qualitative data analysis. 2 in this section, we will discuss: how to load data files. how to use...

Qualitative Data Analysis

2

In This Section, We Will Discuss:

How to load data files.

How to use Signal Options for data display.

How to apply annotation to your chromatogram.

Ways to identify components in your sample.

How to check the purity of a chromatographic peak.

3

Preferences – Signal Options

4

Display Files in Navigation Table

Double-click on sequence or Single Runs to display associated data files.

5

Load Signals using Navigation Table

Right click the mouse

anywhere on the row to activate menu

Click on the ‘+’ to activate signal details

Turn on/off Navigation

Table

Grouping can be customized

Data review tools

6

Load Signals using Menu

7

Signal Options

Use Signal Options... to define how you want a chromatographic signal to look when it is loaded or reproduced.

8

Signal Options Tools

Graphics Task Tool

Same Scale and each in Full ScaleCompound Names

Retention Times

Object TitlesAxis

Separate and Overlay of Signals

Baseline

Print Window

Copy to Clipboard

9

Use the Signal Tool set to graphically work with your chromatogram, then select one of the following:Align the x-axis of multiple signals

Align the y-axis of multiple signals

Reset the alignment of your signals

Create a 3D overlay of signals

Mirror signals

Subtract signals

Integrate all chromatograms

Signal Manipulation

10

Annotation

Options:Font and StyleFont SizeColor, Justification, Rotation

1. Select New Annotation.2. Click where you want the annotation to appear.3. Select Options.4. Type in text.5. Press OK.

11

Edit Annotation

Add Annotation

Draw Line in Window

Move Annotation

Delete Annotation

Annotation with Tools

12

Peak Identification

• Compare k’ values of the unknown with standards.

• Spike the sample with a standard.

• Use a fraction collector to obtain the unknown.

• Use a hyphenated technique such as LC-MS.

• Use a diode array to compare spectra of unknowns with standards.

13

Spike Sample

Analytical Education Center

H5929A 5-04

0 10 20 30 min.7 15 25

* Insulin?

* Spiked with Pure Standard of Insulin

Identification

Not Insulin*

0 10 20 30 min.7 15 25

0 10 20 30 min.7 15 25

14

Displaying Spectra

Display Spectra

15

Displaying Spectra

Selected Spectrumand Reference Spectra

Resulting Background Subtracted Spectrum

16

Spectral Task Tools

Select Spectrum at any time position

Select Spectrum at Peak Apex Position

Average a selected set of spectra

Select a set of Spectra of a peak

Select Spectrum to set as first reference

Select Peak to display Purity

Select Spectrum to set as second reference

17

Take peak spectrum

Compare with library

Print match factor

Match: 998

Chlortoluron ?

250 300

W a v e l e n g t h (nm)

250 300

W a v e l e n g t h (nm)

*Library Searching may be performed in an automated fashion.

Spectral Libraries: Principle

18

Unknown

Standard

Correlation = 0.92399

numerator =

Abs( (2 Abs Abs- * N

/

denominator = *

Match Factor = 1000 * numerator / denominator

1,i 1,i 1,i

Abs = Absorbance

of spectrum 1 at wl i1,i

Abs = Absorbance of spectrum 2 at wl i

2,i

N = number of wavelengths

i=1

i=n

Abs(

(2 Abs Abs- * N

/

2,i 2,i 2,i

i=1

i=n

i=1

i=n

i=1

i=n

i=1

i=n

i=1

i=n

Abs Abs Abs- **1,i 1,i2,i

i=1

i=n

i=1

i=n

i=1

i=n

Abs2,i

2

N o

r m

a l

i z e

d

100

80

60

40

20

0

250 300

W a v e l e n g t h (nm)

S t

a n

d a

r d

(m

Au)

1200

1000

800

600

400

200

0

U n k n o w n (mAu)0 50 100 150

Match Factor: Definition

19

UV spectra not very characteristic

UV spectra dependent on mobile phase (pH)

Non-separated compounds give wrong results

Overlay of sample matrix gives wrong results

No spectral libraries available

UV/VIS Spectral Libraries: Current Limitations

20

Creating a Library

Create a newLibrary in

Data Analysis

21

Search Results

Unknown and Library Spectrum

Overlaid

22

Peak Purity

Assess chromatographic purity by comparing spectra across the peak.

23

Analysis Indicates Peak Purity

Threshold Curve

Similarity Curve

Iso-Plot of Spectra for Selected Peak

3-D Display for Selected Peak

Purity Information

SpectralOverlay

Print

Exit

24

Purity Information - Peak Pure

25

Analysis Indicates Impurity

Three or more diamondsin red band

SimilarityCurve crossesThreshold Curve

26

Purity Information - Peak Impure

27

Compound Specificity Spectral Absorption of Matrix Compounds Spectral Noise Level Spectra Chosen for Comparison

Match factor influenced by:

For best results:

Use an intense lamp and clean flow cell Select the appropriate flow cell and slit. Collect an adequate number of spectra. Set the peakwidth on the diode array detector screen to the

width of the narrowest peak of interest. Use a sample concentration within the linear range of the

detector.

Peak Purity Performance