You probably use UV absorption detection for most of your
routine HPLC methods. Indeed, it is simple to use and maintain, and sensitivity
is often suitable for many typical analyses. Not every compound can be detected
by UV, however. If a compound lacks chromophores, detection can’t be achieved
at any wavelength. In these cases, you will need to use an alternative method.
LC-MS
has become more prevalent in recent years, as advances in technology have
allowed for greater ease of use, reliability, and detection limits. Even so, it
is relatively sophisticated and expensive instrumentation, and many QC
laboratories might find it more than is required for routine assays where great
sensitivity is not required. A good example of this is the food and beverage industry,
where refractive index might be more suitable for typical analysis goals. In
these applications, levels of ingredients are often relatively high, and the
high sensitivity of LC-MS may not be needed. Although refractive index
generally has much lower sensitivity, it is often suitable for these food and
beverage applications. Unlike the complex apparatus used in LC-MS, all that is
required for refractive index is to flush the reference cell with the mobile
phase and to use a thermostat to avoid baseline drift.
I
investigated the use of refractive index detection for a taurine application. I
was able to observe a nice peak of the standard and obtain good retention. This
latter point is important because taurine is very polar and hence difficult to
retain by reversed phase methods. With the Cogent Diamond Hydride column, the
compound could be readily retained by an ANP mechanism. I demonstrated the ANP
behavior by comparing retention times at 70 and 80% acetonitrile; retention
increased at higher organic content.
So I
used two strategies to address the analysis of taurine. The first was the use
of a “universal detector,” refractive index, suitable for detection of any type
of compound, whether UV-absorbing or not. The second strategy dealt with the
retention. Here, I used ANP chromatography to readily retain a compound that
might be poorly retained by traditional retention modes.
