April 20, 2001

Quantitative Chromatography

Methods Development

• (focus on GC but some examples are more applicable to HPLC)

Method Development

• This includes selection of column, detector, temperature, flow rate, detector type and settings
• It also includes the ways of processing the sample prior to injection
• It also includes the ways the data is interpreted, including the use of blanks and standards
• The method is often developed by regulatory agency (EPA) or by an industry group. It is expected that analyses will conform to this method.
• Sometimes. method development is simply finding what works best for you with your instruments and types of samples.
• Invariably, a critical element is testing the method for accuracy and reproducibility.

Sample Processing

Simplest cases:

• sample is a powder or a liquid with narrow range of possibilities
• sample is dissolved, diluted, injected
• comparison is to a set of standards prepared similarly from known pure compounds
• calculations are simple: area is proportional to amount of material,
• proportionality constant fixed by the standards

• uncertainty in weights and dilutions (problem with small samples; 0.1% typically)
• sample size (syringe): 5% with syringe, perhaps 2% with autosampler or training
• if instrument parameters change (inevitable in GC), may need fresh standardization
• realistically, peak area and calculations are 1%
• may have too many components for good analysis
• may include highly unwanted components that damage column

• solvent extraction: separatory funnel, water sample, hexane extraction
  • often pH seriously alters extraction (can use to advantage)
  • if hexane=20 ml and sample =200 ml there is 10X enhancement in concentration
    • may further concentrate by solvent evaporation
    • often want a small drying column or treatment (water removal)
    • may be able to eliminate problem species
  • probably need to dilute to volume

• more steps, each contributing to some sample loss (85-95% retention is typical)
• final volume measurement often 10% (we want tiny vol like 0.5 ml)
• still have syringe errors superimposed

• can use tubes of solid (diatomaceous earth)
• water soaks in, but stays put
• water proof filter at bottom (hydrophobic polymer)
• then draw water insoluble solvent through tube
• collect only the organic layer
• very simple mechanical process-- almost as quick to do multiple samples

• typically small tubes, plastic, volume of 3-10 ml
• also available as disks (like small filters)
• contains 1 g of HPLC column packing (or other solid)
  • 1. draw alcohol to wet the solid
  • 2. draw sample through
    • trap the organics typically
    • drain off water, ionics, ...
  • 3. rinse and draw a little air
    • option of new solvent to remove unwanted species
    • presumably analyte remains in tube
  • 4. Extract with suitable solvent
    • methanol, hexane
    • collect most organics
    • some species (long retention times) may remain in column
    • these would have been serious problems in HPLC or GC column
    • these go into the garbage
  • 5. extract (typically 3-5 ml)
    • dilute to volume and inject
    • perhaps evaporate to concentrate
    • redissolve and dilute to volume, inject

• adsorbant on small quartz fiber
• fiber immersed in sample; collects analyte
• use fiber to deliver entire sample to GC
  • (in needle like holder, inject and desorb)

Role of Internal Standards

• Multiple step processing can introduce 5-20% error
• Many ways to lose part of sample, uncertainty in volume
• Add 5% syringe errors for GC
  • (Not in HPLC-- why?)
• So relationship between peak area and amount is subject to 25% error
  • often, not that serious

• Accurately prepare mixture of analyte and IS
• Dilute and run Chromatograph
  • Results A(1)/ amt(1) = F(1,2) A(2)/amt(2)
  • that is, different response for the two species
  • evaluate F(1,2)
• Add species 2 (IS) at known amounts to samples (at earliest possible stage)
  • Process and then measure chromatogram
  • A(1) / amt(1) = F(1,2) A(2) / amt (2)
    • we know all but amt(1), calculate it now
• Advantage: F(1,2) generally not very sensitive to day to day variations
  • can use calibration data for weeks or months
  • may even be same for similar instruments
• Advantage: Loss and routine errors like syringe precision, dilution
  • same for both species, cancels out