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14

Computing absorbance

The detector calculates absorbance values before transmitting the data to the 

Empower or MassLynx database. It does so as follows:

•

Computes the absorbance at each diode using the dark current and 

reference spectrum (see 

“Calculating absorbance” on page 4

).

•

Averages the absorbances at a particular wavelength, as specified in the 

spectra-per-second sample rate, and reports the average as a single data 

point (see 

“Resolution” on page 15

).

•

Also, the detector can apply a filter when calculating absorbance (see 

“Filtering data” on page 10

).

Dark current

Photodiodes produce thermally excited charge even when not exposed to 

light. The amount of thermally excited charge produced is called dark 

current.
When a dark current update is necessary, the detector closes the shutter 

to take a dark-current reading for each diode. The shutter closes after 

the exposure time is calculated and stays closed for the same interval as 

the exposure time.
The detector subtracts the dark-current values from the current values 

recorded during absorbance measurements for both the sample and the 

reference spectra.

Reference spectrum

Immediately after the dark-current measurement, and before any 

components elute, the detector records a reference spectrum. The 

reference spectrum is a measure of lamp intensity and mobile phase 

absorbance that ideally represents the initial mobile phase. With the 

shutter open, the reference spectrum is determined over the interval 

specified in the exposure time.
For extremely long exposure times, the dark current and reference 

spectrum readings can take several seconds to finish.

Содержание ACQUITY UPC2

Страница 1: ...ACQUITY UPC 2 Photodiode Array Detector Overview and Maintenance Guide Revision A Copyright Waters Corporation 2012 All rights reserved...

Страница 2: ...change without notice and should not be construed as a commitment by Waters Corporation Waters Corporation assumes no responsibility for any errors that may appear in this document This document is b...

Страница 3: ...gnal connector 26 Connecting to the electricity source 26 Starting the detector 27 Monitoring detector LEDs 29 About the detector control panel 30 Using a cuvette 31 Shutting down the detector 34 Main...

Страница 4: ...as vectors 55 Spectral contrast angles 57 Undesirable effects 60 Error messages and troubleshooting 64 Startup error messages 64 Error messages preventing operation 67 Detector troubleshooting 70 Spec...

Страница 5: ...sLynx software can control the detector for LC MS and LC applications With a photodiode array of 512 photodiodes and an optical resolution of 1 2 nm the detector operates within the range 190 to 800 n...

Страница 6: ...r position dark counts are measured at each pixel and subsequently subtracted from observed signal counts to give true signal counts Open Allows light to pass into the flow cell It is the normal setti...

Страница 7: ...he contribution of second order diffraction of UV light less than 370 nm to the light intensity observed at visible wavelengths greater than 370 nm Photodiode array An array of 512 pixel photodiodes a...

Страница 8: ...law Beer s law is expressed as A lc where A dimensionless quantity measured in absorbance units constant of proportionality known as the molar extinction coefficient l path length in centimeters 1 0 c...

Страница 9: ...Overview 5 Absorbance as a function of concentration Concentration Absorbance Ideal Actual Working range Background absorbance...

Страница 10: ...ll The Waters TaperSlit flow cell so called because of the shape of the flow cell exit face matches the shape of the spectrograph slit Compared with a conventional flow cell with a cylindrical shape t...

Страница 11: ...absorption peaks Benzene spectrum at 1 2 nm resolution Measuring light at the photodiode array The photodiode array detector measures the amount of light striking the photodiode array to determine the...

Страница 12: ...e array and process the data The minimum exposure time is 5 msec You can set exposure time from 5 to 500 msec For example if an exposure time is set to 50 milliseconds the detector performs as follows...

Страница 13: ...tent with not saturating due to overexposure Calculates exposure time at the start of a sample set based on maximum light intensity within the selected wavelength range Limits the exposure so that no...

Страница 14: ...to minimize noise The filter is a digital finite impulse response filter that creates peak height degradation and enhances the filtering of high frequency noise The behavior of the filter depends on...

Страница 15: ...broaden peaks For the highest resolution select an appropriate sampling rate for your separation and choose a fast filter time constant For the highest sensitivity select an appropriate sampling rate...

Страница 16: ...index of the data point closest to the end time minus the index of the data point closest to the start time to calculate the Points Across Peak value for each integrated peak in the chromatogram The...

Страница 17: ...of mobile phase gradient separations that demonstrate gradual compositional changes Note that it should not be applied in cases where abrupt gradient changes such as steps are evident Generally the f...

Страница 18: ...current When a dark current update is necessary the detector closes the shutter to take a dark current reading for each diode The shutter closes after the exposure time is calculated and stays closed...

Страница 19: ...n data points in an acquired spectrum The detector s lowest resolution setting is 1 2 nm For example in 3D mode the detector averages three adjacent diodes for each reported wavelength when you specif...

Страница 20: ...Ensures wavelength accuracy Fixed second order filter Filters UV wavelengths above 370 nm Full diagnostic capability Supports built in diagnostic tools to optimize functionality and performance One c...

Страница 21: ...y contact the shipping agent and your local Waters representative Thermal wander management To mitigate thermal instability caused by ambient temperature changes the detector s insulation ensures air...

Страница 22: ...nd submitting claims see Waters Licenses Warranties and Support Services Installing the detector To install the ACQUITY UPC 2 PDA detector 1 Place the detector atop the column manager ensuring that th...

Страница 23: ...ng the detector 19 Proper placement for drip management system 2 Attach the solvent bottle tray so it is next to the convergence manager Guides for feet placement Drain routing hole for drip managemen...

Страница 24: ...20 ACQUITY UPC 2 PDA detector in an ACQUITY UPC 2 system TP03453 Detector Convergence manager Sample manager fixed loop Binary solvent manager Column manager Solvent bottle tray...

Страница 25: ...for use in liquid chromatography If the detector is already powered on in the console select PDA Detector from the system tree and click the lamp icon to extinguish the lamp To plumb the detector 1 O...

Страница 26: ...s on the front alignment pin 4 Continue to insert the flow cell until the three thumbscrews align with their holes in the optics bench Caution To prevent the flow cell from binding and to ensure that...

Страница 27: ...Plumbing the detector 23 5 Finger tighten the thumbscrews and then secure them with a 1 4 inch flat blade screwdriver TP03458 Lamp Flow cell ID connector Thumbscrews Tubing outlet Tubing inlet...

Страница 28: ...s more than one detector or additional components such as dual CM Aux modules plus a CM A module you must install one or more multi detector drip trays If you have two detectors the trays should be po...

Страница 29: ...pack and install the preconfigured workstation 2 Connect one end of one Ethernet cable to the network switch and then connect the other end to the Ethernet card on the workstation Tip On preconfigured...

Страница 30: ...connected near the system ACQUITY UPC 2 PDA detector analog event connections Signal connections Description Inject Start Start injection Event Out Output switch to trigger external devices Analog Ou...

Страница 31: ...electrical cable to the power strip on the back of the cart Finally connect the power strip s cable to a wall outlet operating on its own circuit Starting the detector Starting the detector entails p...

Страница 32: ...lows Each system instrument s power LED shows green During initialization each system instrument s status LED flashes green After the instruments are successfully powered on all LEDs show steady green...

Страница 33: ...e of functioning Power LED The power LED to the left hand side of the detector s front panel indicates when the detector is powered on or powered off Lamp LED The lamp LED to the right of the power LE...

Страница 34: ...unning and shutter position You cannot edit detector parameters while the system is processing samples The following table lists the items in the detector control panel Modifiable detector control pan...

Страница 35: ...uvette holder which you then place in the detector flow cell assembly ACQUITY UPC 2 PDA Detector cuvette holder with the cuvette inserted Shutter Displays the shutter position Open Closed or Erbium La...

Страница 36: ...h quartz cuvette and matched pairs from the same manufacturing lot of quartz cuvettes for your zero and sample measurements Before beginning a measurement using the cuvette fill the flow cell by flush...

Страница 37: ...it does not become dislodged when you are replacing the cuvette holder 4 Gently guide the cuvette holder back into the flow cell assembly until it sits in position securely To prevent invalid subsequ...

Страница 38: ...252 4752 If you are located elsewhere phone the Waters corporate headquarters in Milford Massachusetts USA or contact your local Waters subsidiary Our Web site includes phone numbers and e mail addres...

Страница 39: ...when you handle solvents change tubing or operate the system Know the physical and chemical properties of the solvents you use See the Material Safety Data Sheets for the solvents in use Warning To a...

Страница 40: ...guish the lamp while leaving the detector running but idle Note however that you should do so only when the lamp will remain extinguished more than 4 hours If you use a mobile phase with additives flu...

Страница 41: ...ush the system with 100 methanol for 10 minutes Maintaining the leak sensor A leak sensor in the drip tray continuously monitors the detector for leaks The sensor stops system flow when it detects acc...

Страница 42: ...hat the leak sensor detected a leak Tip If a leak is detected a Leak Detected error message appears 2 Open the detector door gently pulling its right hand edge toward you 3 Locate the source of the le...

Страница 43: ...pulate the leak sensor after removing it from its reservoir detach the leak sensor connector from the front of the instrument see Replacing the detector s leak sensor on page 42 5 Use a nonabrasive li...

Страница 44: ...e liquid from the leak sensor reservoir and its surrounding area 7 With a cotton swab absorb any remaining liquid from the corners of the leak sensor reservoir and its surrounding area Rolled up lint...

Страница 45: ...sensor into place 9 If you detached the leak sensor connector from the front of the instrument reattach it 10 In the console select your detector from the system tree 11 In the detector information wi...

Страница 46: ...pulling its right hand edge toward you 2 Press down on the tab to detach the leak sensor connector from the front of the instrument Warning The leak sensor and its reservoir can be contaminated with b...

Страница 47: ...Maintaining the detector 43 3 Remove the leak sensor from its reservoir by grasping it by its serrations and pulling upward on it 4 Unpack the new leak sensor Serrations...

Страница 48: ...from the system tree 8 In the detector information window click Control Reset to reset the detector Maintaining the flow cell The flow cell requires maintenance when the reference spectrum changes th...

Страница 49: ...s flush with 10 mL of UPLC grade solvent then flush with 10 mL of a low surface tension solvent such as methanol Tip Ensure that the solvent is miscible with the previous mobile phase 2 Stop the solve...

Страница 50: ...of pressurized liquid CO2 which is at its coldest temperature when it first converts to its gaseous form Additionally such a release of decompressed liquid CO2 which increases in volume by a 1 100 ra...

Страница 51: ...from their appropriate connections ACQUITY UPC 2 PDA detector flow cell 5 Using a 1 4 inch flat blade screwdriver loosen the three captive thumbscrews on the flow cell assembly s front plate 6 Grip th...

Страница 52: ...from the column outlet to the flow cell inlet Finger tighten then use the right angle tool nut extender to tighten 1 4 turn 13 Connect the outlet tubing from the outlet of the flow cell to the vent va...

Страница 53: ...ecord table Change the lamp when it repeatedly fails to ignite or when the detector fails to undergo calibration To remove the lamp 1 Power off the lamp To power off the lamp manually click PDA Detect...

Страница 54: ...he lamp to cool for 30 minutes or 15 minutes with the fan running and then open the door gently pulling its right edge toward you 4 Detach the lamp power connector from the detector Warning To avoid b...

Страница 55: ...line with the alignment pin on the lamp housing Warning To avoid laceration injuries use care when disposing of the lamp The lamp s gas is under a slight negative pressure so take care not to shatter...

Страница 56: ...he flow cell can affect reading lamp energy Ensure that the flow cell is clean before you read lamp energy To read the lamp energy 1 In the console select PDA Detector from the system tree 2 In the PD...

Страница 57: ...loaded fuse holder which is above the power entry module on the rear panel of the detector With minimum pressure withdraw the spring loaded fuse holder 3 Remove and discard the fuses 4 Make sure that...

Страница 58: ...ns such as noise photometric error or solvent effects Comparing absorbance spectra When measured at specific solvent and pH conditions the shape of a compound s absorbance spectrum characterizes the c...

Страница 59: ...ross the collected wavelength range Vector direction contributes to the identification of a compound since the direction is a function of the absorbance compound s spectrum The ability of spectral vec...

Страница 60: ...d of the vector for Compound A lies at the intersection of the absorbance values for Compound A at the two wavelengths represented by each axis The remaining vector is similarly derived from the spect...

Страница 61: ...fferent directions Spectral contrast angles Spectra of identical shape have vectors that point in the same direction Spectra of varying shapes have vectors that point in different directions The angle...

Страница 62: ...pectra with similar shapes In the following figure the absorbance spectra of two compounds A and B are similar They therefore produce a small spectral contrast angle 3 0 Spectral contrast angle 62 3 C...

Страница 63: ...bit slight differences because of detector noise photometric error high sample concentration or variations in solvent conditions The spectra in the next figure for example show how instrument noise ca...

Страница 64: ...olvent composition These sources of spectral variation can cause chemically pure baseline resolved peaks to exhibit a small level of spectral inhomogeneity You can assess the significance of spectral...

Страница 65: ...significant source of spectral inhomogeneity To minimize the effects of photometric error for all spectral contrast operations the maximum spectral absorbance of a compound should be less than 1 AU K...

Страница 66: ...general a spectral contrast angle less than its threshold angle indicates that shape differences are attributable to nonideal phenomena alone and that no evidence exists for genuine differences betwe...

Страница 67: ...ectral contrast theory 63 Effects of solvent concentration on the absorbance spectrum of p aminobenzoic acid Effect of concentration Note that position of maxima can be shifted Wavelength nm Absorbanc...

Страница 68: ...r on the detector They verify the proper operation of the detector electronics If one or more of the tests fail the detector beeps and displays an error message For serious errors it displays the word...

Страница 69: ...e data point loss Possible problem with the data system PC or personality card 1 Cycle power to both the detector and the data system PC 2 If problem persists contact Waters Technical Service Configur...

Страница 70: ...device not detected Unable to electronically communicate to lamp memory device Replace lamp with one that has the memory device attached Leak detector Enabled Leak detector has been enabled Informati...

Страница 71: ...c fuse failed Fuse failure was detected 1 Cycle power to the detector 2 Contact Waters Technical Service Instrument error messages Error Message Description Corrective Action Both lamp and flow cell m...

Страница 72: ...Technical Service Lamp failure Lamp indicates Off when it should be On 1 Check lamp icon 2 Cycle power to the detector 3 Replace lamp Lamp lighting failure The lamp failed to ignite 1 Cycle power to t...

Страница 73: ...2 If the problem persists contact Waters Technical Service to replace the personality card Thermal controller disabled Thermal controller was disabled 1 Cycle power to the detector 2 Contact Waters T...

Страница 74: ...Change in reference spectrum Air bubbles trapped in flow cell Reseat and check alignment of flow cell see page 46 Flush the flow cell see page 45 or apply slight backpressure on the detector waste out...

Страница 75: ...low cell gasket Replace the flow cell see page 46 Leak from flow cell inlet and outlet fittings Check fittings for overtightening or undertightening and replace fittings if necessary Status light blin...

Страница 76: ...35 0 pounds Environmental specifications Attribute Specification Operating temperature 15 to 28 C 59 to 82 4 F Operating humidity 20 to 80 noncondensing Shipping and storage temperature 30 to 60 C 22...

Страница 77: ...circuits which may produce a reduction of dielectric strength or surface resistivity Degree 2 refers only to normally nonconductive pollution Occasionally however expect a temporary conductivity caus...

Страница 78: ...Order filter Fixed 370 nm to 800 nm Photodetector 512 element selective access photodiode array Wavelength accuracy 1 0 nm tested with Erbium Perchlorate Wavelength calibration filter Erbium filter us...

Страница 79: ...the UV cutoff the wavelength at which the absorbance of the solvent is equal to 1 AU for some common chromatographic solvents Operating at a wavelength near or below the cutoff increases baseline noi...

Страница 80: ...System Guide Ethanol 210 Petroleum ether 210 Ethyl acetate 256 Pyridine 330 Ethyl ether 220 Tetrahydrofuran 230 Ethyl sulfide 290 Toluene 285 Ethylene dichloride 230 Xylene 290 UV cutoff wavelengths...

Страница 81: ...acquisition auto exposure parameter 9 exposure time parameter 9 data filtering 10 derived vectors 57 detector absorbance calculations 14 control panel using 30 dark current 14 flushing 37 fuses replac...

Страница 82: ...nstalling 51 LED 30 removing 49 replacing 49 turn on turn off control 30 lamp energy reading 52 leak sensor maintaining 37 replacing 42 LED lamp 29 30 monitoring 29 power 29 M maintaining flow cell 44...

Страница 83: ...sical 72 spectra derived vectors 57 differences between 59 different shapes 57 same shapes 58 spectral shape differences 60 vectors 55 spectral contrast angle 57 derived vectors 57 spectral shape diff...

Страница 84: ...Index 4...

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