UPLC column

[landa]

Hello,
I am trying to transfer a HPLC method to UPLC
Current HPLC method requires Xterra Phenyl 4.6X50mm, 5um
when I transferred to UPLC I did use BEH Phenyl 30X2.1 1.7um but i am getting a huge fronting
can someone help me with any ideas ???

Thank you

[tom jupille]

Some possibilities:

Too much extra-column volume?

Too large an injection?

If it's a gradient, inaccurate scaling of the flow rate column dimensions, and gradient time?

A bad column?

[IsotopeC12]

I had a similar problem when first attempting UPLC. I changed the needle in the source probe, cleaned the curtain plate (was very dirty) and also got rid of all my metal tubing. I used metal fittings on the ends of normal red plastic tubing and made sure the length of the tubing from the autosampler to the Mass Spec was as short and direct as possible. I learned that you only need to worry about the pressure before the column. After the column you can use the normal plastic finger tights on your tubing. My UPLC peaks were very sharp and you will see greater sensitivity at a lower injection volume.

[IsotopeC12]

Also your gradient has alot to do with it...dont be afraid to use an aggrressive gradient. I have one method that has a 1 min. run time with a 20 sec. gradient.

[carls]

Did you reduce your injection volume and/or sample concentration proportionately (~8x lower)?

[landa]

Thank you all for your responses !!!!

[DSP007]

C'mon guys scare the girl.

The fact that the column is short - means that the division is close to perfect.
In 50 mm HPLC column length of =>48 mm sorbent column.
Grain size of 5 mm, its maximum theoretical efficiency of about 9000 theoretical plates, the real about 4000.
In the 30 mm column length UPLC=> column sorbent of about 28 mm. Grain size 1,7 mm of its theoretical efficiency as about 16500, real, too, about 5000.
Practical difference (if the sorbent is the same) - no.
There is really a one zamorochka ( unlogical problem) . Hydrostatic pressure of the UPLC column higher almost three times. Do not exceed the hydrostatic pressure silica gel strength of 300 bar.

[unmgvar]

DSP007
sorbent is not the same, so you do have a practicle difference
Xterra is not BEH, are based on the same concept but BEH is a change from Xterra and does present a difference in selectivity
these are not 100% silica columns but a special waters combination technology, which one main features is the capability to work at pH up to 12.
the 300 bars limit really does not apply to columns that can make it safely above 800 up to 1000 bars, even if they are made of 100% silica support. there are enough UHPLC column out there going well above 300 bars

Landa
because there are a few details it is hard to exactely see what is wrong with the transfer of method.
apart for the other good suggestions,
it could also be the difference in the column type
it could be also the mobile phase itself.
i know of a ion pairing method also that was transfered to UHPLC and the fronting in the method that was almost non apparent around 0.95 became 0.85 with a 1 min 100% base peak width ( crazy for a 3 min. UHPLC method)
remeber UHPLC is more sensitive, and sensitivity is a double edge sword

[DSP007]

1) X-Terra isn't BEH? Yes of course, but X-Terra Phenyl and BEH -Phenyl is one chemical group (radical) and use some chemical mechanisms of separation.

2) 300 or 1000 bar? Instant compressive strength of silica gel - a few tons per square millimeter, but the long-term resistance to compression (to the "fluidity" sayd the language of engineers ) - is much lower. In addition, at ultrahigh pressures come into work "usually not taken into account" chemical factors, for example the solubility of silicagel in water. Therefore, we must choose, or if you use the column under very high pressure, but not for long, or the pressure is less than its long-term strength, then it works unlimited long without formation of cavities and channels.

[Mattias]

Are you using a Waters Acquity system?

If so, the "weak needle wash" must not be stronger than your initial mobile phase. The weak needle wash is introduced with your sample on the column.

[unmgvar]

DSP007
1. so like C-18 is C-18, and all C-18 columns are the same, with the same selectivity? I could elaborate with more details, but we know that it is not necesssary.
2. I am hearing you there, so best is not to use UHPLC columns above the pressure of 300 bars for longer and better performance.
of course, this means we should use lesser flow rates or far higher temp in order to decrease pressure and most probably work well outside the optimum range of the Van Deemter curve of the UHPLC columns,
so then what's the point of using the technology?
wait but columns are packed at actually greater pressures, and vendors have found the way to improve silica particle strenght for more performant UHPLC columns
and let's not forget the case at hand with hybrid technology of BEH made not of only silica, which was designed to wistand those high pressures to begin with.
but maybe more relevant, is that the points are irrelevant to the present case of the fronting in the application

[Flappytango]

I agree with Mattias. Careful with the wash solvents.

Depending on what kind of HPLC system you were using and your current wash solvents you may have a very different sample solvent composition upon injection.

As others have said, be sure to scale your injection volume and watch out for extra column volume. Double check your column connections, especialy the head of the column. The waters UPLC ferrule/collect/reusable fitting combination wants to lock into positon. You must release it and reposition if you ran a non Aquity column previously to account for different insert depths.

[DSP007]

DSP007
1. so like C-18 is C-18, and all C-18 columns are the same, with the same selectivity? I could elaborate with more details, but we know that it is not necesssary.
2. I am hearing you there, so best is not to use UHPLC columns above the pressure of 300 bars for longer and better performance.
of course, this means we should use lesser flow rates or far higher temp in order to decrease pressure and most probably work well outside the optimum range of the Van Deemter curve of the UHPLC columns,
so then what's the point of using the technology?
wait but columns are packed at actually greater pressures, and vendors have found the way to improve silica particle strenght for more performant UHPLC columns
and let's not forget the case at hand with hybrid technology of BEH made not of only silica, which was designed to wistand those high pressures to begin with.
but maybe more relevant, is that the points are irrelevant to the present case of the fronting in the application

Well -
1) what is different selectivity whith L1- Nucleosil - C18 and Cromasil C18 ( regular spheric granules , 120-130 A , 15-20% C , cross which metylsilane by silicagel) ?
/Special phase type (small cross of C18 5-8% , cross-link C18, uncross metylsilane in silicagel , semihydrophobic , high porous) we do not consider - it is clear that they are special /
2) You remember the graphic expression of the Van Demeter law ?
http://www.wikipremed.com/image_archive ... m=1&itbs=1
For 2mm x 50 mm 2 mkm column pressure 1000 bar = 0,8-1 ml per minute , pressure 300 bar = 0,25-0,3 ml per minute. Rules UPLC systems have vary small dead volume (cuvete and tube communications) and speed 0,1 or more set in a right part of Van Demeter curve.
..................................................................................................................
Thus the use of high flow rate is not justified in terms of chromatography division.
High speed has other benefits - reducing the analysis time and increase productivity.
But these benefits are not free. The price paid for them - a very quick death of the column (1000 tests). As they say, this stick with two ends

Therefore, if the time of suffering - it makes sense to get the test result is not 30 seconds, but after 1.5 minutes, reducing the pressure and achieving unlimited lifetime of the column. This approach is quite acceptable for most scientific problems and research.

Of course in medical toxicology and accompanied by chemical processes in the production had to use a different approach - "speed is above all" and "when talking about the family's honor - a dispute about money is not relevant."
Because if a candidate for the dead ate a pack of clonidine or broke a pair of grams of heroin - is to live it without resuscitation (reanimation) one hour and your analysis of it may simply not be forthcoming.

[unmgvar]

Dear DSP007,
i first like to remind myself that our debate that contributes nothig to this post, is taking place thanks to your irrelevant remarks in this posting

1. is it good that you ask of nucleosil and kromasil column and that you point out the fact that apart from being C-18 all other specs are different, just like for the phenyl columns of xterra and BEH.
please see the link provided
http://www.mac-mod.com/pdf/ColumnComparisonGuide.pdf
It will explain very nicely to you the intereting differences between columns that to the normal eye would look the same. I enjoyed reading it, very instructive and graphic displays that save a lot of writting

2. you really run a 2X50mm 2dp column at 0.8-1.0ml/min? why? such a column has the optimal of the van-deemter at about 0.5ml/min, at worst case be at 400 bars or so, with a 1.7dp you can get already to 600-700 bars easy. in any cases, UHPLC columns nemesis, is not the pressure, that they can nicely handle but the clogging whose symtom is seen with the increase of clogging.
anyway productivity and column life time is not I think in the amount hour of use used, but actually in the number of time injected, it has been shown by pharma and other labs that in relation to 5u analytical colunms a good UHPLC column whose user has taken the necessary usage of sample and mobile phase and system care can run as many samples on them and achieve the same amount of productivity. yes it can meant that the column lasted less days, but that is irrelevant, and the money saved on time of usage system usage, man power, solvent consuption and handling is huge.

as you should know from Van-deemter there are 2 reasons to increase of pressures for UHPLC columns.
the smaller the dp then the higher the pressure, of course,
but also van deemter shows us that with all dimensions equals except for the dp then to reach the optimum you need to increase the linear velocity, flow rate, and so increase working pressure.
cut velocity then you loose efficiency fast. you can even find yourself using a 2.2/1.8u column no better then a 5u with your approach

you will like reading this link as well
with a small remark that silica can stand up to 1000 bars these days:
http://www.chromacademy.com/resolver-may2010.asp

you also talked of justification of use, as if chromatography by HPLC is the goal in itself,
and here I was thinking it was a very usefull tool for many purposes and needs (with justification in the eye if the beholder)

anyway still irelevant to the debate of this posting,
but I liked your dramatic example, someone with ties to holliwood should use it