Hello!
I ´m in problems.
My columns go continously throug high pressure.
I suspect that are the antibacterials which i´m working with; Could anyone tell me how to clean the column to get them out in the case that this were adsorbed in the silica surface?

What column is it?
What mobile phase?
What antibacterials are you using?

You need to give as much information as
possible or there will be little chance of you
recieving helpful advice from the
chromatographers participating in this site!!!

Sorry, It´s a C18;
b-lactamics, (basic compounds), and i dont know their pKa,and my mobile phase is of ACN and kh2Po4
PH =7


Thanks and don´t be ungry with me, I just couldn´t see other thing than my problem, and i didn´t realice you didn´t got all the information....!!!

I would have thought that washing with water
100%, then organic 100% then 50/50, then
your mobile phase should do it. The organic
wash should get rid of any organic molecules,
the water wash, any salts deposited.

You should start each wash with a very slow
flow rate, i.e. 0.1 mL/min, and ramp up as the
pressure settles down. Also, each wash
should be for quite a long time.

Run a strip chart (or your system's equivalent)
so that you can see if any big peaks come off,
or whether the baseline settles down before
going to the next wash. I have used this
method to clean C18's before and it has
allways worked for me.

However, I am sure that there are plenty of
chromatographers out there with more
experiance than me, who might have a better
method.

Don't panic!

There is already a discussion passed, about cleaning RP-18 columns, maybe you should take a look at this.

Forget about cleaning your column! Use a guard column!

That's all well and good no-name2, but
sometimes there is a precipitation on the
column, how's a guard column going to help
there, eh, eh!

In an environment where not everyone knows
what they are doing, and they come and use
your system without asking, you'd be
supprised some of the things I've had to deal
with!

I do get a guard-column; The way i use to wash the column is :
The time of washing calculated by 40 times the volume of the column is 30 minutes; I spend 20 minutes with water (5%AcN)at 0.2mL/min,after this, i go to 100% and stay there for 10 minutes, and after, I equilibrate in 65%.Totaly it takes me 102 minutes.

And everyday preasure goes 20bar over.
The salt in the mobile phase is soluble until 75% AcN, and i never pass of 70%.

It´s the fourth column that goes bad, and i don´t know what to do...


Thanks again ..

The only thing I can think of would be to do the
water and organic washes for several hours,
maybe overnight at a slower flow rate. If this
doesn't get rid of whatever is causing the back
pressure, you might have to look at what is in
the samples being injected, and how they are
prepared.

Do you see any precipitate formed in the
sample vials when they are left for a long
time?

Have you tried removing the guard column
and seeing if that is causing the pressure
build-up? You could try running the system
without the guard column and see what the
pressure does (but don't inject anything!).

You still have not told us about the analytes´ matrix, is it biological? The matrix is as important as your analytes in developing a method. There have been discussions on getting rid of proteins, etc., etc., before. Usually a replacement of frits cures the problem, a sledge hammer is to wash with a solution of Li-dodecylsulfate containing dithiothreitol (don´t remember conc., am not in my lab as the PC there does not allow getting into the forum, presently).
What happens if you reverse the column (or the guard, separatly)?

H W Mueller, as I mainly work with normal
phase columns I don't know that much about
C18 columns. Is it OK to reverse a C18, does
it not mess up the order of the alkyl chains
(like back combing hair)? Or was someone
winding me up when they told me that?

Cheers,

Will.

Will, the one who told you not to reverse a C18 column should take you out for diner to the most expensive restaurant in your city. Anonymus: can you change to another buffer? What kind of C18 do you use, an endcapped C18 or not endcapped? Are you working at ambient column temperature? Did you try other pH values? What sample preparation you do? Do you prepare your buffer solution daily? Sorry for all these questions. Is it only a backpressure problem, or do you see shifts in retention time, or peak shape changes? Gerhard

Mmmmm, Chinese food!!!!

Ah well, it did seem a bit far fetched. You don't
learn unless you ask!

Good Morning to everybody!

I have to tell you several things:
The analyte´s matrix is not biological; I just dissolve them in 50%AcN/KH2PO4,pH=7, and I dont observe any precipitation, and I filter by 0.22um.

I wash the column after have tooken the guard column off and instaid of it, put a precolumn-filter of 0.22um.

I´ve proved that the pressure is from the column and from nowhere else.But I have to change the precolumn silica each three days.


Grehard, The C18 column is endcapped,I prepare the buffer everyday and filter it by 0.22um.I´m filtering also AcN. The sample preparation is just by disolving the substances ; is not necesary any other manipulation.It´s not the frist column which happens this to me
.I think the analytes are adsorbing in the silica.And I´d like to know if something exist to elute them .


I´m really gratefull to all of you; Just for talking about it is important for me,; you are confirming to me that I´m not doing anything wrong...

Balto, does the same thing happen when you have the pre-filter on there only? Replacing the prefilter restores column performance?
What are your anti-bacterials? Do they polimerize on air contact?
Is there a possibility that you dissolve some polymer with ACN or pick up some other dirt which may go through a microfilter but coagulate on frits? Do you dissolve your microfilter?
Do you pick up dirt after the microfilter?
Are there particulates coming out of the injector? etc. etc.

Will, tell the funny guy that the column worked fine in revese, after putting hair lotion into it.

You may want to check that you are truly washing all of the phosphate buffer off of the column, as well as out of all of your tubing, injectors, pumps, etc. A little phosphate can go a very long way. By the way, are you sure about that phosphate solubility in ACN? Is there any way that the ACN concentration is inadvertantly going up?

Balto, what Chris says is, that in many cases, not in all, with Phophate buffer systems, backpressure problems are observed. I had to fight with such problems also, and when I changed to another buffer, the problem was solved. Sometimes an organic buffer, volatile, is much better than phosphat. Did you tried to use another buffer, and can you change the method to another buffer. The problem is, that Phosphat buffers can clog the column packing. Sometimes it was helpful to rinse the column with hot water at 50°C. But please disconnect your detector, and rinse the column in reversed mode, without hair lotion! Take one of your used columns and try it. If you can see, that after 30 min of rinsing with hot water backpressure is decreasing when you go back to your initial conditions, than the problem is clear. Good luck. Gerhard

Have you already tried if your sample matrix dissolves into the mobile phase, maybe its just a simple, stupid problem.

Yes, I´ve tried several mixtures of phosfate buffer in AcN and I know that It´s possible to reach 70%Acn without any problem (in a tube).The only possibility of have increased the % of AcN, is that the pump was working badly; but i´ve just changed of chromatographyc system, and with the other columns, I worked in others..I measured the flow the other day, but i´m going to chech it again...

We are thinking in change the buffer; We´ll try borates...

Everyday, I put out of the line 20mL of salt before to start flushing water .I´ve calculate and I think 20 mL are enough to have a guarantie of being washing with water and not with KH2PO4...

And yes , the sample matrix is the mobile phase,and I think the substances are soluble; In the other hand, i filter the sample (0.22um).


This days I washed with water(10%AcN), at 40ºC....Today I flushed with H2O(pH=3), and Pressure descended, but when I tried with water(10%AcN), increased again....


I dont know what else I can do...


It´s more than a year that I´m working in this , and I can not advance because of this problem..
I couldn´t get my ( I´m not sure how do you say it in English),graduate tesis..in June, neither i´m being able in September..

Well, I´m not going to continue crying to night..

Thanks,thanks, thanks....

Balto

Balto, nice to hear that you can change your buffer system. I would recommend an organic buffer system, like MOPSO, for pH7 (this was discussed in an earlier discussion)! Please take one of your old columns and open the top endfitting. Check the frit. If the frit shows some dirt, than clean it in an ultrasonic bath. Next step is than your acetonitrile. What grade are you using. HPLC grade or "for analysis"? When you wash the column with water at 40°C or 50°C don't add Acetonitrile. And do it in the reversed mode, and disconnect the column outlet capillary from the detector. After this step, wash with pure cold water, and than with 100% Acetonitril. Than turn the column back and start with a low flow rate with water/acetonitril 50/50 before using your buffer system. And than increase flow up to flow rate written in the methode. And than check backpressure and flow rate. Than we will see. Good luck. Gerhard

Balto,
I am not sure if I read this correctly. Are you using a silica precolumn? Please explain the precolumn. Is it home made? Using a large particle size silica? Or is it something different?

Thanks, and best regards
Uwe Neue

Hello, the precolumn is Perisorb RP-18 and Particle are 30-40um.

The bottle says : "for liquid chromatography under pressure"


Today i´m going to try with KH2PO4 5 mM and adding after each run a step of 95%AcN; Tomorrow I´m going to try with a buffer of tris 25mM.
I´m writting from the job, so I can´t write too much..
Thanks again....
Balto

Balto;

You have heard a lot of information in the previous messages. I would like to tell you that in my experience 0.05M (NH4)2PO4 can not be mixed with ACN at higher levels than about 60% ACN. KH2PO4 is expected to have lower solubilities under similar conditions.

I know you are using lower molarities and therefore it is likely that the salt stays in solution at higher ACN levels. It is possible however, that you are getting slow precipitation and therefore a small gradual increase in back pressure. lowering the molarity will solve this problem.

I also know that KH2PO4 at pH 7 is a perfect culture media for bacterial growth. In less than 24 hrs a 0.02M of such salt will look cloudy if open to the atmosphere. Normally a small amount of organic modifier is expected to prevent bacterial growth, but I am not sure how long the mobile phase can be kept. Perhaps you should try refrigerating the mobile phase when not in use.

Phosphate salts at near neutral pH values also have a bad effect on some Silica B type columns such as Zorbax. This is not likely to be your problem since apparently you only see an increase in backpressure but not a change in efficiency or retention, at least nothing has been mentioned.

Good Luck;

Benjamin

I think that the problem is with the precolumn. With this particle size, you do not get a protection of the main column, and you actually my be plugging your analytical column with the fines that elute from the precolumn. If this is the case, see if you can replace the inlet filter of your analytical column. I bet that the junk sits there, and not in the analytical column itself. If your analytical column does nto allow you to replace the precolumn filter, you are doomed to buy a new one. If you need to do this, I recomend to go to a real guard column packed with the same amterial as in the analytical column.

The last comment by Benjamin may have a point as well. I did not see the buffer concentration in your previous e-mails. 50 mM may be high, but we are using 30 mM phophate buffer pH 7 routinely at 65% MeOH.

We have had occasional difficulties with bacterial growth as well. It can be fixed by adding a bacteriocide such a sodium azide.

I prepare the buffer everyday, so i think it´s not going to be the cause of the problem...

When I fill a precolumn, I spend one or two hours flushing it to expulse the finer particulates before conecting it to the column..

Long time ago, I didn´t use a precolumn and instaid of it , i used a 0.2um prefilter, and i had the same problem, so I think the precolumn is no the problem.

Today I´m trying TRIS buffer 25mM,I know i´m not going to have problems with solubility and I´m prying a lot..!


You´ve helped me a lot. thanks for hearing me.

Again, does the flow normalize if you replace the first (entrance) frit on your guard-column after you had this pressure increase?
Does the flow normalize if you remove the precolumn.....?
If not, does the flow normalize if you replace the initial frit of the column.....?

the pressure comes from the column; if i remove the precolumn,decreases, but just a little.

I´ve never tried to replace the inlet frit of the column..I´m going to tell my boss to do this with one of the old damaged columns i got..I thinf it´s a good idea, but also dangerous..We colud produce a void volume..


IT´S BEEN A NICE SURPRISE FOR ME TO FIND SO MANY PEOPLE WANTING TO HELP ME...JUST A YEAR AGO I DIDN´T KNOW ANYTHING ABOUT CHROMATOGRAPHY, AND NOW, WHAT I KNOW IS THAT THERE ARE A LOT OF THINGS TO LEARN .

I´ve tried with tris buffer, and today i´m going to spend the journey wahsing the column...perhaps the "rubish" is from last days ...I´m not going to loose hopeness...


Balto

Do not dismiss things too easily. You prepare fresh buffer everyday, but your solvent inlet lines may have caught a bug, and you won't see it.

The flushing of the precolumn does not necessarily remove all the fines. Every time you inject, you create a pulse on the precolumn, which may shake loose some fines that had been stuck before.

When you used the 0.2 micron precolumn filter, did you see pressure buil-up on the filter, or only on the main column?

When i used the filter, the pressure went up in both,the filter and the column, but never so fast as now...It´s true that i´ve changed of equipment too..


Everyday I "change" the liquid content of the lines.I take 30 mL before starting flushimg water to clean the column: What I don´t do is flushing it with MeOH or something organic ...I ´ll do it once a week to avoid weeds...

How could I prevent the effect of the fines of the precolumn?

You really need to find the source of the problem. However, to protect your expensive analytical column, you should use a precolumn (also called guard column) packed with the same particles (and particle size) as your analytical column. These things are a bit more expensive than the home-made precolumn with the large particles, but they will save your analytical column, and protect you from all evil..:-)

You are wright, Uwe.
But I got good News to everybody...The pressure has get stable.I changed the buffer for Trys, (instead of kh2po4), and this one, permited me to include a step of 100% ACN after every run; I ´ve achieved that pressure did´t increase, but the column is not very "healthy" and it has more pressure than it has to have.But I can work now and next column is not going to get dirty...


Thanks to everyone...