Calibration

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Calibration

• Terminology
• Method Validation
• Accuracy and Precision
• Detection Limit
• Calibration Curves
• Method of Standard Additions
• Internal Standards

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Quality Assurance (QA)

• How do we ensure a quality analytical result?
• Check the calibration curve (validate it), using
  a separate check standard of known concentration
  from a source different than your standards.
• This is often called a QC (quality control or
  check) standard.
• Analyze replicate samples
• Should get the same result
• Analyze spiked samples
• Added analyte should produce more signal
• Analyze Reference Materials (e.g. SRMs)
• A valid method will return the certified
  concentration
• Use control charts to monitor instrument,
  chemist, and method performance.
• Review section 5-1 on your own.

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Terminology

• A method blank contains all reagents used in the
  analysis and is carried through the whole
  procedure, but without the analyte!
• A reagent blank contains all of the reagents used
  in the analysis but not the analyte and is not
  carried through the whole procedure. It is
  generally less valuable than a method blank.
• A field blank is similar to a method blank but is
  has been taken to the sampling site, preserved
  and held the same as the samples, etc.
• A calibration blank is the calibration standard
  containing no added analyte, but presumably all
  the other components in the standards.
• A spike is an aliquot of sample that the analyte
  has been added to to see if the response of the
  instrument increases.

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Terminology

• Specificity refers to the ability of a method to
  distinguish the analyte from other components. It
  is sometimes called selectivity.
• Linearity refers to the linear nature of the
  calibration curve and how it fits the points that
  originate from the analysis of the standards. A
  way of expressing it is the r-squared
  (correlation coefficient squared) value for the
  linear fit to the calibration curve.
• A calibration curve, regardless of its sub-type
  is a plot of Signal (y axis) versus Concentration
  or Amount (x axis).

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Ideally, your method has a linear response.

• The signal produced by the analyte is directly
  proportional to the amount or concentration of
  the analyte.

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Analytical Figures of Merit.

• Calibration Sensitivity- the slope of the
  calibration curve.
• A steeper curve responds more to slight changes
  in concentration.
• Linear Dynamic Range
• The range over which the calibration curve is
  linear
• Consider Beers Laws failure at high
  concentrations

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• Detection Limit (DL, LOD)
• The concentration at which the analyte can be
  reported as being present in the sample, at some
  level of confidence.
• The choice of k is arbitrary in some cases, but
  usually 3 is chosen. Why 3?
• If the standard deviation of the blank is in
  concentration units. The DL is in the same
  concentration units
• A detection limit should be calculated using
  standards made with a similar matrix to the
  samples. Unfortunately, sometimes instrument
  manufacturers use only ideal samples, reporting
  unrealistically low detection limits.

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• Limit of Quantitation (LOQ)
• The concentration at which the analyte can be
  determined with some level of confidence and
  accurately.
• k 10 ensures this signal or concentration is
  above that expected for any blank.
• MUCH more valuable than limits of detection!

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If you have a linear response.

• You can use a comparator method if you know the
  response is VERY linear over a VERY broad range,
  with few errors.
• OR, if an accurate result is not as important as
  a rapid analysis
• Comparator methods use a single standard

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• IF this relationship is not related without a
  correction, we normally use a response factor (F)

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• A standard containing caffeine at 100 ppm
  produces a HPLC peak area of 100,000 units. 5.0
  mL of a sample is diluted to 25.0 mL and analyzed
  by the same method on the same HPLC. It produces
  a peak area of 28,000 units. What is the
  concentration of caffeine in the original,
  undiluted sample?

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Calibration Curves

• Usually, however, you need to be sure your
  calibration range is linear (nearly proportional
  response). One then uses a calibration curve
  (most commonly with an external standard).
• The concentration range of the analyte is
  estimated.
• The matrix conditions of the sample are
  evaluated.
• A set of standards is prepared with the
  approximate matrix conditions you evaluated,
  covering a range of concentrations close to the
  estimate. One uses SERIAL DILUTION!!
• A blank is prepared containing no added analyte.
• The standards are analyzed and a calibration
  curve is plotted.
• Normally, one uses a calibration blank at least
  3 standards to prepare a curve.
• You want the standard concentrations to bracket
  the analyte concentration

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R-squared is a statistical evaluation of the
quality of the fit. The higher the R-squared
value (to 1), the better the fit (e.g. the data
is more linear compared to a fit with a lower
R-squared value). Emission Intensity 4968.5 x
(ng Pb/mL) 1143.4
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Other Calibration Methods

• Method of Standard Additions
• Used when the sample matrix is not well
  understood or varies from sample to sample.
• Standards are actually added to the samples,
  creating a new type of calibration curve
• Signal vs. Concentration of Added Analyte
• The X-intercept is the concentration of the
  analyte in the sample
• Covered more in CHEM 362!

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Internal Standards

• Used to correct for errors that alter the analyte
  signal unpredictably
• Usually these are instrumental errors (e.g.
  instrument instability)
• A known amount of a compound is added to each
  solution and kept constant
• The compound is an internal standard, and it
  should not be present in the sample prior to its
  addition
• The signal for the analyte vs. the internal
  standard is what is plotted for the calibration
  curve and the samples!

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