Saturday, December 28, 2019

Why phosphate buffer not used in LC-MS

LC-MS (Liquid chromatography-mass spectrometry) is an analytical technique in the chemistry that combines the physical separation abilities of LC with the capabilities of MS. The high-performance liquid chromatography separates the sample mixtures of molecules and mass spectrometry gives structural identification of each molecule with high detection sensitivity and molecular specificity.
It is suggested that the phosphate buffer should avoid in the mobile phase of LC-MS analysis due to their low volatility. A huge amount of buffers passed in ESI that are complicated to eliminate and large amounts of salt is formed in MS. This is the main reason to avoid this type of buffer. If your analyte contains a phosphate group, this is not the issue, because we use the analyte concentration in nanograms. You can use trifluoroacetic acid (TFA) as an alternative to the buffer as it is acidic and volatile.


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What are the causes of peak tailing and fronting

The gas chromatography and high-performance liquid chromatography aim to achieve good separation and symmetrical peak shape. Peak tailing and fronting are the most regular chromatographic peak shape issues in method development. Every chromatographer needs to find out how to fix these issues but first, let's identify what are the causes of peak tailing and fronting.
The common reasons for Peak fronting are given below.
  • Overloading of sample or column fouling:
Peaks fronting shows if the analytical column exceeds the sampling capacity, which can occur in both GC and HPLC separations.
  • Poor sample/peak capacity:
If the analyte does not have sufficient retention on the HPLC column, resulting in no proper separation taking place.
  • Saturation of the Detector:
Just as the shape of the peak may change with overloading the column, overloading the measurement range of the detector can also result in loss of signal saturation and accuracy.
Poorly packed column, channeling in a packed column, dead volume inflow path and mobile phase too weak are also causes of peak fronting
The common reasons for Peak tailing are given below
  • Flow path Diffusion:
Poorly fitted connectors/fittings, a column with a wrongly sized capillary connection line can all cause peak tailing.
  • pH dependence for ionizable molecules:
If there is a difference of 2 pH units between the pKa of the sample and the mobile phase, and when the sample is easily ionized, the peak tailing may result.
  • Overloading of sample or column fouling:
If the column is not washed properly after each analysis, it can form over time and change the surface chemistry of the stationary phase.
There can be many reasons for tailing and fronting. Some possible causes are:
  • Extra column effects such as overloading of column 
  • The flow rate of the mobile phase is too low
  • The void at the column inlet
  • If the concentration of buffer is too low
  • Change in the mobile phase composition
  • Blocked frit
  • Contaminated column
  • Sample reacting with active sites
  • Incorrect pH of the mobile phase /buffer

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How to increase peak response in HPLC

As we know that the high-performance liquid chromatography is a separation technique in analytical chemistry. In HPLC the system suitability parameters such as tailing factor, theoretical plate number, and resolution are most significant. The peak response is also important because the peak area is used to determine the concentration of molecules. Response factors are significant while using HPLC for quantitative analysis. There are different types of detectors are used in HPLC such as UV, PDA. The output signal of the detector is a function of the concentration of molecules passing through the detector cell.
Here are some points that help to increase the peak response.
  • Injecting more amount of samples or higher concentrations of the sample into the HPLC injector is one of the simplest techniques to increase the peak response.
  • The wavelength of the component should be confirmed using a UV/VIS spectrophotometer since if we select a correct wavelength it can increase the peak response in HPLC.
  • Peak volume is associated with column volume therefore a smaller column will also decrease peak volume and proportionally increase the peak height. An easy technique to decrease column volume is to decrease the diameter of the column and keep other column properties the same. An additional way to decrease the peak width is to use a column with a higher theoretical plate number. This is most easily done by particle size reduction.
  • Increasing the flow rate of the system will reduce the detector response therefore the flow rate should be appropriate.

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How to Determine Loading Capacity

Loading in chromatography refers to the quantity of material or sample introduced in an analytical column. The highest possible loading for baseline separation varies for each run and relies on the specific retention factor and separation factor, so it is not possible to determine the exact number based on how high the sample number.
To determine load capacity, load the largest volume of sample at a rapid rate until the analyte of interest no longer has the purity we require. Calculate the loading capacity by dividing the mass of the sample component (gm) by the mass of the media bed in the analytical column. After determining the loading capacity (mg or gm) slash by 10% and do again the test.