Lesson 6: Detectors for HPLC

The actual separation of each component in the sample is carried inside a column; however this separation needs to be “collected” for us to be able to see it. The detectors are used for this purpose. The separated components are monitored and expressed electronically. There is no universal detector that can monitor all compounds and there are many detectors used for LC analysis. Some are listed below.

Detector Type

Abbreviation

Chemiluminescence

CL

Conductivity

CD

Electro Chemical

EC

Evaporative Light Scattering

ELS

Fluorescence

FL

Mass Spectrometer

MS

Multi Angle Laser Light Scattering

MALS

Optical Rotation

        OR

Photo Diode Array

PDA

Refractive Index

RI

Ultra Violet

UV

Visible

VIS

1. UV, VIS, and PDA Detectors

The UV, VIS, and PDA detectors are categorized as absorbance detectors. They provide good sensitivity for light-absorbing compounds at ~pg level. They are easy to operate and provide good stability. UV detector is a very commonly used detector for HPLC analysis. During the analysis, sample goes through a clear color-less glass cell, called flow cell. When UV light is irradiated on the flow cell, sample absorbs a part of UV light. Thus, the intensity of UV light observed for the mobile phase (without sample) and the eluent containing sample will differ. By measuring this difference, the amount of sample can be determined. Since the UV absorbance also differs depend on what wavelength is used, it is important to choose an appropriate wavelength based on the type of analyte. A standard UV detector allows user to choose wavelength between 195 to 370 nm. Most commonly used is 254 nm. Compared to a UV detector, a VIS detector uses longer wavelength (400~700 nm). There are detectors that provide wider wavelength selection, covering both UV and VIS ranges (195~700 nm) called UV/VIS detector.

PDA detects an entire spectrum simultaneously. UV and VIS detectors visualize the obtained result in two dimensions (light intensity and time), but PDA adds the third dimension (wavelength). This is convenient to determine the most suitable wavelength without repeating analyses.

 

2. Refractive-Index Detector

 

RI detector measures change in reflex index. A glass cell is divided into two chambers (cells). The effluent from LC column flow through the “sample cell”, while other cell called “reference cell” is filled with only mobile phase. When the effluent going through the sample cell does not contain any analyte, the solvent inside both cells are the same (Figure 1A). When a beam is irradiate on the cells, the observed beam will be straight in this case. However, in a case the effluent contains any components other than mobile phase; bending of the incident beam occurs due to the reflex index difference between the two solvents (Figure 1B). By measuring this change, the presence of components can be observed.

 


Figure 1

 

RI detector has lower sensitivity compared to UV detector, and that’s the main reason why RI is not as commonly used as UV. However there are some advantages over UV detector.

    • It is suitable for detecting all components. For an example, samples which do not have UV absorption, such as sugar, alcohol, or inorganic ions obviously cannot be measured by a UV detector. In contrast, change in reflective index occurs for all analyte, thus a RI detector can be used to measure all analyte.
    • It is applicable for the use with solvent that has UV absorbance. A UV detector cannot be used with solvent which has UV absorbance. Sometimes the organic solvent used for GPC analysis absorbs UV, and thus UV detector cannot be used.
    • It provides a direct relationship between the intensity and analyte concentration.

The amount of UV absorbed depends on each analyte, thus the intensity of UV detector peak does not provide information on the analyte concentration. While intensity observed by a RI detector is comparable to the concentration of analyte.

Because of those advantages, RI is often used for the detection of sugars and for SEC analysis.

3. Evaporative Light Scattering Detector

ELSD provides good sensitivity for non-volatile analytes at ng level. The column effluent is nebulized and then evaporated to make it form fine particles. The analyte is then radiated with a laser beam and the scattered radiation is detected. The target sample includes lipids, sugar, and high molecular weight analytes. It is used in the similar way as a RI detector, but can provide more sensitive detection with stable base line. Another advantage is that ELSD can be used for the gradient method whereas RI cannot.

 

4. Multi-Angle Light Scattering Detector

For the SEC analysis, MW of analyte is estimated from the calibration curve drown using a set of known standards. However, by using a MALS, MW can be determined directly without the need of calibration curve. Also MALS can provide an absolute MW of the analyte with very low detection limit.

 

5. Mass Spectrometer

The analytes are detected based on their MW. The obtained information is especially useful for compound structure identification. However, its use is not limited to structure identification and can be used to quantify very low detection limit of elemental and molecular components.

 

6. Conductivity Detector

Solutions containing ionic components will conduct electricity. Conductivity detector measures electronic resistance and measured value is directly proportional to the concentration of ions present in the solution. Thus it is generally used for ion chromatography.

 

7. Fluorescence Detector

The advantage of fluorescence method is its high sensitivity for selective groups of compounds at ~fg level. By using a specific wavelength, analyte atoms are excited and then emit light signal (fluorescence). The intensity of this emitted light is monitored to quantify the analyte concentration. Most pharmaceuticals, natural products, clinical samples, and petroleum products have fluorescent absorbance. For some compounds which do not have fluorescence absorbance or low absorbance, they can be treated with fluorescence derivatives such as dansylchloride. The system is easy to operate and relatively stable.

 

8. Chemiluminescence Detector

Similar to FL, but instead of using a light source to excite the analyte atoms, the excitation is initiated by chemical reaction. Since it is not relied on the external excitation source, the noise is small, results in high signal to noise ratio, i.e. it provides even higher sensitivity than FL.

 

9. Optical Rotation Detector

Specific for the optical isomer measurement. The column can separate R- and L- type optical isomers, but the general detectors (e.g., UV) cannot distinguish which is R nor L. OR detector provides this information.

 

10. Electro Chemical Detector

There are several different types of ECs. The detection is based on amperometry, polarography, coulometry, and conductrometry. They offer high sensitivity, simplicity, convenience, and wide-spread applicability. It is especially suitable for the use with semi-micro or capillary type system.

 

 

References
Analytical Chemistry 7th (Seventh) Edition by Skoog 1999
Ekikurono Kotsu Detector (in Japanese) by Hiroshi Nakamura 2006