Selecting the best HPLC wavelength(s) to monitor during an analysis method for use in quantitation and/or purity determination requires both knowledge and careful attention. Here is the basic procedure to use:
Step 1. Create the Method. To determine which UV/VIS
detector wavelength(s) should be chosen for the analysis of your sample, you
will first need to create a general HPLC Method which retains and resolves the compound(s)
of interest on the column (goal is a K prime of >2.0, less than 10.0). Be
sure and utilize a scanning diode array detector in full scan mode (often
referred to as a photo-diode array detector, PDA or DAD) to scan all relevant
wavelengths of your samples (e.g. 210 to 450nm). Note: Your choice of mobile phase and detector settings will effect the S/N values.
Step 2. Determine the lambda max of the sample's spectra using the Data analysis software. Once you have completed the analysis, review the
spectral data to determine which prominent peak wavelengths have the
maximum signal to noise (S/N) ratio. These “peaks” can be used as the individual
wavelengths for integration and purity determination. By sure and double check
that any detector options which use a “reference wavelength" are turned ‘OFF’ when
running these methods (more info on “reference wavelengths” can be found on
this blog in another post). With the wavelength selected, chose an appropriate bandwidth for use (narrow).
Step 3. Edit the HPLC method to use the discreet wavelengths found in step 2. Whenever you run a real sample, continue to use the
full scanning mode of the detector so you will know about any other components
which absorb at wavelengths far away from and/or near the peak wavelengths. These
compounds can add or subtract signal from the main peak making it appear to be
more or less concentrated (or more or less pure) than it actually is. If you
only monitored the sample with a single wavelength detector, then you would
miss this vital information and make errors in your purity or concentration
determinations.
Conclusion. (1) Using a multi-wavelength, scanning HPLC detector such as a DAD is one of
the most important tools you can use to create accurate and reliable chromatography
methods. Always use a scanning DAD for method development to prevent errors. (2) Learning how to correctly use and set up the detector's settings, parameters, special features and
options may prevent false
or misleading results. Only after you have developed a reliable and repeatable method with good sample retention and peak shape can you begin to report accurate integration and concentration values (and/or make UV/VIS "purity" determinations).
What is we have two samples which use the same wavelength. How do we detect one from the other?
ReplyDeleteUse good chromatography fundamentals and resolve the two peaks apart first (e.g. baseline resolution > 1.5). This is the goal of good method development. Additionally, be sure and look at the spectral data obtained for each peak. They may be different, and if so, can aid in identification.
ReplyDeleteOn selecting the proper wavelength can I first make A1% conc. by spectrophotometer and determine the lambda max and take this as my wavelength
ReplyDeleteIF your compound is a 100% chemically pure standard, then yes. However, we are normally running HPLC methods because a sample contains a mixture of components. Placing a sample containing a mixture of compounds into the spectrophotometer will result in a mixed spectra, not a pure spectra of one compound. So that would not provide the lamdba max for the sample. As stated above; "To determine which UV/VIS detector wavelength(s) should be chosen for the analysis of your sample, you will first need to create a general HPLC Method which retains the compound(s) of interest on the column".
DeleteAnalyzing a mixture in a spectrophotometer cell before developing an HPLC method (or Scanning Diode-Array-Detector...) can provide some useful information. You can read about that right here in a related article, "Method Development Hint: Use your HPLC Diode Array Detector (DAD or PDA) as a Spectrophotometer"; Link: [ http://hplctips.blogspot.com/2013/09/method-development-hint-use-your-hplc.html ].
How can i check my detector is ok
ReplyDeleteRun the detector's Operational Qualification (OQ).
DeleteWhat does lambda max refer to?? Is it Specific for the compound (through which i can make sure that this peak is of certain compound ) or it refers to the concentration of the compound and will change with the concentration change???
ReplyDelete"lambda max" is the term which refers to the wavelength of maximum absorbance for the compound. This can be determined by scanning all wavelengths and viewing the resulting spectral data.
Delete