Refraction and Mass Spectrometry

Refractive Index Detector (RID)

• Universal response and low sensitivity to dirt and air bubbles in the cells
• Can cover the entire refractive index range from 1.000 to 1.750 RI with a single, easily balanced cell
• Relatively low sensitivity and difficult to remove and clean or replace the cell when filming or clogging occurs

Mass Spectrometry Detector (MSD)

• Separates bunches or streams of ions according to their individual mass-to-charge (m/z) ratio and transfers by deflection through a magnetic field for counting and quantitation
• Detector generates a signal from incident ions by either generating secondary electrons, which are further amplified or by inducing a current generated by a moving charge
• All ions of a given m/z would impact at the same place on the photographic plate making a spot

Components of Mass Spectrometer

• Ionisation: Atom or molecule is ionised by knocking one or more electrons off to give a positive ion
• Acceleration: Ions are accelerated so that they all have the same kinetic energy
• Deflection: Ions are then deflected by a magnetic field according to their masses
• Detection: The beam of ions passing through the machine is detected electrically

Detecting Ions

• Ion stream most deflected will have the smallest value of m/z (the mass–to-charge ratio)
• Larger m/z value (the heavier ions if the charge is +1) are brought to the detector by deflecting using a larger magnetic field
• Magnetic field is varied, each ion stream can be brought in turn to the detector to produce a current which is proportional to the number of ions arriving
• Mass of each ion being detected is related to the size of the magnetic field used to bring it to the detector

Mass Spectrometer Output

• Output from the chart recorder is usually simplified into a "stick diagram" showing the relative current produced by ions of varying mass/charge ratio
• Vertical scale is related to the current received by the chart recorder - and so to the number of ions arriving at the detector
• Mass is measured on the 12C scale

Factors Influencing HPLC Performance

• Internal diameter of column: smaller in diameter, the higher in sensitivity
• Pump pressure: higher in pressure, the higher in separation
• Sample size
• Polarity of sample, solvent, and column
• Temperature: higher in temperature, the higher in separation

Quantitation

• Each peak for quantification must be identified so standards can be made
• Peaks must be sharp, narrow, well-separated from each other (resolved)
• Baseline should be relatively stable
• Peaks should be symmetrical (see peak tailing and broadening)
• Detector response must be linear in the quantitation range