Chem. 231 – 2/18 Lecture

Announcements

• Set 2 Homework – Due Wednesday• Quiz 2 – Next Monday• Set 1 Labs

– should be switching instruments today (or after today)

– HPLC – instructions on setting up sequence for more efficient use of time

• Extraction Labs– SPE/HPLC lab completed (hand out)– I hope to have SPME/GC lab ready soon

HPLC – Using Sequences

• Demo showed how to analyze samples, one sample at a time

• Using autosampler to run a sequence is useful to:– run multiple samples in a defined way (e.g.

calibration standards after method optimization is done)

– run samples while doing other work (preparing GC lab samples or going home)

– if left running unattended, must use working “Exit” program

HPLC – Using Sequences

• Sequences will be kept in Sequence Folder

HPLC – Using Sequences

• Start a new sequence

HPLC – Using Sequences

• Sequence Parameters and Table Need Setting

HPLC – Using Sequences

• Sequence Parameters gives:• Data Location and names• Shut down can be set here (but we have

done this using an Exit program)

HPLC – Using Sequences

• Sequence Table gives:– auto sampler location/sample name– method– No. injections per sample– Info if using ChemStation Calibration

Add Exit method on last line to shut down

Questions IOn extractions, low performance LC, and

integration1. Fatty acids are being trapped using a C18 SPE column

and then analyzed by HPLC with a C18 column (both under acidic conditions). The elution order observed on the column is: C12, C16:1, C14, C18:2, C16, C18:1, C18. Efficiency tests with C14 show 98% recovery. Will all other fatty acids be retained with similar recovery? If not, which will have a risk of reduced recover?

2. Furans are recovered on a diol type SPE cartridge out of transformer oil diluted in hexane. They are eluted using 50% methanol, 50% water. They are then analyzed using reversed phase HPLC. How can you use the HPLC elution order to predict poor trapping or elution efficiency on the SPE cartridge.

3. If in the SPE analysis in question 2, the chemist forgot to wash the SPE cartridge with hexane after sample application, what would be the consequence?

Questions IIOn extractions, low performance LC, and

integration1. Under which of the following conditions would you want to

use low performance LC?a) separation of chemically similar natural products from a plant sample to obtain mg quantitiesb) separation of mg quantities of reaction products from chemically dissimilar reactants and side productsc) analysis of ng quantities of pesticides from food samplesd) separation of g quantities of reaction products from chemically dissimilar reactants and side products

2. When is it important to set the integration criteria carefully to distinguish between noise and peaks?a) when peaks have small signal to noise ratiosb) when many peaks overlapc) when peak width changes over the chromatogram

Questions IIIOn extractions, low performance LC, and

integrationLooking at the chromatogram to the right, and integrated as showna) Which peak has the largest percent integration error? b) Is the true area for this peak larger or smaller?

A

B

Quantitation in Chromatography

Levels of Detection and Quantification• Limit of Detection (LOD):

– minimum detectable signal can be defined as S/Npeak-to-

peak = 2 to 3.3σ – minimum detectable concentration = concentration

needed to get S/Npeak-to-peak = 2 or S/σ = 3.3– Calculate as 2N/m where m = slope in peak height vs.

conc. calibration plot– Minimum detectable quantity = (minimum detectable

conc.)(injection volume)• Limit of Quantification (LOQ):

– Calculated in similar fashion as LOD– Lowest concentration to give an “reasonable” conc. (e.g.

can be “auto-integrated” using software)– Typically 5∙LOD

Quantitation in Chromatography LOD/LOQ example

• For LOD and LOQ calculations, the peak height must be used as signal (not peak area)

• Determine the LODs and LOQ for the following example. Determine it for the 4.6 min peak if the concentration is 0.4 ng μL-1. Use the 3.3 and 2N LOD defintions.

Quantitation in Chromatography

Calibration• Software for Calculations

– Instrument Software (e.g. ChemStation)• typical method used in industry• requires setting up all work before running standards• often allows checks of calibration quality before

running samples

– Excel• we will use here for a number of reasons (only have

to learn one software program, example spreadsheets often available in texts, more data processing flexibility)

Quantitation in Chromatography

Calibration• Calibration Methods – using Excel

– External Standard – linear calibration• Standards prepared• Standards and Samples analyzed on same day• Use Excel spreadsheet in HW Set 1, problem 3 as an

example• Also, see p. 301-302 of Miller and p. 87-91 of Harris

(Quantitative Chemical Analysis) for examples– Non-linear Calibration

• With some detectors (S – FPD and ELSD) or when operating at high concentrations, non-linear calibration curves are needed

• Mostly can use Y = a + bx + cx2 (2nd order polynomial fit) or power fit (logY = b + mlogC or Y = 10bCm) methods

• Statistics to find uncertainty is much more complex

Quantitation in Chromatography

Calibration• Example of Power Fit

– Data for maltoheptaose (7 glucoses linked together) run with HPLC-CAD

– Linear fit appears not bad, but also not good

– Power fit shows clear non-linear exponent

Quantitation in Chromatography

Calibration• Calibration Methods – using Excel

– Internal Standard• See p. 303-304 of Miller and p.

109 - 110 of Harris Text• Needed for GC with manual

injection• Best results when external

calibration plot is replaced with area ratio vs. conc. ratio

– Surrogate Standard Method• See p. 300-301 of Miller• Used when no standard is

available• Will only work when response is

predictable (e.g. NPD with known #N atoms in surrogate standard and unknown)

• Predictable means either same response or same response on some basis (area/#Ns per molecule in above example)

Concentration

AX/AS

Conc. X (constant conc. S)

QuantitationOne Question

1. A scientist is using GC-NPD to quantitate hydrocarbons. The NPD is expected to generate equal peak areas for equal numbers of nitrogen in a sample. Determine the concentrations of compounds X and Y based on the calibration standard (pyridine).

Compound

C5H5N X = C6H6N2

Y = C8H10N4O2

Area 3520 299 1839

Conc. (ug mL-1)

10.0 ? ?