|
Column Selection Criteria
There is considerable opportunity
to improve your separation with a new and different HPLC column.
But, before you change, consider
how the following may be affected:
Selectivity - Resolution
- Peak Shape - Efficiency - Sensitivity
Run Time - Reproducibility
- pH Stability - Availability - Cost Effectiveness
Column
Criteria
Length
Efficiency
Direct correlation;
double the length/double the efficiency per column
Resolution
Direct
correlation; reduce length by one-half, resolution will decrease
(but often less than one-half)
Retention
Direct correlation;
double the length/double the retention time (with same flow rate)
ID (Diameter)
Efficiency
3/4.6mmID
typically have the highest efficiency. Microbore (1mmID), Narrowbore
(2mmID), and preparative columns (³ 10mmID) are generally
lower. Check manufacturer specifications.
Peak Shape
Wall effects on 1mm/2mmID
columns can cause some loss of peak symmetry.
Sensitivity
Decrease in
I.D. increases sensitivity by ratio of cross-sectional area.
((4.6)2mm/(3) 2mm = 21.16/9
= 2.35; thus 3mmID yields 2.35 times the mass sensitivity of
a 4.6mmID (same packing material: same sample load: assuming
no sample overload)
Packing
Material Criteria
Particle Size
Efficiency
Smaller particles
provide higher efficiencies.
Backpressure
Length
Longer columns
will give higher backpressures ( same packing material, same
ID, same flow rate)
ID(Diameter)
Smaller
IDs give higher backpressures if run at the same flow rate. The smaller
the
internal diameter of
the column, the less the solvent it will consume and the greater
the mass sensitivity. For example, you can expect a 2.0 mmID
column to consume about 80% less solvent, and have mass sensitivity
increase of 180% compared to a 4.6mmID column for the same application.
Surface Area
Higher surface area materials often give higher backpressures.
Particle Shape
Irregularly-shaped
materials often give higher backpressures than spherically-shaped ones.
Particle Size
Smaller particles
typically give higher backpressures. Case in point: a 3m C18
250 x 4.6mm column will usually
give unacceptably high backpressure; yet 5m C18
250 x 4.6mm columns are probably
the most common is use today.
Packing Material Column
backpressure will vary with different packings. Contact your supplier
for specifics.
Column
Selection Criteria
Bonded Phase
C18 (ODS),
C8, and C4 are usually the easiest to reproduce. Phenyls, amino-propyl
(NH2), and cyano-propyl (CN) are more difficult. Some "
"base-deactivated" and other specialty materials require techniques
or steps that can complicate reproducibility. We do not recommend
either NH2 or CN phases for reversed phase chromatography. In
the presence of water, these groups hydrolize, shortening the
lifetime of your column. However, they are fine materials for
normal phase chromatography.
Supplier
The manufacturer
of the HPLC column is responsible for column-to-column reproducibility
of peak symmetry and efficiency under standard Q.C. test conditions.
They are also responsible for packing the right material into
the column. Look for column-to-column reproducibility and keep
track of material lot numbers on the data sheet sent with the
column.
Peak Shape
Even properly
packed materials offer varying degrees of symmetry for ionized compounds.
Desired results can often be found by changing mobile phase, mobile phase
modifier, or ion pairing reagent. For assistance, please contact our technical
help department.
Retention
Higher surface area
and/or higher carbon loading typically increase retention times for
reversed phase separations, but
other factors can be affected.
Availability
Consider
: What scale of column is needed (1mm, 2mm, 3mm,4.6mm, 10mm,
³ 20mmID); What is standard delivery time?; Where can you
get it?; Is it cost-effective?
|
