Advanced Techniques for PDA Analysis

Advanced Techniques for PDA Analysis

Typical Extracted Chromatogram

How Does PDA Determine Purity?
Identifying and extracting co-eluters is not easy, it requires: good optics bench and a good software algorithm. Peak purity or peak homogeneity analysis compares spectra within the same peak. All the spectra from peak liftoff to touchdown are analyzed for spectral differences compared against the apex spectrum which acts as a reference spectrum. Each of the other spectra are compared to the apex spectrum. The actual number of spectra compared is dependant on the peak width and the sampling rate used in the analysis (eg. 2 spectra per second). In our simplified example shown here, we compare the apex to the leading and tailing edge of this peak. If the spectra are all similar, the peak is spectrally pure or homogeneous. (This is not proof of chemical purity.) The peak purity analysis produces a single value, the Peak Purity Angle. This is the weight mean average of the spectral differences across the chromatographic peak. The spectra with the most absorbance affect the Purity Angle more than the spectra with very little absorbance. A Purity Angle of 0 degrees indicates spectrally pure. A Purity Angle of 90 degrees is maximal difference. Purity angle values can then be compared to a sample of the spectral noise, (the noise at every wavelength monitored by the Waters 2996) When the angle is larger than the threshold, its is an indication the the peak is not spectrally homogenous.

Utilizing the power of the PDA

Outline Mobile Phase Hardware Parameters Peak Purity
Spectral Libraries

Solvent Information All wavelengths reported in nm.

Solvent Information 0.1% H3PO4 190 0.1% TFA pH 1.92 205 1% TEA 230
1% HOAc 232 acetate pH phosphate pH tris pH borate pH All wavelengths reported in nm.

Blank Gradient Mobile Phase Blank - Milli-Q water Blank - DI water
Poor Water Quality Blank Gradient Blank - Milli-Q water Blank - DI water 100% H2O to 100% ACN

Spectra of Mobile Phase Components
nm 200 240 280 0.4 0.0 0.8 1.2 1.6 2.0 Acetonitrile Methanol 254 nm 1 AU 2.4 0.1% Phosphoric Acid 1% Acetic Acid Some solvents have significant absorbance below 254 nm. Problems occur when methods developed at 254 nm are altered to a low er wavelength in an attempt to gain greater sensitivity. If the background absorbance is too high there is a loss of linearity and increase in baseline noise. Solvent bottles are labeled with a "UV Cutoff". This is the wavelength where there is 1 AU of absorbance. Highly transparent solvents should be chosen based on wavelength requirements for the analyte. Acids, bases and buffers may have significant absorbance. Their absorbance will often be pH dependent. Always choose the most UV transparent solvent for the best sensitivity.

Compound and Solvent Spectra
2 Absorbance Wavelength 190 350 Compound Mobile phase Observed The compound and the mobile phase have absorbance At low wavelengths the observed AU is out of detector range When there is mobile phase absorbance, that background is added to the absorbance of the compound to produce the observed spectrum (top line).

Effect of Concentration on Spectra
Absorbance 190 210 230 250 270 290 nm 2.0 1.0 0.5 1.5 Different concentrations Spectral shape changes with concentration Linearity also affects spectral analyses. The shape of the spectrum of a pure compound can be affected by its concentration. At the lowest concentration the wavelength maxima at the 195 nm and 227 nm are in a 2:1 ratio. At the highest concentrations the ration is 1:1. These spectra are different for the same pure benzoic acid. If you are using the spectra for compound identification, at high concentrations you could misidentify the peak because the spectra appear different Peak purity analysis actually is spectral homogeneity. If the spectra appear different, the software will flag the peak as impure, even when it is chemically pure.

Summary Make sure that the solvents are filtered
Remember the highest UV Cutoff of the mobile phases you plan on using The observed spectra is a combination of mobile phase and analyte Concentration of analyte will give a different spectral profile

Mobile Phase Hardware Parameters Peak Purity Spectral Libraries

Photodiode Array The diagram show the generic layout of a photo diode array detector. All of the energy from a broad spectrum lamp (or lamps) usually covering both the ultarviolet and visible wavelength range is passed through a flow cell. The light is then split into its component wavelenghts on a grating. A series of diodes measures the light intensity over the entire wavelenght range. The result is two types of data, a traditional chromatogram (time vs absorbance) at any wavelentgh in the operating range, and a spectrum (wavelength vs absorbace) at any given time point. Data collected from a PDA detector is often refered to as 3 dimensional data.

2996 Operation in 3D mode 3D mode for spectral information. Used for peak purity and library matching. Set Wavelength range appropriately Select Sampling Rate Resolution Values 1.2 to 24nm Select Auto Exposure Set Digital Filter Filter Response Range 0.1 to 3.0 The 2996 can be operated in the 3D mode OR the 2D mode. It cannot collect 3D data when set for the collection of discrete 2D channels. This is something to be aware of when in a competitive situation with Agilent. The 1100 DAD can collect 5 channels of 2D data and 3D data at the same time. However, the 1100 DAD has the very large disadvantage of dependence upon a reference wavelength. Agilent employs the reference wavelength as a means to eliminate refractive index effects and to improve baseline noise. The reference wavelength opens the user up to the real possibility of deleting sample peaks.

2996 Operation in 2D mode 2D mode allows up to 8 discrete channels of data. Ease of use. Do not have to extract a wavelength Bandwidth for each 2D channel of data can be entered. Vary bandwidth for best signal to noise For developing methods being transferred to TUV The 2996 can be operated in the 3D mode OR the 2D mode. It cannot collect 3D data when set for the collection of discrete 2D channels. This is something to be aware of when in a competitive situation with Agilent. The 1100 DAD can collect 5 channels of 2D data and 3D data at the same time. However, the 1100 DAD has the very large disadvantage of dependence upon a reference wavelength. Agilent employs the reference wavelength as a means to eliminate refractive index effects and to improve baseline noise. The reference wavelength opens the user up to the real possibility of deleting sample peaks.

Collect the data Multiple runs for 3D mode at different resolution values 2D mode 1 run with various resolution values Collect 3D data for spectral information which will be used to optimize wavelength settings for your analysis HPLC: Alliance 2695 with degasser, column heater and sample chiller Detector: 2996 PDA 210 to 400nm Resolution: 1.2nm 5 pts Digital filter: 0.5 Column: 3.0 x 50mm Xterra RPC °C. Mobile Phase: 60%Water40% Acetonitrile on line mixing Flow: 1.2 mL/min. Sample: Waters PQ mix. 10uL Analysis were run in triplicate.

Influence of bandwidth (resolution) upon signal intensity
Notice that as bandwidth in increased the signal decreases. This occurs because you are averaging the diode of lambda max with ones of less intensity. W nm-1.2 W nm-35.0 Shown is an overlay of 8 channels of data collected from the same injection of the pq mix. As the bandwidth is increased the signal intensity is reduced. This occurs because you are averaging the diode of lambda max with ones of less intensity. However, as the bandwidth is increased the noise is being averaged out at the same time.

Optical Resolution (Bandwidth)
Benzene 230.00 250.00 270.00 nm Absorbance 1.2 nm 3.6 190 6.0 Spectral resolution (or Bandwidth) is the wavelength interval (in nm) between data points in an acquired spectrum. The minimal resolution of the 2996 detector is 1.2 nm. As an example, in 3D mode, the 2996 averages 3 adjacent diodes for each reported wavelength when the spectral resolution is set to 3.6 nm. Less resolution at 3.6 nm vs. 1.2 nm UV maxima shifted Good optical resolution gives good quality spectral information

Influence of bandwidth (resolution) upon Spectral Resolution
Spectral Resolution is also reduced as the bandwidth is increased 3.8nm 2.4nm 1.2nm As the bandwidth is increased the fine spectral differences are lost.

Resolution Versus Chromatographic Performance
Higher resolution => higher number of data points (over the same wavelength range) High resolution => better linearity Lower resolution => lower data file size Lower resolution => lower spectral resolution 14.4 nm Spectrum of Caffeine 1.2 nm Many people select lower resolution (a larger bandwidth) hoping to increase signal strength. This is not always the case due to data averaging. In some cases, however S/N may be improved using the larger bandwidth.

Influence of bandwidth (resolution) upon baseline noise
Noise is also reduced as the bandwidth is increased. As the bandwidth is increased the noise is being averaged out. Noise decreases.

Impact of bandwidth (resolution) upon baseline noise and area
The noise falls off at a faster rate than the signal. The result is the optimization of the signal to noise at a bandwidth which loosely corresponds to the width at ½ height. A wide bandwidth has the advantage of reducing noise by averaging absorbance over a wavelength range. As the bandwidth is increased the signal intensity is reduced (peaks become smaller). This occurs because you are averaging the diode of lambda max with ones of less intensity. However, as the bandwidth is increased the noise is being averaged out. The noise falls off at a faster rate than the signal. The result is the optimization of the signal to noise at a bandwidth which loosely corresponds to the width at ½ height.

Impact of bandwidth (resolution) upon signal to noise
Signal to Noise vs Area 1.50E+03 2.00E+03 2.50E+03 3.00E+03 3.50E+03 4.00E+03 4.50E+03 5.00E+03 1.2 2.4 5 7.5 10 12.5 15 20 22.5 25 27.5 30 32.5 35 37.5 bandwidth signal to noise 240000 260000 280000 300000 320000 340000 360000 380000 400000 area uV at 242nm area at 242nm Note that the signal to noise rapidly increases then plateaus before falling off again. Select bandwidth in this plateaus region for most sensitive analysis. Notice that the signal to noise plateaus

Compare the data Channel Name Baseline Noise Area S/N
W nm -1.2 0.0450 3.89E+05 2.74E+03 W nm -2.4 0.0355 3.90E+05 3.45E+03 W nm -5.0 0.300 3.88E+05 3.96E+03 W nm -7.5 0.0285 3.85E+05 4.00E+03 W nm -10.0 0.0270 3.80E+05 4.03E+03 W nm -12.5 0.0255 3.74E+05 4.39E+03 W nm -15.0 0.0265 3.67E+05 4.07E+03 W nm -20.0 0.0250 3.49E+05 4.06E+03 W nm -22.5 3.27E+05 3.62E+03 W nm -25.0 3.16E+05 3.53E+03 W nm -27.5 0.0275 3.05E+05 3.48E+03 W nm -30.0 2.93E+05 3.32E+03 W nm -32.5 0.0290 2.82E+05 3.23E+03 W nm -35.0 2.71E+05 3.11E+03

Summary Increasing the bandwidth (resolution) results in a decrease in both signal and noise. The noise falls off faster allowing determination of optimum bandwidth for best signal to noise. Increasing the bandwidth (resolution) also results in a decrease in spectral resolution. The impact of bandwidth optimization is dependent upon the spectra of the compound. Other compounds will show larger or smaller gains in S/N depending upon the spectra of the compound. When analyzing compounds with very different spectra, optimize wavelength and bandwidth for each compound.

In a perfect world, all peaks would be separated chromatographically.
Peak Purity In a perfect world, all peaks would be separated chromatographically.

Peak Purity Unfortunately, the world is not perfect and occasionally co-elution's occur.

Extracting Co-Eluters
Absorbance Elution Time A B Sum Identifying and extracting co-eluters is not easy, it requires: good optics bench good software algorithm Here you see a co-elution of two peaks detected at a single wavelength Co-elution is the sum of absorbances of peaks A and B with the assumption that absorbencies are additive

Peak Purity (Homogeneity) Analysis
PDA acquires spectra at every point in time depending on acquisition rate Peak Purity algorithm analyzes all spectra within a peak The Apex Spectrum is the reference spectrum that it compares to. Absorbance Spectra per Run Time 200.00 240.00 280.00 320.00 nm peak Millennium also Interpolate lift-off and touch-down spectra to obtain a baseline spectrum for each sampling time within a peak. This allows the software to easily subtract baseline spectra from the corresponding absorbance spectra within a peak.

Comparing Spectra Ethylparaben EthylPaba 200.00 240.00 280.00 320.0 nm
Absorbance Ethylparaben

Spectral Contrast The Spectral Contrast measures the shape difference between two spectra. Spectra are converted into a vector in n dimensional space. The vectors are moved into a two dimensional plane and the angle between them is measured.

PDA Spectral Contrast™
200.00 240.00 280.00 320.00 nm EthylPaba Absorbance V1 Wavelength = Direction Absorbance = Length

200.00 240.00 280.00 320.00 nm EthylPaba Absorbance Wavelength = Direction Absorbance = Length V2

200.00 240.00 280.00 320.00 nm EthylPaba Absorbance V1 Wavelength = Direction Absorbance = Length V2

200.00 240.00 280.00 320.00 nm EthylPaba Absorbance Resultant Vector V1 + V2

200.00 240.00 280.00 320.00 nm EthylPaba Absorbance Ethylparaben Spectrum A q Spectrum B The shapes of Spectrum A and Spectrum B are represented by vectors The differences between spectral shapes are defined by  is the Spectral Contrast Angle The vector length is proportional to Absorbance

PDA Spectral Contrast Angle
Each compound has a unique spectrum that is represented by a vector in space. Spectral Contrast Angle is the angle between vectors, “the differences”. A value of zero degrees the vectors overlay and suggest that the two spectra are equivalent. A value of 90 degrees demonstrates maximum differences in the two spectra The Spectral Contrast technique also estimates the instrument noise and solvent contribution to determine the significance of the difference between two spectra.

Spectral Differences Spectral Contrast of 53 degrees
200.00 240.00 280.00 320.00 Wavelength (nm) Ethyl-PABA Ethylparaben Absorbance 230.00 250.00 270.00 290.00 310.00 Wavelength (nm) Theophylline Dyphylline Absorbance Absorbance Absorbance Spectral Contrast of 53 degrees Spectral Contrast of 10 degrees-typically observed for structurally related compounds

Spectral Differences Absorbance Wavelength (nm)
200.00 240.00 280.00 320.00 Wavelength (nm) Methylparaben Ethylparaben Absorbance Spectral contrast of 0.5 degrees Very similar spectra, CH2 difference PDA Algorithm was capable of distinguishing the difference between the two By setting up the correct parameters that takes into account electronic noise and solvent contribution on the spectra.

Influence of noise on peak purity and identification
Generally, every time a Spectral Contrast Angle (Purity Angle or Match Angle) is computed, an associated Threshold Angle is also computed. The Threshold Angle is used to assess the significance of spectral homogeneity in purity or Library Matching measurements. To calculate noise, you must specify a noise interval. The noise interval is a region of the baseline where no compound-related spectral absorbance occurs. The noise interval must meet the following criteria: Length of time - Two to four peak widths of the narrowest peak of interest (peak width measured at half height), typically 0.5 to 1.0 minute. The minimum noise interval must contain at least 12 spectra Note: If the noise interval contains fewer than 12 spectra, the Millennium³² software does not calculate a Match Angle or a Purity Angle. Also, if the noise interval is not within the runtime of the chromatogram, the software does not calculate a Match Angle or a Purity Angle. Chromatographic features - No chromatographic peaks that have UV/Vis absorbance in the acquired wavelength range. (The Max Plot channel helps you to visualize any peaks in the noise interval.)

Influence of noise on peak purity and identification
Threshold Angle is the area of uncertainty. The Purity Threshold is the sum of the Solvent Angle and the Noise Angle when applied. If the noise angle is greater than the angle between the two spectra, they cannot be distinguished Generally, every time a Spectral Contrast Angle (Purity Angle or Match Angle) is computed, an associated Threshold Angle is also computed. The Threshold Angle is used to assess the significance of spectral homogeneity in purity or Library Matching measurements. To calculate noise, you must specify a noise interval. The noise interval is a region of the baseline where no compound-related spectral absorbance occurs. The noise interval must meet the following criteria: Length of time - Two to four peak widths of the narrowest peak of interest (peak width measured at half height), typically 0.5 to 1.0 minute. The minimum noise interval must contain at least 12 spectra Note: If the noise interval contains fewer than 12 spectra, the Millennium³² software does not calculate a Match Angle or a Purity Angle. Also, if the noise interval is not within the runtime of the chromatogram, the software does not calculate a Match Angle or a Purity Angle. Chromatographic features - No chromatographic peaks that have UV/Vis absorbance in the acquired wavelength range. (The Max Plot channel helps you to visualize any peaks in the noise interval.)

Peak Purity Results If Purity Angle < Purity Threshold
within the noise of the system the peak is spectrally homogeneous or spectrally pure. If Purity Angle > Purity Threshold there is something within the peak that can not be explained by noise. The peak is not spectrally pure, the Purity Flag field is checked. If any errors occurred during peak purity determination, the Purity Errors field is checked.

Purity Parameters Spectral Contrast Parameters
Control how spectra are compared for Peak Purity and spectrum Library Matching Two Parameters are entered Wavelength limit: Use this parameter to exclude from Spectral Contrast calculations regions of a spectrum where there is no component absorbance or where the solvent strongly absorbs Noise Interval Time: a region of the baseline where no compound-related spectral absorbance occurs. Estimates the noise and/or solvent contributions to the Purity Angle of a spectrum during Peak Purity testing, and flags purity results that exceed this threshold. For details on using Threshold Criteria, click . Available choices: · AutoThreshold – Millennium³² software calculates the Noise Angle (based on the Noise Interval you specify) and the Solvent Angle (based on the absorbance of the peak), then adds the two angles together to automatically compute the Threshold Angle for each peak. · Noise + Solvent – Adds the Solvent Angle to the Noise Angle. If you select this choice, you must enter a value in the Solvent Angle field (as described below). · Noise – Reports and uses only the Noise Angle. The noise angle is calculated automatically for each peak based on total system performance and the peak’s height. It does not take into account solvent contribution. · Solvent – Reports an uses only the Solvent Angle. If you select this choice, you must enter a value in the Solvent Angle field (as described below).

Purity Parameters The noise interval must be 2 to 4 peak widths long (peak width at half-height). The minimum noise interval must contain at least 12 spectra. For example, if the data was acquired at a rate of 1 spectrum/second, the noise interval must be at least 12 seconds long. If you cannot identify a peak-free region that is at least 2 to 4 peak widths (peak width at half-height), select an area with the lowest UV absorbance and that contains at least 12 spectra. Selecting a noise interval that contains UV absorbance of elutors will increase Purity and Match Threshold values. Estimates the noise and/or solvent contributions to the Purity Angle of a spectrum during Peak Purity testing, and flags purity results that exceed this threshold. For details on using Threshold Criteria, click . Available choices: · AutoThreshold – Millennium³² software calculates the Noise Angle (based on the Noise Interval you specify) and the Solvent Angle (based on the absorbance of the peak), then adds the two angles together to automatically compute the Threshold Angle for each peak. · Noise + Solvent – Adds the Solvent Angle to the Noise Angle. If you select this choice, you must enter a value in the Solvent Angle field (as described below). · Noise – Reports and uses only the Noise Angle. The noise angle is calculated automatically for each peak based on total system performance and the peak’s height. It does not take into account solvent contribution. · Solvent – Reports an uses only the Solvent Angle. If you select this choice, you must enter a value in the Solvent Angle field (as described below).

Noise Interval Time Estimates the noise and/or solvent contributions to the Purity Angle of a spectrum during Peak Purity testing, and flags purity results that exceed this threshold. For details on using Threshold Criteria, click . Available choices: · AutoThreshold – Millennium³² software calculates the Noise Angle (based on the Noise Interval you specify) and the Solvent Angle (based on the absorbance of the peak), then adds the two angles together to automatically compute the Threshold Angle for each peak. · Noise + Solvent – Adds the Solvent Angle to the Noise Angle. If you select this choice, you must enter a value in the Solvent Angle field (as described below). · Noise – Reports and uses only the Noise Angle. The noise angle is calculated automatically for each peak based on total system performance and the peak’s height. It does not take into account solvent contribution. · Solvent – Reports an uses only the Solvent Angle. If you select this choice, you must enter a value in the Solvent Angle field (as described below).

Noise Interval Time Selecting a bad region in the chromatogram can yield erroneous Peak Purity results. Here are some spectra obtained from different Noise Intervals. Noise Interval Spectrum Taken at 11.5 to 13 min. Taken at 0.05 to 0.5 min. Taken at 7.0 to 10 min. Estimates the noise and/or solvent contributions to the Purity Angle of a spectrum during Peak Purity testing, and flags purity results that exceed this threshold. For details on using Threshold Criteria, click . Available choices: · AutoThreshold – Millennium³² software calculates the Noise Angle (based on the Noise Interval you specify) and the Solvent Angle (based on the absorbance of the peak), then adds the two angles together to automatically compute the Threshold Angle for each peak. · Noise + Solvent – Adds the Solvent Angle to the Noise Angle. If you select this choice, you must enter a value in the Solvent Angle field (as described below). · Noise – Reports and uses only the Noise Angle. The noise angle is calculated automatically for each peak based on total system performance and the peak’s height. It does not take into account solvent contribution. · Solvent – Reports an uses only the Solvent Angle. If you select this choice, you must enter a value in the Solvent Angle field (as described below).

Importance of Noise Interval
Spectral Purity Peak 1= Fail Peak 2= Fail Peak 3= Fail Noise Interval Spectrum Taken at 0.05 to 0.5 min. The noise interval must be 2 to 4 peak widths long (peak width at half-height). The minimum noise interval must contain at least 12 spectra. For example, if the data was acquired at a rate of 1 spectrum/second, the noise interval must be at least 12 seconds long.

Spectral Purity Peak 1= Pass Peak 2= Pass Peak 3= Pass Noise Interval Spectrum Taken at 7.0 to 10 min.

Spectral Purity Peak 1= Fail Peak 2= Fail Peak 3= Fail Noise Interval Spectrum Taken at 11.5 to 13 min.

Purity Parameters Purity Parameters Parameters
control the Peak Purity measurement Four Parameters can be entered: Purity Passes: Specifies the number of times the software performs Peak Purity testing on a peak. Active Peak Region Specifies the region of each peak over Peak Purity testing is to be performed. Threshold Criteria: Estimates the contributions of: Noise and Solvent Noise Solvent Estimates the noise and/or solvent contributions to the Purity Angle of a spectrum during Peak Purity testing, and flags purity results that exceed this threshold. For details on using Threshold Criteria, click . Available choices: · AutoThreshold – Millennium³² software calculates the Noise Angle (based on the Noise Interval you specify) and the Solvent Angle (based on the absorbance of the peak), then adds the two angles together to automatically compute the Threshold Angle for each peak. · Noise + Solvent – Adds the Solvent Angle to the Noise Angle. If you select this choice, you must enter a value in the Solvent Angle field (as described below). · Noise – Reports and uses only the Noise Angle. The noise angle is calculated automatically for each peak based on total system performance and the peak’s height. It does not take into account solvent contribution. · Solvent – Reports an uses only the Solvent Angle. If you select this choice, you must enter a value in the Solvent Angle field (as described below).

Threshold Choices AutoThreshold – Millennium³² software calculates the Noise Angle (based on the Noise Interval you specify) and the Solvent Angle (based on the absorbance of the peak), then adds the two angles together to automatically compute the Threshold Angle for each peak. Noise + Solvent – Adds the Solvent Angle to the Noise Angle. If you select this choice, you must enter a value in the Solvent Angle field (as described below). Noise – Reports and uses only the Noise Angle. The noise angle is calculated automatically for each peak based on total system performance and the peak’s height. It does not take into account solvent contribution. Solvent – Reports and uses only the Solvent Angle. If you select this choice, you must enter a value in the Solvent Angle field.

Solvent Angle Solvent Angle set to 1.0
Estimates the solvent contribution to Spectral Contrast. Determined by Injecting replicates of a chemically pure standard at the highest concentration that is to be routinely used (<1 AU). Estimate the Solvent Angle by Recording the Purity Angle calculated for each replicate standard injection. Enter the Solvent Angle that is equal to the highest Purity Angle obtained. Solvent Angle set to 1.0

Noise Angle Noise only Takes the spectra from a user-defined noise interval of the baseline. Combines the spectra extracted from the noise interval to yield the root-mean-square (RMS) noise absorbance at each wavelength Compares the noise spectrum to the difference between a normalized spectrum and a baseline-corrected spectrum. Noise Angle is inversely proportional to analyte concentration.

AutoThreshold AutoThreshold Automatically calculates the Solvent Angle for each chromatographic peak by taking the baseline-corrected spectra at the peak apex (with maximum spectral absorbance). Takes user defined Noise Interval to compute the Noise Angle. Computes the Threshold as the sum of the Noise Angle and the Solvent Angle

Criteria for Peak Purity testing
Use the smallest resolution (1.2 nm) to distinguish between similar spectra. For the most sensitive detection of spectral impurities, choose the wavelength range that includes all the absorbance bands of the analytes and the likely impurities. Avoid using wavelengths at which neither the analytes nor the likely impurities absorb. The wavelength range must be at least ten times your resolution for peak purity. If possible, do not use the shortest wavelengths (190 to 210 nm) within the available wavelength range this usually tends to contribute more noise and variability to the Purity Angle determinations. If possible, avoid using wavelengths where solvent absorbance occurs. Recommended to work with absorbencies less than 1.0 A.U Using proper Integration is important since Peak purity is determined from peak lift-off to peak touch-down. Avoid using wavelengths at which neither the analytes nor the likely impurities absorb. Using such wavelengths increases the Noise Angle , data storage requirements, and processing time without increasing the Purity Angle and desensitizes the measurement of spectral impurities.

Criteria for Peak Purity testing – How to setup the PDA
Make sure to have 5-6 replicates of the std’s for Peak Purity analysis Have replicates of the std’s at 25%, 50%, 75%, 100% and 125% for spectral matching (Library) Create a method set that uses a MaxPlot channel Apply to all of the runs Check every chromatogram for quiet baseline interval Minimum of 0.5min to assign Noise Interval Create PDA Processing Method Set wavelength range Set to range of MaxPlot channel Set Noise interval Set criteria for Threshold Determination Absorbance 190 210 230 250 270 290 nm 2.0 1.0 0.5 1.5

Purity Results Purity Results found in the Review Table Window

Purity Results Purity Plot At peak apex purity angle is 0.00
A graphical display of the calculation. Impurity (M) found where Purity angle exceeds Threshold angle At peak apex purity angle is 0.00

Multi-component Peak Purity
The first pass is identical to Peak Purity testing. Subsequent passes use both the peak apex spectrum and the maximum impurity spectra from previous passes to compare with all spectra in the peak. Use Multi-component Peak Purity testing to determine the number of spectrally distinct components within a single chromatographic peak. On each purity pass, the following occurs: On the first pass, the apex spectrum is the reference point and the Purity Angle, Purity Threshold and point of maximum impurity are determined as previously discussed. On the second pass, the new point of reference is the additive spectrum of the apex spectrum and the spectrum from the point of maximum impurity from the first purity pass. All other spectra across the peak are compared to this reference spectrum and a new Purity Angle, Purity Threshold and point of maximum impurity are determined. On the third pass, the new point of reference is the additive spectrum of the apex spectrum, the maximum impurity spectrum from the first pass and the maximum impurity spectrum from the second pass. All other spectra across the peak are compared to this reference spectrum and a new Purity Angle, Purity Threshold and point of maximum impurity are determined.

Peak Purity Measurements
Impurity must have some absorbance Major component and impurity must exhibit some degree of spectral difference Some degree of chromatographic resolution between major component and impurity must exist The impurity must exist at a detectable level Technique CANNOT guarantee that a peak is chemically pure! A PDA detector CANNOT guarantee that any peak is pure. Rather, it can help determine whether an “impurity” has co-eluted with the main peak. This is possible if the following criteria are met: 1) Some degree of chromatographic resolution between major component and impurity must exist. 2) Major component and impurity must exhibit some degree of spectral difference. 3) The impurity must exist at a “detectable level” (e.g., >1:100 compared to major peak).

Peak Purity Measurements
Empower & Millennium³² PDA software will Fail to detect: Multiple, spectrally distinct components in a peak if: Two (or more) components do not have a significant difference in their UV absorbance spectra. There is not a significant chromatographic resolution between components. An insufficient amount of the impurity is co-eluting. Right-click within the Spectrum Index plot to access the properties of this group: In the Spectral Types section, you select which spectra, from within the peaks, are to be overlaid. In the Spectral Characteristics section, you can customize how the spectra are labeled. Selecting Spline applies a cubic spline algorithm to the selected spectra. Splining enhances the spectral display. Select Baseline Correct to: Interpolate lift-off and touch-down spectra to obtain a baseline spectrum for each sampling time within a peak. Subtract baseline spectra from the corresponding absorbance spectra within a peak. Select the derivatives of spectra to be viewed from the Derivative drop-down list. Up to the fourth derivative can be viewed. Select a smoothing factor from the Smoothing drop-down list. Noisy spectra can be mathematically smoothed by choosing a smoothing factor. Use the lowest factor possible to avoid smoothing out spectral features. Consult Millennium32 Online Help for more information. In Spectral View, you can sets the wavelength range over which the spectra will be displayed.

Optimized Separation?

Peak Purity

Optimized Separation? Let’s look at these three peaks. Peak shape can sometimes be used to help determine whether a co-elution has occurred. Based upon this criteria, one might think that both peaks were ‘pure.” But let’s look further with the assistance of the Waters 2996 PDA and Empower.

Peak Purity – Apex

Peak Purity – Peak Inflections

Peak Purity – Peak Offsets

Peak Purity – Peak Valleys

Peak Purity – Peak 2 (zoomed)

Peak Purity- Maximum Impurity -1st Pass

Peak Purity- Maximum Impurity -2nd Pass

Peak Purity – Peak 2 (zoomed)

Peak Purity- Maximum Impurity -3rd Pass

Spectrum Review You can select other spectra from within the peaks to be overlaid as follows: Using the Spectrum Index menu, you can make only one selection at a time. Using the Spectrum Index tab of the Plot Properties dialog box, you can make multiple selections at a time.

Spectrum Review First pass impurity found and the spectrum is shown
Spectrum Review allows you to bring in spectra from spectrum Index, of selected peak;includes impurity spectra,Apex, Valleys and Inflection points Select Spectrum Review from the Spectrum Index drop-down menu to bring the spectra from the Spectrum Index window into the Spectrum Review window. The three spectra from the Spectrum Index window are overlaid in a non-normalized plot.

Spectrum Review Reference spectrum taken after dark current measurement and before the sample is injected. This displays the lamp spectrum and the Exposure time can be found in the 2D Channel Table Noise Spectrum Is taken from the values entered in the Noise Interval Time and each value plotted is the root-mean-square noise at each wavelength in the spectrum. Reference Noise

Spectral Libraries Use spectral matching to:
Confirm peaks are spectrally identical to known peaks Detect co-elution You can search against spectral libraries Interactively Automatically Use pure standards Collect library and sample spectra with the same parameters Collect data at different concentrations All peaks < 1 AU By building spectral libraries of known compounds, you can perform spectral matching to: Confirm that peaks are spectrally the same as known peaks. Detect coelution, which is covered in Section 8, “Peak Purity.” Millennium32 software matches extracted data file spectra against libraries as follows: Interactively, in the Spectrum Review window. Automatically, during processing either in QuickSet, Run and Process mode, or in post-run processing. Follow these guidelines when building a spectral library: Use pure standards to create libraries. If a standard does not exist, use a representative batch of material. Collect all library and sample spectra with: The same instrument method parameters, such as the wavelength range, sampling rate, resolution and filter response The same mobile phase, including organic modifier and pH The same column and column temperature The same sample diluent The same detector calibration Because the spectral shape of a compound may change at different concentrations, include spectra for compounds at various concentrations in the spectral libraries. Keep all peaks under 1 AU across the collected wavelength range.

Creating Spectral Libraries
Bring the standards into Review In an automated library search, every time a data file is processed, the apex spectra of all integrated peaks are subject to a library search. To automate library searching, add the library search to the processing method: 1) Click the Processing Method tool. 2) Click the PDA Library Search tab. 3) You can set the following match criteria in the processing method: Derivative: specifies the derivative to be calculated. Smoothing: specifies the mathematical smooth factor. Search Threshold: sets a maximum for the Match Angle. Search Depth: reports the best possible matches, from 1 to 3. Threshold Criteria: sets how the threshold angle will be calculated. The choices are: Noise, Solvent, and Noise + Solvent. Retention Time Presearch and Wavelength Presearch: limits the search to library spectra with retention times similar to the data file spectra.. Search Libraries: sets the search against multiple libraries. Check the boxes for the appropriate libraries. 4) Make six injections of a chemically pure standard at the highest concentration that is to be used. The peak height should be less than 1 AU across the collected wavelength range. 5) Determine the noise interval. 6) Create a spectral library with the apex spectrum from one of the injections. 7) Create a processing method. Set the Threshold Criteria to Noise. 8) Process the six injections and record the Match Angles. 9) Set the Threshold Criteria to Noise + Solvent. 10) Enter the highest calculated Match Angle into the processing method as the Solvent Angle. The solvent angle is now optimized for all concentrations of this compound lower than the standard.

Apply the Method Set In an automated library search, every time a data file is processed, the apex spectra of all integrated peaks are subject to a library search. To automate library searching, add the library search to the processing method: 1) Click the Processing Method tool. 2) Click the PDA Library Search tab. 3) You can set the following match criteria in the processing method: Derivative: specifies the derivative to be calculated. Smoothing: specifies the mathematical smooth factor. Search Threshold: sets a maximum for the Match Angle. Search Depth: reports the best possible matches, from 1 to 3. Threshold Criteria: sets how the threshold angle will be calculated. The choices are: Noise, Solvent, and Noise + Solvent. Retention Time Presearch and Wavelength Presearch: limits the search to library spectra with retention times similar to the data file spectra.. Search Libraries: sets the search against multiple libraries. Check the boxes for the appropriate libraries. 4) Make six injections of a chemically pure standard at the highest concentration that is to be used. The peak height should be less than 1 AU across the collected wavelength range. 5) Determine the noise interval. 6) Create a spectral library with the apex spectrum from one of the injections. 7) Create a processing method. Set the Threshold Criteria to Noise. 8) Process the six injections and record the Match Angles. 9) Set the Threshold Criteria to Noise + Solvent. 10) Enter the highest calculated Match Angle into the processing method as the Solvent Angle. The solvent angle is now optimized for all concentrations of this compound lower than the standard.

Select “New Library” In an automated library search, every time a data file is processed, the apex spectra of all integrated peaks are subject to a library search. To automate library searching, add the library search to the processing method: 1) Click the Processing Method tool. 2) Click the PDA Library Search tab. 3) You can set the following match criteria in the processing method: Derivative: specifies the derivative to be calculated. Smoothing: specifies the mathematical smooth factor. Search Threshold: sets a maximum for the Match Angle. Search Depth: reports the best possible matches, from 1 to 3. Threshold Criteria: sets how the threshold angle will be calculated. The choices are: Noise, Solvent, and Noise + Solvent. Retention Time Presearch and Wavelength Presearch: limits the search to library spectra with retention times similar to the data file spectra.. Search Libraries: sets the search against multiple libraries. Check the boxes for the appropriate libraries. 4) Make six injections of a chemically pure standard at the highest concentration that is to be used. The peak height should be less than 1 AU across the collected wavelength range. 5) Determine the noise interval. 6) Create a spectral library with the apex spectrum from one of the injections. 7) Create a processing method. Set the Threshold Criteria to Noise. 8) Process the six injections and record the Match Angles. 9) Set the Threshold Criteria to Noise + Solvent. 10) Enter the highest calculated Match Angle into the processing method as the Solvent Angle. The solvent angle is now optimized for all concentrations of this compound lower than the standard.

Name the new Library In an automated library search, every time a data file is processed, the apex spectra of all integrated peaks are subject to a library search. To automate library searching, add the library search to the processing method: 1) Click the Processing Method tool. 2) Click the PDA Library Search tab. 3) You can set the following match criteria in the processing method: Derivative: specifies the derivative to be calculated. Smoothing: specifies the mathematical smooth factor. Search Threshold: sets a maximum for the Match Angle. Search Depth: reports the best possible matches, from 1 to 3. Threshold Criteria: sets how the threshold angle will be calculated. The choices are: Noise, Solvent, and Noise + Solvent. Retention Time Presearch and Wavelength Presearch: limits the search to library spectra with retention times similar to the data file spectra.. Search Libraries: sets the search against multiple libraries. Check the boxes for the appropriate libraries. 4) Make six injections of a chemically pure standard at the highest concentration that is to be used. The peak height should be less than 1 AU across the collected wavelength range. 5) Determine the noise interval. 6) Create a spectral library with the apex spectrum from one of the injections. 7) Create a processing method. Set the Threshold Criteria to Noise. 8) Process the six injections and record the Match Angles. 9) Set the Threshold Criteria to Noise + Solvent. 10) Enter the highest calculated Match Angle into the processing method as the Solvent Angle. The solvent angle is now optimized for all concentrations of this compound lower than the standard.

Add to the new Library In an automated library search, every time a data file is processed, the apex spectra of all integrated peaks are subject to a library search. To automate library searching, add the library search to the processing method: 1) Click the Processing Method tool. 2) Click the PDA Library Search tab. 3) You can set the following match criteria in the processing method: Derivative: specifies the derivative to be calculated. Smoothing: specifies the mathematical smooth factor. Search Threshold: sets a maximum for the Match Angle. Search Depth: reports the best possible matches, from 1 to 3. Threshold Criteria: sets how the threshold angle will be calculated. The choices are: Noise, Solvent, and Noise + Solvent. Retention Time Presearch and Wavelength Presearch: limits the search to library spectra with retention times similar to the data file spectra.. Search Libraries: sets the search against multiple libraries. Check the boxes for the appropriate libraries. 4) Make six injections of a chemically pure standard at the highest concentration that is to be used. The peak height should be less than 1 AU across the collected wavelength range. 5) Determine the noise interval. 6) Create a spectral library with the apex spectrum from one of the injections. 7) Create a processing method. Set the Threshold Criteria to Noise. 8) Process the six injections and record the Match Angles. 9) Set the Threshold Criteria to Noise + Solvent. 10) Enter the highest calculated Match Angle into the processing method as the Solvent Angle. The solvent angle is now optimized for all concentrations of this compound lower than the standard.

Name the Spectrum In an automated library search, every time a data file is processed, the apex spectra of all integrated peaks are subject to a library search. To automate library searching, add the library search to the processing method: 1) Click the Processing Method tool. 2) Click the PDA Library Search tab. 3) You can set the following match criteria in the processing method: Derivative: specifies the derivative to be calculated. Smoothing: specifies the mathematical smooth factor. Search Threshold: sets a maximum for the Match Angle. Search Depth: reports the best possible matches, from 1 to 3. Threshold Criteria: sets how the threshold angle will be calculated. The choices are: Noise, Solvent, and Noise + Solvent. Retention Time Presearch and Wavelength Presearch: limits the search to library spectra with retention times similar to the data file spectra.. Search Libraries: sets the search against multiple libraries. Check the boxes for the appropriate libraries. 4) Make six injections of a chemically pure standard at the highest concentration that is to be used. The peak height should be less than 1 AU across the collected wavelength range. 5) Determine the noise interval. 6) Create a spectral library with the apex spectrum from one of the injections. 7) Create a processing method. Set the Threshold Criteria to Noise. 8) Process the six injections and record the Match Angles. 9) Set the Threshold Criteria to Noise + Solvent. 10) Enter the highest calculated Match Angle into the processing method as the Solvent Angle. The solvent angle is now optimized for all concentrations of this compound lower than the standard.

Using Spectral Libraries
Setting up Match Parameters In the Library menu bar, Match parameters can be selected. In an automated library search, every time a data file is processed, the apex spectra of all integrated peaks are subject to a library search. To automate library searching, add the library search to the processing method: 1) Click the Processing Method tool. 2) Click the PDA Library Search tab. 3) You can set the following match criteria in the processing method: Derivative: specifies the derivative to be calculated. Smoothing: specifies the mathematical smooth factor. Search Threshold: sets a maximum for the Match Angle. Search Depth: reports the best possible matches, from 1 to 3. Threshold Criteria: sets how the threshold angle will be calculated. The choices are: Noise, Solvent, and Noise + Solvent. Retention Time Presearch and Wavelength Presearch: limits the search to library spectra with retention times similar to the data file spectra.. Search Libraries: sets the search against multiple libraries. Check the boxes for the appropriate libraries. 4) Make six injections of a chemically pure standard at the highest concentration that is to be used. The peak height should be less than 1 AU across the collected wavelength range. 5) Determine the noise interval. 6) Create a spectral library with the apex spectrum from one of the injections. 7) Create a processing method. Set the Threshold Criteria to Noise. 8) Process the six injections and record the Match Angles. 9) Set the Threshold Criteria to Noise + Solvent. 10) Enter the highest calculated Match Angle into the processing method as the Solvent Angle. The solvent angle is now optimized for all concentrations of this compound lower than the standard.

Setting up Match Parameters can be performed: In the Library menu bar, Match parameters can be selected. Derivativized or Smooth spectra can also be added to libraries. In an automated library search, every time a data file is processed, the apex spectra of all integrated peaks are subject to a library search. To automate library searching, add the library search to the processing method: 1) Click the Processing Method tool. 2) Click the PDA Library Search tab. 3) You can set the following match criteria in the processing method: Derivative: specifies the derivative to be calculated. Smoothing: specifies the mathematical smooth factor. Search Threshold: sets a maximum for the Match Angle. Search Depth: reports the best possible matches, from 1 to 3. Threshold Criteria: sets how the threshold angle will be calculated. The choices are: Noise, Solvent, and Noise + Solvent. Retention Time Presearch and Wavelength Presearch: limits the search to library spectra with retention times similar to the data file spectra.. Search Libraries: sets the search against multiple libraries. Check the boxes for the appropriate libraries. 4) Make six injections of a chemically pure standard at the highest concentration that is to be used. The peak height should be less than 1 AU across the collected wavelength range. 5) Determine the noise interval. 6) Create a spectral library with the apex spectrum from one of the injections. 7) Create a processing method. Set the Threshold Criteria to Noise. 8) Process the six injections and record the Match Angles. 9) Set the Threshold Criteria to Noise + Solvent. 10) Enter the highest calculated Match Angle into the processing method as the Solvent Angle. The solvent angle is now optimized for all concentrations of this compound lower than the standard.

Setting up Match Parameters can be performed: In Process window it can be found under PDA Library Search Derivativized or Smooth spectra can also be applied In an automated library search, every time a data file is processed, the apex spectra of all integrated peaks are subject to a library search. To automate library searching, add the library search to the processing method: 1) Click the Processing Method tool. 2) Click the PDA Library Search tab. 3) You can set the following match criteria in the processing method: Derivative: specifies the derivative to be calculated. Smoothing: specifies the mathematical smooth factor. Search Threshold: sets a maximum for the Match Angle. Search Depth: reports the best possible matches, from 1 to 3. Threshold Criteria: sets how the threshold angle will be calculated. The choices are: Noise, Solvent, and Noise + Solvent. Retention Time Presearch and Wavelength Presearch: limits the search to library spectra with retention times similar to the data file spectra.. Search Libraries: sets the search against multiple libraries. Check the boxes for the appropriate libraries. 4) Make six injections of a chemically pure standard at the highest concentration that is to be used. The peak height should be less than 1 AU across the collected wavelength range. 5) Determine the noise interval. 6) Create a spectral library with the apex spectrum from one of the injections. 7) Create a processing method. Set the Threshold Criteria to Noise. 8) Process the six injections and record the Match Angles. 9) Set the Threshold Criteria to Noise + Solvent. 10) Enter the highest calculated Match Angle into the processing method as the Solvent Angle. The solvent angle is now optimized for all concentrations of this compound lower than the standard.

Derivative Number of times the software applies the derivative algorithm to a spectrum before it performs Library Matching. Choices: None, First, Second, Third, or Fourth. Default: None. Smoothing Length of the Savitsky-Golay filter applied to the spectra for both smoothing and derivatives. Search Threshold (Degrees) Threshold for the reported Match Angle . Empower software does not report any matches with a Match Angle greater than the Search Threshold value you choose. Entries: 0.00 to Default: 10.0. Search Depth Number of matches reported for a library search. For example, with a search depth of 3, the software performs all possible searches, then selects and reports the best 3 matches. Threshold Criteria – Same as in Purity Solvent Angle – Same as in Purity In an automated library search, every time a data file is processed, the apex spectra of all integrated peaks are subject to a library search. To automate library searching, add the library search to the processing method: 1) Click the Processing Method tool. 2) Click the PDA Library Search tab. 3) You can set the following match criteria in the processing method: Derivative: specifies the derivative to be calculated. Smoothing: specifies the mathematical smooth factor. Search Threshold: sets a maximum for the Match Angle. Search Depth: reports the best possible matches, from 1 to 3. Threshold Criteria: sets how the threshold angle will be calculated. The choices are: Noise, Solvent, and Noise + Solvent. Retention Time Presearch and Wavelength Presearch: limits the search to library spectra with retention times similar to the data file spectra.. Search Libraries: sets the search against multiple libraries. Check the boxes for the appropriate libraries. 4) Make six injections of a chemically pure standard at the highest concentration that is to be used. The peak height should be less than 1 AU across the collected wavelength range. 5) Determine the noise interval. 6) Create a spectral library with the apex spectrum from one of the injections. 7) Create a processing method. Set the Threshold Criteria to Noise. 8) Process the six injections and record the Match Angles. 9) Set the Threshold Criteria to Noise + Solvent. 10) Enter the highest calculated Match Angle into the processing method as the Solvent Angle. The solvent angle is now optimized for all concentrations of this compound lower than the standard.

Library Search To search a library interactively:
1) Select one spectrum to be searched against the library. In this example, the spectrum from peak 1 in the sample Mixture is the data file spectrum.

1) Select one spectrum to be searched against the library. In this example, the spectrum from peak 1 in the sample Mixture is the data file spectrum. 2) From the Spectrum Review menu, select Match against library Mixtures.

1) Select one spectrum to be searched against the library. In this example, the spectrum from peak 1 in the sample Mixture is the data file spectrum. 2) From the Spectrum Review menu, select Library, then select Match against library Impurity. The spectrum matches to a spectrum based on the PDA Match Parameters you set In this case it found a Match.

Library Search Click the Library Match tab to view more information about the matches: Match Angle  Match Threshold The spectra match. Match Angle  Match Threshold There is a significant difference between the spectra and they do not match. Match Angle: the Spectral Contrast Angle. Match Threshold: the largest Spectral Contrast angle due to nonidealities, as discussed Section 6, “Spectral Contrast Theory.” Match Spect. Name: the name of the library spectrum. Match Lib Name: the name of the library containing the library spectrum. Match Ideal: both the data file spectrum and the library spectrum were collected with the same instrument method parameters. Match Error: checked if an error occurs during matching. Match Flag: checked if it is a poor match. Match Wvln RMS: displays the root-mean-square spectral differences between the data file spectrum and the library spectrum at all wavelengths. This value indicates how well the actual wavelengths of both spectra line up. A value of 0 indicates that the wavelengths are well aligned. Note. Match Threshold is reported only when spectra are acquired at the same resolution and wavelength range.

Comparing Library Match
When comparing spectra with different Ideal wavelength parameters the following must be considered: Comparing spectra with the same spectral resolution but with different wavelength ranges will yield: Meaningful Match Angle can be obtained where spectra from regions of interest overlap. No Match Threshold is generated. Comparing spectra with different spectral resolution and different wavelength ranges may yield: Erroneous Match Angle. Examine the spectra and use your own judgment to determine if the no Match Threshold condition is an indicator of a problem.

Spectral Libraries Results of Library Matches can be viewed in several locations. In Review Main Window Table in the Peaks Table In the Spectrum Review in Library Match Table In Results in Library Match Table In Results in Match Plot and Triple Plot The Triple Plot Library Match illustrates if there are distinct differences between the acquired and library spectra Here is an example of two similar spectra, however the differences between the acquired and library spectra is obviously distinct PDA Match Ideal Indicates (when selected) that both spectra in the comparison have the same wavelength range and resolution. If the match is not ideal, no PDA Match Threshold is reported. PDA Match Error Indicates (when selected) that an error condition occurred, preventing the calculation of PDA Match Angle and PDA Match Threshold, for up to three matches. See the Message Center for the specific error. PDA Match Flag Indicates the size of the PDA Match Angle relative to the PDA Match Threshold, for up to three matches. If the PDA Match Angle is greater than the Match Threshold, the check box for this column is selected. If the PDA Match Angle is less than the PDA Match Threshold, the check box remains clear. PDA Match Wvln RMS Displays the root-mean-square (RMS) difference at all wavelengths, for up to three matches. The RMS value indicates how well the actual wavelengths of both spectra line up. It is reported only for comparisons between spectra with the same ideal wavelength parameters . A value of 0 indicates that the wavelengths are well aligned.

PDA Advantages: Library Matching
Match Angle – A measure of the difference in spectral shapes between an acquired spectrum and a library spectrum. Match Angle can range from 0 to 90 degrees. Small values indicate that spectra are similar. Large values indicate greater degrees of spectral difference.

Single Wavelength Quantitation: Risk of Incorrect Identification
Minutes 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 2.1 mAU Unspiked Grape Juice PDA at 288 nm 7.596 Thiabendazole 5.689 Carbendaizm

Single Wavelength Quantitation: Risk of Incorrect Identification
Based on Retention time, peak 2 may be thiabendazole. PDA at 288 nm 7.596 Thiabendazole PDA Peak Match Results indicate identified peak is Not a Match to Thiabendazole 5.689 Carbendaizm 2.1 mAU Unspiked Grape Juice Carbendazim + Thiabendazole Std 200 ppb Standard 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 Minutes

Comparison of Spectra Confirms Differences
Thiabendazole Standard Misidentified Peak 232.4 298.4 14 mAU 200.00 250.00 300.00 350.00 nm PDA Library Matching Indicates Different Spectra

Chromatographic Resolution and Coelution Detection
R=0 Purity Angle not effective; Match Angle useful R=0.3 to R=0.7 Purity & Match Angle useful R>0.7 Match Angle not useful

For Chromatographic Resolution of 0.3
Impurity Detection For Chromatographic Resolution of 0.3 R = 0.3 Analyte concentration is constant; impurity to analyte ratio variable Unknown Purity Angle is greater than Standard = impurity

Importance of Spectral Quality For:

Combining Peak Purity with Library Match to aide in Peak Identity
Fail Pass Intermediate Co-elution At Peak Apex Tailing or Fronting Homogenous Spectra

New 2998 PDA Detector

2998 New Features

New control console

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Advanced Techniques for PDA Analysis

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