[ CARE AND USE MANUAL ]
PIC Reagents
CONT ENT S
I. INT RODUC T ION
II. PAI R E D -ION C H ROMATOG RA P H Y
III. S E L EC T ING T H E R EAG ENT
FO R YOU R S E PA RAT ION
IV. USING AND STO RING WAT E RS P IC R EAG ENT S
V. O RD E RING INFO RMAT ION
I. INT RODUC T ION
An easy solution to the problem of analyzing samples containing
ionic species, PIC allows ionic compounds to be separated by
reverse-phase chromatography while eliminating problems of
precise pH and temperature control, reproducibility and short
column life associated with ion exchange. PIC’s versatility can save
time by allowing simultaneous assay of acids, bases, amphoteric
and neutral compounds.
Waters PIC ® reagents are:
1. Purified and quality controlled by liquid chromatography to
guarantee consistent results
2. Preformulated for ease of use
3. Buffered to an appropriate pH for most acidic and basic compounds
Paired-Ion Chromatography (PIC)
Use Low UV PIC to analyze ionic compounds at low UV (200nm).
Low UV PIC is specially formulated to eliminate background
interference in the low UV range and provides:
■■
Methods development opportunities for ion pairs which do not
have a chromophore in the working range of regular PIC reagents
■■
Increased sensitivity potential for compounds ordinarily used
with existing PIC reagents
■■
Potential for selectivity changes due to new buffers as compared
to regular PIC reagents
Use PIC A for separation of acids.
Use the PIC B series to separate bases.
Use Reagent D4 as a mobile phase modifier when separating basic
pharmaceutical compounds to improve peak shape and reduce
retention times.
Low-UV PIC reagents are also available, for use in separations of
compounds that have chromophores below 240 nm.
[ CARE AND USE MANUAL ]
Table 1. PIC Reagents
Reagent
Compound
PIC A
Tetrabutylammonium phosphate
Low UV PIC A
Tetrabutylammonium hydrogen sulfate
PIC B5
Pentane sulfonic acid
PIC B6
Hexane sulfonic acid
PIC B7
Heptane sulfonic acid
PIC B8
Octane sulfonic acid
Reagent D4
Dibutylammonium phosphate
III. S E L EC T ING T H E R EAG ENT
FO R YOU R S E PA RAT ION
Use PIC A Reagent to increase the retention of acids. Use the
PIC B Reagent to increase the retention of bases. Which of the four
B reagents to use depends upon your sample. Retention increases
with increasing reagent chain length. It is usually best to start
with PIC B7. If more retention is needed, use PIC B8. If less is
needed, use PIC B5 or B6.
You can also use the concentration of organic modifier in your
mobile phase to manipulate the retention of all compounds,
including neutral compounds.
II. PAI R E D -ION C H ROMATOG RA P H Y
■■
Increases the retention of oppositely charged solutes
■■
Decreases the retention of equally charged solutes
■■
Does not affect the retention of uncharged solutes
For a detailed description of the retention mechanism, see
B.A. Bidlingmeyer, S.N. Deming, W.P. Price, B. Sachok and
M. Petrusek, “Retention Mechanism for Reversed-Phase
Ion-Pair Liquid Chromatography”, Journal of Chromatography
186 (1979), 419-434.
PIC Separation of Water-Soluble B Vitamins
3
With
PIC B5 reagent
2
With
PIC B6 reagent
2
1
1
4
10 min.
3
4
Inject
exchange separations on a reversed-phase column. Hydrophobic
paired-ion reagents added to the mobile phase are adsorbed
onto the stationary phase. There they enhance the interaction of
oppositely charged sample components with the stationary phase.
A paired-ion reagent:
Inject
Paired-ion chromatography is a technique for performing ion
10 min.
Column:
μBondapak C18, 3.9 x 300 mm steel column
Part number:
WAT027324
Mobile phase:
Methanol/water 50:50 with PIC Reagent
Flow rate:
2.0 mL/min
Detection:
254 nm
Sample:
1. Niacinamide
2. Pyridoxine
3. Thiamine
4. Riboflavin
PIC Reagents
2
[ CARE AND USE MANUAL ]
IV. USING AND STO RING WAT E RS P IC R EAG ENT S
Table 2 suggests the PIC reagent to use to separate the
components of some typical compounds on a C18 column with a
water/methanol eluent.
Waters PIC Reagents are premixed and buffered. Add 1 vial of
reagent to 1 L of mobile phase and filter.
Table 2. Reagents to Use for Common Compounds
Compound or Class
Reagent Compound or Class
Alkaloids
Reagent
Antitussives
B
Cephaeline
B
Vitamins
Emetinee
B
Calcium Leucovorin
A
Ergot alkaloids
B
FD&C Dyes
A
Folic Acid
A
Analgesics/Antipyretics
B5/B7
A
Aspirin
A
Benzalkonium
Chlorine
B
Codeine
B
Cetylpyridinium
Chloride
B
Indomethacin
A
Imides
A
Meperidine
B
Methotrexate
A
Methadone
B
Petroleum Sulfonates
A
Morphine
B
Surfactants
Propoxyphene
B
Cephalosporins
A or B
Penicillins
A or B
Antihistamines
Store unopened reagent for up to one year at room temperature.
Mobile phase containing the reagent can be stored at 4 °C
indefinitely without loss of effectiveness.
Germicides
APC
Antibiotics
Note: Crystals may form when vials are exposed to lower
temperatures. If crystals have formed, gently warm vial until
crystals go back into solution.
Quaternary amines
B
Sulfonates
A
Sympathomimetics
(Decongestants)
Amphetamine
B
Ephedrine
B
Chlorpheniramine
B
Epinephrine
B
Diphenhydramine
B
Isoproterenol
B
Methapyrilene
B
Naphazoline
B
Phenindamine
B
Nylidrin
B
Pyrilamine
B
Phenylephrine
B
Tripelannamine
B
Phenylpropanolamine
B
Pheniramine
B
PIC Reagents
3
[ CARE AND USE MANUAL ]
V. O RD E RING INFO RMAT ION
Table 3. PIC Reagents
Description
Workable UV Range in
Mobile Phase (nm)
Quantity
(vials/pkg)
Part. No.
200+
240+
5
5
WAT084189
WAT085101
200+
240+
5
5
WAT084198
WAT085110
200+
240+
5
5
WAT084199
WAT085140
200+
240+
5
5
WAT084282
WAT085103
200+
240+
5
5
WAT084283
WAT085142
215+
5
5
WAT085144
WAT085466
PIC A (tetrabutylommonium hydrogen sulfote)
Low UV PIC A
PIC A (tetrobutylommonium phosphate)
PIC B5 (pentane sulfonic acid)
Low UV PIC B5
PIC B5
PIC B6 (hexane sulfonic acid)
Low UV PC B6
Pic B6
PIC B7 (heptane sulfonic acid)
Low UV PIC B7
PIC B7
PIC B8 (octane sulfonic acid)
Low UV PIC B8
PIC B8
PIC Sample Kit (One vial each: PIC A, B5, B6, B7 and B8)
Reagent D4 (dibutylammonium phosphate)
Waters, T he Science of W hats Possible, and Waters PIC are registered trademarks of Waters Corporation.
All other trademarks are the property of their respective owners.
©2016 Waters Corporation. Produced in the U.S.A. May 2016 WAT020065EN Rev 4 IH/ PDF
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F: 1 508 872 1990
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