Chromatography Forum

meillid, do you believe or know that everybody used the anhydrous version?
If I am not mistaken you have not told us how you degas the mobile phase. (Looks like we all assumed you degas sufficiently).

[sorry, back in rant mode]

I'm still not convinced about specifying solutions in g/mL. If you're going to go that way, you also have to specify the exact product you intend to use, and all too often at some stage the instructions get cut.

Also I've come across too many solutions in my time (admittedly in labs that don't have strong QC) which were worked out once, wrongly, and copied for years without anyone noticing. Worse still, there are the situations where a method is used in the lab in g/L, but published incorrectly translated into molarity, so anyone copying it is doomed from the word go. SOPs are great, but I still feel SOP-plus-braincells is a safer option.

It also worries me that people who analyse things can't handle molarity calculations. I wouldn't buy an apple from a greengrocer who couldn't recognise it without a label. Molarity calculations used to be taught in school chemistry before the age of 16 in the UK. This isn't the stuff of PhDs. It's fundamental, basic knowledge.

To answer the degas question,

We usually stir our buffer solution very long time during the preparation. Then we relie on on line degaser for the degas. (water system). Thanks.

What's the pressure reading on your instrument? The 50C column temp will reduce the viscosity of your eluent -
but 2.5um hybrid particles results in 2-fold pressure increase compared to a well packed 3um (silica) column.

too much pressure = more leaks = more precipitated inorganic salts = more headaches on conventional HPLC system.

A 3.5um hybrid column of the same dimension will be more rugged (provided you can obtain adequate resolution / sensitivity).

To my surprise, I have found some QC chemists who are born English speaker, educted in the USA don't know how to transfer ppm to ppb or how to make dilutions. For example, the procdure says add 100uL sample, 50 uL IS, and 850 ul solvent to make a 10x dilution. However, when comes to 20x dilution and if there is no detailed instruction, it is made as adding 50 uL sample, 50 ul IS and 850 uL solvent. very close? yeah!

An analytical lab filled with people who don´t know what molarity is?

H.W.: you're in Europe; this is not uncommon in U.S., at our own QC labs and especially at contract manufacturers' QC labs. At the latter, not uncommon to find workers who are not English-speaking, or have poor English skills, being born elsewhere, which contributes to the communications issues. Needless to say, few of those have college Chemistry or degrees. I'm in R&D, department of six, we have 2 Summa cum Laude and 1 Magna cum Laude employees, two of which are Phi Beta Kappa (no PhDs). But we need to write our procedures so they are as bulletproof as possible. And yes, many of those facilities manufacture pharmaceutical and EPA regulated products, and their employee qualifications are out of our control.

To my surprise, I have found some QC chemists who are born English speaker, educated in the USA don't know how to transfer ppm to ppb or how to make dilutions.

I have observed similar. Sad.

Apparently, to paraphrase Yogi Berra: "There are degrees of degrees"

meillid, maybe you degasser doesn´t work. What happens if you use thouroughly vacuum degassed phase, just as a test on pump prime?

On lab sins: In an ~1996 article in J Chrom B (don´t have the citation here) I wrote that the most careful cortisol determination via HPLC was done by authors who analyzed cow´s blood. (If you mess up on cows you may land behind bars quite quickly . . . . cows cost something, a human being does not).

Thanks for everybody's effort! Now I have to share with you about the how the problem is resolved.

After our metrology changed the check valve of one systerm, it works. It seems this method (not a good method, but we don't have the freedom to change it) is very demanding on the system.

For the topic of drafting method/QC chemist, sometime I believe the language itself is not strong enough to convey all the subtle differences to make everything error proof, although everyone is doing their best. That why you alway have to train your new empoyee even they are experienced, because there are some "rules" not on the paper, but each person practice them. Do you dare to claim that you have put everything on SOP?

Another point, even among the best employee, brain is most smart one, but not nesessaryly the most reliable one, each person have the occasion of overlook somthing/slip of mind something, it needs the whole group work to make final results ZERO error for GMP.

Thanks.

Do you flush the system with high aqueos mobile phase without buffer if the instrument is going to be shut down for a few hours? This should help if the problem of buffer precipitation.