What is a good test compound for normal phase HPLC in Hexane:Ethyl Acetate? I'm looking for a good test mix to make from common things in the lab for this to use on a silica column. I never used normal phase HPLC before. Also when I used HPLC grade ethyl acetate in my mobile phase I saw that it absorbs at 300nm but the literature value for the UV cutoff is 256nm, I wonder what is happening?
Hi Sasha,
here are some test mixtures from different vendors:
http://www.perkinelmer.de/CMSResources/Images/44-152609APP_Normal_Phase%20_Test_Mix_%20Brownlee_Analytical_Silica.pdf
http://www.restek.com/catalog/view/6111
https://books.google.de/books?id=6huy4XaToD8C&pg=PA98&lpg=PA98&dq=test+mixture+silica+normal+phase&source=bl&ots=Iwmx6uROPj&sig=EO5MK3uYi7ej_TXBLno_UwH-1qw&hl=de&sa=X&ved=2ahUKEwiXzIrq1MbcAhUDU1AKHeY2AoE4ChDoATAPegQICRAB#v=onepage&q=test%20mixture%20silica%20normal%20phase&f=false
When you are doing NP-HPLC be careful with the water content of your solvents. Fluctuating water content can lead to unreproducible results. Therefore, check the water content regularly with KF titration. Water traces can be removed quantitatively with 2,2'-dimethoxypropane. It reacts with water to methanol and acetone.
Regards Markus
O-,m-,p- nitroanilines.
Mixture of aromatic hydrocarbons with different number of benzene rings + saturated hydrocarbon+ olefin.
Regards
G
Ethyl acetate does not absorb 100% at 300 nm. It is relative to your reference. It usually leaves too little dynamic headroom at 256 nm and below for use in analyzing samples. We use it for TLC and in some Prep LC applications, but it sees very limited use as a mobile phase for NP analytical LC applications where UV/VIS detection is used. Best to select a more appropriate mobile phase solvent. Be sure and consult a proper table of HPLC solvent properties first: Link: " LC SOLVENT PROPERTIES " http://www.hplctools.com/lcsolvent.htm
"HPLC COLUMN TEST MIXTURE SOLUTIONS; NP & RP EXAMPLES"; https://hplctips.blogspot.com/2017/07/hplc-column-test-mixture-solutions-np.html
Hi William can you pleas explain what is dynamic headroom? I do not know this concept.
Thanks!
Hello Sasha. Yes, in simple terms, it refers to the remaining "space" available that can actually be used. In this context, it refers to the fact that ethyl acetate does absorb a lot of light in the low UV range (hexane does not). This has the effect that very little light is transmitted through the flow cell of the UV/VIS detector. The usable range of most UV/Vis detectors is under 1.4 AU (this value varies, but is approximate). If the ethyle acetate absorbs 1 AU of this, then the remaining linear range may only be 0.4 AU.
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