Instrumental Analysis: Detectors

Detectors in Instrumental Analysis

• Many components have absorption in the ultraviolet or visible region, with lmax selected for determining absorbance of required analyte.

UV/Vis Detector

• Uses absorption of UV/Vis radiation to detect analytes.
• Diode Array Detector (DAD) differs from UV-Vis detectors in that light from lamps is shone directly onto the flow cell, dispersed by the diffraction grating, and estimated for each wavelength in photodiode arrays.

Fluorescence Detector

• Analyte atoms are excited by a specific wavelength and emit light signals (fluorescence).
• The intensity of the emitted light is monitored to quantify the analyte concentration.
• Has higher sensitivity than UV/Vis detector.

Refractive Index (RI) Detector

• Monitors the difference in RI between the column eluent containing analyte and a reference stream containing mobile phase only.
• A universal detector in HPLC, detecting any solute with a different RI from the mobile phase.
• Advantageous for substances with no UV/Vis absorbance (e.g., aliphatic amines).

Mass Spectrometry (MS) Detector

• Operates by converting analyte molecules to a charged (ionised) state, with subsequent analysis of ions based on their mass-to-charge ratio (m/z).
• Offers higher selectivity, higher sensitivity, and detects compounds with no UV/Vis absorbance.

Chromatogram

• A plot of detector response versus elution time.
• Peak position on the time axis is used for identification, while peak area provides a quantitative measure.

System Suitability Parameters

• Calculated to prove the system is working perfectly, ensuring the validity of analytical results.
• Parameters include retention time (tR), retention factor (K), selectivity factor (α), tailing factor (T), number of theoretical plates (N), and height equivalent to theoretical plate (HETP), and resolution (Rs).

Void Volume and Retention Time

• Void volume (tm or t0) is the volume of mobile phase required to elute a molecule with zero retention in the stationary phase.
• Retention Time (tR) is the time from injection to peak maximum.

Retention Factor and Capacity Factor

• Retention factor or capacity factor (K) measures the retention of an analyte on the chromatographic column.
• A high K value indicates the sample is highly retained and has spent significant time interacting with the stationary phase.

Selectivity Factor and Separation Factor

• Selectivity factor or separation factor (α) measures how well the column separates two analytes.
• α = K2/K1 > 1, where K1 and K2 are the retention factors of peak 1 and 2.

Tailing Factor and Asymmetry Factor

• Tailing factor or asymmetry factor (T) measures peak tailing.
• Good peak shape is defined by a tailing factor of 1.0, with tailing caused by sample overload or secondary interactions between solute and stationary phase.
• T = (a+b)/2a, where a and b are front and tail widths at 5% of peak height.

Number of Theoretical Plates and Column Efficiency

• Number of theoretical plates (N) measures the efficiency of the column.
• A higher N value indicates a more efficient column.