Hey Labrats! Long time lurker, first time poster here. I have a unique HPLC problem that I can't seem to solve. I'm looking at you, HPLC pros (or specifically Agilent pros)... Okay, let's get on with it.
I am using an Agilent 1260 Infinity (version I) for HPLC. The setup includes quat pump, standard auto-sampler, sample/column heating block, and DAD. It's my first time using this machine, or doing HPLC for that matter, and it has been a struggle.
I got the machine running, wiped the dust out, made buffers, installed a new column, and everything seems to me to be working great, but something is not quite right. My gradients don't seem to align with my injection. So I started digging into what could be wrong.
It seems that my main problem is dwell volume, but I don't think it can be improved. I've looked through manuals for all of the pieces and the entire unit and found that the dwell volume we are seeing (~1.5mL) is pretty standard for the setup we have.
My second thought was to add a delay to the auto-sampler. This seems like a great idea, but it's not a simple input to control the injection. I would LOVE to find a manual on how to program the auto-sampler, but it seemingly doesn't exist. I even called Agilent and they were at a loss.
If anyone has experiencing programming this specific auto-sampler, or knows some secret way to reduce dwell, please let me know! Otherwise, this outlet was cathartic and I'll take an upvote.
TLDR; Frustrated research tech asks for help manually programming the autosampler on Agilent 1260 Infinity.
I was wondering what fields would have entry level positions where I could become accostomed to using HPLC and GCMS? Many places I have worked in the past don't like new hires touching those machines (quite reasonable due to their cost) and I find myself in a situation where I need experience to get the jobs out there that involve chromatography, but I need to work those jobs to get experience. I find the whole process and idea behind this kind of machinery fascinating and think getting a good amount of experience could open doors in the future. What kind of entry level job could get me some experience? Do most places with QA/QC teams in food and pharma have them?
Does anyone have any experience with HPLC? I'm wondering how long frozen serum samples (never thawed) can be stored before running HPLC to gain reliable results - they're about three years old *cringe*. We're interested in looking at vitamin D levels.
I know sources are not allowed here, but I don't think it pertains to testing cause technically that's NOT a source and it's for harm reduction purposes, so I think it should be OK.
I want to get a purity (like HPLC test) and heavy metals test for Emylcamate. I already have NMR, but this doesn't tell the purity. This research material is used in large quantities, so I think it's important.
Is there any company that will test something like this in the US? Maybe a cannabis company would be more chill haha? Or could I just send it to a normal lab and not have any problems?
What about in-lab chromatography tests? Can anyone explain how that's done or if they've tried it before? I want to be clear, I'm not just looking for a test that determines if it's the correct chemical - I know that - I want the purity.
Hey, this is a weird one but I find the sound of a couple of HPLCs running (valve clicks and the pump noise) really soothing and wondered if anyone had a recording of it since my lab is a no phone zone... Like f*ck your '8 hour rain sounds for sleep' I wanna drift off listening to a 45 injection run.
I'm looking to create a cocktail of fat-soluble vitamins to run on our RP-HPLC for students to have a look at as a standard to then compare isolated samples against.
I've never prepared standards before but will be looking to order the following from Sigma
- Retinol (Vita A)
- Ergocalciferol (Vita D)
- Alpha tocopherol (Vita E)
- Phytonadione (Vita K)
As most of these are fat-soluble i was hoping to use something like Acetonitrile and water as the mobile phase.
Does anyone have suggestions on how to prep standards from a powder? Would I mix them in an aliquot of the mobile phase?
Any other thoughts greatly appreciated!
Hello everyone,
Im working with a shimadzu lc2030c plus. A few days ago, i installed new stainless steel tubing from the HPV to the column inlet. Ever since, I have had this PERSISTENT noise that occurs between 18 to 25 minutes. I'm reluctant to remove the tubing because the end that is screwed into the HPV is really freakin stuck in there (mainly because I used a hybrid ferrule and not a stainless steel ferrule)
Anyway, do yall think the stainless steel tubing is a red herring? Otherwise it has to be the culprit as it is the only thing that has changed.
I've excluded my column as the culprit
flushed the system by replacing the column with a union using a number of different solvents
i let the system flush overnight at a low flow rate
I dont see a bubble in the flow cell
I'm thinking I've largely ruled out contamination as possible source of noise.
Any thoughts?
Found a drawer of old used HPLC columns we used. We follow strict SOPs, but I think these are good for several more uses. Not sure what to do with them. Donate to a school maybe?
I’m in the process of developing a normal phase method for quantitation of vitamin A as trans-retinyl palmitate. My parameters are 100% hexane mobile phase on a phenomenex Luna NH2 column (150x4.6mm), 25C, 1 mL/min, 10 uL injection, detection at 325nm. Samples and references are extracted in hexane.
The issues I’m experiencing are:
- poor separation between the isomers at approx 3 min. My expected RTs are 2.7 and 3.0 mins for cis and trans, respectively, but the cis peak tends to elute slightly later than expected, affecting the resolution.
- slight tailing on the trans peak is failing my efficiency parameters (Tailing <= 2.0; experimental=2.1) (EDIT: TAILING HAS IMPROVED TO 1.8 AFTER RECONDITIONING COLUMN, STILL EXPERIENCING MERGING)
Does anyone have experience with normal phase separation of fat soluble vitamins such as this? I’ve attempted using IPA to rinse the column before hexane is added, which improves the peak shape, but still leaves me this these issues. Thanks in advance for your help!
Hey guys so I'm applying to a role where they will ask me for troubleshooting questions when it comes to HPLC/UPLC. I would like to hear your examples of an issue you had with your run/system and what were your steps to correct that issue. Thank you!
I’m troubleshooting some HPLC weirdness, so I decided to switch from acetonitrile/water mobile phase to methanol/water. One of the problems I’m having is solvent mixing between pumps, so I pre-mixed degassed water with methanol (we don’t use an in-line degasser for this setup, just vacuum degas.) everything looked okay at first, but after about 15 minutes, I noticed a ton of bubbles forming in the solvent bottle. Luckily I caught it before they got sucked into the pump, but does anyone know what happened/how to prevent it? Should I be degassing my methanol as well? Thanks!
HPLC Operator
MH Global LLC
Garden Grove, CA 92841
Employer actively reviewed candidates 3 days ago
Job details
Salary
From $17 an hour (... to be fair, the wage is from $17... Minimum wage is: $13.00/hour to $14.00/hour depending if 26 employees or more)
Job Type
Full-time
Number of hires for this role
1
Qualifications
- Doctorate (Preferred)
- Laboratory Experience: 2 years (Preferred)
Full Job Description
Job Overview
We are looking for a dedicated Chemistry Lab Assistant to be responsible for conducting projects. The responsibilities include preparing test solutions, calibrating and troubleshooting the machines, and collaborating on quality tests.
To be a successful Lab Operator, you should be analytical, detail-oriented, and logical. You should be dedicated to safety issues and be able to work with hazardous chemicals.
Responsibilities and Duties
- Perform laboratory tests according to written procedures.
- Provides routine care and maintenance for instruments such as HPLC. This includes cleaning glassware and equipment.
- Maintains a clean laboratory environment.
- Identify results that require further investigation or disc
What is HPLC?
HPLC is short for “High-performance liquid chromatography”. This is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. HPLC is used in manufacturing, research, and medical fields [1]. In regards to kava, HPLC is used to separate and quantify the different kavalactones in a sample [2].
How does HPLC work?
Simple Graphical Depiction of HPLC System
In HPLC a pump forces a solvent mixed with the testing sample through a metal cylinder that is packed full of adsorbent material called a “column”. Adsorption is the adhesion of atoms, ions or molecules from a gas, liquid, or dissolved state to a surface [3]. At the outgoing end of the metal tube or “column” there will be placed a detector which computes sample quantities and specific molecules [4]. This process is highly automated and very sensitive.
Retention Time.
Retention time is a measure of time that it takes for the sample and solvent to enter from one side of the column to reach the detector at the opposite end of the column. Each compound will have differing retention times based on its composition [5].
Kava Chemotype.
Chemotype is responsible for the quality of kava’s physiological effect. When we refer to chemotypes we express them in 6 digit strings. For example the variety “Kelai” from Vanuatu is seen as having a 423156 chemotype. Each one of the digits corresponds to a different kavalactone in a descending ratio. What this means is that numbers towards the front of the 6 digit string will be higher in quantity than those at the end. A note to keep in mind is that this only represents their relative proportion to each other, and the numbers can vary from each other by only fractions of a percent [6].
How the different kavalactones got their numbers.
It’s been said that these numbers were given arbitrarily by researchers, however this is certainly not the case. Each kavalactone was assigned it’s number based on the increasing time it took for i
... keep reading on reddit ➡Hello everyone. I have a question I'd appreciate some help. My HPLC peaks haven't perfectly separated. That's not ideal obviously but I followed the protocols and don't have the opportunity to repeat the tests again. I had a good R^(2) though, this mean that my measure of analyte is out? If it is, How can I identify how much it is out theoretically? What would you do and what is the theory on this?
Hey LabRats! I am getting ready to run HPLC on an alkaloid extraction from plant tissue. I'm aware that acetonitrile is the go-to mobile phase, but is 0.1% formic acid also needed? The videos I watched all had formic acid in the mobile phase buffers, but they were also on MS-coupled HPLCs whereas ours is not. Is formic acid necessary for HPLC only?
I have convinced the management to upgrade the lab I work in and they are letting me replace the Flexar HPLC they have had since like 2012 (It's been out of service since 2017). I'm thinking of convincing the management to purchase an Agilent1260 Infinity II LC System and I'm trying to broker a discount for trading in our Flexar to recycle it. Anyone have any experience with this system or recommendations on any other HPLC systems? I have minimal experience with HPLC right now, but I am trying to take advantage of this opportunity because it will be a large improvement for our lab and I would like experience with HPLC. I am in cosmetics and the management has no idea so I'm on my own here. I think we could utilize the HPLC for our cosmetic products that contain actives such as Zinc Pyrithione and relaxer cremes which would make our stability testing significantly faster and safer. The company I work for is run horribly so I'm curious to see how this will pan out, I will probably be the only person using it once we get it. The management is very cheap too so I'm worried about that, but they told me I can replace the HPLC, once I have a quote they may want me to find a cheaper one. I don't want the new HPLC to become unused like the Flexar either, so I will definitely take initiative in qualifying it and using, I'm just curious what advice people who already use HPLC have on this endeavor. I have been doing some of my research, but I'm curious what actual HPLC users have to say. My goal is to try to have the system installed before September.
I think a lot of us can relate to having to do things so far outside of our job description; but when equipment goes down and your PhD progress relies on it... you don't have much choice.
So this isn't anything groundbreaking, but I cannot express the relief that comes with finally getting equipment back up and running. Our computer that runs the HPLC system (using Empower 3) died. Its essential for our work so I've had a 2 week research hiatus trying to get it all working again. Its taken buying a new computer, dealing with software license issues, downgrading the operating system and installing drivers, configuring networks, and reading through technical manuals but it's finally back up and communicating with all of the equipment properly. I see why Waters charges 300 USD/hr for software support, because these things are so difficult to make sense of.
I'm currently running HPLC on an Agilent 1260 Infinity, and I just started having an issue where the autosampler arm won't drop the sample vial after an injection. It begins to drop the vial and then immediately lifts it back up, still holding the vial, resulting in an error. I checked to make sure there was no debris in the sample slot and it's completely clear. We replaced the needle seat just before experiencing this issue, but I don't see how it could be related. Any suggestions on what the problem might be would be greatly appreciated.
EDIT: Resolved. Thank you for the help.
The information that I’ve found regarding rt for reverse-phase HPLC seems a little confusing to me. I was reading what I thought was a great section on LibreTexts, but the section on stationary phase polarity seemed to contradict itself. A lot of the info I’ve found online has only deepened my confusion. So I’m looking for as straightforward an answer as I can get.
In reverse-phase HPLC (nonpolar stationary phase), would increasing the polarity of the mobile phase cause rt to increase or decrease?
Specifically, I’m wondering about the biodegradation of TNT and DNT under anaerobic conditions. It results in the creation of hydroxylamino intermediates that are more polar than their parents. Would the rt of these intermediates be longer or shorter than that of their parents?
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Does anyone have any experience with HPLC? I'm wondering how long frozen serum samples (never thawed) can be stored before running HPLC to gain reliable results - they're about three years old *cringe*. We're interested in looking at vitamin D levels.
Does anyone have any experience with HPLC? I'm wondering how long frozen serum samples (never thawed) can be stored before running HPLC to gain reliable results - they're about three years old *cringe*. We're interested in looking at vitamin D levels.
