Wednesday, January 22, 2020

Advantages and disadvantages of universal indicator solution


The pH indicator is a chemical compound that is added to the sample solution to easily determine the pH. The pH is the determination of the concentrations of hydronium ions in aqueous solutions. It is determined on a negative logarithmic scale from pH 0 to 14. Neutral solutions are pH 07, acidic is below pH 07, and basic is above 07 pH. Water is the best example of a neutral pH. The pH can be determined in different ways, such as pH paper, litmus paper, pH indicator and pH meter.
The advantages of universal indicator solution are as follows.
  • Basically, it is used to determine the estimated pH of a solution.
  • It covers the whole range of pH.
  • We can use simple acid-base indicators only to identify the nature of a sample, whether it is a base or an acid. But the universal indicator is used to determine the strength of a sample by indicating its pH value.
  • Like the pH meter, there is no waiting time for equilibrium, it gives fast measurements.
  • The universal indicator gives high accuracy results even with the weak buffer solutions.
  • If we follow the labeled instruction to store, it has good stability.
  • A wide range of colors is produced according to the strength of the acid or base.
  • This allows you to detect more things than an ordinary indicator.
The disadvantages of universal indicator solution are as follows.
  • When using liquid pH indicators, the sample solution should, preferably, be clear and colorless.
  • The pH meter is more accurate than indicators
  • The major disadvantage of the universal indicator solution is that it is non-reusable.
  • This can cause environmental pollution.
  • If the sample is contaminated, the result may be incorrect.

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Advantages and disadvantages of pH indicators

A variety of pH indicators are available which are chemical compounds and that are added to the sample solution to determine the pH. Typically, by adding a pH indicator it detects hydrogen ions and the color of the solution changes according to the pH level. Phenolphthalein, bromothymol blue, methyl red, and Universal indicators are the most commonly used indicators indicating a pH range of about 8 to 10, 6 to 7.5, 4.5 to 6, and 1-14 accordingly.

The advantages of pH indicators are as follows.

  • This technique of determining pH is quick, inexpensive, and easy.
  • They quickly determine the nature of the sample, whether it is acidic or a basic.
  • They save time and money produces fast results.
  • This is quick and easy using than pH meter.
  • The pH indicators are very simple to use, you just have to add a few drops to the sample.

The disadvantages of pH indicators are as follows.

  • The sample solution must be colorless enough to clearly observe the changes in the color of the indicator.
  • An indicator is not functional above its pH range because the indicator does not change color at these pH values.
  • If the substance or sample is contaminated, the color may be wrong.
  • Acid-base indicators show just one or two color changes.
  • Indicators measure pH at low accuracy, they only indicate sample acidity or alkalinity and not exact pH.


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Tuesday, January 21, 2020

How to reduce peak broadening in HPLC?

Peak broadening is an occurrence in high-performance liquid chromatography separation that decreases separation efficiency causes leading to poor resolution of analytes. The ideal peaks in chromatography are symmetric and Gaussian. Peak fronting or tailing appears in the asymmetric peaks. Peak fronting or tailing may occur due to poor quality of the HPLC column or dead volume of the system. 

Peak broadening is caused by a number of factors that are mentioned here, and avoiding them may help reduce peak broadening in HPLC. 

  • The peak broadening is influenced by the flow rate of the mobile phase and particle size of the packing material in the HPLC columns. The smaller the particle with the latter, the better is the peak resolution.
  • Small columns and high amounts of sample lead to peak broadening.
  • Ensure the injected volume and concentration of the target component in the sample.
  • The mobile phase composition in gradient mode is steep towers that change from polar to non-polar in very little time.
  • Precipitation in the sample causes slow dissolution and release of the component; you have to centrifuge and filter before loading into the injector.
  • Check the number of theoretical plates for chromatographic columns and regulate the load accordingly.
  • Use the appropriate wavelength of the analyte.
  • If the current flow rate does not work, then you can work with a higher flow rate to get symmetrical peak shape.
  • Ensure that there is no leakage between the column and the detector.
  • Too long tubing between the column and HPLC detector.
  • Sometimes the tubing ID is too large, which causes the peak to broadening.
  • Make it feasible to inject the sample into the same solvent that is used in the mobile phase.
  • Periodically check the performance of columns; older columns reflect peak tailing and other performance issues.
  • While using just water in the mobile phase, it may be essential to manage the pH to control the charge of molecules of interest.
  • Buffers are considered capable of improving the shape of the peak when selected at the proper pH level to control ionization.
  • You can reduce peak broadening by using different types of buffers such as acetate or phosphate buffer solutions, these adjusted to the suitable pH as per the pKa value of the molecule.
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Sunday, December 29, 2019

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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