ADV06-15_1-412

148 / 413

Easy Transfer of HPLC Methods to UHPLC
Using Fully Scalable Pinnacle' DB Columns
• Methods on Pinnacle' DB columns are easily transferred from 3 and Sum to 1.9Ilm, allowing faster analysis without losing
separation quality.
• Pinnacle' DB columns are 100% Restekmanufactured-from base silica to final packed column.
• Restekoffers the widest selection of stationary phases for UHPLC-more choices mean better selectivity for your analytes.
Ultra High Pressure Liquid Chromatography (UHPLC) is a rap idly growing techFigure 1 Chemical structures for example
nique that produces significantly faster ana lysis times compared to conventional sulfonamides.
HPLC. Wh ile transferring HPLC met hods to UHPLCcan increase sample through­
put, comparable met hod parameters must be used to maintain equivalent separa~~II O
tion s. Here we review which column prop erties and ope rating conditions sho uld H2O' ~S
o
:: NJ
remain consisten t and which need to be optim ized in order to maintain selectivity.
Sulfadiazine HN--<\ ;}
In this example, we will perform a scale-down method trans fer for sulfonamides
o 0b-~O
(Figure 1). For op timal selectivity and faster analysis times, we used a Pinnacle"! ~ /j I b
DB Biphenyl stationary ph ase for this app lication (Figure 2). When performing a 0(S'-., //
scale-down procedure, column pore size, carbon load, and support material must
remain the same . Changes to other para met ers can be made using a few simple cal~ ~ Sulfamethoxazole H2N
culations . Let's go th rou gh them sequentially.
Adjusting Column Size
Th e first calculation de termines the appropriate column length. Keeping the same colum n length while decreasing the par ticle size increases
the number of theoretical plates. Therefore , column length can be shortened with out losing resolutio n. Byadjus ting the column length prop­
erly, using Equatio n 1, we can maint ain the same separation.
Adjusting Injection Volume
Once we have determined the proper colum n length , we can calculate injectio n volume . Decreasing the column internal diameter and length
decreases the overall column volume and sample capacity. Therefore, we must alter the injectio n volume as descr ibed in Equat ion 2. Note
that since overall column volume has decreased, it is important to match the samp le solvent to the starting mobile phase composition.
Mismatched sample solvents can cause irreproducible retention times, efficiencies, and even changes in selectivity.
Adjusting Flow rate
Next, flow rate mu st be adj usted to maintain comparable linear velocity thro ugh a colum n with sma ller interna l diameter. To maint ain the
same linear velocity (which is importa nt in maintainin g efficiencies), flow rates must be decreased. Also, since smaller par ticle sizes give rise
to higher opt imal linear velocities, isocratic flow rates shou ld be calculated with particle size taken int o account. In this example, a gradie nt
elution was used and, th erefore, particle size was not included in the equation. Equation 3 can be used to estima te the adjuste d flow rate need­
ed for equivalent chroma tography. Also, note that since <Zum particle sizes are less affected by flow rate, faster flow rates can be used in iso­
cratic systems witho ut detrimental effects on peak efficiency.
Adjust ing Time Program
After determ ining the proper column length, injectio n volume, and flow rate, we can calculate the time needed for gradient or step elutions.
As an analytical meth od is scaled down, the time pro gram also needs to be scaled down to keep the phase interactio ns the same. Time can
be adjusted using Equation 4.
Figure 2 A 1.91lm Pinnacle' DB Biphenyl column is more selective for sulfonamides than a conventional C18 column.
A. Biphenyl Selectivity
Sample:
Inj.: 10!1L
Cone.: 100!1g/ mL
Sample diluent: startingmobile phase (80:20A:B)
Peak List: 1
l. sulfadiazine
2. sulfathiazole
3. sulfamerazine
4. sulfamethazine
5. sulfachlorpyridizine
6. sulfamethoxazole
7. sulfamethoxine
2
B. (18 Selectivity
Column: A. Pinnacie™ DBBiphenyl
Cal.#:
Dimensions:
Particle size:
Pore size:
Column:
Dimensions:
Particle size:
Pore size:
Conditions:
Mobile phase:
Flow:
Temp.:
Del.:
~
9409565 1,2
150mm x 4.6mm
5!1m
140A
B.Conventional C18
150mmx 4.6mm 5 6
5J.1m
140A
A: 0.1%formic acid in water
B: 0.1%formicacidin acetonitrile
Time(min.) %B
0.0 20
l. 0 20
6.0 80
8.0 80
l.OmL/ min.
300e
UV@ 254nm
\.J '-' ~ '-------'
1" I"""I " " ""' l" "" " 'I"" " " 'I' ;" " " ' j' " " "i ' I""""' I" " " "'I " " " "' j liill"ii'I''' ''''''I"'' ''' 111 "' ....1"'''''''1''..'' " '" i"" ,."", " " I''' '' ''''l
U U U U ~ U ~ U U U ~ ~ U ~ U ~ U ~
Iirre (min) lC_PH0461 Time(min) l C_PH0460
• 6 •
Website : E-mail : TelNo : 03 9762 2034 . . . in AUSTRALIA