Use of Unconventional Solvents as Mobile Phase Additives

Document Sample
Use of Unconventional Solvents as Mobile Phase Additives Powered By Docstoc
					 A Study of the Use of Unconventional Solvents As
Mobile Phase Additives with a Carbon-Clad Zirconia
Stationary Phase: Effects on Retention, Peak Shape,
Plate Count and Selectivity for Steroidal Compounds

                        HPLC 2003
                        Nice, France

  Dwight Stoll and Peter W. Carr, University of Minnesota
     Clayton V. McNeff, ZirChrom Separations, Inc.
                                     Abstract
Graphitic carbon and carbon-clad zirconia reversed-phase materials historically have suffered
from peak tailing, low column efficiency, and irreversible adsorption of some analytes.
Despite recent improvements in carbon-clad zirconia materials, poor performance is still
observed for very strongly retained analytes. In this work we systematically studied the use of
small amounts (0.1-10% (v/v)) of aliphatic alcohols, nitriles, and amines of increasing chain
length as mobile phase additives. The retention, peak shape, plate count, and selectivity for a
number of anabolic and estrogenic steroids were studied as a function of the type and amount
of additive in eluents consisting primarily of water and acetontrile. We show that long chain
alcohols and nitriles significantly decrease the retention, and also improve the peak shape and
plate count of these highly retained steroidal analytes on carbon-clad zirconia phases. In some
cases extraordinary selectivities were achieved in some otherwise very challenging
separations. Furthermore, the use of long chain alcohols and nitriles is advantageous
compared to previously recommended tetrahydrofuran and methyl-tert-butyl ether additives
when working with UV detection at low wavelengths because a substantially smaller amount
is needed and thus the resulting mixtures are more transparent. Finally, the practical utility
of these novel mobile phase additives is demonstrated in the fast (< 5 minutes) separation of
some difficult mixtures of anabolic steroids. The inherent chemical stability of the carbon-
clad zirconia stationary phase allows the use of elevated column temperatures and flow rates
with only modest column backpressures to greatly decrease analysis time.
                               Outline

    Why carbon phases for HPLC?
    Test solutes and solvent additives used in this work
    Solvent effects on retention of steroids
    Solvent effects on peak shape and efficiency of steroids
    Solvent effects on chromatographic selectivity
    UV spectra of eluent mixtures containing solvent additives
    Ultra-fast separations of closely related steroids at elevated
    temperature


Project Goal: To explore alternatives to the use of THF and MTBE
for improving the performance of carbon-clad zirconia phases
Why Carbon Phases for HPLC? Radically
        Different Selectivity!
     1.50

                        ZirChrom®-CARB
     1.00




     0.50

Log k'

     0.00
                                     ODS
     -0.50

                                                                 ZirChrom®-PBD
     -1.00




     -1.50
              1   2     3    4   5   6   7       8   9   10   11 12   13   14 15     16 17   18   19 20   21 22

1. N-benzyl formamide       6. Acetophenone          11. Benzene                  16. Bromobenzene      21. Propylbenzene
2. Benzylalcohol            7. Benzonitrile          12. p-chlorotoluene          17. Naphthalene       22. n-butylbenzene
3. Phenol                   8. Nitrobenzene          13. p-nitrobenzyl chloride   18. Ethylbenzene
4. 3-phenyl propanol        9. methyl benzoate       14. Toluene                  19. p-xylene
5. p-chlorophenol           10. Anisole              15. Benzophenone             20. p-dichlorobenzene
   Selectivity and Shape: Isomeric Analytes

        p-xylene                ethylbenzene
                   αODS=1.03

                   αC-Zr=1.58




C-C-C-C-C-C-C-C-C-C-C-C   C-C-C-C-C-C-C-C-C-C-C-C
       Long Retention and Poor Peak Shape in Traditional
                    Reversed-Phase Solvents
                                                                                                            CH3
   1000
                 ACN
                                                                                                            C
       900       ACN/Butanol                                                                          O           O

       800
                                                                                                      O           CH2
       700
                                                                                                      CH3         OH
       600                                                                             HO
 mAU




       500                                                                              CH3       H

       400
                                                                                              H       H
       300
                                                                           O
       200
       100
                                                                               Hydrocortisone acetate
         0
             0      1          2       3         4         5         6
                                   Time (min.)

LC Conditions: Mobile phase, gradient elution from 10-90% of organic modifier from 0-5 minutes, with 20mM
ammonium carbonate, pH 9.0 buffer; Flow rate, 2.0 ml/min.; Temperature, 80 oC; Injection volume, 1 µl; Detection
at 240 nm; Column, 50 mm x 4.6 mm i.d. ZirChrom®-CARB
  How Can We Overcome the Long Retention
          and Poor Peak Shape?

Historically organic modifiers such as tetrahydrofuran (THF) and
methyl-tert-butyl ether (MTBE) have been used to decrease
retention of strongly retained analytes, and improve their peak
shape, however these solvents have several undesirable
characteristics including:
      High UV-cutoff
      Incompatible with PEEK tubing
      Peroxide formation
      Toxicity

Project Goal: To explore alternatives to the use of THF and
MTBE for improving the performance of carbon-clad zirconia
phases
     Test Solutes Used in this Work
                                    Anabolic Steroids
                           OH                               OH                                   OH
                     CH3                              CH3                                  CH3



       CH3       H                        H       H                          CH3       H



             H       H                        H       H                            H       H


O                               O                                    O


     Boldenone                         Nandrolone                         Testosterone


                                Estrogenic Steroids
                           O                                     O                               OH
                     CH3                                                                   CH3



                 H                                                                     H



             H       H                                                             H       H

                                HO
HO                                                                   HO

      Estrone                            Equilin                           Estradiol
      Solvent Additives Used in this Work
Alcohols                                 Nitriles
   Methanol (HPLC grade, Pharmco)            Butanenitrile (98%, Pfaltz & Bauer)
   Ethanol (HPLC grade, Pharmco)             Hexanenitrile (98%, Pfaltz & Bauer)
   Isopropanol (HPLC grade, Pharmco)         Octanenitrile (98%, Pfaltz & Bauer)
   1-Butanol (HPLC grade, Fisher Sci.)       Octanedinitrile (98%, TCI)
   1-Octanol (HPLC grade, Aldrich)
   Cyclohexanol (99%, Aldrich)
                                         Others
                                            Tetrahydrofuran (HPLC grade, Pharmco)
                                            Methyl-tert-butyl ether (HPLC grade,
                                            Aldrich)
                                            Octylamine (99%, Aldrich)
      The General Experimental Design
The composition of the mobile phase was the main parameter studied in this
work. The mobile phase used in all of the work shown here consisted of a
base of 70/30 ACN/water (v/v). As an example, if 1% octanol was tested as
an additive, 1% of ACN was removed from the base mobile phase such that
addition of the octanol resulted in a mobile phase whose total organic
solvent composition was constant at 70% by volume.
All of the other chromatographic parameters were maintained constant as
follows:
  Column – 50 mm x 4.6 mm i.d. ZirChrom-CARB
  Flow rate – 2.0 ml/min.
  Injection volume, 5 µl
  Sample concentration – 10 µg/ml
  Temperature, 80 oC
  Diode array detection at 240 and 280 nm
                                                Effects of Solvent Additives on
                                                     Retention of Steroids
                                       Comparison of nitrile                                                                            Comparison of alcohol
                        16.0               modifiers                                                                             16.0       modifiers
                                  Boldenone                 Nandrolone                Testosterone                                      Boldenone   Nandrolone   Testosterone
                        14.0                                                                                                     14.0

                        12.0                                                                                                     12.0




                                                                                                     Retention Factor (k')
Retention Factor (k')




                        10.0                                                                                                     10.0


                         8.0                                                                                                      8.0


                         6.0                                                                                                      6.0


                         4.0                                                                                                      4.0


                         2.0                                                                                                      2.0


                         0.0                                                                                                      0.0




                                                                                                                                                           l




                                                                                                                                                          ol
                                                                                                                                                         er




                                                                                                                                                          ol
                                                                                                                                                           l




                                                                                                                                                          ol




                                                                                                                                                          F
                                                                                                                                                           l




                                                                                                                                                           l
                                                                                                                                                       no
                                                                                                                                                       no




                                                                                                                                                       no
                                                                                                                                                       no
                                  er




                                                                                                 F




                                                                                                                                                      TH
                                                                       ile
                                                            ile
                                              ile




                                                                                     ile




                                                                                                                                            1% an
                                                                                                                                                      ifi




                                                                                                                                                     an
                                                                                                                                                     an
                                                                                                TH




                                                                                                                                                    ha
                                                                                                                                                    ha
                               ifi




                                                                                                                                                    xa
                                                                                                                                                    pa
                                                                    itr




                                                                                                                                  od
                                                         itr
                                           itr




                                                                                  itr




                                                                                                                                                   ct
                                                                                                                                                   ut




                                                                                                                                                   ct




                                                                                                                                                  %
                             od




                                                                                                                                                 Et
                                                                                                                                                 et




                                                                                                                                                 he
                                                                  en
                                                     en
                                       en




                                                                               in




                                                                                                                                                 ro




                                                                                                                                                 O

                                                                                                                                                 O
                                                                                                                                 M
                                                                                            %




                                                                                                                                                 B




                                                                                                                                               10
                                                                                                                                               M
                                                                             ed
                            M




                                                                                                                                              op




                                                                                                                                              lo
                                                                                           10
                                                               an
                                                    an
                                      an




                                                                                                                                              %




                                                                                                                                              %
                                                                                                                             o




                                                                                                                                              %
                                                                                                                             N

                                                                                                                                    %




                                                                                                                                           yc
                        o




                                                                          an




                                                                                                                                          10




                                                                                                                                          10
                                                                                                                                          10
                                                                                                                                           Is
                                                             ct
                                    ut



                                                ex
                        N




                                                                                                                                  10




                                                                                                                                         C
                                                            O



                                                                        ct




                                                                                                                                        %
                                  B



                                              H




                                                                       O




                                                                                                                                       %
                                                                                                                                     10
                                                         1%
                               1%



                                           1%




                                                                                                                                    10
                                                                  1%




                        The highest carbon number additive has the largest effect on retention
                                              Effects of Solvent Additives on
                                                   Retention of Steroids
                                   Comparison of different                                                                         Comparison of 8-carbon
                                  concentrations of modifier                                                                            modifiers
                        16.0                                                                                            16.0

                                   Boldenone              Nandrolone             Testosterone                                     Boldenone                 Nandrolone                 Testosterone
                        14.0                                                                                            14.0

                        12.0                                                                                            12.0
Retention Factor (k')




                                                                                                Retention Factor (k')
                        10.0                                                                                            10.0

                         8.0                                                                                             8.0

                         6.0                                                                                             6.0

                         4.0                                                                                             4.0

                         2.0                                                                                             2.0

                         0.0                                                                                             0.0
                                  er




                                                                                       F
                                                                             e




                                                                                                                                  er
                                                 e




                                                               e




                                                                                                                                          ol




                                                                                                                                                                                             F
                                                                                                                                                                                   e
                                                                                                                                                          ile



                                                                                                                                                                       ile
                                                                                      TH
                                                                          in
                                              in




                                                            in




                                                                                                                                                                                in


                                                                                                                                                                                            TH
                               ifi




                                                                                                                               ifi



                                                                                                                                          an



                                                                                                                                                       itr



                                                                                                                                                                    itr
                                                                        am
                                            am




                                                          am




                                                                                                                                                                              am
                             od




                                                                                                                             od




                                                                                                                                                  en
                                                                                                                                        ct




                                                                                                                                                                 in
                                                                                   %




                                                                                                                                                                                        %
                                                                      yl
                                          yl




                                                        yl




                                                                                                                                                                            yl
                            M




                                                                                                                                       O




                                                                                                                                                                ed
                                                                                                                            M
                                                                                 10




                                                                                                                                                                                       10
                                                                                                                                                  an
                                                                    ct
                                        ct




                                                      ct




                                                                                                                                                                          ct
                         o




                                                                                                                        o


                                                                                                                                   1%




                                                                                                                                                            an
                                                                                                                                                ct
                                                                   O




                                                                                                                                                                         O
                                       O




                                                     O
                        N




                                                                                                                        N




                                                                                                                                               O



                                                                                                                                                          ct
                                                               %




                                                                                                                                                                     1%
                                    1%




                                                 0%




                                                                                                                                                         O
                                                                                                                                         1%
                                                              10
                                  0.




                                               1.




                                                                                                                                                   1%
                                Effects of Solvent Additives on Peak
                                          Shape of Steroids
                                Comparison of nitrile                                                                      Comparison of alcohol
                                    modifiers                                                                       1.0
                                                                                                                               modifiers
                  1.0
                             Boldenone                Nandrolone               Testosterone                         0.9   Boldenone   Nandrolone   Testosterone
                  0.9
                                                                                                                    0.8
                  0.8
                                                                                                                    0.7




                                                                                              Symmetry Factor
                  0.7
Symmetry Factor




                                                                                                                    0.6
                  0.6
                                                                                                                    0.5
                  0.5
                                                                                                                    0.4
                  0.4
                                                                                                                    0.3
                  0.3
                                                                                                                    0.2
                  0.2
                                                                                                                    0.1
                  0.1
                                                                                                                    0.0
                  0.0




                                                                                                                                             l




                                                                                                                                            ol
                                                                                                                                           er




                                                                                                                                            ol
                                                                                                                                             l




                                                                                                                                            ol




                                                                                                                                            F
                                                                                                                                             l




                                                                                                                                             l
                                                                                                                                         no
                                                                                                                                         no




                                                                                                                                         no
                                                                                                                                         no




                                                                                                                                        TH
                           er




                                                                                          F
                                                                 ile
                                                     ile
                                       ile




                                                                              ile




                                                                                                                              1% an
                                                                                                                                        ifi




                                                                                                                                       an
                                                                                                                                       an
                                                                                         TH




                                                                                                                                      ha
                                                                                                                                      ha




                                                                                                                                      xa
                        ifi




                                                                                                                                      pa
                                                                                                                     od
                                                              itr
                                                  itr
                                    itr




                                                                           itr




                                                                                                                                     ct
                                                                                                                                     ut




                                                                                                                                     ct




                                                                                                                                    %
                       od




                                                                                                                                   Et
                                                                                                                                   et




                                                                                                                                   he
                                                           en
                                              en




                                                                        in




                                                                                                                                   ro
                                en




                                                                                                                                   O

                                                                                                                                   O
                                                                                                                    M
                                                                                     %




                                                                                                                                   B




                                                                                                                                 10
                                                                                                                                 M
                                                                       ed
                      M




                                                                                                                                op




                                                                                                                                lo
                                                                                    10
                                                         an
                                             an
                               an




                                                                                                                                %




                                                                                                                                %
                                                                                                                o




                                                                                                                                %
                                                                                                                N

                                                                                                                      %




                                                                                                                             yc
                  o




                                                                   an




                                                                                                                            10




                                                                                                                            10
                                                                                                                            10
                                                                                                                             Is
                                                       ct
                             ut



                                         ex
                  N




                                                                                                                    10




                                                                                                                           C
                                                      O



                                                                 ct
                            B




                                                                                                                          %
                                       H




                                                                O




                                                                                                                         %
                                                                                                                       10
                                                  1%
                        1%



                                    1%




                                                                                                                      10
                                                           1%




                            There is considerable peak tailing, even in the best solvents
                                    Effects of Solvent Additives on Plate
                                              Count of Steroids
                                                                                                        Average plate count for three
                         5000
                                                                                                      anabolic steroids in top ten solvents
                         4000
Column Plate Count (N)




                                                                                                    5000




                                                                          Average Plate Count (N)
                         3000                                                                       4000

                                                                                                    3000
                         2000
                                                                                                    2000
                                                      Boldenone
                         1000
                                                      Nandrolone                                    1000
                                                      Testosterone
                           0                                                                            0
                                0        5             10            15




                                                                                                                                                    ol
                                                                                                                                            ol
                                                                                                            E




                                                                                                                                                                            F
                                                                                                                                                              l
                                                                                                                            e




                                                                                                                                                                   e



                                                                                                                                                                                   i le
                                                                                                                  e



                                                                                                                                 i le




                                                                                                                                                          no



                                                                                                                                                                         TH
                                                                                                                          in




                                                                                                                                                                   in
                                                                                                        TB

                                                                                                                  in




                                                                                                                                                 an
                                                                                                                                           an
                                       Retention Factor (k')




                                                                                                                                                                                 itr
                                                                                                                                it r




                                                                                                                                                         xa
                                                                                                                       am




                                                                                                                                                              am
                                                                                                             am
                                                                                                      M




                                                                                                                                                ct
                                                                                                                                       ct




                                                                                                                                                                              en
                                                                                                                            en




                                                                                                                                                                        %
                                                                                                                                                     he
                                                                                                                                            O
                                                                                                                                       O
                                                                                                                     yl




                                                                                                                                                              yl
                                                                                                             yl
                                                                                                    %




                                                                                                                                                                   10

                                                                                                                                                                            an
                                                                                                                           an
                                                                                                                  ct




                                                                                                                                                           ct
                                                                                                            ct




                                                                                                                                                    lo
                                                                                                                                           1%
                                                                                                                                 %
                                                                                                10




                                                                                                                                                                         ct
                                                                                                                       ex
                                                                                                                 O




                                                                                                                                                          O
                                                                                                         O




                                                                                                                                                 yc
                                                                                                                                10




                                                                                                                                                                        O
                          The effect of the type of
                                                                                                                       H
                                                                                                             1%




                                                                                                                                                      0%
                                                                                                     %




                                                                                                                                                C




                                                                                                                                                                    1%
                                                                                                    10



                                                                                                                  1%




                                                                                                                                            %
                                                                                                            0.




                                                                                                                                                     1.
                                                                                                                                           10
                          additive on the plate count is
                          random
                                               Effect of Solvent Additives on
                                                Selectivity for Six Steroids
                                            1.00
 % Decrease in retention upon addition of




                                            0.80
              10% Octanol




                                            0.60



                                            0.40



                                            0.20



                                            0.00
                                                   0   5        10            15            20         25   30
                                                           Original retention factor (k', ACN/Water)


There are only small changes in chromatographic selectivity
over a wide range in retention factor
                              UV Spectra of ACN/Water Eluents
                                   Containing Additives
                           Solvent mixtures containing 65/5/30 (v/v) of ACN/Indicated
                           Additive/Water were prepared and scanned from 200-300 nm.
                  2.0                                                             2.0                        ACN
                                                     ACN                                                     THF
                                                     Methanol
                                                     Ethanol                      1.6
                                                                                                             MTBE
                  1.6
                                                     Isopropanol                                             Octylamine




                                                                     Absorbance (AU)
Absorbance (AU)




                                                     Butanol                                                 Octanenitrile
                  1.2                                                             1.2
                                                     THF                                                     Octanedinitrile
                                                     MTBE
                  0.8
                                                     Octanol                      0.8


                  0.4                                                             0.4


                  0.0                                                             0.0
                     200     220      240     260      280     300                   200   220    240      260     280         300
                                   Wavelength (nm)                                               Wavelength (nm)


                  The relative similarity of the spectra for all mixtures containing HPLC
                  grade solvents indicates that the very strongest solvent additive should be
                  used, so that the relative amount of the additive can be reduced.
       A Fast Separation of Reproductive Steroids

                                                                Gradient Elution Profile
                                                               Time (min.)    %A        %B
      80                                                          0.0         90        10
                                                     5
      70                                                          1.5         10        90
      60                                                          2.0         10        90
      50                               3
                                                               A: 70/30 ACN/Water
mAU




      40                     2                                 B: 85/5/10 ACN/Octanol/Water
      30           1                       4
      20                                                       Flow rate: 5.0 ml/min.
      10                                                       Temperature: 80 oC
      0
                                                               Injection volume: 5 µl
           0.0     0.5           1.0           1.5       2.0
                                                               Detection at 220 nm
                          Time (min.)
                                                               Backpressure: 205 bar
                                                               Sample concentration: 2 µg/ml

       Solutes: 1-Boldenone, 2-Testosterone, 3-Equilin, 4-Estrone, 5-Estradiol
                        Conclusions
The addition of small amounts (0.1-10%) of higher alcohols, nitriles, and
amines to typical RPLC eluents dramatically reduces the retention of highly
retained steroids on carbon-clad zirconia
Octylamine was by far the most potent in terms of reducing the retention of
the molecules tested
Only minor effects on efficiency and peak shape were observed for the
steroid compounds under the conditions of this work
Only minor effects on chromatographic selectivity were observed upon
addition of small amounts of the solvent additives
A major limitation to the practical use of these unconventional solvent
additives is their general unavailability in sufficient purity for use in HPLC
Judicious choice of the correct organic modifier and additive, along with the
use of elevated column temperature, allows very fast separations of closely
related compounds on a carbon-clad zirconia RPLC column
Acknowledgements