Figure 1: Steps in the analytical cycle. Adapted from reference 1.
In her February 2012 column installment titled "It's All About Selectivity," guest columnist Diane Turner introduced the topic
of how selectivity can be incorporated throughout the sample analysis cycle (1). Figure 1, borrowed from her article, nicely
illustrates the workflow in a typical sample analysis. In most analytical processes, chemists are looking for one or perhaps
a few analytes of interest, often in a very complex matrix. Having an analytical method showing sufficient selectivity to
analyze those few compounds of interest with the precision and accuracy required at the concentration level encountered is
the desired outcome of method development. The selectivity can be achieved anywhere within the analytical cycle (Figure 1)
during sampling, sample preparation, sample introduction, analyte separation, at the detector, or even during data analysis.
If the analytes of interest can be determined with good sensitivity, the presence of compounds from the sample matrix can
be tolerated as long as those interferences do not cause harm (short-term or long-term) to the analytical instrument or column
or, if determined to be harmful, they can easily be removed. An example of the latter could be backflushing after each analysis
to remove high-molecular-weight contaminants trapped at the head of a gas chromatography (GC) column. In Turner's column (1),
she gave very nice examples of how selectivity can be achieved at each step of the analytical cycle for GC.
Having less selectivity in one portion of the analytical cycle can be made up for by having greater selectivity in another
portion of the analytical cycle. For example, if an analyst has only a fixed-wavelength UV detector in his or her high performance
liquid chromatography (HPLC) instrument or a thermal conductivity or flame ionization detector for the GC system, there may
not be sufficient detector selectivity to provide the necessary overall method selectivity to measure an analyte of interest
without interference from undesired sample components. Therefore, additional sample preparation or finding a separation column
that provides more selectivity during the separation may be required to make up for the limitations in the detector. In these
cases, the analyst may spend a great deal of time and energy performing one or more sample preparation steps or optimizing
the selectivity of the column and mobile phase system (HPLC) to rid it of potential interferences. On the other hand, if one
has a very sensitive and selective detector, then perhaps spending a great deal of time optimizing the sample preparation
or the analytical separation is unwarranted.
Because achieving selectivity for the separation column is not an easy task to predict, sample preparation often gets the
brunt of the job to remove interferences from the sample of interest. It is sometimes unfortunate to burden analysts with
this job, but there are time-proven sample preparation techniques available. However, with the advent and widespread use of
tandem mass spectrometry (MS-MS) for both HPLC and GC with its high degree of selectivity and sensitivity, sample preparation
as well as the chromatographic separation can sometimes be simplified as long as any interferences carried over from the sample
matrix do not interfere with the separation or detection process. We term this simplified sample preparation process just-enough sample preparation.
This just-enough sample preparation process doesn't always provide the cleanest extract from the sample as more rigorous approaches
such as multimodal solid-phase extraction (SPE) or liquid–liquid back extraction might achieve but as long as the extractables
do not harm the separation or detection (and, of course, the column or instrument), that's okay. In reality, the sample preparation
time can be greatly reduced as long as the final outcome meets the needs of the analyst. Although the mass spectrometer still
represents a much higher priced detector than a UV or flame ionization detector, many laboratories are finding them to be
a cost-effective way to enhance and speed up their analyses, thereby improving overall productivity and lowering costs. Of
course, less-expensive selective detectors such as fluorescence in HPLC and electron capture in GC still allow the practice
of just-enough sample preparation provided the analytes do not need derivatization.
Figure 2: Just-enough sample preparation represents a continuum of methodologies.
The concept of just-enough sample preparation does not imply one is cutting corners or that more sophisticated protocols are
not required. It really represents a continuum of sample preparation procedures as depicted in Figure 2. This figure represents
just a few of the many sample preparation methods that are in widespread use. Starting at the top of the figure with filtration,
centrifugation, and "dilute and shoot" and moving down, the sample preparation protocols become more selective and more complex,
sometimes requiring a greater deal of effort and multiple steps to achieve just enough cleanup to meet the analytical needs.
Minimizing the number of sample handling steps in any analytical technique is desirable since the more times the sample is
transferred, the greater the chance of analyte loss (or modification), thereby resulting in poorer analytical precision and
accuracy. If one or two steps meet the needs of the method that may be sufficient, but, in some cases, additional sample preparation
steps may be needed to get rid of interferences. The need to eliminate or minimize interferences is no greater than that required
for liquid chromatography–mass spectrometry (LC–MS) and LC–MS-MS (see below).
Figure 3: Striking the right balance in sample preparation.
Figure 3 shows a pictorial representation of the just-enough sample preparation concept that actually applies to the entire
analytical cycle, but is emphasized for the sample preparation portion. It is here that many workers are faced with achieving
the bulk of their selectivity enhancement. Ideally, in an analytical method one always wants to achieve the best result with
the least amount of effort and investment. On the other hand, the actual data requirement may not require the ideal result
but rather an acceptable result. For example, in screening hundreds of urine samples for the presence of drugs of abuse, most
samples are negative. Thus, a qualitative analytical method may be sufficient to rule out the presence of an illicit drug.
However, if an illegal drug is spotted during the screening test, then a more careful and perhaps more sophisticated look
at a positive sample is required for quantitative analysis.
There are many other factors that may influence the choice of the sample preparation techniques used to provide just-enough
cleanup. An analyst's skill and knowledge are important. The availability of instrumentation, chemicals, consumables, and
other equipment; the time available to develop a method and to perform the tasks at hand; the complexity and nature of the
matrix; the analyte concentration level and stability; the required sample size; the cost per sample (budget); and the safety
of the sample preparation technique are just a few of the many considerations that must be taken into account. It is the balance
of all of these and other considerations that come into play.
Perspectives in Modern HPLC: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: Seven Common Faux Pas in Modern HPLC