We will be transferring a HPLC method of the following parameters:
C18 column 150 mm x 4.6 mm, 5 μm
Solvent A: 0.5% phosphoric acid in water
Solvent B: 0.5% phosphoric acid in acetonitrile
40 minute run time, 0.6 ml/min, 30 degrees Celsius
Acids detected via UV at 210-230nm
Acids of interest: citric, malic, quinic, shikimic, benzoic
We hope to reduce our run time, which I have read can be done with a 50 mm column pretty simply. However, most recommendations from consumables companies have not mentioned this option.
We have been recommended:
1. Increasing the flow rate on the column received with the instrument (HyperSIL gold C18 column 150mm x 4.6mm, 3 μm) to 1.0mL and introducing solvent B immediately into gradient. I have tried this, but see many peaks in the beginning that are not present in my standards. Also, I have only been able to shorten to 24 minutes with this recommendation.
2. A 150 x 4.6 mm, 3 μm Phenomenex Luna Omega Polar column for higher polar selectivity, with 2.5 pH potassium phosphate buffer.
As a new technician and graduate student, my instinct is to use a 50 mm column with a smaller particle size and maintain the solvent gradient used for conventional HPLC with modifications. However I am quite inexperienced! Are there too many problems associated with transferring a method to a 50 mm column?
Any advice/references from experienced UPLC researchers and technicians would be greatly appreciated.
I suggest you to go for longer column than 50 mm. Why do not you try wirh the original type?
The attached file contains a chart in which you will find a general guideline for choosing your Concise Separations column
Udaya Jayasundara Hi Udaya! I have tried with the original column (150), and I am trying again today a gradient which will bring the run time down to 30 minutes. However, I think we want to cut our run time more significantly than that, down to ten minutes if possible! This is why I thought shorter column may help, but I am learning we may be able to achieve shorter run times with our current column. I can post updates.
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