Chromatography Forum

Hey,

Can someone answer me to few simple (for experienced HPLC Agilent users) questions consider HPLC maintenance because I'm confused with Agilent manual. And I want to do exactly perfect, proper way
1) How to properly prime degasser in pump mode? I mean I read in manual of Agilent Micro Vacuum Degasser 1100 that I should flush degasser with at least 5 ml of water however in Agilent HPLC Maintenance and Troubleshooting I read that I should use isopropanol and in Agilent Pump Manual they recommend flush with 30 ml of, again, water. Some different, other source recommend use just solvent. What is the best? There is some difference in flow rate in flushing degasser and priming the pump?
2) When I use phosphate buffer (0.03 M) how should I clean all capillaries in HPLC system? Again, once they recommend to pump water or isopropanol? If isopropanol what is the ratio for isopropanol:water solution. What i s better? And what is the time period for that? Like 2-3 hours? Or overnight?
3) What is the best way to clean column? If I used for mobile phase ACN:Water should I use only Acetonitrile?
4) How often do you change purge valve frit?

Thank you in advance

• Please consider taking a professional on-site HPLC maintenance class that is specific to your exact model of HPLC system. This is the fastest and best way to learn the correct maintenance procedures for YOUR HPLC system. Consider taking some hands-on formal classes to learn the fundamentals of HPLC as they apply to YOUR HPLC instrument and software to. It takes most users many years just to learn the basics and taking hands-on professional classes can help you understand and apply these concepts more efficiently while you are learning. Please have a more experienced chromatographer help you.

(1) The correct degasser priming/flushing process depends on the exact model of HPLC degasser you have (which you did not provide). When flushing or priming ANY HPLC vacuum degasser (you can use the pump or a syringe), you must first know what the internal channel volume is for that degasser model (plus the tubing volume). Example: For the G1322A, channel volume is between 10 and 13 mLs/channel, each. For the G1379A/B it is 1 mL/channel. To flush ONE channel you need to rinse through it enough liquid to fill and flush it 2 or 3 times, plus the volume of low pressure tubing. So for a G1322A, flush about 30 mLs of liquid through each of the 4 channels when you fill it with mobile phase, change mobile phase bottles or begin to use it after a weekend. Flush about 10-12 mLs through each channel of the G1379A/B series. Please do this for ALL 4 channels, even if you do not plan on using them all. All channels must be filled with liquid at all times. Do not plug an unused channel, the liquid will quickly evaporate inside. Air inside a channel will stress the vacuum system (leading to early failure). Connect unused channels in-series with other channels to keep the 'wet'. Never leave water or buffer sitting in a channel. Critters will grow in it and contaminate your flow path.

(2) Buffers: Any buffers used with your HPLC system (that includes phosphate buffer) should be routinely flushed from the system with high purity water (not IPA). Two types of flushes are used. The column should be flushed with an appropriate solution after use each day (do not store the column in buffer or pure water). The rest of the HPLC flow path must be flushed down to remove buffer. The idea is to flush out the buffer so salt crystals do not form. For RP, start by flushing with pure water, then switch to water mixed with some organic (e.g. MeOH/Water 60/40) to stop corrosion and prevent bacterial, algae or mold growth.

(3) Column cleaning. Basically, first, refer to the column manufacturer's recommendations about what can and what can NOT be used to flush through the column (also, any pressure, pH or temp limits). Next, you flush the column with whatever solution is safe to dissolve one hundred percent of whatever is dissolved or fouling the support. You want to flush any retained material off, w/o damaging the support. The best solution to do this is going to be different for each application and person. IGNORE any suggestions by others of using some 'universal' flushing solution. Any universal washing solution would not address your own applications or use. We have a scientific way of doing things and it starts with information first. What types of samples were run through the column? What solvent is stronger than my mobile phase and will dissolve them 100% to remove them (w/o damage)? Now you have the answer.

(4) The PTFE frit should be changed EVERY MONTH. It is a $5 filter (cheap) and designed to catch normal piston seal debris plus any large particles that might sneak into the system. Normally, it is snow white in color. It is cheap insurance to protect the flow path. Don't forget to change those bottle frits too.

If you are in the USA, please consider my employers professional Agilent specific HPLC training classes. They have taught more than 10K scientists expert techniques and methods to use and service these system over almost 30 years. More info here: http://www.chiralizer.com/hplc-products ... asses.html

Here is an article that may also be of interest to you or anyone interested in performing maintenance on their HPLC system.

Intro:

Periodic "General Maintenance" of the HPLC is one type of service procedure which should be scheduled at a set frequency (Example: Every 6 months) and will serve to provide a time to clean, inspect and repair/replace any parts which are worn due to normal use. Such routine HPLC maintenance is often referred to as a basic "Preventative Maintenance" service (or "PM Service"). Spare parts common to your HPLC system(s) should be on hand to perform these scheduled maintenance procedures as part of a normal PM service.

"HPLC Maintenance & Repair Parts To Have on Hand for HPLC Systems"; https://hplctips.blogspot.com/2013/11/m ... d-for.html

When pumping aqueous out the purge valve at 5ml/min, if the pressure was 3 psi or more, we replaced the purge valve frit. We never replaced them on a timed schedule.

Agilent information then stated the limit was 5 psi.

Thanks for all answers.

But I want to ask something else. So basically I just should set the flow (what is the rate?) Open purge valve and flush degasser. What about the pump? It is connected together but this is the same procedure? I just add that degasser it is G1322A pump G1312A. After I close purge valve should I set up some flow and flush column? I do not a lot of sample, few a weeks and these my next questions - should I power off Agilent devices?

The reason we replace the PTFE Frit every month and not when the backpressure with the PPV open exceeds a specific pressure is that using backpressure alone only checks for plugging/fouling. The secondary concern we have is of contamination over time. The frit is specifically designed to collect many types of contaminated (algae and bacteria "soups" are common types) and reduce the amount of them that enter the flow path. When we perform our monthly inspection of the frit (to replace it). we are provided with an easy and quick opportunity to look at the frit for evidence of contamination. We have taught this task/job as part of the comprehensive service used on all HP/Agilent HPLC systems that utilize the PTFE frit and recommend monthly exchange.

Thanks for all answers.

But I want to ask something else. So basically I just should set the flow (what is the rate?) Open purge valve and flush degasser. What about the pump? It is connected together but this is the same procedure? I just add that degasser it is G1322A pump G1312A. After I close purge valve should I set up some flow and flush column? I do not a lot of sample, few a weeks and these my next questions - should I power off Agilent devices?

"Paul_1002": Based on your questions, it does appear you have no formal HPLC training and may be using an HPLC system by yourself, without any experienced chromatographer on-site to help you (Are you at a school? Please ask your teacher for help). We can not teach you liquid chromatography plus operation of your HPLC system through a web forum like this. This is unrealistic as you may not appreciate what you are asking at this point. Your questions are far too basic to address properly using this format. You require professional training (which will take years to acquire just the basics in). An HPLC system is not like an analytical balance or pH meter where you can be shown a few things, read the manual and start using the tool. HPLC is completely different. There is NO OPERATOR's MANUAL that will tell you how to use the HPLC for your applications, none. The instruments are designed to be used by people who already have the formal training and just need to learn the brand specifics which apply to get started. The instrument, the HPLC system, is just a "dumb" tool by itself. The human is responsible for providing the intelligence and knowledge in how to use the tool. To use it to answer a question requires a great deal of knowledge and training in the fundamentals of chromatography first. Additionally, you will then need training that is specific to your instrument and modules for your applications (probably an HP/Agilent 1100-series system). Did I mention that this takes years? Please look into reading the classic texts on the topic to learn the fundamentals (then you will understand what flow rate to use to prime, flush and run a method) first. Formal training classes will help too (hands-on only!).

Yes I'm still im school I do not operated HPLC by myself
I thought I can ask someone on the forum if I have any doubts to do not bother people on my campus to pass exam but maybe that forum is not for students.
Anyway thanks

You would not be "bothering" the employees on campus at your school. They are paid to try and guide you. No one would expect a student to know how to do these things w/o years of experience. They are there to suggest books to read, classes to take and monitor you. Someone local to you is in the best position to help a new user, esp for classwork.