Study Guide
Bruker Quest Diffractometer Guide
-
University:
Purdue University -
Course:
CHM 62000 | Spectrochemical Instrumentation Academic year:
2022
-
Views:
65
Pages:
37
Author:
kjcarroll01
Standard Operating Procedure – Bruker Quest
Diffractometer with copper wavelength X-ray
microsource
X-ray Crystallography Laboratory
Purdue Department of Chemistry
The following is a guide for collecting data and solving structures using the Purdue Bruker
Quest single crystal diffractometer with a copper wavelength X-ray microsource. It is intended as
a “walk-through” user guide geared especially towards novice users, but also tries to cover more
advanced features of the software where they are important for the collection of normal “simple”
small molecule structural data. A basic knowledge of the fundamentals of diffraction and
crystallography is expected.
This manual is based on an instrument and software produced around 2017 and 2020. Most
of the manual’s content also applies to newer as well as older instrument makes. For the novice
user, changes between different generation instruments are mostly limited to slightly different
program layouts and color schemes. The general procedures described in this manual still apply.
For a more in-depth description of the features of a CCD or CMOS diffractometer, the reader
should refer to the manuals and technique guides on specific topics by the manufacturer of the type
of instrumentation you are using. The gold standard for a more in depth guide towards the use of
Shelxtl for the refinement of single crystal structures is Peter Müller’s book ”Crystal Structure
Refinement”. Every crystallography lab should have at least one copy.
Among the many programs commonly used for crystal structure solution and refinement we
recommend the Bruker Shelxtl package (including XPREP, XS, XM and XT), George Sheldrick’s
refinement program Shelxl2018, the graphical interface Shelxle by Hübschle, Dittrich and
Sheldrick, and Platon by Anthony Spek.
1 Table of Contents
Instrument Overview ...............................................................................................3
Checking the Status and Thermostat .......................................................................4
Starting the System .................................................................................................6
Unloading the Previous Sample ..............................................................................7
Selecting and Mounting of Sample .........................................................................8
Crystal Mounting and Centering ...........................................................................10
Crystal and Compound Description ......................................................................12
Unit Cell Determination ........................................................................................14
Data Collection......................................................................................................22
Data Integration .....................................................................................................28
Scaling and Absorption Correction .......................................................................30
Transferring the Data ............................................................................................37
2 Instrument Overview
The Bruker Quest instrument with a copper microsource consists of:
The diffractometer including the X-ray enclosure, PhotonIII detector, mircosource “IµSCu”
X-ray tube, multilayer optics for monochromatization and collimator, video microscope,
kappa (4-axis) goniometer, electronic controls, power supply and other miscellaneous pieces
of equipment essential for the operation of the instrument.
An Oxford Cryosystems 800 variable temperature unit (pump, controllers and liquid nitrogen
tanks located outside the enclosure) and an Oxford Cryosystems AD61 dry air unit.
The instrument computer: This computer runs the Measurement Server, the Bruker
Instrument Service (BIS), Diffrac.Maintenance and Apex4.
Figure 1, Inside of the Quest X-ray enclosure with microsource X-ray tube, PhotonIII Detector, Variable
Temp Unit and Goniometer. Between X-ray tube and detector are the Optics (Multilayer Optics for
monochromatization, Collimator), the Video Microscope (in back, partially hidden), and the Beam Stop.
On the Goniometer is mounted the Goniometer Head holding the crystal specimen.
3 Having a promising sample of crystals, it is best to start in the following order:
Checking the Status and Thermostat
All but very high melting samples (inorganics, ceramics, etc) should be measured at lower
temperature to avoid extensive thermal motion of the atom cores, to obtain higher angle diffraction
data and to minimize radiation damage to the sample.
The Oxford Cryosystems variable temperature unit can be operated as a stand-alone unit, operated
through its own controllers. More often, it is controlled via Apex3 or BIS. The sample is embedded
in a stream of cold nitrogen gas supplied by a low pressure liquid nitrogen dewar. To avoid buildup
of ice, the sample is insulated from ambient humidity by an outer layer of warm dry air supplied
by an Oxford Cryosystems AD51 dry air unit (located outside the lab in a facilities closet).
The Oxford Cryosystems variable temperature unit is able to achieve temperatures between 375 K
(+102.5°C) down to ca. 85 K (-188.5°C). The recommended temperature for “low temperature”
data collections is 100 K in winter, and 150 K in summer (due to higher humidity levels that can
cause icing around the sample mount).
• Make sure, the tank is properly connected and full enough for the planned experiment.
• The Oxford Cryostream N2 tank (silver dewar behind the diffractometer) is automatically refilled
from the larger low pressure liquid nitrogen tank (between the two diffractometers). Check that
the Liquid Level Controller (the smaller of the two controllers, Fig. 2) is set to
for automatic refills. The fill level displayed on the controller should be between 20 and 80%.
When it is below 20% (despite the controller set to AUTOFILL) the low pressure N2 tank is
empty and needs to be refilled.
4 Figure 2, The external controller (bottom) and the
circulation pump (center), and the liquid level
controller (top) of the Oxford Cryosystems
variable temperature unit.
• Check that no data collection is running (e.g., look at the BIS interface, check for “Frames
left” or “Time left”). If a running data collection can be stopped, abort the data collection (see
below), then make changes to the VT unit.
• Check that the dry air unit providing the shield gas flow is switched on.
• Tap the touchscreen of the Cryostream Controller (larger of the two controllers) to check the
status of the VT unit. If it is running (“Cryostream running” beside the
symbol on the screen),
check if the temperature is appropriate for your experiment. If you need a different temperature,
or if the system is on standby (“Cryostream ready” beside the
symbol on the screen), use the
interface of Apex3 to set the desired temperature (see below, Starting the
System).
• If the unit is switched off it needs to be started. Press the main single button on the front of the
controller. Follow the on screen prompts to switch on the unit. Use the
interface of Apex3 to set the desired temperature (see below, Starting the System).
5 Starting the System
• Start BIS (Bruker Instrumentation System) if it is not already running. This will also start the
measurement server if it is not active already.
Figure 3, The BIS window
• Check if a data collection is still running (e.g., look at the BIS interface, check for “Frames
left” or “Time left”). If a data collection is active but can be aborted, then stop this data
collection first. Navigate to the active copy of Apex4 and click the STOP symbol in the Apex4
interface.
• Minimize any sessions of Apex4 that might be open.
▪
Start a new session of Apex4. No login is required.
▪
Connect to BIS (, then ).
• Via the dropdown menu, either an existing sample (continuation of old
data collection) or
• Start a Sample
▪
Fill in the project name. Avoid using special characters (including dashes and periods) or
overly long project names.
6 ▪
Leave the Group as “Users” and accept the automatically created project path
(D:\frames\guest\ProjectName)
Figure 4, New Sample pop-up window
• If necessary, ramp the temperature:
▪
Under click on
▪
In the column of the table, click on the top row and select
Enter the target temperature (in K). Set the rate to 360 (K/hr). Click the
button. You can mount and center a crystal while the temperature is ramping. For unit
cell measurements and data collections, wait until the temperature has settled at the target
value.
Unloading the Previous Sample
• Before using the video for crystal centering, make sure only one copy of Apex4 is open
(trying to open the crystal centering tab from a second copy of Apex4 can crash the video
feed).
• Under , go to
▪
The video feed will automatically open showing you a life picture of the mounted crystal.
Troubleshooting: If the connection to the video feed fails, do the following: Close all
other open copies of Apex3. Then close the “Center Crystal” subwindow. Disconnect
7 from the Server (Click the dropdown menue, click ). Click
under again. If this also fails, close Apex3 entirely. Open
the Task Manager. Find and close “FrameGrabber.exe” (under “Details”, “End Task”).
Restart Apex3 and try again. Repeat if necessary.
▪
Once the live video feed is running, click in the lower right of the interface.
The angles will drive to a position convenient for mounting a magnetic snap-on mount.
▪
Press the button on the front face of the diffractometer enclosure (to the
right of the front door, below the light button). Doors need to be closed to drive any
mechanical parts other than the phi (spindle) axis of the goniometer.
▪
Carefully dismount the magnetic snap-on mount from the previous experiment. Tilt it
backwards until the magnet comes loose, then take it off.
Figure 5, Goniometer head with magnetic snap on mount and adjustment screw
Selecting and Mounting of Sample
• Crystals can be mounted on glass rods, inside glass capillaries, on Nylon loops, or using mesh
mounts. The Purdue X-ray lab uses Mitegen micromesh mounts for most samples and data
collections.
8 ▪
Two types of micromesh mounts for small (<0.1 mm) and medium and large (0.1 – 0.5 mm)
crystals are stocked in the lab (Fig 6). Crystals collected at low temperature are mounted on
the micromesh with the help of a trace of mineral oil, Parabar 10312 (previous name:
Paratone oil), Fomblin oil (both are perfluorpolyether oils) or polybutene oil (a very viscous
oil to protect extremely oxygen sensitive samples) and flash frozen in the cold stream.
Crystals collected at room or elevated temperature might need to be fixed to the mesh with a
trace of glue to avoid crystal movement during measurement.
Figure 6, Tips of Mitegen Micromesh mounts
as seen through a microscope (Left: small
tipped mount with 15 µm openings for crystals
0.1 mm and smaller. Right: large mesh mount
with 25 µm openings. Mesh is 0.4 mm across).
▪
For typical crystal selection and mounting, place a drop of oil on a glass slide under a
microscope. Place several representative crystals in the oil. Inspect the crystals and select a
suitable candidate.
▪
Crystals on the Quest instrument with copper microsource should ideally be not larger than
0.2-0.3 mm in any direction (the size of the X-ray beam is 0.1 mm). The minimum size
depends on the diffraction intensity of the crystal. Larger crystals, especially if they are
heavily absorbing, are usually more suitable for the Quest instrument with molybdenum
sealed tube X-ray source (the size of its X-ray beam is 0.6 mm). Data collections are much
faster using molybdenum radiation as long as the achievable diffraction is sufficiently
intense. The higher intensity microsource and the intrinsically better diffraction with
copper radiation are ideally suited for small, weakly diffracting crystals that are unsuitable
for molybdenum radiation. Only those samples and organic samples for which absolute
configuration is required should be run on the copper wavelength instrument. For highly
9 absorbing samples limit the size of the crystal (make sure the minimum transmission will be
above 10-20%).
▪
If no single specimen with suitable dimensions can be found, use a sharp razor blade to cut
off a single piece from a larger crystal or cluster. Remove all smaller pieces and dirt from the
crystal (the micromesh mounts can be used to brush off loose pieces from the crystal).
Figure 7, Crystals with well-developed faces on a
microscope slide
▪
Scoop up a crystal with the micromesh mount and place it on the inside of the concave face
of the mount in the center of the mesh. Remove as much excess oil as possible by tipping the
back (convex) side of the mount onto a dry section of the microscope slide until only a
minimum amount of oil remains on the mount and crystal. (If a crystal needs to be glued to
the mount make sure that both crystal and mount are dry without any traces of oil. Dip the
mesh carefully in a small amount of glue and pick up the crystal without touching the glass
slide).
▪
Micromesh mounts are not single use. If necessary, they can be cleaned using e.g. chloroform
(which readily dissolves most of the mounting oils and cryoprotectants)
Crystal Mounting and Centering
▪
Make sure exactly one copy of Apex4 is running (trying to open the crystal centering tab
from a second copy of Apex4 can crash the video feed).
▪
Press the button on the front face of the diffractometer enclosure (to the right of
the front door, below the light button).
▪
Carefully mount the magnetic snap on mount onto the goniometer.
10 ▪
In Apex4, click in the submenue. The crystal centering live video
feed will automatically open (If the live video feed does not start, see “Unloading of Previous
Sample” for troubleshooting). Is the crystal visible on the video screen? If you cannot see the
crystal, its position is further off than usual. Check the position of the mount by eye (is it
aligned with the collimator and the beam stop? Using the crystal centering tool as described
below adjust the position on the goniometer head by eye until the crystal can be seen in the
live video feed.
Figure 8, Crystal Centering Window and live video feed with mounted sample and cross hair.
▪
If the image is too dark to see the crystal well, click the button or place a piece of
paper between the goniometer head and the detector.
▪
There are three adjustment screws (metal pins) on the goniometer head: for up-down, rightleft, and forward-backward. Use the bold end of the adjustment screw driver to turn the pins
to position the crystal in the center of the cross hair of the video feed.
▪
When the crystal is centered in the cross hair, click the large button (in the
window of Apex3) to spin the crystal around the phi axis by 90°. Repeat
11 the centering procedure using the adjustment screws as before. Repeat the process ( followed by centering of the crystal in the cross hair of the video feed) until the crystal
stays positioned in the center of the cross hair in all positions. If the cross hair seems to be
slightly off try to center the crystal so that it “rotates in place”.
For oddly shaped crystals, try to center the mass center of the crystal. For long needles, try to
align them along the axis of the mounting pin.
▪
Measure the dimensions of the crystal: Switch on the measuring tool (double sided arrow
icon on top of window, Fig. 8). Click on one side of the crystal, hold the
left mouse key and drag to the other side. The length of the green line dragged is given in the
bar below the video screen (“vector length”, value in µm). Write down the value and repeat
for the other two directions using .
Crystal and Compound Description
• Under , go to
▪
Fill in all values that apply. In the “compound” line, it is best to give the full name of the
person that provided the sample. Crystal dimensions need to be entered in mm (video
screen values are in µm!).
12 Figure 9, Crystal and compound description
13 Unit Cell Determination
• Under , click on .
▪
Two procedures are available, or .
Figure 10, Starting Window of the Unit Cell determination
If you are a novice user, it is best to use . To do so click ,
this will open the Unit Cell Data Collection Window.
▪
Choose an exposure time (default is 10 seconds). Click .
▪
3 × 20 frames will be collected which will be displayed in the frame window.
▪
By default, the window is scaled to fit the width of the frame. To see the entire frame,
right mouse click on the window and select
▪
The first sweep will be at low angle, with the beam stop visible at the center (Figure 11).
The second and third run (Figure 12) will be at high angle. Make sure that your specimen
diffracts to sufficiently high angle. Use the resolution arc tool (Figure 12) to check the
resolution limit. Only samples that show at least some diffraction above ca. 0.9 Å (with
ten second exposures) are likely to meet the IUCr requirements for publication.
14 ▪
The high sensitivity of the Photon III detector leads to capturing of natural decay events
(γ-rays from radioactive decay), which show up in the diffraction images as very bright
but very sharp signals, often only one pixel in size. They do not affect the final data
quality and are corrected for during data integration via a high energy event
discrimination (HEED) algorithm, but might be harvested during unit cell determination
and reduce the fraction of harvested spots that fit a selected unit cell. These are not
diffraction spots and can be ignored.
Figure 11, Unit Cell window with the first (low angle) sweep running.
15 Figure 12, Unit Cell window with one of the high angle sweeps running and resolution arc tool.
▪
When all 60 frames are collected, the program will automatically proceed to , select an I/sigma(I) cutoff value for the diffraction spots to be used ((circled in
green in the diffraction image). Click on .
16 Figure 13, Unit Cell Harvesting Window
▪
Proceed to , use all default values and click on at the bottom of the
window.
17 Figure 14, Unit Cell Indexing Result Window
▪
Check the unit cells obtained. The positions of the diffraction spots (ignoring natural
decay events, recognizable as very intense but very sharp “spikes”) should agree with the
predicted positions (circled in diffraction images).
▪
If one of the two unit cells looks reasonable, select it and click . This will open
the Refine Window. Use all defaults and click on to run a least squares
optimization of the unit cell against the diffraction spot position. Select more or less
reflections using the sliding bar and repeat, then click .
18 Figure 15, Unit Cell Refinement Window
▪
If there are excess spots not assigned to the cell a non-merohedral twin might be present,
or the specimen is not a single crystal (multi-domain). Try to find a better, more
obviously single crystal. If this is the best crystal that can be found, investigate if it can
be processed as a twin. Depending on the case at hand there are two options:
A) The unit cell appears correct, but there are excess diffraction spots that indicate presence
of a second domain: Proceed to . Click the button (details
not covered in this manual (yet)).
B) The unit cell appears wrong or makes no sense at all: First go back to . At the
bottom of the Index window uncheck the options for and . Select instead. You can narrow down acceptable
unit cells via the option. Click . This will use the
program “Cell Now” to look for unit cells. If a reasonable cell is obtained but there are
19 excess diffraction spots that indicate presence of a second domain: Proceed to
. Click the button (details not covered in this manual (yet)).
C) No reasonable cell is obtained via options A) and B): Export the data as a p4p file using
, then and use the program Cell Now to determine the unit cell and
relationships between domains (see the Purdue Twinning Manual for details).
▪
The remainder of this manual assumes the crystal not to be twinned: Proceed to .
A list of possible Bravais lattices will be displayed with the software's choice highlighted
in green.
Figure 16, Bravais Result Window
The correct choice should have a Figure of Merit (FOM) value significantly higher than
the others. If several solutions have similarly high FOM values, the one with the highest
symmetry is likely to be correct. If you are not sure (e.g. when a high symmetry solution
20 has a high FOM value, but significantly lower than a lower symmetry solution) select the
lower symmetry solution to avoid collecting incomplete data.
▪
Make your choice of Bravais lattice, then click . This will open the second
Refine Window.
▪
Use all defaults and click on to run a least squares optimization of the unit cell
against the diffraction spot positions (constrained to the chosen Bravais lattice). Select
more or less reflections as desired and repeat, then click .
▪
Proceed to , click , then .
▪
This finishes the Unit Cell Determination.
21 Data Collection
• Click < Calculate Strategy> under .
▪
Under Step 1., change the resolution to d = 0.82 Å or smaller. The worst acceptable
value, by IUCr standard, is 0.83 Å. The best resolution that can be set on this instrument
is 0.78 Å. Collecting to 0.78 rather than 0.82 increases the data collection time and is
only recommended for samples that do indeed diffract all the way to the detector edge.
Click if you made any changes.
Figure 17, Strategy Starting Window
▪
Under Step 2., click . In the pop-up window, it is usually safe to
use the default values. Click ,
22 Figure 18, Determine Strategy popup window
▪
Under Step 3., click
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