Chromatography Forum

Could anyone, who used this technique, can explain to me why this is superior to conventional injection? It might precipitates on the column head and along the tubing? what is the advantage? Anyone interest in this technique, please refer to http://www.laboratoryequipment.com/uplo ... hrWAT2.pdf

one other possibility to increase sample loading on a prep column is to go like for flash and use a solid load cartridge
with normal phase applications where the pressure is lower then 20 bars this can be done using the flash plastic cartridges
i have seen a loading pump used for prep or even analytical where the mobile phase or a composition close to initial mobile has very little solubility capacity but does not do separation
then it is use to load on the column a huge amount of sample that his concentrated at the head of the column
after that the compound comes off the column when the gradient is used.
one case that comes to mind is the concentration of pesticides on c-18 column with water as sample solution.

Prep chromatography does not follow a linear scale up model. Generally when flow goes up 10x, sample volume goes up 100x and sample mass goes up 5000x. At-column dliution addresses the fact that a much larger relative amount of solvent is being applied to the column per injection. Since the solvent is typically strong compared to the initial mobile phase condition of a gradient run, the larger the injection volume the more broadening the sample will incur before traditional chromatographic theory starts taking hold.

I actually developed and patented a technique almost indistinguishable for SFC, years before at-column injection was marketed by Waters. My company, Berger Instruments, called it modifier stream injection. Of course Waters owns the vestiges of BI so they have the patent rights to this as well. Basically the technique moves the sample injection valve from mixed mobile phase stream into the strong solvent stream prior to mixing with the main solvent [in HPLC, water; in SFC, CO2]. The sample slug is entrained in the strong solvent stream as if it were just regular slvent and mixes with the main solvent right before the column. Hence, if the gradient starts at 20% strong solvent, the sample gets mixed with 4 parts water before entering the column.

Here's where it can get somewhat dicey. Remember, the concentration is now 1/5th what it was and if the strong solvent can continue to provide solvation in the mixture, you get a great benefit: your sample is delivered at the initial mobile phase strength rather than in pure strong solvent. As far as I can tell, this gives three major advantages. First, the sample behaves much more like an analytical injection. It will begin to elute at approximately the same time, the sides will have a recognizable gaussean shape and the impurities do the same thing. This allows you to use analytical screening to get very close to the conditions you need for prep. Second, the peaks are much narrower. Since the sample can concentrate at the column head, they are focussed. In SFC, I have seen late eluting peaks with volumes only about three fold the initial injection size [of course most of the CO2 has blown of by then]. Finally, no matter how much you inject [for bonded phases; not chiral films], the peaks always start at the same time with a very sharp rise. As you inject more and more volume the peaks get fatter and fatter. Think of them as thousands of overlapped very narrow Gausseans.

As a result, if you are doing large masses of purification, you do a few tracer runs to determine the distance between your peak of interest and the first impurity. These are, say, 100 uL injection on a 2 cm column at 25 mL/min. Now double the volume until the peak stops growing in height and just gets broader. Keep increasing volume until eiter the peaks meet or you see a pressure rise during injection. This is signaling some part of the sample wanting to crash out. If the method is isocratic, you can do this until the two peaks just touch at baseline.

Of course there is the other side of the coin, If you load the sample too much, it can just crash out when it sees water. In SFC we apparently had it easier. We only used methanol as our polar [strong] solvent, and it continues to solvate very well when mixing with CO2. By the time it got to the column, there was a lake of adsorbed methanol on the stationary phase to keep it in solution.

Good luck

KDF

Really appreciate your detailed explanation.
One question regards to your comments. I am really not sure we can increase 5000 times of sample loading capacity if column diameter is increased by 10 times.

Prep chromatography does not follow a linear scale up model. Generally when flow goes up 10x, sample volume goes up 100x and sample mass goes up 5000x. At-column dliution addresses the fact that a much larger relative amount of solvent is being applied to the column per injection. Since the solvent is typically strong compared to the initial mobile phase condition of a gradient run, the larger the injection volume the more broadening the sample will incur before traditional chromatographic theory starts taking hold.

I actually developed and patented a technique almost indistinguishable for SFC, years before at-column injection was marketed by Waters. My company, Berger Instruments, called it modifier stream injection. Of course Waters owns the vestiges of BI so they have the patent rights to this as well. Basically the technique moves the sample injection valve from mixed mobile phase stream into the strong solvent stream prior to mixing with the main solvent [in HPLC, water; in SFC, CO2]. The sample slug is entrained in the strong solvent stream as if it were just regular slvent and mixes with the main solvent right before the column. Hence, if the gradient starts at 20% strong solvent, the sample gets mixed with 4 parts water before entering the column.

Here's where it can get somewhat dicey. Remember, the concentration is now 1/5th what it was and if the strong solvent can continue to provide solvation in the mixture, you get a great benefit: your sample is delivered at the initial mobile phase strength rather than in pure strong solvent. As far as I can tell, this gives three major advantages. First, the sample behaves much more like an analytical injection. It will begin to elute at approximately the same time, the sides will have a recognizable gaussean shape and the impurities do the same thing. This allows you to use analytical screening to get very close to the conditions you need for prep. Second, the peaks are much narrower. Since the sample can concentrate at the column head, they are focussed. In SFC, I have seen late eluting peaks with volumes only about three fold the initial injection size [of course most of the CO2 has blown of by then]. Finally, no matter how much you inject [for bonded phases; not chiral films], the peaks always start at the same time with a very sharp rise. As you inject more and more volume the peaks get fatter and fatter. Think of them as thousands of overlapped very narrow Gausseans.

As a result, if you are doing large masses of purification, you do a few tracer runs to determine the distance between your peak of interest and the first impurity. These are, say, 100 uL injection on a 2 cm column at 25 mL/min. Now double the volume until the peak stops growing in height and just gets broader. Keep increasing volume until eiter the peaks meet or you see a pressure rise during injection. This is signaling some part of the sample wanting to crash out. If the method is isocratic, you can do this until the two peaks just touch at baseline.

Of course there is the other side of the coin, If you load the sample too much, it can just crash out when it sees water. In SFC we apparently had it easier. We only used methanol as our polar [strong] solvent, and it continues to solvate very well when mixing with CO2. By the time it got to the column, there was a lake of adsorbed methanol on the stationary phase to keep it in solution.

Good luck

KDF

My apology. You are correct.
Actual SFC conditions: Analytical: 2.5 mL/min on 4.6 mm column, 5uL injection of 1 mg/mL solution = 5 ug
Prep: 50 mL/min [20x increase] on 21cm column; 1 mL[200 fold increase]
injection of 50 mg/mL in MeOH = 50 mg [10,000 fold increase]

so while my ratios were correct, the actual values were not. You would need a 14.5mm column.
HPLC conditions would be about 2.5x slower for optimized flow in the same columns [4.6 and 21mm respectively]

These days people tend to run 21mm columns superoptimally in prep SFC at flows up to 100 mL.min.

I have put as much as 300 mg [100mg of 3 components in 2mL] with baseline separation on a 21mm column.
Of course, not all compounds are soluble to 50 mg/mL. Again, in SFC with modifier stream injection, it is likely easier.

Even with much more humble scale-up the issues remain. It is easy to solvent overload the column by applying larger and larger injections of strong solvent

KDF

No apology. you already helped me a lot.
Thanks.

My apology. You are correct.
Actual SFC conditions: Analytical: 2.5 mL/min on 4.6 mm column, 5uL injection of 1 mg/mL solution = 5 ug
Prep: 50 mL/min [20x increase] on 21cm column; 1 mL[200 fold increase]
injection of 50 mg/mL in MeOH = 50 mg [10,000 fold increase]

so while my ratios were correct, the actual values were not. You would need a 14.5mm column.
HPLC conditions would be about 2.5x slower for optimized flow in the same columns [4.6 and 21mm respectively]

These days people tend to run 21mm columns superoptimally in prep SFC at flows up to 100 mL.min.

I have put as much as 300 mg [100mg of 3 components in 2mL] with baseline separation on a 21mm column.
Of course, not all compounds are soluble to 50 mg/mL. Again, in SFC with modifier stream injection, it is likely easier.

Even with much more humble scale-up the issues remain. It is easy to solvent overload the column by applying larger and larger injections of strong solvent

KDF