ASTM E1657-98

NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
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Designation: E 1657 – 98
Standard Practice for
Testing Variable-Wavelength Photometric Detectors Used in
Liquid Chromatography
This standard is issued under the fixed designation E 1657; 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 variable-wavelength photomet- 3.1.1 absorbance calibration—the procedure that verifies
ric detector (VWPD) used as the detection component of a that the absorbance scale is correct within 65%.
liquid-chromatographic (LC) system operating at one or more 3.1.2 drift—the average slope of the noise envelope ex-
wavelengths in the range 190 to 800 nm. Many of the pressed in absorbance units per hour (AU/h) as measured over
measurements are made at 254 nm for consistency with a period of 1 h.
Practice E 685. Measurements at other wavelengths are op- 3.1.3 dynamic—under conditions of a flow rate of 1.0
tional. mL/min.
1.2 This practice is intended to describe the performance of 3.1.4 linear range—of a VWPD, the range of concentrations
the detector both independently of the chromatographic system of a test substance in a test solvent over which the ratio of
(static conditions) and with flowing solvent (dynamic condi- response of the detector versus concentration of test substance
tions). is constant to within 5 % as determined from the linearity plot
1.3 For general liquid chromatographic procedures, consult specified in 7.1.2 and illustrated in Fig. 1. The linear range
Refs (1-9). should be expressed as the ratio of the upper limit of linearity
1.4 For general information concerning the principles, con- obtained from the plot to either a) the lower linear concentra-
struction, operation, and evaluation of liquid-chromatography tion, or b) the minimum detectable concentration, if the
detectors, see Refs (10, 11) in addition to the sections devoted minimum detectable concentration is greater than the lower
to detectors in Refs (1-7). linear concentration.
1.5 The values stated in SI units are to be regarded as 3.1.5 long-term noise—the maximum amplitude in AU for
standard. all random variations of the detector signal of frequencies
1.6 This standard does not purport to address all of the between 6 and 60 cycles per hour (0.1 and 1.0 cycles per min).
safety concerns, if any, associated with its use. It is the 3.1.5.1 Discussion—It represents noise that can be mistaken
responsibility of the user of this standard to establish appro- for a late-eluting peak. This noise corresponds to the observed
priate safety and health practices and determine the applica- noise only and may not always be present.
bility of regulatory limitations prior to use. 3.1.6 minimum detectability— of a VWPD, that concentra-
tion of a specific solute in a specific solvent that results in a
2. Referenced Documents
detector response corresponding to twice the static short-term
2.1 ASTM Standards: noise.
E 275 Practice for Describing and Measuring Performance
3.1.6.1 Discussion—The static short-term noise is a mea-
of Ultraviolet, Visible, and Near-Infrared Spectrophotom- surement of peak-to-peak noise. A statistical approach to noise
eters
suggests that a value of three times the rms (root-mean-square)
E 682 Practice for Liquid Chromatography Terms and Re- noise would insure that any value outside this range would not
lationships
be noise with a confidence level of greater than 99 %. Since
E 685 Practice for Testing Fixed-Wavelength Photometric peak-to-peak noise is approximately five times the rms noise
Detectors Used in Liquid Chromatography
(12), the minimum detectability defined in this practice is a
more conservative estimate.
3.1.7 response time (speed of output)— the detector, the
This practice is under the jurisdiction of ASTM Committee E13 on Molecular
Spectroscopy and is the direct responsibility of Subcommittee E13.19 on Chroma-
time required for the detector output to change from 10 % to
tography.
90 % of the new equilibrium value when the composition of
Current edition approved Nov. 10, 1998. Published January 1999. Originally
the mobile phase is changed in a stepwise manner, within the
published as E 1657 – 94. Last previous edition E 1657 – 96.
The boldface numbers in parentheses refer to the list of references at the end of
linear range of the detector.
this practice.
3.1.7.1 Discussion—Because the detector volume is very
Annual Book of ASTM Standards, Vol 03.06.
4 small and the transport rate is not diffusion dependent, the
Annual Book of ASTM Standards, Vol 14.01.
Copyright © ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
E 1657
requires adjusting the gain, damping, and calibration in accor-
dance with the manufacturer’s directions. If additional elec-
tronic filters or amplifiers are used between the detector and the
final readout device, their characteristics should also first be
established.
5. Noise and Drift
5.1 Test Conditions—Pure, degassed methanol shall be
used in the sample cell. Air or nitrogen shall be used in the
reference cell if there is one. Nitrogen is preferred 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-up may result in drift in excess of the actual value for the
detector. The detector wavelength should be set to 254 nm.
5.2 Methods of Measurement:
5.2.1 Connect a suitable device (see Note 1) between the
pump and the detector to provide at least 75 kPa (500 psi) back
FIG. 1 Example of Linearity Plot for a Variable-Wavelength
pressure at 1.0 mL/min flow of methanol. Connect a short
Detector
length (about 100 mm) of 0.25-mm (0.01-in.) internal-diameter
stainless steel tubing to the outlet tube of the detector to retard
response time is generally fast enough to be unimportant. It is
bubble formation. Connect the recorder to the proper detector
generally comparable to the response time of the recorder and
output channels.
dependent on the response time of the detector electrometer
NOTE 1—Suggested devices include (a)2to4mof 0.1-mm (0.004-in.)
and on the recorder amplifier. Factors that affect the observed
internal-diameter stainless steel tubing, ( b) about 250 mm of 0.25 to 0.5
response time include the true detector response time, elec-
mm (0.01 to 0.02-in.) internal-diameter stainless steel tubing crimped with
tronic filtering, and system band-broadening.
pliers or cutters, or ( c) a constant back-pressure valve located between the
3.1.8 short-term noise—the maximum amplitude, peak to pump and the injector.
peak, in AU for all random variations of the detector signal of
5.2.2 Repeatedly rinse the reservoir and chromatographic
a frequency greater than one cycle per minute.
system, including the detector, with degassed methanol to
3.1.8.1 Discussion—It determines the smallest signal de-
remove from the system all other solvents, any soluble mate-
tectable by a VWPD, limits the precision attainable in quanti-
rial, and any entrained gasses. Fill the reservoir with methanol
tation of trace-level samples, and sets the lower limit on
and pump this solvent through the system for at least 30 min to
linearity. This noise corresponds to the observed noise only.
complete the system cleanup.
3.1.9 static—under conditions of no flow.
5.2.3 Air or nitrogen is used in the reference cell, if any.
3.1.10 wavelength accuracy—the deviation of the observed
Ensure that the cell is clean, free of dust, and completely dry.
wavelength maximum from the maximum of a known test
5.2.4 To perform the static test, cease pumping and allow
substance.
the chromatographic system to stabilize for at least1hat room
3.1.11 wavelength precision—a measure of the ability of a
temperature without flow. Set the attenuator at maximum
VWPD to return to the same spectral position as measured by
sensitivity (lowest attenuation), that is, the setting for the
the reproducibility of absorbance values when the detector is
smallest value of absorbance units full-scale (AUFS). Adjust
reset to a wavelength maximum of a known test substance.
the response time as close as possible to 2 s for a VWPD that
has a variable response time (see Note 2). Record the response
4. Significance and Use
time used. Adjust the detector output to near midscale on the
4.1 Although it is possible to observe and measure each of
readout device. Record at least1hof detector signal under
the several characteristics of a detector under different and
these conditions, during which time the ambient temperature
unique conditions, it is the intent of this practice that a
should not change by more than 2°C.
complete set of detector specifications should be obtained
NOTE 2—Time constant is converted to response time by multiplying
under the same operating conditions. It should also be noted
by the factor 2.2. The effect of electronic filtering on observed noise may
that to completely specify a detector’s capability, its perfor-
be studied by repeating the noise measurements for a series of response-
mance should be measured at several sets of conditions within
time settings.
the useful range of the detector. The terms and tests described
5.2.5 Draw pairs of parallel lines, each pair corresponding
in this practice are sufficiently general that they may be used
to between 0.5 and 1 min in length, to form an envelope of all
regardless of the ultimate operating parameters.
4.2 Linearity and response time of the recorder or other
readout device used should be such that they do not distort or
Distilled-in-glass or liquid-chromatography grade. Complete freedom from
otherwise interfere with the performance of the detector. This particles may require filtration, for example, through a 0.45-μm membrane filter.
E 1657
observed random variations over any 15-min period (see Fig. this distance in AU by the cell length in centimeters to obtain
2). Draw the parallel lines in such a way as to minimize the the static long-term noise.
distance between them. Measure the vertical distance, in AU, 5.2.7 Draw the pair of parallel lines that minimizes the
between the lines. Calculate the average value over all the vertical distance separating these lines over the1hof mea-
segments. Divide this value by the cell length in centimeters to surement (Fig. 2). The slope of either line is the static drift
obtain the static short-term noise. expressed in AU/h.
5.2.6 Now mark the center of each segment over the 15-min 5.2.8 Set the pump to deliver 1.0 mL/min under the same
period of the static short-term noise measurement. Draw a conditions of tubing, solvent, and temperature as in 5.2.1-5.2.3.
series of parallel lines encompassing these centers, each pair Allow 15 min for the system to stabilize. Record at least1hof
corresponding to 10 min in length, and choose that pair of lines signal under these flowing conditions, during which time the
whose vertical distance apart is greatest (see Fig. 2). Divide ambient temperature should not change by more than 2°C.
FIG. 2 Example for the Measurement of the Noise and Drift of a VWD (Chart Recorder Output)
E 1657
erbium perchlorate hexahydrate to dissolve the solid. Transfer the contents
5.2.9 Draw pairs of parallel lines, measure the vertical
to a 25 mL volumetric flask and make up to volume with water. While
distances, and calculate the dynamic short-term noise follow-
reasonable care should be observed in transferring the dissolved erbium
ing the procedure of 5.2.5.
perchlorate into the volumetric flask, the final solution is not used
5.2.10 Make the measurement for the dynamic long-term
quantitatively.
noise following the procedure outlined in 5.2.6.
6.2.2 Turn on the detector and allow it to warm up accord-
5.2.11 Draw the pair of parallel lines as directed in 5.2.7.
ing to the manufacturer’s recommendations. Thoroughly flush
The slope of these lines is the dynamic drift.
the detector cell with water preferably from the same source as
5.2.12 The actual noise of the system may be larger or
that to make up the test solution. (If using another test
smaller than the observed values, depending upon the method
compound, be sure to use the same solvent as the test solution.)
of data collection, or signal monitoring of the detector, since
Set the detector wavelength to 250 nm. Zero the absorbance of
observed noise is a function of the frequency, speed of
the detector. (Some detectors will automatically zero the
response, and bandwidth of the readout device.
detector after changing wavelengths.) Flush the cell with at
6. Wavelength Accuracy and Precision
least 1 mL of the erbium test solution. Record the absorbance
reading. Increase the wavelength by 1 nm. Flush the cell with
6.1 The wavelength accuracy and precision of a VWPD are
at least 1 mL of water. Zero the absorbance of the detector.
important parameters for the performance of chromatographic
Flush the cell with the erbium test solution and record the
methods. The wavelength specified in the method may be
absorbance. Repeat the procedure in 0.5 to 1.0 nm increments
critical to the detection of different compounds having different
until reaching 260 nm.
absorption spectra. The stated linear range of the method may
6.2.3 Plot absorbance versus wavelength and determine the
be compromised if the wavelength is inaccurate. Further, the
maximum absorbance. (See Fig. 3) Compare the calculated
precision of adjusting the detector to the same wavelength
maximum to the maximum for erbium perchlorate of 255 nm
should also be known. The wavelength of a VWPD is
(see Note 4). Report the nominal and calculated maximum of
determined by the monochromator and the optical alignment of
the test sample. The calculated maximum should be within the
the detector. The optical alignment is performed by the
manufacturer’s specification for wavelength accuracy. If the
manufacturer and usually does not need readjustment. Some
detector does not meet specifications, service on the detector to
detectors require alignment of the lamp after replacement. This
realign the lamp or the monochromator, or both, is indicated.
procedure verifies that the detector is properly aligned and
meets the manufacturer’s specifications for wavelength accu
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