Questions and Answers
What does a higher α value indicate in chromatography?
What is the term commonly used to describe column efficiency in chromatography?
What is the unit of measurement for tr and w1/2 in calculating N?
What is the primary purpose of chromatography?
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What is the effect of increasing α on resolution?
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What is the value of H that results in a high value of N?
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What is the effect of increasing N on resolution?
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What is the resolution value at which two peaks are considered fully separated?
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What is the term used to describe the distance between two peaks?
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What is the formula to calculate N from a chromatogram?
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Study Notes
Quantitation in Chromatography
- Detector response depends on the concentration of analyte or mass of analyte reaching the detector.
- Most detectors give a linear response over a portion of the detectable range.
- Detector noise is present in all detectors and has high and low frequency types.
Levels of Detection and Quantification
- Noise can have high and low frequency parts, and can be defined in two ways: peak to peak (roughly 5σ) and standard deviation (more accurate).
- Signal is measured as peak height.
- The ability to detect small quantities depends on the signal-to-noise ratio.
Data Smoothing
- Data should be digitized with a frequency of approximately 20/peak width.
- High frequency noise can be removed by filtering, but over-filtering can result in reduction of signal and loss of resolution.
Integration
- Integration of a peak should give peak height, peak area, and peak width.
- Difficulty comes from determining if a peak is a peak (or just noise) and when to start and end the peak.
External Standard
- Known amounts of analytes are run in a separate analysis, and the resulting peak areas are used to obtain calibrated response factors.
- The response factors are stored in a calibration library and used to calculate analyte concentrations in later runs.
Internal Standard
- A known and constant quantity of a compound (not one of the analytes) is added to the sample.
- The ratio of its retention time to the retention times of the analytes is used to calculate the unknown concentrations of the analytes.
- Internal standards should be of the same family as the target compounds but have different retention times.
Efficiency
- Efficiency is a factor that describes peak width, with high efficiency resulting in narrow peaks.
- The term used to describe column efficiency is "number of theoretical plates" or N.
- N can be measured from the peaks on a chromatogram and is calculated as N = 5.54 (tr / w1/2).
Resolution
- Resolution is the separation or distance between two peaks and is a function of retention, selectivity, and efficiency.
- Resolution can be calculated as Rs = ¼ (α-1/α) (k/k+1) N½.
- Increasing α, k, or N increases resolution.
- Resolution can also be calculated from actual measurements of peak retention times and measured peak widths.
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Description
Learn about the importance of reproducible sample injection sizes and detector responses in chromatography quantitation methods. Discover the benefits of using external standard methods with autosamplers.
