ASTM E685-93(2000)
(Practice)Standard Practice for Testing Fixed-Wavelength Photometric Detectors Used in Liquid Chromatography
Standard Practice for Testing Fixed-Wavelength Photometric Detectors Used in Liquid Chromatography
SCOPE
1.1 This practice is intended to serve as a guide for the testing of the performance of a photometric detector (PD) used as the detection component of a liquid-chromatographic (LC) system operating at one or more fixed wavelengths in the range 210 to 800 nm. Measurements are made at 254 nm, if possible, and are optional at other wavelengths.
1.2 This practice is intended to describe the performance of the detector both independently of the chromatographic system (static conditions) and with flowing solvent (dynamic conditions).
1.3 For general liquid chromatographic procedures, consult Refs (1-9).
1.4 For general information concerning the principles, construction, operation, and evaluation of liquid-chromatography detectors, see Refs (10 and 11) in addition to the sections devoted to detectors in Refs (1-7).
1.5 The values stated in SI units are to be regarded as standard.
1.6 This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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An American National Standard
Designation:E685–93 (Reapproved 2000)
Standard Practice for
Testing Fixed-Wavelength Photometric Detectors Used in
Liquid Chromatography
This standard is issued under the fixed designation E 685; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope 3. Terminology
1.1 This practice is intended to serve as a guide for the 3.1 Definitions:
testing of the performance of a photometric detector (PD) used 3.1.1 absorbance calibration—the procedure that verifies
as the detection component of a liquid-chromatographic (LC) that the absorbance scale is correct within 65%.
systemoperatingatoneormorefixedwavelengthsintherange 3.1.2 drift—the average slope of the noise envelope ex-
210 to 800 nm. Measurements are made at 254 nm, if possible, pressed in absorbance units per hour (AU/h) as measured over
and are optional at other wavelengths. a period of 1 h.
1.2 This practice is intended to describe the performance of 3.1.3 dynamic—under conditions of a flow rate of 1.0
the detector both independently of the chromatographic system mL/min.
(static conditions) and with flowing solvent (dynamic condi- 3.1.4 linear range—ofaPD, the range of concentrations of
tions). a test substance in a mobile phase over which the response of
1.3 For general liquid chromatographic procedures, consult the detector is constant to within5%as determined from the
Refs (1-9). linearity plot specified below and illustrated in Fig. 1. The
1.4 For general information concerning the principles, con- linear range should be expressed as the ratio of the highest
struction, operation, and evaluation of liquid-chromatography concentration to the minimum detectable concentration or the
detectors, see Refs (10 and 11) in addition to the sections lowest linear concentration, whichever is greatest.
devoted to detectors in Refs (1-7). 3.1.5 long-term noise—the maximum amplitude in AU for
1.5 The values stated in SI units are to be regarded as all random variations of the detector signal of frequencies
standard. between 6 and 60 cycles per hour (0.1 and 1.0 cycles per min).
1.6 This standard does not purport to address all of the 3.1.5.1 Discussion—Itrepresentsnoisethatcanbemistaken
safety problems, if any, associated with its use. It is the for a late-eluting peak. This noise corresponds to the observed
responsibility of the user of this standard to establish appro- noise only and may not always be present.
priate safety and health practices and determine the applica- 3.1.6 minimum detectability—ofaPD, that concentration of
bility of regulatory limitations prior to use. a specific solute in a specific solvent that results in a detector
response corresponding to twice the static short-term noise.
2. Referenced Documents
3.1.7 response time (speed of output)—the detector, the
2.1 ASTM Standards: timerequiredforthedetectoroutputtochangefrom10%to90
E 275 Practice for Describing and Measuring Performance
% of the new equilibrium value when the composition of the
of Ultraviolet, Visible, and Near-Infrared Spectrophotom- mobilephaseischangedinastepwisemanner,withinthelinear
eters
range of the detector.
E 682 Practice for Liquid Chromatography Terms and Re- 3.1.7.1 Discussion—Because the detector volume is very
lationships
small and the transport rate is not diffusion dependent, the
response time is generally fast enough to be unimportant. It is
generally comparable to the response time of the recorder and
This practice is under the jurisdiction ofASTM Committee E-13 on Molecular
dependent on the response time of the detector electrometer
Spectroscopy and is the direct responsibility of Subcommittee E13.19 on Chroma-
and on the recorder amplifier. Factors that affect the observed
tography.
Current edition approved Feb. 15, 1993. Published April 1993. Originally response time include the true detector response time, elec-
published as E 685 – 79. Last previous edition E 685 – 79.
tronic filtering, and system band-broadening.
The boldface numbers in parentheses refer to the list of references at the end of
this practice.
Annual Book of ASTM Standards, Vol 14.02.
Annual Book of ASTM Standards, Vol 03.06.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E685
where the presence of high-voltage equipment makes it likely
that there is ozone in the air. Protect the entire system from
temperature fluctuations because these will lead to detectable
drift.
5.1.1 The detector should be located at the test site and
turned on at least 24 h before the start of testing. Insufficient
warm-upmayresultindriftinexcessoftheactualvalueforthe
detector.
5.2 Methods of Measurement:
5.2.1 Connect a suitable device (Note 1) between the pump
and the detector to provide at least 75 kPa (500 psi) back
pressure at 1.0 mL/min flow of methanol. Connect a short
length(about100mm)of0.25-mm(0.01-in.)internal-diameter
stainless steel tubing to the outlet tube of the detector to retard
bubble formation. Connect the recorder to the proper detector
output channels.
NOTE 1—Suggested devices include (a)2to4mof 0.1-mm (0.004-in.)
internal-diameter stainless steel tubing, (b) about 250 mm of 0.25 to
0.5-mm (0.01 to 0.02-in.) internal-diameter stainless steel tubing crimped
with pliers or cutters, or (c) a constant back-pressure valve located
FIG. 1 Example of a Linearity Plot for a Photometric Detector
between the pump and the injector.
5.2.2 Repeatedly rinse the reservoir and chromatographic
3.1.8 short-term noise—the maximum amplitude, peak to
system, including the detector, with degassed methanol to
peak, inAU for all random variations of the detector signal of
remove from the system all other solvents, any soluble mate-
a frequency greater than one cycle per minute.
rial, and any entrained gasses. Fill the reservoir with methanol
3.1.8.1 Discussion—It determines the smallest signal de-
and pump this solvent through the system for at least 30 min to
tectable by a PD, limits the precision attainable in quantitation
complete the system cleanup.
of trace-level samples, and sets the lower limit on linearity.
5.2.3 Air or nitrogen is used in the reference cell, if any.
This noise corresponds to the observed noise only.
Ensure that the cell is clean, free of dust, and completely dry.
3.1.9 static—under conditions of no flow.
5.2.4 To perform the static test, cease pumping and allow
4. Significance and Use
the chromatographic system to stabilize for at least1hat room
temperature without flow. Set the attenuator at maximum
4.1 Although it is possible to observe and measure each of
the several characteristics of a detector under different and sensitivity (lowest attenuation), that is, the setting for the
smallest value of absorbance units full-scale (AUFS). Adjust
unique conditions, it is the intent of this practice that a
complete set of detector specifications should be obtained the response time as close as possible to 2 s for a PD that has
under the same operating conditions. It should also be noted a variable response time (Note 2). Record the response time
that to completely specify a detector’s capability, its perfor- used.Adjustthedetectoroutputtonearmidscaleonthereadout
mance should be measured at several sets of conditions within device. Record at least1hof detector signal under these
the useful range of the detector. The terms and tests described conditions, during which time the ambient temperature should
in this practice are sufficiently general that they may be used not change by more than 2°C.
regardless of the ultimate operating parameters.
NOTE 2—Time constant is converted to response time by multiplying
4.2 Linearity and response time of the recorder or other
by the factor 2.2. The effect of electronic filtering on observed noise may
readout device used should be such that they do not distort or
be studied by repeating the noise measurements for a series of response-
otherwise interfere with the performance of the detector. This
time settings.
requires adjusting the gain, damping, and calibration in accor-
5.2.5 Draw pairs of parallel lines, each pair corresponding
dance with the manufacturer’s directions. If additional elec-
to between 0.5 and 1 min in length, to form an envelope of all
tronicfiltersoramplifiersareusedbetweenthedetectorandthe
observed random variations over any 15-min period (see Fig.
final readout device, their characteristics should also first be
2). Draw the parallel lines in such a way as to minimize the
established.
distance between them. Measure the vertical distance, in AU,
between the lines. Calculate the average value over all the
5. Noise and Drift
segments. Divide this value by the cell length in centimetres to
5.1 Test Conditions—Pure, degassed methanol of suitable
obtain the static short-term noise.
grade shall be used in the sample cell.Air or nitrogen shall be
5.2.6 Now mark the center of each segment over the 15-min
used in the reference cell if there is one. Nitrogen is preferred
period of the static short-term noise measurement. Draw a
series of parallel lines encompassing these centers, each pair
correspondingto10mininlength,andchoosethatpairoflines
Distilled-in-glass or liquid-chromatography grade. Complete freedom from
particles may require filtration, for example, through a 0.45-µm membrane filter. whose vertical distance apart is greatest (see Fig. 2). Divide
E685
FIG. 2 Example for the Measurement of the Noise and Drift of a PD (Chart Recorder Output).
this distance in AU by the cell length in centimetres to obtain at least1hof signal under these flowing conditions, during
the static long-term noise.
which time the ambient temperature should not change by
5.2.7 Draw the pair of parallel lines that minimizes the
more than 2°C.
vertical distance separating these lines over the 1 h of mea-
5.2.9 Draw pairs of parallel lines, measure the vertical
surement (see Fig. 2). The slope of either line is the static drift
distances, and calculate the dynamic short-term noise follow-
expressed in AU/h.
ing the procedure of 5.2.5.
5.2.8 Set the pump to deliver 1.0 mL/min under the same
5.2.10 Make the measurement for the dynamic long-term
conditions of tubing, solvent, and temperature as in 5.2.1
noise following the procedure outlined in 5.2.6.
through 5.2.3.Allow 15 min for the system to stabilize. Record
E685
5.2.11 Draw the pair of parallel lines as directed in 5.2.7. 6.1.5 Plot or calculate the detector response (AU) versus
The slope of these lines is the dynamic drift. concentrations (µg/mL) for a test substance of known molar
5.2.12 The actual noise of the system may be larger or absorptivity to find the best-fit line through the origin. Calcu-
smaller than the observed values, depending upon the method late the molar absorptivity, e, of the test solution as follows:
of data collection, or signal monitoring of the detector, since
slope 3 MW
e5 (1)
observed noise is a function of the frequency, speed of
b
response, and bandwidth of the readout device.
where:
6. Minimum Detectability, Linear Range, and
slope = the slope of the linear portion of the plot,AU·µl/µg,
Calibration MW = molecular weight, g/mole, and
b = nominal cell length, cm, as specified by the manu-
6.1 Methods of Measurement—For the determination of the
facturer.
linear range of a PD, (12) for a specific substance, the response
Compare the value of e obtained with an experimentally
to that test substance must be determined. The following
determined value or one from the literature (Note 3). Should
procedure is designed to provide a worst-case procedure.
the values differ by more than 5 %, the PD may require
6.1.1 Dissolve in methanol a suitable compound with an
adjustment. Consult the manufacturer’s directions.
ultraviolet spectral absorbance that changes rapidly at the
wavelength of interest. Choose a concentration that is ex-
NOTE 3—For example, the values of molar absorptivity for uracil in
3 3
pected to exceed the linear range, typically to give an absor-
methanolare7.7 3 10 at254nmand1.42 3 10 at280nm;forpotassium
bance above 2AU. Dilute the solution accurately in a series to
dichromate in 0.01 N sulfuric acid they are 4.22 3 10 at 254 nm and
cover the linear range, that is, down to the minimum detectable 3.60 3 10 at 280 nm.
concentration. Rinse the sample cell with methanol and zero
7. Response Time
the detector with methanol in the cell. Rinse the cell with the
solution of lowest concentration until a stable reading is
7.1 The response time of the detector may become signifi-
obtained; usually rinsing the cell with 1 mL is sufficient.
cant when a short micro-particle column and a high-speed
Record the detector output. After rinsing the syringe thor-
recorder are used.Also, it is possible, by using an intentionally
oughly with the next more concentrated solution, fill the cell
slow response time, to reduce the observed noise and hence
withthesolutionfromeachdilutioninturn.Obtainaminimum
increase the apparent linear range. Although this would have
of five on-scale measurements. Measure under static condi-
little effect on broad peaks, the signal from narrow peaks
tions.
would be significantly degraded. Measure at the highest and
6.1.2 Calculate the ratio of detector response (AU) to
lowest values of the electronic filter if it is variable.
concentration (µg/mL) for each solution and plot these ratios
7.2 Method of Measurement:
versus log concentration (see Fig. 1). The region of linearity
7.2.1 The composition of the mobile phase is changed in a
will define a horizontal line of constant response ratio. At
stepwise manner and the output signal is recorded on the
higher concentrations, there will typically be a negative devia-
highest-speed device available. If the recorder has a response
tion from linearity, while at lower concentrations there may be
timenotsignificantlyfasterthanthedetector,onlytheresponse
deviation in either direction. Draw horizontal lines 5 % above
time of the detector-recorder combination will be obtained, as
and below the line of constant response ratio. The upper limit
it would be when the combination is used to record chromato-
of linearity is the concentration at which the line of measured
grams.
response ratio intersects one of the 5 % bracketing lines at the
7.2.2 Set a flow rate of 2.0 mL/min.
highconcentrationend.Thelowerlimitoflinearityiseitherthe
7.2.3 A stepwise change may be obtained by means of
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