WPI SpectroPipetter SPT User manual

SpectroPipetter SPT & SPT-2
Miniature sample cell for microliter sampling
INSTRUCTION MANUAL
Serial No._____________________
042302
World Precision Instruments, Inc.
www.wpiinc.com

SpectroPipetter SPT & SPT-2
WORLD PRECISION INSTRUMENTS 16
Contents
General Warnings and Cautions ..................................... 1
Instrument Description .................................................. 2
Wetting the sample cell for the first time ........................................................ 4
Proper handling of the pipetter ...................................................................... 5
Keeping the tip clean ..................................................................................... 6
Maintaining stability ........................................................................................ 8
Scattering for UV measurements ................................................................. 10
Sterilization ................................................................................................... 11
Storage ......................................................................................................... 11
Troubleshooting ............................................................ 12
Typical Contamination Effects ...................................................................... 12
Specifications ............................................................... 13
Accessories and Kits.................................................................................... 13
References .................................................................... 13
Warranty ....................................................................... 14
Copyright © 2002 by World Precision Instruments, Inc. All rights reserved. No part of this publication
may be reproduced or translated into any language, in any form, without prior written permission of
World Precision Instruments, Inc.

SpectroPipetter SPT & SPT-2
WORLD PRECISION INSTRUMENTS 1
General Warnings and Cautions
CAUTION: Do not try to dissamble the SpectroPipetter (SPT, SPT-2).
Disassembly by untrained personnel will damage the unit and invalidate the
warranty.
CAUTION: Do not apply any mechanical force to the tip of the SpectroPipetter.
Store it in the stand provided or in a secure place with the protective tip in place.
CAUTION: Clean the SpectroPipetter only with diluted Cleaning Solution
Concentrate (#15807). Do not use concentrated acids or bases; these solutions
may change the optical characteristics of the waveguide.
NOTE: Dilute hydrochloric acid (5-10% w/w) can be used to enhance the
efficiency in the detection range below 300 nm if measurement has fallen by more
than 30% of initial readings. Immediately flush with cleaning solution and then with
distilled water.

SpectroPipetter SPT & SPT-2
WORLD PRECISION INSTRUMENTS 2
Tip retainer — Holds the sample cell in
place.
Sample cell — Sample fluid is drawn into
and expelled from this microchamber by
means of a tiny side opening. A transparent
sleeve (not shown here) protects the sample
cell from accidental breakage when not in
use. See tip diagram, page 2.
Set screw — Two set screws on the
plunger handle control the zero position of
the plunger, and holds the plunger handle to
the main body. It is used mainly for calibra-
tion.
Main body — Serves as a base for the
sample cell and plunger and acts as the
primary handle for the instrument.
Plunger handle — As in a standard
pipetter, pushing and releasing the plunger
handle drives a piston that draws in and
expels fluid from the sample cell.
Instrument Description

SpectroPipetter SPT & SPT-2
WORLD PRECISION INSTRUMENTS 3
WPI’s SpectroPipetter is a spectrophotometry sampling device for spectral
analysis on fluid samples of less than two microliters. In its outward design, the
SpectroPipetter resembles a standard laboratory pipetter. Fluid is taken up and
expelled by means of a manual plunger, just as in a standard pipetter. The plunger
is made of a bundle of fiber optics protected by stainless steel tubing. In addition
to moving the sample in and out, it delivers and collects the light used for
absorbance measurements. The SpectroPipetter is connected to a
spectrophotometer by a fiber-optic cable, which passes through the pipetter’s core
and carries light to the delicate tip module. The tip module (see diagram), which
resembles a standard pipette tip, is
actually a tiny photo-optic component
which serves as the sample cell in
place of a cuvette. The fiber optic
cable from the pipette splits into two
cables each with a SMA connector
termination. The SPT cable labeled
with “Launch” is a 400 micron core
fiber [a bundle of three 200 micron
fibers in the SPT-2] that should be
connected to the light source(s). The
cable labeled with “Return” is a 100
micron core fiber [a 200 micron fiber in
the SPT-2] that should be connected to
the spectrometer. During the measurement, light is launched into the "Launch" fiber
and then transmitted to the pipetter where it passes through fluid held in the
sample cell, strikes a mirror, and reflects back to the “Return” fiber. Returning light
is routed to a fiber optic spectrometer.
In addition to the standard accessories, the optional Ultrasonic Cleaning Kit (WPI
part #UCK) is highly recommended for keeping the mirror clean and for removing
any residual sample fluid or dye from the sample cell. The kit contains an
ultrasonic cleaning bath for the tip module; a special stand for placing the
SpectroPipetter in the ultrasonic bath; and a bottle of concentrated cleaning
solution. The compact ultrasonic bath is specially designed for cleaning the
SpectroPipetter. A built-in timer provides 20 seconds’ cleaning with a high
intensity ultrasonic wave each time it is activated.
SpectroPipetter
tip
Piston is part of fiber
optic pathway Protective
metal sheath Mirror
Cell opening
Sample
After passing through sample,
reflected light returns to the
spectrometer
Quartz guide

SpectroPipetter SPT & SPT-2
WORLD PRECISION INSTRUMENTS 4
Wetting the sample cell for the first time
The SpectroPipetter is shipped dry and the sample cell requires a special wetting
procedure before its first use. To wet the sample cell, gently depress the plunger
handle with the thumb until the piston just touches the end of the sample cell. Do
not use force. When you feel resistance, the piston is in contact with the mirror.
Immerse the pipette tip in distilled water, and slowly release the plunger. Water
enters the sample cell through a side opening near the tip. Using a laboratory
tissue, blot the tip end while slowly depressing the plunger to expel the water
droplet. Refill and examine as described in “Air bubbles in the sample cell”
(page 8). If air bubbles persist, re-insert the tip into water while holding the plunger
handle depressed and lightly tap the main body to free bubbles clinging to the
inner surface, then slowly release the plunger.
Removing the air completely may be difficult at first, and these tactics may need to
be repeated several times. After this first wetting procedure, however, you will find
less trapped air when filling the tip.
Once the inner surface of the sample cell has been wetted and all air has been
expelled, keep the sample cell wet at all times by storing the pipetter in its stand
with its tip immersed in distilled water. If left exposed to air, the sample cell will dry
out very quickly, leaving a film on the mirror. This film, which will cause light
scattering, impairs the instrument’s sensitivity. A dry film such as this is difficult to
re-dissolve and may require extended cleaning in an ultrasonic cleaning bath. It is
important, therefore, to replace the pipetter in its stand (with the tip submerged in a
vial of distilled water or sample solution) as soon as one spectrum has been taken,
and to keep the sample cell continuously filled between one spectrum and the
next.
NOTE: If you plan to use the instrument over a period of several days, it is good
practice to keep the tip wet throughout this time to avoid repeatedly dealing with air
bubbles.

SpectroPipetter SPT & SPT-2
WORLD PRECISION INSTRUMENTS 5
Proper handling of the pipetter
The sample cell and piston, which make micro-volume measurements possible,
form the heart of SpectroPipetter. Although simple in appearance, they are delicate
optical components and should be handled carefully. Replacing these precision
components is very costly and will not be covered by WPI warranty if damage
occurs through abuse or improper usage.
The following precautions should be
observed:
The SpectroPipetter is shipped with a clear plastic protective sleeve. Remove
the sleeve and keep the pipetter in its stand with tip immersed in distilled water
between samples and when not in use. Save the protective sleeve — you will
need it when transporting or storing the SpectroPipetter.
After initial wetting, keep the sample cell wet.
Do not disassemble the pipetter or tamper with its adjustments. Contact WPI if
you are experiencing problems with the device.
Avoid dropping or bumping the pipetter. Although it can withstand a light touch,
bumping the sample cell or dropping the instrument onto a hard surface will
cause permanent damage.
Never force the piston. When filling or emptying the sample cell, depress the
plunger only until you feel resistance, indicating that the piston has touched the
mirror at the tip end. Excess force can crack the piston, scratch the mirror, or
break the tip.
Use safe, appropriate cleaning solutions. Cleaning solutions safe to use with
SpectroPipetter include distilled water, detergent, alcohol, acetonitrile, and
properly diluted WPI Cleaning Solution Concentrate (in the optional Ultrasonic
Cleaning Kit, part #15807). See below for further precautions.
Rinse the sample cell in distilled water between one sample solution and the
next. This helps to reduce contamination.
Use the ultrasonic cleaning bath supplied with your system to remove any
residue that does not rinse out easily with cleaning solution. To maintain
sensitivity and consistency and to prevent residue accumulation in the sample
cell, clean the pipetter tip in the ultrasonic cleaning bath before switching
between different solutions. See also the manual supplied with your ultrasonic
cleaner.

SpectroPipetter SPT & SPT-2
WORLD PRECISION INSTRUMENTS 6
Use the ultrasonic cleaning bath supplied with your system to remove any
residue whenever cross-contamination must be strictly avoided.
Never fill or immerse the sample cell in strong acid or strong alkaline solutions.
These will damage the metallic coating of the piston.
Never fill the sample cell with solutions containing solid particles. Solids may be
very difficult to remove and may become trapped in the sample cell. Solids
entering the sample cell may abrade the optical surface and permanently impair
sensitivity. Solutions should be carefully filtered before use in SpectroPipetter.
 Never fill the sample cell with solvents containing fluoride (such as artificial
blood)! Contact WPI if you have any doubt about how the solvent you plan to
use will affect the sample cell.
Never fill or immerse the sample cell in hydro fluoride acid. This will corrode the
optical fiber.
Keeping the tip clean
A small amount of residual fluid remains in the sample cell each time a sample is
expelled. This is generally less than 5% by volume and can be detected by
checking the absorbance baseline with the sample cell filled with reference
solution ( i.e. , distilled water). A small shift from zero generally indicates residual
fluid in the sample cell.
Routine rinsing with distilled water: Since most fluids are easily rinsed away,
form the habit of routinely rinsing the sample cell between one spectrum and the
next when there is any change in sample fluid. The simplest rinsing method
involves immersing the pipetter tip in clean, distilled water and drawing in and
expelling water from the sample cell. It is recommended that you repeat this 3-5
times, which takes only a few seconds.
Each rinse with cleaning solution reduces the residue by at least a factor of ten.
After five rinses, the residue will be less than 10-5 by volume of the sample
concentration.
To correct a baseline shift caused by residual fluid, set the spectrometer to scan in
repeat mode. Immerse the sample cell in distilled water. Draw in and expel water
from the sample cell repeatedly, while monitoring the absorbance baseline on
screen. You will notice that the baseline returns to its original level. Once the

SpectroPipetter SPT & SPT-2
WORLD PRECISION INSTRUMENTS 7
baseline shift is corrected, the sample cell is clean and ready to be used for testing
another sample.
Using alcohol or acetonitrile as cleaning solutions: Certain residues, of course,
cannot be removed easily with distilled water. These primarily include molecules
that are neutral in charge and/or are only slightly polarized, as, for example,
Brilliant Green dye. The best way to remove residues of this type is to substitute
alcohol or acetonitrile for distilled water. To do this, expel the sample fluid, then
draw in and expel alcohol or acetonitrile. Repeat from three to five times, then use
the on-screen display to recheck the baseline in reference solution.
Cleaning organic solvents: If the SpectroPipetter is used with non-water soluble
organic solvents such as acetone, THF, benzene, etc., a thin layer of solvent (or
chemicals that dissolved in the solvent) will remain on the surface of the cell when
it is used later for aqueous solution sample. That thin layer can severely reduce the
transmission of the light especially in the ultraviolet range. It may also affect the
baseline stability of the spectrum. To remove this layer, use the ultrasonic cleaner
to clean the pipette in an organic solvent that can dissolve both the thin layer and
the water. Acetonitrile, DMF and DMSO are good solvents for this purpose. Another
cleaning method is to gradually increase the polarity of the washing solvent in
several steps.
When to use the ultrasonic bath: Sample residue that persists after rinsing can
be removed by using the ultrasonic bath. This device sends ultrasonic waves
through the pool of cleaning solution, causing implosion of microscopic bubbles in
the cleaning solution. The collapsing bubbles create a vacuum that draws
contaminants away from the surfaces being cleaned. Residues are removed from
the mirror and inner surface of the sample cell. The ultrasonic bath should be used
whenever a residue cannot be removed by simple rinsing, or whenever cross-
contamination must be eliminated completely.
Any of the solvents mentioned previously (distilled water, detergent, alcohol,
acetonitrile, or diluted WPI ultrasonic cleaning solution) can be used with an
ultrasonic cleaner. We have found that properly diluted WPI Cleaning Solution
Concentrate is effective for most applications.
Setting up an ultrasonic bath: Put the diluted cleaning solution into a 20 mL glass
scintillation vial, and set the vial in the ultrasonic tank which is partially filled with
distilled water. (Follow manufacturer’s directions for diluting commercial solutions.)

SpectroPipetter SPT & SPT-2
WORLD PRECISION INSTRUMENTS 8
In this way, you can quickly change cleaning solution by switching one vial with
another if the first should become contaminated or prove to be unsuitable.
Using the ultrasonic bath: To use the ultrasonic cleaner, turn it on (
i.e. , pulsing)
and set spectrometer to scan in repeat mode. Immerse the sample cell into the
cleaning solution, wait a moment, and then depress and release the plunger
several times. The spectrum will appear quite erratic. This is because the
ultrasonic cleaner creates air bubbles which cause instability in the
SpectroPipetter. After a few seconds, turn off the ultrasonic bath and draw in
cleaning solution. Examine the spectrum. If it has reached its previous baseline
level, the tip is clean and ready to be used to measure another sample. If it has not
reached its previous baseline level, repeat the procedure. The tip can usually be
cleaned in less than 10 seconds. However, at times, removing a residue may take
several minutes.
Note: Depressing the reset button activates the ultrasonic cleaner for about 20
seconds before it automatically shuts off.
Maintaining stability
The SpectroPipetter’s unique optical system entails challenges to stability that are
very different from those encountered in conventional spectrophotometry.
Controlling the baseline shift is of first importance to SpectroPipetter users.
Instability arising from conditions within the pipetter can be pronounced if the
instrument is poorly understood or improperly used.
Understanding the various factors contributing to baseline shift can help you to
avoid them for the most part and can help you to diagnose and correct any
problems that do occur. Very often, multiple factors are involved. To aid you in
troubleshooting stability problems, we have included the following discussion of
possible causes. This list of contributing factors is not all-inclusive, however. In all
likelihood, as applications for SpectroPipetter continue to be developed, more
factors contributing to baseline shift will be documented.
Air bubbles in the sample cell: The main characteristic of air bubble-induced drift
is that it is unstable — the baseline shifts by different amounts each time the
plunger is pumped. Larger air bubbles can cause a baseline shift of more than
1AU. Large air bubbles can be removed by tapping the body of the pipette while
lightly depressing the plunger against the mirror, when the sample cell is