Generic Method Development Strategies in Chiral HPLC, for Analytical & Process Methods on QuikPrep™ CelCoat™ RBC-1
1) Understanding the CelCoat™ Chiral HPLC phase
In common with historic, and clones of historic cellulose coated, silica based, CHIRAL HPLC phases, CelCoat™ uses macro-porous silica, which has bonded to it a polar, cationic short-carbon chain entity, with amine functionality. This matrix then has alkylphenyl-carbamoyl derivatised cellulose precipitated on to its surface and into its porous matrix structure.
The derivatised cellulose entity is not bonded, but coated onto the surface; therefore care has to be taken to avoid solvents, which would dissolve this coating. In particular DMF, Acetone, THF and chlorinated solvents should not be used.
O
ne
advantage of a coating to a bonding of the cellulose derivative, for chiral
HPLC, is that the coated structure is more easily deformable, which enhances
it's chiral, and general spatial recognition abilities as compared to more
rigidly bonded, historic cellulose or amylose derivatives.If one considers the functionalities involved, which range from silanols on the silica, amine on the bonding and those coated cellulose / alkylphenyl-carbamoyl entities, many and varied ionic, non-ionic and spatial bonding interactions are feasible in cellulose coated, chiral HPLC.
For certain ionic molecules it is reasonable to predict that infinite retention could occur without the use of competing anions / cations or ion-pairs.
For generic methods, many favour limiting solvent matrices to organic solvents without ionic or ion pair additives, but by definition, reference the above, these will always be of more limited generic chiral HPLC application, than those with ionic / ion-pair functionalities present.
2) Choice of solvent for CHIRAL HPLC
Solvents than must not be used, this list is not all-inclusive and users should never use a solvent know to dissolve cellulose.
DMF, Acetone, THF, Dichloromethane, Chloroform
Solvents that should be limited to 0 to 5 %
Ethyl Acetate
Solvents that should be limited to 0 to 60%
Note : If using in Polar Organic Mode add IPA so the total combined mix of Ethanol and/or Methanol does not exceed 60%. For Normal Phase make appropriate allowance for the limited miscibility in Ethanol or Methanol in n Hexane etc.
Methanol, Ethanol
Solvents suitable for 0 to 100%
n-Hexane, iso-hexane, cyclo-hexane, heptane, IPA, Acetonitrile ( For Acetonitrile be aware of its very limited miscibility in n Hexane etc ). Liquid CO2 (SFC) best non-polar solvent.
Preferred solvent for NON-POLAR, ORGANIC NORMAL PHASE CHIRAL HPLC
Preferred generic gradient with no ionic / ion-pair agent
(A) Solvent : your choice of non-polar solvent ( preferred CO2 )
(B)
Solvent : 50% IPA & 50% Ethanol; 50% IPA & 50% Methanol; Ethanol
& 50% Methanol mixes, or less preferred, 100% Ethanol and least preferred,
100% IPA,
Typical Gradient; 99.6% (A) to 60% (A) + 40% (B)
Extended Gradient; 99.6% (A) to 99.6% (B) for……..50% IPA &
50% Ethanol, 50% IPA & Methanol, 100% IPA,
BUT LIMITED TO…… 99.6% (A) to 50% (A) + 50% (B) for 50% Ethanol
& 50% Methanol & 100% Ethanol owing to the limited miscibility
of Ethanol & Methanol in Hexane etc
In general for the
non polar solvent choice n-Hexane, iso-hexane, cyclo-hexane,
heptane will all give somewhat similar chiral HPLC selectivity, cases
will occur where one solvent will have enhanced selectivity to another.
Heptane is generally considered a less hazardous solvent to use than hexanes,
but on several occasions with CelCoat™ RBC-1, n-hexane etc has given
preferable resolution / cost / availability advantages.
For the polar modifier choice, ethanol to a maximum of 50% in non polar,
or IPA up to 100% are possibilities, as are mixes.
The traditional preferred choice of polar modifier is 0.4 to 40% IPA on
historic cellulose coated phases. The low backpressure capability of historic
cellulose coated phases would greatly reduce the flow at 100% IPA to maybe
a fraction of an ml / min for 4.6 x 250, 5 micron hplc columns.
The 2000 psi capability of CelCoat™ means that extended chiral HPLC
gradients from 99.6% non polar to 100% IPA at 0.5 to 0.6 ml /min are possible,
on 4.6 x 250 mm CelCoat 5 micron columns, but the pump should be set at
2000 psi maximum cut-out to avoid over pressurising the column.
We have completed numerous cycles of this gradient over 30 to 60 minutes
duration per gradient with reproducible chromatographic performance for
sequential runs.
In general a truncated gradient from 99.6% non polar to 60% non polar
/ 40% IPA is adequate for most compounds, and then a flow of 1 ml / min
can be used (but always with a pump set at 2000 psi maximum cut-out to
avoid over pressurising the column).
It is the high backpressure generated by the use of IPA that is a limiting
factor, combined with its poorer chiral HPLC selectivity and greater tailing
that favours the use of 100% Ethanol and mixes over 100% IPA.
By making the polar modifier a mix of IPA & ethanol, the backpressure
generated can be very substantially reduced.
For reasons as yet undefined, we usually find a mix of polar modifier
of 50% IPA & 50% Ethanol, or 50% IPA & 50% Methanol as the B solvent
in a gradient often CONSIDERABLY IMPROVES RESOLUTION & REDUCES TAILING
AS WELL AS REDUCING BACKPRESSURE as compared to 100% IPA. A 100% Ethanol
is usually preferable to 100% IPA, but owing to its limited solubility
in Hexane etc a truncated gradient to 50% Hexane 50% Ethanol is required.
Please see our Application Note on Generic Chiral HPLC Gradients for an
example.
Preferred
solvent for NON-POLAR ORGANIC NORMAL PHASE CHIRAL HPLC with ionic or ion-pair
additives
As
stated above the ionic nature of the CelCoat™ phase can mean that
infinite retention of certain ionic compounds may occur without the presence
of an ionic or ion-pair modifier.
CelCoat™ has been design to work within the range of total ionic
/ ion-pair concentration of 0.01% to 0.1% v/v ( 0.2% v/v maximum ). This
can be as a single compound or as the total of a mix of compounds.
In general we would recommend for the acid, TFA. Other acid options are
Formic Acid & Acetic Acid. For the base TEA is often preferred. Other
base options include; di-ethyl amine ( DEA ) & di-n-butyl amine (
DBA ).
In the rare circumstance where the functionality of the compound is unknown,
or the base for base / acid for acid principle has been unsuccessful;
try acid for base, or base for acid or a mix of approximately 0.05% TFA
+ 0.05% TEA in both (A) and (B) solvents is recommended.
Because the solubility of TFA & TEA in non-polar solvents is limited,
we suggest adding 0.2% IPA as well to the (A) solvent to facilitate dissolution
of the TFA & TEA.
TFA
& TEA and other solvent may have significant UV absorbance’s
at various longer wavelengths, therefore when using generic gradients,
we recommend slight modifications of concentrations of TFA & TEA in
both the (A) & (B) solvent to accurately equate the absorbance of
these start solvents at the UV / Visible wavelength you are about to work
at (simply us a syringe to fill UV spectrophotometer cell, monitor (A),
compare to (B) and change content of TFA & TEA to equate (A) to (B)).
Care must be taken
with the use of ACID / BASE as CelCoat™ like historic cellulose
coated phases can be modified after exposure to either. It is recommended
to set a column aside for no ACID or BASE exposure and keep separate from
those that have had this treatment.
Preferred
solvent for POLAR ORGANIC NORMAL PHASE CHIRAL HPLC with ionic or ion-pair
additives
(A) Solvent : Preferred;
40% IPA & 60% Ethanol, 40% IPA & 60% Methanol; 100% Acetonitrile
(wash
with IPA or IPA/Ethanol if coming from Hexane mix for for the latteroptions),
less preferred 100% IPA……. TO BE AVOIDED more than 60% total;
as separate or as a mix of Methanol or Ethanol(B)
Solvent : Your chosen above, plus acid or base modifier
Run gradient from (A) to (B). A small amount of Ethyl Acetate may be added
to (A) to equate the UV / Visible absorbance to (B) at the chosen wavelength
(as above)
Preferred solvent for REVERSE PHASE CHIRAL HPLC
A CelCoat™ chiral column can be converted
to aqueous reverse phase by flowing 20 bed volumes of 50% IPA & 50%
Ethanol through the column prior to use of any water.
Once the CelCoat™ has had any water passed through it, we highly
recommend that the column is then exclusively kept for aqueous reverse
phase use.
DMF / THF MUST NOT BE USED AS A MODIFIER IN AQUEOUS
REVERSE PHASE FOR CELCOAT™
Most Polar solvent (A) : Water with 10% + of
Polar Organic Modifier
Less Polar solvents (B) : 10% to 100% Acetonitrile preferred,
also 10% to 60% Ethanol or 10% to 60% Methanol or mixes of all former.
A less preferred option would be, 10% to 100% IPA ( take care with backpressure,
2000 psi max )
To
date Acetonitrile, Ethanol and Methanol, plus in particular mixes of Acetonitrile
/ Ethanol / Methanol have proven the most effective (B) solvent modifiers
for CelCoat™ RBC-1
Again the use of ionic / ion-pairs is recommended, TFA / Formic Acid /
Acetic Acid / Phosphoric Acid etc for Acids & TEA / DEA / DBA etc
for Bases at typically 0.025 to 0.1% v/v. The absolute maximum v/v concentration
singly or as a combined total is 0.15% v/v.
Ammonium Formate or Ammonium Acetate at 10 to 20 mM range may also be
utilised.
The maximum pH range is a pH 2 to 8 inorganic buffer range.
3) Scale Up from ANALYTICAL to PROCESS CHIRAL HPLC
Certain users of historic cellulose coated silica have
informed us that the chiral selectivity on 5 & 10micron phase may
vary compared to 20 micron phase.
CelCoat™ uses identical silica specifications, coating etc for 5
& 10 & 20 & 20-45 micron (15 micron is a custom option)
We have not to date noted any significant selectivity differences between
the particle sizes/targets examined.
5, 10, 20 and 20-45 can all be custom supplied in trial 4.6 x 250 columns
and in preparative S/Steel
HPLC column hardware, 10, 21,
30, 50,
101 mm id S/Steel HPLC columns.
For CHIR
AL
ProcessPrep™ we offer 20, 20-45 micron BULK PHASE direct to end
users and CelCoat™ can also be custom packed into a fully patented
design of axial & radial polypropylene cartridge, which range in capacity
from 80g up to 4500g per cartridge.
The use of the QuikPrep™’s, ProcessPrep™ pre-packed
cartridges for chiral process hplc ha
s
the very significant advantage that you, the Process Chromatographer,
no longer has to endure the safety risks associated with your self-packing
traditional process HPLC columns with loose, very fine, bulk silica based
phase.
This may also ease your requirements/difficulties regarding the writing
and implications of Heath & Safety Directives. We believe that to
date, we are UNIQUE in offering a
patented, pre-packed cartridge ProcessPrep™ concept chiral and non-chiral
process HPLC options.
Four sizes of S/Steel cartridge holders that provide the axial and radial
compression, are available as standard, these are the…….
ProcessPrep™ Jumbo 50 for 50 x100 &
50 x 250 cartridges
ProcessPrep™ Jumbo 80 for 400 & 800g cartridges
ProcessPrep™ Jumbo 140 for 2350g cartridges.
ProcessPrep™ Jumbo 200
for 4500g cartridges.
Larger cartridges and cartridge holders can be custom-made for very large
scale, chiral process hplc.
For Contact Details Click Here
Ordering
Information
While many of our competitors’ chiral columns retail at 3 to 4 times
the cost of quality reverse phase HPLC analytical columns, we sell CelCoat™
5 micron, analytical columns at approximately the same cost as the higher
cost, higher quality, reverse phase HPLC columns.
Distributed in USA and
Canada by:
Manufactured by:
3015 Barrow Drive, Raleigh, NC 27616 PO
Box 80, Bridgend, United Kingdom, CF31 4XZ Phone:
919.872.5700 Phone:
UK 1656 782 985
Toll Free: 866.4Chrom1 (424.7661) Fax:
UK 1656 789 282
Fax 919.872.5738 For Contact Details Click Here
techsupport@4chrom.com www.chiral-hplc.com
www.4chrom.com www.ccc4labprep.com
