Need help/advice with HPLC operation

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

8 posts Page 1 of 1
Hi, I am a HPLC novice here. Have been doing some reading up online but could not find answers to some of my doubts so would appreciate any help in answering some questions regarding HPLC operation (am using Shimadzu HPLC btw) :mrgreen: :

1) About column conditioning - I was told by a colleague to do manually i.e. set the desired mobile phase % on control panel, turn on pump and wait for desired duration. There should be a more automated way of doing this?? Is it possible to create a separate method file for column conditioning then execute it as a single analysis run? However, when i tried doing so i do not see a command for system not to inject sample, does this mean i will need to insert an empty vial? --- sorry if my qn sounded stupid.

2) The mobile phases i am using are ammonium acetate buffer and ACN. However, in our lab, flow line A has been dedicated to water already and since i am going to use water for washing i decided to leave it. So now, i am dedicating flow lines B and C to ACN and buffer respectively. Since I will only use solvents B and C for my method run, is it correct to write my time program this way:

Time Module Command Value (%)
0.01 Pumps Solvent B conc 8
0.01 Pumps Solvent C conc 92
13.00 Pumps Solvent B conc 67
13.00 Pumps Solvent C conc 33
13.20 Pumps Solvent B conc 8
13.20 Pumps Solvent C conc 92
15.00 Pumps Solvent B conc 8
15.00 Pumps Solvent C conc 92
15.00 Controller Stop

3) Qn about column flushing. Understand that water should be used for column washing to remove buffer salts. What is the recommended duration for washing? The buffer conc i am using is 0.01M (which is not considered high?). Also, should i disconnect the column from the detector during washing or can I just flush the entire system? I am thinking of creating a "column flushing" method and executing it at the end of my sequence but not sure if this is advisable?

Thank you for taking the time to read & answer my questions! Your help would be greatly appreciated!!
Hi Penxx,

I was actually in your exact shoes about a year ago, with the exact same system and software! Thankfully, through online resources like this one, my knowledge and love for the instrument has grown parabolically.

When it comes to column conditioning, I would recommend a few things. At the start of the day, or whenever you plan on starting the instrument, you should wash your columns and lines with specific method files that can both clean and equilibrate your column. On my instrument, this is all compiled into a single method (binary pumps) that can run through a wash sequence and, depending on the column and flowrate, execute an equilibration for (my preference) ten column volumes. If it is asking you to inject a sample, this setting can actually be turned off or you could just manually click start on the window.

There is also a setting (for example if you run your HPLC every day, for the same amount of time) called start-up (as well as shut-down), that ensures your machine is started (which you can link a method to) before you even enter the lab (requires the instrument to be on). I'm a little confused regarding the latter half of your question however. Are you executing all of these from an autosampler? If so, simply keep a "wash" vial that has amenable/miscible liquids that can serve as a placeholder for the wash run in your first autosampler vial port.

As for your method/gradient, that seems like a fine way to write it out. Albeit, a little redundant if you already stated that your mobile phase is buffered. Typically, when writing our gradient profiles, A is assumedly your aqueous phase, while B is your organic. However, as long as it's easy to read and well understood, I'm sure your way of writing it will not cause any issues.

As for column flushing, I would wash the column for as long, if not a little longer, than you would for equillibrating the column at the start of the day. Essentially, you want to ensure that whatever liquid that is on your column is exactly what you need for analysis (or for this case, shut-down). So a flush of roughly ten column volumes should leave the column clean-enough and buffer free. Remember to flush any/all lines that use the buffer, including the column and all the detector! If not, you have a chance for your salts to precipitate onto the column which will lower it's lifespan over time, and this effect could happen realistically anywhere along the way if the right conditions are met. This includes having a high% organic portion of a gradient or wash sequence and you should always make sure that your buffer works at all of your gradient's concentrations. For example (and I don't expect this to be the case currently), when your method dips to a lower buffer percentage (~33% at 13min), you should assure that the salts are still soluble in the ACN/buffer mixture.

I hope some of this helped you!
Tyler
Hi Penxx,

I was actually in your exact shoes about a year ago, with the exact same system and software! Thankfully, through online resources like this one, my knowledge and love for the instrument has grown parabolically.

When it comes to column conditioning, I would recommend a few things. At the start of the day, or whenever you plan on starting the instrument, you should wash your columns and lines with specific method files that can both clean and equilibrate your column. On my instrument, this is all compiled into a single method (binary pumps) that can run through a wash sequence and, depending on the column and flowrate, execute an equilibration for (my preference) ten column volumes. If it is asking you to inject a sample, this setting can actually be turned off or you could just manually click start on the window.

There is also a setting (for example if you run your HPLC every day, for the same amount of time) called start-up (as well as shut-down), that ensures your machine is started (which you can link a method to) before you even enter the lab (requires the instrument to be on). I'm a little confused regarding the latter half of your question however. Are you executing all of these from an autosampler? If so, simply keep a "wash" vial that has amenable/miscible liquids that can serve as a placeholder for the wash run in your first autosampler vial port.

As for your method/gradient, that seems like a fine way to write it out. Albeit, a little redundant if you already stated that your mobile phase is buffered. Typically, when writing our gradient profiles, A is assumedly your aqueous phase, while B is your organic. However, as long as it's easy to read and well understood, I'm sure your way of writing it will not cause any issues.

As for column flushing, I would wash the column for as long, if not a little longer, than you would for equillibrating the column at the start of the day. Essentially, you want to ensure that whatever liquid that is on your column is exactly what you need for analysis (or for this case, shut-down). So a flush of roughly ten column volumes should leave the column clean-enough and buffer free. Remember to flush any/all lines that use the buffer, including the column and all the detector! If not, you have a chance for your salts to precipitate onto the column which will lower it's lifespan over time, and this effect could happen realistically anywhere along the way if the right conditions are met. This includes having a high% organic portion of a gradient or wash sequence and you should always make sure that your buffer works at all of your gradient's concentrations. For example (and I don't expect this to be the case currently), when your method dips to a lower buffer percentage (~33% at 13min), you should assure that the salts are still soluble in the ACN/buffer mixture.

I hope some of this helped you!
Tyler
TylerSmith123 wrote:
Hi Penxx,

I was actually in your exact shoes about a year ago, with the exact same system and software! Thankfully, through online resources like this one, my knowledge and love for the instrument has grown parabolically.

When it comes to column conditioning, I would recommend a few things. At the start of the day, or whenever you plan on starting the instrument, you should wash your columns and lines with specific method files that can both clean and equilibrate your column. On my instrument, this is all compiled into a single method (binary pumps) that can run through a wash sequence and, depending on the column and flowrate, execute an equilibration for (my preference) ten column volumes. If it is asking you to inject a sample, this setting can actually be turned off or you could just manually click start on the window.

There is also a setting (for example if you run your HPLC every day, for the same amount of time) called start-up (as well as shut-down), that ensures your machine is started (which you can link a method to) before you even enter the lab (requires the instrument to be on). I'm a little confused regarding the latter half of your question however. Are you executing all of these from an autosampler? If so, simply keep a "wash" vial that has amenable/miscible liquids that can serve as a placeholder for the wash run in your first autosampler vial port.

As for your method/gradient, that seems like a fine way to write it out. Albeit, a little redundant if you already stated that your mobile phase is buffered. Typically, when writing our gradient profiles, A is assumedly your aqueous phase, while B is your organic. However, as long as it's easy to read and well understood, I'm sure your way of writing it will not cause any issues.

As for column flushing, I would wash the column for as long, if not a little longer, than you would for equillibrating the column at the start of the day. Essentially, you want to ensure that whatever liquid that is on your column is exactly what you need for analysis (or for this case, shut-down). So a flush of roughly ten column volumes should leave the column clean-enough and buffer free. Remember to flush any/all lines that use the buffer, including the column and all the detector! If not, you have a chance for your salts to precipitate onto the column which will lower it's lifespan over time, and this effect could happen realistically anywhere along the way if the right conditions are met. This includes having a high% organic portion of a gradient or wash sequence and you should always make sure that your buffer works at all of your gradient's concentrations. For example (and I don't expect this to be the case currently), when your method dips to a lower buffer percentage (~33% at 13min), you should assure that the salts are still soluble in the ACN/buffer mixture.

I hope some of this helped you!
Tyler


Hi Tyler,

You have no idea how touched I am at seeing your reply to my post T_T Thank you so much for your valuable inputs!! :mrgreen: I would like to clarify on a few more points:

If I were to incorporate a wash sequence at the end of the sample run and executed an "auto shut-down" of system after this and I come back the next day to start on another run (using the same analysis method), I have to purge the system first before starting right? My understanding from a colleague was that purging has to be done at the start of every analysis...

Regarding the method written in my previous post, I wanted to check if it is the correct way to write the time program for my method if I want to command the system to use only mobiles B and C through the whole analysis? Since other users in my lab mostly use water as their mobile phase A, I want to save them the trouble of switching the phases if i were to replace that with my buffer (i prefer to dedicate a flow line for buffer MP :lol: ) However, I do see that by default, users would assign A as aq solvent and B as organic solvent so in the time program, the command would state only the B conc.

Well noted on your point on the flushing! After flushing, I will store my column in 100% ACN (as recommended in manual) if not in use for several days or longer. So for my next use, will I have to condition my column first before starting to equilibrate with my mobile phases (buffer/ACN)? If so, do I condition with the same set of gradient ratios used in my analysis but without buffer (i.e. replace buffer with water) since there is risk of buffer precipitating out if I deliver it through the column (that contains 100% ACN) straightaway?

Thank you very much once again for your advices! God bless you! :mrgreen:
Hi penxx,

I'm glad I can help in any way possible! So for my auto shut-down procedure, I usually am dealing with samples that go through an auto-sampler and are included within a batch queue. The batch queue/batch sequence has an option to turn on the shut-down sequence, which then allows you to choose a method file yourself to implement during that shut-down procedure. After that everything will "turn off".

As for the purging prior to starting, my lab does not do this practice. This may be unique to me as I use the HPLC almost exclusively, so I understand every solvent and mobile phase on the column prior to using it. The mobile phases I use are quite tame as well so leaving a line for a little while (such as the purge-line) should not damage them (at least I have seen no issues or changes regarding my purge cycles). But if you have frequent mobile phase changes, and if you're using a buffer, it is always a good idea to flush each line to ensure they're ALL clean. It would also be wise to just purge the pumps if you don't know exactly what the last mobile-phase used was. I think purging at the start of EVERY analysis seems quite excessive though and a small waste of solvent.

Your method looks great, and yes it is quite smart to keep the buffer on the other line as it seems like your instrument is used by a variety of people. In this case, it does sound important to purge prior to analyses just incase of unknown mobile phases/other uses.

If you are taking any column out of long time storage then it could be smart to recondition using the recommended methods with a low-flow and without any buffer. You are quite right that when that buffered mobile phase contacts the 100% ACN, you will see some precipitation on the column. After you've conditioned and washed the column clean of ACN, then I would swap to my mobile phases and eq in that. A few recommendations for conditioning would be to select a low flow-rate method that has a long eq time at the end that matches your starting gradient (of the analysis you want to achieve next). But, for example when EQing after conditioning, this method does not HAVE to be your analyses' method. It could be a simple modified wash sequence with a long EQ that matches up to that of your starting gradient and has the same flow-rate.

Good luck on your chromatography!
Tyler
TylerSmith123 wrote:
Hi penxx,

I'm glad I can help in any way possible! So for my auto shut-down procedure, I usually am dealing with samples that go through an auto-sampler and are included within a batch queue. The batch queue/batch sequence has an option to turn on the shut-down sequence, which then allows you to choose a method file yourself to implement during that shut-down procedure. After that everything will "turn off".

As for the purging prior to starting, my lab does not do this practice. This may be unique to me as I use the HPLC almost exclusively, so I understand every solvent and mobile phase on the column prior to using it. The mobile phases I use are quite tame as well so leaving a line for a little while (such as the purge-line) should not damage them (at least I have seen no issues or changes regarding my purge cycles). But if you have frequent mobile phase changes, and if you're using a buffer, it is always a good idea to flush each line to ensure they're ALL clean. It would also be wise to just purge the pumps if you don't know exactly what the last mobile-phase used was. I think purging at the start of EVERY analysis seems quite excessive though and a small waste of solvent.

Your method looks great, and yes it is quite smart to keep the buffer on the other line as it seems like your instrument is used by a variety of people. In this case, it does sound important to purge prior to analyses just incase of unknown mobile phases/other uses.

If you are taking any column out of long time storage then it could be smart to recondition using the recommended methods with a low-flow and without any buffer. You are quite right that when that buffered mobile phase contacts the 100% ACN, you will see some precipitation on the column. After you've conditioned and washed the column clean of ACN, then I would swap to my mobile phases and eq in that. A few recommendations for conditioning would be to select a low flow-rate method that has a long eq time at the end that matches your starting gradient (of the analysis you want to achieve next). But, for example when EQing after conditioning, this method does not HAVE to be your analyses' method. It could be a simple modified wash sequence with a long EQ that matches up to that of your starting gradient and has the same flow-rate.

Good luck on your chromatography!
Tyler


Hi Tyler,

Thank you again for your reply! It has helped clarify a lot of my doubts! May I know the rationale behind conditioning at a low flow rate for a long eq? How low a flow rate do you mean? (0.5ml/min and below?) and by long equilibration, does 30 mins suffice?

For the column conditioning, I am planning to run at the starting gradient but replace buffer with water (eg. 92% water/8% ACN; in my method starting gradient is 92% buffer/8% ACN) at my method flow rate of 1mL/min for 30 mins? Following that, I will equilibrate with my mobile phases (92% buffer/8% ACN) for another 30mins. Will this be a sound plan? Appreciate your advice on this.

Thanks so much & wising you a good day ahead! :mrgreen:
Hi Penxx,

I condition at a lower flow-rate just because I read it out of some manual or online resource somewhere (probably out of a column manufacturer's booklet or something), but, and I do mainly prep LC at 20ml/min, I like to reduce my flow by half. For example, when using an older column that may have been stored for months or even years, I like to condition on a low-flow just in case something bad did occur during storage. Particularly in my lab, the last tech did NOT keep any notes whatsoever regarding mobile phase, column storage conditions, standard runs, etc. It was both tedious and scary as I was re-assessing some of these columns, in fact one of them was stored totally improperly and I could tell immediately from the low-flow pressure reading. As for equilibration, the timing really depends on the volume of your column and flow-rate. I would should for between 8-10 column volumes. If you don't know your column volume and want a good estimate, shoot a non-retained compound through and see how long it takes to elute. This will give you the dead time it takes for something to run through your column (or close enough) and allow you to estimate the length in time of those 8-10 column volumes. For example, I usually get away with roughly 10-15 mins eq on some of my analytical columns with perfect retention and no chromatographic differences on subsequent runs. The prep LC is a little more variable as the goal is more focused to collecting product, rather than perfecting retention or theoretical plates.

As for your conditioning, it looks quite fine. Depending on your conditions/columns you may be able to shorten the eq. time by quite a bit, but just remember to finish equilibrating (or equilibrate the entire time) in the starting mobile phase conditions of your actual analysis. You want the column's mobile phase to be homogenous every time for replicable runs. Please update me on how everything is going! I'm sure I could learn a thing or two from what you're trying.
TylerSmith123 wrote:
Hi Penxx,

I condition at a lower flow-rate just because I read it out of some manual or online resource somewhere (probably out of a column manufacturer's booklet or something), but, and I do mainly prep LC at 20ml/min, I like to reduce my flow by half. For example, when using an older column that may have been stored for months or even years, I like to condition on a low-flow just in case something bad did occur during storage. Particularly in my lab, the last tech did NOT keep any notes whatsoever regarding mobile phase, column storage conditions, standard runs, etc. It was both tedious and scary as I was re-assessing some of these columns, in fact one of them was stored totally improperly and I could tell immediately from the low-flow pressure reading. As for equilibration, the timing really depends on the volume of your column and flow-rate. I would should for between 8-10 column volumes. If you don't know your column volume and want a good estimate, shoot a non-retained compound through and see how long it takes to elute. This will give you the dead time it takes for something to run through your column (or close enough) and allow you to estimate the length in time of those 8-10 column volumes. For example, I usually get away with roughly 10-15 mins eq on some of my analytical columns with perfect retention and no chromatographic differences on subsequent runs. The prep LC is a little more variable as the goal is more focused to collecting product, rather than perfecting retention or theoretical plates.

As for your conditioning, it looks quite fine. Depending on your conditions/columns you may be able to shorten the eq. time by quite a bit, but just remember to finish equilibrating (or equilibrate the entire time) in the starting mobile phase conditions of your actual analysis. You want the column's mobile phase to be homogenous every time for replicable runs. Please update me on how everything is going! I'm sure I could learn a thing or two from what you're trying.


Hi Tyler,

Thanks for your detailed explanations! The information you've provided are certainly of great help to me. I have a clearer idea on how to go about doing my analysis now. Will get down to setting things up and I shall update you again on how it goes soon. Stay safe and take care! :mrgreen:
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