1/1/2023 0 Comments Reverse phase hplc![]() ![]() Today, however, intermediates and final products are increasingly polar. Historically, normal-phase flash chromatography has been the “go-to” purification tool for synthetic intermediates. Honestly, it sounds like you are not cleaning your column properly after each run (or poor sample prep), but only someone physically in your lab would be able to check everything to know for sure.An interesting question for synthesis chemists as the need for reversed-phase flash chromatography for both intermediates and final compounds increases. You need training to better understand what is happening and to learn yourself how to run and check a method. A colleague can help you with such basic issues better than a web group can. Is there someone at your place of work with experience ? Ask them for help. To the original poster: Get some help and training with the HPLC basics first. Try a 50 mm (5 cm) column." ?-? These just confuse the issue as such statements are wildly inaccurate and of no value in this case. Weird comments such as, "You do not use a gradient with a 250 mm column! It is far to sluggish to changes in the current mobile phase composition. It sounds like the user is also just starting out with HPLC so they may not even know what questions to ask or what information should be shared. Monitor at 214 nm and also 230 nm and 280 nm if you have a diode-array detector.Ħ1 is so often the case with these posts, not enough information is provided to render any intelligent responses. After 2-3 of those injections, carry out a blank gradient, injecting 10 uL of 25:75 ACN/water or the sample solvent used for the bolus injections.Īssuming you're not seeing too much carryover, try injecting 10 uL of 1 mg/mL of your protein in an appropriate sample diluent. A gradient from 10:90 to 80:20 ACN/water (with 0.1% TFA in each, or 0.08% in B and 0.1% in A) in 10 minutes with a hold for 5 minutes at 80:20, should be fine. Ensure that the protein is soluble in your sample solvent by dissolving it in 25:75 or 50:50 ACN/water with 0.1% (v/v) TFA (if you're using TFA as additive). Set the column temperature at at least 40 C or even 50 or 60 C, if possible.įor a column of your size, I'd recommend something like 100 uL of 1-2 mg/mL protein. What are your analysis conditions for this method? Mobile phase A? Mobile phase B? Temperature?įrequently with a brand new column that has never seen protein at all or the protein of interest, it is beneficial to make several bolus injections onto the column with a fast up and down gradient. ![]()
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