behavior?

[per_oxid]

I am new user on agilent 1290 system with dad detector. I had a problem with my coffeine system test. My peak was eluting in the void volume when i use the needle wash port that should wash needle outside after sample draw and before inject. I used 100%methanol for wash since it should not enter column. Gradient was 10%meoh to 99,% meoh. When i dont use needle wash method works fine RT of 5min uhplc c18.

To my understanding from the manual the flush port wash only wash outside of needle in the wash port position before the injection, but it appears that the a significant amount of meoh wash solvent gets into syringe causing my analyte to elute in the front. The wash solvent system use its own pump and is fully separated from lc gradient flow.

Could someone used to these system explain to me how this can happen? There are no options for the needle wash more than on/off and vial wash/wash port wash so cant see that there is an error in the method.

Of course i could just use a weaker wash solvent but still would like to understand why this happens. Its not just fronting peak is really in the void volume with no retention so amount of meoh must be large.

[JI2002]

What’s the injection volume?

[per_oxid]

What’s the injection volume?

Its 2ul

Manual says needle wash is usually 50-100% meoh. My gradient starts at 15% 0.5ml/min so wash is a lot stronger. I will test a weaker wash. I thought it would not go into column and would like to know why it apparently does.

[Multidimensional]

A few comments:
Agilent offers many types of 1290-series A/S modules. Sure would help us if you provided the exact model of A/S you are using and HOW you have programmed it.
The "needle wash" feature is part of the available programming set for the injector and it will do exactly what you tell it to do. But why are you using it? Is your sample super sticky and sticking to the stainless steel?
In 99.9% of the cases NO NEEDLE WASH would ever be needed for most method types, none. *This is due to the flow through design of the Agilent autoinjector which uses a high-pressure pump, not a glass syringe. The flow path is always washed clean (continuously washed clean with mobile phase, unless you program the system to by-pass the injector flow path during a run, which is not the default) so a "needle wash" is only useful if you need to rinse the outside of the needle for std samples.
If you really did have wash solution going into the column, then that implies that the entire autoinjector flow path is continuously contaminated. The most likely reason for this rare failure would be a worn out autoinjector PUMP seal or worn out needle seat seal.

[per_oxid]

A few comments:
Agilent offers many types of 1290-series A/S modules. Sure would help us if you provided the exact model of A/S you are using and HOW you have programmed it.
The "needle wash" feature is part of the available programming set for the injector and it will do exactly what you tell it to do. But why are you using it? Is your sample super sticky and sticking to the stainless steel?
In 99.9% of the cases NO NEEDLE WASH would ever be needed for most method types, none. *This is due to the flow through design of the autoinjector which uses a high-pressure pump, not a syringe. The flow path is always washed clean so a "needle wash" is only useful if you need to rinse the outside of the needle for std samples.
If you really did have wash solution going into the column, then that implies that the entire autoinjector flow path is continuously contaminated. The most likely reason for this rare failure would be a worn out autoinjector PUMP seal or worn out needle seat seal.

Thank you for the advice! I am new to this system so I appreciate the advice. I am using external needle wash since the manual recommends that it should be used to prevent contamination of the needle seat. This sounded like good advice since it seems reasonable that the external of the needle could contaminate injector port . I am used to waters and cleaning the outside of the needle is standard practice. I was asking for advice from more experienced users if there is any disadvantage of using the external needle wash. Agilent recommends that it is used but when expert user say it is not necessary most of the time I will listen . So what is the disadvantage of using needle external wash besides the time it takes to inject is increased?

I have now changed to 20%meoh as needle wash and then i dont have any clear problem , peak shape is perfect.

[Multidimensional]

Waters users often make this mistake (due to lack of professional training on how to use the new, different HPLC system). Agilent's autoinjector design is far superior to any of the designs used by Waters. It is important to note that each manufacturer has their own way of doing things and has variations in their flow paths (so never assume that all HPLC systems are the same). SInce the late 1980's, Agilent has used a much more expensive autoinjector design than other manufacturers. A high pressure pump is used as the "syringe" (in fact, it is the same pump as used in the PUMP, but with only one chamber). When a low pressure syringe is used to inject sample through an autoinjector, residual sample is often left inside the syringe and tubing that connect it to the flow path (unavoidable, per design). Low pressure injectors require a range of tricks to attempt to flush out this residual liquid and material, some with poor results. The low pressure design means it must always be isolated from the high pressure flow path. A low pressure syringe (glass) design is used in many HPLC system autoinjectors because it costs less to build. To truly flush the glass syringe of sample requires that the plunger be removed from the syringe to backflush solution through the syringe and connected tubing (some A/I actually do this. The HP 1090 HPLC has this rare feature, but this requires a lot of extra parts, valves and money). If you do not do this, residual liquid is left in the syringe, and optionally the needle if it does not get placed back in the flow path during the run (Many Waters system place the needle back in the flow path and refer to their systems as having a clean flow path, but this is obviously not true. The syringe and connecting tubing are left contaminated on the low pressure side). In all of these low-pressure auto-injector designs, the NEEDLE WASH feature must be used to reduce contamination of the entire HPLC system. Since there is no way to rinse or clean the low pressure syringe, the needle wash must be programmed to have the syringe draw up and then expel solution, often many times, to manually flush the syringe and tubing of sample material and solution. If you do not do this after each injection, then the flow path may be contaminated. Most HPLC systems work this way, but this is not the case with the Agilent series of Auto-injectors.

Incorporating a true high pressure HPLC injector system into the fow path means it must withstand the same pressure as the HPLC flow path, so costs far more money. However, a high-pressure injector allows it to be positioned directly into the flow path from the pump to the column allowing the entire flow path to be continuously flushed with running mobile phase (cleaning the flow path so no needle or separate syringe cleaning is needed). Carryover is super rare with this true high-pressure design as by design, contamination can not come from the needle or syringe. The only time we ever use a "needle wash" on an Agilent HPLC system is when the sample is super sticky (e.g. some proteins) and a concern exists where the sample may stick to the outside of the needle, then be deposited onto the needle seat (where it might enter the flow path during the next injection cycle). In that case, the needle can be programmed to do a "dip" in a separate vial filled with a wash solution (no cap on vial) to remove any residue.

"Needle Wash" systems, and the users who are not trained in how and when to properly use them accounts for many HPLC analysis problems (which are training problems, not the fault of the instrument unless the untrained user has also forgotten to perform the needed maintenance and service on the injector too). In your case, please do not use Needle Wash, esp for your sample type. It just introduces more possible errors.

[Multidimensional]

"Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems"; https://hplctips.blogspot.com/2015/02/c ... on-in.html

[per_oxid]

"Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems"; https://hplctips.blogspot.com/2015/02/c ... on-in.html

Thank you multidimensional! I am new to this system so my questions here are my attempt to become proficient and trained. If you have time please explain the problems that using the needle wash can cause. Agilent manual recommends to use his function so it would be helpful for me to understand why it should not be used.

[Multidimensional]

"If you have time please explain the problems that using the needle wash can cause."

- I already did. Did you read the article? Contamination, peak shape problems, poor reproducibility, erroneous peak contamination etc. All from using needle wash incorrectly. Once you understand how it works, the problems are many. We never use any injector needle wash mode unless we have to.
The inclusion and suggestion to use needle wash often in some of the manuals (Agilent manuals) is a byproduct of poor training of their authors. Mostly the fact that they do not understand how it works. So many Waters, Shimadzu, Dionex/Thermo users are unfamiliar with it, but use it to feel comfortable using a familiar feature so they can transfer methods without making changes. IOW: IGNORANCE drives its use. Training and understanding make it clear that it should not be used most of the time.

Please keep in mind that it takes a LONG TIME to learn the basics of HPLC operation and analysis. LONG TIME. You can not learn HPLC from any class or website. You can not learn it from these web forums (with so many novices who pretend to know something, but do not). For most scientists, five years of full-time training in an industrial lab with mentoring allows them to achieve a "BASIC" level of skill. Many more years of training are needed to reach an intermediate level, and only when training continues (as most people just keep making the same mistakes, over-and-over, without learning). Employers are partly to blame for this as most do not invest in their employees. They do not pay to have experts brought in regularly to provide on-site, hands-on training to help them learn about what they are and are not doing right (proper training is needed to learn new skills).

[per_oxid]

Thank you for sharing the knowledge and I have read many of you articles. Yes you stated the problems but did not describe the mechanism how flushing of the outside of the needle cause these problems. Sorry I was not clear in my question. Does wash solvent go into the needle for example and why, diffusion?

But I understand that generally I dont need to use external wash on agilent infinity injectors, thanks!
All injectors i have used before have had external needle wash CTC PAL and waters to name two.

Found interesting article that evaluated this problem of wash solvent on needle outside gets injected.
https://www.waters.com/nextgen/us/en/li ... R135122508

[JI2002]

Nice article explaining the effect of the wash solvent on the HPLC method performance. I believe solvent wash mechanism for Agilent 1290 is the same as System x mentioned in the article. So that explains the issue you had.

I asked the injection volume because the smaller the injection volume, the larger effect of the wash solvent. If the injection volume of your method is higher, e.g., 10 ul instead of 2 ul, the effect is expected to be much smaller. You can also use a weaker solvent as you suggested.

[per_oxid]

Nice article explaining the effect of the wash solvent on the HPLC method performance. I believe solvent wash mechanism for Agilent 1290 is the same as System x mentioned in the article. So that explains the issue you had.

I asked the injection volume because the smaller the injection volume, the larger effect of the wash solvent. If the injection volume of your method is higher, e.g., 10 ul instead of 2 ul, the effect is expected to be much smaller. You can also use a weaker solvent as you suggested.

When you think about it it is pretty obvious that the film of wash fluid will accumulate at the tipp by the gravity force when needle moves to inject port. For uhplc columns the volume is large enough to make a difference if to strong wash is used. Not a big limitation but shows that one needs to understand different designs. I think recommended wash solvent could have been better in manual but now i know.

Indeed the agilent system I have use the system x wash design.

[Multidimensional]

Pure sales misinformation. Are you aware of the fact that the 'Waters' Alliance article you read is just a SALES article, biased, designed to ignore following good chromatographic fundamentals? These white papers are produced by all the vendors and the goal for each is the same.

• To make "their" system appear better than the "other" system (They are not going to provide an article that shows how inferior their system is).

The article makes no attempt to show why the difference in results occurred between the systems (they leave out vital info and operate the other system incorrectly). No details of the flow path or design differences between the systems is mentioned. Instead, it jumps to hide a known weakness in the Waters system and operates a different system as if it too was a Waters type system. In fact, this commonly happens when users only have experience with Waters brand samplers, as they are unaware of the design differences which may be found in other models. Not taking the time to receive professional training in how to use these DIFFERENT systems may result in the same errors and same wrong conclusions found in the article. Since the other system is NOT a Waters type system, but instead uses a different injector design, it was not optimized or even operated correctly.

See how clever the sales people are to turn a weakness in their design and make it appear as an advantage? They know that 99% of chromatographers have no idea how their auto-injectors operate or what the differences are in their flow paths. Information, which is vital to performing scientifically correct comparisons. This is called 'Marketing' and is directed to fool you into accepting the explanation offered. For those with an understanding of how the two different systems operate, the error they made operating "System x' is obvious [Incorrect use of the wash system].

The article demonstrates how choosing to ignore learning about how to use an auto-injector (A/I) properly may result in invalid experiments and false conclusions. A valid comparison would require that we know the details of the second system too. They intentionally operate the "other" system in a manner that is incorrect (using a wash vial), implying it is inferior. If the "System X" was an Agilent A/I, then no needle wash would be needed, so no problem would be observed. Making the process more complex to sokve a problem that does not exist is not the solution. Additionally, in the very rare case where a Agilent needle wash (or a 'dip' in this case) is desirable, we use a vial that still has a loosely fitted cap with septa fitted for the wash (vs. a cap-less vial, which are also used). Using a capped wash vial allows the needle to wick off any liquid as it is withdrawn. Waters did this with the Alliance system they used, but not with "System X".

Please be more skeptical when reading in-house articles whose main purpose is to discredit competitors and make their product(s) appear superior. BTW: I do not, nor have I even worked for Waters or Agilent. My interest is to encourage people to use critical thinking and seek out professional knowledge before drawing conclusions.

[per_oxid]

You are kicking in open doors. I never criticized or passed judgement on the different brands. I agree with your comment that knowing how a system works is key for good results and systems are different. An article posted by waters is of course going to be biased but it can still be informative. I had a question and I feel that I have found an answer and solution, thanks for the input! I never blamed the design of the system I just wanted to understand it inorder to use it correctly.

[Peter Apps]

Pure sales misinformation. ..............

Please be more skeptical when reading in-house articles whose main purpose is to discredit competitors and make their product(s) appear superior. BTW: I do not, nor have I even worked for Waters or Agilent. My interest is to encourage people to use critical thinking and seek out professional knowledge before drawing conclusions.

Which company do you work for ?