Column Evaluation

[00:00:02]every column should be tested for packing quality an efficiency test can evaluate the performance of the packed column it's an excellent tool to check and document the quality of a column over its lifetime it is also an effective diagnostic tool when performance problems occur this test can be done by running low molecular weight non-interacting solutes such as acetone or sodium chloride through the column and calculating the number of theoretic plates and the peak symmetry using these equations n / M = 5.54 quantity ve over W12 quantity squared by 1,000 l n equals the number of theoretical plates ve equals Peak illusion or retention volume W2 equals Peak width at 1/2 the peak height L equals bed height in millimet a s = b/ a as s equals a symmetry Factor b equals second half Peak width at 10% Peak height a equals first half Peak width at 10% Peak height this test may be done for all types of packed chromatography columns containing both absorptive and nonabsorptive media to perform this test you will need a 1% % volume per volume solution of acetone in distilled water equilibrate the column in distilled water apply a sample volume of between 0.5 to 1% of the total bed volume if using a standalone UV monitor with a flow cell diameter of 1 cm or less set it to between 0.1 and 1 absorbance units and run the column at the normal operational flow rate for the media you are using if using a standalone chart recorder set it to a speed that will generate an adequately sized Peak such as 1 cm per minute UV analysis and Peak presentations are automated in integrated chromatography systems such as the ecta the Unicorn system software for the ecta system will allow you to make efficiency calculations automatically if for application reasons such as in a regulated pharmaceutical production process acetone cannot be used a 0.8 molar solution of sodium chloride is a suitable alternative use a sample volume of between 0.5 to 1% of the total bed volume and detect the peak using a conductivity Monitor equilibrate and run the column As Above in 0.4 molar sodium chloride or a buffer that suits the media type you are using always refer to the instructions to to calculate the number of theoretical plates measure the retention volume ve and the peak width at 1/2 the peak height W2 the retention volume is measured from the point of injection to the center of the eluted peak to measure the peak width at 1/ half the peak height divide the peak in half from the averaged Baseline to the top of the peak measure the width at 50% of this height be sure that the values for retention volume and Peak width at 1/ half height have identical units that is measure in either volume or distance also measure the bed height in millimeters these values will allow you to generate the number of theoretical plates per meter or n / M this value allows you to determine how well a column is packed independent of column length the efficiency of a column is directly related to the bead size the smaller the bead size the higher the efficiency and the higher the number of theoretical plates per meter most media supplied have a number to which you can refer for evaluating the column's performance the instructions or product literature may say something like for a well-packed column you should expect 10,000 plates per meter for example if your number is significantly off of this value say less than 50% of the value then you may want to consider repacking the column a column will lose efficiency with age please note however that efficiency is a measure of all components in a system and not necessarily the packed column these variables include things such as tubing diameter and length the quality of the pump temperature and even how the numbers for the efficiency test are generated to measure the asymmetry Factor split the column in half lengthwise from the apex of the peak measure the column height at 10% off of the averag Baseline measure the width of the front half of the peak this is a and the back half of the peak this is B divide B by a and determine the asymmetry factor a symmetry is a direct measure of peak shape and can be used to determine among other things if a column has been over or underpacked that is if too much or too little pressure has been applied during packing a column is considered well packed when the asymmetry value is between 0.8 and 1.3 this range serves as a general guideline however a column May perform well with an asymmetry value outside these values if your asymmetry value is very far from this range and the peak appears highly asymmetric you may have to repack the column column packing is more of a science than an art this means that there are certain guidelines that must be followed to produce good results it also means that with the right raw materials adequate preparation and some practice anyone should be able to generate a well-packed column the chromatography column should not be an additional variable in your scientific experiments investing the time to prepare a well-packed column will provide you with ample reward in your purification efforts