Good day, I’m planning to start a new project which involves recovering samples from Franz diffusion cells after permeation from pig’s skin. The amount of my sample at each sampling time is just 20uL, which may be difficult to filter before the HPLC analysis. Someone advised that since I’m using column guard I can skip the filtration step and directly place my samples in the autosampler after its recovery.
I will be using Shimadzu HPLC system (picture attached)
Is it recommended to do that? Pls advice
Not really. Even a 20 uL injection can 'foul' an HPLC and lead to crippling backpressure. How many injections were you thinking of making? Have to thought of high speed centrifugation or even diluting the sample (1/10) in filtered mobile phase and increasing the injection volume?
I recommend filtration before injection as sample without filtration may damage the hplc column.
As with many things in Life, some people will provide you with bad advice. You were wise to ask here.
- Proper chromatography technique suggests that you never inject anything into an expensive HPLC system which is likely to damage, foul or plug it. All samples should be fully dissolved in the mobile phase and filtered before injection. Filtering exceptions may include highly soluble, pure compounds.
The HPLC guard column is not there to perform sample prep for you. Best to always properly dissolve and filter the samples before placing in to vials for injection. Respect the column and give it the best chance to retain, resolve and elute your samples by always performing any needed sample prep before use. BTW: Guard columns are often a waste of money and not needed for most types of LC separations. However, very dirty samples may have components which may precipitate out of solution right after injection and a guard column acts as a sacrificial column, saving your main column. In such cases the guard column allows the main column to last a bit longer.
Not really. Even a 20 uL injection can 'foul' an HPLC and lead to crippling backpressure. How many injections were you thinking of making? Have to thought of high speed centrifugation or even diluting the sample (1/10) in filtered mobile phase and increasing the injection volume?
I understand that you do not want to filter the sample because you have only 20 uL. The fix is to put an in-line filter between the column and the guard column. You can order from many vendors such as Thermo, Phenomenex, Upchurch. They will filter particulates and if it gets clogged, youbjust replace it as needed. It is cheaper than a guard column.
Hi,
Thank you for asking. What is the expected concentration of the sample to be analyzed? If possible you can dissolve first the sample and filter the obtained solution prior to injection. It is virtually not possible to inject your sample when you have only 20 ul. As a consequence, a dilution of your sample to a fixed volume is mandatory.
Best of luck,
JPM.
The typical 3 or 4 mm diameter, HPLC grade syringe filter has a hold up volume of about 8 ul. That may or may not be too large for your application (depends on the sample concentration which you did not indicate). * "Syringe Filter Selection for HPLC or LC/MS samples"; https://hplctips.blogspot.com/2014/11/syringe-filter-selection-for-hplc-or.html
As mentioned, a run through a micro-ultracentrifuge might reduce the chances of any particulate matter being transferred (do not inject the whole volume). It will not stop anything from precipitating out though.
Thank you all for your valuable and rational suggestions, i really appreciate that. @ Bruce and William, yes I'm considering micro-centrifugation prior to the hplc analysis in order to settled any potential debris.
**200 uL not 20 uL: sorry for the typing error, the sample volume is 200uL(0.2mL), which i initially considered as too little to filter with hplc syringe filters. But as mentioned by Prof. William, if only 8 uL may be held by the filter i think there shouldn't be a big problem right?
@Prof. Narong, i will enquire about this hplc in-line filters.
You will have no problem filtering it through a high quality 3 or 4 mm syringe filter. 200 ul is very different from 20 ul!. Please refer to the article in my post for more information.
@William, i will definitely refer to the article and find the most suitable filter for my samples. Many thanks indeed.
@ Jean-Pierre, i have no idea about the sample concentration yet, and yes, u may be right. 20 uL is impracticable to process and directly analysed. But since it's 200 uL i will try and do as advised. Many thanks indeed
Hi @Nafiu,
200 ul are sufficient to have a sample for hplc (with the possibility to use special vial). When you find the suitable filters as suggested by Prof William, it should be fine to run HPLC. Once more good luck!
I will be more worried about the shift valve that selects solvent/sample and the sample needle itself for making it dirty. Prefilter or guard column are extra meausures to avoid damage to the column, but i would always avoid a dirty sample.
Google miniprep vial thomson and you will see all-in-one filter and autosampler vial with max volume of 300 uL and it will fit your need.
My recommendation is to play safe. I always use a guard column. Why? Because exchanging a guard always is less expensive than throwing away the separator. It prolongs the lifetime of the separator, thus saving your time when it comes for reevaluating the new column. Then I would recommend using a high pressure inline filter. The suggestion was already made above. You can have such filters with pore sizes down to 0,5 µm, so that it is a very good protection of the guard column... (protecting the guard, funny thought....)
As these high pressure inline filters come with exchangeable filter plates, you can simply monitor the instrument back-pressure (it will go up) and then decide based on the increase when it is time to exchange. The filter manufacturers will provide you with some "acceptable" increase deltas. In my laboratories we used a typical threshold of + 500 to 800 psi, as a decision point when to exchange the filter plate. It is a rather broad range, depending on whether you use stainless steel filters (higher delta) or PEEK (lower delta)... I haven't kept an eye on the recent developments, so that the back-pressure tolerance of such high pressure inline filters might even haven further increased. Hope this helps.
I would like to recommend to utilize the pre-column line-filter (please see file; JCB Fucoidan transport). Sample filtration or centrifugation at 1,500 x g for 10 min is indispensable in the HPLC analysis.
Skin sample is very difficult to handle with (standing on ice bath induces aggregation of skin samples due to collagens), and dilution by suitable solution may be necessary (please see file; J Chrom B Rat BIN LIP Km). After adequate dilution of skin sample, filtration or centrifugation is able to be performed. Therefore, I have safely and firstly shown that upper labial skin has higher biotinidase activity as compared to lower labial skin and abdominal skin. Thus, sensitive HPLC-protein determination method is indispensable (please see file; HPLC-Surfactant-SEC protein determination method).
In the case of skin samples, I would recommend manual injector to develop stable analytical conditions. When stable condition has been developed, then please use auto-sampler.
Further, RP-HPLC is very weak to sulphur-containing proteins, and please defend the cystein residues by pyrydylethylation (please see file; PTH Roma RP-HPLC).
- Badges
- Science method