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Questions and Answers
What does a higher α value indicate in chromatography?
What does a higher α value indicate in chromatography?
What is the term commonly used to describe column efficiency 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 unit of measurement for tr and w1/2 in calculating N?
What is the primary purpose of chromatography?
What is the primary purpose of chromatography?
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What is the effect of increasing α on resolution?
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?
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?
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?
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?
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?
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.
