as a fundamental characteristic of chemical substances , the phenomenon of chirality also exists in natural products .
for instance , isatidis radix ( root of isatis indigotica fort . ) is one of the most commonly used traditional chinese medicines in china , whose indications include pestilence and seasonal toxin , fever and sore throat , macula and papule caused by warm toxin , mumps , scarlatina , erysipelas facialis , erysipelas , and swelling abscess . antiviral activity of this herb has drawn great attention due to its successful application in epidemics like severe acute respiratory syndrome ( sars ) and avian influenza [ 24 ] .
results of in vivo studies indicated that epigoitrin ( ( + ) -(r)-goitrin ) was the therapeutic agent [ 57 ] .
however , neither of the epigoitrin used in the studies was optically pure when the source of the material used in their reports was investigated . by testing their optical rotation values
, it was found that they were mixtures of unequal amounts of ( + ) -(r)-goitrin ( epigoitrin ) and ( )-(s)-goitrin ( goitrin ) , which could not be resolved by regular c18 column . here
, the mixture was named as  ( r , s)-goitrin .  to address this issue , a normal phase hplc ( nplc ) based methodology was developed by the authors to separate r - goitrin and s - goitrin and was applied in chiral analysis of isatidis radix .
the structure of the enantiomers can be seen in figure 1 . over the past decades
, chiral separation has become an important research topic in the analytical science arsenal [ 911 ] .
individual enantiomers of chemical drugs may evince very different bioactivities and/or biotoxicities , so do natural products .
while one isomer possesses a therapeutic effect , its enantiomer may be inactive and antagonistic or even has reverse effect . with this perspective in mind
, it is imperative to investigate pharmacology and toxicology of ( r)-goitrin and ( s)-goitrin , respectively , which means large amount of resolved enantiomers need to be prepared for tests in vivo and in vitro . generally , hplc is the most frequently used technique for preparative separation [ 1215 ] .
but when the established nplc method was scaled up into a high - throughput way , things became less efficient due to long analytical time ( about 30  min ) , large solvent consumption ( especially non - environmentally friendly solvent such as n - hexane ) , and heavy solvent recovery work .
luckily , industrial application of supercritical fluids offered the chemists a new option [ 16 , 17 ] .
recently supercritical fluid chromatography ( sfc ) has been viewed as a preferred approach showing advantages in shorter time and lower cost [ 1822 ] .
major part of the solvent used in the mobile phase of sfc is supercritical co2 , low viscosity and high diffusivity of which allow higher flow rate than hplc .
, sfc was adopted to develop a more efficient method for separation of ( r , s)-goitrin , broadening its application in chiral investigation of tcm .
the analytical method was validated and compared to the former work in chromatographic performance . and the preparative method was used in purification of ( r)-goitrin and ( s)-goitrin .
all the solvents were hplc grade and were purchased from merck ( darmstadt , germany ) .
( r , s)-goitrin ( product number 111753 ) was from national institutions for food and drug control ( beijing , china ) .
analytical and preparative sfc separations were performed on a waters investigator sfc system controlled by chromscope software ( waters , milford , ma , usa ) .
the system consisted of a fdm10 fluid control module , an alias autosampler , a 10-port thar columns oven , and a 2998 photodiode array ( pda ) detector .
the daicel chiralpak adh , chiralpak ash , chiralcel odh , chiralpak ia , and chiralpak ic analytical columns ( 4.6  mm  250  mm , 5  m ) and the preparative scale chiralpak ic column ( 20  mm  250  mm , 5  m ) were purchased from daicel chiral technologies ( shanghai , china ) . the mobile phase was comprised of carbon dioxide and cosolvent .
the scan range of pda was from 190  nm to 380  nm and the compensated reference was set from 270  nm to 380  nm .
injection volumes were 10  l and 100  l for analytical and preparative separation , respectively . for analytical sfc , type and ratio of modifier / mixed modifier , column temperature , flow rate , and back pressure
were examined to adjust the mobile phase elution strength . for milligram sfc , ratio of modifier and flow rate
( r , s)-goitrin was prepared with 100% methanol for both analytical and preparative separation .
the proposed analytical method was validated as per the recommendations laid down by international conference on harmonization ( ich ) guideline .
for linearity test , stock standard solution was prepared by dissolving 20.0  mg of ( r)-goitrin and ( s)-goitrin in 10  ml methanol ( 2.0  mg / ml ) , respectively . for precision test , the intraday and interday variation for determination were carried out at three different concentration levels : 0.0003 , 0.1 , and 2.0  mg / ml .
the limit of detection ( lod ) and limit of quantitation ( loq ) were separately determined at a signal to noise ratio ( s / n ) of 3 and 10 .
initial chiral screening was automated on sfc using 5 different polysaccharide csps ( chiralpak adh , chiralpak ash , chiralcel odh , chiralpak ia , and chiralpak ic ) cross - matched with 5 different organic modifiers ( meoh , etoh , acn , ipa , and 50  :  50 meoh / etoh solvent ) .
for each combination , the percentages of the modifier were set at 5% , 10% , and 20% . though clear separation of r - goitrin and s - goitrin could be observed either on chiralcel odh coupled with meoh or on chiralpak ic coupled with acn , the greatest potential for preparative purification was demonstrated on the latter combination .
therefore , further sfc conditions were optimized on chiralpak ic column using acetonitrile as cosolvent .
four parameters , namely , modifier concentration , column temperature , flow rate , and back pressure , were chosen as optimization factors . retention time ( rt ) and resolution ( rs ) were used as criteria to evaluate the separation quality . as can be found in tables 14 ,
composition of the mobile phase shows dominant influence on rt and rs , while the other 3 factors have relatively weaker impact on separation .
it is obvious that when less acetonitrile is used , larger resolution will be obtained .
finally , the condition that gave sufficient resolution and relatively shorter analysis time was chosen . at 35c , 15% acn ( 3.5  ml / min ) , and 100  bar , satisfactory resolution ( rs = 3.75 ) and quick separation ( within 5  min ) were achieved .
the total analysis time represented a tenfold increase in speed , compared to the authors ' previous established nplc method ( see figures 2 and 3 ) .
interestingly , the elution order of ( r)-goitrin and ( s)-goitrin was reversed on the same column in sfc and hplc ( figures 2 and 3 ) . in the range of 0.00032.0 
mg / ml , the linearity of calibration graphs and adherence of the system to beer 's law were validated by high value of correlation coefficient .
the repeatability of sample application and measurement of peak area were expressed in terms of relative standard deviation ( rsd ) .
the lod and loq reached ng degree , much lower than those of the nplc method .
this improvement benefited from reference wavelength compensation technique we used in data acquisition , which collects wideband absorbance data in a region where the analytes have minimal or no absorption .
the detector calculates the compensation value by averaging the absorbance values within the selected range of wavelengths .
the averaged value is then subtracted from the absorbance value . since the main absorbance ( 190  nm to 380  nm ) includes the reference bands ( 270  nm to 380  nm ) , noises from mechanical and thermal noise can be effectively reduced .
all the method validation data are summarized in table 5 . while the analytical sfc condition produced adequate peak resolution in minimal run time , peak broadening
slight modifications were required for the preparative purification ( 20% modifier at a total flow of 9  ml / min ) . under these conditions ,
5  mg of ( r , s)-goitrin could be injected at 3  min intervals using injection stacking ( see figure 4 ) .
aliquots from the preparative collection vessels were removed and the individually isolated peaks were examined for chiral purity using the original analytical method ; each was found to be greater than 99.0% pure by diode array detection . optical rotation ( [ ]d ( c , 1.0 ; chloroform ) ) of the separated ( r)-goitrin and ( s)-goitrin was + 68.3 and 66.2 , respectively , which was close to the reported value .
the yield of the sfc preparation exceeded 90% , based on the total amount of the two isomers .
in summary , a novel analytical and preparative sfc separation method was developed for the separation of ( r , s)-goitrin . under optimized condition , ( r)-goitrin and
( s)-goitrin could be completely separated within 5  min , 10 times faster than the previous established nplc method .
in addition , preparative resolution of the enantiomers with sfc showed unequivocal advantages in shorter time , easier removal of solvent , and much higher throughput .
finally , the separated material is now available in pure enantiomeric form for use in the pharmacodynamics comparison to illustrate the effective substance in isatidis radix .
 like chemical drugs , research and development of herbal medicine also have a need to resolve enantiomers . 
 to help illustrating the antiviral bioactivity of isatidis radix , a widely used traditional chinese medicine ( tcm ) , supercritical fluid chromatography ( sfc ) was used for analytical and preparative separation of ( r , s)-goitrin , which was reported as the active ingredient of the herbal . improved resolution was achieved on chiralpak ic column , using acetonitrile as the organic modifier , representing a tenfold increase in speed , compared to the previous normal phase hplc ( nplc ) method . 
 the newly developed chromatographic method was validated in terms of linearity , precision , limit of detection ( lod ) , and limit of quantitation ( loq ) . 
 scale - up purification of ( r)-goitrin and ( s)-goitrin was performed on a preparative column with > 90% total recovery . the absolute stereochemical assignment of the purified isomers was determined through optical rotation study . 
 this attempt explored sfc 's application in chiral research of traditional chinese medicine . 
1. Introduction 
2. Experimental
3. Results and Discussion
4. Conclusion