For difficult separations such as retinols and retinyl esters, a sub-ambient temperature programme has proven an effective method to achieve more efficient peaks and resolution
Typically, a solvent gradient is used to resolve all forms of vitamin A in a single HPLC analysis as the polarity range is large.
The Polaratherm total temperature controller from Selerity Technologies has demonstrated optimised results in the separation of vitamin A in biological samples by using a sub-ambient temperature programme, says the company.
A new application note (request no 813) is available free of charge from Selerity Technologies to describe the improvements in separation time and performance by using sub-ambient temperatures.
For difficult separations such as retinols and retinyl esters, a sub-ambient temperature programme has proven an effective method to achieve more efficient peaks and resolution.
Retinyl acetate and retinyl palmitate are both used for food fortification and vitamin A supplementation.
Retinyl acetate does not occur naturally in biological tissues, but is often used as an internal standard in HPLC analysis of vitamin A because it has the same absorbance characteristics, is inexpensive and readily available.
In biological samples, vitamin A exists as the free alcohol (retinol) and its esters with long-chain fatty acids (predominantly retinyl palmitate and retinyl stereate).
In most biological tissues, for example liver and kidney, the esters dominate, whereas high ester concentration in plasma is diagnostic of hypervitaminosis A.
One separation was done at ambient temperature and one was performed using a sub-ambient temperature programme.
The temperature programme was started at 0C, holding for one minute, ramping to 35C over six minutes, holding for 0.1 minutes, then ramping to 45C over ten minutes, and holding at 45C until the end of the run.
Using the temperature programme, a 60% reduction in analysis time and much sharper peaks for all analytes were attained.
Selerity's Polaratherm is a HPLC column compartment capable of controlling the temperature from sub zero to 200C benefiting from two modes of operation: temperature programming, and isothermal.
It is said to be the first column compartment that allows and regulates high, ambient, low or dynamic temperatures for selectivity tuning and improved HPLC separations.
This equipment is applicable to most vendor HPLC systems and can be deployed in any laboratory already operating HPLC at little additional cost, says Selerity.
