An HPLC ate my Protein

[Jbw312]

I am using a XBridge BEH300 Prep C4 10 um column to purify proteins (~8-16 KDa). We have had the column for two months and just very recently the pressure jumped from 700 psi (95:5 water:acetonitrile with 0.1% TFA at 15 mL/min) up to ~2000 psi. With various washing conditions the pressure returned to normal. However, now when I inject purified protein standards dissolved in water I observe no eluted protein throughout the entire run. The UV detector is functioning properly and there is no protein in the injection peak (there are no real peaks during the run either). I would be extremely grateful to anyone who might have suggestions or by some chance encountered this problem before.

[badger]

It would be helpful to know some more details such as, column size specifications and the manner in which you are injecting samples, i.e. manually or with an autoinjector. What concentration are your samples, what wavelength(s) are you monitoring. Given that flow rate I assume you are working with a ~21 mm I.D. column. I don't have a huge amount of experience with C4, but the first few things that jump out to me about your set up...

First...5% acetonitrile is pretty low, I've observed on occasion that too low of a % of organic modifier in your mobile phase results in collapse of the stationary phase which can be regenerated by flushing with several column volumes your organic solvent (ACN in this case). I would recommend not going any lower than 10% if you are uncertain how your system will behave.

Second...assuming your column, pumps, and detector are behaving properly (no leaks at any connections, pressure is steady, and the detector signal changes when you flush by hand with a UV absorbing solution through the flow cell) and your sample is also prepared properly, the lack of peaks is usually indicative of an injection problem in my experience. It appears you are doing prep scale HPLC so I assume your set up has a 6-port valve assembly that your sample is delivered into. If your set-up allows it you should watch the needle load the sample onto the loop. Autosamplers will extract your sample from a vial with a needle and then load the volume you specified into the samle loop, during this process the same volume is displaced out of the loop and into the waste port. If the waste port is clogged the needle assembly can't really fight the backpressure and your sample just leaks all over the injection port resulting in no sample being injected, and hence no peaks.

If none of these suggestions work and everything seems to be working properly then report back and we can figure out some other things. Good luck, I've been in your situation before.

[Jbw312]

I believe it is a 21 mm column. We have been running with 5% ACN at the starting gradient and our proteins generally elute between 26-40% ACN depending on the protein. For the most part the proteins are previously purified using Ni affinity columns. Concentrations are generally 1-2 mg/mL and we monitor at 214 and 280 nm. The injection is manual. However I can rule out any error in the tubing between the valve assembly and the column (if I inject a sample in GnHCl there is a sharp strong signal for the salt but still no elution of protein).

What makes this more interesting is we have a second column that had previously been used for ~2 years and also has the same problem with protein not being eluted. The columns both have seen injections containing the following salts/buffers/ or compounds in moderate to high concentrations:

urea, GnHCl, 3-Mercaptopropionic acid, TFA, possible traces of nickel ions, EDTA, and on occasion cleared E. coli lysates

My thoughts are (but I do not know what interactions any of the above would have) that one or more have interacted with the column resin and now protein is being "stuck". Are there stringent ways to clean these types of matrices and to regenerate them to factory specs?

Thanks for the reply!! (I've been working for 4 days non-stop trying to rescue this column)

[badger]

What size are your proteins? Have you successfully observed them before with a different column? It may be possible they are not retained and eluting at the void time with the GnHCl. You said you are doing gradient elution, do you have a flush of 95% ACN at the end of your gradient? What is your injection volume? If you are injecting too much onto the column everything might be coming off in the void.

If you have a column you know works for these proteins I would use that to ensure nothing else is wrong with your instrument. I'm not entirely sure what you would do for an Xbridge columns but I use the following solvents to clean out my stable bond columns

mobile phase ---> acetonitrile ---> isopropanol ---> dichloromethane ----> isopropanol ---> acetonitrile ---> mobile phase

Also, are you analyzing your crude samples via some other means prior to injection? In particular are you 100% sure there is protein in your sample to begin with.

[Jbw312]

What size are your proteins? Have you successfully observed them before with a different column? It may be possible they are not retained and eluting at the void time with the GnHCl. You said you are doing gradient elution, do you have a flush of 95% ACN at the end of your gradient? What is your injection volume? If you are injecting too much onto the column everything might be coming off in the void.

If you have a column you know works for these proteins I would use that to ensure nothing else is wrong with your instrument. I'm not entirely sure what you would do for an Xbridge columns but I use the following solvents to clean out my stable bond columns

mobile phase ---> acetonitrile ---> isopropanol ---> dichloromethane ----> isopropanol ---> acetonitrile ---> mobile phase

Also, are you analyzing your crude samples via some other means prior to injection? In particular are you 100% sure there is protein in your sample to begin with.

Proteins are 8-16 KDa and with this same column only a few days ago the purification went without any problem. Since the problem arose I have been injecting pure protein standards and cheking samples via UPLC (they show uo very nicely). The void volume and injection peak have been confirmed to contain to protein (via UPLC and LCMS).

[JMB]

Proteins are 8-16 KDa and with this same column only a few days ago the purification went without any problem. Since the problem arose I have been injecting pure protein standards and cheking samples via UPLC (they show uo very nicely). The void volume and injection peak have been confirmed to contain to protein (via UPLC and LCMS).)

What size (KDa) are your protein standards ?

Do you inject them singly or as a mixture ?

Do they all give the expected response (height or area) ?

Are their RT values "normal" ?

JMB

[JMB]

Your pressure increase (700 to 2000 psi) suggests a partially plugged column; either the frit or the packing (or both) at the head of the column may be involved.

Suggest a reverse-flow at high water concn. with repetitive injection of slugs (50-100 uL) of DMSO to possibly remove anything at head of column; monitor backpressure to determine if this has any worthwhile effect.

JMB

[danko]

The void volume and injection peak have been confirmed to contain to protein (via UPLC and LCMS).

If you see your protein/s in the injection peak/void volume then the HPLC didn’t ”eat” them – did it?
So either the column’s completely destroyed or the mobile phase' composition is not right.
1. Check your eluents (A and B) – are they connected to the right channels and/or are they the right eluent at all? Is the pump method unchanged?
2. Replace the column with another one (preferably the same brand/type etc. but if you don’t have another one of exactly the same column, just find some similar) and check whether you see the same picture or there is som retention of the protein/s

Best Regards

[Jbw312]

The void volume and injection peak have been confirmed to contain to protein (via UPLC and LCMS).

If you see your protein/s in the injection peak/void volume then the HPLC didn’t ”eat” them – did it?
So either the column’s completely destroyed or the mobile phase' composition is not right.
1. Check your eluents (A and B) – are they connected to the right channels and/or are they the right eluent at all? Is the pump method unchanged?
2. Replace the column with another one (preferably the same brand/type etc. but if you don’t have another one of exactly the same column, just find some similar) and check whether you see the same picture or there is som retention of the protein/s

Best Regards

Sorry that should have read "no protein"

[tom jupille]

If this were my problem, at this point I would just get a new column so I could get back up and running. Then, at my leisure, figure out what went wrong with the first column.

[Jbw312]

Thanks to all for the replies and suggestions. It seems the column is dead and beyond regeneration. At this point I'd like to find out if there are recommendations for a C4 prep column that will handle routine daily injections of protein samples containing 1-2% TFA, 1-20% acetic acid, and/or cleared cellular lysates. We are injecting 5-15 mg of proteins at a time and doing this up to 6 injections a day. Thanks again for the suggestions.

[badger]

Try this guy, it's a C5 but should work the same. Our lab has had great success with this column.

http://www.sigmaaldrich.com/analytical- ... re-c5.html