HPLC-08

Know about Void volume and Dead volume

Void volume
The volume of mobile phase in the column is Void volume. If the stationary phase occupies 45% of total column volume, the void volume would be 55% of total column volume.

Dead volume
The volume between the injection point and detection point excluding the stationary phase in the column. It is also known as Extra-column volume.

HPLC-07

S/N Ratio & Baseline Noise 

S/N- Signal to Noise Ratio
The signal for analyte peak and baseline noise both contribute to S/N ratio.

Baseline Noise: It is the variation of baseline from the straight one caused by many factors like electrical signal fluctuations, lamp instability, temperature fluctuations, etc..

General info-10

Difference between Deamidation and Deamination

Deamidation
It is the removal or convertion of amide functional group in the side chain of amino acids like asparagine or glutamine to aspartic acid or glumatic acid.

Deamination
It is the process in which amine group is removed and converted to ammonia.

HPLC-06

Charged Aerosol Detector (CAD)

CAD is the universal detector used to measure the chemical compounds by producing charged aerosols which are detected by electrometer. Here the particles are measured based on the size of aerosols.

HPLC-05

Know the difference between UV/VIS, PDA and DAD detectors

 Don't think all these three detectors work on different principle. (refer HPLC-03 post for abbreviations) PDA and DAD are actually UV/VIS detectors and they have an array of photodiodes. Photodiode is used to convert light into electrical signal.The UV/Vis detector can detect  single wavelength at one time while the DAD/PDA can scan a wavelength range (200-800 nm). If you want to scan a wavelength range DAD/PDA are better or If you know the particular absorbance wavelength, go with UV/VIS dtector. So UV/VIS (PDA or DAD) are the most commonly used detectors in HPLC analysis because it is also very easy to analyse/interpret the peaks in the chromatogram.

HPLC-04

Principle of UV Visible detector

UV VIS detector works on the principle of Beer Lambert's law. It means the Absorbance is directly proportional to the concentration of the analyte. 

HPLC-03

Detectors used mostly in HPLC

The most commonly used detectors in High Performance Liquid Chromatography:

• UV Visible detector
• Diode Array Detector (DAD)
• Photo Diode Array detector (PDA)
• Charged Aerosol Detector (CAD)

HPLC-02

Peak Broadening

The tendency of a chromatographic peak to broaden as it passes through the column. It is also  known as peak spreading or peak dispersion. The peak width or the number of theoretical plates in the column (N) is measure of peak broadening.

General info-09

UV cutoff wavelength

Every solvent has a UV-vis absorbance cutoff wavelength. UV cutoff means the wavelength at which the solvent has an absorbance of 1 AU for 1 cm path length by using water in the reference cell.

Example: UV cutoff for acetonitrile and water is 190 nm.

HPLC-01

Why Column Temperature is the important factor in HPLC?

The maintenance of optimum temperature plays a vital role in the separation of analyte of interest from a sample. Temperature of column directly influences with the viscosity of the mobile phase. Increase in temperature of column decreases the viscosity of the mobile phase. Therefore the viscosity gets decreased, the flow rate becomes fast and it also reduces longitudinal diffusion. So, Optimum temperature (It shouldn't be very high or low) should be maintained for the better separation of peaks.